Flow Cytometric Methods for Assessment of Cell-Mediated Immune Responses

FLOW CYTOMETRIC METHODS FOR ASSESSMENT OF

CELL-MEDIATED IMMUNE RESPONSES

KARINA GODOY RAMIREZ

STOCKHOLM 2005

From Microbiology and Tumorbiology Center (MTC), Karolinska Institutet and the Swedish Institute for Infectious Disease Control, Stockholm, Sweden

FLOW CYTOMETRIC METHODS FOR ASSESSMENT OF CELL-MEDIATED IMMUNE RESPONSES

Karina Godoy Ramirez

STOCKHOLM 2005

Karina Godoy Ramirez: Flow cytometric methods for assessment of cell-mediated immune responses. Karolinska Institutet, Stockholm, 2005.

The previously published papers were reproduced with permission from the publisher.

Repro Print AB, Stockholm

ABSTRACT The aim of this thesis was to develop and improve flow cytometry-based methods for assessment of cell-mediated immune responses (CMI) induced by natural infection or vaccination. Traditionally, the measurement of antigen-specific cellular immune responses has been based on the use of complex, labour-intensive, and less informative bulk-results assays (51Cr-release, 3H-thymidin uptake and Limiting dilution assay, LDA). Alternative techniques for assessment have been developed (tetramer- staining, ELISspot, intracellular cytokine staining, ICS), with pros and cons for each system. We have focused on the assessment of cytokine production and cytolytic capacity, two activities of importance for evaluation of CMI to viral infections, especially HIV. Flow cytometry technology offers the possibility for simultaneous analysis of multiple characteristics and functions of single cells at high rate. We have developed two lytic assays for the assessment of natural killer (NK) cell mediated and HIV-antigen-specific T-cell mediated cytotoxicity (FC-CTL). In the NK cell assay, the cell populations are separated by post-culture immunophenotyping and cell death is detected by uptake of propidium iodide (PI), which permits the combined assessment of NK cell lysis and conjugate formation between target and effector cells. The assay is sensitive, very easy to perform and more informative than other lytic assays. The FC-CTL is based on dual staining with 5-(and –6)- carboxyfluorescein diacetate succinimidyl ester, CFSE (for tracing target cells) and PI (for cell-death), which permits identification of several cell populations including viable and dead target cells at different stages of cell-death; live and dead effector cells; and also enumeration of lysed ‘not- detectable’ target cells. Determination of HIV-specific cytolysis is based on the enumeration of viable target cells rather than percentage of dead cells as commonly used for other assays. The FC-CTL is well correlated to, but more sensitive than the conventional 51Cr-release assay. The standard procedure for ICS is based on short-term (6h) activation of PBMCs or undiluted whole blood. We performed kinetic studies of cytokine production with regard to incubation time and dilution of whole blood samples for specific and non-specific activation. We consistently recorded higher frequencies of responding T-cells in samples of diluted blood (1/5-1/10) cultured for several days (long-term (LT-ICS), as compared to short-term incubation. The increased levels of cytokines, in particular IFN-γ with peak detection at 72h, are due to expansion of the antigen specific cytokine-producing cells. The new assays (LT-ICS and FC-CTL) and conventional methods (ELIspot and Cr-release) were subsequently applied for a study of HIV-1 infected (>5 years) treatment-naïve individuals with CD4+ T-cell counts >400 and with low or high viral load. The levels of IFN-γ responses and cytolytic activity were consistently higher in subjects with low viral load, against all HIV-antigen tested (Gag, Nef and avipox-based antigens), as assessed by all methods employed. By the LT-ICS (48h), the difference between the two patient-groups was statistically significant for CD4+ T-cell responses against all HIV-antigens tested. The strong HIV-specific cell-mediated immune responses found in subjects with stable CD4+ T-cell counts and low viral load, support the idea that strong HIV-1-specific cellular immunity is protective. Both cytolytic assays and the LT-ICS can be further expanded for simultaneous assessment of several different molecules expressed on cell-surface or present intracellularly, especially if the 3-4- laser flow cytometers with ability to detect up to 18 colours are utilized. The new assays presented in this thesis will be valuable tools for research regarding evaluation of CMI against viral infections such as HIV and for monitoring in vaccine studies, in particular if immune responses may appear at low levels. The NK cell cytotoxicity assay has been further developed for use of whole blood and is now employed for a study of the role of innate immunity for control of herpes simplex infection, and the LT-ICS will be used as one of the assays for evaluation of CMI in a phase I HIV-1 vaccine trial in Stockholm.

Keywords: flow cytometry, intracellular cytokine staining (ICS), cytotoxicity, HIV-1

THANKS TO LIFE (by Violeta Parra a Chilean poetess and singer, 1917-1967)

Thanks to life that has given me so much It has given me two eyes, that when I open them I clearly distinguish the black from the white And in the infinite sky, its starry depths, And from the crowds of people, the friend that I love.

Thanks to life that has given me so much It has given me hearing that in all its breadth Night and day records crickets and canaries, Hammers, turbines, barking, splashes, And the tender voice of my loved ones.

Thanks to life that has given me so much It has given me sound and the alphabet And with it the words to think and speak Mother, friend, sister, and the light that brightens The path of the soul of my loved ones.

Thanks to life that has given me so much It has kept my tired feet walking With them I walked through the cities and the puddles, Beaches and deserts, mountains and plains And to your house, your street and your courtyard.

Thanks to life that has given me so much It has given me laughter and it has given me tears With them I can tell the difference between joy and pain, The two things that make up my song, And your song that is everyone's song that is my own song.

Thanks to life.....

To my parents Ana and Jorge

ORIGINAL PAPERS AND MANUSCRIPTS

This thesis is based on the following original papers and manuscript, which will be referred to in the text by their Roman numerals:

I. Godoy Ramirez K, Franck K, Gaines H. A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi- parameter flow cytometry. J Immunol Methods 239, 35-44, 2000.

II. Godoy Ramirez K, Mäkitalo B, Thorstensson R, Sandström E , Biberfeld G, Gaines H. A novel assay for assessment of HIV-specific cytotoxicity by multi-parameter flow cytometry. Submitted to Cytometry.

III. Godoy Ramirez K, Franck K, Mahdavifar S, Andersson L, Gaines H. Optimum culture conditions for specific and non-specific activation of whole blood and PBMC for intracellular cytokine assessment by flow cytometry. J Immunol Methods 292, 1-15, 2004.

IV. Godoy Ramirez K, Mäkitalo B, Heideman B, Thorstensson R, Sandström E , Biberfeld G, Gaines H. HIV-specific CD4+ and CD8+ T-cell responses associated with low viral load in treatment-naïve HIV-infected persons. Manuscript.

CONTENTS

THE IMMUNE SYSTEM…………………………………………...…………………….……3 The innate immune system…………………………………………..…………...……….....3 The adaptive immune system………………………………………………….………….....3 Humoral immune response……………..…………………………………………….....4 Cellular immune response……………..…………………………………………….….4 Antigen recognition and activation……………..…………..…………………………….….5 Antigen processing and presentation……………..………………………………………….5

FLOW CYTOMETRY………………………………………………………………………….8 Introduction…………………………………………………………………………………..8 History………………………………………………………………………………….……8 Principle…………………………………………………………….……………………..…9 Applications…………………………………………………………………………….……9 Disadvantages……………………………………………………...………………….……10

ASSESSMENT OF CELLULAR IMMUNE RESPONSES…………………...……………12 Phenotypic/structural assessment…………………….……………………………….……12 Markers associated with activation, differentiation and function………………..……13 Tetramer-staining………………………………...……………………………………13

Functional assessment…………………………………………...…………………….……14 T-cell proliferation…………………………………………….………………….…….…14 Cytolytic activity………………………………………………………………….………15 Cytokine production…………………………………………………..…………………..17

HUMAN IMMUNODEFICIENCY VIRUS INFECTION……………………………..……20 The HIV/AIDS pandemic……………………………………………………….………….20 Immunodeficiency viruses…………………………………………………………….……20 The course of infection………………………………………………………………..……22 The immune responses……………………………………………………………..….…...24 Humoral immune response……………………………...…………………….…….…24 Cellular immune response………………………………………………….…….……25

AIMS OF THE THESIS………………………………………………..…………….….……27

RESULTS AND DISCUSSION………………………………………………………….……28 I. Development of cytotoxicity assays for assessment of natural killer cells and HIV-specific cytotoxic T-lymphocyte activity (Paper I, II and IV)….………………..28 II. Intracellular cytokine staining (Paper III and IV)…………………………………….……45 III. Assessment of HIV-1 specific cell-mediated immune responses (Paper IV)……………...49

GENERAL COMMENTS AND CONCLUDING REMARKS…………………….….……54

ACKNOWLEDGEMENTS………………….…………………………………….…….……56

REFERENCES………………….……………………….……………………….…………....59

APPENDIX (PAPERS I-IV)

LIST OF ABBREVIATIONS

ADCC Antibody-ependent Cellular Cytotoxicity APC: Antigen resenting cell CFSE 5-(and –6)-carboxyfluorescein diacetate succinimidyl ester CMI cell-mediated immune response CTL: cytotoxic T lymphocyte ELISA: enzyme-linked immunoassay ELISPOT: enzyme-linked immunospot FACS fluorescence-activated cell sorter FC flow cytometry FC-CTL flow cytometric cytotoxic T-lymphocyte assay HIV-1 Human Immunodeficiency Virus Type one ICS: intracellular cytokine staining IFN-γ interferon-gamma LDA limiting dilution assay LT-ICS: long-term intracellular cytokine staining MHC: major histocompatibility complex Mab monoclonal antibody NK: natural killer PI propidium iodide PBMC: peripheral blood mononuclear cells TCR T-cells receptor WB: whole blood

THE IMMUNE SYSTEM The immune system consists of a complex network and strategies developed during evolution, to defend the organism against infectious microbes, such as bacteria, viruses and parasites, and also against tumour-transformed cells that could threat the host survival. The immune system is traditionally divided into an innate and an adaptive (acquired) compartment, where the later is further divided into humoral and cell-mediated responses but all the systems co- operate to provide an effective defence against potential pathogens.

THE INNATE IMMUNE SYSTEM Besides the physical barriers, the innate immune system is the first line of host defence, and it is specialized for the control of invading pathogens during the primary phase of infection. The responses are generated within hours after infection/activation of the involved cells, do not require previous antigen exposure to effectuate a good response and do not develop memory. The innate immune system involves macrophages, granulocytes, dendritic cells and γδ+ T- cells, that recognize structures such as polysaccharides and other conserved bacterial components by their binding to pattern recognition receptor (PRR) or toll like receptors (TLR). Another important component of the innate immune system are the natural killer (NK) cells (Kiessling 1975). NK cells form a significant third group of lymphocytes (5-10%) that differs from T and B cells in surface phenotype, target cell recognition, and function. They have two relevant functions, related to the innate immune response against pathogens: the first is cytotoxicity, mediated by the recognition and lysis of virus-infected cells or mediated by antibodies (antibody-dependent cell-mediated cytotoxicity, ADCC). The second function is the production of cytokines, mainly IFN-γ, and β-chemokines that can modulate innate and specific immune responses. NK cells can also mediate antibody-dependent cell-mediated cytotoxicity (ADCC) together with virus-specific antibodies. NK cells are important for the defence against certain types of viruses such as cytomegalovirus (CMV) (Biron 1989), Epstein-Barr virus (EBV) (Joncas 1989), and herpes simplex virus (HSV) (Ching 1979).

THE ADAPTIVE IMMUNE SYSTEM The adaptive arm of the immune system is composed of highly specific responses for each different invading microbe, mediated by B-cells producing immunoglobulins or by T-cells recognizing the antigen by their specific T cell receptors (TCR) (Janeway and Travers 2005). Immune responses imply the interaction of different cell types, and humoral and cell-mediated

systems are not entirely separated. In contrast to the innate immunity, the adaptive immune system generates immunological memory to prevent re-infection by the same pathogen.

Humoral immune responses B-lymphocytes are activated by antigen or antigen presenting cells in the presence of cytokines such as IL-4 and IL-5 which through cooperation with CD4+ T-helper cells stimulate their proliferation and differentiation into antibody-secreting plasma cells. The antibodies produced in response to viral infection may act by several anti-viral mechanisms including binding to the virus which may prevent initial infection (neutralization); activation of the complement system, induction of ADCC, and inhibition of virus released from infected cells.

Cellular immune responses The cellular immune response is mediated by T-lymphocytes that can be subdivided into several populations but the two major subtypes are: T-helper cells, which express the surface marker CD4, and cytotoxic T-lymphocytes (CTL), which express the surface marker CD8. Both T-helper cells and CTLs are critical for the protection against infectious microbes (Mills 1989).

The CD4+ T-cells play a crucial role in the regulation of virtually all immune responses, providing help for antibody production by B cells, and growth factors for B-cells, CD8+ T- cells, and other cell types. CD4+ T-cells are also known to secrete antiviral IFN-γ and chemokines, to mediate cytolysis, and to activate macrophages for the killing of intracellular pathogens.

CD8+ T-cells are important for the resistance against intracellular infections with certain viral, protozoan and bacterial pathogens, and against cancer tumors. Activation of CTLs leads to the production of cytokines (IL-2, IFN-γ, TNF-α), chemokines (RANTES, MIP-1α, MIP-1β) and antigen-specific lysis of MHC class I matched target cells. The target killing can be mediated by the release of granules containing cytolytic effector molecules such as perforins, granzymes and lysin or by interaction of Fas and Fas-ligand or TNF and TNF-receptor (Janeway and Travers 2005).

Cytokines regulate the initiation and maintenance of the immune response and are essential to determine the nature of the immune response and what effector mechanisms should be selected and propagated (Cher 1987).

Antigen recognition and activation T-cell activation is initiated when the antigen-specific T-cell receptor (TCR) recognises antigens presented by the major histocompatibility complex (MHC) class I or class II molecules on the antigen-presenting cell. This interaction triggers a cascade of intracellular reactions, which in conjunction with simultaneous co-ligation of either CD4 to MHC class II molecules or CD8 to MHC class I, finally results in the activation of three different intracellular signalling pathways. To obtain a complete T-cell activation, further co- stimulatory signals are required, such as the interaction between CD28, on T-cells and CD80, on APCs. Activation of CD4+ T-cells may lead to cytokine secretion (Arai 990; Scott 1990; Finklemann 1995), expression or up-regulation of cell adhesion molecules, ligands for molecules expressed on other cells, and cytokine receptors (Kaye and Janeway 1984; Andersson 1994; Jeannin 1999), and eventually also to cell division (proliferation) of antigen- specific clones (Vasseur 1999) (Figure 1).

Antigen processing and presentation Antigen is processed by antigen-presenting cells in two major pathways, the endogenous and the exogenous pathway (Figure 2) (Germain 1994). In the endogenous pathway, newly synthesized proteins by the APC are cleaved by proteasomes into peptides consisting of ∼20 amino acids. The peptides are then transported via the transporter complex (TAP) to the endocytoplasmic reticulum (ER), where they encounter and bind to newly formed MHC class I molecules. The peptide-MHC-I complex is then transported to and expressed on the cell surface where it is recognised by T- lymphocytes expressing the CD8 molecules. In the exogenous pathway, extra-cellular soluble antigens are taken up in endocytic vesicles and further processed in endosomes (intracellular acidic vesicles). The endosomes interact with vesicles that are transporting MHC class II molecules to the cell surface. The peptide- MHC-I complex is then recognised by T- lymphocytes expressing the CD4 molecules.

5 LT-ICS ELISpot NK cell lys FC-CTL Cr-release

R1 R3 R2

R3 R1 QuickTime och en Foto - JPEG-dekomprimerare krävs för att kunna se bilden. NK R2

10 0 10 1 10 2 10 3 10 4 0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 FL1-H FL3-H FL1-H

HIV IFN- CD4 T-cell APC

NK-cell

IFN-γ-

IFN- IFN-

IFN-γ- CTL-

CD8 T-

Figure 1. Interaction between an antigen-presenting cell (APC) and HIV-specific CD8+ T-cell (low), CD4+ T-cell (left) and Natural killer (NK) cell (right), leading to antigen IFN-γ-production and cytolysis. IFN-γ-production is detected by intracellular cytokine staining (ICS) or ELISpot. Antigen-specific cytolysis is assessed by flow cytometry (FC-CTL) or by Cr-release and NK-cell lysis by the NK cell assay.

The nature of the process by which T-cells recognise antigens, only if they are presented by self-MHC molecules (MHC-restriction), has important implications for the design of assays to measure T-cell functions.

Class II-peptide CD4 Class II pathway complex (exogenous) lysosome Cli p CD8 endosome

Ag uptake in Golgi endocytic exocytic Class II Class I-peptide vesicles E.R. vesicle MHC complex Nucle Class us IMH * Class I pathway (endogenous)

newly synthesized proteasome viral protein Adapted from micro 289 lecture 11.ppt

Figure 2. Degradation and transport of antigens that bind to histocompatibility complex (MHC) class I (endogenous pathway) or class II (exogenous pathway) molecules.

FLOW CYTOMETRY

Introduction A flow cytometer is an instrument that identifies cells, or other particles, as they pass in a stream (flow) individually through a laser beam. The illuminated cells display several types of signals which are detected, converted to digital data, and further analysed by a computer. A flow cytometer equipped to separate (sort) the identified cells is called a Fluorescence Activated Cell Sorter (FACS). The technology of flow cytometry relies on fluidics, optics and electronics that enables simultaneous analysis of multiple characteristics of single cells at a very high rate as well as separation of viable and pure cell populations for further characterization.

History As it applies for most inventions, there is controversy about who developed the first “flow cytometer”. Recently, two major books have been published that summarize the historical aspects of the technology (Shapiro, 2003; Keating, 2003). Both books emphasize that flow cytometry is the result of numerous innovations and of multidisciplinary collaboration between engineers, biophysicists, biochemists, histopathologists, molecular cytologists, haematologists, immunologists, and quality controllers. However, the instrument described by Moldavan in 1934 (Moldavan 1934) is generally acknowledged to be an early prototype, although it was never built. Intense work by several groups in the following decades (Gucker 1947; Cornwall 1950; Coulter 1956; Bierne 1957; Kamentsky 1965; Fulwyler 1965; Van Dilla 1969; Hulett 1969) resulted in an instrument that principally was rather similar to the cytometers of today (Shapiro, 2003; Melamed 2001), although with less capacity, e.i. fewer parameters and lower analysis rate. The first commercial cytometer launched in the early 1970s by the Herzenberg laboratory was actually a cell sorter (FACS). The earliest instrument used a single fluorescence and scatter signals to measure size and shape of the cells, and the earliest studies were referred as “one-colour” experiments. These “one-color” experiments captured the interest of immunologists whereby the earliest successful sorting was the isolation of antibody secreting B-cells (Hulett 1969). Soon, it became obvious that only one fluorescence was not enough to obtain additional information of T- and B-cell subsets and gradually, more fluorochromes coupled to antibodies were developed. Fifteen years later (1984), the most modern laboratories were conducting 4-colour experiments, although 10 more years passed before 4-colour instruments were available in most research and clinical

laboratories. Modern commercially available four-laser cytometers provide the ability to detect uo to 18 colours, at analysis rates of >10.000 cells per second.

Principle A schematic picture of a flow cytometer is shown in Figure 3. A cell suspension is injected through a nozzle whereby the cells are focused into the center of a columnar flow by mechanical properties of the fluid (hydrodynamics). The cells in the single-cell stream, passes through a light source usually generated from a laser beam or a mercury arc lamp. Each cell then scatters the laser light, which gives information about the size (forward scatter light, FSC) and granularity of the cell (side scatter light, SSC). If the cell that passes through the laser beam has been labelled with a fluorescent reagent (fluorochrome) it emits fluorescence. Fluorescence may also be emitted by the cells naturally (autofluorescence). Scattered and fluorescent light is collected by the optics, transferred through specific filters to photodetectors (phomultiplier tubes, PMTs) and further digitalised and analysed by a computer. Fluorochromes absorb light of certain specific colour and then emit light of a different colour, usually at longer wavelength. These fluorochromes may be coupled to antibodies, which are directed to molecules expressed on the cell surface or to intracellular markers. Thus, the fluorescence of a cell is proportional to the amount of molecules to which the antibody has bound. The fluorochromes may also be applied directly to the cell whereby they to a membrane or intracellular component of the cell. Cells can be scanned at a high rate (500 to >5000 per second) and a large number of cells can be enumerated and characterized individually upon their specificity, frequency, function, state of activation, differentiation, viability etc, which may give some information of the phenotype and function of different cell subsets.

Applications The ability to simultaneously analyse multiple parameters of the cells at a high rate has enormous advantages. Naturally, the diversity of applications and number of studies based on flow cytometry has increased remarkably during the last decades (Figure 4). Initially, by the 1970s, blood cells (leukocytes), cultured cells or cell lines were the main particles analysed. Later, bacteria (Alberghina 2000), sperm (Fugger 1998) and plankton (Reckerman 2000) have also been studied. In addition, flow cytometry has been used to analyse viruses (Marie 1999),

nuclei (Hedley 1989), chromosomes (Gray 1990), DNA fragments (Yan 2000), and latex beads (Carson 1999). The AIDS epidemic had a clear impact on the diffusion of flow cytometry into the research and clinical area. It has also driven the development of the multicolour technique, culminating in the current 18-colour instrument (Perfetto 2004). Simultaneous measurements of multiple markers is an invaluable tool for both basic and applied immunology research and might lead to clinically relevant findings, specially regarding the quality of immune responses (Betts 2005).

Principle of Flow Cytometry PMT 4

PMT Dichroic 3 SSC Flow cell Filters PMT 2 Bandpass PMT Filters 1

Laser 488

FSC

R1 R3 R2

FL1 SSC

0 200 400 600 800 1000 100 101 102 103 104 FSC-H FL3FL3-H FSC

Figure 3. In the flow cytometry system described here, there are six photodetectors (PMT) which detect forward scatter light (FSC), side scatter light (SSC), and fluorescence light in blue, green, orange and red wavelengths ranges.

Disadvantages A disadvantage of flow cytometry is that particles and cells have to be “single”. Aggregates or adherent cells can be treated mechanically or enzymatically and then analysed as single cells.

However, some important individual cell functions can be altered and the information about tissue structure and cell distribution can be lost. In addition, due to the rather low sensitivity of the technique it is not always possible to detect rare populations, such as antigen-specific cells, which are normally present at very low frequencies. For instance, the detection of a cell subset in whole blood present at the level of 0.01% of CD4+ T cells, requires the identification of 10 positive cells out of about 500,000 other cells including lymphocytes, monocytes and granulocytes.

Figure 4. The increased number of references to “flow cytometry” in the Medline database (adapted from Methods in Molecular Biology: Flow Cytometry Protocols, 2nd ed Hawley T and Hawley R, Humana Press Inc, Totowa, NJ).

ASSESSMENT OF CELLULAR IMMUNE RESPONSES

It is generally agreed that T-cells play a central role in the generation of adaptive immune responses against many viral and also bacterial infections. In the field of HIV, intensive research has been performed during the last years to determine the nature of the T-cells that are believed to mediate efficient and protective immune responses for many years in some patients such as the long-term non-progressors. Recently, it has been proposed that multi- functional T-cells most likely may prove to be the best predictors of immune protection to chronic viral infections (Roederer 2004). There are numerous techniques for quantification of T-cell responses including both conventional and recently emerged assays. Ideally, a T-cell assay should be sensitive, specific, reliable, reproducible, simple, rapid, cheap, require small sample volumes, and, most important, should provide results that correlate well with clinical outcome in terms of protection or susceptibility. Unfortunately, this far in infectious disease immunology, no such assay has been developed, at least not for universal use in any situation.. Here, I will give a short presentation of some of the conventional and novel assays for detection of antigen- specific T-cell activity, and the pros and cons of each assay system. Some of the assays measure only structural markers of memory T-cells, while others measure functional activities.

PHENOTYPIC/STRUCTURAL ASSESSMENT Assessment of surface markers on cells (immunophenotyping) is generally simple and rapid to perform. However, immunophenotyping alone cannot, of course, be employed to detect specific immune responses, but can be a valuable complement to other methods based on tetramer technology or activation assays. Although some surface markers may correlate with cellular functions, it is often not possible to determine the function of a cell subset just by surface staining. Immunophenotypically similar cells, may exert very different functions as seen in the case of CD4+ T-cells, which by their cytokine profiles can be further characterized into Th0, Th1 and Th2 cells with separate and distinctive purposes. Further, surrogate markers may only give a hint of the function that the cells are capable to perform. For instance, CD8+ cytotoxic T-cells that degranulate in response to activation may not necessarily have the potential to kill a target cell. Thus, immunophenotyping should generally be used in combination with other assays.

Markers associated with activation, differentiation and function The expression of a variety of surface molecules on lymphocytes is up-regulated following T- cell activation and can be analysed by flow cytometry. These activation markers include: CD69 (activation antigen), CD25 (IL-2 receptor), CD71 (transferrin receptor), HLA-Dr (a subtype of human MHC class II molecule), and many more. In HIV-infection, there are reports of incomplete or skewed maturation of CD4+ and CD8+ T- cells into dysfunctional effector phenotype. The cells appear to be deficient in the expression of CD45RA (Chen 2001; Mueller 2001). Recently, memory T-cells have been further characterized into T effector-memory (CD27-, CD62L-, CCR7-) and T central- memory cells (CD27+, CD62L+, CCR7+), with different functions and migratory properties (Esser 2003). The effector memory population is present in the peripheral tissue, produce IFN-γ, and has high potential for immediate killing of virus infected cells. In contrast, the central memory T-cells reside within lymphoid organs, produce IL-2 and divide rapidly upon stimulation. Assessment of the surface molecules CD107a/b on CD8+ T-cells have recently been proposed to identify CTLs capable of exerting cytolytic function The CD107a/b are lysosomal membrane proteins, which are transported to the cell surface following degranulation of the CTLs in response to antigen stimulation (Betts 2003). Measurement of intracellular perforin or granzyme content in HIV-specific CD8+ T-cells is used as a surrogate marker for potential cytotoxicity activity (Maher 2002). Pros and Cons: The assessment is very easy to perform, since a simple surface staining using a specific antibody is enough to identify the specific cells. A major limitation for the use of some of the markers is that they may be non-specifically upregulated to a certain level, can be expressed by subpopulations of resting cells (e.g. CD25) or display high base-line levels, as for perforin content.

Tetramer staining Tetramer staining is based on the visualization of antigen-specific T-cells by the use of fluorescently labelled, multimeric peptide-MHC complex that binds to their specific T-cell receptor. Tetramer staining has been used to enumerate, characterize, and purify peptide- specific CD8+ T-cells (Altman 1996; Murali-Krishna 1998). Pros and Cons: The primary advantage of tetramer staining is that it does not require in vitro antigen stimulation and culture. The disadvantages include the lack of functional correlate,

unless simultaneous additional immunophenotyping, staining for cytokines or cytotoxic effector molecules is carried out (Appay et al., 2002). The need of previous knowledge of the subjects HLA haplotype (and that corresponding tetramers must be available), as well as the need for information on what peptides can be employed are other potential drawbacks. Moreover, all antigen-specific cells will be identified including non-responsive (anergic) T- cells (Lee 1999).

FUNCTIONAL ASSESSMENT A functional assay is based on the capacity of the analysed cells to react in response to an activator (mitogen or antigen). The interaction of the antigen with a proper receptor on the targeted cells starts a cascade of events eventually leading to a response detectable in a sensitive system. Thus, a functional assay can be developed at measuring any parameter/event participating in the events downstream the activation site. A variety of functional assays for detection of different variables of cell-mediated immune responses have been developed over the years based on the detection of cytolytic ability, lymphoproliferative ability, or cytokine- production.

T-cell proliferation The lymphoproliferative assay (LPA), has traditionally been employed as a measure of CD4+ T-cell function assessed as in-vitro lymphoproliferative capacity of CD4+ T-cells. This assay has been the standard method for measurement of antigen-specific memory CD4+ T-cell responses for many years and it is still widely used (Valentine 1990; Rosenberg 1997). LPA results are influenced by the following parameters: 1) antigen processing and presentation; 2) the ability of T-cells to proliferate in culture, and, 3) the initial frequency of antigen-specific (memory) T-cells (Gehrz 1979; Valentine 1998).

Conventional method: 3H-thymidine assay T-cell proliferation has traditionally been measured by incorporation of a radioactive tracer (3H-thymidine), incorporated in newly synthesized DNA of dividing cells. Pros and Cons: The assay provides information of the total amount of DNA-synthesis in bulk culture but provides no information on the frequency of antigen-specific T-cells, nor of the phenotype of proliferating cells.

Alternative methods: Several flow cytometry-based methods have been described to measure proliferative T-cell responses including the detection of intracellular markers of cell division such as Ki67 (Sikora 1998), incorporation of the thymidin-analogue bromodeoxyuridine (BrdU) (Mehta 1997) and staining with fluorescent dyes, such as CFSE and PKH26 (Fulcher 1999; Lyons 1999). CFSE or PKH26 is divided between daughter cells upon cell division whereby the fluorescence intensity of the cells reveals the number of divisions that have occurred. Another LPA, the Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA), developed by us, employs diluted whole blood samples, cultivated for 7 days in the presence or absence of stimulators and results are assessed as the number of antigen- specific lymphoblasts generated (Gaines 1996; Svahn 2003). Pros and Cons: The assessment of proliferative responses based on flow cytometry and whole blood samples is very advantageous over the 3H-thymidine assay since the assay is easy and simple to perform, displays high sensitivity and specificity, is suitable for large-scale studies; and it allows simultaneous phenotyping of the responsive T-cells. It can also be combined with characterisation of the cytokine profile of responding cells using intracellular staining or examination of culture supernatants.

Cytolytic activity Just as proliferation has been employed as a measure of CD4+ T-cell function, the cytolytic activity has been a traditional measurement of CD8+ cytotoxic T-cell function, as well as NK cell function. Cytolytic activity of human NK cells is routinely measured using an NK cell sensitive human erythromyelocytic leukemia cell line, K562, and PBMC, as effector cells. Daudi, a human Burkitt’ s lymphoma cell line, which is resistant to lysis by NK cells, is used for assessment of cytotoxicity mediated by IL-2 activated NK cells (LAK). For assessment of MHC class I-restricted antigen-specific lysis, infected or antigen-loaded autologous B-cell lines (BCL) or similar are used as target cells, and fresh or expanded antigen-specific CTLs, as effector cells. The cytolytic process consists of a number of steps, including: 1) recognition and binding of the cytotoxic cell to the target cells; 2) delivery of the lethal hit to the target cells; and 3) target cell lysis and recycling of the effector cell (Figure 5).

CTL-Target CTL-cytoplasmic CTL-Target conjugate Target cell Granule rearrangement conjugates

R3 CTL Target cell R1 CTL- CTL granule Exocytosis R2

Membran damage 100 101 102 103 104 FL1-H Cell debris Flow cytometric assessment 15min-4h of cytotoxicity

Figure 5. CTL-mediated killing. The T-cell receptor on a CTL interacts with antigen-MHC-I-complex on a target cell leading to formation of a CTL-target-cell conjugate complex. The granule in the CTL reorient toward the target cells and the granule contents (perforin, granzymes, lysin) are released by exocytosis. Following degranulation a cascade of events occurs inside the target cell which finally leads to cell death (lysis).

Conventional method: 51Cr-release assay The standard method employed for assessment of MHC class I-restricted HIV-antigen- specific lysis has been the chromium (51Cr) release assay, (Brunner 1968). The assay involves 51 radiolabelling of target cells with sodium chromate (Na2 CrO4), followed by incubation with effector cells. Once the target cells are lysed, the 51Cr is released into the supernatant and can be quantified to provide a measure of target-cell lysis. Pros and Cons: This assay has several disadvantages including the use of radioactive reagents, poor labelling, and high spontaneous release by some cell lines, low sensitivity and difficulty to accurately quantitate the cells involved in the cytolytic process or to distinguish them phenotypically. Furthermore, the method is extremely complex and labour-intensive.

Alternative methods A couple of lytic and non-lytic flow cytometry based assays have been developed using different approaches for discrimination of the cell populations and for direct or indirect assessment of lytic activity. These assays are discussed in greater detail under “Results and discussion”. Pros and Cons: Multi-parameter flow cytometry based cytotoxicity assays provide several advantages, such as no need for radioactive reagents, detection of cytotoxicity at different stages of the killing process and at the single-cell level, and the possibility to further characterise the cells by immunophenotyping.

Cytokine production Cytokines play an important role in determining the strength and nature of antigen-specific immune responses. They can also influence the expression of ligands and receptor molecules on cell-surfaces.Numerous techniques have been developed for measuring cytokine responses, such as ELISA (Ledur 1995), ELISpot (Klinman 1994), LDA (Sharrock 1990), PCR (Reiner 1993), ISH (Hoefakker 1995) and ICS (Picker 1995), and there are advantages and drawbacks attached to each assay system. While the ELISA measures total amount of secreted cytokine and the PCR measures levels of mRNA of a specific gen, the ELISpot and ICS identify cytokine-producing cells at the population and single-cell level and the ISH can localize the cytokine producing cells in tissues.

Conventional method: ELISA Until recently, cytokine secretion by T-cells was usually analysed by enzyme-linked immunoassay technique (ELISA). Pros and Cons: The assay provides no information on the number or phenotype of cells that secrete the measured cytokine but samples can be frozen until analysis making it a convenient method for large-scale testing.

Alternative methods Intracellular cytokine staining Intracellular cytokine staining (ICS) is a relatively new flow cytometry-based method that requires culture in the presence of a protein transport inhibitor, to retain and accumulate the produced cytokines inside the cells; fixation, to maintain structure of the cells; and permeabilization, to allow the entry of cytokine-antibodies into the cells. This method allows determination of several cytokines in combination with enumeration and identification of the responding lymphocyte population, CD4+ or CD8+. The ICS has become a valuable tool for monitoring of cellular immune responses in vaccine trials, supplementing or even replacing the earlier more commonly used ELISpot assay. Pros and Cons: The ICS is the method of choice for the detection of simultaneous production of different cytokines by the same cell. However, most cytokines are not intracellularly present at high enough concentrations to be detected by ICS and, furthermore, frequencies below 1/1000 cells are difficult to accurately detect.

ELISpot The enzyme-linked immunospot, ELISpot, detects cells that secrete cytokines (e.g., IFN-γ) following antigen stimulation (Herr W, 1997). PBMCs are cultured with antigen in plates, which have been coated with anti-cytokine antibodies. The secreted cytokine is captured by the antibodies and subsequently revealed with a second enzyme-conjugated antibody. The secreted cytokine molecules form spots, each one corresponding to a cytokine-secreting cell. The spots assay permits the enumeration of cytokine-secreting cells and results are expressed as spot-forming cells (SFC/106 PBMC). Pros and Cons: This assay is a robust and highly sensitive method that has been used as a major screening assay for evaluation of cellular immune response in vaccine trials. However, the assay is not able to define responder cells phenotypically (unless CD4+ or CD8+ T-cell

depletion is applied), thus the measured IFN-γ response represents the total responses of CD4, CD8 and NK cells. ELISpot-analysis is essentially equivalent to a one-color assay (cytokine secretion only).

MULTIPLE ASSAY SYSTEMS Recently, several systems have been developed for simultaneous quantitation of multiple soluble molecules using microsphere-based assays flow cytometric detection. The. Cytometric Bead Array (CBA) system, based on flow cytometry, uses beads of different sizes or colors as carriers for antigens or antibodies. The corresponding analyte is captured and detected through binding of a fluorescent antibody or antigen. The beads are identified by one channel and the analyte by a second fluorescent signal. The Lumixe xMAP technology, uses distinct color-coded polystyrene beads conjugated with capture antibodies and a biotinylated detection antibody. Pros and Cons: A uniform feature of multiplex immunoassays is that they can be employed for the simultaneous measurement of concentrations of many cytokines using smaller volume of samples than traditional approaches.

HUMAN IMMUNODEFICIENCY VIRUS INFECTION

THE HIV/AIDS PANDEMIC The HIV epidemic emerged in the last quarter of the 20th century, and now two decades later has affected more than 190 countries, infected a cumulative total of more than 65 millions and killed over 20 millions (UN 2004). Unfortunately, the pandemic is still expanding and during the last year 5 million people became infected, which is more than in any single year since the crisis began. Worldwide, the number of adults and children living with HIV was estimated to almost 40 million people at the end of 2004 (UN). Although the proportion of HIV-infected men and women is almost equal today, the number of women and girls infected with HIV increases rapidly in most regions, which in turns means increased number of mother-to-child transmission. Sub-Saharan Africa remains the worst affected region, with 25 million HIV- infected people; although the steepest increase at the moment occurs in East Asia (UN 2004). Long-term programmes such as development of vaccine and other preventive strategies are of great urgency to control the pandemic in the long run. Meanwhile, a wide scale introduction of antiretroviral therapy would mean improved quality of life for the HIV-infected subjects and also a reduction of AIDS incidence and number of deaths due to HIV-infection, as that seen in Western countries following introduction of antiretroviral drugs (Carpenter 1998).

IMMUNODEFICIENCY VIRUSES The human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) and the simian immunodeficiency viruse (SIV) belongs to the lentivirus genus of the Retroviridae family. Lenti (lat. Lentus = slow) refers to the gradual course of the disease caused by the virus, and retrovirus to the fact that the viral genetic material RNA has to be reverse-transcribed into DNA to be inserted into the host cell’s genome. HIV-1 is the predominant virus worldwide, while HIV-2 is found mainly in West Africa but occurs also in Europe, India and North America. The structure of virus and genomic organization of HIV-1 is shown in Figure 6. HIV infection begins with the interaction of the glycoprotein, gp120 with the CD4-molecule and a co-receptor on the target cell. Once in the cell cytoplasm the viral RNA is copied by viral reverse transcriptase (RT) into DNA that enters the nucleus and integrates into host cell DNA (provirus). At this point, the integrated provirus can be transcribed, leading to the production of new HIV-1 virions, or may become dormant for a long period of time. CD4 is

expressed on T cells, monocytes, macrophages, dendritic cells, some rectal lining cells and microglia in the brain. The β-chemokine receptors, CXCR4 and CCR5, have been identified as two major co- receptors for the entry/fusion process of HIV. CXCR4, the receptor for stromal cell derived factor (SDF-1), is expressed on T-lymphocytes and mediates entry of T-cell tropic HIV- strains (X4 virus) (Feng 1996; Oberlin 1996), while CCR5, the receptor for RANTES, MIP- 1α and MIP-1β is expressed on monocytes and lymphocytes, and mediates entry of M-trophic HIV-strains (R5 virus) (Alkhatib 1996). It has been shown that during the early stage of infection and during the asymptomatic phase the M-trophic R5-virus predominate (Connor 1997), whereas the T-cell tropic X4 virus emerges during progression to advanced disease stages (Connor 1993; Roos 1995), and is associated with a rapid progression to AIDS (Schuitemaker 1992).

Reverse gp120 (env) transcriptase (pol) gp 41 (env)

Protease Lipid bilayer

RNA matrix

Integrase p24 (gag)

Figure 6a. The mature HIV-1 virion is surrounded by a viral envelope consisting of a lipid bilayer membrane with spikes, which are build up of transmembrane (gp41) and surface (gp120) glycoproteins. Inside the viral envelope there is a viral core, composed of the capside protein (p24), encapsulating two strains of viral RNA, proteinase, reverse transcriptase, and integrase. The accessory proteins vif, vpr and nef, are also found inside the capside.

HIV-1 TAT VIF GAG REV NEF POL

U U 3 ENV 3 VPR VP R U5 R U5

Figure 6b. The genome of HIV-1 consists of three major structural genes (env, gag, and pol) encoding for structural proteins and six minor genes (tat, rev, nef, vif, vpr and vpu), encoding for regulatory and accessory proteins.

THE COURSE OF HIV-1 INFECTION A typical natural course of HIV infection with different “phases” is generally accepted, although individual development patterns occur. Three distinct phases are distinguished throughout the HIV infection: primary HIV-infection (PHI), clinical latency, and clinically apparent disease (Figure 7). The PHI-phase may be associated with an influenza-like illness, which may occur 2 to 4 weeks following infection (Gaines 1988). This phase is characterized by a significant decline in the numbers of circulating CD4+ T-cells (lymphopenia) and high levels of virus in peripheral blood (viremia) (Gaines 1988; Albert 1987). Following the appearance of cytotoxic T-lymphocytes (CTLs), the plasma viral load subsequently falls to a level of steady-state (setpoint), which is believed to predict the rate of disease progression to AIDS (Fauci 1996; Mellors 1997). After PHI, there is a period of clinical latency that varies greatly between individuals. However, HIV viral replication is continuing constantly during this period and the immune system is slowly deteriorating. The rate of progression to disease varies from person to person and depends on a number of both viral and host factors including virulence, host genetics, host immune response, and cytokine milieu. Based on the duration of the asymptomatic period and kinetics of virologic and immunologic events, three distinct rates of development towards HIV-disease are observed. “Typical progressors” consist of 80%- 90% of the HIV-infected persons whose immune system show gradual deterioration i.e decreasing CD4 T-cell counts and increasing viral load, for a median period of 10 years till development of disease (Haynes 1996). 5%-10% of people infected with HIV is referred as “rapid progressors” since their CD4+ T-cells decline rapidly and they progress to AIDS within

3-4 years (Sheppard 1993). The third group defined as “long-term non-progressors” (LTNP), consists of about 5% of the HIV-infected individuals. They remain asymptomatic and have normal CD4+ T-cell counts and low or undetectable viral load for many (Buchbinder 1999). The advanced stage of HIV infection is characterized by severe impairment of immune function and the appearance of AIDS-related symptoms.

The introduction of highly active retroviral treatment (HAART), has led to dramatic changes in the course of the infection with prolonged asymptomatic phase attributable to decreased viral load and increased CD4+ T-cell counts. Thus, the number of deaths due to AIDS has been dramatically reduced in areas where anti-HIV treatment is available (Carpenter 1998). However, despite the initial success following the initiation of therapy, there are adverse effects and toxicities frequently occurring as a result of the treatment. In addition, poor treatment adherence has been shown to lead to development of resistance (Walsh 2000).

Primary infection Latent infection Progression to AIDS

CTL

Ab CD4 T

Viral

0 weeks 10 1 2 3 4 5 6 7

Figure 7. Kinetics of viremia, CD4+ T-cell counts, CTL and neutralizing antibodies in typical HIV-1 infection.

THE IMMUNE RESPONSES Innate immune response The role of the innate immune responses in the control of HIV infection has been far less studied compared to the adaptive responses. Nevertheless, there is evidence that it plays a critical role during the early phase of infection (Lehner 2003). Beside epithelia and the complement system, the innate immune system consists of a number of cells such as neutrophils, macorophages, natural killer cells and dendritic cells. These cells may act by phagocytosis, killing or secretion of chemokines and cytokines, to block HIV transmission and replication in the mucosal tisues. The β-chemokines (RANTES, MIP-1α, MIP-1β) may also attract immune cells to the infection site. Natural killer cells play an important role in the innate defence against viral infections (Kägi 1996; Biron 1999) and may act against HIV by lysing HIV-infected cells and/or by the secretion of chemokines and cytokines (Wallace 1996). However, the real importance of NK cellsin HIV infection has not been completely established (Dines 2004). Normal or decreased NK cell activity has been found in HIV-infected subjects, although NK-cell numbers are not significantly affected until the latest stages of HIV-infection (Levy 2001; Brenner 1993).

Humoral immune response A strong antibody response to HIV is developed from 2-4 weeks after infection against several viral antigens (Lindbäck 2000), with predominance to the envelope glycoproteins (gp120 and gp40) and core/matrix proteins (gag p24 and p17). These HIV specific binding antibodies can be detected by various immunoassays for diagnosis of HIV-infection. Serum antibodies against HIV envelope exhibiting virus neutralisation can usually be detected 2-4 weeks after PHI (Albert 1990), and they mainly target the HIV envelope. However, their neutralizing efficacy during the early stage of HIV infection has been questioned since the control of the viral replication occurs before their appearance (Wyatt and Sodroski 1998). In addition, due to the emergence of new HIV variants, neutralizing antibodies rapidly become ineffective in most infected individuals (Moore 1995; Montefiori 1997). Several studies have shown broader and higher titers of neutralizing antibodies in LTNPs as compared to subjects with progressing disease (Burton and Montefiori 1997). Thus, neutralizing antibodies may be important in controlling viral replication over time. Further, experimental passive transfer of neutralizing antibodies has been shown to protect against systemic and mucosal challenge

with HIV-2, SIV and SHIV in primate models (Putkonen 1991; Haigwood 1996; Mascola 2000). Non-neutralizing antibodies that mediate antibody dependent cellular cytotoxicity (ADCC) (Connick 1996; D’Souza 1996) or activate complement (Frank 1991) may also be important during the early phase of HIV-infection.

Cellular immune response Both CD4+ T-cells (T-helpers) and CD8+ T-cells (CTLs) are thought to play crucial roles for the control of HIV-1 replication and slowing disease progression (Bollinger 1996; Matloubian 1994; Kalams 1998). T-helpers are a fundamental part of our immune system and they mainly provide help for CD8+ and B cells (Hogan 2001). CTLs have multiple effector functions that finally results in lysis of virus-infected cells (Walker 1987; Borrow 1994; Koup 1994; Rowland-Jones 1997). Lately, it has been shown that the CD4+ T cells are important for the maintenance or maturation of CD8+ T cell function and the maintenance of CD8+ T cell memory (Altfeld 2000).

HIV-specific T-helper dysfunction has been observed early in the infection when CD4+ T-cell counts are still within the normal range, thereby affecting both cellular and humoral immunity (Shearer 1991; Miedema 1994; Musey 1999, Altfeld 2000; Wilson 2000). CD8+ T-cell dysfunction involves impaired cytolytic activity (Appay 2000; Shankar 2000) and reduction in cytokine production (Goepfert 2000; Vogel 2001).

The impairment of HIV-specific lymphocyte proliferation is a relatively early functional defect of cell-mediated immunity. HIV-specific proliferative capacity is weak or undetectable in the majority of chronically HIV-1 infected untreated individuals but is more frequently detected in LTNPs and in patients treated early during primary HIV-1-infection (PHI) than in subjects with progressive disease (Wahren 1987; Rosenberg 1997; Malhotra 2000; Oxenius 2000; Rosenberg 2000; Wilson 2000; Altfeld 2001; Imami 2002). HIV-specific proliferative response in LTNPs has been associated with low viral load (Rosenberg 1997) and strong CTL responses (Kalams 1999). The presence of HIV-1-specific CD4+ T-cells in HIV-infected subjects at different disease stages has been reported when using intracellular cytokine staining (ICS) (Pitcher 1999; Wilson 2000, Betts 2001; Ostrowski 2001; Boaz 2002; Harari

2002), although with conflicting data in relation to viral load and/or disease progression or lymphoproliferation.

Evidence of CD8+ T-cell mediated control of HIV-1 replication include the appearance of virus-specific CD8+ coincident with the fall in viremia during primary HIV-infection (Koup 1994; Borrow 1994), the selection of CTL escape viral variants (Phillips 1991; McMichael 1998) and in experimental macaque studies the rapid increase of SIV load after experimental depletion of CD8+ T-cells during acute or chronic SIV-infection (Jin 1999; Schmitz 1999). HIV-1-specific CD8+ T-cells are in general readily detectable but there are conflicting data regarding the association between viral load/disease progression and HIV-1-specific CD8+ T- cell responses. CD8+ T-cells can also suppress HIV-replication by non-cytolytic mechanisms (Walker 1986; Levy 1993), involving inhibition of virus entry by β-chemokines, such as RANTES, MIP-1α and MIP-1β (Cocchi 1995), and suppression of HIV transcription by CD8+ non-cytotoxic T- cell antiviral factor (CAF) (Walker 1986; Klinter 1996; Le Borgne 2000). Cytokines play an important role in determining the strength and nature of the antigen-driven immune responses and can affect the expression of HIV (Romagnani 1997).

AIMS OF THE THESIS

The general aims of this study was to develope, improve, establish and introduce flow cytometric methods for assessment of cell-mediated immune responses induced by vaccination or following natural infection, in particular for HIV.

Specific aims

To develop methods for assessment of natural killer cell mediated and cytotoxic T-cell mediated cytolytic activity (paper I and II).

To establish intracellular cytokine staining based on long-term culture of diluted whole samples (paper III and IV).

To employ both the new and conventional assays for study of treatment-naive HIV-infected individuals’ HIV-specific cellular immune responses in relation to viral load (paper IV).

RESULTS AND DISCUSSION

I DEVELOPMENT OF CYTOTOXICITY ASSAY FOR ASSESSMENT OF NATURAL KILLER CELLS AND HIV-SPECIFIC CYTOTOXIC T-LYMPHOCYTE ACTIVITY (PAPER I AND II AND IV)

The effector mechanism responsible for killing virally infected and tumour transformed cells by the perforin/granzyme pathway, is cell-mediated cytotoxicity executed by either MHC class I restricted antigen-specific T-cells (CTLs) or by MHC non-restricted NK-cells. For many years, the standard method for assessment of cytolytic function has been the chromium (51Cr) release assay (Brunner 1968), although the assay suffers from many disadvantages, which have been described under “functional assays”. Flow cytometry-based cytotoxicity assays offer several advantages over the 51Cr-release tests, but the main power of the technique is the single-cell analysis that permits essential discrimination between different cell populations and offers detailed information about the phenotype and functionality of the cells.

The purpose of this part of the study was to establish a flow cytometry-based NK cell cytotoxicity assay and use it as a model for further development of a HIV-specific cytotoxicity assay. There are some important technical and biological factors and restrictions to consider for the design of a successful FC-based cell-mediated cytotoxicity assay: 1) the effector cells must be clearly distinguished from the target cells; 2) the killing will vary depending on the target cell, the effector cell and its state of activation, and the culture conditions; 3) the dead cells that disintegrate will not be detected; and 4) the kinetics of killing differ between individuals and killing system. Quantitation of cytolysis generally involves the assessment of release or retention of a fluorochrome in pre-labelled target cells, or uptake of a vital dye by which the target cell death is measured at the end of the cytolytic reaction in cells that have lost membrane integrity. Previous to our work, several approaches for discrimination of target and effector cells and detection of cell death have been suggested: 1) the use of differences in light scatter properties (Vitale 1989); 2) the use of one or two fluorochromes loaded into the cell cytoplasm, either alone or combined with scatter characteristics for the cells (McGinnes 1986, Lebow 1986, Cavarec 1990); 3) labelling of target and/or effector cells with cell-membrane binding

fluorochromes, and the detection of cell death by incorporation of vital dyes into the DNA of the compromised target cells (Slezak 1989, Radosevic 1990, Chang 1993, Hatam 1994, Papadopoulos 1994). However, some of these methods are associated with problems such as morphological and spectral overlap, poor dye stability, and high spontaneous release of the dyes, especially during lengthy incubations.

Evaluation of fluorochromes for discrimination purposes We decided to evaluated three membrane-binding fluorochromes with different spectral properties, DiO-275 (3,3’-dioactadecyloxacarbocyanine perchlorate (DiOC18)) (green fluorescence), PKH2 (green fluorescence) and PKH26 (red fluorescence), in conjunction with two DNA-binding dyes for detection of cell death, PI (propidium iodide) and 7-AAD (7- amino-actinomycin). In our hands the membrane dyes were difficult to solve resulting in great variations of the final dye concentration, and the staining procedure was difficult to standardise. In addition, some of the dyes are solved in rather toxic compounds, such as DMSO, which may increase the spontaneous cell death, alter the cell-to-cell contact or affect the function of the effector cells, especially in CTL-systems, where the target cells are already affected by the infectious agent to be studied. Spectral overlap between PKH26 and PI or 7- AAD was a main problem, requiring adjustment of the instrument at each experiment occasion. At that time we worked on a FACScan, a 3-color flow cytometer, and spectral overlap would limit the possibility to further analyse the target and/or effector cells. In view of these problems, we decided to try another approach for the separation of target from effector cells, namely the use of monoclonal antibodies.

Evaluation of monoclonal antibodies for discrimination purposes Several Mabs were screened for specific and non-specific binding to the target cells (K562 or Daudi cell line) and effector cells (PBL). Both the isotype of the antibody and the attached fluorochrome seemed to influence the non-specific binding and autofluorescence displayed by the stained cultures, especially the target cells. The anti-CD56 antibody, which initially seemed to be an obvious choice for an NK cell assay, did bind non-specifically to the K562 cell line. Finally, the anti-CD45 Mabs was chosen as the discriminatory tool since it displayed the best separation between the cell populations. We further decided to stain the cells with CD45 after incubation of samples (post-assay immunophenotyping), given that pre-assay staining could alter or hinder the cell-to-cell binding or affect the function of the cells. In

addition, binding of Mab to the target cells could result in an ADCC-reaction. Since the post- assay immunophenotyping staining procedure is very simple, the cells are little altered resulting in low spontaneous cell death. Furthermore, using Mabs with standard conjugation, adjustment of fluorescence signals or compensation of the instrument was not needed, since no spectral overlap between the fluorochromes used was observed. A possible limitation of this approach is that monoclonal antibodies for discrimination of the cell populations must be evaluated when establishing a new cytotoxic system. In our system CD45 worked perfectly when used in the cytotoxicity assay consisting of K562 and PBMCs or LAK cells. However, when the Daudi cell line was used as target cells the best separation between the cell populations was obtained with the CD2 Mabs.

The NK cell assay (paper I) The developed NK cell assay is based on discrimination of target and effector cells by post- assay immunostaining using either anti-CD45 (K562 killing) or anti-CD2 (Daudi killing) and detection of cell death by incorporation of PI (Figure 8). We initially conducted kinetics experiment to determine an appropriate incubation time. Although the levels of NK cell killing obtained by the FC-assay after 2 h of culture were similar or higher than those obtained by the Cr-release assay at 4hr, we decided to use 4 hr incubation for both assays given that this is the conventional culture time for Cr-release assays. A comparison of the assays using K562 cultured with effector cells at different ratios (50:1, 25:1, 12:1 and 6:1), demonstrated that the assays were significantly correlated (r=0.82; p>0.001), but the levels of cytotoxicity detected by the FC-assay were significantly higher (p>0.001). We also evaluated the cytolysis mediated by IL-2 activated NK cells (LAK, Lymphokine activated Killer cells) using K562 and Daudi cells as target cells. The Daudi cell line is resistant to NK cell-mediated killing but sensitive to LAK-mediated lysis. The LAK- mediated cytolysis of K562 target cells was almost 100% at a ratio of 50:1, while the NK cell- mediated killing was approximately 50%. By contrast, there was no killing of Daudi cells mediated by the NK cells whereas the LAK cells killed about 30% of the Daudi cells, at ratio of 12:1. This demonstrates that the FC-assay may be adapted to other cytotoxicity systems. Lastly, we performed a 4-color staining with Mabs directed to different lymphocyte subtypes in order to demonstrate the possibility of studying the nature of the conjugate-forming effector cells (Figure 9).

Target cells ( K562 ) Propidium iodide uptake

R1 3% R3 M1 M2

10 0 10 1 10 2 10 3 10 4 FL3-H

10 0 10 1 10 2 10 3 10 4 FL1-H Effector cells ( PBL )

R1 R3 M1 M2

10 0 10 1 10 2 10 3 10 4 FL3-H

25% 10 0 10 1 10 2 10 3 10 4 FL1-H M1 M2 Target and effector cells

10 0 10 1 10 2 10 3 10 4 FL3-H

5% M1 M2 R3 R1

10 0 10 1 10 2 10 3 10 4 FL3-H

1% M1 M2 R2

10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FL1-H FL3-H

Figure 8. NK cell-mediated cytotoxicity using anti-CD45 (FL 1) and scatter profile (SSC) to identify different cell populations and PI for detection of cell death. Target cells are clustered by region one (R1), effector cells by region 2 (R2) and conjugates by region 3 (R3). Percentages of cell death in different population are calculated from histograms displaying PI- positive cells.

<1% of all conjugates are CD19+ 21% of all conjugates are CD3-CD16/56+ 79% of all conjugates are CD3+

<1% of CD19+ cells are conjugated 11% of CD3-CD16/56+ cells are conjugated 7% of CD3+ cells are conjugated

Figure 9. Multi-parameter analysis of conjugate formation between PBLs and K562. PBLs were stained with a cocktail of Mabs and incubated for 2h with target cells at a ratio of 25:1. The Mabs were directed to leukocytes (CD45), T-cells (CD3), NK cells (CD16/56) and B-cells (CD19). CD45+ effector cells and conjugates were selected from R2 and R3 (figure 8), respectively, on a dotplot of SSC vs. CD45 and further displayed on dotplots of SSC vs. CD19, CD16/56 or CD3. The respective conjugates are found within R3.

The developed FC-NK cell assay has several advantages over the earlier methods: 1) labelling of cells prior to the assay is not required and the possible influence on cellular function or cell-to-cell contact is avoided; 2) the method can be used for the assessment of cell death of target and effector cells but also the earlier steps of the cytotoxic process, namely the conjugate-formation between target and effector cells and for further examination at the single-cell level by simultaneously carried out additional immunophenotyping; and 3) no washing steps are required after immunophenotyping avoiding unnecessary disturbance of the sensitive cells. Just previous to our paper two groups reported the assessment of NK cell cytotoxicity using monoclonal antibodies (Zamai 1998; Goldberg 1999). Zamai and co-workers described an NK cell assay in which the cell populations can be identified on the basis of pre-culture, indirect staining of effector cells using a CD56-antibody, combined with target cells autofluorescence, and the detection of cell death based on changes in scatter characteristics of the target cells. Although this method is useful in the selected system, there are major drawbacks such as: staining of effector cell-surfaces may affect cell-function; conjugates cannot be detected; indirect staining requires washes, complex preparation and is difficult to combine with additional staining; autofluorescence is not a reliable marker for identification; and the model cannot be applied for systems including other target or/and effector cells with other profiles of light scatter and/or autofluorescence. Goldberg and co-workers, described the use of two-colour flow cytometry for annexin-V detection of dead or dying target cells in combination with staining of effector cell-lines with anti-CD8 and anti-CD56, respectively, for the measurement of CTL and NK cell-mediated cytotoxicity. Annexing V binds to phosphatidylserine expressed on the cell surface early during apoptosis. In initial studies we found certain correlation between annexin V staining and propidium iodide uptake. However, the staining procedure includes several washing steps after assay incubation, which seems to affect the sensitive, compromised target cells. In addition, the binding buffer used for annexin V staining altered the light scatter characteristics of the target cells.

Limitations of the NK cell assay A general limitation of flow cytometric measurement of cytotoxicity, including our assay, is that lysed cells i.e. already disintegrated cells, are not detected. By the time we developed the NK cell assay we thought that the problem of lysed cells would mainly affect the long-term assays, such as toxicity assay, which are run for several days. Nevertheless, in later

experiments we studied the impact of the cell loss on the calculation of cytolysis. NK cell cytotoxicity was performed using CD45 and PI, as previously described. The main difference in this set of experiments relies on the mode of sample acquisition and calculation of the cell death. For acquisition, the flow rate of the calibrated instrument (FACSCalibur) was set on “high”, which corresponds to a collection rate of 1 µl /sec, and the collection criterion was set on time (32 sec.) instead of events, to collect a tenth of the total sample volume (320 µl). Thus, the total number of a cell subset in a culture was obtained by multiplying the number of acquired cells with a factor of 10. The target cell death was determined either by calculation of the percentage PI-positive target cells or by the disappearance of target cells (Figure 10). Detection of cell death by PI-uptake may correlate well with the assessment of cell loss if the numbers of lysed cells are low. However, if a great cell loss has occurred when the determination of PI-uptake is done, the two approaches of cell death calculation may differ significantly. We concluded that the number of lysed target cells in NK cell cytotoxicity assays should be considered for a proper determination of cell death. This approach was further applied for the development of the HIV-antigen specific cytotoxicity assay presented below and for the development of a whole blood N NK cell cytotoxicity assay currently used in large scale studies (not shown).

Targets only Targets only Targets only R1 R2 n=1564 n=4 4% PI+ 0% PI+

M1 M1

10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FL3-H FL4-H FL3-H

Effectors only Effectors only Effectors only n=80 R1 R2 n=91 2% PI+ 1% PI+

M1 M1

0 1 2 3 4 10 10 10 10 10 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FL3-H FL4-H FL3-H

Effectors and targets Effectors and targets Effectors and targets n=926 R1 R2 n=336 31% PI+ 8% PI+

M1 M1

0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 FL3-H FL4-H FL3-H

Figure 10. Assessment of NK cell cytotoxicity using post-assay staining with anti-CD45 (FL4) and PI (FL3) following 4h incubation of targets only (top lane), effectors only (middle lane), and effectors and targets at a ratio of 25:1 (bottom lane). The same numbers of targets were added to target culture and mixed culture, and the same numbers of effectors were added to effector culture and mixed culture. Single targets are included in region 1 (R1) and conjugates between effectors and targets are included in region 2 (R2), and the total numbers of cells in R1 and R2 are displayed in left and right histograms, respectively, where the percentages of dead (PI+) cells are also given. In the mixed culture, a total of 1262 (926+336) cells were detected, although 1739 (1564+4+80+91) cells were expected from numbers identified in the target and the effector control cultures. Thus, 477 (1739-1262) cells are absent.

Development of HIV-specific CTL (FC-CTL) (paper II) Assessment of CD8+ T-cell activity is of significant importance for evaluation of cellular immune responses in HIV infection and a variety of recently developed techniques have been used to determine the frequencies and function of virus-specific CD8+ T-cells. However, none of these methods do directly assess the cytolytic function.

We wanted to employ the NK cell assay as a model for development of an HIV-specific cytotoxicity assay considering the cell loss. Although the NKs and CTLs appear to use similar cytolytic mechanisms in the elimination of their respective target cells (Henkart 1985), the assessment of antigen-specific cytotoxicity is more complex and difficult to perform than NK cell-mediated cytolysis due to important differences between the two assay systems, such as, the target cells (a well established cell line, K562, vs. autologous EBV-transformed B-cell lines), the effector cells (PBMCs vs. antigen-specific expanded PBMCs); and the effector mechanisms (MHC class I restricted vs. non-restricted killing). The generation and handling of effector and target cells for a CTL-assay is more time-consuming and complex, and gives rise to more pitfalls. Thus, we encountered several problems due to the basic differences between the NK cell and CTL killing systems. Firstly, the cell cultures involved in CTL-assays are in general very heterogeneous and variable, with a relative high degree of dead cells in the target cell cultures and the expanded CTLs. Secondly, the spontaneous cell death of target and effector cells, and the non-specific killing, give rise to high background signals, which may interfere with detection of specific lysis. Thirdly, discrimination between target and effector cells by post- assay CD45-staining, as for the NK cell assay, was not possible since the CD45 marker is expressed on both cell populations. Fourthly, the discrimination between target and effector cells by post-assay staining with Mabs directed to the target cells (anti-CD19 or anti-CD20), was not suitable since B-cells are also present in the effector cell population. Moreover, the surface markers on B-cells (CD19 and CD20) may be altered on the differentiated B-cell lines. Lastly, the use of HIV-positive samples, makes the system even more complex since these samples are known to be more prone to apoptosis. However, in contrast to the 51Cr- release assay, flow cytometry single-cell analysis permits detailed studies of different cell populations providing the possibility to control different parameters influencing the assays.

Previous to our study, a couple of lytic and non-lytic assays for assessment of cytolytic activity had been proposed. These assays include: fluorometric assessment of antigen-specific T-cell mediated lysis using CFSE and PKH26 staining (Sheehy 2001); assessment of CD95-mediated lysis using CFSE and 7-AAD staining (Lecour 2001); assessment of apoptosis, using Annexin V (Vermes 1995; Goldberg 1999); detection of CTL-induced caspase activation within target cells (Liu 2002); and a non-lytic assay based on the expression of CD107 on the surface of effectors cells after degranulation (Betts 2003). However, a major disadvantage of most of the reported lytic methods is the lack of dissection of the complex and heterogeneous target cell population always present in cytotoxicity assays, and the lack of calculation of disrupted target cells.

CFSE is an uncharged fluorescein derivate that readily crosses cell membrane of viable cells that becomes charged inside the cell and is retained as long as the cell membrane is intact. We evaluated CFSE regarding solubility, staining stability and spectral characteristics using the K562 cell line in NK cell assay. CFSE seemed to be stable, easy to employ and without serious problem of spectral overlapping with PI-staining. Then, we performed a pilot study of HIV-specific cytotoxicity for the evaluation of different staining strategies for discrimination between target and effector cells including, pre-assay and/or post assay staining using CFSE, anti-CD3, anti-CD20 and anti-CD45 and, for the detection of cell death, target cell loss of CFSE and/or post-assay PI uptake. Following the analysis of a large amount of staining combinations we concluded that: 1) pre-assay staining of either target or effector cells with Mabs was not possible since the Mabs seemed to be released from cells (probably dying cells) and non-specifically bound to other cells; 2) staining with CFSE was rather stable and reproducible also in the antigen-specific CTL assay; and 3) the importance of keeping the cell cultures in optimal condition prior to the cytotoxicity assay. Assessment of the viability within a cell suspension may be important to determine optimum culture conditions and to obtain optimal function of the cells especially if they are used for functional analysis. For example it is known that dead or dying cells may bind antibodies non- specifically (Dangl 1982; Loken 1982; Wing 1990), may alter expression of surface antigen (Schmid 1992), and often display increased autofluorescence (Zamai 1993).

We developed an assay for assessment of HIV-antigen specific cytotoxicity in which we focused on the live-target cells rather than the dead or dying cells since they are very heterogeneous and difficult to assess.

The assay consists of CFSE-stained target cells (pulsed with Gag or Nef peptide pools), which are cultured with specific or non-specific effector cells and stained with propidium iodide (PI). The dual staining permits the identification of several cell populations including viable target and effector cells and dead target and effector cells. Thus, the HIV-specific cytolysis is determined by the enumeration of viable target cells in the test sample, containing target and matched effector cells, as compared to corresponding cells in the control sample, containing target and mismatched effector cells (Figure 11).

Specific effector cells ( E gag) Target cells ( T gag) Non-specific effector cells ( E nef) (Gag-expanded CTLs) (Gag-pulsed B-cells) (Nef-expanded CTLs)

R3 R3 R3

3 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 0 10 1 10 2 10 3 10 4 FL3-H FL3-H FL3-H C F S Specific killing Non-specific killing E ( T gag+ E gag) ( T gag+ E nef)

R3 R3

795 1104

10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FL3-H FL3-H PI-staining

Figure 11. Flow cytometric assessment of antigen-specific T-cell mediated cytotoxicity using staining of target cells with CFSE and PI to identify the viable target cells selected by region R3 in control and test samples. The mean percentage of specific lysis is calculated as follows:

(Viable Target Cells Control sample – Viable Targets Cells Test sample ) x 100

Viable Target Cells Control sample

A comparison with the chromium-release showed good correlation (r=0.79; P<0.05) and concordance (Figure 12, upper) between the assays but the sensitivity of the flow cytometric assay was significantly higher (P<0.05). So, what do we measure with flow cytometry and Cr- release, respectively, that might explain the differences in the magnitude of reactivity? By the FC-CTL five different target cell populations can be assessed including CFSE+PI- viable target cells, CFSE+PI+, CFSE-PI+, CFSE-PI- dying/dead but detectable target cells, and lysed but quantifiable target cells (Figure 13). The Cr-release assay detects only disrupted target cells through their release of 51Cr. If we assume that the CFSE and chromium have similar kinetics of release, it means that the Cr-release assay fails to detect dying/dead cells with relatively intact cell membrane. In summary, this new assay has several advantages over earlier described perforin-based cytotoxicity assays: 1) the calculation of antigen-specific cytotoxicity is based on the absolute enumeration of live target cells, rather than on percentage of not accurately enough detected dying/dead target cells; 2) focusing on live cells excludes the disturbing background signals; 3) the exclusion of the background reactivity displayed by dying cells enhances the sensitivity and specificity of the assay; and 4) exactly the same type of target cell is used in both sample test and control tube, providing a more accurate enumeration of the target cell population of interest. In summary, the FC-CTL showed a good correlation with the Cr-release assay but also higher sensitivity. Moreover, the assay displayed good specificity when samples from HIV-negative blood donors were analysed. This assay can be a valuable tool for evaluation of the cytolytic function in vaccine trials as well as in HIV-infected subjects, in particular when such responses are present at low levels. In addition, the present cytolytic assay can be further developed using multi-colour flow cytometry for simultaneous assessment of intracellular cytokines or cytotoxic effector molecules (perforin, granzymes) as well as for phenotypic characterisation of the effector or target cell population and conjugate formation.

FC-CTL - + Total

- 325

+ 099

Total 31114

Concordance: 12/14 (86%)

FC-CTL FC-CTL - + Total - + Total

- 022 - 156

+ 31013 + 279

Total 31215 Total 31215

Concordance: 10/15 (67%) Concordance: 8/15 (53%)

Cr-CTL Cr-CTL - + Total - + Total

- 101 - 325

+ 4812 + 358

Total 5813 Total 6713

Concordance: 9/13 (69%) Concordance: 8/13 (61%)

Figure 12. 2x2 tables comparing the concordance of HIV-specifi (Gag or Nef) reactivity between the methods.

Cytotox Cr-CTL FC-CTL

no no no (CFSE+PI-) CFSE + ±) R1 yes no yes (CFSE PI R3 R2 yes no yes (CFSE+ PI+)

yes yes yes* (CFSE- PI+)

yes yes yes* (CFSE- PI-)

yes yes yes* (absent) 100 101 102 103 104 FL3-H PI

(lysed cells)

Figure 13. Flow cytometric detection of target-cells, in mixed culture of targets and effectors, displaying different stages of the cytotoxic process ranging from live to dying/dead and finally lysed cells.Cytotox ’yes’ indicates that cells are dying or already dead (all of these display much lower forward scatter - due to shrinkage - than Cytotox ’no’ cells). Cr-CTL ’no’ has been defined assuming that crom is released at the same time as CFSE, and * indicates that, although these cells are not possible to distinguish from effectors in mixed populations, they can be properly estimated by comparison with controls with targets only.

Future studies Cytotoxic T-lymphocytes carry out one of the most important effector functions, being responsible for destroying virally infected cells by the perforin/granzyme pathway. In HIV- infection CTL responses have been extensively studied and CD8+ T-cell activity can generally be detected in most HIV-infected individuals. However, in general cytotoxicity assays measure mainly the terminal stage of the cytolytic process and little work has been done to directly address other aspects of the lytic function, such as differences in the rate of killing or conjugate formation. It would be interesting to assess the relative ability of different effector

cell subsets to conjugate to target cells and correlate this binding capacity to the ability of mediating cytotoxicity. Thus, there is a need to assess or quantitate cell-mediated cytotoxicity at the different stages of the cytolytic reaction. Further, although many types of cytolytic cell populations are capable of cell recognition and binding to target cells, only a minority of these are capable to mediate the ”lethal hit”. For example it is known that different lymphocyte subsets can actively bind to K562 (Lebow 1986; Vitale 1991) but it is believed that the NK cells are the actual killers of K562 in-vitro. Nonetheless, IL-2 vitro-activated CTL can kill by both MHC-restricted and MHC non-restricted mechanisms (Whiteside 1989).

Other cytolytic assays Recently, two cytolytic assays based on the use of fluorochromes for identification of the cell populations and assessment of cell death, have been presented (Sheehy 2001; Lecour 2001). Sheehy and co-workers have used target cells stained with PKH-26 and CFSE whereby the specific lysis is measured by the loss of CFSE in the PKH-26-positive targets. Lecour and co- workers (2001) have used CFSE and 7-AAD targets, although this method has so far been limited to detection of Fas-ligand (CD95)-mediated cell death in CD95-transfected murine cells or in human cell lines susceptible to CD95-induced cell death. However, none of the assays considers the target cell loss for the calculation of specific cytolysis. Further, the combination of CFSE and PKH-26 may result in significant spectral overlap requiring compensation setting of the instrument, which can be complex and time-consuming. Another group (Liu 2002) has recently presented an alternative approach to assess apoptosis, based on the detection of caspace activation in target cells, an early occurring event in the cytolytic process. Although this is an interesting approach for assessment of apoptosis, there are some technical drawbacks with this assay, including high background reactivity and problems with compensation settings. It is also important to consider that the level of cleaved caspases is not the same as active caspases.

Non-lytic assays for CTL-assessment The complex, time-consuming and labour-intense characteristics of cytotoxicity assays restrict their use, although these assays measure an important function of cellular immunity, namely the ability of effector cells to kill infected target cells. Therefore, big efforts have been made to develope more simple and rapid approaches such as ELISpot, ICS and phenotypic staining of effector T-cells.

Several markers have been proposed as surrogates for antigen-specific CTL functions including, IFN-γ production (Suni 2001; Ghaneker 2001); TNF-α production (Ratner 1993; Derby 2001); expression of a degranulation marker (CD107) on the surface of effector cells (Betts 2003), and intracellular content of perforin and granzyme (Maher 2002). However, it is worthwhile to note that detection of these markers does not imply cytolytic activity, although good correlation has been shown by some groups between the markers and cytotoxicity in a number of cases. But if the CTLs do not contain perforin, which has been shown to occur in HIV-infection (Andersson 2002), the cells would not be capable of killing despite that degranulation occurs. Detection of IFN-γ production has been proposed and generally accepted as a reliable marker for antigen-specific CTL function, although the association between cytolytic activity and IFN-γ expression is not completely established. IFN-γ-production can be assessed by the enzyme- linked immunospot (ELIspot) or by intracellular cytokine staining (ICS), which will be discussed later in greater details. Some comparative studies of the ELISpot and Cr-release, using influenza- (Scheibenbogen 1997), malaria- (Schneider 2001), papillomavirus- (Michel 2002) or HIV-specific activation (Xu 2003) suggest a close correlation between results by the two methods, while others found no such relation (Appay 2000; Goepfert 2000). In our study, the levels of IFN-γ responses displayed by the ELISpot correlated well with the cytolytic activity detected by the FC-CTL (r=0.64; P<0.05) and the Cr-release assay (r=0.61; P<0.01). The concordance of positive or negative results was similar between the ELISpot and ICS responses in relation to each cytolytic assay (Figure 12). Yet, it is not clear to what extent IFN-γ production by ELISpot and ICS correlates with cytolytic activity since few comprehensive comparative studies including all three assays have been done. Moreover, in HIV-infection the use of IFN-γ production as a cytolytic marker must be employed by caution since it is well known that different effector functions of CD8+ T-cell are dysfunctional in HIV-infected subjects (Appay 2000; Goepfert 2000; Shankar 2000; Migueles 2002).

II INTRACELLULAR CYTOKINE STAINING (PAPER III AND IV)

Development of the ICS-technique Intracellular cytokine staining is a relatively new technique initially introduced for detection of intracellular cytokines in tissue-sections by indirect immunofluorescence following fixation, permeabilisation and staining of the cells (Andersson 1988). The method was rapidly adopted for flow cytometric assessment after the introduction of monensin and brefeldin A (Jung 1993) which block the cytoplasmic protein transport causing accumulation of cytokines in the Golgi complex, thereby increasing the intracellular concentration of cytokines (Prussin and Metcalfe 1995; Openshaw 1995; Picker 1995; North 1996; Waldrop 1998). This was employed in a standard procedure, in which T cell responses to specific antigens are assessed in secretion- inhibited conventional cultures of isolated PBMCs after brief incubation (6 h). However, the number of antigen-specific cytokine responding T cells is usually low and thereby difficult to detect. Therefore, several approaches have been employed in order to enhance the detection of these responses, including antigenic expansion of the memory T cell population followed by restimulation with PMA-IO (Bradley 1993; Evans 1998; Kallas 1999), prolonged incubation time using PBMCs (Rostaining 1999), staining with anti-CD69 (activation marker) for more easily detection of activated cytokine producing cells, and recently the addition of antibodies to co-stimulatory molecules, CD28/CD49d during incubation with antigen (Suni 1998). In the majority of the studies using assessment by ICS, PBMCs have been used (North 1996; Waldrop 1998; Kallas 1999; Mascher 1999; Rostaing 1999; Asanuma 2000; Collins 2000; Baran 2001) but whole blood assays have also been employed by several groups (Jason 1997; Waldrop 1997; Maino 1998; Suni 1998; Pitcher 1999; Asanuma 2000; Mendez 2000; Nomura 2000; Bueno 2001; Maecker 2001).

Whole blood assays A number of investigators have shown that the use of whole blood has several advantages over the use of PBMC cultures, for the detection of proliferative responses and should be preferred whenever possible (Gratzner 1982; Gonchoroff 1985; Carayon and Bord 1992; Gaines 1996; Gaines and Biberfeld 1998). The employment of whole blood is simple, avoids time-consuming separation steps (potentially responsible for selective losses, cellular activation and contamination), and offers an environment close to the in-vivo situation. Further, we and others (Leroux 1985; Weir 1994; Gaines 1996) have earlier reported that whole blood must be diluted

1/5 or 1/10 to permit culturing for longer periods of time when measuring cell-mediated immune responses.

The whole blood long-term ICS In this study we investigated the influence of the degree of dilution of whole blood and the duration of the incubation period on intracellular cytokine production by CD4+ and CD8+ T lymphocytes. Whole blood was diluted 1/1, 1/2, 1/5 and 1/10, and cultured for 6, 24, 48, 72 and 120 h in the presence of anti-CD28 and CD49d, and, during the last 4 h of culture, in the presence of brefeldin A. The samples were cultured in the absence or presence of mitogens (PMA/IO, PHA), superantigens (SEA/B) or specific antigens (CMV, VZV). Our results showed consistently higher frequencies of responding T cells in cultures where blood diluted 1/5 or 1/10 was incubated for several days. The optimum conditions for detection of IFN-γ positive cells following activation with PMA/IO, PHA and SEAB, were 72 h of culture in blood diluted 1/10, while activation with CMV and VZV induced peak levels at 120 h. On the other hand, the frequency of cells producing TNF-α, IL-2, IL-4, IL-10 or IL-13 were generally much lower than those producing IFN-γ, and in some cases difficult to distinguish from background levels. Kinetic studies of cytokine production by PBMCs were also performed showing a reactivity pattern similar to that of whole blood with peak levels of IFN- γ-producing cells at 72 h. We suggest that the increased levels of IFN-γ production after culture of whole blood or PBMC for 72 h are due to an expansion of the numbers of cytokine-producing cells responding to a specific stimulus (Gaines 1996; Gaines and Biberfeld 2000). However, an assay requiring in vitro amplification of responding cells is not able to quantify antigen-specific effector cells. In addition, since memory T-cells rather than effector cells will be expanded in vitro, the cell population identified by long-term ICS is qualitatively different to populations detected by short-term assays. Nevertheless, an expansion of responding cells in cultures in order to reach a level of possible detection must, in most cases, be more important than aiming strictly at precursor cell frequencies and failing to detect any response at all. However, further evaluation of the long-term whole blood ICS-assay and comparative studies with the ELISpot are needed, in order to determine a reliable relation between the assays.

Comparison of ICS and ELISpot Lately, the ICS and ELIspot have become the assays of choice for assessment of antigen- specific responses in clinical trials evaluating vaccines candidates against HIV-1. The Elispot is a robust and very sensitive method for detection of extracellular cytokine production but a major disadvantage of the assay is the inability to define responder cells phenotypically (unless CD4+ or CD8+ T-cell depletion is applied), while the ICS assay provides precise enumeration and identification of the responding lymphocyte population as well as simultaneous detection of several cytokines. Furthermore, the ICS is most frequently performed on PBMCs but, in contrast to the ELISpot, whole blood preparations may also be used. In my last study (paper IV) HIV-specific IFN-γ responses were determined in HIV-1 infected, treatment-naive individuals with different levels of viral load, using ELISpot and ICS. The ICS results obtained following peptide (Gag or Nef) stimulation were expressed as number of IFN- γ+CD4+ and IFN-γ+CD8+ lymphocytes per million in order to compare with responses obtained by the ELISpot, expressed as spot-forming cells (SFC) /106 PBMC. The IFN-γ+ responses obtained by ELIspot and ICS for T-cells were correlated for Gag (r=0.56; p<0.05) but not for Nef, and the concordance of reactivity was relatively good for Gag-responses (10/13 samples) (Figure 14, upper lane) but poor for Nef-responses (5/12) samples. Further, the magnitude of Gag-specific IFN-γ responses, expressed as IFN-γ-positive cells/106 PBMCs for both assays, was in relative good agreement (Figure 14, bottom lane). There are several factors that might contribute to the differences we observed between the two assays when all the results (Gag-, Nef- and vCP-specific) were considered including: sample material (PBMC vs whole blood), co-stimulation molecules (presence or absence), sample preparation, and detection approach (population or single cell level). However, in this particular study of samples from HIV-infected individuals, the incubation time (16 h vs. 48 h) is probably the main reason for discordant results between the assays. This will be further discussed in the next section (III). Comparative studies have generally shown that the frequencies of IFN-γ producing cells detected with either method correlate fairly well (Goulder 2000; Assemisen 2001; Ghanekar 2003; Currier 2002; Karlsson 2003; Maecker 2003; Sun 2003; Horton 2004; Maino 2004). However, there are discrepancies in respect to the limit or range of detection by each assay. While some groups observed that the frequency of responses was similar by the two assays (Goepfert 2000; Sun 2003; Maino 2004), another group found that the ICS was less likely to detect low-level reactivity than the ELISpot (Karlsson 2003). Further, Karlsson and co-workers

(2003) found that values by the ICS were generally higher than values by the ELISpot, although no consistent conversion factor between the results could be determined. Although considerable progress has been made in the optimisation and validation of each of these assays for vaccine trials (Rusell 2003), few comparative studies have been reported.

ICS

- + Total

- 000

+ 31013

Total 31013

Concordance: 10/13

Elispot ICS-CD4 ICS-CD8

2600 2400 2200 2000 1800 PBMC

6 1600 1400 1200

cells/10 1000 + 800

IFNg 600 400 200 0 ICS ICS ICS ICS ICS ICS ICS ICS ICS ICS ICS Elispot Elispot Elispot Elispot Elispot Elispot Elispot Elispot Elispot Elispot Elispot

I II III IV V VI VII VIII IX X XI

Figure 14. HIV-1 Gag-specific responses by LT-ICS and ELISpot, upper lane shows a 2x2 table for concordance of reactivity and bottom lane, shows the magnitude of the responses,

expressed as number of IFN-γ-positive cells/106 PBMC.(two samples are exclude since they were not analysed in parallel)

III ASSESSMENT OF HIV-1 SPECIFIC CELL-MEDIATED IMMUNE RESPONSES (PAPER IV)

Both CD4+ T-helper cells (Th) and cytotoxic T lymphocytes (CTL) have been shown to be important for protection from progressive disease against HIV and SIV infection (Gruters 2002; Heeney 2002). Antigen-specific T-cell responses to HIV-1 in relation to viral load and/or disease progression have been extensively studied by several techniques including conventional bulk assays (H-thymidine and Cr-release assays) and more recently developed single-cell assays (MHC-peptide tetramers, ELISpot and ICS). However, these studies have given partly conflicting results depending on the study population and the assays employed. Thus, positive, negative and no correlation has been found between virus load/disease progression and HIV-specific CD8+ T-cell responses (Ogg 1998; Betts 2001; Edwards 2002; Addo 2003; Boaz, 2003) or HIV-specific CD4+ T-cell responses (Pitcher 1999; Wilson 2000; Betts 2001; McNeil 2001; Ostrowski 2001; Harari 2002; Palmer 2002). The various methods currently available raise the question of which technology is most appropriate to assess virus specific T-cell responses induced by natural HIV-1-infection or by vaccine immunogens.

The purpose of this study, was to examine the HIV-1-specific cell-mediated immune responses in HIV-1 infected treatment-naive individuals in relation to viral load, by assessment of IFNγ responses and cytolytic activity, using our recently developed and established assays, the long-term ICS (LT-ICS) and FC-CTL, as well as the conventional assays ELISpot and Cr-release-CTL.

The subjects included in our study consisted of seventeen HIV-1 infected individuals with >400 CD4+ T cells/µl for at least 5 years, which were divided according to their HIV-1 RNA levels into two groups: patients with low (<5000 copies/ml) (n=9) and high (>5000 copies/ml) viral load (n=8). The IFNγ and cytolytic responses were assessed following activation with peptide pools (Gag and Nef) and avipox virus-based antigen (ALVAC vCP 300). Although the CD4+ T-cell counts and RNA levels differed between the two groups at study entry, the patients represent a selective group of HIV-infected individuals who are relatively healthy and

some of them meet the criteria for LTNPs. The patients were followed-up for a median of 2.4 years and pre-study recorded CD4+ T-cell counts and viral load during five years were also examined. The levels of HIV-1-specific IFNγ- production and cytolytic activity were consistently higher in subjects with low viral load, reaching statistical significance for CD4+ T-cell responses to all antigens tested by ICS, to ALVAC vCP 300 by ELISpot, and of border-line significance for the Gag-specific cytolytic responses. Furthermore, Gag-specific CD8+ T-cell IFN-γ-production was significantly inversed correlated to viral load. Although the CD4+ T-cell counts in the subjects with high viral load had been relative stable during the five years prior to study entry, 6 of 8 patients in this group displayed declining CD4+ T-cell counts to <400 during the follow-up time and 5 of 8 were subsequently started on antiretroviral therapy. In contrast, among patients with low viral load, only one subject displayed CD4+ T-cell counts <400 during the follow-up period and treatment was initiated in 1 of 9 individuals.

Comparison of different assays for HIV-specific responses If we focus on the methodological part of the study and for simplicity on Gag-specific responses (Figure 15), we can conclude that the responses obtained by LT-ICS and the cytotoxicity assays, were better correlated to viral load than those obtained by the ELIspot. Although the sample size is small, there are some technical and functional differences between these assays, which may explain the discordant results. Short-term assays, like the ELIspot or 6hr-ICS, are believed to assess mainly the effector function of T-cells while long-term assays, such as LT-ICS and LPA, are suggested to provide additional information on long-term memory as well as the proliferative capacity of the T-lymphocytes. Wu and co-workers (2002) have shown that secretion of IFN-γ is typical of the early effector phase of immune response while IL-2 secretion is characteristic for the long-term memory T-cell response. Furthermore, Harari and co-workers have shown a good correlation between HIV-specific CD4+ T-cell proliferation and IL-2 production (Harari 2004). We have previously pointed out that the enhanced detection of antigen-specific cytokine-producing cells observed by the LT-ICS may be explained by the fact that during long-term incubation a certain degree of proliferation and cell division may occur. It is known that cells from non-progressors have a greater capacity to proliferate than those from progressors (Rosenberg 1997; Imami 2002; Iyasere 2003; Harari 2004). Thus, the higher levels of ICS-IFN-γ production and cytolytic activity observed in cells from patients with low viral

load, as compared to subject with high viral load., are probably due to the greater proliferative capacity of the cells. Using short-term ICS assays a few investigators have found that the frequency of HIV-1 Gag- specific double positive IFN-γ+IL-2+ CD4+ T-cells was significantly higher in patients with non- progressing disease (LTNPs) as compared to that in slow-progressors (Boaz 2002; Harari 2004), and this response was inversely correlated with viral load. In contrast, other groups, assessing only IFN-γ+ responses, have found higher frequencies of CD4+IFN-γ+ T-cells in patients with non-progressive disease, as compared to progressors, but no correlation between CD4+IFN-γ responses and viral load (Pitcher 1999; Betts 2001; Harari 2002), whereas other investigators found either no differences in the magnitude of CD4+ IFN-γ responses between the two patient groups (Wilson 2000; McNeil 2001), or no relationship between HIV-specific CD4+ T-cell responses and viral load (Wilson 2001; Betts 2001; McNeil 2001; Ostrowski 2001; Palmer 2002). To conclude, our results showed that long-term IFN-γ ICS is a simple and sensitive assay to measure HIV-1-specific CD4+ and CD8+ T-cell responses in HIV-1-infected individuals and demonstrate that subjects with stable CD4+ T-cell counts and low viral load have significantly stronger HIV-specific cellular immune responses than patients with higher load and declining CD4+ counts, supporting a protective role for specific cellular immunity in HIV-infection.

GAG-specific immunere

p<0.01 n.s p<0.01 n.s

2000 100

1800 90

1600 80

PBMC 1400 70 6

10 1200 60 ty (%)

1000 50 ci

ive cells/ 800 40 Cytotoxi

posit 600 30 Ng-

F 400 20 I

200 10

0 0 RFDJPCH ALBGKE RFDJPCH ALBGKE RFDJPCH ALBGKE RNFMDJPCH ALSBGKEO RDPCH AGK RDPC AGK low VL high VL low VL high VL low VL high VL low VL high VL low VL high VL low VL high VL ICS (CD4+ T cells) ICS (CD8+ T cells) ICS (T cells) ELISPOT FC-CTL Cr-CTL

Figure 15. HIV-1 Gag-specific IFN-γ-responses and cytolytic activity in apteints with low orhigh viral load. The IFN-γ- production (expressed as number of IFN-γ-positive cells/106 PBMC) detected by LT-ICS for CD4+ (circles), CD8+ (triangles) and (CD4+ + CD8+) T-cells (rhomb), and by ELISpot (circles); and the cytotoxicity (%) assessed by the FC-CT (triangles) and Cr-release (squares) assays.

Possible role for the long-term whole-blood ICS assay There are currently many old and new techniques available for the assessment of cell- mediated immune responses induced by natural infection as well as following vaccination and all of these can of course not be employed for every study. While some methods may be the best choice for some investigations, other assays can be more suitable in other situations. On one hand, the tests should be as simple and rationale as possible, but on the other hand, they should provide accurate and reliable information needed for the particular objective investigated. I believe that the long-term whole blood ICS can be a valuable tool for monitoring cell-mediated immune-responses for several reasons. First, the use of whole blood eliminates technically complex and time-consuming sample processing needed for PBMC assays, and is thus more convenient for large-scale studies in particular for areas with less extensive laboratory facilities. Additionally, whole blood offers an environment more close to the in-vivo situation, and possible selective cell-losses and other sources of variation are avoided. Second, ICS can be combined for the simultaneous measurement of several different molecules both on cell-surfaces and intracellularly, including not only cytokines but also perforin, granzymes and chemokines, especially if now available 3-4-laser flow cytometers with the ability to detect up to 18 colours are ulitized. By contrast, the widely used ELISpot assay is essentially equivalent to a one-colour assay. In the case of HIV, it has lately become clear that although HIV-specific CD8+ cell- responses can be detected in may patients, these cells are frequently dysfunctional and techniques that can measure ‘polyfunctional’ CD8+ cells must be applied to detect immunity associated with control against disease progression (Appay 2000; Harari 2004; Roederer 2004). Third, the long-term (48-72h) culture provides opportunity for cellular expansion and more sophisticated activation of effector mechanisms than short-term (e.g. 6-12h) cultures, and thus the response and the concentration of the various molecules that need to be detected for the succesful demonstration of the polyclonal nature of that response, have enough time to reach a sufficient level to be dectected. Hence, the long-term whole blood ICS can be recommended for many purposes.

GENERAL COMMENTS AND CONCLUDING REMARKS

It is important to study and delineate the immune responses during natural course of infection to identify correlates of protective immunity, which may help to define effective treatment and vaccine approaches. The development of assays, technologies, and reagents for detection of immunological responses is an essential part of this process. There are numerous assays based on different approaches for the study of cell-mediated immune responses (CMI), which raises the inevitable question of what technology should be employed to assess antigen-specific T-cell responses. The spontaneous answer would be that it depends on the purpose of the study, although there are several clinical and methodological issues to consider when choosing an assay to evaluate CMI. The clinical aspects would be related to the infectious agent, the clinical status, if CMI will be evaluated during early or late phase of natural course of infection, and if antimicrobial treatment or prophylactic or therapeutic vaccines have been administered. The methodological aspects would be related to which parameter to assess: the frequency of antigen responding cells; the effector or long- term memory function such as cytokine production or proliferative and cytolytic capacity; or surrogate markers for such functions. Additional methodological aspects include: the detection approach (assessment at the population vs. single cell level); the sample material (PBMC vs. whole blood); the culture time (no vs. short-term vs. long-term); activation (no vs. specific vs. non-specific activation), the use of co-stimulating molecules (CD28/CD49d) (present vs. absent), and several aspects concerning the method of sample preparation. Thus, results obtained from a study may be related not only to biological parameters but also to the design of the study, and the choice of materials and methods should be given proper and careful attention do provide results that will be meaningful for the objective investigated. In the last years, extensive and successful efforts have led to the development of new methods/techniques such as tetramer-staining, ELISpot, ICS and assessment of surrogate markers of CMI. However, in infectious disease immunology there is so far no assay of T-cell function that can be universally employed for any study, and if a certain assay has not been validated and established for a particular objective, techniques that enable simultaneous assessment of different functions and markers should be chosen, or different methods have to be combined.

The long-term whole blood assay can be a valuable tool for monitoring CMI for several reasons: the assay is simple to perform; it requires only small amounts of blood; complex and

time-consuming sample processing as for PBMC cultures is not needed; it is suitable for large-scale studies; it is more sensitive than short-term ICS; and it provides more information than the ELISpot. Furthermore, the LT-ICS can be combined with simultaneous measurement of several different molecules present intracellularly or at the cell surface, especially if a 3-4- laser flow cytometer with ability to detect up to 18 colours is utilized.

Cytotoxic T-lymphocytes carry out one of the most important effector functions for control of viral infections, since they are responsible for destroying virally infected cells. Therefore, assessment of CD8+ T-cell activity is of significant importance for evaluation of cellular immune responses to viral agents. In view of how complex, labour-intensive and time- consuming the 51Cr-release cytotoxicity assay is to perform, and yet how little information it provides (only percentage cytotoxicity), it is generally desirable to use more simple and rapid methods for assessment of antigen-specific CD8+ T-cell responses, but these more convenient assays have to be properly validated to deliver reliable and relevant results for the question studied.

The FC-CTL is just as difficult to perform as the Cr-release assay, but it appears to be more sensitive, and it has several advantages since it can be further expanded for simultaneous assessment of important features of the cytotoxic process in greater detail such as the presence and concentration of effector molecules like perforin, granzymes and CD107. In addition, the FC-CTL can be employed for the detection and functional comparison of different effector subsets in the same culture concerning binding capacity and conjugate formation as well as the ability to mediate cytotoxicity.

The new assays developed and presented in this thesis can be valuable tools for research regarding evaluation of CMI against viral infections. The NK cell cytotoxicity assay has been further developed for use of whole-blood and is now employed for a study of the role of innate immunity for control of herpes simplex infection, and the LT-ICS will be used as one of the assays for evaluation of CMI in a phase I HIV-1 vaccine trial in Stockholm.

ACKNOWLEDGEMENTS

We all know that the accomplishment of this thesis is a results of a team-work to which you have contributed in one way or another. I whish to express my sincere gratitude to all of you, in particularly I would like to thank:

Hans Gaines, my supervisor, for letting me fly freely, though sometimes it would have been proper to stop me, for your invaluable guidance, trust, support and encouragement. For been generous with your great knowledge and new ideas.

Gunnel Biberfeld, my co-supervisor, for introducing me to research in such a concrete way as the HIV-field studies in Kagera, Tanzania. For sharing your great knowledge and experience, for being so serious, devoted, and enthusiastic in your work, and for always having time for small and big discussions.

Rigmor Thorstensson, my mentor, for being a wise “multi”-women. For showing interest in my “flow-work”, for good advises and contributions to my last papers and for devoting time to discuss big and small, scientific and non scientific issues despite your hectic schedule as head of the department.

Kristina Franck, my soul mate in the lab throughout the years, for always being helpful, supportive, and meticulous with your work and for your beautiful way of approaching people.

Barbro Mäkitalo and Raija Ahmed, my dear colleagues and friends for all these years as “doktorander”. For incredible support and encouragement whenever I needed, and good scientific and non-scientific exchange.

Katarina Karlén and Charlotta Nilsson for their invaluable contributions to the last papers, and for always being helpful. Thank you Lotta for the good pre-postdocs talks.

Eric Sandström at Venhälsan, for his interest in my work and invaluable comments, and for giving a clinical dimension to my work; Bo Hejdeman for his contribution to the last paper.

Former, present and new members of the “Immunologen-family”, for contributing to such a nice working atmosphere, for always been helpful, supportive and for all the good and bad discussions during the coffee breaks. Shahnaz Mahdavifar, our Persian queen for her big heart and concern for everybody in the lab; Irene Silhammar for her wonderful sense of humour and recommendations of “good” books, Lena Anderson, Tehesti Tecleab, Maria Lagrelius, Pertra Jones, Helen Linder, Andreas Mörner, Hélène Fredlund, Eva Olausson-hansson, Maria Widfelt, Karin Winberg and Marianne Diursson.

Our coordinators, Lena Vetterli and Marlene Quesada-Rolander, for always helping me out with practical problems.

Members of the “vaccin programmet”: Margareta Ljungman, Abdolreza Advani, Declan Donelly, Marie Henriksson and Hans Hallander, for all joy and good laughs.

The new members of our group: Markus Maeurer, for such an enthusiastic approach to research and encouraging words during the process of writing this thesis, Isabelle Magalhaes and Nalini Kumar Vudattu for their nice attitude and good working spirit in the lab.

Members of he hepatitis group, Tatjana Tallo, Marjan Mohseni and Shahin Sendi for good company and nice talks during the long weekends and late nights. Tajana, we are lucky, there are no reports about “mad-chicken”-disease.

The Tanzania-group in Sweden: Anita Östborn, Katarina Karlsson and Ulla Bredberg (former member); Anita Sandström and Maria Emmelin (at Umeå university); and our Tanzanian colleagues in Tanzania, Fred Mhalu, Willy Urassa; Eligius Lyamuya, Japhet Killewo and Gideon Kwesigabo, for sharing the inspiring and interesting but sometimes tiring work with the HIV-project in Kagera and Dar es Salaam. Ii gave me an invaluable experience and a humanist dimension to my work.

My friends outside the lab: Lilian Walther and Amadou, for true friendship and Lilian for always being at the same “wavelength” no matter the distance or time; Inger and Jan Ohlsson, for sincere friendship and support throughout the years, our generous friends Åsa Wallgren and Cristian for sharing good and bad times as “små-barns föräldrar”; Vivi Altamirano, for your kindness and

tenderness and “ crazy” philosophical discussions; Beatriz and Favio Sanchez deep friendship and Fabio, for your interest in my work and genuine devotion to science, Jolanta (Karina) my first friend in Sweden for close friendship throughout the years.

My beautiful and exceptional “Chilean family”: My parents, Ana and Jorge, for their deepest and unconditional love, support, and devotion to each one of the family members, for their interest in my work. My wonderful and special sisters, Alicia, Vania and Katia, with their families, for their unconditional help and support and big joy in my life.

My boys at home, Leandro our 2 years old sunshine, for reminding me that small and simple things are the most important in life; Andreas my pearl, for being so especial, just being you!!!!; Mauricio and Roberto, for letting me take place in your lives and for being such wonderful big brothers and good guys, and Juan Carlos, for always standing by me, sharing the experience as a PhD-student, for being caring, supportive and patient….. You are unique!!

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