UNIVERSITY OF CINCINNATI

Date:__10/23/2007______

I, __Stella A. Nicolaou______, hereby submit this work as part of the requirements for the degree of: Ph.D in: Pathobiology and Molecular Medicine It is entitled: K+ channel trafficking in the immunological synapse of human T cells in health and autoimmunity

This work and its defense approved by:

Chair: Dr. Laura Conforti Dr. Sean Davidson Dr. Alexandra Filipovich Dr. Robert Franco Dr. Judith Heiny

K+ channel trafficking in the immunological synapse of

human T cells in health and autoimmunity

A dissertation submitted to the

Graduate School

of the University of Cincinnati

In partial fulfillment of the

requirements for the degree of

DOCTOR OF PHILOSOPHY

In the Department of Pathobiology and Molecular Medicine

of the College of Medicine

By

STELLA A. NICOLAOU

B.Sc., Medical Technology

University of Indianapolis, 2002

Committee Chair: Dr. Laura Conforti

THESIS ABSTRACT

T cell receptor (TCR) engagement by an antigen presenting cell (APC) results in

reorganization of intracellular and membrane molecules at the T/APC interface, forming a “signalosome”, the immunological synapse (IS). An early event associated with T/APC interaction is Ca2+ influx. K+ channels, Kv1.3 and KCa3.1, modulate Ca2+ signaling in

human T cells. Resting and activated human T cells express both channels, albeit to

different degrees. Kv1.3 channels modulate Ca2+ in resting, while KCa3.1 channels do so

in activated, T cells. Although these channels play such an important role in Ca2+ homeostasis, very little is known about their localization in the IS. Furthermore, aberrant

T cell responses in IS formation and Ca2+ influx have been documented in T cells from

patients with systemic lupus erythematosus (SLE). The potential involvement of K+ channels in the etiology and progression of SLE remains unknown.

Herein we determined K+ channel membrane distribution in resting and activated human

T cells following TCR engagement and performed comparative studies with SLE T cells

to decipher the role of K+ channels in the Ca2+ response anomaly. Our data show that in

SLE T cells, Kv1.3 channels constitute the dominant channel and are functionally

identical to their normal counterparts. We also found that resting SLE T cells show faster

Kv1.3 kinetics out of the IS as compared to healthy T cells and comparable to healthy pre-activated T cells. However, normal pre-activated T cells recruit and maintain KCa3.1 channels in the IS after Kv1.3 channels leave, while SLE T cells do not express the appropriate KCa3.1 channel number to support this activated phenotype. This Kv1.3 mobility defect appears to be specific to SLE and not other autoimmune diseases, as it

was not observed in rheumatoid arthritis (RA) patients.

Further, transcription factor activation and gene expression relies on the shape of the Ca2+ response. Although SLE T cells demonstrate abnormal transcription factor regulation and gene expression, the potential contribution of the Ca2+ responses to these abnormalities

remains unknown. We performed single T cell Ca2+ response analysis to better define the

Ca2+ shape in SLE T cells. Our data suggest that there is an increase in cells with more

sustained Ca2+ response in SLE as compared to normal and RA T cells, while a transient,

short duration, Ca2+ response is more pronounced in normal T cells. We also found that

during and upon termination of the Ca2+ response, Kv1.3 channels are retained in the IS

in healthy T cells, implying a role for Kv1.3 in the termination of the Ca2+ response.

We conclude that altered localization of Kv1.3 channel in the IS of SLE T cells is at least

in part responsible for more sustained Ca2+ response in SLE T cells. This phenotype, in

turn, supports T cell hyperactivity in SLE T cells. Therefore we propose that Kv1.3

channels may offer novel therapeutic targets for SLE.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my advisor Dr. Laura Conforti whom I had the pleasure and honor of working with for the past four years. She has been a great and inspiring mentor and her guidance has been of tremendous value. Her ethical approach to science has taught me how to be a successful scientist myself. She pushed me to achieve more than I could have ever imagined and for that I thank her.

In addition, I would like to thank my thesis committee members, Drs. Sean Davidson,

Alexandra Filipovich, Robert Franco and Judith Heiny for all their input, guidance and assistance in completing this degree.

Next, I would like to thank all the past and current members of the Conforti lab. Personal thanks to Lisa Neumeier, the senior research assistant in the lab, for her help and support.

It has been a real pleasure working with her. I next wish to thank all of our collaborators whom without this work would not have been possible: Susan Molleran Lee, Dr. Heather

Duncan, Dr. Shashi Kant, Dr. Anne Barbara Mongey, Dr. Daniel Devor (University of

Pittsburg) and Dr. Koichi Takimoto (University of Pittsburg).

Special thanks to my sister, Soulla, who was always there after a tough day, and my parents, Eleni and Antoni Nicolaou, and my brother, Nicos, for their encouragement and support. This work was supported by National Institute of Health Grant CA95286 to L. Conforti and a pre-doctoral fellowship from the American Heart Association, Southern and Ohio

Valley Affiliate 0615213B to S. A. Nicolaou.

TABLE OF CONTENTS

Page Committee Approval i

Title Page ii

Thesis Abstract iii

Acknowledgements v

List of Tables 4

List of Figures 5

Abbreviations 8

CHAPTER I: Background and Thesis Scope 10

1.1 T cell activation, the Immunological Synapse and Calcium Signaling 11 1.1.1 Ion Channels in T cell Activation 14 1.1.2 T cell proliferation and K+ channel expression 16 1.2 Properties, structure and pharmacological characteristics of Ca2+ and K+ 18 channels 1.2.1 The CRAC channel. 19 1.2.2 The Kv1.3 channel 20 1.2.3 The KCa3.1 channel 25 1.3 K+ channels and disease 29 1.3.1 The Kv1.3 channel 29 1.3.2 The KCa3.1 channel 33 1.4 Systemic Lupus Erythematosus 35 1.4.1 The role of T cells in the etiopathogenesis of SLE. 37 1.4.2 T cell activation and Immunological Synapses in SLE T cells 38 1.4.3 Contribution of K+ channels in the pathophysiology of SLE 40 1.5 Scope of Thesis 40 1.5.1 Hypothesis 41 1.5.2 Organization of thesis 42 1.6 References 43

CHAPTER II: Altered dynamics of Kv1.3 channel 62 compartmentalization in the immunological synapse in systemic lupus erythematosus

- 1 - 2.1 Abstract 63 2.2 Introduction 63 2.3 Materials and Methods 66 2. 4 Results 72 2.4.1 Determination of T cell phenotype 72 2.4.2 Biophysical and Pharmacological characteristics of Kv1.3 channels 74 in SLE T cells 2.4.3 Native Kv1.3 channels are recruited in the immunological synapse 77 upon activation of healthy and SLE T cells 2.4.4 Kv1.3 channel compartmentalization in the immunological synapse 79 is altered in SLE T cells. 2.4.5 The kinetics of Kv1.3 redistribution in the immunological synapse 86 of SLE T cells resemble those of pre-activated normal T cells 2.4.6 T lymphocytes from patients with SLE display a K channel 90 phenotype similar to healthy resting T cells. 2.5 Discussion 92 2.6 References 97

CHAPTER III: The Ca2+-activated K+ channel KCa3.1 103 compartmentalizes in the immunological synapse of human T lymphocytes

3.1 Abstract 104 3.2 Introduction 105 3.3 Materials and Methods 106 3.4 Results 112 3.4.1 Electrophysiological and pharmacological profile of the cloned 112 YFP-KCa3.1 channel in HEK 293 cells matches the native KCa3.1 channel in human T cells 3.4.2 Overexpression of functional YFP-tagged KCa3.1 channels in 114 human primary T lymphocytes 3.4.3 KCa3.1 channels and F-actin redistribute to the T cell and anti- 117 CD3/CD28 antibody coated bead contact site 3.4.4 Calcium influx during antigen presentation and its regulation by 119 KCa3.1 channels 3.4.5 Redistribution of KCa3.1 channels at the immunological synapse 122 3.5 Discussion 127 3.6 References 132

CHAPTER IV: Differential calcium signaling in T lymphocytes 135 from patients with systemic lupus erythematosus

4.1 Abstract 136 4.2 Introduction 137

- 2 - 4.3 Materials and Methods 140 4.4 Results 149 4.4.1 Pattern of [Ca2+]i signaling in SLE T cells 150 4.4.2 Magnitude of [Ca2+]i in SLE T cells 153 4.4.3 Biophysical and pharmacological profile of pEGFP-Kv1.3 channels 157 in HEK 293 cells 4.4.4 Overexpression of functional pEGFP-Kv1.3 channels in human 160 primary T cells 4.4.5 Redistribution of Kv1.3 channels in the IS in Jurkat T cells 162 4.4.6 Kv1.3 trafficking to the IS and [Ca2+]i in human T cells 163 4.6 Discussion 166 4.7 References 171

CHAPTER V: General Discussion 176

5.1 K+ channel trafficking in the immunological synapse in healthy T cells 177 5.1.1 Membrane distribution of Kv1.3 channels in healthy resting T cells 177 5.1.2 Membrane distribution of Kv1.3 and KCa3.1 channels in healthy 179 activated T cells 5.2 K+ channel trafficking in the immunological synapse and Ca2+ mobilization 181 in SLE T cells 5.2.1 Membrane distribution of Kv1.3 channels in SLE resting T cells 181 5.2.2 Ca2+ signaling in SLE T cells 182 5.3 Future Directions 185 5.4 Clinical Relevance and Therapeutic Indications 186 5.5 References 187

- 3 - LIST OF TABLES Page CHAPTER I: Background and Thesis Scope

Table 1.1: Summary of T cell signaling defects in SLE 39

CHAPTER II: Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus

Table 2.1: K+ channel expression in SLE and normal T lymphocytes 76

Table 2.2: Details of SLE and RA patients in the microscopy studies 80

CHAPTER IV: Altered calcium mobilization in T lymphocytes from patients with systemic lupus erythematosus

Table 4.1: Quantitative analysis of [Ca2+]i in Normal, SLE and RA T cells 154

- 4 - LIST OF FIGURES

Page Chapter I: Background and Thesis Scope

Figure 1.1: Organization of a mature immunological synapse 12

Figure 1.2: Calcium and potassium channels in T cell activation 14

Figure 1.3: Differential expression of Kv1.3 and KCa3.1 channels in T cell 18 subsets

Figure 1.4: Side view of Kv1.3 main α subunit 21

Figure 1.5: Side view of KCa3.1 main α subunit 26

Chapter II: Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus

Figure 2.1: Expression of T cell subsets in SLE, RA and normal donors 73

Figure 2.2: Electrophysiological and pharmacological properties of Kv1.3 75 channels in SLE T cells

Figure 2.3: Kv1.3 channels are recruited at the interface between CD3/CD28 78 beads and T cells

Figure 2.4: Differential kinetics of Kv1.3 channel reorganization in the IS 82

Figure 2.5: Determination of specificity of anti-Kv1.3 antibodies 83

Figure 2.6: APC-T cell activation induces differential reorganization of Kv1.3 85 channels in the IS formed with resting healthy and SLE T cells

Figure 2.7: Kv1.3 channel recruitment in the IS in activated healthy T cells 86 parallels SLE T lymphocytes

Figure 2.8: Comparison of the rates of Kv1.3 channel compartmentalization in 88 the IS in normal and SLE T cells

Figure 2.9: The expression of KCa3.1 channels in SLE T cells is comparable to 91 that in resting healthy T cells

- 5 -

Chapter III: The Ca2+-activated K+ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

Figure 3.1: Determination of specificity of anti-KCa3.1 antibodies 113

Figure 3.2: Functional and pharmacological properties of recombinant YFP- 115 tagged KCa3.1 channels

Figure 3.3: Expression of functional YFP-tagged KCa3.1 channels in human T 116 cells

Figure 3.4: KCa3.1 channels and F-actin localize at the T cell/bead contact point 118

Figure 3.5: SEB pulsed B cell interaction with T cells induces an increase a 120 KCa3.1-dependent increase in cytoplasmic calcium in activated T cells

Figure 3.6: KCa3.1 channel redistribution in the immunological synapse 124

Figure 3.7: KCa3.1 channel redistribution in live T cells 126

CHAPTER IV: Altered calcium mobilization in T lymphocytes from patients with systemic lupus erythematosus

Figure 4.1: Determination of excitation of pEGFP-Kv1.3 with Ti-Sa laser 148

Figure 4.2: Representative example of the four patterns of [Ca2+]i observed 151

Figure 4.3: Detailed analysis of [Ca2+]i pattern in NL, SLE and RA T cells 152

Figure 4.4: Quantitative analysis of continuous [Ca2+]i pattern 154

Figure 4.5: Power spectra of B cell-induced [Ca2+]i oscillations 156

Figure 4.6: Conjugation of EC-anti-Kv1.3 antibody with Alexa Fluor 488 158

Figure 4.7: Functional and pharmacological properties of recombinant pEGFP- 160 tagged Kv1.3 channels

Figure 4.8: Overexpression of pEGFP-Kv1.3 channels in human T cells 161

Figure 4.9: Time-lapse imaging of Kv1.3 channel redistribution to the IS 163

- 6 - Figure 4.10: Time-lapse imaging of Kv1.3 channel redistribution and [Ca2+]i in 165 human T cells

CHAPTER V: General Discussion

Figure 5.1: Proposed mechanism of abnormal Ca2+ signaling in SLE T cells 184

- 7 - ABBREVIATIONS

AA African American AF488 Alexa Fluor 488 AICD activation induced cell death AP1 activation protein 1 APC antigen presenting cell

BF brightfield

C Caucasian [Ca2+]i Cytosolic Ca2+ CaM calmodulin CaMKII CaM-dependent protein kinase II CaMKIV CaM-dependent protein kinase IV CD40L CD40 ligand CRAC Ca2+ release-activated Ca2+ channel CRACM1 CRAC modulator 1 CREM cAMP response element modulator

DIC differential interference contrast

EAE experimental autoimmune encephalomyelitis EBV Epstein Barr virus EC extracellular EF-hand helix (E), loop and another helix (F) motif ER endoplasmic reticulum

FBS fetal bovine serum

GFP Green fluorescent protein

HEK human embryonic kidney cells hdlg-1 Human homologue of drosophila discs large tumor suppressor protein HP holding potential

IC intracellular Ik-2 Ikaros-2 IL Interleukin IP3 Inositol 1,4,5 triphosphate IS immunological synapse ITAM immunoreceptor tyrosine-based activation motif

LFA-1 Leukocyte function-associated antigen-1

MFR mean fluorescent ratio

- 8 - MHC major histocompatibility complex min minutes MS multiple sclerosis

NF-AT Nuclear factor of activated T cells NK Natural killer NL normal

PBMC peripheral blood mononuclear cell PMCA plasma membrane calcium ATPase PFA parafolmaldehyde PHA phytohemagglutinin PLCγ phospholipase C γ PKA protein kinase A PKC protein Kinase C

RA rheumatoid arthritis rbc red blood cell REST repressor element 1-silencing transcription factor

SD standard deviation SE standard error SEB staphylococcal enterotoxin B sec second SLE systemic lupus erythematosus SLEDAI SLE disease activity index SMAC supramolecular activation cluster c-SMAC central-SMAC d-SMAC distal-SMAC p-SMAC peripheral-SMAC STIM-1 stromal inducing molecule 1

TCM central memory T cell TCR T cell receptor T1DM type 1 diabetes mellitus ΤΕΜ effector memory T cell TM transmembrane ΤΝFα tumor necrosis factor α

VLA-4 very late activation antigen-4

YFP yellow fluorescent protein

- 9 -

CHAPTER I: Background and Thesis Scope

- 10 - 1.1 T cell Activation, the Immunological Synapse and Calcium Signaling

In order for the immune system to fight pathogens it utilizes two arms of defense, the

innate and the adaptive responses. The adaptive immune response is further divided into

the cellular immune response, which is mediated by T lymphocytes, and the humoral

immune response, which is mediated by antibody-producing B lymphocytes. The focus

of this thesis dissertation is the T lymphocyte, an integral component of the adaptive

immune response. To illustrate, once a pathogen enters the immune system it is taken up by an antigen presenting cell (APC) such as a B cell or a dendritic cell and is processed into smaller peptide fragments. Subsequently these fragments are presented to the T cell receptor (TCR) via the major histocompatibility complex (MHC) and a number of pathways are elicited to fight the pathogen at hand. Importantly, it is not only pathogenic peptides that are presented to T cells but also self-peptides. Therefore it is important for the T cells to be able to distinguish self from non-self i.e. to exhibit self-tolerance. If self-

tolerance is breached then autoimmunity develops.

Numerous studies have shown that upon TCR engagement the TCR, CD3 complex,

adhesion molecules (1), intracellular signaling molecules and lipid rafts (2) are organized

into supramolecular activation clusters (SMAC) at the point of contact between the T cell

and APC forming the immunological synapse (IS) (3, 4) (Fig.1.1 A). The first report of

distinct patterning during T cell activation was documented in 1998 by Monks and

colleagues and has gained enormous recognition ever since (5). More recently it was

revealed that preceding the formation of a mature IS the TCR and associated adaptor

molecules form microclusters that within minutes coalesce to form a mature synapse (6,

- 11 - 7). The mature synapse is organized in a central-SMAC (c-SMAC) that includes the TCR complex, a peripheral-SMAC (p-SMAC) that consists mainly of adhesion molecules (3)

and the distal-SMAC (d-SMAC) where the phosphatase CD45 and other large

glycoproteins such as CD43 and CD44 reside (Fig. 1.1 B) (6, 8). The formation of an

organized IS has been documented in vitro as well as in vivo further validating its

existence and significance (9).

A. B. c-SMAC

d-SMAC p-SMAC

Figure 1.1: Organization of a mature immunological synapse. (Modified from Kupfer et al, Seminars in Immunology, 2003)

The role of this structure is gradually being elucidated and it is currently believed to have

several roles including (i) signal transduction, as it allows signaling molecules to be in

close proximity (ii) orientation of cytokine and chemokine secretion, since chemokine

receptors were shown to accumulate at the IS (11), (iii) provision of a site for

degradation, as TCR degradation takes place there via a ubiquitin mediated pathway (10),

as well as facilitation of the ultimate termination of the immune response (6) and (iv)

creation of docking sites, such as phosphorylation sites, for protein-protein interactions

that are otherwise not possible (12). As a result of IS formation, multiple signal

transduction pathways are elicited and enhanced, resulting in the generation of mitogenic

signals. IS formation is therefore a dynamic process thought to facilitate signaling

- 12 - through the TCR and to fine-tune the ultimate outcome of TCR engagement. In addition

it allows the cell to act in a highly cooperative and directional way in the generation and

the termination of the T cell activation process.

The T cell activation process culminates with the induction of transcription factor activation including, among others, the Nuclear Factor for Activated T cells (NF-AT), that mediates gene expression of interleukin (IL)-2, IL-4 and tumor necrosis factor-α

(TNFα) (13, 14), NF-κB that regulates a number of genes encoding cytokines, adhesion molecules, growth factors and other membrane proteins (13, 15) and Oct/OAP involved in IL-2 transcription (13). IL-2 is one of the most important cytokines produced as it promotes clonal T cell expansion resulting in the effective targeting of specific pathogens. The function of these transcription factors relies either directly or indirectly on intracellular Ca2+. Interestingly, 75% of all T cell activation genes are in some way

dependent on Ca2+ signaling (16).

It is thus not surprising that the onset of T cell activation is marked by an increase in

intracellular Ca2+ that occurs immediately upon TCR engagement by the APC/antigen.

This increase in Ca2+ must be sustained for a long time before IL-2 and other cytokines

and chemokines are produced and activation becomes antigen independent. As a result a

sustained [Ca2+]i is necessary for T lymphocyte activation and gene expression (17).

Importantly, Ca2+ does not simply promote gene expression but it can drive differential

gene expression depending if it presents as an oscillatory or a continuous response (18,

19). Ca2+ oscillations contribute to the efficiency and specificity of gene expression by

- 13 - reducing the threshold required to induce expression (18). Specifically, rapid oscillations

activate NF-AT, NF-κB and Oct/OAP whereas infrequent oscillations activate only NF-

κB (18, 20). NF-AT expression is also promoted by a sustained continuous Ca2+ response

(19). Calcium oscillations depend on Ca2+ influx/efflux, uptake and release from

intracellular stores as well as membrane potential which in turn relies on ion channels

expressed in T cells (21).

1.1.1 Ion Channels in T Cell Activation

Ca2+ and K+ channels are integral components of the activation process. Specifically

(Fig.1.2), upon antigen binding (orange) to the T cell receptor complex (yellow)

phospholipase Cγ (PLCγ) is activated leading to the formation of the second messenger

inositol 1,4,5 triphosphate (IP3). IP3 then binds to the IP3 receptor on the surface of the

2+ + Ca K K+

TCR/ CRAC Kv1.3 KCa3.1 CD3

PLC Em~-50 mV

IP3R IP3 ER Ca2+

CaM NF-AT Calcineurin

IL-2 gene expression proliferation

Figure 1.2: Calcium and potassium channels in T cell activation. Other ion channels are also found on the membrane of T cells but for the sake of clarity are excluded from this diagram. (Modified from Panyi et al, Trends in Immunology 2004(22)).

- 14 - endoplasmic reticulum (ER) and Ca2+ is released. Once the ER Ca2+ stores are depleted,

stromal interaction molecule 1 (STIM1) proteins sense the emptying of the stores through

an EF-hand (a helix (E), loop and helix (F) motif) and relocalize to the plasma membrane

to activate the Ca2+ release-activated Ca2+ channels (CRAC) resulting in Ca2+ influx (23).

However, to maintain the Ca2+ influx the K+ channels (Kv1.3 and KCa3.1) need to allow

K+ ions to exit the cell and as such balance the positive charge. Maintenance of the Ca2+

signal is critical as it results in the activation of calmodulin (CaM) which activates

calcineurin leading to gene expression and proliferation. Indeed, blockers of Kv1.3 and

KCa3.1 channels are immunosuppressive. A very limited number of studies have shown

that recombinant Kv1.3 channels co-localize with CD3 and are recruited at the IS (22, 24,

25). However, the time-dependent trafficking of the Kv1.3 channel is still to be

determined. Moreover, at present nothing is known regarding KCa3.1 membrane

distribution during T cell activation. Owing to the importance of these K+ channels a

more detailed determination of their trafficking during T cell activation is warranted.

Although CRAC channels appear to be the single most important mechanism that allows

Ca2+ entry in human T cells, Kv1.3 and KCa3.1, which modulate the function of CRAC

and hence Ca2+ influx, take dominance according to their expression level which relies

heavily on T cell subtypes. Therefore to fully grasp the contribution of each channel to T

cell activation one must appreciate (i) the differentiation of the T cells in specific sub-

types after antigenic stimulation and (ii) the differential expression of the Kv1.3 and

KCa3.1 channels in these sub-types.

- 15 - 1.1.2 T cell proliferation and K+ channel expression

When an antigen that had not been previously encountered by the organism is encountered a primary immune response is elicited resulting in the initiation of proliferative pathways (13). As early as 24 hr later these cells differentiate into acutely activated T cells and undergo clonal expansion via a recently proposed asymmetric cell division mechanism (26) and further differentiation into effector helper (CD4+) and cytotoxic (CD8+) T cells. From these the “effector” T cells work to eliminate the pathogen at hand. Once the threat has been dealt with some of these cells revert to a

“memory” phenotype and return to a long-lived resting state in preparation for a similar future pathogenic attack (27). These effector memory cells (TEM) provide protective memory and readily migrate to inflamed peripheral tissues to fight pathogens. In addition to TEM cells, T cells differentiate into another memory phenotype: the central memory T cells (TCM). These cells home to secondary lymphoid organs and have no effector functions. However, they provide reactive memory by readily differentiating in TEM cells in response to a similar pathogenic attack (28). As a consequence, when the organism is faced with the same pathogenic threat it can then elicit a secondary immune response which is faster than the primary immune response by undergoing enhanced proliferation.

To differentiate among the different T lymphocytes mentioned above a number of surface markers are enlisted. First, CD3 is used as the pan T cell receptor as it is strictly expressed on T cells and thymocytes. Second, T cells are broadly categorized as CD4+ or

CD8+ according to whether they express the corresponding coreceptor. Third, T cells can

- 16 - be further categorized according to the surface expression of the homing chemokine receptor CCR7 and the CD45RO (low MW) or CD45RA (high MW) phosphatase isoform expression. Particularly, naïve T cells, the cells that never encountered an antigen before, are CCR7 +/ CD45RA+ / CD45RO-, TEM cells are CCR7 - / CD45 RA -

CD45RO+ and TCM cells are CCR7 + /CD45RA - /CD45RO+. Also, acutely activated T cells can be differentiated from naïve or resting TEM and TCM by the expression of

CD25 (IL-2Rα chain).

The expression pattern of Kv1.3 and KCa3.1 change drastically as T cells transition from naïve to activated to memory phenotypes (Fig. 1.3). In resting cells (naïve, TEM and

TCM) Kv1.3 channels constitute the predominant K+ conductance with the expression

ranging from 200-400 channels per cell while KCa3.1 channel expression is low, ranging

from 0-30 channels per cell. Kv1.3 remains the dominant channel (about 1500 channels)

when TEM cells are activated. KCa3.1 channels are instead up-regulated when naïve and

TCM cells are activated, to about 500 channels, and control Ca2+ influx in these cells, despite the slight up-regulation of Kv1.3 channels (29, 30). To demonstrate the differential contribution of Kv1.3 and KCa3.1 in Ca2+ modulation in different T cell types

proliferation studies using [3H] thymidine incorporation were performed in the presence

of blockers targeting either Kv1.3 or KCa3.1. It was shown that while Kv1.3 blockers

prevent T cell proliferation in resting cells they do not have the same effect on activated

T cells. Instead, blocking KCa3.1 in activated T cells prevented T cell proliferation as

well as IL-2 production, while Kv1.3 blockers did not have the same effect (29). This differential pattern of expression can prove beneficial as it may allow for targeting of

- 17 - KCa3.1

KCa3.1

KCa3.1

KCa3.1

KCa3.1

KCa3.1

Figure 1.3: Differential expression of Kv1.3 and KCa3.1 channels in T cell subsets. (Diagram from Wulff et al, 2003, JCI (31)). The number of Kv1.3 (red) and KCa3.1 (blue) channels for each T cell subset is indicated as a number in the same color. The terms Kv1.3 high and KCa3.1 high indicate the dominant channel for each T cell subtype. TCM = central memory T cell and TEM = effector memory T cell.

specific T cell subtypes by abolishing the function of the corresponding dominant K+ channel, allowing the rest of the T cell population to remain unaffected.

1.2 Properties, structure and pharmacological characteristics of Ca2+ and K+ channels

Each ion channel displays specific characteristics, its “biophysical fingerprint”, that differentiate it from closely related channels. These include: ion conductivity, gating and pharmacological inhibition. This section will provide the reader with a basic

- 18 - understanding of each of the major ion channels involved in T cell activation focusing

largely on the two K+ channels Kv1.3 and KCa3.1 but also briefly discussing the major

channel involved in Ca2+ influx, the CRAC channel.

1.2.1 The CRAC channel

The CRAC channel is a store-operated calcium channel. As the name implies, the

emptying of intracellular Ca2+ stores acts as a trigger that promotes the opening of the

channel allowing Ca2+ to flow into the cell. Under physiologic conditions, in human T

cells the depletion of Ca2+ from the intracellular stores occurs after TCR engagement. As

ER stores become depleted STIM1 proteins from the ER membrane relocate to the

plasma membrane and act as the calcium sensor to facilitate the activation of CRAC channels (23). Beside Ca2+ stores, the activity of this channel also relies on the

maintenance of a hyperpolarized (negative) membrane potential (the resting potential of

T cells is approximately -50 mV). Both Kv1.3 and KCa3.1 channels oppose

depolarization by allowing K+ to flow out of the cell and effectively counteract the

positive influx due to Ca2+. As such these channels facilitate the function of CRAC

channels and modulate Ca2+ influx inside the cell.

The molecular identity of the CRAC was a mystery for a long time until recently when,

almost simultaneously, several groups proposed that the CRAC channel is composed of

CRAC modulator 1 (also known as Orai1) subunits (32-36). Although significant

progress has been made towards understanding the molecular identity and regulation of

the CRAC channel, the structure of the channel is still under investigation.

- 19 - 1.2.2 The Kv1.3 channel

The Kv1.3 channel is the product of the Kv1.3 gene located on chromosome 1p13.3 of the human genome (37-40). The existence of the Kv1.3 channel was documented over two decades ago in 1984 (41, 42) and it was cloned a few years later, in 1992 (43, 44).

This allowed the channel to be characterized and classified as a normal or n-type K+ channel belonging to the Shaker family of voltage gated K+ channels (Kv1), and it later

became known as Kv1.3 (45). Currently there exist 75 known members in the Kv family

but Kv1.3 is the only Kv channel expressed on human T cells where it is expressed as a

homotetramer (46). Besides T cells these channels are also expressed in B cells,

macrophages, microglia, osteoclasts, platelets, oligodendrocytes and the olfactory bulb

(47-55). In the brain they can also form functional heterotetramers with other members of

the Kv1 family (56). During T cell activation Kv1.3 channels increase in number.

However, the modulation of expression of new Kv1.3 channels is not very well

understood. Even though there is increase in channel number there is no corresponding

increase in mRNA suggesting that the regulation is posttranscriptional (29, 57).

Structurally the Kv1.3 channel in T lymphocytes is composed of four identical pore-

forming alpha subunits of about 500 amino acids (aa) each (58) and an auxiliary β

subunit at the amino terminus. Each α subunit is composed of six putative

transmembrane (TM) helices (S1-S6) that are connected by intra- and extracellular loops

(Fig. 1.4). S1-S3 helices are thought to scaffold the activation gate of the channel and

provide the lipid-protein interface of voltage gated K+ channels (58-61). It was generally

- 20 -

OUT

IN

Figure 1.4: Side view of Kv1.3 main α subunit (Modified from Cahalan et al, J. Clin. Immunol., 2001(62)).

believed that the S4 helix, which contains positively charged aa residues (lysine or

arginine) appearing at every third or fourth position, served as the “voltage sensor”,

however X-ray crystallography data indicated that parts of S3 and S4 together form a

voltage-sensor paddle and voltage sensing is most probably mediated through these two

helices (63). The pore of the channel is formed by S5 and S6 helices. The ion-conducting

pore is constructed of an inverted teepee composed of the selectivity filter, the narrowest outermost part of the pore, which is responsible for K+ ion specificity while the “bundle”

crossing of S6 helix directly follows. The selectivity filter carries carbonyl oxygens that

act as surrogate water to facilitate K+ crossing (64).

The N-terminus (T1 domain) does not only carry the β subunit but it also contributes to

the formation of the Kv1.3 tetramer (65). The auxiliary β subunit was first isolated from

the leukemic T cell line, Jurkat, and was speculated to have a regulatory role on Kv1.3

- 21 - function based on PKA phosphorylation data (66, 67). In addition coexpression of the

Kvβ1.2 subunit with Kv1.3 in HEK cells was shown to decrease the time constant of

inactivation and in its absence the channels had reduced sensitivity to PKA and PKC

modulation (68). In addition, the presence of Kvβ2, the most abundant Kvβ subunit in

human T cells, accelerates the formation of Kv1.3 channels, stabilizes the tetrameric

protein and provides docking sites to signaling molecules (69-72). Further, some Kvβ

subunits are hypothesized to be involved in folding of nascent α-subunits of Shaker

channels (71).

The biophysical properties of Kv1.3 have been well established using the whole-cell

clamp technique (73-75). Kv1.3 is an outward rectifying K+ channel with a single

channel conductance (a measure of the number of ions conducted through the pore) of 11

pS (76). As already stated the Kv1.3 channel is voltage gated so membrane

depolarization steeply induces the opening of the channel, with a threshold membrane

potential of -50 to -60 mV and maximal opening at -10 to 0 mV (73, 74). However,

prolonged depolarization can promote inactivation of the channel. The Kv1.3 channels

inactivate by a C-type mechanism (75). During C-type inactivation the pore of the

channel undergoes conformational changes resulting in occlusion of the pore and K+ ions are no longer conducted out (77).

In addition to membrane depolarization, other factors have been shown to influence inactivation kinetics including temperature, phosphorylation by receptor and non-receptor tyrosine kinases as well as ionic composition of the solution in which the cells are bathed

- 22 - (78-83). Further, Kv1.3 differs from other members of the Shaker family in that

inactivation of this channel is slowed down by acidic pH (84). Also, hypoxia inhibits the

function of Kv1.3 (85). The gating of the channel is also affected by lipids in the plasma

membrane such as cholesterol and ceramide. Specifically, cholesterol enrichment

increased the inactivation time while cholesterol depletion had no effect on the gating of

the channel (86). On the other hand, ceramide enrichment (seen in lipid rafts during T

cell activation) resulted in inhibition of the channel (87). Other regulatory mechanisms

are more controversial. For instance, whole cell patch clamp recordings have shown that

in resting and acutely activated human T cells Kv1.3 currents are blocked by high Ca2+

(1.0-1.1 µM) (88, 89). Contrary to this, cell-attached recordings (a configuration which maintains the cytoplasmic milieu intact) showed that Ca2+ had no effect on channel

function (90).

As mentioned previously Kv1.3 is regulated by phosphorylation. There have been

conflicting reports as to the effect of the src tyrosine kinase p56lck on the channels’

function. In Jurkat T cells and HEK 293 cells that were transfected with the channel, it

was shown that the src tyrosine kinases play an inhibitory role. However, in human

primary T cells it was demonstrated that the opposite is true, that is, the src tyrosine

kinase p56lck has a positive regulatory role (76, 91, 92). Kv1.3 has been shown to be

physically linked to p56lck through human homologue of drosophila discs large tumor

suppressor protein -1 (hdlg-1) as well as the type-II Ca2+/CaM-dependent protein kinase

(CaMK II), the later providing a link for Ca2+ sensing in T cells (89, 93). Moreover

within the Kv1.3 sequence lies a number of potential serine/threonine phosphorylation

- 23 - sites which would be ideal sites for PKA, PKC as well as CaMKII phosphorylation

activity (94). This serine/threonine phosphorylation involves an elaborate interplay

between PKA and PKC. Interestingly, individual kinases induced an increase in current

while sequential administration showed no difference (67, 95).

Inhibition of the channel can also be induced pharmacologically. Classical Kv1.3

blockers included margatoxin, kaliotoxin and charybdotoxin. However these toxins were

not specific enough for Kv1.3 as they also blocked other Kv1 channels or KCa3.1 (30) so

the search for other blockers continued. To that end, Kv1.3 blocking peptides have been

isolated from scorpions, snakes, sea anemones and marine cone snails. One of the most promising blockers is ShK, a peptide isolated from the sea anemone Stichodactyla

heliantus (96). Structural modifications of this blocker gave rise to even more selective blockers of the channel such as ShK-Dap22 (97). In general these blockers block the channel like a cork in a bottle, effectively blocking the pore of the channel (98). Another category of blockers, that originated from the leaves of the Ruta graveolens, the Common

Rue, are Psora-4 and its analogue PAP-1. These blockers bind the C-type inactivated

state of the channel conferring added specificity for the homotetrameric Kv1.3 channel

(99, 100). These blockers have become integral tools in the study of ion channels as they

contribute to the identification and differentiation of specific ion channels.

Throughout this section we have discussed several structural, biophysical, and pharmacological characteristics of the Kv1.3 channel. So now it becomes important to converge on the specific features of this channel which make it an important modulator of

- 24 - membrane potential and Ca2+ influx in T cells. Firstly, since the channel is activated by

membrane depolarization even small fluctuations, for instance due to Ca2+ entry in the

cell, will activate the channel leading not only to the maintenance of Ca2+ influx but also to sustaining the membrane potential. Secondly, since repeated depolarizing pulses can inactivate the channel, its alternating opening and closing states can lead to Ca2+ oscillations, a feature of T cells discussed previously. And thirdly, these channels are so effective in maintaining membrane potential that even the opening and closing of a few channels can uphold the resting membrane potential (62). This is made possible by the high electrical resistance of T cells (10-20 GΩ).

1.2.3 The KCa3.1 channel

The KCa3.1 channel is a calcium regulated K+ channel and it is the product of the hKCa4

gene (101). The first reports of this channels’ existence came from Wilbrant in 1940

when he observed K+ efflux in red blood cells (102). It was not until the late ‘50s that its

calcium dependence was discovered by the Hungarian scientist, Gardos (103). Finally

patch clamp experiments determined that the K+ efflux was due to a channel that came to be known as the Gardos channel (102, 104, 105). Since then the KCa3.1 channels have also been discovered in lymphocytes (106, 107), vascular endothelium (108), secretory

epithelia (109), pancreas, (110), colon (111), placenta (102) as well as enteric neurons

(112). Currently there are eight known KCa channels that have been further classified in

two groups based on (i) genetic homology, (ii) calcium signaling and (iii) single channel

conductance (113). KCa3.1 is a member of the KCNN gene family and members of this

family have ~40 % sequence homology with the pore and proximal C terminus having

- 25 -

KCa3.1-CaM

OUT

IN

Figure 1.5: Side view of KCa3.1 channel main alpha subunit (Modified from Cahalan et al, J. Clin. Immunol., 2001(62)). Red arrow indicates where CaM binds. CaM is depicted with four Ca2+ ions bound. CaM = calmodulin.

the greatest level of homology (114, 115).

Structurally KCa3.1 channels resemble Kv1.3 in that they also composed of four 6TM

spanning α subunits (Fig, 1.5) and in that they have similar pore architecture,

demonstrated by peptide toxin interactions (96). Trafficking of newly formed KCa3.1

proteins from the ER to the plasma membrane depends on a lysine residue on the S4-S5

linker domain (116). The S4 helix of KCa3.1 channels has fewer positively charged aa

(as compared to Kv1.3), rendering this channel voltage independent (101, 110). However,

this channel shows exquisite sensitivity to Ca2+ while its α subunits lack any Ca2+ binding sites such as EF-hand domains, C2 domains or Ca2+ “bowls” (115, 117, 118). Instead it is

permanently associated to CaM at its proximal C-terminus (95 aa Ct1 domain) immediately following the S6 helical domain (115). The association involves four CaM

- 26 - proteins for each homotetrameric channel (119). It has been proposed that the “gating” of

the pore of the channel involves four apposed Ct1 regions that constrict the pore in the

absence of Ca2+ (119). Interestingly, X-ray crystallography data have demonstrated that

in another channel of the family, SK2 (also known as KCa2.2), CaM has the ability to

link two α helices from each proximal domain forming a channel dimmer when Ca2+ is bound and as such opening the pore of the channel and allowing K+ efflux (120). Not surprisingly CaM has thus been termed the Ca2+ sensor of these channels (119). Besides

its role in Ca2+ sensing and opening of the channel pore CaM also mediates the assembly

of newly formed homotetramers (121).

The biophysical characteristics of KCa3.1 have been described using electrophysiological

studies at the single channel and whole cell level (107, 122, 123). These studies have

shown that the KCa3.1 channel is an intermediate conductance channel (11pS) (107).

Further, KCa3.1 is a Ca2+ activated channel and as such a rise of intracellular Ca2+ above approximately 100 nM will evoke K+ efflux due to the opening of the channel. Half

maximal activation is achieved at about 450 nM and maximal activation at ~1 µM (107,

122). Therefore it can be inferred that at basal Ca2+ levels, less than 100 nM in T cells,

these channels are silent but become readily available to sustain the Ca2+ influx upon

TCR engagement.

Pharmacologically, KCa3.1 channels are sensitive to charybdotoxin (ChTx), which also blocks Kv1.3 with similar potency, and clotrimazole but are insensitive to apamin, allowing for differentiation from small conductance SK channels (101, 107, 115, 122).

- 27 - Clotrimazole was one of the most promising KCa3.1 blockers for the treatment of disease, as discussed later. However, one of the major limitations of this blocker is that it has high affinity for cytochrome P450-dependent enzymes which leads to increased side- effects (124, 125). To avoid toxicity new blockers have since been designed to target

KCa3.1 using clotrimazole as a template. The TRAMs are one of these groups of clotrimazole analogues that show therapeutic potential (126). The most promising of this group, TRAM-34, binds KCa3.1 from the cytoplasmic side of the S5-P-S6 region of the channel and it is speculated that it fits in the pore just below the selectivity filter (127).

Expression of the channel is modulated at the transcriptional level by several transcription factors. The hKCa4 gene contains a functional repressor element 1-silencing transcription factor (REST) binding site and it is repressed by REST (128). In smooth muscle cells REST differentially regulates expression of the KCa3.1 channels.

Specifically, KCa3.1 is only expressed in proliferating, but not contractile, smooth muscle (129). During T cell activation KCa3.1 up-regulation is driven mainly through

PKC but also through Ca2+ mediated pathways (29). This results in newly formed channels as indicated by a ~10 fold increase in mRNA in activated T cells, compared to almost undetectable mRNA levels in resting peripheral blood lymphocytes (101, 115).

The major transcriptional factor involved in this process is activation protein-1 (AP1) but also Ikaros-2 (Ik-2) albeit to a lesser extent (29). Interestingly, although KCa3.1 has two

NF-AT consensus motifs a deletion of both these motifs did not lead to decreased promoter activity and KCa3.1 expression (29). Besides regulation at the transcriptional level, KCa3.1 channels have also been shown to be modulated by PKA and PKC, while

- 28 - reports have been conflicting as to whether they have a positive or negative regulatory

role (130-132).

In this section the key structural, biophysical and pharmacological features of KCa3.1

have been discussed. As previously stated, KCa3.1 channels do not contribute

significantly in T cell activation of resting T cells, owing to the small numbers expressed.

However, they exert a powerful effect in activated T cells where they take dominance over Kv1.3 channels in regards to Ca2+ modulation. These modulator properties of the

channel stem from its exquisite sensitivity to intracellular Ca2+ which acts as a trigger for

the opening of the channel leading to K+ efflux and the ultimate shaping of the Ca2+ response in acutely activated naïve and TCM cells.

1.3 K+ channels and disease

Throughout the previous sections it has been pointed out that Kv1.3 and KCa3.1 are

integral components of the T cell activation process. Currently, both channels are heavily

researched as potential therapeutic targets, not only in T cell mediated diseases but also in

a plethora of other diseases as well. The goal of this section is to highlight the therapeutic

indications of each K+ channel in a variety of disease processes while focusing on T cell

mediated diseases.

1.3.1 The Kv1.3 channel

At present, millions of people suffer by T cell mediated autoimmune diseases and

although immunotherapy has helped immensely, still most of these therapies have serious

- 29 - side-effects (133-136). For that reason the search for more efficient and less toxic pharmacologic targets/agents is on the rise. As aforementioned Kv1.3 channels regulate the function of naïve and activated TEM cells by sustaining the membrane potential.

Further, Kv1.3 channels participate in volume regulation as well as adhesion and migration (137, 138). As such they can be a valuable target in diseases where TEM cells play a central role. Interestingly, TEM cells, which exhibit a Kv1.3 high phenotype upon stimulation, have been implicated in the pathogenesis of several inflammatory and autoimmune diseases such as periodontal disease, multiple sclerosis (MS), type 1 diabetes mellitus (T1DM), inflammatory arthritis and psoriasis (31, 139-145).

The immunosuppressive effects of Kv1.3 blockers, including margatoxin, kaliotoxin,

PAP-1 and ShK, are now intensely researched with quite promising results. Koo and colleagues administered margatoxin to mini swine and effectively inhibited a delayed type hypersensitivity response to tuberculin PPD (146). They subsequently examined whether margatoxin can block cytokine production and T cell proliferation and it potently blocked both events (147). Further, the Kv1.3 blocker kaliotoxin was shown to reduce bone resorption in rats, seemingly by blocking TEM cells, and thus ameliorating periodontal disease, a disease characterized by bone loss and subsequent loss of teeth

(140). However, both margatoxin and kaliotoxin block other members of the Kv1 family and this poses as a disadvantage. PAP-1, a small molecule that blocks Kv1.3, was recently used in the treatment of allergic contact dermatitis in a rat model and both topical and oral administration were effective (148).

- 30 - In the past decade a new family of peptide Kv1.3 blockers, from the sea anemone

Stichodactyla helianthus, have shown tremendous therapeutic potential in autoimmune diseases including MS, T1DM and rheumatoid arthritis (RA). Rats afflicted with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, were administered the Kv1.3 blocker ShK-Dap22 as treatment after the onset of the disease and alleviation of the clinical course of the disease was observed (149). Beeton and colleagues also demonstrated that repeated stimulation of TEM cells from MS patients with myelin antigens, but not non-MS specific peptides, resulted in activated TEM cells with Kv1.3 high expression supporting the notion that it is through blockade of these effector cells that disease progression was halted. This further suggested that specific targeting of this cell type, while sparing naïve and TCM cells, could be an ideal therapy for MS patients (31). Interestingly, there is evidence that Kv1.3 is highly expressed in T cell inflammatory infiltrates of MS brain tissue as well as on T cells from cerebrospinal fluid. These results in combination with the efficacy of treatment in EAE rats promotes

Kv1.3 blockers as potential therapeutic agents (149, 150).

The effectiveness of Kv1.3 blockers was also demonstrated in rat models of RA and

T1DM. For the treatment of RA, SL5 (149), a Shk analogue, was used at the first signs of arthritis. As a result of treatment the animals exhibited significant improvement in joint damage, however, rheumatoid factor was not significantly reduced so the authors suggested a combination therapy with TNF-α blockers as a more effective combination therapy (144, 151). Moreover, treatment of MHC class II-restricted DP-BB/W rats, a model of T1DM, with PAP-1 resulted in decreased intraislet T cell and macrophage

- 31 - infiltration and reduced β cell destruction via an immunomodulary mechanism (144).

Also, as for MS T cells, repeated antigenic stimulation with autoantigens from RA and

T1DM resulted in formation of Kv1.3 high TEM that were once again suppressed by

Kv1.3 blockers while sparing naïve and TCM cell proliferation (144). The toxicity profile of both PAP-1 and SL5 was promising without any major histopathological or hematological effects, except for skin irritation at the SL5 administration site (144).

Besides autoimmune diseases Kv1.3 channels have shown therapeutic potential in other

diseases. Specifically, Kv1.3 knock-outs provided insight into a number of diseases

including anosmia (loss of smell) as well as obesity and diabetes. To illustrate, upon loss

of Kv1.3 the mice became “super-smellers” (152) implying that by blocking Kv1.3 the

elderly population that suffers from a deteriorating sense of smell would benefit. Further,

when these mice were fed a high fat diet they gained less weight and exhibited increased

peripheral insulin sensitivity, which was attributed to enhanced translocation of

transporter 4 to the plasma membrane, an effect induced by insulin (153, 154). The same

group also showed that pharmacologic abrogation of Kv1.3 function with margatoxin

stimulated glucose uptake in the adipose tissue and skeletal muscle (155). A more recent

study, investigating the effect of Kv1.3 polymorphisms in humans, found a variant in the

promoter of the Kv1.3 gene that conferred impaired glucose tolerance and lower insulin

sensitivity but had no effect on body weight or body mass index (BMI), providing more

solid evidence in the potential role of Kv1.3 channels in these diseases (156).

In summary, it becomes apparent that Kv1.3 channel blockers show tremendous

- 32 - pharmacologic potential for the treatment of autoimmune and inflammatory T cell mediated diseases such as psoriasis, MS, RA and T1DM. Still there remain open questions of the potential contribution and pharmacologic indications of K+ channel blockers in other autoimmune diseases such as systemic lupus erythematosus (SLE).

1.3.2 The KCa3.1 channel

One of the functions of KCa3.1 is to promote cell proliferation. In the differentiated smooth muscle cells the expression of KCa3.1 is repressed by REST (128). However when the smooth muscle undergoes proliferation then the repression is lifted and KCa3.1 channel replaces KCa1.1 (a large conductance BK channel), which is normally expressed on differentiated smooth muscle cells (129, 157). Patients that undergo angioplasty develop restenosis and KCa3.1 facilitates this process by promoting smooth muscle cell proliferation (157). Pharmacologic inhibition of KCa3.1 channels with TRAM-34, clotrimazole or charybdotoxin stunted smooth muscle cell proliferation and TRAM-34 treatment in rats that underwent balloon catheter injury significantly reduced restenosis

(157-159). Further, KCa3.1 has been implicated in the proliferation of a number of cancer lines including prostate, glioblastoma, breast, pancreatic and endometrial cancer cell lines

(160-164). A recent study in mice with endometrial cancer has shown that treatment with

TRAM-34 and clotrimazole slows the tumor growth (164). These data suggest that

KCa3.1 is a viable pharmacologic target for certain cancers.

KCa3.1 is also involved in volume regulation in red blood cells (114, 165). Under physiological conditions, in normal red blood cells, the channel remains silent. However,

- 33 - in disease states, such as sickle cell disease, upon a stress, such as hypoxia, the channel

promotes K+ efflux and water loss further aggravating the sickling process (166, 167).

Currently, KCa3.1 is pursued as a therapeutic target for sickle cell disease as KCa3.1

blockade by clotrimazole was shown to prevent sickling (168). In addition to

clotrimazole another promising blocker, ICA-17043 is under investigation as a potential

treatment for sickle cell disease (169). Beside red blood cells KCa3.1 is also involved in

volume regulation in T lymphocytes (114, 115).

Since KCa3.1 channels regulate the function of acutely activated naïve and TCM T cells

they make excellent therapeutic targets for immunosuppression in disease states that

involve acute immune responses that are centered on these T cell subtypes. In vitro

experiments have shown that TRAM-34 can selectively inhibit the proliferation of human

naïve and TCM cells (31). Also, KCa3.1 blockers have been proposed as promising therapeutic targets in diseases such as RA, transplant rejection, asthma and primary biliary cirrhosis (102). The literature so far supports this view. Specifically, an eight week clinical trial performed with RA patients using clotrimazole demonstrated this to be a superior treatment to ketoprofen, an anti-inflammatory, non-steroidal drug (170). Further,

patients receiving transplant face a dual danger of transplant rejection as well as

cytotoxicity from cyclosporine treatment. Promising proliferation studies, where TRAM-

34 was used in conjunction to lower doses of cyclosporine, suggested a synergistic effect

between the two drugs thus providing a viable alternative which warrants further

investigation (126).

- 34 - Thus, it becomes clear that these channels hold promise for the cure of a number of diseases. Interestingly, the impending role of these channels in the pathophysiology of the

autoimmune disease SLE remains to be determined.

1.4 Systemic Lupus Erythematosus

By definition lupus means “wolf” and erythematosus means “redness”. The term

systemic lupus erythematosus was coined in 1851 by doctors that believed that the facial

rash, that often accompanies SLE, looked like the bite of a wolf, although nowadays is

more commonly referred to as a “butterfly rash”. According to the Lupus Foundation of

America over 1.5 million Americans suffer with a form of lupus 70% of which are

systemic. This disease affects predominantly women in their child bearing years and is

more prevalent in African American women over any other race. It has a highly variable

and unpredictable progression and this is probably the reason why about 50% of these

patients see at least three doctors before diagnosis.

SLE is the prototype autoimmune disease characterized by multi-organ involvement and

a broad variety of clinical symptoms such as rashes, glomerulonephritis and central

nervous system impairment. To determine the activity of SLE physicians often employ

the American College of Rheumatology Criteria which include detailed examination of

specific organs including skin manifestations, neurologic disorders, hematologic

disorders, immunologic disorders and anti-nuclear antibody formation among others

(171). Each manifestation is then given a score (1-8), depending on the severity, and

those patients with a SLE Disease Activity Index (SLEDAI) > 3 are considered to have

- 35 - an active disease (172). An example of the SLEDAI evaluation form is shown below

(172).

The etiology of the disease remains largely unknown although genetic, environmental, hormonal and stochastic factors have been implicated (173). Even though no single

“lupus” gene has been identified there exist several candidates. Not surprisingly gene

- 36 - association studies focused largely on immune regulatory genes of innate, adaptive and apoptotic immunity (173). Despite having inherited the genetic defects an individual does not immediately present with the disease and the exact point when tolerance is breached is not well defined. It has been speculated that the combination of genetic, environmental and stochastic factors eventually culminate in a pool of auto-antigens. Inability of the host to induce or maintain self-tolerance, due to numerous defects in immunity, forms the basis of autoimmunity in SLE patients (173). Further, other triggers such as sunlight, stress, viruses and certain drugs may contribute to the disease. At present, there is no cure for lupus.

The primary defect in these patients lies in impaired humoral and cellular immune responses to self-antigens resulting in the production of autoantibodies. This is indicative of a breach in tolerance which subsequently leads to autoimmunity. Many different autoantibodies are found in SLE patients including anti-chromatin and anti- ribonucleoprotein antibodies, a serologic hallmark of these patients, as well as antibodies against the TCR (174, 175). These autoantibodies form antigen-antibody complexes and deposit to various organs propagating chronic inflammation and eventually destroying organ parenchyma, ultimately leading to organ failure (176).

1.4.1 The role of T cells in the etiopathogenesis of SLE

Despite extensive studies the precise etiology of SLE remains elusive. What is more puzzling is that virtually every aspect explored in regards to antigen has been shown to be abnormal. Accumulating evidence suggests that T cells play a central role in the

- 37 - pathogenesis of SLE by mediating these altered immune responses (176). T cells in SLE

patients display a greater degree of apoptosis and are resistant to activation-induced cell

death (AICD) (173, 177). Furthermore, there exists an imbalance in T cell subsets.

Particularly CD4 T cells have an exaggerated response while CD8 T cells show a diminished response (178). In addition to CD8 T cells, natural killer (NK) cells also

exhibit defective functions (179). This creates a feedback loop which allows otherwise

prohibited B cell clones to produce autoantibodies (180).

The formation of these autoantibodies was primarily attributed to B cell hyperactivity.

However, T cell involvement was shown when the B cell receptor, of the B cells producing anti-nuclear antibodies, was investigated revealing that they had undergone T

cell mediated affinity maturation. Moreover, nucleotide substitutions, typical of antigen-

driven T cell dependent immune responses in murine as well as human B cells, which

effectively promoted class switch recombination and autoantibody production, were

documented (174, 181). Distinctively, T cells obtained from patients with active SLE

promoted antibody synthesis in the absence of antigens or mitogens (182).

1.4.2 T cell activation and Immunological Synapses in SLE T cells

This led to the well supported belief that SLE T cells, and not only B cells, are also

hyperactive. This proposition was strengthened by data indicating that the threshold for

TCR induced activation in SLE T cells is lowered (183). From a biochemical standpoint,

increasing evidence suggests that freshly isolated human SLE T cells display a “TEM

signaling phenotype” as indicated from a switch from the canonical

- 38 - TCR/CD3/CD3ζ/ZAP-70 receptor complex, in normal T cells to TCR/CD3/FcRγ/Syk,

which is observed in TEM (184). Notably SLE T cells show reduced expression of TCRζ

chain while up-regulating the more potent FcRγ chain which is believed to facilitate, in

part, the hyperresponsiveness of SLE T cells (184). Indeed, transfection of the FcRγ

chain to normal cells conferred a hyperexcitable phenotype accompanied by a reduction

in TCRζ chain (185). Moreover, lipid rafts are pre-clustered in TEM and SLE but not resting T cells, also pointing to a more active phenotype in these cells (186).

Along with the more obvious switch in signaling a number of more subtle signaling abnormalities have been documented in SLE T cells (reviewed in Table I). Among these molecules, several have been shown to accumulate in the IS upon TCR engagement. As a result one would anticipate abnormal TCR mediated events that would encourage this hyperresponsiveness. However, contrary to their activated T cell phenotype, SLE T cells show reduced production of IL-2 which is attributed to a defect at the transcriptional level

(187).

Table 1.1: Summary of T cell Signaling Defects in SLE

Defect Reference (s) • Exaggerated intracellular free calcium levels (188) • Increased activity of p56lck (189, 190) • Enhanced phosphorylation of intracellular molecules (189, 190) • Increased expression of lipid rafts (191) • Decrease in TCR ζ chain and up-regulation of Fcγ receptor chain (192) • Decreased activity of CD45 (193) • Decreased PKC expression (178) • Decreased PKA activity (194) • Increased NF-AT (195)

- 39 -

1.4.3 Contribution of K+ channels in the pathophysiology of SLE

Of particular interest is that upon T cell activation T cells from SLE patients presented

with a higher, more sustained Ca2+ response, as compared to normal controls, which was unrelated to disease activity or organ involvement pointing to an intrinsic defect in these

T cells (188, 196). This abnormality has been attributed to the switch from TCRζ to FcRγ

chain as well as the presence of lipid rafts. In support of these views transfection of SLE

T cells with TCRζ and disruption of lipid rafts corrected the Ca2+ flux anomaly (186,

197). As such, one may speculate that there is not a single factor to justify the abnormal

Ca2+ response.

Therefore, it is also possible that the modulators of Ca2+ influx such as Kv1.3 and KCa3.1

are the source of the underlying defect that induces this Ca2+ hyperactive response. On

the other hand, it is possible that the defect lies in abnormal regulation of Kv1.3 and

KCa3.1 channels in SLE T cells. As previously discussed K+ channels are regulated by

several kinases including p56lck (Kv1.3), PKC and PKA (Kv1.3 and KCa3.1), which

have been shown to be abnormal in SLE T cells (Table I). So it is quite possible that K+ channels could be either directly or indirectly implicated in the etiopathogenesis of SLE.

To our knowledge, there are no studies addressing the possible involvement of K+ channels in the etiology of SLE.

1.5 Scope of Thesis

Although our understanding of the molecular dynamics of the IS has improved

- 40 - considerably over the past few years there remain many gaps in knowledge and basic questions related to the ionic mechanisms involved in IS formation are still unresolved. In particular the membrane distribution of K+ channels during T cell activation remains largely unknown. As mentioned previously, K+ channels are important modulators of T lymphocyte function as they set the membrane potential and support Ca2+ influx upon T cell stimulation and facilitate the activation process. In fact by blocking K+ channels T lymphocyte activation is suppressed. Despite their significance, the mechanisms that regulate Kv1.3 channels and their role in the mechanics of TCR-mediated activation are not well understood. These questions pose important problems because they prevent the better understanding of how T cell activation occurs which is essential for the application of this knowledge to disease processes.

In fact this information would be crucial for autoimmune diseases where autoreactive / hyperactive T cells pose a serious threat to the organism. Specifically, increasing evidence suggests that T cell signaling at the IS is abnormal in patients with the autoimmune disease SLE ((191), Table I). In the past few years our knowledge of the aberrant molecular responses in T cells from SLE patients has advanced rapidly.

However, despite this wealth of knowledge at present nothing is known about ion channel activity and regulation in T cells from patients with SLE. Interestingly, K+ channels have been used as targets for specific immunomodulation in other autoimmune diseases such as MS, RA and T1DM (31, 142-144).

1.5.1 Hypothesis

- 41 - The overall goal of this thesis is to delineate the trafficking of the two K+ channels found

on human T cells, Kv1.3 and KCa3.1, during T cell activation in normal T cells. Further,

we will perform comparative studies with T cells from normal donors and patients with

SLE in order to decipher any abnormalities associated with K+ channel trafficking to the

IS, focusing on the dominant K+ channel in these cells. Following, we will explore the functional implications of K+ channel trafficking. To address these questions the

following general hypothesis will be tested:

General Hypothesis: K+ channel redistribution in the immunological synapse upon T cell stimulation affects the overall process of T cell activation, thus altered dynamics of

K+ channel transition in the immunological synapse account for the abnormal, more

pronounced activation response observed in SLE T cells.

1.5.2 Organization of thesis

This thesis comprises of five chapters. Chapter 1 provides a comprehensive background and a review of the classical and current literature of the subject matter. Chapters 2-4 are presented as separate manuscripts which will include an abstract, introduction, materials and methods, results, discussion and references. Chapter 2 focuses on comparative studies investigating the K channel phenotype of normal and SLE T cells followed by imaging studies to decipher anomalies regarding Kv1.3 channel compartmentalization in the IS. Data reported in this chapter are published in the Journal of Immunology (198).

Chapter 3 focuses on KCa3.1 channels and their membrane distribution in activated T cells. Data reported in this chapter are published in the American Journal of Physiology-

- 42 - Cell Physiology (199). Chapter 4 is centered on functional studies and determination of

K channel involvement in the etiology of SLE. Finally, Chapter 5 discusses the novel findings of this thesis, provides future directions and discusses the clinical implications of the data presented herein.

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- 61 -

CHAPTER II: Altered dynamics of Kv1.3 channel

compartmentalization in the immunological synapse

in systemic lupus erythematosus1

1 The data presented in Chapter II are published in the Journal of Immunology, Nicolaou et al, J Immunol. 2007 Jul 1;179(1):346-56. The link for the article is: http://www.jimmunol.org/cgi/reprint/179/1/346

- 62 - 2.1 ABSTRACT

Aberrant T cell responses during T cell activation and immunological synapse (IS)

formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium

channels are expressed in T cells where they compartmentalize at the IS and play a key

role in T cell activation by modulating Ca2+ influx. Although Kv1.3 channels have such

an important role in T cell function, their potential involvement in the etiology and

progression of SLE remains unknown. This study compares the K channel phenotype and

the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells.

IS formation was induced by 1-30 min exposure to either anti-CD3/CD28 antibody- coated beads or EBV-infected B cells. We found that, while the level of Kv1.3 channel

expression and their activity in SLE T cells are similar to normal resting T cells, the

kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy

resting T cells, Kv1.3 channels are progressively recruited and maintained in the IS for at

least 30 min from synapse formation. In contrast, SLE, but not rheumatoid arthritis, T

cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out

of the IS by 15 min after activation. These kinetics resemble pre-activated healthy T cells,

but the K+ channel phenotype of SLE T cells is identical to resting T cells, where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells.

2.2 INTRODUCTION

Systemic lupus erythematosus (SLE) is a rheumatic autoimmune disease characterized by abnormal T cell function (1-3). A variety of signaling alterations have been identified in

- 63 - SLE T cells (3). In particular, T cells from SLE patients, but not patients with other

autoimmune diseases, display an exaggerated response to antigen stimulation (2). The

hallmark of this T cell “hyperactivity” is a more pronounced and more sustained increase

2+ 2+ in intracellular Ca levels ([Ca ]i) following T cell receptor (TCR) ligation as compared

to healthy T cells (4-6). This sustained influx of Ca2+ is essential for the activation of

downstream signaling events and ultimately T cell function (7). Thus, impaired

2+ regulation of [Ca ]i appears to contribute to altered T cell functions in SLE T cells.

However, the precise mechanisms underlying this aberrant Ca2+ response in SLE T

2+ lymphocytes have not yet been identified. Although the increase in [Ca ]i has been

attributed in part to an increased release of Ca2+ from intracellular stores, membrane

related processes have also been implicated (5, 6).

The TCR mediated influx of Ca2+ in T cells occurs through Ca2+ release activated Ca2+

(CRAC) channels and is regulated by various membrane channels and signaling molecules (8, 9). Briefly, engagement of the antigen to the TCR activates phospholipase

Cγ (PLCγ) and induces the release of Ca2+ from intracellular stores. Depletion of Ca2+ from intracellular stores causes the CRAC channels to open and Ca2+ to flow into the

cells. This sustained influx of Ca2+ is essential to activate T cells, regulating both

proliferation and cytokine production. The necessary electrochemical driving force for

Ca2+ influx is provided by the cation efflux through K+ channels. The two major K+ channels expressed in T cells are the voltage-dependent Kv1.3 channel and the Ca2+- activated K+ channel KCa3.1. Along with allowing initiation of the Ca2+ influx, the

crosstalk between these and other channels shapes the overall Ca2+ response, i.e.

- 64 - amplitude and frequency of Ca2+ oscillations which can determine specificity of gene

expression (10). Recently, it has been shown that the expression of these K+ channels

depends on the immune cell activation state (11, 12). Kv1.3 channels constitute the

predominant K+ conductance and regulate Ca2+ influx in resting naïve and central

memory (TCM) as well as resting and activated effector memory (TEM) T cells. KCa3.1

2+ are instead upregulated when naïve and TCM cells are activated and control Ca influx in these cells (13).

Very recently, a limited number of studies have shown that Kv1.3 channels redistribute in the immunological synapse (IS) during TCR engagement (14-16). At present nothing is known regarding KCa3.1 membrane distribution during T cell activation. The IS is a tight and highly organized interactive signaling zone localized at the point of contact between

T cell and antigen presenting cell (APC) and it contains membrane molecules (e.g. TCR,

CD3 and CD28) as well as signaling components (e.g. Lck and PKCθ) (17). Functionally,

the process of IS formation is thought to facilitate signaling through the TCR and to fine-

tune the ultimate outcome of TCR engagement. The structure of the IS is very dynamic,

with molecules entering and leaving at different times. However, the process of Kv1.3

channel re-localization in the IS is not yet understood. Furthermore, no information is available on potential alterations in Kv1.3 channel redistribution at the IS in pathological conditions. SLE T cells display certain features that can affect the formation of the IS:

SLE T cells possess greater capacity to generate lipid rafts than normal T cells in response to activation, faster kinetics of lipid raft clustering and polarization, and faster kinetics of actin polymerization and depolymerization (6). In particular, it has been

- 65 - shown that cross-linking of lipid rafts evokes faster and more pronounced Ca2+ response

in SLE T cells, indicating that early structural rearrangements in the T cell membrane

contribute to the increased activity of SLE T cells.

The purpose of our study was to investigate whether the expression and activity of key

regulators of Ca2+ homeostasis, such as Kv1.3 and KCa3.1 channels, is altered in SLE T

cells. Furthermore, we have investigated whether abnormalities in the process of

translocation of Kv1.3 channels in the IS that forms upon TCR binding occur in SLE T

cells. We found that K+ channel phenotype in SLE T cells resembles resting T cells from

normal donors in regards to expression of Kv1.3 and KCa3.1 channels. Further, our

results indicate that, while the biophysical and pharmacological properties of Kv1.3

channels in SLE T cells are identical to normal T cells, the dynamics of Kv1.3 channel compartmentalization in the IS of SLE T cells are altered. These alterations in TCR

activated membrane rearrangements might underlie the downstream functional

abnormalities of SLE T cells.

2.3 MATERIAL AND METHODS

Human subjects: Twenty patients with SLE fulfilling at least four of the American

College of Rheumatology classification criteria for SLE were included in this study: 3 males and 17 females, 4 Caucasian (C), 14 African American (AA) and 2 Hispanic, age

24-68 years (18, 19). Eleven had lupus nephritis of whom two required dialysis. In our

cohort, 17 patients had a SLE Disease Activity Index (SLEDAI) >3, indicative of active

disease, and 18 were being treated with immunosuppressive therapy (20). Control groups

- 66 - consisted of 5 patients with Rheumatoid Arthritis (RA) who fulfilled the American

College of Rheumatology classification criteria for RA and 26 healthy individuals. The

RA group consisted of 5 females, 2C and 3AA, with age range of 40-68 years. The healthy control group consisted of 6 males, 17 females and 3 unknown, 21C, 2AA and 3 unknown, age 30-54 years. The study was approved by the University of Cincinnati

Institutional Review Board.

Cells: Peripheral blood mononuclear cells (PBMC), CD3+, CD4+ and CD8+ lymphocytes were isolated from venous blood collected from consenting donors by Ficoll-Paque density gradient centrifugation (ICN Biomedicals, Aurora, OH, USA) and E-rosetting

(StemCell Tech., Vancouver, Canada) as previously described (21). The homogeneity of the T cell populations was determined by FACS (21).Cells were maintained in RPMI medium supplemented with 10% pooled male human AB serum (Intergen, Milford, MA,

USA), 200 U/ml penicillin, 200 µg/ml streptomycin, 1 mM Hepes. Pre-activated T cells were obtained by exposure to 4 µg/ml PHA (Sigma-Aldrich, St. Louis, MO) for 48-72 hrs in the presence of autologous PBMCs. Epstein-Barr Virus (EBV) infected-B cells were cultured in RPMI 1640 supplemented with 20% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin.

Flow Cytometry: Freshly obtained peripheral blood was stained with the following antibodies: CD3-FITC, CD4-PerCP, CD8-APC-Cy7, CD45RA-PE-Cy7 (BD Biosciences,

Mountain View, CA), CCR7-PE and CD45RO-APC (Pharmingen, San Jose, CA). The cells were stained for 20 min at room temperature followed by red cell lysis with

- 67 - FACSlyse solution (BD Biosciences, Mountain View, CA) for 10 min. The resultant

white cell pellet was washed with PBS and fixed in 1% paraformaldehyde (PFA) prior to

analysis by 4- or 6-color flow cytometry (FACSCalibur or FACSCanto flow cytometer,

Becton Dickinson, San Jose, CA). Side scatter, CD3 and CD4 staining were used to

distinguish for CD4+ and CD4- populations, which were then used as gates for an analysis

of CCR7 vs CD45RO or CD45RA staining. Lymphocyte subsets were analyzed using

MultiSet reagent cocktail (BD Biosciences).

T Cell Stimulation and Immunocytochemistry: T cells were stimulated using either anti-

CD3/CD28 or anti-CD19 antibody-coated beads (Dynal Biotech, Lake Success, NY)

(22). Alternatively, T cells were stimulated with EBV-B cells pre-pulsed with 7 µg/ml

staphylococcal enterotoxin B (SEB) (Sigma-Aldrich), for 2 hr at 37oC and labeled with 5

µM cell tracker blue CMAC (Molecular Probes, Inc., Eugene, OR). T cells were mixed with either beads or B cells at a ratio of 1:1.5 and spun briefly at 100 g. After stimulation, they were maintained in a humidified incubator at 37oC for 1-30 min and plated onto

poly-L-lysine coated coverslips. Attached cells were fixed with 4% PFA for 20 min,

blocked using 10% normal goat serum or horse serum, permeabilized with 0.2% Triton

X-100, and incubated overnight with primary antibodies followed by the appropriate

fluorescent secondary antibodies (Molecular Probes Inc.). The primary antibodies used

for detecting Kv1.3 proteins were either a rabbit polyclonal anti-Kv1.3 antibody against

an epitope on the C-terminal domain of the protein (Alomone, Jerusalem, Israel) or an

extracellular epitope (Sigma-Aldrich). The latter was used for labeling “live” Kv1.3

channels in T lymphocytes before interaction with the EBV-B cells. F-actin and GM1

- 68 - were stained using Alexa Fluor 546 phalloidin and Alexa Fluor 555 cholera toxin B,

respectively (Molecular Probes Inc.) and CD3ε was stained with a goat anti-CD3ε

antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Determination of antibody specificity and reactivity: To determine the specificity and

reactivity of Kv1.3 antibodies for intacellular (IC) or extracellular (EC) epitopes of the

protein (both polyclonal rabbit antibodies from Alomone, Jerusalem, Israel) a fusion

protein, specific for the corresponding antibody, and the anti-Kv1.3 antibody, were

incubated at a ratio of 1 µg (antibody) to 3 µg (fusion protein) for 1 hr at 4oC and centrifuged at 10,000 g for 5 min. Next, the preadsorbed antibody was added to human T cells, plated on poly-L-lysine coated coverslips and fixed with 4% PFA, overnight at room temperature. T cells were also incubated with non-preadsorbed antibody, treated as the preadsorbed antibody, and in the absence of primary antibody. Cells were then washed with PBS and incubated with a donkey anti-rabbit secondary antibody (1:1500)

(Molecular Probes Inc.) for 1 hr to allow fluorescent visualization. Coverslips were subsequently mounted on glass slides and viewed by widefield fluorescence microscopy using a Nikon Microphot FXA microscope coupled to a Spotcam camera and using the appropriate filters. Images were obtained using the same exposure time and gain to allow for direct comparison between groups.

Fluorescence and Confocal Microscopy: Protein accumulation was detected by fluorescence microscopy using either a Nikon Microphot FXA or a Zeiss Axioplan

Imaging 2 infinity-corrected upright scope coupled to an Orca-ER cooled camera

- 69 - (Axioscope, Carl Zeiss, Microimaging Inc.), Plan-Apochromat 60X-100X oil immersion

objectives and the appropriate filters. For co-localization studies a Zeiss LSM510 laser

scanning confocal microscope (Axioscope) equipped with an Ar ion laser, a HeNe laser

and a Plan-Apochromat 63X oil immersion objective was used. The “Multi Track” option

of the microscope was used to exclude cross-talk between detection channels.

Quantitation of fluorescence images: Kv1.3 accumulation at the bead/T cell point of

contact was analyzed as previously described (23). Briefly, boxes of equal area were

drawn around the IS and in the area most representative of the membrane outside the IS.

The mean fluorescence ratio (MFR), indicative of protein recruitment, was calculated as

follows: MFR = [Mean Fluoresce Intensity (MFI) at the IS–background]/[MFI outside

the IS–background]. More than 50 T/bead conjugates were analyzed for each donor at

each time point except for one RA patient for which 33 conjugates were analyzed for the

5 min time point. For the analysis of activated cells we used the increase in cell size as a

marker of activation and excluded those cells that showed a resting phenotype (diameter

≤0.5 µm). To determine colocalization confocal image stacks of 0.8-1.5 µm thick optical slices were collected and single optical slices of doubly labeled cells were then evaluated.

For quantitation of polarization in B/T cell conjugates a region was drawn around the T/B

cell contact area and another region was drawn around the entire T cell. The fluorescence

intensity was calculated for both regions. If the contact fluorescence was >50% of the

total the T/B cell conjugate was scored as positive for protein recruitment into the IS (24).

The data were analyzed using the Metamorph computer software.

- 70 - Electrophysiology: K+ currents were recorded in whole-cell configuration. The external

solution for activating and recording KCa3.1 currents had the following composition (in

mM): 160 NaCl, 4.5 KCl, 2.0 CaCl2, 1.0 MgCl2, and 10 HEPES, pH 7.4. The pipette

solution was composed of (mM): 145 K-Aspartate, 8.5 CaCl2, 10 K2EGTA, 2.0 MgCl2, and 10 HEPES, pH 7.2, with an estimated free [Ca2+] of 1 µM (25). KCa3.1 current was

measured in voltage-clamp mode by ramp depolarization from –120 mV to +40 mV, 200

ms duration, every 10 s, -80 mV holding potential (HP). Data were corrected for a liquid junction potential of –10 mV (21). The slope conductance of the KCa3.1 current was measured between –100 mV and –60 mV. Kv1.3 currents were induced by depolarizing voltage steps from -80 mV HP and applied every 30 s, unless otherwise indicated. The external solution for recording of Kv1.3 currents had the following composition (in mM):

150 NaCl, 5 KCl, 2.5 CaCl2, 1.0 MgCl2, 10 glucose and 10 HEPES, pH 7.4. The pipette

solution was composed of (mM): 134 KCl, 1 CaCl2, 10 EGTA, 2 MgCl2, 5 ATP-sodium,

and 10 HEPES, pH 7.4 (estimated free Ca2+ concentration 10 nM) (26). The number of

Kv1.3 and KCa3.1 channels per cell was determined by dividing the channel maximum

conductances for their corresponding single channel conductances. The Kv1.3 single

channel conductance was determined by us to be 11 pS (27). For KCa3.1 we used the

single channel conductance determined by Grissmer et al. and used by others to calculate

the number of KCa3.1 channels in T cells in similar experimental conditions (11, 28).

Membrane potential was measured by current-clamp with the same solutions used to

record Kv1.3 currents (21). The cell surface area was determined from the cell

capacitance based on the approximation that 1 pF=100 µm2 (13). Data were collected

- 71 - using the Axopatch200A amplifier and analyzed with pClamp8 software (Axon

Instruments, Foster City, CA, USA).

Statistical Analysis: All data are presented as means ± SEM, unless otherwise indicated.

Statistical analyses were performed using Student’s t-test (paired or unpaired); p<0.05 was defined as significant.

2.4 RESULTS

2.4.1 Determination of T cell phenotype. We have performed comparative studies aimed to identifying differences in the expression and activity of K+ channels in normal T

lymphocytes and T lymphocytes from patients with SLE that could explain the enhanced

Ca2+ response of the latter cells. Throughout this manuscript we report data collected

from SLE T cells within 24 hr from isolation from the blood. The time interval between

blood collection and analysis is critical since it has been shown that after 24 hr in culture

SLE T cells lose their peculiar characteristics (abnormalities in Lck, CD45 and lipid rafts)

and revert to a normal T cell phenotype (29). Since the addition of patients’ serum

during the in vitro incubation did not prevent this reversion of cell phenotype, it was

suggested that interaction with other cell types might be responsible for the alteration in

proximal signaling pathways (29). The T cell phenotype of the SLE patients enrolled in

this study was analyzed by flow cytometry. SLE patients displayed a significant

reduction in CD4:CD8 ratio (Fig. 2.1A) as compared to healthy donors and RA patients,

attributed to a significant decrease in CD4+ and an increase in CD8+ cells (Fig. 2.1B) and

in agreement with previous reports (29, 30). Furthermore, SLE patients also display a

- 72 - p=0.07 A p=0.0002 4 60 p=0.02 SLE p=0.002 3.5 50 NL p=0.00001 3 RA p=0.0001 40 2.5 2 30

1.5 20 CD4+/CD8 1 % lymphocytes 10 0.5 0 0 CD4+ CD8+

C NL 100 SLE 100

80 80 +

+ 60 60

40 % CD8 40 % CD4 20 20

0 0 naive TCM TEM naive TCM TEM p=0.08 p=0.4 p=0.04 p=0.2 p=0.1 p=0.03

Figure 2.1: Expression of T cell subsets in SLE, RA and normal donors. Flow cytometry analysis using anti-CD4 and anti-CD8 antibodies as gating antibodies of SLE patients (n=18), healthy controls (n=16) and RA patients (n=4). (A) CD4/CD8 ratio indicates the relative proportions of T cells expressing CD4 or CD8. (B) Levels of expression of CD4 and CD8 in the 3 populations. C. Relative levels of naïve, TCM and + + TEM cells in CD4 (left) and CD8 (right) lineages for SLE (n=18) and normal (n=16) individuals. Normal and SLE T cell populations were further characterized as follows: + - + + - + naïve (CCR7 CD45RO ), TCM (CCR7 CD45RO ), and TEM (CCR7 CD45RO ). There + + was an increase in CD4 TEM cells accompanied by a decrease in CD8 TEM populations in SLE as compared to healthy donors. The levels of significance within the T cell subsets are indicated at the bottom.

- 73 - + - + + significant increase in CD4 TEM (CCR7 CD45RO ) cells but a decrease in CD8 TEM cells (Fig. 2.1C). This demonstrates that CD4+ cells exist in a more active state in SLE

patients as previously reported (30, 31).

2.4.2 Biophysical and Pharmacological characteristics of Kv1.3 channels in SLE T

cells. Currently nothing is known about the expression and activity of ion channels in T

cells from patients with SLE. Electrophysiological experiments were thus performed to

characterize Kv1.3 channels in ex vivo SLE T cells. We found that Kv1.3 currents in SLE

T cells were voltage-dependent with a V1/2 (voltage at which half of the channels are

activated) of –27±1 mV (n=18) similar to that of normal resting T cells (-25±1 mV, n=6,

p=0.27) (12, 32) (Fig. 2.2A). The activation and inactivation time constants,

physiologically important characteristics, of Kv1.3 currents measured at +50 mV in SLE

T cells were also similar to those of Kv1.3 current in healthy resting T cells. The

activation time constants were 163±13 ms (n=52) in healthy T cells and 186±29 ms

(n=20; p=0.5) in SLE T cells. The time constants of inactivation in healthy and SLE

resting T cells were 207±19 ms (n=52) and 247±16 ms (n=20, p=0.1), respectively.

Furthermore, Kv1.3 channels in SLE T cells display cumulative inactivation, a

characteristic property of these channels, as indicated by the progressive decrease of the

maximal current amplitude upon application of consecutive depolarizing pulses every

second (Fig. 2.2B) (8). Additional studies established that the Kv1.3 currents recorded in

SLE T cells were completely and reversibility blocked by the selective Kv1.3 inhibitor

ShK-Dap22 (10 nM, Fig. 2.2C) and this concentration of ShK-Dap22 induced a 23±4 mV

(n=4, Fig. 2.2D) membrane depolarization in these cells (33). We have also estimated

- 74 - A 1.0

0.8

0.6 200 pA 0.4 G/Gmax G/Gmax 500 ms 0.2 V (mV) 0.0 -60 -40 -20 0 20 40 C 100 pA B 200 ms wo 0 200 pA 22 20 ms +ShK-Dap 1 D 20 0 2 -20 MP (mV) -40 100 ms

Figure 2.2: Electrophysiological and pharmacological properties of Kv1.3 channels in SLE T cells. A. Kv1.3 currents were recorded in whole-cell configuration and were elicited by depolarizing voltage steps from -60 mV to +40 mV (10 mV increments) from -80 mV HP every 30 sec. The conductance-voltage curve (constructed from current amplitudes such as those shown) was fitted to a Boltzman function and the voltage at which half of the channels are activated (V1/2) calculated. B. Kv1.3 channels’ cumulative inactivation was induced by consecutive depolarizing pulses applied every second. The maximal current amplitude progressively decreased with each successive pulse (indicated by progressive numbers). C. Effect of Shk-Dap22 on Kv1.3 current in SLE T cells. Currents were recorded in whole cell configuration in physiologic solution before application of Shk-Dap22 (-ShK-Dap22), after Shk-Dap22 (10 nM) inhibition and after drug wash-out (wo). D. Membrane potential (MP) measured by current-clamp before and after ShK-Dap22 (10 nM) addition. The time of ShK-Dap22 introduction is indicated by an arrow. The SLEDAI of the patients for this study ranged from 2-12.

- 75 - whether T lymphocytes from SLE patients expressed the same number of Kv1.3 channels

as healthy resting T cells. The total number of Kv1.3 channels/cell was determined by

dividing the channel maximum conductance by its corresponding single channel conductance (11 pS) (27). SLE T cell express on average 349±30 channels/cell (n=20),

ranging between 178 and 675 channels/cell. This is similar to the number of channels

expressed by normal resting T cells (308±16, n=52; p=0.2) (Table 2.1).

Table 2.1 K+ channel expression in SLE and normal T lymphocytes

Normal SLE Resting Activated Capacitance (pF) 1.01 ± 0.04 4.46 ± 0.19*** 1.49 ± 0.08***,$$$ n625540 current density (pA/pF) 501 ± 36 263 ± 27*** 416 ± 40$ total channels (#channels /cell) 308 ± 16 786 ± 83*** 349 ± 30$$$ Kv1.3 channel density (#channels/µm2) 3.50 ± 0.25 1.84 ± 0.19*** 2.91 ± 0.28$ n522320 conductance (nS) 0.33 ± 0.03 2.55 ± 0.27*** 0.34 ± 0.03$$$ $$$ KCa3.1 total channels (# channels /cell) 29.7 ± 3.3 232.0 ± 24.9*** 30.7 ± 2.5 channel density (#channels/µm2) 0.24 ± 0.03 0.57 ± 0.07** 0.21 ± 0.02$$$ n103220

** p<0.01 vs resting $ p<0.005 vs activated *** p<0.0001 vs resting $$$ p<0.0001 vs activated

Taken together these data demonstrate that SLE T cells expressed the same number of

Kv1.3 channels as resting T cells from healthy donors and that these channels share

identical biophysical and pharmacological properties with their healthy counterparts.

Moreover, Kv1.3 channels control the membrane potential in SLE T cells as indicated by

the depolarization induced by Kv1.3 channel blockade.

- 76 - 2.4.3 Native Kv1.3 channels are recruited in the immunological synapse upon

activation of healthy and SLE T cells. Since the biophysical properties of the channel

remained unaltered we wanted to investigate whether other alterations in the Kv1.3

channel behavior might be encountered in SLE T cells. Previous studies have shown that recombinant Kv1.3 channels are recruited in the IS (14-16). However, the process by which native Kv1.3 channels transition into the IS is still to be defined. Furthermore, possible alterations of this process in diseased T cells have never been investigated. To address this question, we first investigated Kv1.3 channel polarization to the synapse in human CD3+ T cells from healthy donors. To induce T cell activation, and synapse formation, we used anti-CD3/CD28 antibody coated beads as surrogate APCs. This is a well validated system to study membrane reorganization and downstream functional events triggered by TCR binding (21, 22). Our results indicate that upon stimulation with

CD3/CD28 beads, Kv1.3 channels partition to the T cell/bead contact area and colocalize

extensively with F-actin and the glycosphingolipid GM1, a marker of lipid rafts (Fig.

2.3A bottom panels). Both F-actin and GM1 are known to reorganize and accumulate at

the IS (17, 34). In contrast, Kv1.3 channels are evenly distributed on the membrane of

resting T cells not exposed to beads (Fig. 2.3A top panels). In the same way, SLE and RA

T cells recruit Kv1.3 channels in the cell/bead contact interface upon activation with the

CD3/CD28 beads (Fig. 2.3B-C, lower panels) while the channels remain evenly

distributed in the absence of beads (Fig. 2.3B-C, upper panels). To exclude that Kv1.3

channel relocalization occurs because of simple cell to bead contact, to establish the

variability of our technique and to determine the threshold for a significant Kv1.3 channel

accumulation in the synapse, we performed identical experiments using beads coated

- 77 - A NL Kv1.3 F-actin Merge Kv1.3 GM1 Merge

X

X X

X

B SLE C RA Kv1.3 F-actin Merge Kv1.3 F-actin Merge

X X

X

E 6 D Kv1.3 F-actin CD3/CD28 CD19 X X X 4 CD19 No Beads

conjugates 2 Number of bead/cell X X X 0 CD3/CD28

0.0 0.5 1.0 1.5 2.0 2.5 MFR

Figure 2.3: Kv1.3 channels are recruited at the interface between CD3/CD28 beads and T cells. A-C. Normal (A), SLE (B) and RA (C) human CD3+ cells were stimulated with CD3/CD28 beads for 5-15 min at 37oC. Resting (non exposed to beads, top panels) or bead activated T cells (bottom panels) were fixed, permeabilized, immunolabelled for Kv1.3 (green) and either F-actin (A, left panel, B and C) or GM1 (A, right panel) and visualized with confocal microscopy. F-actin and GM1 were identified by fluorescence- labeled phalloidin and fluorescence-labeled cholera toxin B, respectively (red). Areas of co-localization of Kv1.3 channels and F-actin or GM1 are shown in yellow in the right panels (merge). D. T cells were exposed to either CD3/CD28 or CD19 beads for 15 min at 37oC. Resting (non exposed to beads, top left panel), CD19 exposed T cells (top middle and right panels) or CD3/CD28 activated T cells (bottom panels) were fixed, permeabilized, immunolabelled for Kv1.3 (green) and F-actin (red) and visualized with fluorescence microscopy. Beads are marked with an X. Scale bar = 5 µm. E. Distribution of the mean fluorescence ratio (MFR) in T/bead conjugates that form in presence of either CD19 or CD3/CD28 coated beads.

- 78 - with an antibody against CD19 (a component of the B cell receptor complex) (22). In

contrast to CD3/CD28 beads, CD19-coated beads did not have a significant effect on

Kv1.3 or F-actin localization to the cell/bead contact interface (Fig. 2.3D). The degree of

protein accumulation at the IS was indicated by the mean fluorescence ratio (MFR),

calculated as described in the method section. The distributions of the MFRs in T cells exposed to CD3/CD28 and CD19 coated beads are reported in Fig. 3E. The cells

stimulated with CD19 and CD3/CD28 beads had a MFR of 1.04 (SD 0.20, n=49) and

1.78 (SD 0.24, n=126), respectively. As a result, T cell/bead conjugates that displayed a

MFR >1.5 (>2 folds the SD of the average MFR in CD19 experiments) were scored

positive for Kv1.3 channel polarization in the IS. Based on these results we were able to

study the kinetics of Kv1.3 accumulation in the IS. The process of IS formation is quite

dynamic with different proteins transition in the synapse at different times. Thus specific

kinetics of a protein transitioning in the IS might guarantee its coming in contact with

signaling molecules present at the IS and thus its proper regulation and function. The

time frame of Kv1.3 compartmentalization in the IS is not known in either normal or SLE

T cells.

2.4.4 Kv1.3 channel compartmentalization in the immunological synapse is altered

in SLE T cells. We studied the process of Kv1.3 channel translocation in the IS in SLE,

RA and normal donors. Fourteen SLE patients were included in the following

microscopy studies: 11 females and 3 males, 10AA and 4C, age, 38.0 + 3.1 years (p=0.3

vs. healthy individuals); range 24-67 years. These patients’ SLEDAI ranged from 2-12.

As controls we used nine normal subjects: 5 females, 2 males and two unknown, 2AA,

- 79 - 5C and 2 unknown, age 43.3 + 3.1 years; range 33-53 years and five RA patients: 5 female, 3AA and 2C, age 57 + 6.0 and one unknown years; range 40-68. Disease activity and medication regime of the patients used in the microscopy experiments is shown in table 2.2.

Table 2.2 Details of SLE and RA patients in the microscopy studies

Pt # SLEDAI MEDICATIONS Kv1.3 Abnormality SLE patients 1 12 HCQ + MMF + Pred.* Yes 2 12 Azathioprine + Pred.* Yes 3 5 HCQ + Pred.* Yes 4 10 Pred. Yes 5 4 HCQ + Pred.* Yes 6 3 HCQ Yes 7 2 HCQ + MMF Yes 8 10 HCQ + Pred. + MMF No 9 8 Pred. + MMF Yes 10 2 None Yes 11 4 None Yes 12 4 Pred.* Yes 13 10 HCQ + Pred. Yes 14 4 HCQ + Pred. + Yes Azathioprine RA patients 1 Pred. No 2 HCQ + Pred. * No 3 HCQ + MTX. No 4 HCQ No 5 HCQ + MTX + Pred.* No

HCQ = Hydroxychloroquine (Plaquenil); MMF = Mycophenolate Mofetil (CellCept); MTX = Methotrexate; Pred.= Prednisone, * <10mg.

- 80 - We first examined the kinetics of Kv1.3 channel recruitment into the IS in resting T cells

from healthy individuals by exposing them to CD3/CD28 beads for 1, 5, 15 and 30 min.

Cell conjugates formed between CD3/CD28 beads and T cells were then fixed and

immunostained with anti-Kv1.3 antibody. The assessment of the time-dependent

distribution of Kv1.3 channels in the IS was done by establishing the number of T

cell/bead conjugates with polarized Kv1.3 proteins over the total number of conjugates

for each time point. Fig. 2.4A indicates that Kv1.3 channel redistribution in the IS of resting healthy T cells occurs after only 1 min of exposure to the beads and progressively

increases over time. Overall Kv1.3 recruitment in the IS is maintained for at least 30 min

from synapse formation. Still at 1 hr there was sustained recruitment. The percentage of

Kv1.3 polarized conjugates at 1 hr was 53+5% (n=2). By 2–5 hr the Kv1.3 channel was

removed from the synapse with only 25+4% conjugates showing Kv1.3 recruitment

(n=2). Similar experiments were performed with SLE T cells and we observed that in 7

out of eight patients the kinetics of Kv1.3 channel compartmentalization in the synapse

are quite different (Fig. 2.4B). Specifically, Kv1.3 polarization in primary SLE T cells is

maximal at 1 min after TCR engagement and progressively declines over time, indicating

that Kv1.3 channels either re-distribute on the plasma membrane outside the IS or that

they are internalized and degraded. This defect appears to be restricted to SLE as it was

not observed in RA patients (Fig. 2.4C and Table 2.2). Although some degree of

variability was observed in individual RA patients, we never encountered Kv1.3 kinetics

matching SLE T cells and on average, Kv1.3 channels are recruited in the IS of RA T

cells within 1 min and are maintained there for at least 30 min.

- 81 - A NL BF Kv1.3 F-actin Merge p=0.02 70 1 60 50 5 40 30

15 conjugates 20 Time (min) % Kv1.3 polarized 10 30 0 1 5 15 30 Time B SLE BF Kv1.3 F-actin Merge p=0.01 60 p=0.04 1 50 40 5 30 20 15 conjugates Time (min) 10 30 % Kv1.3 polarized 0 1 5 15 30 Time RA C BF Kv1.3 F-actin Merge 70

) 1 60 50 min ( 5 40 30 20 15 conjugates Time 10 % Kv1.3 polarized 30 0 1 5 15 30 Time

Figure 2.4: Differential kinetics of Kv1.3 channel reorganization in the IS. Left panels: Representative fluorescent images for resting normal (A) SLE (B) and RA (C) T cells after 1-30 min activation with CD3/CD28 beads. T cells were activated with CD3/CD28 beads for 1, 5, 15 and 30 min, fixed, permeabilized and stained with phalloidin conjugated to Alexa fluor 546 (to visualize F-actin) and Kv1.3 antibody followed by a fluorescent secondary antibody. Scale bar = 5 µm. Right panels: Quantitative analysis of Kv1.3 channel recruitment in the IS was performed as described in the methods section. The % of cells that display Kv1.3 accumulation in the IS at different times of exposure to beads is reported as % of cells with Kv1.3 polarized at the site of contact relative to the number of cells that made contact with beads. The data shown are the average responses collected from 6 healthy individuals (2 AA and 4 C, n=4 donors for 1min), 7 SLE patients (7 AA, SLEDAI 2-12) (n=6 for 1 min) and 5 RA patients (3AA and 2C).

- 82 -

This differential kinetics of Kv1.3 translocation into the IS in healthy and SLE T cells was also observed to occur at the interface between T cells and APCs (Fig. 2.6). T cells were incubated with EBV-infected B cells in the presence or absence of SEB for 1 and 30 min and the accumulation of Kv1.3 and CD3ε at the IS was determined. Control experiments were performed using EBV-infected B cells in the absence of SEB. To study the compartmentalization of Kv1.3 channels in the contact area between T cells and SEB- pulsed EBV-infected B cells we labeled the T cells “live” with an anti-Kv1.3 antibody against an extracellular (EC) epitope of the Kv1.3 channel protein before encounter with the APCs. This allowed selective labeling of the Kv1.3 channels in the T cell membrane

A B EC anti-Kv1.3 Preadsorbed antibody IC anti-Kv1.3 antibody Preadsorbed EC anti-Kv1.3 IC anti- Kv1.3 antibody antibody EC anti-Kv1.3 antibody with beads

Figure 2.5: Determination of specificity of anti-Kv1.3 antibodies. Human T cells were incubated with either an anti-Kv1.3 antibody for an IC (A) or an EC (B) epitope of the Kv1.3 protein that was or was not preadsorbed. Both antibodies were specific as fluorescence was observed when the antibody followed by a fluorescent secondary antibody was used (A, bottom, left panel and B, top panels) but no fluorescence was observed with the preadsorbed antibody followed by a fluorescent secondary antibody (A, top panels and BV, middle panels). Comparable effectiveness of both anti-Kv1.3 antibodies in detecting Kv1.3 channel accumulation in the IS upon anti-CD3/CD28 bead stimulation was observed (A, bottom right panel and B, lower panels).

- 83 - as determined by the lack of fluorescence signal after pre-adsorption of the antibody to

the corresponding antigen (Fig. 2.5) and it can be used alternatively to the antibody for an

intracellular (IC) epitope of the Kv1.3 channel that we have used in T cell/bead

experiments. Similar results were obtained using the two antibodies (Fig. 2.5). The

accumulation of Kv1.3 channels in the IS was determined as described in the method

section and B/T cell conjugates that displayed a fluorescence at the synapse ≥ 50% of the

total fluorescence were defined as polarized Kv1.3 conjugates. Our results indicate that in the absence of SEB, Kv1.3 and CD3ε were evenly distributed on the plasma membrane

of healthy T cells in the majority of the conjugates while in the presence of SEB Kv1.3

and CD3ε concentrated at the IS (Fig. 2.5B). Overall, normal resting T cells showed

Kv1.3 polarization to the IS at 1 min and the channels were maintained in the IS for at

least 30 min (Fig. 2.5B and D). All the cells that recruited Kv1.3 also recruited CD3ε. A

different pattern of translocation into the IS that forms with APCs was instead observed

with SLE T cells (Fig. 2.5C and E). Kv1.3 polarization in SLE T cells is maximal at 1

min after TCR engagement and is decreased by 30 min. These results substantiate, in a

more physiological model of T/APC interaction, the observations made with the

CD3/CD28 coated beads.

- 84 - A NL DIC B cell Kv1.3 CD3ε Merge

T -SEB

+SEB T

B SLE DIC B cell Kv1.3 CD3ε Merge

T ) 1 min (

30 T Time NL SLE C p=0.05 D 3 1.2 p=0.02 2.5 1 2 0.8

1.5 0.6 1 0.4

Relative Kv1.3 0.5 0.2 polarized conjugates 0 0 1 30 1 30 Time (min) Time (min)

Figure 2.6: APC-T cell activation induces differential reorganization of Kv1.3 channels in the IS formed with resting healthy and SLE T cells. T cells were either mixed with the beads and immunolabeled, after fixation, with anti-Kv1.3 IC antibody or labeled live with the EC anti-Kv1.3 before interaction with the beads. CD3/CD28 beads are marked by an X. A. Accumulation of Kv1.3 and CD3ε in the IS. Healthy resting T cells were incubated with EBV-infected B cells that had been exposed to medium with (bottom panels) or without (top panels) SEB. B cells were labeled with CMAC cell tracker blue. B. Accumulation of Kv1.3 and CD3ε in the IS formed between APCs and SLE T cells after 1 and 30 min interaction. Scale bar = 5µm. C-D. Time-dependent recruitment of Kv1.3 channels in the IS in healthy and SLE T cells. T/B cell conjugates were quantitatively evaluated for the recruitment of Kv1.3 channels as described in the methods section. The data are reported as average of the relative percentage of Kv1.3 polarized conjugates (normalized for Kv1.3 polarized conjugates at 1 min, note the y-axis scale difference attributed to SLE T cell recruitment being maximal at 1 min and depicted as such to reflect this). The histograms represent 3 healthy donors (3 C) and 3 SLE

- 85 - patients (1 AA and 2 C, SLEDAI range: 2-8). At least 10 T/APC conjugates were evaluated for each donor per time point.

2.4.5 The kinetics of Kv1.3 redistribution in the immunological synapse of SLE T cells resembles those of pre-activated normal T cells. It is generally believed that SLE

T cells exist in an active state (36). Accordingly, it is possible that the different dynamics of Kv1.3 compartmentalization in SLE T cells might reflect a more activated T cell phenotype. We have thus studied the process of Kv1.3 compartmentalization upon TCR- engagement in PHA pre-activated healthy T cells (Fig. 2.7). While resting T cells display

A p= 0.003 B p= 0.02 BF Kv1.3 F-actin Merge 1.2 p=0.02 70 p= 0.02 No 1.0 beads 60 50 0.8 1 40

) 0.6 30 min

( 0.4 20 Relative Kv1.3 Kv1.3 Relative 30 conjugates 10 0.2 polarized conjugates conjugates polarized % Kv1.3 polarized % Kv1.3 polarized

Time 0 0 1 5 15 30 1 30 Time (min) Time (min)

Figure 2.7: Kv1.3 channel recruitment in the IS in activated healthy T cells parallels SLE T lymphocytes. A. T cells were pre-activated by exposure to PHA (4µg/ml) in the presence of autologous PBMCs for 72 hr. Pre-activated T cells were or were not stimulated with CD3/CD28 beads for 1-30 min. Left panels: representative photomicrographs. Right Panels: Quantitative analysis of Kv1.3 channel recruitment in the IS of activated T cells was performed as described in the methods section. The histogram shows the percentage of cells showing Kv1.3 accumulation at the IS at different time points (1-30 min). The data are the average of >50 cells/donor from seven healthy donors except 5 min, six donors. B. Activated healthy T cells were incubated with SEB-pulsed EBV B cells for 1 or 30 min. T/B cell conjugates were quantitatively evaluated for the recruitment of Kv1.3 channels as described in the methods section. The histograms represent an average of the relative percentage of Kv1.3 polarized conjugates (normalized for the recruitment of Kv1.3 polarized conjugates at 1 min) in 3 healthy donors. At least 10 T/APC conjugates were evaluated for each donor per time point.

- 86 - a long-lasting recruitment of Kv1.3 channels in the IS (Figs. 2.4 and 2.6), pre-activated T

cells display a different time-course: Kv1.3 channels moved rapidly to the IS with maximal recruitment at 1 min and progressively moved out of the synapse by 30 min

(Fig. 2.7). Instead, in the absence of stimulation, Kv1.3 channels remained evenly distributed around the membrane. Consistent results were obtained using either

CD3/CD28 beads or SEB-pulsed B cells as APCs. Overall, the dynamics of Kv1.3 compartmentalization in healthy activated T cells parallel the Kv1.3 recruitment observed in resting SLE T cells.

The process of Kv1.3 channel translocation in the IS during activation with CD3/CD28 beads was quantitatively summarized by determining the rate of change in percentage of

Kv1.3 polarized conjugates over time using a linear regression model. The “slope” of the model, indicative of the rate of formation of Kv1.3 polarized conjugates, was compared in SLE patients and normal controls (Fig. 2.8A). Overall we observed similar negative slopes in 7 out of 8 SLE patients, indicating that in these patients the localization of

Kv1.3 in the IS is short-lived. Similar slopes were observed in activated healthy T cells and they were significantly different from those determined in healthy resting T cells.

This behavior appeared to be unrelated to the disease activity and immunosuppressive regime. When we grouped all the SLE patients that were used in the microscopy studies, using either CD3/CD28 coated beads or APCs, we observed this Kv1.3 mobility defect in all except one patient. The outlier (SLE patient #8, Table 2.2) displayed kinetics similar to normal resting T cells. This patient also showed a particularly immunosuppressed T cell phenotype as indicated by a percentage of naïve CD4+ and CD8+ cells well above

- 87 - A 1.5

1

0.5

0.0

-0.5 formation -1

-1.5 rate of Kv1.3 polarized conjugate rate of Kv1.3 -2 NL-r NL-a SLE p=0.0002 p=0.3

p=0.0004 B 1.2

0.8 0.4

0.0 -0.4

formation -0.8 -1.2

-1.6 rate of Kv1.3 polarized conjugate rate of Kv1.3 -2.0 SLE CD4+ CD8+ p=0.5 Figure 2.8. Comparison of the rates of Kv1.3 channel compartmentalization in the IS in normal and SLE T cells. A. The rate of Kv1.3 polarized conjugate formation induced by activation with CD3/CD28 beads was determined in normal and SLE T lymphocytes by linear fitting of the time courses shown in figs 4C-D and 6B. The slope of the model is plotted for each group: normal lymphocytes (NL), resting ( ○ ) and activated ( ● ), and SLE ( ∆ ) T cells. A negative slope is indicative of rapid Kv1.3 channel redistribution outside the IS. B. Rate of Kv1.3 polarized conjugate formation in CD4+ and CD8+ cells from SLE patients. CD4+ and CD8+ cells were separated from the same individual and studied in parallel. A total of 4 SLE patients were studied: 3AA and 1C, SLEDAI range 4-10. T cells were activated by exposure to CD3/CD28 beads for 1 and 30 min. The number of Kv1.3 polarized conjugates for each time point was determined as described in the legend of Fig. 4 and plotted against time. The slope obtained by linear fitting of the time-course is reported. The symbols indicating each donor are conserved among the two groups.

- 88 - healthy controls (70% and 84%, respectively). This raised the possibility that the abnormal Kv1.3 behavior in SLE T cells might be determined by the more activated state

of their CD4 lineage (Fig. 2.1). If this is the case, we would expect that CD4+, and not

CD8+, display these characteristic kinetics. Experiments were thus performed to compare the rate of Kv1.3 polarized conjugate formation in CD4+ and CD8+ cells from 4 SLE

patients. We observed, on average, no differences between these T cell subsets (Fig.

2.8B).

Overall, these results establish that the general SLE T cell population displays faster

kinetics of Kv1.3 channel translocation in and out of the IS as compared to healthy

resting T cells. This defect occurs independently of disease activity as it was observed in

patients with a SLEDAI ranging from 2-12. It also occurs independently of

immunosuppressive therapy as 3 SLE patients who were not under immunosuppressive therapy (allow <10mg Prednisone/day) display this defect. Moreover, freshly-isolated

SLE T cells behave like healthy blast T cells in regards to Kv1.3 transitioning into the IS.

It has been shown that KCa3.1 channels, and not Kv1.3 channels, control Ca2+ homeostasis in activated cells (25). So it is possible that the rapid dynamics of the Kv1.3 translocation into the IS in pre-activated T cells are compensated by the presence of

KCa3.1 channels. The expression of KCa3.1 channels in SLE T cells has yet to be determined. Experiments were thus performed to determine whether the K channel expression in SLE T cells matches pre-activated healthy T cells.

- 89 - 2.4.6 T lymphocytes from patients with SLE display a K+ channel phenotype similar

to healthy resting T cells. Whole-cell voltage-clamp experiments were performed to determine the expression of KCa3.1 channels in SLE T cells and compare it to that of healthy T cells (Fig. 2.9 and Table 2.1). The healthy T cells studied consisted of both

resting (freshly-isolated) and mitogen pre-activated T cells obtained by prolonged

exposure to PHA. This intervention has been shown to activate human T cells and

increase their cell capacitance, a measure of cell size, and KCa3.1 conductance (25, 37).

These cells also showed a faster Kv1.3 channel compartmentalization upon TCR

activation (Fig. 2.7). Membrane capacitance measurements indicated that the activated T

cells we studied were indeed activated. The membrane capacitances of mitogen pre-

activated and resting (freshly-isolated) CD3+ cells were 4.46+0.19 pF (n=55) and

1.01+0.04 pF (n=62; p<0.001), respectively (Table 2.1). Similar capacitance values have

been reported for quiescent and pre-activated human T cells (38). Interestingly, we found

that resting SLE T cells have membrane capacitance higher than healthy resting T cells,

but less than pre-activated T cells. This indicates that resting SLE T cells are bigger than

resting healthy T cells with an average cell surface area of 149 µm2 and 110 µm2, respectively (Fig. 2.9B). The cell surface area was determined from the cell capacitance based on the approximation that 1 pF=100 µm2. This might indicate that SLE T cells are

partially activated or are ‘frozen’ in an early stage of activation as previously suggested

(36). Yet, the KCa3.1 conductance in SLE T cells is identical to normal resting T cells,

suggesting that the number of channels is the same (Table 2.1). Indeed, the KCa3.1

channel number/cell in SLE T cells is similar to that of primary resting T cells (Fig. 2.9C

and table 2.1). In contrast, healthy pre-activated T cells have an eightfold increase in

- 90 - A SLE NL-resting NL-activated

600 200 150 I (pA) I (pA) I (pA) 400 100 100 200 50

-80 -40 +20 -80 -40 +20 -80 -40 +20 V (mV) V (mV) V (mV)

B p<0.0001 C p<0.0001 p<0.0001 NL-resting

50 NL-activated 30 ) p<0.0001 2 SLE m 25 µ 40 ( 20 30 15 p<0.00 20 10 10 5 KCa3.1 channels/cell Cell surface area Cell surface 0 0

Figure 2.9: The expression of KCa3.1 channels in SLE T cells is comparable to that in resting healthy T cells. A. KCa3.1 channel currents were recorded in whole cell configuration, voltage-clamp mode by ramp depolarization from -120 mV to +40mV (200 ms duration, every 10 sec, HP= -80mV). Characteristic traces are shown for resting SLE (left), normal resting (middle) and activated (right) T lymphocytes (NL). B. Cell surface area for SLE (n=40) and healthy, resting (n=62) and activated (n=55) T cells. C. Expression levels of KCa3.1 channels in SLE and healthy (resting and activated) T cells. The total number of KCa3.1 channel/cell was calculated by dividing the KCa3.1 maximum conductance (determined by fitting of the linear portion of the KCa3.1 current measured between –100 mV and –60 mV) for the KCa3.1 unitary conductance. The SLEDAI of the patients for this study ranged from 2-12.

- 91 - KCa3.1 conductance which translates to an eightfold increase in channel numbers. When

normalized for cell size, SLE T cells have KCa3.1 channel density similar to resting T cells and significantly lower than healthy pre-activated T cells (Table 2.1). Similarly, the

Kv1.3 channel density in SLE T cells is comparable to healthy primary T cells. The

Kv1.3 and KCa3.1 channel composition in the mixed population of normal (resting and activated) and SLE T cells is summarized in Table 2.1. These results indicate that the number of functional Kv1.3 and KCa3.1 channels expressed in SLE T cells is similar to that of healthy resting T cells. Thus, Kv1.3 channels constitute the main K+ conductance

in SLE T cells and as such are the main regulators of membrane potential and Ca2+ homeostasis in these cells.

2.5 DISCUSSION

In this study we examine for the first time K+ channels in SLE T cells and provide

evidence of significant differences in Kv1.3 channel dynamics of translocation in the IS

between resting healthy and SLE T lymphocytes. We also find that in SLE T cells the

movement of the Kv1.3 channel on the plasma membrane during presentation of the

antigen and formation of the IS resembles that of healthy activated T cells. Despite that,

SLE T cells lack the corresponding K+ channel make-up that is integral in regulating the activation response in normal activated T cells. These discrepancies might account for

the hyperactivity and exaggerated response of SLE T cells to antigen presentation.

K channels have been shown to be key regulators of T cell activation as they control the

membrane potential and the influx of Ca2+ triggered by antigen presentation (8). As such

- 92 - K channels could play a role in the abnormal Ca2+ response to antigen stimulation that

has been reported to occur in human SLE T lymphocytes (5, 6). Nevertheless, their activity and function in SLE T cells has never been investigated. We have conducted various studies aimed at dissecting the properties of K+ channels in SLE T cells. These

studies were conducted on a cohort of SLE patients with a T cell phenotype characteristic

of this disease as indicated by the low CD4/CD8 ratio (29, 30). This low CD4/CD8 ratio

might have been exacerbated by the presence of patients with lupus nephritis and patients

under corticosteroid treatment (39). Furthermore, the SLE patients display an activated

+ + CD4 memory phenotype with CD4 TEM levels higher than healthy individuals as

+ previously described (40). This is accompanied by a decreased expression of CD8 TEM cells and it is in agreement with the common consent that the altered immune response in

SLE is mediated by an imbalance in the functions of T cell subsets: exaggerated activity of CD4+ helper cells and diminished function of CD8+ suppressor/cytotoxic cells (2).

When we analyzed this mixed population of SLE T cells for the expression of K+ channels we found that SLE T cells display a K+ channel phenotype similar to normal

resting T cells with Kv1.3 channels constituting the main K+ conductance and controlling

the membrane potential. Although we showed no differences in the biophysical and

pharmacological properties of Kv1.3 channels in SLE T cells as well as in their number

as compared to normal resting T cells, our data indicate that there are fundamental

differences in the process of Kv1.3 channel translocation in the IS. The accumulation of

Kv1.3 channels in the IS in healthy resting T cells occurs progressively and it is sustained

for a long time, well beyond the onset of signal transduction (i.e. the onset of Ca2+ influx)

- 93 - (41). This is consistent with the long time necessary for resting T cells to form mature

synapses (42). In contrast, SLE T cells show a faster recruitment of Kv1.3 channels into

the IS and redistribution outside the synapse. Interestingly, the process of Kv1.3

recruitment in SLE T cells parallels the process observed in healthy pre-activated T cells.

Indeed it has been shown that synapse maturation occurred much faster in T cell blasts

than resting T cells (42). This behavior of SLE T cells is consistent with the view that T

cells from SLE patients resemble activated T cells (36). T cells from patients with SLE

display various characteristics of activated T cells: they exhibit a loss of CD3ζ chain

which is replaced by FcRγ chain and Syk recruitment to the TCR complex. These alterations indicate a switch from the TCR/CD3/CD3ζ/ZAP-70 receptor complex of

resting T cells to the TCR/CD3/FcRγ/Syk receptor complex of effector T cells (36).

Functionally SLE T cells are primed for activation and respond more rapidly to antigenic

triggers than do T cells from normal individuals (29). Furthermore, they react more

rapidly than healthy T cells to antigen presentation in terms of reorganization of elements

known to accumulate at the IS such as F-actin and lipid rafts (6). Along this line, the

significantly larger size of SLE T cells we have measured during our electrophysiological

experiments indicates that in these patients T cells exist in a partially activated state as

previously suggested (36). Similar to our findings all these alterations were found in

freshly isolated peripheral blood T cells from SLE patients independent of their disease

activity, thus suggesting a constant activation state of SLE T cells (6, 30).

However, although SLE T cells circulate as activated (or partially activated) T cells they

do not display the K channel make-up characteristic of activated T cells. SLE T cells

- 94 - express ca. 300 Kv1.3 and ca. 30 KCa3.1 channels/cell. Similar values are reported in the

literature for resting naïve, TCM and TEM cells of the CD4 and CD8 lineages (13).

Although we were measuring a mixed population, we never encountered SLE T cells

with either high Kv1.3 channel number (≥1,500/cell), indicative of the activated TEM phenotype, or high KCa3.1 channel number, indicative of activated naïve and TCM cells

(13). Overall, freshly isolated peripheral blood SLE T cells express a number of Kv1.3 and KCa3.1 channels equal to resting healthy T cells and, likewise, Kv1.3 channels constitute the main K+ conductance in SLE T cells. As such, they modulate SLE T cell

membrane potential. Thus alterations in Kv1.3 channel behavior might have important consequences in the Ca2+ homeostasis of SLE T cells. It is possible that alterations in

dynamics of Kv1.3 localization in the IS contribute to the pronounced and sustained

TCR-mediated Ca2+ influx of SLE T cells. This exaggerated Ca2+ response was observed

in both SLE CD4+ and CD8+ subsets, although higher in CD4+ (5). Consistently, we

showed that both CD4+ and CD8+ SLE T cells display faster dynamics of Kv1.3 translocation in and out of the IS. Furthermore, we showed that this defect is not present in T cells from RA patients. This is consistent with the fact that RA T cells do not display an exaggerated Ca2+ response to antigen presentation (5, 43).

The functional consequences of this differential dynamics of Kv1.3 protein localization in the IS of the SLE T cells are unclear at present. However, it has been suggested that ion

channel localization in the IS might be necessary for guaranteeing the channel proximity

to signaling molecules that control the channel’s function (44). The data we have

presented indicating that in healthy resting T cells the Kv1.3 channels are maintained in

- 95 - the IS for about 2 hr are consistent with the notion that a prolonged interaction of naïve T cell with APC lasting 2 hr or more is required for cell division and IL-2 production/release from the cell (42, 45, 46). Although it has been shown that tyrosine phosphorylation activation mechanisms and the initial Ca2+ influx occur early upon T cell

contact with the antigen (within 2-15 min), other signaling systems such as those

involving Ca2+ or serine/threonine phosphorylation have been suggested to be critical

during the later stages of activation. Since Kv1.3 channels are known regulators of Ca2+ homeostasis in human T cells and their activity is modulated by serine/threonine kinases it is very likely that they constitute key components of the late activation signaling complex. The prolonged time Kv1.3 channels reside in the IS may indeed be necessary for the channels to come in close proximity with signaling molecules also recruited at the

IS thus facilitating the regulation of their activity and consequently the control and termination of the Ca2+ response. It has been shown that various elements that accumulate

at the IS such as cholesterol and lipid rafts as well as Lck, PKC and PKA can modulate

Kv1.3 channel function (27, 47-52). Furthermore these kinases move into the IS at different times after antigen presentation, with PKA and PKCθ still present at the IS well after a mature synapse is formed (53-55). Our results suggest a model in which a proper time-dependent localization of Kv1.3 in the IS is necessary for its regulation. In normal resting T cells the Kv1.3 channel remains in the IS for the time necessary for its regulation. This process responsible for bringing Kv1.3 channels into close physical proximity with signaling molecules would have particular biological relevance in the setting of SLE where there is a documented decrease in the expression and activity of multiple kinases (2). Unfortunately, since Kv1.3 channels in SLE T cells leave the IS

- 96 - prematurely they might not be properly regulated and an abnormal Ca2+ response might

develop. On the other hand, a prolonged localization of Kv1.3 channels is instead not

necessary in normal activated T cells because they also express high levels of KCa3.1

channels that could control Ca2+ homeostasis (25).

The data presented herein raise the possibility that Kv1.3 channels might be involved in

the pathophysiology of SLE. Given the availability of pharmacological agents altering

these channels, these data may lead to the discovery of new therapeutic targets for this

disease.

Still, there remains an open question that was not addressed by these studies. This

involves the membrane distribution of KCa3.1 during T cell activation. Based on our

results we hypothesized that the presence of KCa3.1 channels in healthy activated T cells

compensates for the early exit of Kv1.3 from the IS. This information would significantly

enhance our understanding of K+ channel trafficking in human T cells and help us understand better the defect observed in SLE T cells, namely the early exit of Kv1.3 from the IS without a corresponding up-regulation of KCa3.1. As such in the next chapter we will focus on studies aimed at elucidating KCa3.1 channel trafficking during T cell activation in activated human T cells.

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38. Beeton, C., H. Wulff, S. Singh, S. Botsko, G. Crossley, G. A. Gutman, M. D. Cahalan, M. Pennington, and K. G. Chandy. 2003. A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes. J Biol Chem 278:9928-9937.

39. Horwitz, D. A., J. D. Gray, K. Ohtsuka, M. Hirokawa, and T. Takahashi. 1997. The immunoregulatory effects of NK cells: the role of TGF-beta and implications for autoimmunity. Immunol Today 18:538-542.

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52. Payet, M. D., and G. Dupuis. 1992. Dual regulation of the n type K+ channel in Jurkat T lymphocytes by protein kinases A and C. J Biol Chem 267:18270-18273.

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55. Zhou, W., L. Vergara, and R. Konig. 2004. T cell receptor induced intracellular redistribution of type I protein kinase A. Immunology 113:453-459.

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CHAPTER III: The Ca2+-activated K+ channel

KCa3.1 compartmentalizes in the immunological

synapse of human T lymphocytes1

1 The data presented in Chapter III are published in part in the American Journal of Physiology-Cell Physiology, Nicolaou et al, 2007 Apr;292(4):C1431-9. The link for the article is: http://ajpcell.physiology.org/cgi/reprint/292/4/C1431

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3.1 ABSTRACT

T cell receptor engagement results in the reorganization of intracellular and membrane

proteins at the T cell-antigen presenting cell interface forming the immunological

synapse (IS), an event required for Ca2+ influx. KCa3.1 channels modulate Ca2+ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding

KCa3.1 membrane distribution during T cell activation. Herein we determined whether

KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels.

These channels showed identical electrophysiological and pharmacological properties as wild-type channels. IS formation was induced using either anti-CD3/CD28 antibody coated beads for fixed microscopy experiments, or Epstein Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments T cells were also immunolabeled for F-actin or CD3ε that served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that upon T cell activation KCa3.1 channels localize with F-actin and CD3ε to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15-30 min. Interestingly, pre- treatment of activated T cells with the specific KCa3.1 blocker, TRAM-34, blocked Ca2+ influx but channel re-distribution to the IS was not prevented. These results indicate that

KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

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3.2 INTRODUCTION

T cell receptor (TCR) engagement by an antigen presenting cell (APC) carrying a foreign antigen results in T cell activation. The process is initiated by reorganization of membrane and cytosolic proteins at the T cell-APC contact interface forming a

“signalosome”, the immunological synapse (IS) (1). As a result of IS formation multiple signal transduction pathways are elicited and enhanced leading to the generation of mitogenic signals.

The onset of T cell activation is marked by an increase in intracellular Ca2+ that occurs

immediately upon TCR engagement by the APC/antigen. Moreover, increased

intracellular Ca2+ levels must be sustained for a long time before interleukin-2 (IL-2) is produced and activation becomes antigen independent (2). A sustained intracellular Ca2+ concentration is thus necessary for T cell activation and gene expression (3, 4). Calcium signaling in human T lymphocytes is modulated via two K+ channels, the voltage-gated

K+ channel, Kv1.3, and the calcium-activated K+ channel, KCa3.1. Kv1.3 channels

regulate the membrane potential in resting T cells where they represent the dominant

conductance (2). However when naïve and central memory T cells are exposed to an

antigen and become activated the expression of KCa3.1 channels is strongly enhanced

compared to a modest increase in Kv1.3 channels, and KCa3.1 channels become the

major regulators of membrane potential in these cells (5, 6). Via regulation of the membrane potential these channels provide the driving force for Ca2+ entry since the

efflux of K+ ions assists in maintaining the necessary electrochemical gradient (2).

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Interestingly, although recent evidence suggests that Kv1.3 channels localize in the IS in

T cells, nothing is known regarding KCa3.1 channel ability to compartmentalize in the IS

(7). In the present study we investigated KCa3.1 channel distribution on the plasma

membrane upon T cell activation. By utilizing electrophysiological methods and

fluorescence microscopy we demonstrate that KCa3.1 channels redistribute to the IS

upon TCR binding and become part of the IS signaling complex that facilitates T cell

activation.

3.3 MATERIALS AND METHODS

Cells and transfection: CD3+ and CD4+ lymphocytes were isolated from healthy donors

by E-rosetting (StemCell Tech., Vancouver, Canada) and Ficoll-Paque density gradient centrifugation (ICN Biomedicals, Aurora, OH, USA) and maintained as previously described (8). Freshly isolated human T cells were pre-activated with 4 µg/ml phytohemmaglutinin (PHA, Sigma-Aldrich, St. Louis, MO) and transfected 18-24 hours later with YFP-KCa3.1 with the Amaxa Nucleofector technology (Amaxa Biosystems,

Cologne, Germany) using 10x106 cells, 5 µg DNA, and program T20 according to the

manufacturer’s instructions. For ratiometric Ca2+ imaging experiments human T cells

were activated with 4 µg/ml PHA for 48-72 hr allowing for sufficient expression of the

native KCa3.1 channels (5). This became necessary due to a low transfection efficiency

that did not allow us to use the transfected T cells (ca. 10%). Blood was obtained from either healthy volunteers or healthy blood bank donors (unutilized blood units from the

Hoxworth Blood Bank Center). The blood collection was approved by the IRB of the

University of Cincinnati. Epstein Barr virus (EBV)-infected B cells (gift of A.H.

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Filipovich) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum

(FBS), 2 mM glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Human

embryonic kidney (HEK 293) cells (American Tissue Culture Collection, Manassas, VA)

were cultured in Dulbecco’s modified eagle’s medium (Invitrogen, Carlsbad, CA)

supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml

streptomycin. HEK 293 cells were transfected with YFP-KCa3.1 using Lipofectamine

2000 (Invitrogen, Carlsbad, CA) according the manufacturer’s instructions at a ratio of

DNA to Lipofectamine of 1:5.

Determination of antibody specificity and reactivity: We determined antibody specificity

and reactivity of three anti-KCa3.1 antibodies (i) a polyclonal anti-rabbit antibody, for an

intracellular (IC) epitope at the C-terminus, aa 350-363 (Alomone, Jerusalem, Israel), (ii)

a polyclonal anti-goat antibody, for an IC epitope corresponding to aa 60-110 and (iii) a

polyclonal anti-goat antibody, for an IC epitope corresponding to aa 200-250 (Santa Cruz

Biotechnology, Santa Cruz, CA). To determine antibody specificity a fusion protein,

specific for the Alomone antibody, or a peptide, specific for the two Santa Cruz

antibodies, were incubated at a ratio of 1 µg antibody to 3-5 µg (fusion protein or

peptide) 1-12 hr at 4oC and centrifuged at 10,000g for 5 min. Next the preadsorbed

antibody was added to activated T cells, plated on poly-L-lysine coated coverslips and

fixed with 4% PFA, for 1 hr at either room temperature or 4oC. T cells were also

incubated with non-preadsorbed antibody, which was treated as the pre-adsorbed antibody, and in the absence of primary antibody. Next, the appropriate secondary antibodies were added for 1 hr to allow fluorescent visualization (donkey anti-rabbit

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Alexa Fluor 488, or donkey-anti-goat Alexa Fluor 555, Molecular Probes). Coverslips

were then mounted on glass slides and viewed by widefield fluorescence microscopy using a Nikon Microphot FXA microscope coupled to a Spotcam camera and using the appropriate filters. Images were obtained using the same exposure time and gain to allow for direct comparison between groups.

Molecular Biology: YFP was appended onto the C-terminus of KCa3.1 via the addition of

SalI and BamHI sites to the N- and C-terminus, respectively in a single step PCR reaction resulting in the removal of the stop codon in KCa3.1. This PCR product was subcloned in-frame into the pEYFP-N1 vector (Clontech, Mountain View, CA). The fidelity of this construct was confirmed by sequencing (ABI PRISM 377 automated sequencer,

University of Pittsburgh) and subsequent sequence alignment (NCBI BLAST) with

KCa3.1 (GenBankTM accession number AF022150).

Measurement of intracellular calcium: Resting and PHA pre-activated T cells were plated on poly-L-lysine coated coverslips and loaded with 1 µM Fura-2/AM (Molecular probes, Eugene, OR) for 35 min at room temperature (22-24oC) in RPMI and rinsed with

2+ 0.5 mM Ca Ringer solution containing (in mM): 155 NaCl, 4.5 KCl, 2.5 MgCl2, 10

o HEPES, 10 glucose and 0.5 CaCl2, pH 7.4. Cells were stored at 37 C in the dark for up to

2 hr before use. All cell imaging experiments were performed on a Cyt-Im2 Ca2+ imaging

system (Intracellular Imaging, Cincinnati, OH) using a ratiometric method as previously

described (8, 9). Cells were imaged on a Nikon inverted epifluorescence microscope

equipped with a heated microscopy chamber, a 20 X objective and a xenon arc lamp,

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which was used for the alternative excitation of Fura-2 at 340 and 380 nm. Emitted light

passed through a 535 WB35 emission filter and intensity values averaged over either 10

sec (experiments with CD3+ T cells) or 0.5-0.7 sec intervals (experiments with CD4+ T cells) for analysis. For activation with EBV-infected B cells, B cells were pre-pulsed for

2 hr at 37oC with 7 µg/ml staphylococcal enterotoxin B (SEB) (Sigma-Aldrich, St. Louis,

MO), centrifuged and re-suspended in 1 ml 0.5 mM Ca2+ Ringer solution and stored at

37oC. For experiments with the KCa3.1 blocker TRAM-34, cells were pre-incubated

with 1 µM TRAM-34 (in 0.5 mM Ringer solution) for 15 min before recording. Fura-2

loaded T cells were recorded while bathed in 0.5 mM Ca2+ Ringer solution for 2 min

before addition of SEB-pulsed B cells. The cells were then allowed to interact for 15 min

before 1-2 µM ionomycin was added as a positive control. Visual inspection showed

formation of APC/T cell stable conjugates in the bath. Initial experiments were

performed in CD3+ T cells but since SEB only activates CD4+ T cells subsequent

experiments were performed in CD4+ T cells to increase the proportion of responding T

cells. Cells that had an increase in 340/380 ratio >0.1 ratio units were regarded as cells responding to antigen presentation. This value was well above two standard deviations of

the average background noise: 0.023+0.024 ratio units as determined in three separate experiments from 440 cells (>110 cells per experiment) that showed no apparent response. In addition, to obtain the average of the 340/380 ratio the cells that exhibited a calcium response were synchronized to reflect initiation of Ca2+ influx.

T Cell Activation: Transfected T cells were stimulated with 4.5 µm polystyrene beads coated with anti-CD3/CD28 antibodies (Dynal Biotech, Lake Success, NY) as formerly

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described (10). Transfected T lymphocytes were mixed with the beads at a ratio of 1:1.5 and centrifuged for 5 min at 100 x g. The cells were then incubated at 37oC for 30 min,

resuspended and plated onto poly-L-lysine (Sigma-Aldrich, St. Louis, MO) coated

coverslips and allowed to attach for 3-5 min. Activation experiments were also performed

using EBV-infected B cells as APCs. EBV-infected B cells were pulsed with SEB (7

µg/ml for 2 hr) and loaded with 1 µM DDAO Far Red Cell Tracker (Molecular Probes,

Inc., Eugene, OR) for 20 min. T and B cells were then mixed at a ratio of 1:1.5, spun

briefly at 1100 rpm and incubated at 37oC for 1-30 min. Finally the cells were plated onto

poly-L-lysine coated coverslips.

Immunocytochemistry: Immunolabeling was carried out, in part, as previously described

(11). Cells attached onto poly-L-lysine coated coverslips were washed with PBS and

fixed with 4% paraformaldehyde (PFA) for 20 min. To double label with CD3ε the cells

were blocked using 10% fetal bovine serum (FBS), permeabilized with 0.2% Triton X-

100, incubated for 1 hr with goat anti-CD3ε antibody (Santa Cruz Biotechnology, Inc.,

Santa Cruz, CA) washed and incubated with donkey anti-goat Alexa Fluor 546

fluorescent secondary antibody (Molecular Probes, Inc., Eugene, OR) for one hour. To

stain for F-actin Alexa Fluor 546 phalloidin (Molecular Probes, Inc., Eugene, OR) was

added for 20 min. Finally the cells were washed and mounted onto glass slides. Samples

were visualized by confocal microscopy (Axioscope, Carl Zeiss, Microimaging Inc)

using a 63X oil objective lens. The fluorescent probes were excited using an Ar ion laser

and a HeNe laser. Data were obtained using the “Multi Track” option of the microscope

to exclude cross-talk between detection channels.

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Image quantification: To evaluate co-localization of proteins and their position within the

IS, unprocessed images were analyzed using the linescan function of the MetaMorph

program (Molecular Devices, Downingtor, PA 19335). Briefly, a reference line was

drawn along the T/B cell contact site. The software calculates the mean red and green

fluorescence intensities along the reference line for 4 pixels of width and plots the

measurements in respect to their position within the selected portion of the membrane.

To determine the percentage of T/B cell conjugates that formed in the presence or absence of 1 µM TRAM-34 we counted the number of T/B cell conjugates/total number of T cells. Eight random fields were analyzed for each donor and each treatment condition.

Time-lapse microscopy: CD4+ T cells were transfected with YFP-tagged KCa3.1

channels and used for live microscopy experiments 6 hr after transfection. EBV-infected

B cells were pre-pulsed with SEB and loaded with 1 µM Far Red DDAO cell tracer

(Molecular Probes, Inc., Eugene, OR). T cells were seeded into a heated microscopy

chamber (37oC) on poly-L-lysine coated coverslips. Next, B cells were added and time-

lapse images were recorded using a Plan- Apochromat 60 X oil immersion objectives on

a Nikon Microphot FXA inverted microscope coupled to an Orca-ER cooled camera

(Axioscope, Carl Zeiss, Microimaging Inc). Images were processed using the Metamorph

software.

Electrophysiology: Experiments were performed in the whole cell configuration using an

Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) at room

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o temperature (22 C). The external solution was (in mM): 140 NaCl, 4.5 KCl, 2 CaCl2, 1

MgCl2, 10 Hepes, pH 7.4. The pipette solution was (in mM): 145 K aspartate, 8.5 CaCl2,

2+ 10 EGTA, 2 MgCl2 and 10 Hepes, pH 7.2, with an estimated free Ca concentration of 1

µM (5). All solutions were 290–310 mOsm. The cells were continuously perfused at a

constant rate of 2 ml min-1. Electrodes were pulled from TW150F-4 glass micropipettes

(World Precision Instruments, Sarasota, FL) on a horizontal pipette puller (model P-97,

Sutter instrument CO., USA) and had a resistance of 4-6 MΩ. KCa3.1 current was

measured in voltage-clamp mode and induced by ramp depolarization from -120 mV to

+40 mV, 200 ms duration, every 10 s, –80 mV holding potential (HP). Data were corrected for a liquid junction potential of -10 mV (8). KCa3.1 slope conductance was measured between -100 mV and -60 mV. The digitized signals were stored and analyzed using pClamp 9 software (Axon Instruments).

Statistical Analysis: All data are presented as means ± SEM. Statistical analyses were performed using Student’s t-test (paired or unpaired); p<0.05 was defined as significant.

3.4 RESULTS

3.4.1 Electrophysiological and pharmacological profile of the cloned YFP-KCa3.1

channel in HEK 293 cells matches the native KCa3.1 channel in human T cells.

Initially we designed studies to decipher the trafficking of native KCa3.1 channels in human T cells using anti-KCa3.1 antibodies. As such, we tested three different antibodies and determined the specificity of each by staining the cells with non-preadsorbed antibody, preadsorbed antibody, secondary antibody only and unstained cells (Fig. 3.1

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two antibodies shown). As an additional control we also incubated the antibodies with

resting T cells that express very low levels of KCa3.1. We found that the fluorescence

intensity in these cells was comparable to activated T cells. As a result we found no

specific commercially available anti-KCa3.1 antibody. Accordingly we decided to

transfect human T cells with YFP-KCa3.1 channels to enable us to perform our studies.

But first we needed to characterize these recombinant KCa3.1-YFP channels.

A B Epitope: aa 350-363 Epitope: aa 200-250

Anti-KCa3.1 antibody

Preadsorbed Anti-KCa3.1 antibody

No primary

Figure 3.1: Determination of specificity of anti-KCa3.1 antibodies. Activated human T cells were incubated with either an anti-KCa3.1 antibody corresponding to an epitope of aa 350-363 (A) or for an epitope corresponding to aa 200-250 (B) that was or was not preadsorbed to the corresponding antigen. Both antibodies were non-specific as similar fluorescence was observed when the antibody was used (A, top, left panel and B, top panels) but also with the preadsorbed antibody (A, middle panels and B, middle panels). This was not due to the secondary antibodies as the no primary antibody controls showed minimal fluorescence (A, bottom panels and B bottom panels).

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To that end, YFP-tagged KCa3.1 channels were expressed in HEK 293 cells, which lack

endogenous KCa3.1 channels and would allow unbiased evaluation of our clone. Their

pharmacological and electrophysiological properties were investigated and compared to

native KCa3.1 channels previously described in the literature (5). KCa3.1 currents were

recorded using the whole cell configuration and with a pipette solution (intracellular) of 1

µM [Ca2+] that allows KCa3.1 activation (5). Ramp pulse depolarization induced K+ currents with a reversal potential of -79.0+0.3 mV (n=6) (Fig. 3.2A). This was indicative

of a K+-selective current. Mock-transfected HEK 293 cells transfected with the empty

YFP-vector displayed very small background K current (Fig 3.2A). Overall the KCa3.1

conductance was significantly higher in YFP-KCa3.1 transfected HEK 293 cells as

compared to mock transfected cells (Fig. 3.2B). Furthermore, KCa3.1 current in YFP-

KCa3.1 transfected cells was blocked by the specific KCa3.1 blocker TRAM-34 (kind

gift of K.G. Chandy) (Fig. 3.2A-B) (12). Collectively these electrophysiological and

pharmacological studies confirm that transfection of HEK 293 cells with the YFP-

KCa3.1 clone resulted in the expression of functional KCa3.1 channels which are

functionally identical with their native counterparts as their characteristics are in

agreement with previous reports (5).

3.4.2 Overexpression of functional YFP-tagged KCa3.1 channels in human primary

T lymphocytes. Next, functional YFP-KCa3.1 channels were expressed in primary

human T lymphocytes. To achieve this, pre-activated human T cells were transfected

with YFP- tagged KCa3.1 channels and subsequently used for whole-cell voltage-clamp

experiments six hours after transfection. Our results revealed an up-regulation of KCa3.1

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A 80 YFP-KCa3.1 60 YFP-KCa3.1 40 + TRAM-34 I (pA) 20 Control 0 -80 -40 0 V(mV)

B 6 # 5

4 3 2

Conductance (nS) 1 ** 0 Control - + TRAM-34 YFP-KCa3.1

Figure 3.2. Functional and pharmacological properties of recombinant YFP-tagged KCa3.1 channels. A. Representative traces shown were obtained in HEK 293 cells transfected with YPF-KCa3.1 and YFP-vector (pEYFP-N1, control). YFP-KCa3.1 currents were blocked by 1 µM TRAM-34. Currents were induced by ramp depolarization from -120 mV to +40 mV (-80 mV HP). The theoretical EK was -88 mV. B. The KCa3.1 conductance increased significantly in YPF-KCa3.1 transfected HEK 293 cells compared with mock-transfected cells (n=6, #p=0.003). The YFP-KCa3.1 currents decreased significantly after application of 1 µM TRAM-34 (n=6, **, p=0.02).

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A 1.6 * 20 YFP-KCa3.1 1.4

15 1.2 1.0 ) 10 Control 0.8

I (pA 5 0.6 0.4 0 -80 -60 -40 -20 0 Conductance (nS) 0.2 -50 V (mV) 0.0 Control YFP-KCa3.1

B -TRAM-34 30

20

+TRAM-34

I (pA) 10

0 -80 -60 -40 -20 0 20 40 V (mV) -100

Figure 3.3. Expression of functional YFP-tagged KCa3.1 channels in human T cells. A. Top panel: Representative current-voltage relationships obtained from YPF-KCa3.1 transfected T cells and non-transfected (control) T cells are shown. Control T cells have undergone the same transfection procedure as YFP-KCa3.1 channel transfected T cells. Currents were obtained by ramp depolarization as described in the legend of figure 1. Inset: Representative images of a control (top panel) and transfected (lower panel) cell are shown. Both cells were from the same donor and in the same image. Scale bar: 5µm. Bottom panel: There was a significant increase in KCa3.1 conductance in YPF-KCa3.1 transfected T cells compared with non-transfected T cells which corresponds to an increase in the number of functional KCa3.1 channels/cell. The data are the average of 5 cells from 2-4 donors. *p=0.02. B. KCa3.1 currents decreased significantly after application of 1 µM TRAM-34. Currents, elicited by ramp depolarization as described in the legend of Fig. 1, were recorded continuously before (-TRAM-34) and during exposure to the drug (+TRAM-34).

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current in transfected as compared to non-transfected T cells (Fig. 3.3A). Similar

membrane capacitance, a measure of cell surface area, was measured in transfected and

non-transfected cells: 1.10+0.10 pF (n=5) and 1.08+0.11 pF (n=5, p=0.89), respectively

(13). This indicated that there was a significant increase in the number of functional

KCa3.1 channels/cell expressed in transfected as compared to non-transfected T cells.

However, the degree of this increase may be accentuated or diminished if differences in channel open probability were great between the control and transfected groups.

Moreover YFP-KCa3.1 channel transfected T cells were visualized with confocal microscopy (Fig. 3.3A inset) and these data showed that KCa3.1 channels were evenly distributed on the plasma membrane although we did observe some intracellular expression of YFP-tagged KCa3.1 channels in some cells (data not shown). Further experiments indicated that YFP-KCa3.1 channels in T cells are sensitive to TRAM-34 as exposure to 1 µM TRAM-34 induced 68+8% inhibition of KCa3.1 current (n=6,

p=0.009) (Fig. 3.3B) (12). Overall these data demonstrate that functional YFP-tagged

KCa3.1 channels were successfully expressed on the plasma membrane of human T

lymphocytes and this enabled us to use these cells for colocalization studies.

3.4.3 KCa3.1 channels and F-actin redistribute to the T cell and anti-CD3/CD28

antibody coated bead contact site. After we confirmed that functional KCa3.1 channels

were expressed on human T cells we used these cells to explore the possibility that

KCa3.1 channels translocate to the IS upon T cell activation. To achieve this we used

anti-CD3 and anti-CD28 antibody coated beads to induce T cell activation. This is a well

validated system shown by us and others to induce Ca2+ influx and molecular

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DIC KCa3.1 F-actin Merge Control

Activated XZ projection

Figure 3.4. KCa3.1 channels and F-actin localize at the T cell/bead contact point. YFP-KCa3.1 transfected T cells were activated with anti-CD3/CD28 antibody coated beads for 30 min, fixed, permeabilized and stained with phalloidin Alexa fluor 546 (to visualize F-actin). T cells not conjugated with beads display uniform distribution of KCa3.1 and F-actin around the membrane (top panels) whilst KCa3.1 and F-actin localize at the T cell/ bead contact interface upon conjugation (bottom panels). Scale bar: 5µm. The 3D T/bead interface reconstruction (xz projection) is shown under the corresponding 2D image. The reconstructed portion of the T/B cell complex is indicated by a box in the 2D merged image. Scale bar for the xz projection=2 µm for both x and z axes.

reorganization, both indicative of a functional T cell activation (8, 10). Further, upon T cell activation extensive cytoskeletal reorganization takes place resulting in F-actin accumulation at the contact point and as such it can serve as a marker of IS formation

(14, 15). The YFP-KCa3.1 channels were expressed in pre-activated human T cells and were prepared for immunocytochemistry experiments six hours after transfection. This time frame was sufficient for channel expression and insertion in the plasma membrane as demonstrated from the electrophysiological and microscopy experiments described in the previous section (Fig. 3.3). Moreover the cells were used at this early time point in

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order to avoid overexpression that could possibly affect the function of the T cells. As shown in Fig. 3.4 (lower panels) KCa3.1 channels and F-actin reorganize at the T cell- bead contact point after 30 min of conjugation whilst both maintain an even distribution when no beads are attached (Fig. 3.4, upper panels). Interestingly it appears that F-actin is concentrated mostly on the periphery of the T cell/bead contact point after 30 min of activation as previously shown, whilst KCa3.1 channels acquire a more central localization (Fig. 3.4 XZ projection) (15, 16). These studies indicate that KCa3.1 channels reorganize at the T cell-bead interface upon conjugation. Still further studies are needed to elucidate the location of KCa3.1 channels in the supramolecular activation complex (SMAC) of the IS and to define the kinetics of their recruitment in the IS. In order to induce the formation of the SMAC we used superantigen-loaded B cells that closely mimic the antigen presenting cells found in vivo.

3.4.4 Calcium influx during antigen presentation and its regulation by KCa3.1 channels. EBV-infected B cells that were pre-pulsed with the superantigen SEB were used as APCs to induce T cell activation and IS formation. To ensure that SEB pulsed B cells were competent APCs we investigated their ability to induce elevation of intracellular Ca2+, indicating a productive activation. To that end we examined the Ca2+ response at the single-cell level in PHA-activated T cells loaded with the ratiometric calcium dye Fura-2 (9). T cells were maintained in a heated chamber and the 340/380 ratio was monitored throughout the experiment. After a short equilibration period, SEB- loaded B cells were added in the chamber. Activated human T cells responded to B cell

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B 1.2 A 60 1 -TRAM-34 +TRAM-34 50 0.8 B 0.6 40

340/380 340/380 0.4 0.2 30 responding cells responding + 0 2 20

10 % of Ca % of 1.2 0 Resting 1 Activated 0.8 C 0.6 B Control

340/380 340/380 0.8 0.4 0.2 0.7 0 0.6 0.5 TRAM-34

340/380 340/380 0.4 0.8

0.3 0.6 B 0 200 400 600 800 0.4 Time 340/380 340/380 0.2 D -TRAM-34 0 50 +TRAM-34

40 1.2 1 30 0.8 B I 0.6 20 340/380 340/380 0.4 0.2 10 % B/T cell conjugates 0 0

Figure 3.5. SEB pulsed B cell interaction with T cells induces an increase a KCa3.1- dependent increase in cytoplasmic calcium in activated T cells. A. Human T cells were loaded with the ratiometric dye Fura-2 and stimulated with SEB pulsed B cells for 15 min. Each panel shows a representative trace of cytoplasmic Ca2+ in individual cells. The point of introduction of the B cells into the bath is indicated by an arrow. T cells coming in contact with a B cell displayed differential responses including a sustained increase of intracellular Ca2+, a transient response and an oscillatory response. These data

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are representative of a total of 35 activated T cells. Unstimulated cells (cells not contacting a B cell) showed little or no response. Yet they responded to ionomycin (I, 1- 2 µM). Scale bars correspond to 200 s. Experiments were performed at 29oC. B. Resting and activated CD4+ T cells were or were not pre-treated with 1 µM TRAM-34 before interacting with B cells. The numbers of T/B cell conjugates showing a significant increase in intracellular Ca2+ were normalized for the total number of T cells. Resting T cells showed no significant difference between untreated and pre-treated cells (n=4, >70 cells/experiment and n=4, >50 cells/experiment, from 3 donors; p=0.3). However activated T cells showed a significant decrease in the number of cells that responded when treated with the blocker (untreated: n=4, >40 cells/experiment, treated: n=5 >40 cells/experiment, from 2 donors; p=0.002). C. Average increase in intracellular Ca2+ in control (-TRAM-34) and TRAM-34 treated cells that responded to antigen stimulation (same experiment as panel B). The average cytoplasmic Ca2+ levels for control (-TRAM- 34, 118 cells from 4 separate experiments) and cell treated with TRAM-34 (47 cells from 5 separate experiments) were obtained by alignment of the traces so that the times of onset of the Ca2+ response corresponded. Experiments in panels B and C were performed at 34.7+0.2 oC (n=16). D. The number of T/B cell conjugates that form in control and TRAM-34 (1 µM) pre-treated activated T cells was determined in fixed micrographs and reported as percentage of total T cells counted. *p=0.2.

2+ 2+ stimulation with an increase in intracellular Ca concentration ([Ca ]i, Fig. 3.5A). A

large heterogeneity was observed in regards to calcium signaling in these cells including

continuous, transient and oscillatory responses (Fig. 3.5A). These data indicate that SEB-

pulsed B cells act as APCs and form conjugates similar to genuine T cell–APC pairs.

It has been shown that KCa3.1 channels regulate calcium signaling in human activated T

cells (5). Accordingly, we observed that TRAM-34, a specific KCa3.1 channel blocker,

inhibited the Ca2+ response in these cells (Fig. 3.5B-C). While on average 46+6 % (n=4,

>40 cells/experiment, from 2 donors) of activated T cells showed an increase in

intracellular Ca2+ upon exposure to APCs, pre-treatment with 1 µM TRAM-34 induced a

significant decrease in the number of responding T cells with only 16+3 % of cells

showing a Ca2+ response (n=5, >40 cells/experiment, from 2 donors, p=0.002) (Fig.

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3.5B). Furthermore, the TRAM-34 pre-treated cells that still responded to antigen

stimulation displayed a blunted Ca2+ response as compared to control cells (Fig. 3.5C).

As a control we performed identical experiments with resting T cells. In these cells Kv1.3

channels, and not KCa3.1 channels, regulate the membrane potential and the Ca2+ influx

and therefore blocking of KCa3.1 channels should not significantly alter the Ca2+ response (2). Indeed a similar number of resting T cells displayed a Ca2+ response upon

antigen presentation both in control and TRAM-34 pre-treated cells: 14.5+6.4% and

24.9+6.4 %, respectively (Fig. 3.5B). To ensure that the decrease in number of activated

T cells responding to antigen presentation was not due to a decrease in T/B conjugate

formation we compared the number of T/B cell conjugates that form in activated T cells

in presence and absence of TRAM-34. Comparable numbers of T/B cell conjugates form

in the two groups: 28+10% in control and 36+10% in treated cells (n=3 experiments for

each group from 3 different donors, >30 T/B conjugates per donor, p=0.2) (Fig.3.5D).

Overall these data confirm in our experimental setting that KCa3.1 channels control Ca2+ homeostasis in activated but not in resting T cells (5). Moreover they indicate that blockade of KCa3.1 channels, although inhibiting the Ca2+ response did not preclude the

physical association between B and T cells. It still remains to be determined whether

KCa3.1 channels are recruited in the IS that forms with APCs and if KCa3.1 blockade

affects the formation of the IS and the KCa3.1 recruitment at this site.

3.4.5 Redistribution of KCa3.1 channels at the immunological synapse. Confocal

microscopy experiments were performed to study the KCa3.1 channel distribution upon

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contact with APCs. The YFP-KCa3.1 channels were expressed in human T cells and the

T cells were prepared for immunocytochemistry experiments six hours after transfection.

It is known that the TCR and associated molecules redistribute to the T cell-APC contact

interface upon T cell activation (1). CD3ε is part of the TCR complex that localizes in the

center of the mature IS, thus it can be used as a marker of IS formation (17). Our data

indicate that KCa3.1 channels redistributed at the T cell-APC interface and colocalized

extensively with CD3ε upon conjugation (Fig. 3.6B-C) but both remained evenly distributed on the membrane when the T-APC were not conjugated (Fig. 3.6A) or in the absence of SEB (Fig. 3.6B, top panel). Notably, KCa3.1 channels acquired a central localization within the contact interface early upon IS formation and this localization is

maintained for at least 30 min of conjugation. This localization within the IS as well as

the co-localization with CD3ε is clearly visible in the XZ projection of the T/B cell

interface and it was further confirmed by the position of the peak fluorescence intensities

in the linescan graphs (Fig. 3.6). Interestingly, despite pre-treatment with 1 µM TRAM-

34, a concentration already demonstrated to block channel current (Figs 3.2 and 3.3) and

the Ca2+ response (Fig. 3.5B-C), KCa3.1 channels were still recruited in the IS and they

also maintained the same central location within the IS (Fig. 3.6C, lower panel). Overall

KCa3.1 blockade did not seem to affect IS formation since CD3ε was still recruited and

its localization within the IS, together with that of the channel, remained unchanged. Our

data therefore suggest that functional KCa3.1 channels are not required for IS formation

and maintenance nor for the channel membrane trafficking.

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DIC B cell KCa3.1 CD3ε Merge A

B 192 128 F 64 SEB - 0 1 2 3 4 Position 1min 256

192 +SEB 128 F XZ 64 projection 0 1 2 3 4 Position

C 256

192

128 F

64

-TRAM-34 XZ

30min 0 projection 1 2 3 4 Position

256

192

128 F

+TRAM-34 64 XZ 0 projection 1 2 3 4 Position

Figure 3.6. KCa3.1 channel redistribution in the immunological synapse. KCa3.1 channel transfected human T cells were incubated with EBV-infected B cells that had been exposed to medium with or without SEB at 37oC. B cells were labeled with Cell Trace FarRed DDAO (pseudocolored blue). T cells, pre-treated or not pre-treated with TRAM-34 (1 mM) for 15 min, were mixed with B cells and incubated for 1-30 min at 37oC, plated on coverslips, fixed, permeabilized and stained with anti-CD3ε antibody. Representative images are shown for A. T cells that did not form a stable conjugate with SEB infected B cells, B. T cell conjugated with B cells in the absence or presence of SEB after 1 min and C. T cells un/treated with TRAM-34 after 30 min conjugation with SEB- pulsed B cells. Scale bar: 5µm. The three-dimensional T/B cell interface reconstructions are shown under the corresponding 2D images. The area used for the reconstruction is marked in the 2D merged images by a box. The linescan analyses of KCa3.1 (green) and

- 124 -

CD3ε (red) fluorescent intensity (FI) over the T/B cell contact area are shown as right panels adjacent to the corresponding micrographs. These images are representative of the results obtained from 5 donors for control experiments and 2 donors for pre-treatment experiments. Scale bar for the xz projection=2 µm for both x and z axes.

Overall, these results indicate that KCa3.1 channels quickly redistribute at the IS upon

antigen presentation and therein they aggregate at the center of the IS; a process independent of the activation state of the channels.

To more precisely define the kinetics of KCa3.1 channel distribution in the IS we used time-lapse microscopy to directly image live KCa3.1 channel translocation in T cells as it develops upon encounter with APCs. Activation and IS formation of transfected T cells was induced by SEB-pulsed EBV-infected B cells. The results obtained in these studies confirmed the observations in the fixed microscopy studies, although a certain degree of variability in the kinetics of KCa3.1 channel translocation to the T/B interface was also observed. In the majority of conjugates imaged the channels were recruited at the IS within 40 sec-2 min (5 out of 7 conjugates) (Fig. 3.7). Only in two conjugates was a longer time necessary for channel recruitment. Interestingly, in some experiments we observed that the channels were recruited upon contact with an APC but then readily relocalized to a second APC upon contact (data not shown) (n=3). It is not uncommon for

T cells to undergo serial stimulation by APCs during the activation process (18). But

because of the multiple encounters and transient nature of this polarization, these

experiments were not included in the overall analysis. In addition we observed two

distinct patterns of retention in the IS. Specifically, in 43% of the cells the channels

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resided in the IS throughout the duration of the whole experiment at least 14-30 min

(n=3), whilst in 57% of the conjugates the channels stayed in the synapse for a shorter time (7:04+0:35 min, n=4). Moreover the channels appear to obtain a central localization in 86% of the conjugates studied, and only in 14% maintained a more peripheral distribution (n=7). These data further define the dynamics of KCa3.1 channel recruitment in the IS.

0:00 0:42 2:42 3:22 9:22

14:02 21:04 24:51 28:42 35:05

Intensity of KCa3.1 0 256

Figure 3.7: KCa3.1 channel redistribution in live T cells. Human T cells transfected with YFP-KCa3.1 were seeded on a heated microscopy stage and imaged whilst interacting with SEB-pulsed EBV-infected B cells. In the top row, brightfield images are shown and on the bottom row the corresponding images of KCa3.1 staining intensity are shown using a pseudocolor scale. The location of the B cells is indicated by a white line around the cell membrane. Snapshot sequence corresponds to Supplemental Movie S1. We observed that at time 0:00, before the transfected T cell comes in contact with an APC, KCa3.1 channels are uniformly expressed all along the cell membrane. After 0:42 min, an APC comes in contact with the T cell and, upon contact, the channels begin to accumulate at the IS that forms at the T cell/APC interface where they remain for over 25 min. At 28:42 min the channels began to redistribute along the cell membrane and they acquire a uniform distribution by 35:05 min. Scale bar=5 µm.

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3.5 DISCUSSION

Sustained TCR engagement by an APC leads to the formation of a highly organized structure, the IS. This is characterized by an extensive reorganization of the actin cytoskeleton as well as specific membrane (TCR and adhesion molecules) and signaling proteins to the T cell/APC contact interface (1, 15, 19). In the present study we demonstrate that KCa3.1 channels are evenly distributed on the membrane of human T lymphocytes but translocate to the IS upon encounter with an APC and become part of the signaling complex that facilitates T cell proliferation and cytokine production. To our knowledge this is the first report showing KCa3.1 channel redistribution at the IS in human T cells upon antigen stimulation.

Experiments were performed on human T cells expressing YFP-tagged KCa3.1 channels.

This was made necessary by the fact that, to our knowledge, no specific anti-KCa3.1 antibodies are commercially available. The availability of a specific antibody would have allowed us to study the behavior of native KCa3.1 channels that exist at the appropriate conformational and phosphorylation state as well as in association with other cellular components. Furthermore, from a technical point of view, this would have excluded the limitation inherent in the low efficiency of transfection of primary T cells (i.e. low number of transfected cells available for the experiments). Recombinant channels instead carry the risk of overexpression, which we avoided as much as possible by using the cells as soon as expression was seen. In our experiments the transfected T cells expressed on average a KCa3.1 conductance of 1.3 nS/µm2. This is comparable to the level of expression of native KCa3.1 channels in human T lymphocytes reported after 2 days

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activation with PHA (1.12 nS/µm2)(5). It is also possible that the YFP tag could have hindered other molecular interactions, although it did not compromise the ability of the channel to enter and exit the IS. Furthermore, a more cytoplasmic distribution has been observed in GFP-tagged channels as compared to native K channels (20). Still, a YFP protein is extremely photostable, thus allowing detailed and prolonged live cell imaging experiments minimizing the risk of photobleaching. Furthermore, these studies using recombinant channels set the stage for future structural-functional studies that will allow the determination of the channel protein sequence/s necessary for its recruitment in the IS and thus the possible mechanisms driving this process.

Electrophysiological experiments indicated that the YFP-KCa3.1 channels displayed biophysical properties identical to their native counterparts and were inhibited by the specific KCa3.1 blocker TRAM-34 (5). This allowed us to express these channels in primary human T lymphocytes and perform localization experiments to demonstrate that

KCa3.1 channels localize at the IS. Two methods were used to induce IS formation and T cell activation: CD3/CD28 coated beads and SEB-pulsed EBV-infected B cells. The former have been used as surrogate APCs and they have been shown to induce re- organization of F-actin and accumulation of structural proteins at the bead/T cell contact area (10). We have also shown that CD3/CD28 beads were able to induce a productive activation in human T cells as they can elicit an increase in [Ca2+]i upon contact (8).

However, stimulation of T cells with superantigen-loaded B cells more closely resembles the “in vivo” situation where the antigen is presented to the T cell by either B or dendritic cells (1). The association between T cells and these APCs involves adhesion and other

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co-stimulatory molecules not provided by the CD3/CD28 beads (1). We have confirmed

that SEB-pulsed EBV-infected B cells can be used as effective APCs by monitoring the

Ca2+ response induced in T cells upon binding (Fig. 4). These experiments were

performed in human T cells pre-activated by exposure to PHA for 72 hr. This

intervention was shown to induce expression of KCa3.1 channels in human T cells and,

in these cells, the Ca2+ response becomes dependent on these channels (5). Different patterns of Ca2+ response were elicited in individual T cells by exposure to the SEB-

pulsed B cells. Similar heterogeneity to TCR stimulation was previously observed and

described using soluble antigens and CD3/CD28 beads (8, 21, 22). This reflects the

mixed T cell population that comprises T cells freshly isolated from the blood and

includes T cells at different degrees of activation and development.

Overall, the results presented reveal that KCa3.1 channels moved into the IS immediately

upon its formation and they localized with F-actin and CD3ε. Furthermore, fixed

microscopy experiments, representative snapshots of the process, revealed that KCa3.1 channels are rapidly recruited at the center of the IS where they reside for at least 30 min after stimulation. We observed that KCa3.1 channels co-localized with CD3ε and were surrounded by F-actin. This is in agreement with F-actin forming a peripheral ring within the IS while the TCR accumulates at the core of the IS (15, 16).

To further substantiate our data we also performed time-lapse microscopy experiments.

KCa3.1 channels are recruited early on in the IS in the majority of the cells imaged.

Intriguingly we also noted two patterns of recruitment: a sustained recruitment and a

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shorter lived one. This might reflect the variability in activation and differentiation state

of our mixed T cell population, an observation also demonstrated by the variability of our

Ca2+ response.

KCa3.1 localization in the IS could have important implications on the channel activity

and overall T cell function. It is well known that in activated T cells KCa3.1 channels

regulate the membrane potential and as a consequence, Ca2+ influx as well (5, 6). In

agreement with the literature, we observed that blockade of KCa3.1 channel activity

inhibits the TCR-mediated Ca2+ response in these cells (Fig. 4). Notably, blocking Ca2+ increase does not prevent the formation of tight T/B cell interfaces and accumulation of adhesion molecules at site of contact (23). Interestingly, we observed that when KCa3.1 channels were blocked neither formation of T/B cell conjugates nor KCa3.1 and CD3ε transition to the IS was prevented. So our results suggest that KCa3.1 channel transition to the IS is not dependent on Ca2+ influx. Furthermore it suggests that the functionality of

the channel is not integral to its migration to the IS. Similarly, we have observed that

Kv1.3 channels can also translocate to the IS when their activity is pharmacologically abrogated (data not shown).

The functional consequences of KCa3.1 channel translocation in the IS might reflect on the Ca2+ response that is triggered upon antigen presentation. It is generally accepted that

following TCR engagement the magnitude and pattern of the Ca2+ signaling is in part

regulated by the activity of Kv1.3 and KCa3.1 channels (24). It has already been shown

that Kv1.3 channels translocate to the IS in human T cells and in the present study we

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show that KCa3.1 channels also move to the IS upon T cell activation (7). Moreover, it is

commonly believed that formation of the IS occurs in order to provide close proximity

between various elements of the T cell activation machinery and thus more efficient

signaling among them (1). Interestingly, signaling molecules such as protein kinases A

(PKA) and C (PKC), which are known regulators of KCa3.1 channel function, have been

shown to accumulate in the IS upon T cell activation as well (25-30). Investigation of the

reorganization of PKCθ during IS formation reveals that this PKC isoform sustains a

central localization in the IS supramolecular activation complex (31). Furthermore, PKA

also moves into the IS 30 min after activation and it partially colocalizes with the

TCR/CD3 complex to facilitate the termination of the activation process (29). Thus the

spatial and temporal distribution of PKCθ and PKA allow for access to the KCa3.1

channels and as such could provide a regulatory mechanism affecting the channel’s

activity. As a result it is quite possible that recruitment of KCa3.1 channels could lead to

differential regulation of these channels. Consequently, modulation of KCa3.1 channel

activity will determine the magnitude and duration of the Ca2+ response triggered by

antigen presentation as it contributes to the driving force for Ca2+ influx.

In view of this we propose that the functional relevance of KCa3.1 channel translocation to the IS could be to facilitate the better regulation of the channel by signaling molecules recruited at this site during T cell activation with the ultimate goal to shape the Ca2+ response which is integral for differential gene expression (4).

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3.6 REFERENCES

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3. Negulescu, P. A., N. Shastri, and M. D. Cahalan. 1994. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci U S A 91:2873-2877.

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6. George Chandy, K., H. Wulff, C. Beeton, M. Pennington, G. A. Gutman, and M. D. Cahalan. 2004. K+ channels as targets for specific immunomodulation. Trends Pharmacol Sci 25:280-289.

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9. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440- 3450.

10. Xavier, R., S. Rabizadeh, K. Ishiguro, N. Andre, J. B. Ortiz, H. Wachtel, D. G. Morris, M. Lopez-Ilasaca, A. C. Shaw, W. Swat, and B. Seed. 2004. Discs large (Dlg1) complexes in lymphocyte activation. J Cell Biol 166:173-178.

11. Conforti, L., I. Bodi, J. W. Nisbet, and D. E. Millhorn. 2000. O2-sensitive K+ channels: role of the Kv1.2 -subunit in mediating the hypoxic response. J Physiol 524 Pt 3:783-793.

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12. Wulff, H., M. J. Miller, W. Hansel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. 2000. Design of a potent and selective inhibitor of the intermediate- conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci U S A 97:8151-8156.

13. Wulff, H., P. A. Calabresi, R. Allie, S. Yun, M. Pennington, C. Beeton, and K. G. Chandy. 2003. The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. J Clin Invest 111:1703-1713.

14. Cannon, J. L., and J. K. Burkhardt. 2002. The regulation of actin remodeling during T-cell-APC conjugate formation. Immunol Rev 186:90-99.

15. Das, V., B. Nal, A. Roumier, V. Meas-Yedid, C. Zimmer, J. C. Olivo-Marin, P. Roux, P. Ferrier, A. Dautry-Varsat, and A. Alcover. 2002. Membrane- cytoskeleton interactions during the formation of the immunological synapse and subsequent T-cell activation. Immunol Rev 189:123-135.

16. Bunnell, S. C., V. Kapoor, R. P. Trible, W. Zhang, and L. E. Samelson. 2001. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14:315-329.

17. Ehrlich, L. I., P. J. Ebert, M. F. Krummel, A. Weiss, and M. M. Davis. 2002. Dynamics of p56lck translocation to the T cell immunological synapse following agonist and antagonist stimulation. Immunity 17:809-822.

18. Friedl, P., and M. Gunzer. 2001. Interaction of T cells with APCs: the serial encounter model. Trends Immunol 22:187-191.

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20. Kupper, J. 1998. Functional expression of GFP-tagged Kv1.3 and Kv1.4 channels in HEK 293 cells. Eur J Neurosci 10:3908-3912.

21. Hess, S. D., M. Oortgiesen, and M. D. Cahalan. 1993. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. J Immunol 150:2620-2633.

22. Verheugen, J. A., and H. P. Vijverberg. 1995. Intracellular Ca2+ oscillations and membrane potential fluctuations in intact human T lymphocytes: role of K+ channels in Ca2+ signaling. Cell Calcium 17:287-300.

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23. Wulfing, C., M. D. Sjaastad, and M. M. Davis. 1998. Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels. Proc Natl Acad Sci U S A 95:6302-6307.

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25. Gerlach, A. C., N. N. Gangopadhyay, and D. C. Devor. 2000. Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275:585-598.

26. Neylon, C. B., T. D'Souza, and P. H. Reinhart. 2004. Protein kinase A inhibits intermediate conductance Ca2+-activated K+ channels expressed in Xenopus oocytes. Pflugers Arch 448:613-620.

27. Del Carlo, B., M. Pellegrini, and M. Pellegrino. 2003. Modulation of Ca2+- activated K+ channels of human erythrocytes by endogenous protein kinase C. Biochim Biophys Acta 1612:107-116.

28. Skalhegg, B. S., K. Tasken, V. Hansson, H. S. Huitfeldt, T. Jahnsen, and T. Lea. 1994. Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex. Science 263:84-87.

29. Zhou, W., L. Vergara, and R. Konig. 2004. T cell receptor induced intracellular redistribution of type I protein kinase A. Immunology 113:453-459.

30. Bi, K., Y. Tanaka, N. Coudronniere, K. Sugie, S. Hong, M. J. van Stipdonk, and A. Altman. 2001. Antigen-induced translocation of PKC-theta to membrane rafts is required for T cell activation. Nat Immunol 2:556-563.

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Chapter IV: Differential calcium signaling in T

lymphocytes from patients with systemic lupus

erythematosus1

1 The data presented in Chapter IV are not published and are part of a manuscript in preparation.

- 135 - 4.1 ABSTRACT

T lymphocytes from patients with systemic lupus erythematosus (SLE) present with a

plethora of signaling anomalies during T cell activation and immunological synapse (IS)

formation. Among others, these cells present with altered compartmentalization of Kv1.3 channels in the IS and abnormal Ca2+ response. Since Kv1.3 channels modulate Ca2+ influx, the Ca2+ defect might be associated with defective Kv1.3 localization in the IS.

Further, the shape and magnitude of the Ca2+ response are associated with transcription

factor regulation that is altered in SLE. The potential involvement of Ca2+ in these

aberrant responses is unknown in SLE T cells. This study compares single T cell

quantitative Ca2+ responses from SLE, normal and rheumatoid arthritis (RA) donors. T

cell activation, and Ca2+ influx, was induced using EBV-infected B cells. Further, we

employ a two-photon imaging technique to correlate cytosolic Ca2+ ([Ca2+]i) and Kv1.3

trafficking in the IS in healthy T cells. We found that SLE, but not RA, T cells have

altered Ca2+ patterns. SLE T cells are associated with more sustained Ca2+ responses as

compared to normal and RA T cells. Further, oscillating cells in SLE have longer

periodicity as compared to normal T cells. Finally, we demonstrate that in live T cells increase and subsequent decline of [Ca2+]i is accompanied by Kv1.3 localization and

maintenance in the IS. Based on our results, we propose that the altered [Ca2+]i shape

observed in SLE T cells depends on a Kv1.3 channel trafficking defect and is at least in

part responsible for altered activation of transcription factors in SLE T cells.

- 136 - 4.2 INTRODUCTION

Systemic lupus erythematosus (SLE) is a chronic rheumatic autoimmune disease, which

implies that immune tolerance is breached resulting in the production of numerous

autoantibodies that eventually lead to organ damage (1). In these patients, T cells provide

support to autoreactive B cell clones propagating the disease (2). Besides assistance to B

cells, T cells are also hyperresponsive following antigenic stimulation (3). This

hyperactivity manifests during T cell activation in the early events following T cell

receptor (TCR) engagement by antigen presenting cell (APC) / antigen. Indeed, a

plethora of signaling defects have been documented during IS formation in SLE T cells

(4). Importantly, SLE T cells show an increased and more sustained Ca2+ response that is

attributed in part to increased release from intracellular stores (5).

In human T cells, the shape of the Ca2+ response relies on Ca2+ and K+ channels on the

plasma membrane. Briefly, TCR engagement results in Ca2+ influx through CRAC

channels. However, CRAC relies on a hyperpolarized membrane potential to allow Ca2+

influx. As such, K+ channels, and specifically Kv1.3 and KCa3.1, drive K+ efflux,

allowing Ca2+ influx to continue. This process eventually leads to downstream signaling

pathways which ultimately result in gene expression and proliferation.

Notably, approximately 75% of all T cell activation genes rely, either directly or indirectly, on Ca2+ (6). Furthermore, gene expression can be differentially modulated

based on the shape and amplitude of the Ca2+ response. To illustrate, NF-AT activation

that leads to the production of IL-2 and other cytokines and chemokines (7, 8), is

- 137 - promoted when there is a steady-state Ca2+ in the cell and also by frequent oscillations (9,

10). Other transcription factors such as NF-κB, which regulates a number of genes encoding cytokines and adhesion molecules, among others (7, 11), requires less frequent

Ca2+ oscillations or a transient Ca2+ rise to be activated (9, 12). Notably, the differences in the Ca2+ requirements between NF-AT and NF-κB correlate with the regulation of these transcription factors. Specifically, NF-AT nuclear localization relies on Ca2+ dependent phosphorylation while exit from the nucleus depends on dephosphorylation.

These events occur within minutes, and as such to maintain NF-AT in the nucleus frequent or sustained [Ca2+]i input is required. On the other hand, NF-κB nuclear localization relies on degradation of the inhibitory subunit associated with NF-kB and re- synthesis of this subunit to inhibit it. This process takes tens of minutes and as such only a brief change in [Ca2+]i is sufficient to maintain NF-κB nuclear localization (13).

Interestingly, SLE T cells present with increased NF-AT nuclear localization (14) and diminished NF-κB activity (15). Increased NF-AT activation, in particular, leads to overexpression of CD40 ligand (CD40L) in SLE T cells, which in turn supports B cell differentiation and autoantibody production through the engagement of CD40 (16). At present the potential role of [Ca2+]i in the abnormal expression and regulation of transcription factors, and ultimately gene expression in SLE, remains elusive.

As aforementioned, the shape of the Ca2+ response shows a dependence on K+ channels and membrane potential, as CRAC channels rely on a hyperpolarized membrane potential to function. As a result, any anomalies associated with K+ channels could significantly

- 138 - contribute to abnormal Ca2+ signaling. We have shown that although the function and expression of Kv1.3 and KCa3.1 channels is not altered in resting SLE T cells, there are abnormalities in the trafficking of the Kv1.3 channel during T cell activation.

Specifically, in normal T cells Kv1.3 channels are recruited early in the IS and are maintained there for at least 30 min. In contrast, in SLE T cells, the channels move out of the IS within 15 min of conjugation (17). This could be due to the more active phenotype of SLE T cells in regards to IS formation (18). Faster Kv1.3 kinetics were also observed in normal pre-activated T cells. However, while normal pre-activated T cells up-regulate

KCa3.1 channels that are recruited and maintained in the IS for at least 30 min and possibly take over after Kv1.3 is gone, SLE T cells do not up-regulate KCa3.1 channels to compensate for this rapid exit.

The rapid exit of Kv1.3 channels from the IS in SLE, associated with abnormalities of known regulators of the channels such as PKC and PKA, could explain the abnormal

Ca2+ response (4, 19-21). Overall, it is possible that by leaving the IS prematurely Kv1.3

channels escape down-regulation by elements recruited in the IS, resulting in a more

sustained Ca2+ response.

In this study we aimed to characterize the shape of the Ca2+ response in single SLE T cells and compare it to normal and RA T cells using quantitative Ca2+ imaging

techniques. Moreover, we wanted to examine Kv1.3 localization in the IS in conjunction

with cytosolic Ca2+ ([Ca2+]i) in live human T cells from normal controls and patients with

SLE. To achieve that, we used two-photon microscopy. Our results indicate a shift in the

- 139 - shape of the Ca2+ response in T cells from SLE patients as compared to normal donors

and patients with RA, favoring a more sustained Ca2+ response. Moreover, we have

optimized a challenging two-photon live cell imaging technique that allows simultaneous recordings of Kv1.3 trafficking and [Ca2+]i. The latter will allow us to establish a

correlation between Kv1.3 trafficking in the IS and Ca2+ homeostasis.

4.3 MATERIALS AND METHODS

Human Subjects: All SLE patients included in this study fulfilled the American College of Rheumatology classification criteria for SLE (22, 23). Our study included 6 SLE patients, 2 male (M) and 4 female (F), 6 African American (AA), of whom 3 were on dialysis. As disease controls 4 RA patients, 4F, 3AA and 1 Caucasian (C) were included.

These patients fulfilled the American College of Rheumatology classification criteria for

RA. Also, 7 normal individuals were included in this study, 6C (2M and 4F) and 1 unknown. Studies and informed consent forms were approved by the University of

Cincinnati Institutional Review Board.

Cells: CD4 + T cells were isolated from venous blood collected from consenting normal,

SLE and RA donors using E-rosetting (StemCell Tech., Vancouver, Canada) followed by

Ficoll-Paque density gradient centrifugation (ICN Biomedicals, Aurora, OH, USA).

Memory T cells were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using fluorescence activated cell sorting (FACS). CD4 cells were first isolated from whole blood using RosetteSep Human CD4+ T Cell Enrichment Cocktail

(Stem Cell Technologies, Vancouver, BC). The purified CD4 cells were then stained with

- 140 - CD45RA-FITC and CD8-APC (BD Biosciences, San Jose, CA), and sorted based on forward and side scatter and double-negative staining (FACSVantage flow cytometer,

BD Biosciences, San Jose, CA). The purity of the TEM population was determined by

flow cytometry. The cells were maintained in RPMI medium supplemented with 10%

pooled male human AB serum (Intergen, Milford, MA, USA), 200 U/ml penicillin, 200

µg/ml streptomycin and 1 mM Hepes as previously described (24). Jurkat T cells were

maintained in RPMI 1640 medium supplemented with 10 % FBS (Fisher Scientific,

Pittsburgh, PA), 200 U/ml penicillin, 200 µg/ml streptomycin and 1% of 1 M Hepes.

Epstein-Barr Virus (EBV) infected-B cells were cultured in RPMI 1640 supplemented

with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml

streptomycin. Human embryonic kidney (HEK 293) cells (American Tissue Culture

Collection, Manassas, VA) were cultured in Dulbecco’s modified eagle’s medium

(Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin and 100

µg/ml streptomycin.

Molecular Biology: The Kv1.3 gene was subcloned into the mammalian expression

vector pcDNA3, and Hind III/Xho I restriction sites were introduced outside the coding

Kv1.3 gene region. The restriction enzymes Hind III/Xho I were then used to digest

pcDNA3-Kv1.3 and pEGFP-C1 plasmids (pEGFP-C1 plasmid contains a brighter

fluorescing GFP variant). The Kv1.3 fragment was then isolated and ligated to Hind

III/Xho I-digested pEGFP-C1 plasmid to create a pEGFP-Kv1.3 N-terminus-directed

fusion construct. The plasmid was then amplified in DH5α cells, column purified, and

sequenced across the entire amplified region to exclude any stray mutations.

- 141 -

Cell Transfection: For the transfection of HEK 293 cells with pEGFP-Kv1.3 plasmid

DNA and pEGFP vector DNA, HEK 293 cells were plated in growth medium without

antibiotic such that they are confluent the next day. After 24 hours of growth, the medium was changed to Optimem (Invitrogen Corp. Carlsbad, CA) without serum. DNA

was combined with Lipofectamine-2000 (Invitrogen Corp.) at a ratio of 1:2 in Optimem

and added to the confluent HEK 293 cells. After 24 hours, cells were lifted from the

dishes using trypsin and plated on coverslips in growth medium. Fluorescence was

observed 24 hours later. Jurkat T cells were transfected using the Amaxa Nucleofector

technology (Amaxa Biosystems, Cologne, Germany) using 10x106 Jurkat T cells, 5 µg

DNA and program C-16, according to the manufacturer’s instructions. Cells transfected

were < 10th passage. Freshly isolated human CD4+ T cells were transfected using the

Amaxa Nucleofector technology using 5x106 T cells, 5 µg of pEGFP-Kv1.3 or vector

plasmid DNA and program U-14, according to the manufacturer’s instructions.

Measurement of Intracellular Calcium in Single Cells: CD4+ T cells were plated on

poly-L-lysine coated coverslips and loaded with 1 µM Fura-2/AM (Molecular probes,

Eugene, OR) for 35 min at room temperature (22-24oC) in RPMI and rinsed with 0.5 mM

2+ Ca Ringer solution containing (in mM): 155 NaCl, 4.5 KCl, 2.5 MgCl2, 10 HEPES, 10

o glucose and 0.5 CaCl2, pH 7.4. Cells were stored at 37 C in the dark for up to 2 hr before

imaging. All cell imaging experiments were performed on a Cyt-Im2 Ca2+ imaging system (Intracellular Imaging, Cincinnati, OH) using a ratiometric method as previously described (24, 25). Cells were imaged on a Nikon inverted epifluorescence microscope

- 142 - equipped with a heated microscopy chamber, a 20 X objective and a xenon arc lamp,

which was used for the alternative excitation of Fura-2 at 340 and 380 nm. Emitted light

passed through a 535 WB35 emission filter and intensity values were averaged over 1-2

sec intervals for analysis. For activation with EBV-infected B cells, B cells were pre-

pulsed for 2 hrs at 37oC with 7 µg/ml SEB (Sigma-Aldrich, St. Louis, MO), centrifuged

and re-suspended in 0.5 mM Ca2+ Ringer solution and stored at 37oC. Experiments were

performed at 34.3 + 0.2oC (n=29). Fura-2 loaded T cells were recorded while bathed in

0.5 mM Ca2+ Ringer solution for 2 min before addition of SEB-pulsed B cells. The cells

were then allowed to interact for 15 min before 1-2 µM ionomycin was added as a

positive control. Visual inspection showed formation of APC/T cell stable conjugates in

the bath. Absolute [Ca2+]i values were obtained using the formula : [Ca2+]i = Kd (R-

Rmin)/(Rmax-R) * F380min/F380max (25), where Kd= effective fura-2 dissociation

constant, R= 340/380 ratio, Rmin= R at 0 Ca2+, Rmax= R of maximum saturating Ca2+ and F380min/F380max is the ratio of the 380 nm intensity of Fura-2 at minimum and maximum saturating Ca2+.

Fura-2 Calibration: For analysis of quantitative [Ca2+]i we empirically derived the

effective Fura-2 dissociation constant (Kd) in situ using human CD4+ T cells and

calibration buffers with estimated free [Ca2+]i of 0-30 µM (Molecular Probes, Inc.,

Eugene, OR).We found that the Kd = 250 + 7 (n=2) at 34.7 + 0.1 oC (n=2) which is in

agreement with previous reports (26). Rmin was measured after every experiment using

2+ 0.0 mM Ca Ringer’s solution containing (in mM): 155 NaCl, 4.5 KCl, 1.0 MgCl2, 10

HEPES, 10 glucose and 5 EGTA in the presence of 3 µM Ionomycin. Rmax was also

- 143 - measured after every experiment using 2.5 mM Ca2+ Ringer’s solution containing (in

mM): 155 NaCl, 4.5 KCl, 0.5 MgCl2, 10 HEPES, 10 glucose and 2.5 CaCl2. We observed

no difference in Rmax when using 10 mM Ca2+ Ringer solution.

Analysis of Ca2+ Responses:

Pattern of Ca2+ signaling: To determine the distribution of Ca2+ responses we

categorized the cells as (i) Non-responding: no increase in Ca2+ after B cell addition, (ii)

Transient: a brief Ca2+ response that returns to baseline within the 15 min duration of the experiment, (iii) Continuous: a Ca2+ response that is followed by a sustained plateau

within the 15 min duration of the experiment and (iv) Oscillatory: a Ca2+ response that

includes three or more transient responses. The number of cells displaying a specific

response was reported as % of total responding cells.

Analysis of [Ca2+]i Oscillations: Data analysis was performed using the program

Microcal Origin 5.0 (Microcal Software Inc., Northampton, MA) and Microsoft Office

Excel as previously described (27). When B cells were added in the bath they made contact with T cells at various time points. To allow for direct comparison, time points

prior to 50 sec before the first oscillation was observed were excluded. The resulting data

were imported to Microcal Origin (Microcal Software Inc.) and fitted using a third order

polynomial function. This eliminated low frequency components unrelated to Ca2+ oscillations. Next, the data were analyzed using the fast fourier transform (FFT) algorithm, which sums the squares of the values of a set of uniformly spaced points and normalizes by the number of data points. The FFT data were then used to determine the power spectral density (PSD), which describes how the power of the signal is distributed

- 144 - with frequency. The PSD function ultimately provides information regarding oscillation

frequency and amplitude. Any cell with a frequency of <3 mHz was not included in order

to eliminate cells with less than 3 oscillations.

Conjugation of EC-Kv1.3 Antibody With Alexa Fluor 488: The EC anti-Kv1.3 antibody is

packaged, in lyophilized form, with PBS, pH 7.4 and 1% bovine serum albumin (BSA).

BSA disrupts the conjugation reaction with the fluorophore. To remove the BSA, the

Swell Gel Blue Albumin removal kit (Pierce Inc., Rockford, IL) was used. For the kit to be effective the antibody needs to be in low salt solution, so it was dialyzed in 25 mM

Tris, 25 mM NaCl, and pH 7.5 overnight. The next day albumin was removed according to the manufacturer’s instructions and protein concentration was determined using a

BioPhotometer (Eppendorf North America Inc., Westbury, NY) and absorbance was determined at 280 nm (A280). Protein concentration was then determined using the

formula: protein concentration (M) = (A280 x MW x dilution factor) / ε x path length where MW= the molecular weight of the antibody (150,000 Da), ε = the molar extinction coefficient (203,000) and path length = 1. At this point our antibody was diluted in 25 mM Tris, pH 7.2, but before the conjugation kit can be used the antibody must be in PBS so microdialysis was used overnight to substitute the buffer. Finally the antibody, free of

BSA and in the appropriate buffer, was conjugated to Alexa Fluor 488 (AF488)

(Molecular Probes, Inc., Eugene, OR). Conjugation was performed according to the manufacturer’s instructions. The degree of labeling was determined by

spectrophotometry. Readings were obtained at A280 and A494 and the degree of labeling

was determined using the formula: Moles dye per mole protein = (A494 x dilution factor) /

- 145 - 71,000 (ε of dye) x protein concentration (M). The yield was low ranging from 10-20 %

of the original antibody concentration and the degree of labeling was approximately 8

moles of dye per mole of antibody, which is optimal per the manufacturer’s

recommendations.

T cell Activation and Immunocytochemistry: The primary antibody used for detecting

Kv1.3 proteins in non-transfected T cells was a rabbit polyclonal anti-Kv1.3 antibody

against an extracellular epitope of the Kv1.3 protein that was or was not conjugated to

FITC or AF488 (Sigma-Aldrich). The unconjugated antibody was detected using a

donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes). The

antibodies were used for labeling Kv1.3 channels in “live” T lymphocytes before

interaction with the EBV-B cells. Then pEGFP-Kv1.3 transfected or antibody labeled

non-transfected T cells were stimulated with either anti-CD3/CD28 antibody coated

beads or EBV-infected B cells loaded with either Cell Trace Blue CMAC or Far Red

Tracker DDAO dyes (Molecular Probes). EBV-infected B cells were pulsed with SEB (7

µg/ml for 2 hr). T and B cells were then mixed at a ratio of 1:1.5, spun briefly at 1100

rpm and incubated at 37oC for 1-30 min. Finally the cells were plated onto poly-L-lysine

(Sigma-Aldrich, St. Louis, MO) coated coverslips and allowed to attach for 3-5 min.

Cells attached onto poly-L-lysine coated coverslips were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. Finally the cells were washed and mounted onto glass slides. Protein accumulation was detected by fluorescence microscopy using a

Zeiss Axioplan Imaging 2 infinity-corrected upright scope coupled to an Orca-ER cooled camera (Axioscope, Carl Zeiss, Microimaging Inc.), a Plan-Apochromat 60X oil

- 146 - immersion objective and the appropriate filters. Alternatively, protein accumulation was

detected by confocal microscopy using a Zeiss LSM510 laser scanning confocal microscope (Axioscope, Carl Zeiss, Microimaging Inc) using a 63X oil objective lens.

The fluorescent probes were excited using an Ar ion laser and a HeNe laser. Data were

obtained using the “Multi Track” option of the microscope to exclude cross-talk between

detection channels.

Confocal Time-Lapse Microscopy: Jurkat T cells were transfected with pEGFP-Kv1.3 as described above and used for live cell imaging experiments 6-16 hr after transfection.

EBV-infected B cells were pre-pulsed with SEB and loaded with 1 µM Far Red Tracker

DDAO (Molecular Probes, Inc., Eugene, OR). T cells were seeded into an Attofluor

chamber (Molecular Probes) and placed on a heated microscopy chamber (37oC) on poly-

L-lysine coated coverslips. Next, SEB-pulsed B cells were added and time-lapse images

were recorded. Images were obtained using a Plan- Apochromat 60 X oil immersion

objective on a Nikon Microphot FXA inverted microscope (Axioscope, Carl Zeiss,

Microimaging Inc). Z-stacks of 1 µm intervals were captured every 30-60 sec. For

excitation an Ar and a HeNe laser were used and images were obtained using the “Multi-

Track” option.

Two-Photon Time-Lapse Microscopy: pEGFP-Kv1.3 transfected CD4+ T cells were

loaded with 2 µM Fura-2 for 35 min in suspension in RPMI in the dark, washed with

RPMI and stored at 37oC for up to 3 hr before imaging. Coverslips were coated with poly-L-lysine and mounted onto an Attofluor chamber (Molecular Probes) and placed in

- 147 - the microscope stage that was enclosed by a dark environmental chamber (Solent

Scientific Ltd, Segensworth, UK) to allow 37°C control and keep stray light out of

detectors. CD4+ T cell data were recorded using a Zeiss Axiovert 200 inverted

microscope (Carl Zeiss, Microimaging Inc) and a Plan-Apochromat 63 X water immersion objective. Z-stacks of 1 µm intervals were captured every 30-60 sec. For green fluorescence excitation an Ar laser was used. For fura-2 imaging the Ca2+ free form

was excited at 800 nm using the two-photon microscope laser (Ti-Sa). This excitation

was chosen to avoid excitation of the Ca2+ bound Fura-2 (28). Therefore an increase in

Ca2+ is reflected by a corresponding decrease in fluorescence. Also, at this wavelength,

our clone was not excited (Fig. 4.1). The “Multi Track” option was used to avoid cross-

talk between channels.

Two-photon

Excitation 488 700 725 750 775 800 825 850 875 900 λ (nm) X

X

Figure 4.1: Determination of excitation of pEGFP-Kv1.3 with Ti-Sa laser. T cells were transfected with pEGFP-Kv1.3 and allowed to interact with anti-CD3/CD28 antibody-coated beads (indicated by X). The cells were then fixed and mounted onto glass slides. Accumulation of Kv1.3 (green) at the T/bead contact interface is seen at 488 nm excitation (far left panels). pEGFP-Kv1.3 was also excited with the two-photon laser at 700-900 nm at 25 nm increments. The lack of fluorescence indicates that our clone is not excited at any wavelength in this range.

- 148 - Image Analysis: Images were processed using the Metamorph software as previously

described (29, 30). Since optical sectioning was required to obtain all the green signal,

the most representative section and its corresponding DIC image were isolated (29). For

clarity, Kv1.3 fluorescence is shown in green while Fura-2 is shown in an arbitrary blue-

red pseudocolor range. To analyze [Ca2+]i in live cell imaging experiments, a region was

drawn around the cell of interest and the fluorescence was quantified. Subsequently, F0-

F/F0 was used to obtain a pseudoratio where F is the fluorescence intensity and F0 is the averaged prestimulus fluorescence (30).

Electrophysiology: K+ currents were recorded in whole-cell configuration. Kv1.3 currents

were induced by depolarizing voltage steps from -80 mV holding potential (HP) to +50

mV applied every 30 s. The external solution for recording of Kv1.3 currents had the

following composition (in mM): 150 NaCl, 5 KCl, 2.5 CaCl2, 1.0 MgCl2, 10 glucose and

10 HEPES, pH 7.4. The pipette solution was composed of (mM): 134 KCl, 1 CaCl2, 10

2+ EGTA, 2 MgCl2, 5 ATP-sodium, and 10 HEPES, pH 7.4 (estimated free Ca concentration 10 nM) (31). Kv1.3 cumulative inactivation was induced by consecutive depolarizing pulses applied every second. Data were collected using the Axopatch200A amplifier and analyzed with pClamp8 software (Axon Instruments, Foster City, CA,

USA).

Statistical Analysis: All data are presented as means ± SEM. Statistical analyses were

performed using Student’s t-test (paired or unpaired); p<0.05 was defined as significant.

- 149 - 4.4 RESULTS

4.4.1 Pattern of [Ca2+]i signaling in SLE T cells. Previous studies indicated that the

general SLE T cell population presents with a more pronounced and sustained Ca2+ response as compared to normal T cells (5, 32). However, to our knowledge, there are no detailed quantitative single cell Ca2+ studies in SLE T cells. Single cell [Ca2+]i studies

have been previously performed in T cell lines as well as human T cells and point to

diverse responses of cells upon stimulation (33, 34). Similar to other studies, we observed

that activation of human T cells with APCs induces differential patterns of responses

including transient, continuous and oscillatory (35). We wanted to investigate the patterns

of Ca2+ influx in SLE T cells. To that end, we designed quantitative Fura-2 imaging

experiments aimed at dissecting these responses in SLE T cells. Six SLE patients were

included in the following microscopy studies: age 42.8 + 4.7 years; range 24-54 years. As

controls we used six normal subjects: age 40.5 + 5 years; range 25-55 years and four RA patients: age 54 + 7 years; range 33-64 years. The normal and SLE donors were age and sex matched as age related Ca2+ alterations have been previously documented (36). In

addition, although our RA patients were not precisely age matched to our SLE patients,

still the age between the two groups was not statistically different (p= 0.2).

We examined single cell Ca2+ responses in SLE, normal and RA T cells that were loaded

with the ratiometric dye fura-2 and allowed to interact with SEB-loaded EBV-infected B

cells. The signal was recorded for 15 min after addition of the APCs. The cells were then

categorized according to the response pattern as (a) non-responding, (b) transient, (c)

continuous or (d) oscillatory (for details refer to materials and methods). SLE, normal

- 150 - and RA T cells exhibited all aforementioned types of responses (Fig. 4.2A). The shape of

the Ca2+ response is important in that it can direct specific transcription factor activation

and ultimately gene expression to meet the different needs of the cell during activation

(Fig. 4.2B) (9, 10, 37). Interestingly, we found that SLE, but not RA, T cells had a

significant increase in the percentage of responding cells presenting with a continuous

response, accompanied by a corresponding decrease in the percentage of cells showing a

B cells 1-2 µM Ionomycin

1400

1200 1000

] (nM) 800 2+ 600

[Ca 400 200 0 200 sec 1000

800 600 ] (nM)

2+ 400

[Ca 200 0 700 600 500

] (nM) 400 2+ 300

[Ca 200 100 0 600

500 400 ] (nM)

2+ 300 200 [Ca 100 0

Figure 4.2: Representative example of the four patterns of Ca2+ response observed. Resting human T cells were loaded with the ratiometric dye Fura-2 and stimulated with SEB- pulsed B cells for 15 min. Each panel shows a representative trace of cytoplasmic [Ca2+]i in individual cells. The point of introduction of the B cells and 1-2 µM ionomycin into the bath is indicated by an arrow. Cells that did or did not contact a B cell and showed little or no response still responded to ionomycin (top panel). T cells coming in contact with a B cell displayed differential responses including a transient response, a continuous increase or an oscillatory Ca2+ response. These data were from one SLE patient.

- 151 - transient response (Fig. 4.3). Moreover, in SLE T cells the continuous response is three

fold higher as compared to transient or oscillatory responses, making it by far the

dominant response, while normal T cells have equal numbers of continuous and transient

responses. Instead, T cells from RA patients showed decreased continuous responses. No

significant difference was observed in the number of cells showing oscillatory responses.

These results raised the question as to whether specific Ca2+ patterns are exclusively

associated with specific T cell subtypes. In the current study we show that the mixed

population of resting T cells exhibits all three responses. Since SLE T cells have a higher

percentage of TEM as compared to normal donors (15) we isolated TEM CD4+ cells

p= 0.0006

p= 0.02 p= 0.01

70 p= 0.003

60 Transient p= 0.02 50 Continuous Oscillatory 40

30

20 % cells showing response 10

0 Normal SLE RA

Figure 4.3: Detailed analysis of [Ca2+]i pattern in normal, SLE and RA T cells. Cells were activated as described in the legend of fig. 4.2. The percentage of responding cells exhibiting transient, continuous or oscillatory responses was determined from 6 normal (number of cells per response T: 91, C: 127, O: 56), 6 SLE (number of cells per response T: 57, C: 162, O: 52) and 4 RA (number of cells per response T: 54, C: 36, O: 36) donors.

- 152 - from one of the normal donors included in these studies and performed similar

experiments. However, we found no unique Ca2+ response associated with TEM cells;

instead they display all three types of responses. Specifically, 33% of TEM cells

displayed a transient response, 49% a continuous response and 18% an oscillatory

response.

Overall, these experiments demonstrate that in SLE T cells there is a shift in the shape of

the Ca2+ response to a more continuous and sustained Ca2+ response as compared to

normal donors. Moreover, this appears to be unique to SLE T cells as it was not observed

in T cells from RA patients. As a consequence these data support the notion that

differential gene expression in SLE T cells may be mediated by altered [Ca2+]i shape.

4.4.2 Magnitude of [Ca2+]i in SLE T cells. Although ratiometric Ca2+ studies have been

performed in SLE T cells (5, 32) at present, there exist no quantitative [Ca2+]i measurements in SLE T cells. Here [Ca2+]i in SLE T cells was compared to that of

normal donors and RA patients. Specifically, we looked at the baseline and peak [Ca2+]i in continuous, transient and oscillatory responses described above. In cells that exhibited a continuous response we also included the [Ca2+]i magnitude after 5 min to investigate

[Ca2+]i decay.

In the continuous response we found no differences in baseline, peak or 5 min values in

SLE as compared to normal donors (table 4.1 & Fig. 4.4). However, RA T cells showed a

trend for diminished [Ca2+]i signal upon stimulation as previously described (38).

- 153 - 2+ Table 4.1: Quantitative analysis of [Ca ]i in Normal, SLE and RA T cells

Normal SLE RA

n=6 n=6 n=4

Base Peak Base Peak Base Peak

Continuous [Ca2+]i (nM) 161 + 42 645 + 154 168 + 61 508 + 134 114 + 54 319 + 65

Transient [Ca2+]i (nM) 146 + 33 460 + 93 153 + 64 466 + 157 106 + 56 289 + 71

Oscillatory [Ca2+]i (nM) 134 + 35 499 + 88 149 + 70 468 + 138 113 + 49 335 + 56

1200 1000 800

] nM 600 2+

[Ca 400 200

0 NL SLE RA NL SLE RA NL SLE RA

BASELINE PEAK 5 MIN

Figure 4.4: Quantitative analysis of continuous [Ca2+]i pattern. Baseline, peak and 5 min [Ca2+]i were measured in the responding T cells that exhibited a continuous response in normal (NL) (◊), SLE (■) and RA (▲) donors. Cells were activated as described in the legend of fig. 4.2. Baseline [Ca2+]i was similar between normal (NL), SLE and RA donors. Peak and 5 min [Ca2+]i was similar in NL and SLE T cells. In this study 6 NL (total number of cells (n) =127), 6 SLE (n = 162 cells) and 4 RA (n = 36 cells) donors participated.

- 154 - Further, we found no difference between baseline or peak responses in normal and SLE T

cells that exhibited a transient or an oscillatory response (table 4.1). RA patients showed

a trend for diminished peak [Ca2+]i responses as compared to normal T cells while the

baseline was the same (table 4.1). Furthermore, the duration of the transient Ca2+ response, that is the time from initial [Ca2+]i rise to signal termination, was not different

between the three groups (normal: 204 + 13 sec, SLE: 231 + 19 sec, RA: 215 + 18 sec).

Subsequently we investigated the frequency of oscillations in cells that exhibited oscillatory responses. This is important as the pattern of oscillations determines differential gene expression (9). The frequency characteristics were analyzed using power spectral density (PSD) analysis as described in the materials and methods section. The

PSD analysis allowed us to evaluate the shape, amplitude and periodicity of oscillations.

For each cell the frequency was determined by using the PSD maximum value. Power spectra for a normal and SLE donor with the corresponding original trace are shown in fig. 4.5. The median response for SLE T cells was 4.6 mHz (periodicity = 217 sec), while for normal T cells it was 5.7 mHz (periodicity = 175 sec). That is, the majority of SLE T cells displayed longer periodicity of oscillations as compared to normal T cells. The detailed distribution is shown in the frequency histogram in fig. 4.5. The oscillation pattern in RA patients closely mirrored SLE T cell responses with a median frequency response of 4.6 mHz. Interestingly, 33 % of normal T cells have a periodicity of < 100 sec while only 11 % of both SLE and RA T cells have this feature.

- 155 - A. NORMAL B. SLE 400 800

350 300 700 250 600 500 ]i nM

200 ]i nM 400 2+

150 2+ 100 300 200 [Ca

50 [Ca 100 0 200 sec 0 200 sec 6 1.6x10 5.0 x 106 1.4 4.0 1.2 /HZ) /HZ)

2 1.0 2 3.0 0.8 0.6 2.0 0.4 1.0 0.2 PSD (nM 0.0 PSD (nM 0.0 5 10 15 20 25 30 5 10 15 20 25 30 Frequency (mHz) Frequency (mHz) 45 45 40 40 35 35 30 30 25 25 20 20 15 15 10 10 5 5 %of oscillating cells

0 %of oscillating cells 0 2 4 6 8 10 12 14 16 18 20 22 2 4 6 8 10 12 14 16 18 20 22 Frequency (mHz) Frequency (mHz)

Figure 4.5: Power spectra of B cell-induced [Ca2+]i oscillations. The frequency of oscillations was determined using PSD in single cells in normal (A) (n=6 donors, 42 cells) and SLE (B) (n=6 donors, 47 cells) T cells. For specific criteria for determining oscillatory activity refer to materials and methods section. A trace representative of the median response frequency is shown in the top panels for normal (A) and SLE (B) donors. Directly below is the corresponding PSD analysis for that cell. The lower panels show the distribution of oscillation frequencies for normal and SLE donors. Results are presented as percentage of cells, within the oscillating population, displaying a particular frequency.

Overall we found no significant differences in the magnitude of [Ca2+]i between SLE and

normal T cells in continuous, transient or oscillatory responses. However, RA T cells

showed a trend for diminished [Ca2+]i upon TCR engagement. In addition, the majority

of oscillating T cells from SLE and RA patients have longer periodicity than normal T

- 156 - cells. It is possible that the altered [Ca2+]i observed in SLE T cells in this and the

previous section is due to faster kinetics of Kv1.3 out of the IS. As such, experiments

aimed at correlating Kv1.3 trafficking to the IS with the shape of the Ca2+ response are

described in the following section.

4.4.3 Biophysical and pharmacological profile of pEGFP-Kv1.3 channels in HEK

293 cells. To support our hypothesis that a specific duration of Kv1.3 localization in the

IS influences the development of the Ca2+ response, we designed experiments targeted at

correlating Kv1.3 movement in and out of the IS and [Ca2+]i in real time. Initially we

attempted to visualize the native channels in T cells. To that end, we used a specific

rabbit polyclonal anti-Kv1.3-FITC antibody for an extracellular epitope of the Kv1.3 channel (EC anti-Kv1.3-FITC). This antibody was previously used successfully for both

fixed and live cell staining and T/B cell conjugation experiments ((17), Fig. 4.6A, top

panels). We immunolabeled T cells live with this antibody and attempted to perform live

cell imaging experiments. However, the fluorescence was quite dim and did not allow us

to visualize the channels sufficiently to perform our experiments. We rationalized that we

were unable to see the channels due to inherent limitations of the fluorescence of the

FITC flourophore. In reality, the FITC fluorophore was not very bright in fixed

microscopy experiments either and it required longer exposure times. Still, owing to

Kv1.3 segregation at the IS and the lack of serial exposures, that would be required for

live cell imaging, we were able to use it.

- 157 - For the current experiments we decided to conjugate the EC anti-Kv1.3 antibody with a

brighter fluorophore, namely Alexa Fluor 488 (AF488). We were able to successfully

conjugate the EC anti-Kv1.3 antibody to AF488. This allowed us to visualize the

channels localize to the T/B cell contact point in fixed imaging experiments (Fig. 4.6,

middle panels). Encouragingly, the fluorescence was as strong as using a secondary

antibody to amplify the signal (Fig. 4.6, lower panels). Next, human T cells were

incubated with the conjugated antibody and were used for live cell imaging experiments.

Unfortunately, we were once again unable to visualize the channels sufficiently to

DIC B cell Kv1.3 Merge

EC-Kv1.3 -FITC

EC-Kv1.3 -AF488

EC-Kv1.3 + 2o antibody

Figure 4.6: Conjugation of EC-anti-Kv1.3 antibody with Alexa Fluor 488. A. Human T cells were labeled live with an EC anti-Kv1.3-FITC antibody (top panels), an EC anti- Kv1.3-Alexa Fluor 488 antibody (middle panels) or an EC anti-Kv1.3 antibody followed by a secondary antibody (bottom panels). The cells were then allowed to interact with SEB-pulsed B cells (blue), fixed and visualized with fluorescence microscopy. Comparative effectiveness of detection of Kv1.3 localization in the IS was observed with EC anti-Kv1.3-AF488 antibody and EC anti-Kv1.3 antibody followed by a secondary antibody. Longer exposure times were required for the EC anti-Kv1.3-FITC antibody. Scale bar = 5 µm

- 158 - perform these experiments even with this brighter fluorophore. It is possible that we were

unable to visualize the channels in live, unstimulated T cells due to the even distribution

of the channels around the membrane as compared to the segregation of numerous Kv1.3

channels at the T/APC interface in the fixed cell experiments.

Based on these results we decided to transfect human T cells with a pEGFP (a brighter

form of GFP) -tagged Kv1.3 clone. To establish that our clone resulted in Kv1.3 channels

that share biophysical and pharmacological characteristics identical to their native

counterparts, we transfected HEK 293 cells that do not have endogenous Kv1.3 channels

and would thus enable us to study the channels’ characteristics. Kv1.3 currents were

recorded using the whole cell configuration and were induced by depolarizing voltage

steps from -80 mV HP to +50 mV in mock and pEGFP-Kv1.3 transfected HEK 293 cells.

A family of currents was elicited as a result and mock transfected cells had low

background current 98 + 52 pA (n=8) compared to pEGFP-Kv1.3 transfected cells, 9972

+ 1202 pA (n=7). Kv1.3 current was reversibly blocked by the specific blocker ShK-

Dap22 (851 + 590 pA, n=7) (Fig. 4.7A) (39). Another feature of Kv1.3 is that it undergoes cumulative inactivation upon application of repeated depolarizing pulses every second. As seen in figure 4.7B, upon application of this protocol there is progressive decline of current, further substantiating that the current recorded is due to Kv1.3

- 159 - AB.

pEGFP-Kv1.3 500 pA 0 20 msec 2 nA

200 msec 1 wo 2 3

200 msec ShK-Dap22

Figure 4.7: Functional and pharmacological properties of recombinant pEGFP- tagged Kv1.3 channels. A. Currents were recorded in whole-cell configuration and were elicited by depolarizing from -80 mV HP to +50 mV every 30 sec in mock (n=8) (A, left) and pEGFP-Kv1.3 (n=7) (A, right) transfected HEK 293 cells. Representative traces are shown. Kv1.3-GFP currents were reversibly blocked by 20 nM Shk-Dap22 (washout: wo) (n=7). B. Cumulative inactivation was induced by consecutive depolarizing pulses applied every second. The maximal current amplitude decreased with each successive pulse (indicated by progressive numbers 0-3).

channels. The properties of Kv1.3 channels described herein are in agreement with previous reports (40-42).

These experiments demonstrate that in an expression system our pEGFP-Kv1.3 clone produces functional channels with identical biophysical properties as their native counterparts. Still this needs to be confirmed in human T cells.

4.4.4 Overexpression of functional pEGFP-Kv1.3 channels in human primary T cells. Subsequently, we transfected freshly isolated human T cells with pEGFP-Kv1.3 channels to ensure that functional channels were inserted in the plasma membrane. As such, we performed whole-cell voltage-clamp experiments after 16-24 hr of transfection.

All experiments were performed within 24 hr of T cell isolation as SLE T cells revert to a

- 160 - A B DIC pEGFP-Kv1.3 pEGFP-Kv1.3 20 pA T 200 msec 1 nA T Mock 200 msec

wo T

ShK-Dap22

Figure 4.8: Overexpression of pEGFP-Kv1.3 channels in human T cells. A. Kv1.3 currents in vector (mock, n=8, left panel) and pEGFP-Kv1.3 transfected T cells (n = 11, right panel) were recorded as described in legend of figure 4.7. pEGFP-Kv1.3 channels were reversibly blocked by 20 nM Shk-Dap22 (n = 7). B. Human T cells transfected with pEGFP-Kv1.3 were incubated with (bottom panels) or without (top panels) SEB-pulsed EBV infected B cells. Channels were expressed and evenly distributed around the plasma membrane in the absence of stimulation (top panels) but redistributed to the T/B cell contact point upon activation (bottom panels). Scale bar = 5 µm.

more normal phenotype after 24 hr (43). Our results indicate an up-regulation of Kv1.3

current in transfected as compared to vector (mock)-transfected T cells (3515 + 256 pA,

n=8 and 107 + 46 pA, n=11 respectively, p= 3x10-9) (Fig. 4.8A). Membrane capacitance

was comparable between groups: vector transfected cells 0.5 + 0.03 pF (n=8) and

pEGFP-Kv1.3 cells 0.6 + 0.05 pF (n=11, p=0.2). As with native Kv1.3 channels, the current was reversibly blocked by ShK-Dap22: 3502 + 372 pA before ShK-Dap22 to 418 +

108 pA after ShK-Dap22 (n=7, p=4x10-6) and the current was recovered after wash out

(wo) (Fig. 4.8A).

Moreover we investigated pEGFP-Kv1.3 membrane distribution in human T cells using

confocal microscopy. We found that pEGFP-Kv1.3 channels are expressed evenly on the

T cell membrane in the absence of stimulation (Fig. 4.8B, top panels) and retain the

- 161 - ability to relocalize to the IS upon stimulation by SEB-pulsed EBV-infected B cells (Fig.

4.8B, bottom panels).

Taken together, our electrophysiological and microscopy data demonstrate that our Kv1.3 clone results in the expression of functional channels that are inserted in the plasma membrane and have identical biophysical and pharmacological characteristics as native channels. Further, these channels retain the ability to relocalize to the IS upon TCR engagement. These experiments set the stage for live cell imaging experiments.

4.4.5 Redistribution of Kv1.3 channels in the IS in Jurkat T cells

Initial live cell imaging experiments were performed using Jurkat T cells (a leukemic T cell line) to investigate Kv1.3 trafficking. These cells were transfected with pEGFP-

Kv1.3 and were followed by confocal time-lapse microscopy for at least 30 min as they interacted with SEB-pulsed B cells. The results obtained in these studies supported the data previously obtained in fixed cell experiments. Specifically, prior to stimulation the channels were uniformly expressed around the T cell membrane but rapidly relocalized to the T/B cell contact point upon conjugation (Fig. 4.9). Remarkably, Kv1.3 channels moved from one contact point to another when a B cell that presumably provided a stronger stimulus was encountered. Next, we set out to perform live cell imaging experiments in human T cells to investigate the functional implications of Kv1.3 relocalization to the IS by correlating it to [Ca2+]i.

- 162 - 0:00 min 1:12 min 4:49 min 6:25 min 11:38 min 19:07 min

3 3 1 1 1 1 1 2 1 2 2 2 2 3 3

Figure 4.9: Time-lapse imaging of Kv1.3 channel redistribution to the IS. Jurkat T cells transfected with pEGFP-Kv1.3 were allowed to interact with SEB-pulsed B cells loaded with the Far Red DDAO dye. Cells were followed live using confocal microscopy. Top panels show the DIC images and lower panels show the corresponding merged Kv1.3 (green) /B cell (blue) images. B cells are numbered in the DIC image in sequence of interaction with the T cell. Initially the Kv1.3 channels are evenly distributed around the membrane (0:00 min) and early upon contact with B cells 1 and 2 redistributed to the contact point (1:12 min). After 4:49 min B cell 3 makes contact and at 6:25 min it crawls over the T cell and makes a tight contact (11:38 min). At this point the Kv1.3 channels relocalize again to the new contact point and are maintained there for the duration of the experiment. Scale bar = 5 µm.

4.4.6 Kv1.3 trafficking to the IS and [Ca2+]i in normal human T cells. Since our

Kv1.3 clone is pEGFP tagged and is excited at 488 nm and emits at 510 nm (green), initially we used the non-ratiometric Ca2+ dye calcium orange, a dye that is excited at 550

nm and emits at 575 nm (red), to avoid any cross-talk between the channels. We

encountered several problems with this dye including organelle compartmentalization,

leaking and low fluorescence emissions. The last problem is an inherent limitation in our

system as we are attempting to visualize [Ca2+]i at the 200-400 nM level which is quite

low.

- 163 - Following, we investigated the possibility of using Fura-2, one of the most commonly

used Ca2+ dyes. However, the limitation of this dye is that it is excited at a UV range (the

Ca2+ bound form is excited at 340 nm and the Ca2+ free form at 380 nm) and the confocal

microscope available to us was not equipped with a UV laser. So instead we decided to

use two-photon microscopy to overcome this problem. Still, to enable us to do that we

had to use Fura-2 as a non-ratiometric dye and use only the Fura-2 Ca2+ free excitation at

800 nm. This was due to the fact that at lower excitation wavelengths (700-780 nm) both

the Ca2+ bound and free Fura-2 forms are excited, albeit to different degrees.

Consequently, Fura-2 was excited at 800 nm which avoids excitation of the Ca2+ bound

Fura-2 (28). This is important as although both Ca2+ bound and Ca2+ free Fura-2 have

different excitation wavelengths, they both emit at approximately 510 nm. Of note, since

the Ca2+ free Fura-2 emission is recorded, a decrease in fluorescence is indicative of an increase in [Ca2+]i.

Our data indicate that there is a steep increase in [Ca2+]i, as seen by the decrease in

fluorescence, upon conjugation of a healthy T cell with a B cell (Fig. 4.10A) and that

Kv1.3 channels relocalize to the IS early upon T/APC conjugation. Notably, [Ca2+]i remains elevated for about 40 min (n=3) and Kv1.3 channels are maintained at the T/B cell contact point for the same time period. Interestingly, in two out of three T cells imaged the Fura-2 signal is recovered after about 45 min, indicating that [Ca2+]i returns

to baseline while Kv1.3 channels are still maintained at the T/B contact interface (Fig.

4.10B).

- 164 - A 1:48 2:12 7:23 8:51

16:13 27:04 46:19 53:01

Intensity of Fura-2 staining

0 256 High [Ca2+]i Low [Ca2+]i B 0.4

0.3 0.2

Fo-F/Fo 0.1 0 10 20 30 40 50 60 Time (min)

Figure 4.10: Time-lapse imaging of Kv1.3 channel redistribution and [Ca2+]i in human T cells. A. Human T cells were transfected with pEGFP-Kv1.3 and loaded with Fura-2. The cells were then allowed to interact with SEB-pulsed B cells. Top panels show

- 165 - the DIC image, middle panels show Kv1.3 distribution (green) and lower panels show [[Ca2+]i using a pseudocolor scale. Initially the Kv1.3 channels are evenly distributed around the membrane (1:48 min) and upon contact with B cell (2:12 min) redistribute to the T/B interface. At 8:51 min there is a decrease in fluorescence of Fura-2 indicating an increase in [Ca2+]i. The Kv1.3 channels remain in the IS for the duration of the experiment whilst at 27:04 min the Ca2+ signal starts to be recovered reaching baseline at 53:01 min. B. The Fura-2 fluorescent intensity was quantified and the F-F0/F0 ratio was obtained as described in the materials and methods section. Although an increase in [Ca2+]i is accompanied by a decrease in fluorescence the estimated ratio inverts the data to allow for comparison with previous experiments. Therefore, in the depicted graph, an increase in [Ca2+]i is represented by a rise in the curve. Scale bar = 5 µm.

These experiments demonstrate that Kv1.3 channels remain in the IS when [Ca2+]i returns to baseline, supporting the hypothesis that the channels’ regulation facilitates the termination of the Ca2+ response. Further, the experiments described herein clearly show

that we have the ability to perform similar experiments in SLE T cells.

4.5 DISCUSSION

The experimental studies presented herein were designed to elucidate the pattern and

magnitude of [Ca2+]i responses in SLE T cells following TCR engagement, and to

correlate [Ca2+]i with Kv1.3 channel localization in the IS. We provide evidence of significant differences in the pattern of [Ca2+]i signaling that is unique to SLE T cells and

not other autoimmune diseases such as RA. Specifically, SLE T cells have an increased

percentage of cells with a continuous [Ca2+]i response and a decrease in the percentage of cells with a transient response. Further, we find that the majority of the oscillating cells in

SLE have longer periodicity as compared to normal donors. These findings could have important implications in transcription factor activation and gene expression. Finally, we

have optimized a challenging two-photon imaging technique that allows simultaneous

- 166 - visualization of Kv1.3 channel trafficking and [Ca2+]i following TCR engagement,

allowing for future comparative studies with SLE T cells.

Numerous signaling anomalies have been documented in SLE T cells following TCR-

engagement (4). Importantly, SLE T cells are hyperresponsive following antigenic

stimulation (3). One of the defects associated with this T cell hyperactivity is a more

sustained [Ca2+]i response that was documented in both CD4 and CD8 T cell lineages by

non-quantitative flow cytometric Ca2+ techniques (5, 32). Yet, the shape and magnitude

of [Ca2+]i response has never been investigated in single T cells from SLE patients. This

information is critical as nearly 75% of all T cell activation genes show some dependence

on [Ca2+]i (6). Importantly, the shape and magnitude of the [Ca2+]i response guides

specific gene expression (9, 10).

As a result, we have conducted quantitative, single cell [Ca2+]i studies aimed at dissecting

the shape and magnitude of [Ca2+]i in SLE T cells. We analyzed the pattern of [Ca2+]i in

the mixed CD4 T cell population and found that SLE T cells present with a higher

percentage of cells displaying a continuous [Ca2+]i response. Previous reports presented

data indicating that a continuous [Ca2+]i response promotes NF-AT localization in the

nucleus (10). Also, Negulescu and colleagues found that a sustained [Ca2+]i was necessary for the activation of an NF-AT promoter and IL-2 production (37).

Interestingly, a recent study indicated that SLE T cells present with increased localization of NF-AT to the nucleus as compared to normal donors and patients with other autoimmune diseases (14). Thus, our data suggest that this increased NF-AT nuclear

- 167 - localization may be attributed to this increase in SLE T cells having a continuous [Ca2+]i response.

Further, this increase in continuous responses was accompanied by a decrease in transient responses. The transient response we observed can either be interpreted as an isolated rise in Ca2+ that returns to baseline and ends, or as part of an oscillatory response, with

periodicity larger than the 15 min used in the current study. Longer periodicity

oscillations and transient [Ca2+]i spikes can preferentially drive specific transcription

factor activation such as NF-κB. In reality, activation of NF-κB can occur at oscillations

with Ca2+ spikes of up to 30 min apart (9, 12). Importantly, SLE T cells have defective

NF-κB activation (15), a defect that could be in part attributed to the decreased percentage of cells with longer periodicity oscillations as suggested by the decrease in transient responses we have observed in our experiments.

Moreover, after detailed analysis of oscillating cells, cells that exhibited three or more

Ca2+ spikes within 15 min, we found that the majority of oscillating cells in SLE had

longer periodicity as compared to normal donors. This also raises the possibility of

differential gene expression. Since NF-AT activation is promoted through frequent

oscillations (9), it is possible that the oscillating cells are compensating for the increased

number of cells that present with a continuous response and promote NF-AT expression

by increasing the periodicity of oscillations and effectively inhibiting further NF-AT

expression (9, 10).

- 168 - Further, SLE T cells exhibit an altered, more active T cell phenotype with a low

CD4/CD8 ratio and an increase in CD4+ TEM cells (17, 43, 44). This raised a question as to whether this differential Ca2+ pattern observed could be attributed to this altered

phenotype. However, no specific pattern was associated with resting or TEM cells. Still,

SLE T cells are hyperresponsive following antigenic stimulation and can elicit more

pronounced responses than normal T cells (3). Wulfing and colleagues used altered

antigenic peptides that reduced the effectiveness of MHC binding and they reported

variability in Ca2+ responses, from continuous to reduced, partial or transient, reflecting the strength of the antigenic stimulus (45). Specifically, they reported that the more

specific the peptide, the higher the percentage of cells that exhibited a sustained response.

So the altered [Ca2+]i patterns could be a sign of the ability of SLE T cells to respond to

an antigenic stimulus more efficiently than healthy T cells or RA T cells that are

hyporesponsive (38).

This more sensitive and activated phenotype also raises the possibility that the altered

[Ca2+]i response is due to anomalies associated with the modulation of Ca2+ in SLE T cells. In human T cells Ca2+ modulation relies not only on CRAC channels and the two

K+ channels, Kv1.3 and KCa3.1, but also on Ca2+ re-uptake in intracellular compartments

and efflux through plasma membrane calcium ATPases (PMCA). In a previous study we

have shown altered compartmentalization of Kv1.3 upon T/APC contact in SLE T cells.

Specifically, while Kv1.3 resides in the IS for at least 30 min in normal T cells, the

channels are removed from the IS within 15 min in SLE T cells. Thus, the premature exit

of the channel may affect its regulation and as such promote the enhanced Ca2+ pattern

- 169 - we observed. To decipher the role of Kv1.3 in this [Ca2+]i defect we have developed a

two-photon live cell imaging technique that allows simultaneous recordings of Kv1.3

trafficking and Ca2+ influx. Currently we were able to perform experiments in NL T cells

where we observed Kv1.3 trafficking in the IS accompanied by an increase in [Ca2+]i for at least 40 min. Interestingly, when [Ca2+]i returned to baseline Kv1.3 was still localized

in the IS, advocating a role for Kv1.3 in the termination of the Ca2+ response. These

experiments set the stage for future experiments aimed at correlating Kv1.3 trafficking

with [Ca2+]i in SLE T cells. Still, we cannot exclude that defective Ca2+ re-uptake and

efflux may also contribute to the Ca2+ defect observed.

Despite the alterations in the [Ca2+]i shape we report no differences in the magnitude of

[Ca2+]i in SLE as compared to normal T cells at baseline, peak or 5 min intervals.

However, RA T cells did present with a tendency for diminished [Ca2+]i upon stimulation

as previously described (38). Still, due to the limited number of patients tested, no

definitive conclusion was made. Previous studies have pointed to enhanced Ca2+ rise upon stimulation in SLE T cells, albeit small (5). Those studies were performed using antibodies that induce TCR capping and as such T cells do not form a focused IS. We have used a more physiological system by inducing activation using SEB-pulsed B cells.

On the other hand, our system allows the study of a limited number of cells as compared to a flow cytometry technique that takes into account the entire T cell population. Also, we have studied [Ca2+]i in three distinct populations. However, even when we grouped

all responding cells together we still did not observe a difference in [Ca2+]i in SLE T cells

(data not shown). In agreement with our data, other studies using PHA to stimulate Ca2+

- 170 - influx point to normal, or even decreased, Ca2+ influx following stimulation (46, 47). So,

even though the antibody stimulation results in more physiological responses than the

PHA experiments, it is not as applicable as T/APC interactions, thus experimental

differences may account for this discrepancy.

To sum up, data presented herein provide the first evidence of altered [Ca2+]i shape

following TCR engagement in SLE T cells. This pronounced [Ca2+]i response may be responsible for the hyperactivity documented in SLE T cells and may contribute to T cell dysfunction in these patients. Further, these data might have important implications in gene expression and differentiation in SLE. Although initial findings indicate a possible role of defective Kv1.3 trafficking to the IS, further studies are warranted to dissect the mechanisms that promote these changes.

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- 175 -

Chapter V: General Discussion

- 176 - This dissertation is comprised of two tightly interwoven pieces of work. Firstly, we

provide direct evidence of the spatial and temporal redistribution of Kv1.3 and KCa3.1

channels in the immunological synapse upon T cell activation in healthy resting and

activated T cells. Secondly, we perform comparative studies with T cells from patients with SLE and highlight anomalies associated with the localization of Kv1.3 in the IS in T cells from these patients. Kv1.3 localization was pursued primarily because this was the dominant channel in these cells. Finally, we performed functional studies aimed at investigating Ca2+ response in single cells in normal donors and patients with SLE as well as correlating Ca2+ flux with the movement of Kv1.3 in live human T cells. This chapter aims to tie these data together with the relevant literature and highlight the novel work from this study. Since the trafficking of K+ channels in normal T cells by itself was

largely unknown, the first part of the discussion will focus on healthy T cells. Next, we

will examine Kv1.3 trafficking in SLE T cells followed by comparative functional studies

and future directions, and conclude with the clinical implications of this study.

5.1 K+ channel localization in the immunological synapse in healthy T cells

5.1.1 Membrane distribution of Kv1.3 channels in healthy resting T cells

Within the first few minutes of T cell engagement by APC/ag there is general

reorganization of structural (F-actin and lipid rafts) and signaling elements (p56lck and

PKCθ) at the T/APC cell contact interface. Prior to the formation of an organized IS there is release of Ca2+ from the intracellular stores. The emptying of the stores promotes Ca2+

influx through the CRAC channels. To sustain the activation process, K+ channels allow

K+ efflux and maintain a hyperpolarized membrane potential. Resting T cells express

- 177 - high levels of Kv1.3 (200-400) and very low levels of KCa3.1 channels (0-30) (1). As a

result, Kv1.3 channels regulate the membrane potential and shape the Ca2+ response in

resting T cells. A limited number of studies indicated that Kv1.3 channels localize to the

IS upon TCR engagement (2, 3). In Chapter II, we defined the spatial and temporal

distribution of Kv1.3 in the IS and found that within 1-5 min from contact with the APC,

Kv1.3 channels move to the T/APC interface and become part of the early IS signaling

complex. At least in the early stages of IS formation, the function of the channel does not

appear to be relevant to the formation of this complex as blocking Kv1.3 function, using

the specific blocker Shk-Dap22, did not prevent its trafficking to the IS or the formation of

the IS ((4) and our unpublished data). Still, pretreatment of resting T cells with ShK,

another Kv1.3 blocker of the same family, abolished Ca2+ influx.

Soon after, a mature synapse forms with proteins organized in distinct SMACs (5). After

30 - 60 min of T/APC conjugation, well after a mature synapse forms, there is recycling

of the T cell receptor via a ubiquitin mediated pathway in preparation for termination of

the response (6). Interestingly, the Kv1.3 channels still reside in the IS at this time, suggesting a role for Kv1.3 in the termination of the activation response. In support of this idea, in Chapter IV, we observed that in live human T cells Kv1.3 channels localize in the IS upon initiation and termination of the Ca2+ response. One of the benefits of IS

formation is that it allows protein segregation and interaction at specific time intervals. It

is thus possible that this specific spatial and temporal distribution of Kv1.3 in the IS

facilitates the regulation of the channel by molecules that accumulate in the IS at defined

time points. To illustrate, Kv1.3 is known to be regulated by p56lck, PKC, PKA and

- 178 - CaMKII, whose entry and exit in the IS is time specific (7-11). Besides signaling

molecule accumulation, reorganization of lipid rafts at the T/APC contact point could

also result in the regulation of this channels’ function (12, 13).

Taken together these data advocate that the Kv1.3 channels localize to the IS in order to

facilitate the regulation of the channels’ function. Kv1.3 channels in turn regulate

membrane potential and shape the Ca2+ response, facilitating T cell activation and ultimately the termination of the response.

5.1.2 Membrane distribution of Kv1.3 and KCa3.1 channels in healthy activated T

cells

There have been numerous studies aimed at deciphering the structure of the IS and

protein localization during T cell activation. As far as activated T cells are concerned, the studies are far more limited except for studies in cell lines that exhibit an activated T cell phenotype such as Jurkat T cells. To our knowledge this is the first study that investigated

Kv1.3 and KCa3.1 channel localization in the IS during T cell activation in activated human cells (Chapters II & III). In activated T cells KCa3.1 channels, and not Kv1.3

channels, are the dominant K+ channels and as such modulate the membrane potential

and Ca2+ influx (14). In support of this, we have shown that blockade of KCa3.1 by the

specific blocker TRAM-34, halted Ca2+ flux in activated but not resting T cells. We

designed detailed microscopy studies aimed at dissecting the temporal and spatial

distribution of Kv1.3 and KCa3.1 channels in activated human T cells.

- 179 - The early stages of activation, such as TCR translocation and phosphotyrosine related

events, mirror naïve T cells (15). That is where the similarities end. Activated T cells are

better equipped to act towards a pathogen as they have increased lipid rafts which

presumably make the TCR more accessible to MHC dimmers (16, 17). In addition, these

cells up-regulate p56lck which becomes membrane bound and ready to act (18). Our data

demonstrate that early upon TCR engagement and within 1-5 min, both Kv1.3 and

KCa3.1 channels are recruited at the T/APC contact interface with KCa3.1 acquiring a

central localization.

After 15 min of T/APC conjugation Kv1.3 channels start moving out of the IS. In sharp

contrast, KCa3.1 channels are maintained in the center of the IS for at least 30 min after

conjugation. Interestingly, pretreatment of activated T cells with the specific KCa3.1

blocker TRAM-34, did not prevent KCa3.1 trafficking to the IS, nor did it prevent IS

formation. These data imply that KCa3.1 channel activity, like Kv1.3 channel activity, is

not necessary for IS formation and it suggests that K+ channel relocalization to the IS is a common mechanism to facilitate the better regulation of the channel by kinases localized there. Specifically, KCa3.1 channels’ function depends on PKC and PKA and both kinases are recruited in the IS upon stimulation (19-23).

In this section we discussed evidence of K+ channel localization in the IS in normal

resting and activated T cells. These studies provide insight into the differential

mechanisms employed by T cells at different activation stages to combat pathogens.

Importantly, this information significantly enhances our understanding of TCR mediated

- 180 - responses and paves the way for comparative studies with diseased T cells that display

aberrant T cell mediated responses. Such a disease is the autoimmune disease SLE.

Comparative studies with normal and RA T cells will be discussed in the next section.

5.2 K+ channel trafficking in the immunological synapse and Ca2+ signaling in

SLE T cells

5.2.1 Membrane distribution of Kv1.3 channels in resting SLE T cells

Several studies point to a plethora of T cell signaling abnormalities in SLE T cells

(summarized in table 1.1). Notably, SLE T cells present with abnormal, more sustained

Ca2+ response and it is believed that Ca2+ plays a pivotal role in SLE T cell hyperactivity.

Although Ca2+ and K+ channels are the primary regulators of Ca2+ homeostasis, the

impeding role of membrane driven mechanisms in the etiopathogenesis of SLE had not

been previously investigated.

In Chapter II, we set out to determine whether the K+ channel phenotype is altered in

SLE T cells and whether the channels’ function was changed. Our data indicate that in

SLE T cells, Kv1.3 channels have identical biophysical and pharmacological

characteristics as T cells from healthy individuals. Also, freshly isolated SLE T cells had

the same number of Kv1.3 and KCa3.1 channels as healthy resting T cells, with Kv1.3

being the dominant channel. Since the function of the channels was normal we decided to

study the trafficking of Kv1.3 channels in the IS during T cell activation. Examining our

results, we found that while normal resting T cells recruit Kv1.3 and maintain it at the

T/APC interface for at least 30 min, SLE T cells show altered compartmentalization with

- 181 - Kv1.3 channels leaving the IS after 15 min of activation. Notably, this defect was not

seen in RA patients, suggesting that this Kv1.3 mobility defect is unique to SLE and not

shown by other autoimmune diseases. We speculated that the Kv1.3 mobility defect was

due to the more active phenotype of SLE T cells, as healthy pre-activated T cells exhibit

faster kinetics in and out of the IS as observed in SLE. However, while normal pre-

activated T cells up-regulate KCa3.1, which is maintained in the IS for at least 30 min,

SLE T cells do not. KCa3.1 up-regulation relies mainly on PKC mediated pathways that

involve the transcription factor AP-1 (14). SLE T cells have decreased PKC (24) as well

as decreased AP-1 (25). As such, it is quite possible that these cells lack the tools

required to increase KCa3.1 channel expression that accompanies T cell activation.

Therefore, while SLE T cells exhibit an activated phenotype in regards to Kv1.3 kinetics, and allow the Kv1.3 channels to leave the IS rapidly, their K+ channel make-up, and

specifically the lack of KCa3.1, does not support this activated phenotype.

It is possible that this rapid exit of Kv1.3 from the IS may result in abnormal regulation

of Kv1.3 channel activity by avoiding down-regulation, and as such, leading to an

exaggerated Ca2+ response that characterizes these cells (26, 27). Several lines of

evidence suggest defective regulation of Kv1.3 in addition to the Kv1.3 mobility defect.

To name a few, SLE T cells have increased p56lck activity, decreased PKC expression,

decreased PKA activity and decreased CaMKII expression (also refer to table 1.1).

5.2.2 Ca2+ signaling in SLE T cells

- 182 - Interestingly, we observed the Kv1.3 mobility defect in both CD4 and CD8 T cell

lineages and independent of disease activity or drug regime. It has also been reported that

the Ca2+ abnormalities were independent of disease activity and organ involvement as

well (27). Of interest, altered gene expression in SLE can be linked to Ca2+ defects.

Specifically, the expression of a Ca2+ dependent gene, CD40L, is augmented in SLE patients (28). Notably, CD40L supports B cell differentiation through the engagement of

CD40, resulting in autoantibody production, one of the hallmarks of autoimmunity (28).

Importantly, it is not only the occurrence but also the shape of the Ca2+ response that drives specific gene expression (29, 30). To illustrate, optimal NF-AT activation and nuclear localization is achieved with a sustained Ca2+ increase (31). At present there are

no single T cell quantitative studies in SLE T cells. To that end, we performed

comparative studies in single T cells from normal, SLE and RA donors to examine in

detail the magnitude and shape of the Ca2+ response in these cells. We found that SLE T

cells present with a higher percentage of cells exhibiting a continuous Ca2+ response,

accompanied by a corresponding decrease in the percentage of cells exhibiting a transient response. Our data are in agreement with the recent work of Kyttaris and colleagues which indicates that there is increased NF-AT translocation to the nucleus in SLE T cells

(31).

We hypothesized that altered [Ca2+]i is due to altered Kv1.3 kinetics in the IS that result

in abnormal regulation of the channel leading to the exaggerated Ca2+ signal. To support

this hypothesis we optimized a challenging two-photon live cell imaging technique that

- 183 - allows simultaneous recording of Kv1.3 trafficking and [Ca2+]i. Studies reported in

Chapter IV suggest that Kv1.3 localizes in the IS while [Ca2+]i is elevated and after about

45 min, still resides at the T/APC interface while [Ca2+]i returns to baseline. These data

point to a possible role of Kv1.3 in the termination of the [Ca2+]i response. However, in

SLE T cells Kv1.3 is maintained in the IS for only 15 min. Based on our findings and the

current literature we propose a model that facilitates abnormal Ca2+ responses in SLE T

cells (Fig. 5.1). In the proposed model Kv1.3 channels escape down-regulation by

exhibiting altered faster kinetics out of the IS, resulting in sustained Ca2+ influx.

Unstimulated T/APC

1-15 min 15-30 min >30 min

X X

2+ 2+ 2+ NL [Ca ]i [Ca ]i 2+ [Ca ]i [Ca ]i

2+ 2+ SLE [Ca2+]i [Ca ]i [Ca ]i [Ca2+]i

Kv1.3 LFA-1 CRAC TCR &CD3

Kinases MHC/ag

Figure 5.1: Proposed mechanism of abnormal Ca2+ signaling in SLE T cells. Unstimulated cell: Prior to TCR engagement Kv1.3 channels (green) are evenly distributed around the membrane in both normal (NL) and SLE T cells and exist in close proximity to CD3 chains (yellow). [Ca2+]i is at resting state and shown by a straight line.

- 184 - 1-15 min: Upon TCR engagement through MHC (dark green)/ ag (red circle), CRAC channels (red) allow Ca2+ influx and Kv1.3 moves to the T/APC interface and becomes part of the IS signaling complex in both normal and SLE T cells. At this point there is an increase in [Ca2+]i as indicated by the curve. The dotted line indicates the Ca2+ shape at previous times. Also, adhesion molecules (LFA-1, orange) are recruited in the IS to stabilize the structure. Later on, a mature IS is formed. 15-30 min: While Kv1.3 channels are still in the IS in normal T cells they exit the IS prematurely in SLE T cells. [Ca2+]i is still elevated. >30 min: In normal T cells Kv1.3 is still in the IS and its function is inhibited by kinases (blue) localized in the IS leading to termination of Ca2+ influx and return to baseline. However, since SLE T cells already excluded Kv1.3 from the T/APC interface the Kv1.3 channels avoid down-regulation and the Ca2+ response persists.

5.3 Future Directions

The studies described in this thesis define the temporal and spatial distribution of Kv1.3

and KCa3.1 in healthy T cells. Further, they point to defects associated with Kv1.3

localization in the IS and altered shape of the Ca2+ response in SLE T cells. These findings contribute to the understanding of membrane mechanisms involved in IS formation and the defects that accompany abnormal behavior in diseased cells and could be exploited pharmacologically. Future studies could be directed towards determining the precise mechanisms that facilitate K+ channel trafficking in T cells and additional

functional studies.

An important question to address is whether the Kv1.3 mobility defect documented in

SLE T cells is directly attributed to Kv1.3 or another element which is physically

associated with the channel. To better define the role of Kv1.3 associated elements,

mutant Kv1.3 channels could be constructed targeted at disrupting protein association and

transfected into human T cells. Then microscopy techniques could be enlisted to investigate whether the channel’s distribution to the IS is disrupted. Also of interest

- 185 - would be to determine the fate of the channels once they exit the IS. Do the channels

redistribute around the membrane or are they internalized and degraded? To that end, T

cells would be transfected with a photoactivable GFP-tagged channel that could be turned

on at different times and allow observation of the channels’ localization.

Upon completion of these studies in healthy T cells, similar studies could be performed in

SLE T cells to determine altered regulatory mechanisms. Further, we speculated that the

Ca2+ abnormality observed in SLE T cells could be due to the inability of these cells to

up-regulate KCa3.1. As such, detailed electrophysiological studies could be performed to

evaluate the ability of SLE T cells to up-regulate KCa3.1 channels. Also, the two-photon live cell imaging technique presented in Chapter IV could be used for additional experiments designed at correlating Kv1.3 localization and [Ca2+]i in SLE T cells.

5.4 Clinical Relevance and Therapeutic Indications

In the past SLE was considered to be a B cell mediated autoimmune disease. However, the plethora of information emerging supports the view that SLE T cells have a multitude of signaling abnormalities which could promote B cell hyperactivity and autoantibody production. As such, in our time, the immunopathological contribution of T cells in SLE is taking center stage. A lot of the defects in these patients have been linked to abnormal

Ca2+ signaling. Since Ca2+ is one of the main concerns, it follows that our primary target

should be the Ca2+ mediators. That is, Ca2+ and K+ channels on the membrane of T cells.

Our data and the literature suggest that Kv1.3 channels are crucial components of the T

cell activation process and as such pose attractive targets for specific immunomodulation

- 186 - in autoimmune diseases. Therefore, it is not surprising that the search for specific Kv1.3

channel blockers is on the rise. In fact, Kv1.3 channel blockers have been proven

effective in ameliorating other autoimmune diseases such as multiple sclerosis,

rheumatoid arthritis and type I diabetes mellitus (4).

In this thesis we report abnormalities associated with Kv1.3 channel trafficking in the IS

in SLE T cells as well as abnormal [Ca2+]i shape in these cells. As such, the work

presented herein suggests that Kv1.3 channels are involved in the hyperactivity of SLE T

cells and that the channel could be a prime candidate for pharmacologic intervention to

reduce hyperactivity of T cells from patients suffering with SLE, a disease that to date

goes uncured.

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