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Immunomodulation and AutoimmunityDifferentiation: Implications for

Channel Expression during B Cell+K

K. George ChandyHeike Wulff, Hans-Günther Knaus, Michael Pennington and

http://www.jimmunol.org/content/173/2/776doi: 10.4049/jimmunol.173.2.776

2004; 173:776-786; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/173/2/776.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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K� Channel Expression during B Cell Differentiation:Implications for Immunomodulation and Autoimmunity 1

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

Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel ex-pression during differentiation of human B cells from naive to memory cells. Naive and IgD�CD27� memory B cells express smallnumbers 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 Bcells express high levels of Kv1.3 (�2000 channels/cell) and maintain their Kv1.3high expression after activation. Consistent withtheir channel phenotypes, proliferation of naive and IgD�CD27� memory B cells is suppressed by the specific IKCa1 inhibitorTRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memoryB cells is suppressed byStichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells.Therefore, specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets inimmunological disorders. The Journal of Immunology, 2004, 173: 776–786.

T wo K� channels in T lymphocytes, the voltage-gatedKv1.3 channel (also known as KCNA3 (HUGO nomen-clature)) and the Ca2�-activated IKCa1 channel (also

known as KCNN4 (HUGO nomenclature); KCa3.1 (InternationalUnion of Pharmacology nomenclature) (1)), regulate Ca2� signal-ing by controlling the membrane potential of lymphocytes (2–7).During activation, inositol 1,4,5-triphosphate generation inducesthe release of Ca2� from internal stores. Depletion of these Ca2�

stores causes calcium-release-activated calcium (CRAC)3 chan-nels to open in the membrane, and the resulting Ca2� influx sus-tains elevated levels of cytosolic Ca2� (8–11). Ca2� influxthrough CRAC channels is reduced at depolarized potentials, andconsequently, membrane depolarization attenuates the Ca2� signal(10–13). The driving force for Ca2� entry is restored by a coun-terbalancing efflux of K� ions and membrane hyperpolarizationbrought about by the opening of Kv1.3 channels in response tomembrane depolarization, and of IKCa1 channels as a conse-quence of elevated cytosolic Ca2�. The tightly coupled interplaybetween the K� channels and CRAC channels maintains enhanced

cytosolic Ca2� levels in the time frame required for optimal acti-vation. Coordinated activity of Ca2� and protein kinase C-depen-dent signaling pathways leads to new gene expression culminatingin cell proliferation. The relative contributions of Kv1.3 andIKCa1 in regulating this process depend on their expression levelsin the different lymphoid subsets.

Kv1.3 and IKCa1 channels are expressed in T lymphocytes in adistinct pattern that depends upon the state of activation and dif-ferentiation. In the quiescent state, naive, central memory (TCM)and effector memory (TEM) T cells express�250 Kv1.3 and�20IKCa1 channels per cell. The potent Kv1.3 blockerStichodactylahelianthus toxin (ShK) suppresses the proliferation of these cells,whereas the selective IKCa1 blocker TRAM-34 is ineffective (14).Activation induces differential expression of K� channels in thethree T cell subsets, leading to an altered channel phenotype andconsequently to altered responsiveness to Kv1.3 and IKCa1 block-ers. Naive and TCM cells up-regulate IKCa1 to�500/cell uponactivation via transcriptional mechanisms, and as a consequence,these subsets escape further Kv1.3 inhibition and become sensitiveto IKCa1 blockade (14, 15). TEM cells, in contrast, up-regulateKv1.3, and their proliferation is sensitive to Kv1.3 blockers (15).The discovery that the majority of myelin-reactive T cells in pa-tients with multiple sclerosis (MS), an autoimmune disease of theCNS, are Kv1.3high TEM cells that can be suppressed by Kv1.3blockers (15), has raised interest in the therapeutic potential ofKv1.3 blockers in autoimmune disorders. This idea has been suc-cessfully tested in experimental autoimmune encephalomyelitis, ananimal model for the disease (16).

An increasing body of evidence emphasizes the importance ofmemory B cells in the pathogenesis of autoimmune disorders (17–19). As in the T lineage, ionchannels may play functionally impor-tant roles in B cells and may serve as therapeutic targets for autoim-mune disorders. Four human B cell subsets can be distinguished bythe expression of IgD and CD27, a member of the TNFR family(20–22). Mature naive B cells express IgD but not CD27. Acquisitionof CD27 following somatic hypermutation results in a CD27�IgD�

memory B cell subset, and then during Ig class switching, replace-ment of surface IgD with other Ig isotypes yields CD27�IgD� mem-ory B cells. These class-switched memory B cells are a major sourceof pathogenic IgG autoantibodies that contribute to tissue damage in

*Department of Medical Pharmacology and Toxicology, University of California,Davis, CA 95616;†Institute for Biochemical Pharmacology, Medical University Inns-bruck, Innsbruck, Austria;‡Bachem Bioscience, Inc., King of Prussia, PA 19406; and§Department of Physiology and Biophysics, University of California, Irvine, CA92697

Received for publication February 6, 2004. Accepted for publication May 10, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the National Multiple Sclerosis Society (toK.G.C.), the Juvenile Diabetes Research Foundation (to K.G.C.), National Institutesof Health (MH59222; to K.G.C.), Rockefeller Brothers Fund (to K.G.C.), Fonds zurForderung der Wissenschaftlichen Forschung (Fonds zur Forderung der Wissen-schaftlichen Forschung P14954-PHA; to H.-G.K.), and the Cancer Research Coordi-nating Committee of the University of California (to H.W.).2 Address correspondence and reprint requests to Dr. Heike Wulff, Department ofMedical Pharmacology and Toxicology, School of Medicine, University of Califor-nia, Davis, One Shields Avenue, Tupper Hall Room 1311, Davis, CA 95616. E-mailaddress: hwulff@ucdavis.edu3 Abbreviations used in this paper: CRAC, calcium-release-activated calcium; TCM,central memory T; TEM, effector memory T; ShK,Stichodactyla helianthus toxin;MS, multiple sclerosis; PB, peripheral blood; KCa, Ca2�-activated K� current; KV,voltage-gated K� channel; GC, germinal center; MZ, marginal zone; DTH, delayed-type hypersensitivity.

The Journal of Immunology

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MS (17, 19), type-1 diabetes (23), and rheumatoid arthritis (24). Aminor population of IgD�CD27� B cells exists in most donors, buttheir functional role has yet to be defined (21).

Mature naive B cells were reported over a decade ago to expressKV and KCa currents with properties resembling those of Kv1.3and IKCa1. Studies with nonspecific K� channel inhibitors sug-gested a functional role for these channels in B cell mitogenesis(25–29). Because the changes in K� channel phenotype are func-tionally important in the T cell lineage, we were interested in de-termining whether a parallel switch in K� channel expression ac-companies differentiation from naive to memory cells in the B celllineage. Therefore, we examined the expression and functionalrole of Kv1.3 and IKCa1 during the differentiation of human Bcells from naive to memory cells using whole-cell patch-clamprecording in combination with fluorescence microscopy, flow cy-tometry, and specific channel blockers. Our studies demonstratethat a switch in K� channel expression accompanies B cell dif-ferentiation that parallels changes seen in the T cell lineage. Theplasticity of K� channel expression in both the B and T cell lin-eages and its functional ramifications afford promise for specificimmunomodulatory actions of K� channel blockers.

Materials and MethodsChannel blockers

ShK, charybdotoxin, margatoxin, ShK-Dap22, and ShK-F6CA were fromBachem Biosciences (King of Prussia, PA). TRAM-34 and Psora-4 weresynthesized as previously described by our group (30, 31). Dendrotoxin-Iand dendrotoxin-� were purchased from Sigma-Aldrich (St. Louis, MO).

B cell isolation

Peripheral blood (PB) CD19� cells were negatively selected from venousblood of healthy volunteers with RosetteSep B cell enrichment mixture(StemCell Technologies, Vancouver, BC, Canada). Human tonsil sampleswere obtained under an Institutional Review Board-approved protocol.Within 1 h after tonsillectomy, mononuclear cells were prepared by slicingthe sample into pieces and forcing them through a 70-�m filter to get asingle-cell suspension. CD19� cells were isolated using the StemSep BCell Negative Isolation System according to the manufacturer’s instruc-tions. Cells isolated from both the blood and the tonsil were found to be99.2–99.6% CD19� by flow cytometry (PE-conjugated mouse anti-humanCD19 mAb; HIB19; BD Pharmingen, San Diego, CA). IgD� cells werepositively selected from CD19� cells with a biotinylated anti-IgD mAb(IA6-2; BD Pharmingen) followed by an anti-biotin tetrameric Ab complexand magnetic beads (StemSep System; StemCell Technologies). From thecolumn flow-through containing IgD� B cells, we positively selectedIgD�CD27� cells with a custom-made anti-CD27� Ab mixture and mag-netic nanobeads according to the manufacturer’s protocol (EasySep Sys-tem; StemCell Technologies).

B cell culture and activation

CD19� cells from PB or tonsil were cultured in complete RPMI medium(RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM Na�

pyruvate, 1% nonessential amino acids, 100 U/ml penicillin, 100 �g/ml strep-tomycin, and 50 �M 2-ME). The cells were activated with 10 nM PMA and175 nM ionomycin for 48 h to achieve maximal B cell activation (32, 33). Tosimulate T cell-mediated activation of B cells, we cultured various B cellsubsets for 96 h with 1 �g/ml mouse anti-human CD40 mAb (5C3; BDPharmingen) presented by stably CD32-transfected irradiated (10,000 rad)K562 cells (B cells:K562 cells, 4:1) (34). The CD32-transfected K562 cellswere a generous gift from Dr. C. June (University of Pennsylvania, Philadel-phia, PA) (34).

Electrophysiology

Channel expression was studied in the four B cell subsets defined by ex-pression of IgD and CD27 before or after activation with PMA and iono-mycin or with anti-CD40 Ab in the whole-cell mode of the patch-clamptechnique with an EPC-9 HEKA amplifier (HEKA Elektronik Dr. Schulze,Lambrecht, Germany). All experiments were conducted at room tempera-ture, and series resistance compensation was used for Kv currents whenthey exceeded 2 nA. Cells were stained for IgD and CD27 on ice in com-plete RPMI with FITC-conjugated mouse anti-human CD27 mAb (M-

T271) and PE-conjugated mouse anti-human IgD mAb (IA6-2; both BDPharmingen). Cells were washed, put on poly-L-lysine-coated coverslips,kept in the dark at 4°C for 10–30 min to attach, visualized by fluorescencemicroscopy, and patch-clamped in the whole-cell configuration. For ex-periments on CD27�IgA� and CD27�IgG� class-switched B cells, westained CD19� cells with PE-conjugated mouse anti-human CD27 mAb(M-T271; BD Pharmingen) and FITC-conjugated F(ab�)2 of rabbit anti-human IgA or anti-human IgG (both DakoCytomation, Carpinteria, CA).Kv1.3 currents were elicited by repeated 200-ms pulses from a holdingpotential of �80 to 40 mV applied every 30 s unless otherwise stated.Kv1.3 currents were recorded in normal Ringer solution with a Ca2�-freepipette solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA,and 2 mM MgCl2 (pH 7.2; 300 mOsm). Whole-cell Kv1.3 conductancewas calculated from the peak current amplitude at 40 mV.

IKCa1 currents were elicited with voltage ramps from �120 to 40 mVof 200-ms duration applied every 10 s. The pipette solution contained 145mM K� aspartate, 2 mM MgCl2, 10 mM HEPES, 10 mM K2EGTA, and8.5 mM CaCl2 (1 �M free Ca2�) (pH 7.2; 290 mOsm). To reduce chlorideleak currents, we used a Na� aspartate external solution containing 160mM Na� aspartate, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 5 mMHEPES (pH 7.4; 300 mOsm). Whole-cell IKCa1 conductances were cal-culated from the slope of the current-voltage relationship at �80 mV.Kv1.3 and IKCa1 channel numbers per cell were determined by dividingthe whole-cell Kv1.3 or IKCa1 conductance by the single-channel conduc-tance value for each channel: Kv1.3, 12 pS; IKCa1, 11 pS (14, 35). Cellcapacitance, a direct measure of cell surface area, was constantly moni-tored during recording and noted for each cell, and the surface of each cellwas measured by the formula: 1 pF � 100 �m2. To normalize for cell size,we determined Kv1.3 and IKCa1 channel densities for each cell by divid-ing the channel number per cell by that cell’s surface area.

Statistical differences in channel numbers per cell and channel densityper square micrometer between different B cell subsets were determined byone-way ANOVA with a significance level of p � 0.05.

Three-color flow cytometry

Negatively selected CD19� cells from PB or tonsil were stained for CD27,IgD, CD80, CD86, or CD38 immediately after isolation. Cells were incubatedin PBS containing 2% goat serum with a combination of FITC-conjugatedmouse anti-human IgD mAb (IA6-2), PE-conjugated mouse anti-human CD27mAb (clone M-T271), and CyChrome-conjugated mouse anti-human CD80Ab (clone L307.1) or CyChrome-conjugated mouse anti-human CD86 Ab(clone 2331; all BD Pharmingen). A BD Biosciences (San Jose, CA) FACScanwith CellQuest software was used to analyze the stained cells.

Immunohistochemistry

Paraffin-embedded sections from human tonsil and spleen (donor �30years of age) were acquired from the University of California, Irvine, Pa-thology Department, under an Institutional Review Board-approved pro-tocol. Sections were dewaxed with xylene and rehydrated through an al-cohol gradient. Before staining with primary Abs, sections were heatedwith 10 mM Na citrate (pH 6.5) in a microwave for 15 min to retrieveantigenic determinants masked by paraffin embedding. After treatmentwith 1% H2O2 to inactivate endogenous peroxidase activity and blockingwith 5% goat serum in PBS for 12 h, sections were incubated with poly-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-technology, Santa Cruz, CA) for 2 h at room temperature. Bound primaryAbs were detected with a biotinylated goat anti-rabbit secondary Ab (1 h;1:1000; Jackson ImmunoResearch, West Grove, PA) followed by a HRP-conjugated avidin complex (Vectastain Elite ABC kit; Vector Laboratories,Burlingame, CA). After each incubation step, sections were rinsed withPBS three times for 5 min. Peroxidase activity was visualized with 3,3�-diaminobenzidine (DAB substrate kit for peroxidase; Vector Laboratories).Sections were counterstained with hematoxylin (Fisher Scientific, Hamp-ton, NH), dehydrated, and mounted with Permount (Fisher Scientific). Thepolyclonal rabbit Kv1.3 Ab was previously shown to be specific forKv1.3 (36).

[3H]Thymidine incorporation

Tonsillar IgD� or IgD�CD27� B cells (1 � 105 cells per well) wereincubated in complete RPMI medium in 96-well plates in the presence orabsence of 10 nM PMA plus 175 nM ionomycin, and with or without ShK(Bachem Biosciences) or TRAM-34 (30). [3H]Thymidine (1 �Ci/well) wasadded after 24 h to the IgD�CD27� (which responded faster than the IgD�

cells) and after 36 h to the IgD� cells. ShK and TRAM-34 were chosen forthese experiments as Kv1.3 and IKCa1 blockers, because both compoundshave demonstrated therapeutic efficacy in animal models of disease (16).

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[3H]Thymidine incorporation was then determined after a further 12 h.Counts in the presence of blockers were normalized to maximal counts fromthe same experiment using the following formula: blocker cpm � restingcpm/maximal cpm � resting cpm (counts for IgD� cells were typically�80,000 cpm, and for IgD�CD27� cells �10,000 cpm). For anti-CD40 ac-tivation, 5 � 104 IgD� or IgD�CD27� cells were incubated with 2 � 104

irradiated CD32-transfected K562 cells and 1 �g/ml anti-CD40 mAb for 48 h(IgD�CD27�) or 96 h (IgD�) in round-bottom 96-well plates to allow suffi-cient contact. [3H]Thymidine was added for the last 12 h of culture (maximalcounts for IgD� cells were 35,000 cpm, and for IgD�CD27� cells 7,000 cpm).

ResultsK� channel expression pattern of quiescent class-switchedmemory B cells distinguishes this subset from other B cells

Using the pan-B cell marker CD19, we isolated B lymphocytesfrom PB of healthy volunteers (�98% purity) and stained themwith fluorophore-conjugated Abs specific for IgD (orange, PE) andCD27 (green, FITC). The different B cell subsets were visualizedby fluorescence microscopy, and their K� channel expression pat-terns were determined by whole-cell patch clamp. In the exampleshown in Fig. 1a, the patch-clamped IgD�CD27� memory cell isdistinguished from the surrounding naive IgD�CD27� cells by its

FIGURE 1. IgD�CD27� memory B cells express much larger Kv1.3currents than naive B cells. a, Transmitted light (left) and fluorescent image(right) of CD19� B cells stained with PE-conjugated IgD and FITC-con-jugated CD27 Abs. A IgD�CD27� memory B cell is patch-clamped. b, KV

currents in both IgD�CD27� memory (left) and IgD�CD27� naive (right)B cells exhibit the characteristic use-dependence of Kv1.3. Currents wereelicited by 200-ms pulses from �80 to 40 mV applied every 1 s. Thenumbers 1, 2, and 3 above the traces refer to the first, second, and thirddepolarizing pulse. The inset in the right panel shows the current in theIgD�CD27� naive cell on a �18 expanded scale. c, Normalized peak K�

conductance-voltage relationship for the KV current in IgD�CD27� mem-ory (left) and IgD�CD27� naive (right) B cells (n � 3). The line throughthe points was fitted with the Boltzmann equation. d, Blockade of KV

FIGURE 2. IKCa1 currents in IgD�CD27� memory and IgD�CD27�

naive B cells. a, IgD�CD27� class-switched memory B cells dialyzed with1 �M free Ca2� through the patch pipette exhibit a voltage-independentKCa current in voltage ramps from �120 to 40 mV. This current is notpresent when the pipette solution contains 50 nM free Ca2�. The voltage-activated current visible above �40 mV is Kv1.3. b, Effect of increasingconcentrations of the selective IKCa1 inhibitor TRAM-34 on the KCa cur-rent in a IgD�CD27� memory B cell. Kv1.3 was completely blocked by 1nM ShK-Dap22 in this experiment. c, Kv1.3 and IKCa1 currents inIgD�CD27� (left) and IgD�CD27� (right) cells. The current seen at volt-ages below �40 mV is carried by IKCa1, whereas the current at morepositive 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 panelshows the current in the IgD�CD27� naive cell on a �15 expanded scale.

currents in both subsets by ShK. Currents were elicited by 200-ms depo-larizing pulses from �80 to 40 mV applied every 30 s. e, The Kv1.3-specific ShK derivative ShK-F6CA completely inhibits the KV current inboth IgD�CD27� (left) and IgD�CD27� (right) B cells, whereas theKv1.1 blocker DTX-I has no effect on the current at 100 nM. f, Dose-dependent block of KV currents in both subsets by Psora-4, the most potentsmall-molecule inhibitor of Kv1.3. All experiments shown in b–f wereconducted with a Ca2�-free KF-based pipette solution.

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larger size (left) and its green fluorescence (right). Both subsets ex-pressed voltage-gated (KV) and calcium-activated (KCa) K� currentswith the characteristic fingerprints of Kv1.3 and IKCa1 channels(Figs. 1 and 2; Table I). The amplitude of both the Kv1.3 and theIKCa1 current was substantially larger in the IgD�CD27� class-switched memory B cells than in IgD�CD27� naive B cells (Figs. 1and 2). Staining with the anti-IgD and the anti-CD27 Abs did not alterthe properties or the expression of the Kv1.3 and IKCa1 currents (datanot shown).

KV currents in both B cell subsets exhibited use-dependence, aproperty characteristic of Kv1.3, in which rapid repetitive depo-larizing pulses cause a progressive decrease in the KV current am-plitude due to channel trapping in the inactivated state (Fig. 1b).Their half-activation voltage and inactivation time-constant werealso similar to Kv1.3 (Fig. 1c; Table I). The polypeptides ShK(Fig. 1d), ShK-Dap (22), charybdotoxin, and margatoxin blockedthe B cell KV currents with the same potencies as Kv1.3 (Table I).ShK-F6CA, an analog of ShK (37) that exhibits �80-fold selec-tivity for Kv1.3 over closely related KV family channels, and Pso-ra-4 (31), the most potent small molecule inhibitor of Kv1.3, alsoblocked the KV current at concentrations that block Kv1.3 (Fig. 1,e and f; Table I). Dendrotoxin-1 and dendrotoxin-�, two polypep-tides that inhibit the Kv1.1 channel that has been reported in mousethymocytes (38) and mouse T cells (39, 40) but not in human Tcells (6), had no effect on the KV currents in the B cell subsets (Fig.1e; Table I). Taken together, these results show that the biophys-ical and pharmacological properties of the KV channel in human Bcells are identical to those of Kv1.3, and suggest that Kv1.3 is theonly KV channel expressed in human B cells (14–16, 35).

We also observed KCa currents in both B cell subsets whenrecording with a pipette solution containing 1 �M free calcium. Inthe ramp protocol from an IgD�CD27� memory B cell a calcium-dependent, voltage-independent, weakly inwardly rectifying KCa

current is seen that reverses at �80 mV (Fig. 2a). This current wasblocked by the IKCa1-specific inhibitor TRAM-34 (Fig. 2b) and bycharybdotoxin with potencies identical to those of the cloned IKCa1channel (Table I). Together, these results strongly argue that theIKCa1 channel underlies the KCa currents in naive and class-switchedhuman B cells. Furthermore, the complete inhibition of all K� currentby the combination of 1 nM ShK-F6CA and 250 nM TRAM-34 (Fig.2, c and d) demonstrated that Kv1.3 and IKCa1 are the only K�

channels present in circulating human B cells.

The four B cell subsets defined by CD27 and IgD expression areshown in the flow cytometry profile in Fig. 3a. The relative pro-portions of each subset varied in different individuals: naiveIgD�CD27� cells (45–73% in five donors), IgD�CD27� memorycells (3–21%), class-switched IgD�CD27� memory cells(4–20%), and IgD�CD27� cells (2–11%). The activation markersCD86 and CD38 were not expressed or expressed only at verylow levels in all four subsets (data not shown), suggesting thatthese cells were quiescent. The majority (88–95%) of class-switched memory cells expressed cell surface CD80 (CD80 �B7.1 � ligand for CD28), whereas only 20–40% of IgD�CD27�

memory cells expressed this marker and naive B cells were CD80negative (Fig. 3b). Identical results were obtained with tonsillar Bcells.

Representative Kv1.3 and IKCa1 currents of the four major Bcell subsets are shown next to the respective FACS quadrants inFig. 3a. The properties of the KV and KCa currents were identicalin the four B cell subsets. The amplitude of the Kv1.3 and IKCa1currents in quiescent class-switched B cells was significantlylarger than in the other three B cell subsets. We determined chan-nel numbers per cell by dividing the whole-cell conductance foreach channel in each cell with the corresponding single-channelconductance values. The Kv1.3 and IKCa1 channel numbers percell in the four quiescent B cell subsets are shown in Fig. 4a. NaiveB cells expressed small numbers of Kv1.3 (�100/cell) and IKCa1(�5/cell) channels. IgD�CD27� memory B cells expressed moreKv1.3 channels (�250/cell) than naive cells but the same numberof IKCa1 channels (�7/cell). IgD�CD27� cells also expressedsmall numbers of Kv1.3 (�70/cell) and IKCa1 (�18/cell) chan-nels. In contrast, class-switched IgD�CD27� cells expressed10–20 times more Kv1.3 channels (�2400/cell; p � 0.00001 forall three cases) and 4–8 times more IKCa1 channels (�60/cell;p � 0.00001 (naive and IgD�CD27�); p � 0.00234(IgD�CD27�)) than the other three subsets. This channel expres-sion pattern has not been seen before in any quiescent lymphoidsubset. The corresponding B cell subsets in the human tonsil ex-hibited K� channel expression patterns identical with their PBcounterparts (see Fig. 4a). Because class-switched memory B cellsare significantly larger than the other three subsets, we normalizedchannel expression levels for cell size by calculating the channeldensity per square micrometer of surface area. Class-switched B

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

KV IgD�CD27� IgD�CD27�Kv1.3 Human

T Cells Cloned Kv1.3 IKCa1 Human T Cells Cloned IKCa1

V1/2 �32 mV �31 mV �36 mV �35 mV�h 240 58 ms 260 69 ms 178 ms 250 51 msShK 11 2 pM 10 1 pM 10 1 pM 11 2 pMShK-Dap22 58 12 pM 55 9 pM 52 10 pM 25 pMCharybdotoxin 3.0 0.5 nM 3.2 0.5 nM 2.5 nM 2.6 nMMargatoxin 115 8 pM 108 14 pM 110 16 pM 100 pMShK-F6CA 39 8 pM 42 10 pM ND 48 4 pMPsora-4 3.2 0.5 nM 2.7 0.5 nM 3.0 0.3 nM 2.9 0.3 nMDendrotoxin-1 No effect No effect No effect No effectDendrotoxin-� No effect No effect ND No effect

KCa

TRAM-34 20 2 nM 18 3 nM 20 3 nM 20 3 nMChTX 5.5 1 nM 5.0 0.8 nM 3 2 nM 3 nM

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 mVwith 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, 14,15, 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 �80mV 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 humanT cells and cloned IKCa1 are from Refs. 14, 30, and 86.

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cells had 6- to 21-fold higher Kv1.3 ( p � 0.00001 in all threecases) and 2- to 8-fold higher IKCa1 channel densities ( p �0.00012–0.05663) than the other three subsets (Fig. 4a).

Class-switched IgD�CD27� memory cells express other Ig iso-types on the cell surface in place of IgD (20, 22). Therefore, patch-clamp recording was done on IgA�CD27� and IgG�CD27� cells

visualized by fluorescence microscopy. IgA�CD27� (Fig. 4b, left)and IgG�CD27� (middle) cells expressed large Kv1.3 currentsthat exhibited use-dependence. IgG� cells expressed slightlyhigher Kv1.3 channel numbers per cell than IgA� cells ( p � 0.05).Both class-switched subsets expressed 10-to 20-fold more Kv1.3channels than naive or IgD�CD27� cells (Fig. 4b, right).

FIGURE 3. K� channel expression in B cells subsets. a, Flow cytometry profile showing IgD and CD27 expression in quiescent CD19� B cells. Foursubsets are distinguished: IgD�CD27� (naive), IgD�CD27�, IgD�CD27� (class-switched memory B cell), and IgD�CD27�. Representative Kv1.3 andIKCa1 currents are shown next to each FACS quadrant. Kv1.3 currents were recorded with depolarizing steps from �80 to 40 mV every 30 s with KF-basedpipette solution. IKCa1 currents were elicited by 200-ms ramp pulses from �120 to 40 mV with 1 �M free Ca2� in the pipette solution and 1 nM specificKv1.3 blocker ShK-Dap (22, 84) in the bath. b, Flow cytometry profile showing CD80 expression on naive IgD�CD27� (left), IgD�CD27� (middle), andclass-switched IgD�CD27� (right) B cells.

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In summary, differentiation of human B cells from a naive to aclass-switched memory B cell stage is accompanied by a change inK� channel expression. Class-switched B cells express signifi-cantly higher numbers of Kv1.3 and IKCa1 channels than the otherthree B cell subsets. Our flow cytometry data show that the ma-jority of class-switched B cells express the activation markerCD80, the ligand for CD28. Interestingly, the CD27�CD80� sub-

set (class-switched B cells based on our results) has been reportedto effectively present Ag to CD4� T cells without requiring pre-activation, activate at lower thresholds, and differentiate rapidlyinto cells that secrete large amounts of class-switched Abs (41),and has also been implicated in autoimmune pathophysiology (42–45). The Kv1.3high pattern of these cells may position them forrapid activation.

FIGURE 4. Numbers of K� channels expressed per cell in resting and activated cells of the four B cell subsets. a, Scatterplot of Kv1.3 and IKCa1channel numbers per cell in the four B cell subsets from PB in the resting state (E, ‚, �, �) and after activation (F, Œ, f, �). The bar shows the meanof the respective subset. Mean channel numbers per cell (SEM), mean cell capacitance (a measure of cell size), and mean Kv1.3 and IKCa1 channeldensities are shown in a table below the figure. Similar results were obtained with tonsillar B cells: naive resting (63 4 Kv1.3 channels, n � 10; 3 1 IKCa1 channels, n � 6); naive activated (108 10 Kv1.3, n � 15; 742 90 IKCa1, n � 14); IgD�CD27� resting (245 26 Kv1.3, n � 8; 8 1.2IKCa1, n � 5); IgD�CD27� activated (213 20 Kv1.3, n � 13; 880 92 IKCa1, n � 12); class-switched IgD�CD27� resting (1817 124 Kv1.3, n �11; 47 13 IKCa1, n � 7); and class-switched IgD�CD27� activated (2285 232 Kv1.3, n � 14; 80 11 IKCa1, n � 8). A one-way ANOVA testrevealed 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 CD27�IgA� (1648 364 Kv1.3, 3.8 0.2 pF, n � 12) and CD27�IgG�

(1966 234 Kv1.3, 3.7 0.2 pF, n � 12) B cells.

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IKCa1 is up-regulated in naive and IgD�CD27� B cellsfollowing activation, whereas class-switched memory B cellsretain their Kv1.3high expression following activation

We examined the effect of mitogenic activation on channel ex-pression in the four human B cell subsets. PB CD19� B lympho-cytes were stimulated for 30–96 h with anti-CD40 Ab (presentedby CD32-transfected K562 cells (34)) to mimic T cell-mediatedactivation through CD40 (33, 46), or with a combination of PMAplus ionomycin (32). Cells were immunostained for IgD andCD27, and then visualized by fluorescence microscopy and patchclamped. Analysis of multiple cells showed that naive,IgD�CD27� and IgD�CD27� B cells, up-regulated IKCa1 ex-pression following activation, whereas Kv1.3 levels did not change(Fig. 4a). When normalized for cell size, IKCa1 density in acti-vated cells of these three subsets was �45-fold higher ( p �0.000001) than in the resting state, whereas Kv1.3 density was4-fold lower ( p � 0.00003). In contrast, class-switched memory Bcells augmented Kv1.3 levels following activation, but this in-crease was proportionate to the change in size and did not alterchannel density (Fig. 4a). IKCa1 density in these cells did notchange with activation. Similar results were obtained with tonsillarB cells (Fig. 4a). Thus, activation enhances IKCa1 density andreduces Kv1.3 density in naive, IgD�CD27� memory andIgD�CD27� B cells, but has no significant effect on channel den-sity in class-switched IgD�CD27� memory B cells, which retaintheir Kv1.3high pattern.

Kv1.3high B cells reside in memory compartments of humanlymphoid tissues

The number of Kv1.3 channels (�2000 channels/cell) in quiescentclass-switched memory B cells should be sufficiently high to detectKv1.3 protein within the B cell compartments of human secondarylymphoid tissues by immunostaining with Kv1.3-specific Abs. To

determine the regional distribution of K� channels in intact tissuesamples and to compare it with our electrophysiological data, westained paraffin sections of human tonsil and spleen with a poly-clonal Ab that has been previously demonstrated to be specific forKv1.3 (36). In the tonsillar section shown in Fig. 5a, a ring ofIgD� cells is seen in the mantle of the B cell follicles surroundingthe germinal center (GC). Robust Kv1.3 staining was detectedwithin the GC where class-switched memory B cells are found(Fig. 5b) but not in the mantle where IgD� B cells reside. Theexpression pattern in the GC varied—in some cases, Kv1.3� cellswere dispersed throughout (Fig. 5b), whereas in others, they wereclustered at one edge of the GC (d). Additional Kv1.3 staining wasseen outside the GC (Fig. 5c) in a region that has been describedas marginal zone (MZ)-like, because it contains IgD� class-switched memory B cells (47, 48). Serial sections stained forKv1.3 (Fig. 5d) and CD27 (e) confirmed that these markers wereexpressed on the same population of cells in the GC and the MZ-like area. No significant Kv1.3 staining was seen in the T cell zone(data not shown) consistent with the low Kv1.3 expression levelsof resting naive, TCM and TEM cells (15). In the human spleen,Kv1.3 and CD27 staining was detected in the MZ (data not shown)where CD27� B cells are located (49, 50). No staining was ob-served with rabbit control IgG. In summary, Kv1.3high cells arefound in the GC and MZ of the tonsil and spleen where class-switched memory B cells reside or traffic through.

IKCa1 blockade suppresses proliferation of naive B cells andIgD�CD27� memory B cells, whereas Kv1.3 blockadesuppresses proliferation of class-switched memory B cells

The availability of highly specific Kv1.3 and IKCa1 inhibitors andthe subset-specific patterns of Kv1.3 and IKCa1 expression raisethe possibility of using IKCa1-specific inhibitors to suppress theproliferation of naive and IgD�CD27� memory B cells and Kv1.3

FIGURE 5. Localization of IgD�, Kv1.3� and CD27� cells in the human tonsil. a, IgD� cells outline the mantle (M) of the B cell follicles (�40). band c, Positive Kv1.3 staining is seen in the GC (b) and the adjacent region that is consistent with the MZ (c) (both �200). d and e, Kv1.3 (d) and CD27(e) stain the same areas in serial sections from human tonsil (�100). All sections apart from the one shown in a are counterstained with hematoxylin.

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blockers to suppress class-switched Kv1.3high memory B cells. Totest this idea, human tonsillar CD19� B cells were isolated andthen further separated into two subsets, an IgD� subset containingboth naive and IgD�CD27� cells, and a second subset containingclass-switched IgD�CD27� memory B cells. Both subsets wereactivated with anti-CD40 Ab (33, 46) or with a combination ofPMA and ionomycin (32, 33, 51–56) in the presence or absence ofthe IKCa1 blocker TRAM-34 or the Kv1.3 blocker ShK, and[3H]thymidine incorporation was measured 24–72 h later. In keep-ing with our expectation, TRAM-34 suppressed the proliferation ofthe IgD� subset (Fig. 6) with EC50 values of 200 nM (anti-CD40Ab) and 100 nM (PMA plus ionomycin), and ShK had no effect ata concentration (10 nM) that completely blocks Kv1.3 channels.At a 10-fold higher ShK concentration (100 nM) that also affectsIKCa1 channels, 40–50% suppression was observed. Also, as an-ticipated, ShK suppressed the proliferation of class-switchedKv1.3highIgD�CD27� memory B cells with EC50 values of 1 nM(anti-CD40 Ab) and 4 nM (PMA plus ionomycin), concentrationsthat selectively block Kv1.3, whereas TRAM-34 was ineffective(EC50 � 1 �M) (Fig. 6). Similar results were obtained with Pso-ra-4, the most potent small molecule inhibitor of Kv1.3 (31). Pso-ra-4 suppressed the proliferation of IgD�CD27� memory B cellswith an EC50 of 250 nM (data not shown) but had no effect on theproliferation of IgD� B cells at concentrations up to 1 �M. Theseresults indicate that Kv1.3 blockers preferentially suppress the pro-liferation of class-switched memory B cells, whereas IKCa1 block-ers suppress the proliferation of naive and IgD�CD27� B cells.

DiscussionUsing whole-cell patch-clamp recording in conjunction with flu-orescence microscopy, we demonstrate a switch in K� channel dom-inance from IKCa1 to Kv1.3 during differentiation of naive andIgD�CD27� B cells into class-switched memory B cells. As shownin Fig. 7, naive and IgD�CD27� memory B cells up-regulate IKCa1expression during activation, and their proliferation is suppressed bythe specific IKCa1 inhibitor TRAM-34 (6) but not by Kv1.3 blockade.In contrast, class-switched IgD�CD27� memory B cells express highlevels of Kv1.3 channels in the resting state, a pattern not seen pre-viously in any lymphoid subset. The Kv1.3 inhibitor ShK suppressedmitogen-driven proliferation of this subset, whereas the IKCa1 inhib-itor TRAM-34 was much less effective (Fig. 7). These Kv1.3high

class-switched memory cells reside in the GC and MZs of the humantonsil and spleen. The functional dominance of IKCa1 in naive andIgD�CD27� B cells, and Kv1.3 in class-switched memory B cellsoffers a powerful way to manipulate the activity of these subsets withspecific IKCa1 and Kv1.3 inhibitors.

The changes in K� channel expression observed in the B celllineage parallel those reported to be seen in the T cell lineage (Fig.7b). Naive T cells and TCM cells start with 300 Kv1.3 and 10IKCa1 channels/cell, and up-regulate IKCa1 expression to �500–600/cell with little change in Kv1.3 numbers (15, 37). TEM cells,in contrast, up-regulate Kv1.3 upon activation with no change inIKCa1 expression. Thus, naive and early memory (IgD�CD27�;TCM) cells of both lineages up-regulate IKCa1 during activation,whereas late memory (class-switched B cells, TEM) cells of bothlineages up-regulate Kv1.3 (Fig. 7).

Differences in the ratio of IKCa1 and Kv1.3 numbers may con-tribute to differences in Ca2� signaling patterns and thereby influ-ence the function of distinct T and B cell subsets. The shape andthe nature of the Ca2� signal regulate gene expression in responseto antigenic stimulation (57, 58). For example, lymphocytes inwhich IKCa1 predominates (e.g., activated naive and early mem-ory cells) have been reported to have an enhanced tendency toexhibit oscillatory Ca2� signals in response to stimulation, pre-sumably because calcium entry is tightly coupled to the opening ofIKCa1 channels (13, 59, 60). Kv1.3 might promote trafficking oflymphocytes to inflamed tissues via its physical and functionalcoupling to �1 integrins and its reported role in cell adhesion andmigration (61). As suggested by two recent papers on Kv1.3-trans-fected Jurkat and human CTLs, Kv1.3 may also play an importantrole in formation of the immunological synapse (62) and in theinteraction of CD8� cells with their targets (63).

The differential channel expression patterns in naive and earlymemory cells vs late memory cells in both lineages are responsiblefor their differential sensitivities to Kv1.3 and IKCa1 blockers inproliferation assays (Fig. 7b). Because Kv1.3 channels predomi-nate in resting naive T cells and TCM cells, these subsets are ini-tially sensitive to Kv1.3 blockade but escape inhibition within 48 hthrough up-regulation of IKCa1 (6, 14, 15). During reactivation,TRAM-34 suppresses proliferation of these cells, whereas Kv1.3blockers are ineffective. In contrast, the proliferation of naive andearly memory B cells is suppressed by the IKCa1-specific inhibitorTRAM-34 and not Kv1.3, presumably because these cells startwith small Kv1.3 currents, up-regulate IKCa1 rapidly, and takelonger to activate than T cells. In the late memory cell pool, TEM

cells are highly sensitive to Kv1.3 but not IKCa1 blockers becausethey contain more Kv1.3 than IKCa1 channels in the resting stateand augment this difference further during activation (Fig. 7b).Kv1.3 blockers also suppress mitogen-driven proliferation ofclass-switched memory B cells but with 10-fold lower potencythan TEM cells (Fig. 7b), possibly because memory B cells start

FIGURE 6. Suppression of B cell proliferation by K� channel blockers.Effect of TRAM-34 (f) and ShK (u) on anti-CD40 Ab- and PMA-plus-ionomycin-stimulated [3H]thymidine incorporation by IgD� cells (top) vsIgD�CD27� B cells (bottom).

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with 5- to 10-fold higher numbers of Kv1.3 (�2000/cell) channelsin the quiescent state than TEM cells (200–400/cell).

Because IKCa1 is the functionally dominant K� channel in na-ive and early memory B and T cells, IKCa1 blockers might haveuse in suppressing acute immune reactions, for example, duringgraft rejection and during the acute phases of autoimmune dis-eases. Clotrimazole, an IKCa1 blocker, was reported to ameliorate

rheumatoid arthritis in patients, but was abandoned due to adverseeffects resulting from blockade of cytochrome P450 enzymes (64, 65).The newer IKCa1-specific blockers TRAM-34, ICA-17043, and4-phenyl-4H-pyran (66–68), which lack this activity, should beevaluated for therapeutic use in autoimmune disorders. Furthermore,cyclosporin A, an immunosuppressant widely used to prevent graftrejection, synergizes with TRAM-34 in T cell proliferation assays

FIGURE 7. a, Plot of average Kv1.3 vs IKCa1 channel numbers per cell (mean SEM) in resting and activated B cells: IgD�CD27� (E), IgD�CD27�

(‚), IgD�CD27� (f), and IgD�CD27� (�). For each data plot, equal numbers of PB and tonsillar B cells were averaged. b, Diagrammatic representationof 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 ShKand the IKCa1 blocker TRAM-34 are also shown.

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(30), and combining these compounds could reduce the toxicity thatcomplicates cyclosporin A therapy.

Earlier studies showed that the Kv1.3 blockers margatoxin,correolide, and kaliotoxin suppressed delayed-type hypersensitiv-ity (DTH) in miniswine and rats (5, 69, 70). Based on these results,investigators in the field (5, 71) concluded that Kv1.3 blockersinhibited the primary immune response and caused generalizedimmunosuppression. However, our in vitro results, presented bothin this paper and in earlier reports (15, 16), suggest that Kv1.3blockers preferentially target late memory cells of both lineages(TEM and class-switched memory B cells), whereas naive and earlymemory cells, although initially sensitive to Kv1.3 block, up-reg-ulate IKCa1 and escape Kv1.3 inhibition. How might one recon-cile these differing views? Recent evidence indicates that essen-tially all of the T cells isolated from DTH skin in humans exhibitthe TEM memory phenotype (72), and TEM cells have the propen-sity to traffic to the skin (73). It is not surprising then that Kv1.3blockers effectively inhibited DTH in miniswine via preferentialsuppression of TEM cells. Specific Kv1.3 blockers might thereforeconstitute a new class of late memory-specific immunomodulators.

There is an increasing body of evidence implicating late mem-ory cells in the pathogenesis of MS, type-1 diabetes mellitus, rheu-matoid arthritis, psoriasis, Crohn’s disease, and chronic graft-vs-host disease (15–17, 74–77). Ag-specific targeting of pathogenicautoreactive T and B cells is the most desirable therapeutic strat-egy for autoimmune diseases, but Ag-specific strategies may faildue to the phenomenon of epitope spreading. A Kv1.3-based ther-apeutic approach that targets late memory cells and spares the bulkof the adaptive immune response mediated by naive and earlymemory cells that depend on IKCa1 channels might be beneficialunder these circumstances. For example, channel-based suppres-sion of autoantigen-specific memory B cells that have been re-ported to contribute to epitope spreading (78), and effectively func-tion as APCs through enhanced autoantigen capture via their Ag-specific membrane-bound Ig (41, 76, 79, 80), may have therapeuticvalue in autoimmune disorders. In proof-of-concept studies in rats,Kv1.3 blockers have been shown to ameliorate adoptive experi-mental autoimmune encephalomyelitis induced by myelin-specificmemory T cells (16, 70), a model for MS, and to prevent inflam-matory bone resorption in experimental periodontal disease causedmainly by memory cells (81). Several small-molecule Kv1.3 in-hibitors with nanomolar potency have been developed over the lastdecade (WIN-17317, CP-339818, U.K.-78,282, correolide, trans-N-propylcarbamoyloxy-PAC, sulfamidebenzamidoindanes, tetra-phenylporphyrins, dichlorophenylpyrazolopyrimidines, chalcones,and Psora-4 (6, 7, 31, 69, 82, 83)), and the continuing developmentof more selective and potent Kv1.3 blockers may make Kv1.3-based immunomodulation for autoimmune disorders a reality.

AcknowledgmentsWe are deeply indebted to Dr. Edward Monuki for helpful advice and theuse of his camera.

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