Implications for Channel Expression During B Cell +K

K+ Channel Expression during B Cell Differentiation: Implications for Immunomodulation and Autoimmunity

This information is current as Heike Wulff, Hans-Günther Knaus, Michael Pennington and of September 24, 2021. K. George Chandy J Immunol 2004; 173:776-786; ; doi: 10.4049/jimmunol.173.2.776 http://www.jimmunol.org/content/173/2/776 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

:K؉ Channel Expression during B Cell Differentiation Implications for Immunomodulation and Autoimmunity1

Heike Wulff,2* Hans-Gu¨nther Knaus,† Michael Pennington,‡ and K. George Chandy§

Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD؉CD27؉ memory B cells express small -numbers of the voltage-gated Kv1.3 and the Ca2؉-activated intermediate-conductance IKCa1 channel when quiescent, and in crease IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast, quiescent class-switched memory B cells express high levels of Kv1.3 (ϳ2000 channels/cell) and maintain their Kv1.3high expression after activation. Consistent with their channel phenotypes, proliferation of naive and IgD؉CD27؉ memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memory

B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Downloaded from Therefore, speciﬁc Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders. The Journal of Immunology, 2004, 173: 776–786.

wo Kϩ channels in T lymphocytes, the voltage-gated cytosolic Ca2ϩ levels in the time frame required for optimal acti- Kv1.3 channel (also known as KCNA3 (HUGO nomen- vation. Coordinated activity of Ca2ϩ and protein kinase C-depen- clature)) and the Ca2ϩ-activated IKCa1 channel (also dent signaling pathways leads to new gene expression culminating T http://www.jimmunol.org/ known as KCNN4 (HUGO nomenclature); KCa3.1 (International in cell proliferation. The relative contributions of Kv1.3 and Union of Pharmacology nomenclature) (1)), regulate Ca2ϩ signal- IKCa1 in regulating this process depend on their expression levels ing by controlling the membrane potential of lymphocytes (2–7). in the different lymphoid subsets. During activation, inositol 1,4,5-triphosphate generation induces Kv1.3 and IKCa1 channels are expressed in T lymphocytes in a ϩ ϩ the release of Ca2 from internal stores. Depletion of these Ca2 distinct pattern that depends upon the state of activation and dif- 3 stores causes calcium-release-activated calcium (CRAC) chan- ferentiation. In the quiescent state, naive, central memory (TCM) 2ϩ ϳ ϳ nels to open in the membrane, and the resulting Ca influx sus- and effector memory (TEM) T cells express 250 Kv1.3 and 20 ϩ ϩ tains elevated levels of cytosolic Ca2 (8–11). Ca2 influx IKCa1 channels per cell. The potent Kv1.3 blocker Stichodactyla through CRAC channels is reduced at depolarized potentials, and helianthus toxin (ShK) suppresses the proliferation of these cells, by guest on September 24, 2021 ϩ consequently, membrane depolarization attenuates the Ca2 signal whereas the selective IKCa1 blocker TRAM-34 is ineffective (14). ϩ (10–13). The driving force for Ca2 entry is restored by a coun- Activation induces differential expression of Kϩ channels in the ϩ terbalancing efflux of K ions and membrane hyperpolarization three T cell subsets, leading to an altered channel phenotype and brought about by the opening of Kv1.3 channels in response to consequently to altered responsiveness to Kv1.3 and IKCa1 block- ϳ membrane depolarization, and of IKCa1 channels as a conse- ers. Naive and TCM cells up-regulate IKCa1 to 500/cell upon ϩ quence of elevated cytosolic Ca2 . The tightly coupled interplay activation via transcriptional mechanisms, and as a consequence, ϩ between the K channels and CRAC channels maintains enhanced these subsets escape further Kv1.3 inhibition and become sensitive

to IKCa1 blockade (14, 15). TEM cells, in contrast, up-regulate Kv1.3, and their proliferation is sensitive to Kv1.3 blockers (15). *Department of Medical Pharmacology and Toxicology, University of California, Davis, CA 95616; †Institute for Biochemical Pharmacology, Medical University Inns- The discovery that the majority of myelin-reactive T cells in pa- bruck, Innsbruck, Austria; ‡Bachem Bioscience, Inc., King of Prussia, PA 19406; and tients with multiple sclerosis (MS), an autoimmune disease of the §Department of Physiology and Biophysics, University of California, Irvine, CA CNS, are Kv1.3high T cells that can be suppressed by Kv1.3 92697 EM blockers (15), has raised interest in the therapeutic potential of Received for publication February 6, 2004. Accepted for publication May 10, 2004. Kv1.3 blockers in autoimmune disorders. This idea has been suc- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cessfully tested in experimental autoimmune encephalomyelitis, an with 18 U.S.C. Section 1734 solely to indicate this fact. animal model for the disease (16). 1 This work was supported by grants from the National Multiple Sclerosis Society (to An increasing body of evidence emphasizes the importance of K.G.C.), the Juvenile Diabetes Research Foundation (to K.G.C.), National Institutes memory B cells in the pathogenesis of autoimmune disorders (17– of Health (MH59222; to K.G.C.), Rockefeller Brothers Fund (to K.G.C.), Fonds zur Fo¨rderung der Wissenschaftlichen Forschung (Fonds zur Fo¨rderung der Wissen- 19). As in the T lineage, ion channels may play functionally impor- schaftlichen Forschung P14954-PHA; to H.-G.K.), and the Cancer Research Coordi- tant roles in B cells and may serve as therapeutic targets for autoim- nating Committee of the University of California (to H.W.). mune disorders. Four human B cell subsets can be distinguished by 2 Address correspondence and reprint requests to Dr. Heike Wulff, Department of the expression of IgD and CD27, a member of the TNFR family Medical Pharmacology and Toxicology, School of Medicine, University of Califor- (20–22). Mature naive B cells express IgD but not CD27. Acquisition nia, Davis, One Shields Avenue, Tupper Hall Room 1311, Davis, CA 95616. E-mail ϩ ϩ address: [email protected] of CD27 following somatic hypermutation results in a CD27 IgD 3 Abbreviations used in this paper: CRAC, calcium-release-activated calcium; TCM, memory B cell subset, and then during Ig class switching, replace- ϩ Ϫ central memory T; TEM, effector memory T; ShK, Stichodactyla helianthus toxin; ment of surface IgD with other Ig isotypes yields CD27 IgD mem- 2ϩ ϩ MS, multiple sclerosis; PB, peripheral blood; KCa,Ca -activated K current; KV, voltage-gated Kϩ channel; GC, germinal center; MZ, marginal zone; DTH, delayed- ory B cells. These class-switched memory B cells are a major source type hypersensitivity. of pathogenic IgG autoantibodies that contribute to tissue damage in

MS (17, 19), type-1 diabetes (23), and rheumatoid arthritis (24). A T271) and PE-conjugated mouse anti-human IgD mAb (IA6-2; both BD minor population of IgDϪCD27Ϫ B cells exists in most donors, but Pharmingen). Cells were washed, put on poly-L-lysine-coated coverslips, their functional role has yet to be defined (21). kept in the dark at 4°C for 10–30 min to attach, visualized by fluorescence microscopy, and patch-clamped in the whole-cell configuration. For ex- Mature naive B cells were reported over a decade ago to express periments on CD27ϩIgAϩ and CD27ϩIgGϩ class-switched B cells, we ϩ KV and KCa currents with properties resembling those of Kv1.3 stained CD19 cells with PE-conjugated mouse anti-human CD27 mAb ϩ Ј and IKCa1. Studies with nonspecificK channel inhibitors sug- (M-T271; BD Pharmingen) and FITC-conjugated F(ab )2 of rabbit anti- gested a functional role for these channels in B cell mitogenesis human IgA or anti-human IgG (both DakoCytomation, Carpinteria, CA). ϩ Kv1.3 currents were elicited by repeated 200-ms pulses from a holding (25–29). Because the changes in K channel phenotype are func- potential of Ϫ80 to 40 mV applied every 30 s unless otherwise stated. tionally important in the T cell lineage, we were interested in de- Kv1.3 currents were recorded in normal Ringer solution with a Ca2ϩ-free ϩ termining whether a parallel switch in K channel expression ac- pipette solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, companies differentiation from naive to memory cells in the B cell and 2 mM MgCl2 (pH 7.2; 300 mOsm). Whole-cell Kv1.3 conductance lineage. Therefore, we examined the expression and functional was calculated from the peak current amplitude at 40 mV. IKCa1 currents were elicited with voltage ramps from Ϫ120 to 40 mV role of Kv1.3 and IKCa1 during the differentiation of human B of 200-ms duration applied every 10 s. The pipette solution contained 145 ϩ cells from naive to memory cells using whole-cell patch-clamp mM K aspartate, 2 mM MgCl2, 10 mM HEPES, 10 mM K2EGTA, and ␮ 2ϩ recording in combination with fluorescence microscopy, flow cy- 8.5 mM CaCl2 (1 M free Ca ) (pH 7.2; 290 mOsm). To reduce chloride leak currents, we used a Naϩ aspartate external solution containing 160 tometry, and specific channel blockers. Our studies demonstrate ϩ ϩ mM Na aspartate, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,and5mM that a switch in K channel expression accompanies B cell dif- HEPES (pH 7.4; 300 mOsm). Whole-cell IKCa1 conductances were cal- ferentiation that parallels changes seen in the T cell lineage. The culated from the slope of the current-voltage relationship at Ϫ80 mV. ϩ plasticity of K channel expression in both the B and T cell lin- Kv1.3 and IKCa1 channel numbers per cell were determined by dividing Downloaded from eages and its functional ramifications afford promise for specific the whole-cell Kv1.3 or IKCa1 conductance by the single-channel conduc- immunomodulatory actions of Kϩ channel blockers. tance value for each channel: Kv1.3, 12 pS; IKCa1, 11 pS (14, 35). Cell capacitance, a direct measure of cell surface area, was constantly moni- tored during recording and noted for each cell, and the surface of each cell Materials and Methods was measured by the formula: 1 pF ϭ 100 ␮m2. To normalize for cell size, Channel blockers we determined Kv1.3 and IKCa1 channel densities for each cell by dividing the channel number per cell by that cell’s surface area. 22 http://www.jimmunol.org/ ShK, charybdotoxin, margatoxin, ShK-Dap , and ShK-F6CA were from Statistical differences in channel numbers per cell and channel density Bachem Biosciences (King of Prussia, PA). TRAM-34 and Psora-4 were per square micrometer between different B cell subsets were determined by synthesized as previously described by our group (30, 31). Dendrotoxin-I one-way ANOVA with a significance level of p Ͻ 0.05. and dendrotoxin-␬ were purchased from Sigma-Aldrich (St. Louis, MO). Three-color flow cytometry B cell isolation ϩ ϩ Negatively selected CD19 cells from PB or tonsil were stained for CD27, Peripheral blood (PB) CD19 cells were negatively selected from venous IgD, CD80, CD86, or CD38 immediately after isolation. Cells were incubated blood of healthy volunteers with RosetteSep B cell enrichment mixture in PBS containing 2% goat serum with a combination of FITC-conjugated (StemCell Technologies, Vancouver, BC, Canada). Human tonsil samples mouse anti-human IgD mAb (IA6-2), PE-conjugated mouse anti-human CD27 were obtained under an Institutional Review Board-approved protocol. mAb (clone M-T271), and CyChrome-conjugated mouse anti-human CD80 Within 1 h after tonsillectomy, mononuclear cells were prepared by slicing Ab (clone L307.1) or CyChrome-conjugated mouse anti-human CD86 Ab by guest on September 24, 2021 the sample into pieces and forcing them through a 70-␮m filter to get a ϩ (clone 2331; all BD Pharmingen). A BD Biosciences (San Jose, CA) FACScan single-cell suspension. CD19 cells were isolated using the StemSep B with CellQuest software was used to analyze the stained cells. Cell Negative Isolation System according to the manufacturer’s instruc- tions. Cells isolated from both the blood and the tonsil were found to be Immunohistochemistry 99.2–99.6% CD19ϩ by flow cytometry (PE-conjugated mouse anti-human ϩ Paraffin-embedded sections from human tonsil and spleen (donor Ͼ30 CD19 mAb; HIB19; BD Pharmingen, San Diego, CA). IgD cells were ϩ years of age) were acquired from the University of California, Irvine, Pa- positively selected from CD19 cells with a biotinylated anti-IgD mAb thology Department, under an Institutional Review Board-approved pro- (IA6-2; BD Pharmingen) followed by an anti-biotin tetrameric Ab complex tocol. Sections were dewaxed with xylene and rehydrated through an al- and magnetic beads (StemSep System; StemCell Technologies). From the Ϫ cohol gradient. Before staining with primary Abs, sections were heated column flow-through containing IgD B cells, we positively selected Ϫ ϩ ϩ with 10 mM Na citrate (pH 6.5) in a microwave for 15 min to retrieve IgD CD27 cells with a custom-made anti-CD27 Ab mixture and mag- antigenic determinants masked by paraffin embedding. After treatment netic nanobeads according to the manufacturer’s protocol (EasySep Sys- with 1% H O to inactivate endogenous peroxidase activity and blocking tem; StemCell Technologies). 2 2 with 5% goat serum in PBS for 12 h, sections were incubated with poly- B cell culture and activation clonal rabbit primary Abs for Kv1.3 (1:500 of 4.1 ␮g/ml IgG) or IgD (1:500; A0093; DakoCytomation) or CD27 (1:100; H-260; Santa Cruz Bio- ϩ CD19 cells from PB or tonsil were cultured in complete RPMI medium technology, Santa Cruz, CA) for2hatroom temperature. Bound primary ϩ (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM Na Abs were detected with a biotinylated goat anti-rabbit secondary Ab (1 h; pyruvate, 1% nonessential amino acids, 100 U/ml penicillin, 100 ␮g/ml strep- 1:1000; Jackson ImmunoResearch, West Grove, PA) followed by a HRP- tomycin, and 50 ␮M 2-ME). The cells were activated with 10 nM PMA and conjugated avidin complex (Vectastain Elite ABC kit; Vector Laboratories, 175 nM ionomycin for 48 h to achieve maximal B cell activation (32, 33). To Burlingame, CA). After each incubation step, sections were rinsed with simulate T cell-mediated activation of B cells, we cultured various B cell PBS three times for 5 min. Peroxidase activity was visualized with 3,3Ј- subsets for 96 h with 1 ␮g/ml mouse anti-human CD40 mAb (5C3; BD diaminobenzidine (DAB substrate kit for peroxidase; Vector Laboratories). Pharmingen) presented by stably CD32-transfected irradiated (10,000 rad) Sections were counterstained with hematoxylin (Fisher Scientific, Hamp- K562 cells (B cells:K562 cells, 4:1) (34). The CD32-transfected K562 cells ton, NH), dehydrated, and mounted with Permount (Fisher Scientific). The were a generous gift from Dr. C. June (University of Pennsylvania, Philadel- polyclonal rabbit Kv1.3 Ab was previously shown to be specific for phia, PA) (34). Kv1.3 (36). Electrophysiology [3H]Thymidine incorporation Channel expression was studied in the four B cell subsets defined by ex- Tonsillar IgDϩ or IgDϪCD27ϩ B cells (1 ϫ 105 cells per well) were pression of IgD and CD27 before or after activation with PMA and iono- incubated in complete RPMI medium in 96-well plates in the presence or mycin or with anti-CD40 Ab in the whole-cell mode of the patch-clamp absence of 10 nM PMA plus 175 nM ionomycin, and with or without ShK technique with an EPC-9 HEKA amplifier (HEKA Elektronik Dr. Schulze, (Bachem Biosciences) or TRAM-34 (30). [3H]Thymidine (1 ␮Ci/well) was Lambrecht, Germany). All experiments were conducted at room tempera- added after 24 h to the IgDϪCD27ϩ (which responded faster than the IgDϩ ture, and series resistance compensation was used for Kv currents when cells) and after 36 h to the IgDϩ cells. ShK and TRAM-34 were chosen for they exceeded 2 nA. Cells were stained for IgD and CD27 on ice in com- these experiments as Kv1.3 and IKCa1 blockers, because both compounds plete RPMI with FITC-conjugated mouse anti-human CD27 mAb (M- have demonstrated therapeutic efficacy in animal models of disease (16). 778 Kv1.3 IN MEMORY B CELLS Downloaded from FIGURE 2. IKCa1 currents in IgDϪCD27ϩ memory and IgDϩCD27Ϫ naive B cells. a, IgDϪCD27Ϫ class-switched memory B cells dialyzed with 1 ␮M free Ca2ϩ through the patch pipette exhibit a voltage-independent Ϫ KCa current in voltage ramps from 120 to 40 mV. This current is not present when the pipette solution contains 50 nM free Ca2ϩ. The voltage- activated current visible above Ϫ40 mV is Kv1.3. b, Effect of increasing

concentrations of the selective IKCa1 inhibitor TRAM-34 on the KCa cur- http://www.jimmunol.org/ rent in a IgDϪCD27ϩ memory B cell. Kv1.3 was completely blocked by 1 nM ShK-Dap22 in this experiment. c, Kv1.3 and IKCa1 currents in IgDϪCD27ϩ (left) and IgDϩCD27Ϫ (right) cells. The current seen at voltages below Ϫ40 mV is carried by IKCa1, whereas the current at more positive voltages is a combination of Kv1.3 (blocked by 1 nM ShK-F6CA) and IKCa1 (blocked by 250 nM TRAM-34). The inset in the right panel shows the current in the IgDϩCD27Ϫ naive cell on a ϫ15 expanded scale. by guest on September 24, 2021 [3H]Thymidine incorporation was then determined after a further 12 h. Counts in the presence of blockers were normalized to maximal counts from the same experiment using the following formula: blocker cpm Ϫ resting cpm/maximal cpm Ϫ resting cpm (counts for IgDϩ cells were typically ϳ80,000 cpm, and for IgDϪCD27ϩ cells ϳ10,000 cpm). For anti-CD40 activation, 5 ϫ 104 IgDϩ or IgDϪCD27ϩ cells were incubated with 2 ϫ 104 irradiated CD32-transfected K562 cells and 1 ␮g/ml anti-CD40 mAb for 48 h (IgDϪCD27ϩ) or 96 h (IgDϩ) in round-bottom 96-well plates to allow suffi- cient contact. [3H]Thymidine was added for the last 12 h of culture (maximal counts for IgDϩ cells were 35,000 cpm, and for IgDϪCD27ϩ cells 7,000 cpm). Results Kϩ channel expression pattern of quiescent class-switched memory B cells distinguishes this subset from other B cells Using the pan-B cell marker CD19, we isolated B lymphocytes from PB of healthy volunteers (Ͼ98% purity) and stained them with fluorophore-conjugated Abs specific for IgD (orange, PE) and CD27 (green, FITC). The different B cell subsets were visualized by fluorescence microscopy, and their Kϩ channel expression patterns were determined by whole-cell patch clamp. In the example Ϫ ϩ FIGURE 1. IgDϪCD27ϩ memory B cells express much larger Kv1.3 shown in Fig. 1a, the patch-clamped IgD CD27 memory cell is currents than naive B cells. a, Transmitted light (left) and fluorescent image distinguished from the surrounding naive IgDϩCD27Ϫ cells by its (right) of CD19ϩ B cells stained with PE-conjugated IgD and FITC-con- Ϫ ϩ jugated CD27 Abs. A IgD CD27 memory B cell is patch-clamped. b,KV currents in both IgDϪCD27ϩ memory (left) and IgDϩCD27Ϫ naive (right) B cells exhibit the characteristic use-dependence of Kv1.3. Currents were currents in both subsets by ShK. Currents were elicited by 200-ms depo- elicited by 200-ms pulses from Ϫ80 to 40 mV applied every 1 s. The larizing pulses from Ϫ80 to 40 mV applied every 30 s. e, The Kv1.3- numbers 1, 2, and 3 above the traces refer to the first, second, and third specific ShK derivative ShK-F6CA completely inhibits the KV current in depolarizing pulse. The inset in the right panel shows the current in the both IgDϪCD27ϩ (left) and IgDϩCD27Ϫ (right) B cells, whereas the ϩ Ϫ ϩ IgD CD27 naive cell on a ϫ18 expanded scale. c, Normalized peak K Kv1.1 blocker DTX-I has no effect on the current at 100 nM. f, Dose- Ϫ ϩ conductance-voltage relationship for the KV current in IgD CD27 mem- dependent block of KV currents in both subsets by Psora-4, the most potent ϩ Ϫ ory (left) and IgD CD27 naive (right) B cells (n ϭ 3). The line through small-molecule inhibitor of Kv1.3. All experiments shown in bÐf were 2ϩ the points was fitted with the Boltzmann equation. d, Blockade of KV conducted with a Ca -free KF-based pipette solution. The Journal of Immunology 779

Table I. Comparison of the biophysical and pharmacological properties of the KV and KCa currents in human B cells with the KV and KCa currents in human T cells and the cloned Kv1.3 and IKCa1 channelsa

Kv1.3 Human ϩ Ϫ Ϫ ϩ KV IgD CD27 IgD CD27 T Cells Cloned Kv1.3 IKCa1 Human T Cells Cloned IKCa1 Ϫ Ϫ Ϫ Ϫ V1/2 32 mV 31 mV 36 mV 35 mV ␶ Ϯ Ϯ Ϯ h 240 58 ms 260 69 ms 178 ms 250 51 ms ShK 11 Ϯ 2pM 10Ϯ 1pM 10Ϯ 1pM 11Ϯ 2pM ShK-Dap22 58 Ϯ 12 pM 55 Ϯ 9pM 52Ϯ 10 pM 25 pM Charybdotoxin 3.0 Ϯ 0.5 nM 3.2 Ϯ 0.5 nM 2.5 nM 2.6 nM Margatoxin 115 Ϯ 8 pM 108 Ϯ 14 pM 110 Ϯ 16 pM 100 pM ShK-F6CA 39 Ϯ 8pM 42Ϯ 10 pM ND 48 Ϯ 4pM Psora-4 3.2 Ϯ 0.5 nM 2.7 Ϯ 0.5 nM 3.0 Ϯ 0.3 nM 2.9 Ϯ 0.3 nM Dendrotoxin-1 No effect No effect No effect No effect Dendrotoxin-␬ No effect No effect ND No effect

KCa TRAM-34 20 Ϯ 2nM 18Ϯ 3nM 20Ϯ 3nM 20Ϯ 3nM ChTX 5.5 Ϯ 1 nM 5.0 Ϯ 0.8 nM 3 Ϯ 2nM 3nM

a Kv1.3: Compounds were tested at least three times at three to five concentration, and Kd values were determined by fitting the resulting inhibition of peak current at 40 mV with the Hill equation. Dendrotoxin-1 and its Kv1.1-selective derivative dendrotoxin-␬ showed no effect at 100 nM. Values for human T cells and Kv1.3 are from Refs. 2, 14, ␶ Ϫ Downloaded from 15, 31, 35, 37, 84, and 85. V1/2, Half-activation voltage; h, inactivation time constant. IKCa1: Kd values were determined by fitting the reduction of slope conductance at 80 ϩ Ϫ mV to the Hill equation. The measurements on IgD CD27 B cells were performed after activation because the KCa currents are much larger (see Fig. 4a). Values for human T cells and cloned IKCa1 are from Refs. 14, 30, and 86. larger size (left) and its green fluorescence (right). Both subsets ex- The four B cell subsets defined by CD27 and IgD expression are ϩ pressed voltage-gated (KV) and calcium-activated (KCa)K currents shown in the flow cytometry profile in Fig. 3a. The relative pro- with the characteristic fingerprints of Kv1.3 and IKCa1 channels portions of each subset varied in different individuals: naive http://www.jimmunol.org/ (Figs. 1 and 2; Table I). The amplitude of both the Kv1.3 and the IgDϩCD27Ϫ cells (45–73% in five donors), IgDϩCD27ϩ memory Ϫ ϩ IKCa1 current was substantially larger in the IgD CD27 class- cells (3–21%), class-switched IgDϪCD27ϩ memory cells ϩ Ϫ switched memory B cells than in IgD CD27 naive B cells (Figs. 1 (4–20%), and IgDϪCD27Ϫ cells (2–11%). The activation markers and 2). Staining with the anti-IgD and the anti-CD27 Abs did not alter CD86 and CD38 were not expressed or expressed only at very the properties or the expression of the Kv1.3 and IKCa1 currents (data low levels in all four subsets (data not shown), suggesting that not shown). these cells were quiescent. The majority (88–95%) of class- KV currents in both B cell subsets exhibited use-dependence, a switched memory cells expressed cell surface CD80 (CD80 ϭ property characteristic of Kv1.3, in which rapid repetitive depo- B7.1 ϭ ligand for CD28), whereas only 20–40% of IgDϩCD27ϩ by guest on September 24, 2021 larizing pulses cause a progressive decrease in the KV current am- memory cells expressed this marker and naive B cells were CD80 plitude due to channel trapping in the inactivated state (Fig. 1b). negative (Fig. 3b). Identical results were obtained with tonsillar B Their half-activation voltage and inactivation time-constant were cells. also similar to Kv1.3 (Fig. 1c; Table I). The polypeptides ShK Representative Kv1.3 and IKCa1 currents of the four major B (Fig. 1d), ShK-Dap (22), charybdotoxin, and margatoxin blocked cell subsets are shown next to the respective FACS quadrants in the B cell K currents with the same potencies as Kv1.3 (Table I). V Fig. 3a. The properties of the K and K currents were identical ShK-F6CA, an analog of ShK (37) that exhibits ϳ80-fold selec- V Ca in the four B cell subsets. The amplitude of the Kv1.3 and IKCa1 tivity for Kv1.3 over closely related K family channels, and Pso- V currents in quiescent class-switched B cells was significantly ra-4 (31), the most potent small molecule inhibitor of Kv1.3, also larger than in the other three B cell subsets. We determined chan- blocked the K current at concentrations that block Kv1.3 (Fig. 1, V nel numbers per cell by dividing the whole-cell conductance for e and f; Table I). Dendrotoxin-1 and dendrotoxin-␬, two polypep- each channel in each cell with the corresponding single-channel tides that inhibit the Kv1.1 channel that has been reported in mouse conductance values. The Kv1.3 and IKCa1 channel numbers per thymocytes (38) and mouse T cells (39, 40) but not in human T cell in the four quiescent B cell subsets are shown in Fig. 4a. Naive cells (6), had no effect on the K currents in the B cell subsets (Fig. V ϳ 1e; Table I). Taken together, these results show that the biophys- B cells expressed small numbers of Kv1.3 ( 100/cell) and IKCa1 ϳ ϩ ϩ ical and pharmacological properties of the K channel in human B ( 5/cell) channels. IgD CD27 memory B cells expressed more V ϳ cells are identical to those of Kv1.3, and suggest that Kv1.3 is the Kv1.3 channels ( 250/cell) than naive cells but the same number of IKCa1 channels (ϳ7/cell). IgDϪCD27Ϫ cells also expressed only KV channel expressed in human B cells (14–16, 35). small numbers of Kv1.3 (ϳ70/cell) and IKCa1 (ϳ18/cell) chan- We also observed KCa currents in both B cell subsets when Ϫ ϩ recording with a pipette solution containing 1 ␮M free calcium. In nels. In contrast, class-switched IgD CD27 cells expressed Ϫ ϩ ϳ Ͻ the ramp protocol from an IgD CD27 memory B cell a calcium- 10–20 times more Kv1.3 channels ( 2400/cell; p 0.00001 for dependent, voltage-independent, weakly inwardly rectifying K all three cases) and 4–8 times more IKCa1 channels (ϳ60/cell; Ca ϩ ϩ current is seen that reverses at Ϫ80 mV (Fig. 2a). This current was p Ͻ 0.00001 (naive and IgD CD27 ); p ϭ 0.00234 Ϫ Ϫ blocked by the IKCa1-specific inhibitor TRAM-34 (Fig. 2b) and by (IgD CD27 )) than the other three subsets. This channel expres- charybdotoxin with potencies identical to those of the cloned IKCa1 sion pattern has not been seen before in any quiescent lymphoid channel (Table I). Together, these results strongly argue that the subset. The corresponding B cell subsets in the human tonsil ex- ϩ IKCa1 channel underlies the KCa currents in naive and class-switched hibited K channel expression patterns identical with their PB human B cells. Furthermore, the complete inhibition of all Kϩ current counterparts (see Fig. 4a). Because class-switched memory B cells by the combination of 1 nM ShK-F6CA and 250 nM TRAM-34 (Fig. are significantly larger than the other three subsets, we normalized 2, c and d) demonstrated that Kv1.3 and IKCa1 are the only Kϩ channel expression levels for cell size by calculating the channel channels present in circulating human B cells. density per square micrometer of surface area. Class-switched B 780 Kv1.3 IN MEMORY B CELLS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Kϩ channel expression in B cells subsets. a, Flow cytometry profile showing IgD and CD27 expression in quiescent CD19ϩ B cells. Four subsets are distinguished: IgDϩCD27Ϫ (naive), IgDϩCD27ϩ, IgDϪCD27ϩ (class-switched memory B cell), and IgDϪCD27Ϫ. Representative Kv1.3 and IKCa1 currents are shown next to each FACS quadrant. Kv1.3 currents were recorded with depolarizing steps from Ϫ80 to 40 mV every 30 s with KF-based pipette solution. IKCa1 currents were elicited by 200-ms ramp pulses from Ϫ120 to 40 mV with 1 ␮M free Ca2ϩ in the pipette solution and 1 nM specific Kv1.3 blocker ShK-Dap (22, 84) in the bath. b, Flow cytometry profile showing CD80 expression on naive IgDϩCD27Ϫ (left), IgDϩCD27ϩ (middle), and class-switched IgDϪCD27ϩ (right) B cells.

cells had 6- to 21-fold higher Kv1.3 ( p Ͻ 0.00001 in all three visualized by ﬂuorescence microscopy. IgAϩCD27ϩ (Fig. 4b, left) cases) and 2- to 8-fold higher IKCa1 channel densities ( p ϭ and IgGϩCD27ϩ (middle) cells expressed large Kv1.3 currents 0.00012–0.05663) than the other three subsets (Fig. 4a). that exhibited use-dependence. IgGϩ cells expressed slightly Class-switched IgDϪCD27ϩ memory cells express other Ig iso- higher Kv1.3 channel numbers per cell than IgAϩ cells ( p Ͻ 0.05). types on the cell surface in place of IgD (20, 22). Therefore, patch- Both class-switched subsets expressed 10-to 20-fold more Kv1.3 clamp recording was done on IgAϩCD27ϩ and IgGϩCD27ϩ cells channels than naive or IgDϩCD27ϩ cells (Fig. 4b, right). The Journal of Immunology 781 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Numbers of Kϩ channels expressed per cell in resting and activated cells of the four B cell subsets. a, Scatterplot of Kv1.3 and IKCa1 channel numbers per cell in the four B cell subsets from PB in the resting state (E, ‚, Ⅺ, ᭛) and after activation (F, Œ, f, ᭜). The bar shows the mean of the respective subset. Mean channel numbers per cell (ϮSEM), mean cell capacitance (a measure of cell size), and mean Kv1.3 and IKCa1 channel densities are shown in a table below the ﬁgure. Similar results were obtained with tonsillar B cells: naive resting (63 Ϯ 4 Kv1.3 channels, n ϭ 10; 3 Ϯ 1 IKCa1 channels, n ϭ 6); naive activated (108 Ϯ 10 Kv1.3, n ϭ 15; 742 Ϯ 90 IKCa1, n ϭ 14); IgDϩCD27ϩ resting (245 Ϯ 26 Kv1.3, n ϭ 8; 8 Ϯ 1.2 IKCa1, n ϭ 5); IgDϩCD27ϩ activated (213 Ϯ 20 Kv1.3, n ϭ 13; 880 Ϯ 92 IKCa1, n ϭ 12); class-switched IgDϪCD27ϩ resting (1817 Ϯ 124 Kv1.3, n ϭ 11; 47 Ϯ 13 IKCa1, n ϭ 7); and class-switched IgDϪCD27ϩ activated (2285 Ϯ 232 Kv1.3, n ϭ 14; 80 Ϯ 11 IKCa1, n ϭ 8). A one-way ANOVA test revealed that the Kv1.3 and IKCa1 expression levels were not signiﬁcantly different between PB or tonsillar B cells from each subset. b, Kv1.3 currents (left and middle) and Kv1.3 channel numbers per cell (right) in class-switched CD27ϩIgAϩ (1648 Ϯ 364 Kv1.3, 3.8 Ϯ 0.2 pF, n ϭ 12) and CD27ϩIgGϩ (1966 Ϯ 234 Kv1.3, 3.7 Ϯ 0.2 pF, n ϭ 12) B cells.

In summary, differentiation of human B cells from a naive to a set (class-switched B cells based on our results) has been reported class-switched memory B cell stage is accompanied by a change in to effectively present Ag to CD4ϩ T cells without requiring pre- Kϩ channel expression. Class-switched B cells express signiﬁ- activation, activate at lower thresholds, and differentiate rapidly cantly higher numbers of Kv1.3 and IKCa1 channels than the other into cells that secrete large amounts of class-switched Abs (41), three B cell subsets. Our ﬂow cytometry data show that the ma- and has also been implicated in autoimmune pathophysiology (42– jority of class-switched B cells express the activation marker 45). The Kv1.3high pattern of these cells may position them for CD80, the ligand for CD28. Interestingly, the CD27ϩCD80ϩ sub- rapid activation. 782 Kv1.3 IN MEMORY B CELLS

IKCa1 is up-regulated in naive and IgDϩCD27ϩ B cells determine the regional distribution of Kϩ channels in intact tissue following activation, whereas class-switched memory B cells samples and to compare it with our electrophysiological data, we retain their Kv1.3high expression following activation stained paraffin sections of human tonsil and spleen with a poly- We examined the effect of mitogenic activation on channel ex- clonal Ab that has been previously demonstrated to be specific for ϩ Kv1.3 (36). In the tonsillar section shown in Fig. 5a, a ring of pression in the four human B cell subsets. PB CD19 B lympho- ϩ cytes were stimulated for 30–96 h with anti-CD40 Ab (presented IgD cells is seen in the mantle of the B cell follicles surrounding by CD32-transfected K562 cells (34)) to mimic T cell-mediated the germinal center (GC). Robust Kv1.3 staining was detected activation through CD40 (33, 46), or with a combination of PMA within the GC where class-switched memory B cells are found (Fig. 5b) but not in the mantle where IgDϩ B cells reside. The plus ionomycin (32). Cells were immunostained for IgD and ϩ CD27, and then visualized by fluorescence microscopy and patch expression pattern in the GC varied—in some cases, Kv1.3 cells clamped. Analysis of multiple cells showed that naive, were dispersed throughout (Fig. 5b), whereas in others, they were IgDϩCD27ϩ and IgDϪCD27Ϫ B cells, up-regulated IKCa1 ex- clustered at one edge of the GC (d). Additional Kv1.3 staining was seen outside the GC (Fig. 5c) in a region that has been described pression following activation, whereas Kv1.3 levels did not change Ϫ (Fig. 4a). When normalized for cell size, IKCa1 density in acti- as marginal zone (MZ)-like, because it contains IgD class- vated cells of these three subsets was ϳ45-fold higher ( p Ͻ switched memory B cells (47, 48). Serial sections stained for 0.000001) than in the resting state, whereas Kv1.3 density was Kv1.3 (Fig. 5d) and CD27 (e) confirmed that these markers were 4-fold lower ( p ϭ 0.00003). In contrast, class-switched memory B expressed on the same population of cells in the GC and the MZ- cells augmented Kv1.3 levels following activation, but this in- like area. No significant Kv1.3 staining was seen in the T cell zone crease was proportionate to the change in size and did not alter (data not shown) consistent with the low Kv1.3 expression levels Downloaded from channel density (Fig. 4a). IKCa1 density in these cells did not of resting naive, TCM and TEM cells (15). In the human spleen, change with activation. Similar results were obtained with tonsillar Kv1.3 and CD27 staining was detected in the MZ (data not shown) where CD27ϩ B cells are located (49, 50). No staining was ob- B cells (Fig. 4a). Thus, activation enhances IKCa1 density and high reduces Kv1.3 density in naive, IgDϩCD27ϩ memory and served with rabbit control IgG. In summary, Kv1.3 cells are IgDϪCD27Ϫ B cells, but has no significant effect on channel den- found in the GC and MZ of the tonsil and spleen where class- sity in class-switched IgDϪCD27ϩ memory B cells, which retain switched memory B cells reside or traffic through. http://www.jimmunol.org/ their Kv1.3high pattern. IKCa1 blockade suppresses proliferation of naive B cells and Kv1.3high B cells reside in memory compartments of human IgDϩCD27ϩ memory B cells, whereas Kv1.3 blockade lymphoid tissues suppresses proliferation of class-switched memory B cells The number of Kv1.3 channels (ϳ2000 channels/cell) in quiescent The availability of highly specific Kv1.3 and IKCa1 inhibitors and class-switched memory B cells should be sufficiently high to detect the subset-specific patterns of Kv1.3 and IKCa1 expression raise Kv1.3 protein within the B cell compartments of human secondary the possibility of using IKCa1-specific inhibitors to suppress the ϩ ϩ

lymphoid tissues by immunostaining with Kv1.3-speciﬁc Abs. To proliferation of naive and IgD CD27 memory B cells and Kv1.3 by guest on September 24, 2021

FIGURE 5. Localization of IgDϩ, Kv1.3ϩ and CD27ϩ cells in the human tonsil. a, IgDϩ cells outline the mantle (M) of the B cell follicles (ϫ40). b and c, Positive Kv1.3 staining is seen in the GC (b) and the adjacent region that is consistent with the MZ (c) (both ϫ200). d and e, Kv1.3 (d) and CD27 (e) stain the same areas in serial sections from human tonsil (ϫ100). All sections apart from the one shown in a are counterstained with hematoxylin. The Journal of Immunology 783 blockers to suppress class-switched Kv1.3high memory B cells. To Discussion ϩ test this idea, human tonsillar CD19 B cells were isolated and Using whole-cell patch-clamp recording in conjunction with flu- ϩ then further separated into two subsets, an IgD subset containing orescence microscopy, we demonstrate a switch in Kϩ channel dom- ϩ ϩ both naive and IgD CD27 cells, and a second subset containing inance from IKCa1 to Kv1.3 during differentiation of naive and Ϫ ϩ class-switched IgD CD27 memory B cells. Both subsets were IgDϩCD27ϩ B cells into class-switched memory B cells. As shown activated with anti-CD40 Ab (33, 46) or with a combination of in Fig. 7, naive and IgDϩCD27ϩ memory B cells up-regulate IKCa1 PMA and ionomycin (32, 33, 51–56) in the presence or absence of expression during activation, and their proliferation is suppressed by the IKCa1 blocker TRAM-34 or the Kv1.3 blocker ShK, and the specific IKCa1 inhibitor TRAM-34 (6) but not by Kv1.3 blockade. 3 [ H]thymidine incorporation was measured 24–72 h later. In keep- In contrast, class-switched IgDϪCD27ϩ memory B cells express high ing with our expectation, TRAM-34 suppressed the proliferation of ϩ levels of Kv1.3 channels in the resting state, a pattern not seen pre- the IgD subset (Fig. 6) with EC50 values of 200 nM (anti-CD40 viously in any lymphoid subset. The Kv1.3 inhibitor ShK suppressed Ab) and 100 nM (PMA plus ionomycin), and ShK had no effect at mitogen-driven proliferation of this subset, whereas the IKCa1 inhib- a concentration (10 nM) that completely blocks Kv1.3 channels. itor TRAM-34 was much less effective (Fig. 7). These Kv1.3high At a 10-fold higher ShK concentration (100 nM) that also affects class-switched memory cells reside in the GC and MZs of the human IKCa1 channels, 40–50% suppression was observed. Also, as an- tonsil and spleen. The functional dominance of IKCa1 in naive and ticipated, ShK suppressed the proliferation of class-switched IgDϩCD27ϩ B cells, and Kv1.3 in class-switched memory B cells Kv1.3highIgDϪCD27ϩ memory B cells with EC values of 1 nM 50 offers a powerful way to manipulate the activity of these subsets with (anti-CD40 Ab) and 4 nM (PMA plus ionomycin), concentrations specific IKCa1 and Kv1.3 inhibitors. that selectively block Kv1.3, whereas TRAM-34 was ineffective ϩ Downloaded from The changes in K channel expression observed in the B cell (EC Ͼ 1 ␮M) (Fig. 6). Similar results were obtained with Pso- 50 lineage parallel those reported to be seen in the T cell lineage (Fig. ra-4, the most potent small molecule inhibitor of Kv1.3 (31). Pso- 7b). Naive T cells and T cells start with 300 Kv1.3 and 10 ra-4 suppressed the proliferation of IgDϪCD27ϩ memory B cells CM IKCa1 channels/cell, and up-regulate IKCa1 expression to ϳ500– with an EC of 250 nM (data not shown) but had no effect on the 50 600/cell with little change in Kv1.3 numbers (15, 37). T cells, proliferation of IgDϩ B cells at concentrations up to 1 ␮M. These EM in contrast, up-regulate Kv1.3 upon activation with no change in results indicate that Kv1.3 blockers preferentially suppress the pro- ϩ ϩ http://www.jimmunol.org/ IKCa1 expression. Thus, naive and early memory (IgD CD27 ; liferation of class-switched memory B cells, whereas IKCa1 block- ϩ ϩ T ) cells of both lineages up-regulate IKCa1 during activation, ers suppress the proliferation of naive and IgD CD27 B cells. CM whereas late memory (class-switched B cells, TEM) cells of both lineages up-regulate Kv1.3 (Fig. 7). Differences in the ratio of IKCa1 and Kv1.3 numbers may contribute to differences in Ca2ϩ signaling patterns and thereby influ- ence the function of distinct T and B cell subsets. The shape and the nature of the Ca2ϩ signal regulate gene expression in response

to antigenic stimulation (57, 58). For example, lymphocytes in by guest on September 24, 2021 which IKCa1 predominates (e.g., activated naive and early memory cells) have been reported to have an enhanced tendency to exhibit oscillatory Ca2ϩ signals in response to stimulation, presumably because calcium entry is tightly coupled to the opening of IKCa1 channels (13, 59, 60). Kv1.3 might promote trafﬁcking of lymphocytes to inﬂamed tissues via its physical and functional ␤ coupling to 1 integrins and its reported role in cell adhesion and migration (61). As suggested by two recent papers on Kv1.3-transfected Jurkat and human CTLs, Kv1.3 may also play an important role in formation of the immunological synapse (62) and in the interaction of CD8ϩ cells with their targets (63). The differential channel expression patterns in naive and early memory cells vs late memory cells in both lineages are responsible for their differential sensitivities to Kv1.3 and IKCa1 blockers in proliferation assays (Fig. 7b). Because Kv1.3 channels predomi-

nate in resting naive T cells and TCM cells, these subsets are initially sensitive to Kv1.3 blockade but escape inhibition within 48 h through up-regulation of IKCa1 (6, 14, 15). During reactivation, TRAM-34 suppresses proliferation of these cells, whereas Kv1.3 blockers are ineffective. In contrast, the proliferation of naive and early memory B cells is suppressed by the IKCa1-speciﬁc inhibitor TRAM-34 and not Kv1.3, presumably because these cells start with small Kv1.3 currents, up-regulate IKCa1 rapidly, and take

longer to activate than T cells. In the late memory cell pool, TEM cells are highly sensitive to Kv1.3 but not IKCa1 blockers because they contain more Kv1.3 than IKCa1 channels in the resting state FIGURE 6. Suppression of B cell proliferation by Kϩ channel blockers. and augment this difference further during activation (Fig. 7b). Effect of TRAM-34 (f) and ShK (u) on anti-CD40 Ab- and PMA-plus- Kv1.3 blockers also suppress mitogen-driven proliferation of ionomycin-stimulated [3H]thymidine incorporation by IgDϩ cells (top)vs class-switched memory B cells but with 10-fold lower potency Ϫ ϩ IgD CD27 B cells (bottom). than TEM cells (Fig. 7b), possibly because memory B cells start 784 Kv1.3 IN MEMORY B CELLS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. a, Plot of average Kv1.3 vs IKCa1 channel numbers per cell (mean Ϯ SEM) in resting and activated B cells: IgDϩCD27Ϫ (E), IgDϩCD27ϩ (‚), IgDϪCD27ϩ (f), and IgDϪCD27Ϫ (Ⅺ). For each data plot, equal numbers of PB and tonsillar B cells were averaged. b, Diagrammatic representation of Kv1.3 and IKCa1 expression in naive and memory B and T cell subsets. The EC50 values for suppression of proliferation by the Kv1.3 blocker ShK and the IKCa1 blocker TRAM-34 are also shown.

with 5- to 10-fold higher numbers of Kv1.3 (ϳ2000/cell) channels rheumatoid arthritis in patients, but was abandoned due to adverse in the quiescent state than TEM cells (200–400/cell). effects resulting from blockade of cytochrome P450 enzymes (64, 65). Because IKCa1 is the functionally dominant Kϩ channel in na- The newer IKCa1-speciﬁc blockers TRAM-34, ICA-17043, and ive and early memory B and T cells, IKCa1 blockers might have 4-phenyl-4H-pyran (66–68), which lack this activity, should be use in suppressing acute immune reactions, for example, during evaluated for therapeutic use in autoimmune disorders. Furthermore, graft rejection and during the acute phases of autoimmune dis- cyclosporin A, an immunosuppressant widely used to prevent graft eases. Clotrimazole, an IKCa1 blocker, was reported to ameliorate rejection, synergizes with TRAM-34 in T cell proliferation assays The Journal of Immunology 785

(30), and combining these compounds could reduce the toxicity that 5. Koo, G. C., J. T. Blake, A. Talento, M. Nguyen, S. Lin, A. Sirotina, K. Shah, complicates cyclosporin A therapy. K. Mulvany, D. Hora, Jr., P. Cunningham, et al. 1997. Blockade of the voltage- gated potassium channel Kv1.3 inhibits immune responses in vivo. J. Immunol. Earlier studies showed that the Kv1.3 blockers margatoxin, 158:5120. correolide, and kaliotoxin suppressed delayed-type hypersensitiv- 6. Wulff, H., C. Beeton, and K. G. Chandy. 2003. Potassium channels as therapeutic targets for autoimmune disorders. Curr. Opin. Drug Discov. Devel. 6:640. ity (DTH) in miniswine and rats (5, 69, 70). Based on these results, 7. Chandy, K. G., H. Wulff, C. Beeton, M. Pennington, G. A. Gutman, and investigators in the field (5, 71) concluded that Kv1.3 blockers M. D. Cahalan. Potassium channels as targets for specific immunomodulation. inhibited the primary immune response and caused generalized Trends Pharmacol. Sci. In press. 8. Zweifach, A., and R. S. Lewis. 1993. Mitogen-regulated Ca2ϩ current of T lym- immunosuppression. However, our in vitro results, presented both phocytes is activated by depletion of intracellular Ca2ϩ stores. Proc. Natl. Acad. in this paper and in earlier reports (15, 16), suggest that Kv1.3 Sci. USA 90:6295. blockers preferentially target late memory cells of both lineages 9. Lewis, R. S., and M. D. Cahalan. 1995. Potassium and calcium channels in lymphocytes. Annu. Rev. Immunol. 13:623. (TEM and class-switched memory B cells), whereas naive and early 10. Cahalan, M. D., H. Wulff, and K. G. Chandy. 2001. Molecular properties and memory cells, although initially sensitive to Kv1.3 block, up-reg- physiological roles of ion channels in the immune system. J. Clin. Immunol. ulate IKCa1 and escape Kv1.3 inhibition. How might one recon- 21:235. 11. Lewis, R. S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu. Rev. cile these differing views? Recent evidence indicates that essen- Immunol. 19:497. tially all of the T cells isolated from DTH skin in humans exhibit 12. Hess, S. D., M. Oortgiesen, and M. D. Cahalan. 1993. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium the TEM memory phenotype (72), and TEM cells have the propen- influx. J. Immunol. 150:2620. sity to traffic to the skin (73). It is not surprising then that Kv1.3 13. Fanger, C. M., H. Rauer, A. L. Neben, M. J. Miller, H. Rauer, H. Wulff, blockers effectively inhibited DTH in miniswine via preferential J. C. Rosa, C. R. Ganellin, K. G. Chandy, and M. D. Cahalan. 2001. Calcium- activated potassium channels sustain calcium signaling in T lymphocytes. J. Biol. Downloaded from suppression of TEM cells. Specific Kv1.3 blockers might therefore Chem. 276:12249. constitute a new class of late memory-specific immunomodulators. 14. Ghanshani, S., H. Wulff, M. J. Miller, H. Rohm, A. Neben, G. A. Gutman, There is an increasing body of evidence implicating late mem- M. D. Cahalan, and K. G. Chandy. 2000. Up-regulation of the IKCa1 potassium channel during T-cell activation: molecular mechanism and functional conse- ory cells in the pathogenesis of MS, type-1 diabetes mellitus, rheu- quences. J. Biol. Chem. 275:37137. matoid arthritis, psoriasis, Crohn’s disease, and chronic graft-vs- 15. Wulff, H., P. A. Calabresi, R. Allie, S. Yun, M. Pennington, C. Beeton, and ϩ host disease (15–17, 74–77). Ag-specific targeting of pathogenic K. G. Chandy. 2003. The voltage-gated Kv1.3 K channel in effector memory T cells as new target for MS. J. Clin. Invest. 111:1703. autoreactive T and B cells is the most desirable therapeutic strat- 16. Beeton, C., H. Wulff, J. Barbaria, O. Clot-Faybesse, M. Pennington, D. Bernard, http://www.jimmunol.org/ egy for autoimmune diseases, but Ag-specific strategies may fail M. D. Cahalan, K. G. Chandy, and E. Beraud. 2001. Selective blockade of T lymphocyte Kϩ channels ameliorates experimental autoimmune encephalomy- due to the phenomenon of epitope spreading. A Kv1.3-based ther- elitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. USA 98:13942. apeutic approach that targets late memory cells and spares the bulk 17. O’Connor, K. C., A. Bar-Or, and D. A. Hafler. 2001. The neuroimmunology of of the adaptive immune response mediated by naive and early multiple sclerosis: possible roles of T and B lymphocytes in immunopathogen- esis. J. Clin. Immunol. 21:81. memory cells that depend on IKCa1 channels might be beneficial 18. Iglesias, A., J. Bauer, T. Litzenburger, A. Schubart, and C. Linington. 2001. T- under these circumstances. For example, channel-based suppres- and B-cell responses to myelin oligodendrocyte glycoprotein in experimental sion of autoantigen-specific memory B cells that have been re- autoimmune encephalomyelitis and multiple sclerosis. Glia 36:220. 19. Berger, T., P. Rubner, F. Schautzer, R. Egg, H. Ulmer, I. Mayringer, E. Dilitz, ported to contribute to epitope spreading (78), and effectively func- F. Deisenhammer, and M. Reindl. 2003. Antimyelin antibodies as a predictor of tion as APCs through enhanced autoantigen capture via their Ag- clinically definite multiple sclerosis after a first demyelinating event. N. Engl. by guest on September 24, 2021 specific membrane-bound Ig (41, 76, 79, 80), may have therapeutic J. Med. 349:139. 20. Klein, U., K. Rajewsky, and R. Kuppers. 1998. Human immunoglobulin value in autoimmune disorders. In proof-of-concept studies in rats, (Ig)MϩIgDϩ peripheral blood B cells expressing CD27 cell surface antigen carry Kv1.3 blockers have been shown to ameliorate adoptive experi- somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188:1679. mental autoimmune encephalomyelitis induced by myelin-specific 21. Agematsu, K., S. Hokibara, H. Nagumo, and A. Komiyama. 2000. CD27: a memory T cells (16, 70), a model for MS, and to prevent inflam- memory B-cell marker. Immunol. Today 21:204. matory bone resorption in experimental periodontal disease caused 22. Shi, Y., K. Agematsu, H. D. Ochs, and K. Sugane. 2003. Functional analysis of human memory B-cell subpopulations: IgDϩCD27ϩ B cells are crucial in sec- mainly by memory cells (81). Several small-molecule Kv1.3 in- ondary immune response by producing high affinity IgM. Clin. Immunol. hibitors with nanomolar potency have been developed over the last 108:128. decade (WIN-17317, CP-339818, U.K.-78,282, correolide, trans- 23. Atkinson, M. A., and G. S. Eisenbarth. 2001. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 358:221. N-propylcarbamoyloxy-PAC, sulfamidebenzamidoindanes, tetra- 24. Dorner, T., and G. R. Burmester. 2003. 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