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

Channel blockers

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

B cell isolation

Peripheral blood (PB) CD19⁺ cells were negatively selected from venous blood of healthy volunteers with RosetteSep B cell enrichment mixture (StemCell Technologies, Vancouver, BC, Canada). Human tonsil samples were obtained under an Institutional Review Board-approved protocol. Within 1 h after tonsillectomy, mononuclear cells were prepared by slicing the sample into pieces and forcing them through a 70-μm filter to get a single-cell suspension. CD19⁺ cells were isolated using the StemSep B Cell Negative Isolation System according to the manufacturer’s instructions. Cells isolated from both the blood and the tonsil were found to be 99.2–99.6% CD19⁺ by flow cytometry (PE-conjugated mouse anti-human CD19 mAb; HIB19; BD Pharmingen, San Diego, CA). IgD⁺ cells were positively selected from CD19⁺ cells with a biotinylated anti-IgD mAb (IA6-2; BD Pharmingen) followed by an anti-biotin tetrameric Ab complex and magnetic beads (StemSep System; StemCell Technologies). From the column flow-through containing IgD⁻ B cells, we positively selected IgD⁻CD27⁺ cells with a custom-made anti-CD27⁺ Ab mixture and magnetic nanobeads according to the manufacturer’s protocol (EasySep System; 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 μg/ml streptomycin, and 50 μM 2-ME). The cells were activated with 10 nM PMA and 175 nM ionomycin for 48 h to achieve maximal B cell activation (32, 33). To simulate T cell-mediated activation of B cells, we cultured various B cell subsets for 96 h with 1 μg/ml mouse anti-human CD40 mAb (5C3; BD Pharmingen) presented by stably CD32-transfected irradiated (10,000 rad) K562 cells (B cells:K562 cells, 4:1) (34). The CD32-transfected K562 cells were a generous gift from Dr. C. June (University of Pennsylvania, Philadelphia, PA) (34).

Electrophysiology

Channel expression was studied in the four B cell subsets defined by expression of IgD and CD27 before or after activation with PMA and ionomycin or with anti-CD40 Ab in the whole-cell mode of the patch-clamp technique with an EPC-9 HEKA amplifier (HEKA Elektronik Dr. Schulze, Lambrecht, Germany). All experiments were conducted at room temperature, and series resistance compensation was used for Kv currents when they exceeded 2 nA. Cells were stained for IgD and CD27 on ice in complete RPMI with FITC-conjugated mouse anti-human CD27 mAb (M-T271) and PE-conjugated mouse anti-human IgD mAb (IA6-2; both BD Pharmingen). 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 fluorescence microscopy, and patch-clamped in the whole-cell configuration. For experiments on CD27⁺IgA⁺ and CD27⁺IgG⁺ class-switched B cells, we stained 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 holding potential of −80 to 40 mV applied every 30 s unless otherwise stated. Kv1.3 currents were recorded in normal Ringer solution with a Ca²⁺-free pipette solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, and 2 mM MgCl₂ (pH 7.2; 300 mOsm). Whole-cell Kv1.3 conductance was calculated from the peak current amplitude at 40 mV.

IKCa1 currents were elicited with voltage ramps from −120 to 40 mV of 200-ms duration applied every 10 s. The pipette solution contained 145 mM K⁺ aspartate, 2 mM MgCl₂, 10 mM HEPES, 10 mM K₂EGTA, and 8.5 mM CaCl₂ (1 μM free Ca²⁺) (pH 7.2; 290 mOsm). To reduce chloride leak currents, we used a Na⁺ aspartate external solution containing 160 mM Na⁺ aspartate, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES (pH 7.4; 300 mOsm). Whole-cell IKCa1 conductances were calculated from the slope of the current-voltage relationship at −80 mV. Kv1.3 and IKCa1 channel numbers per cell were determined by dividing the whole-cell Kv1.3 or IKCa1 conductance by the single-channel conductance value for each channel: Kv1.3, 12 pS; IKCa1, 11 pS (14, 35). Cell capacitance, a direct measure of cell surface area, was constantly monitored during recording and noted for each cell, and the surface of each cell was measured by the formula: 1 pF = 100 μm². To normalize for cell size, we determined Kv1.3 and IKCa1 channel densities for each cell by dividing the channel number per cell by that cell’s surface area.

Statistical differences in channel numbers per cell and channel density per square micrometer between different B cell subsets were determined by one-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 incubated in PBS containing 2% goat serum with a combination of FITC-conjugated mouse anti-human IgD mAb (IA6-2), PE-conjugated mouse anti-human CD27 mAb (clone M-T271), and CyChrome-conjugated mouse anti-human CD80 Ab (clone L307.1) or CyChrome-conjugated mouse anti-human CD86 Ab (clone 2331; all BD Pharmingen). A BD Biosciences (San Jose, CA) FACScan with CellQuest software was used to analyze the stained cells.

Immunohistochemistry

Paraffin-embedded sections from human tonsil and spleen (donor >30 years of age) were acquired from the University of California, Irvine, Pathology Department, under an Institutional Review Board-approved protocol. Sections were dewaxed with xylene and rehydrated through an alcohol gradient. Before staining with primary Abs, sections were heated with 10 mM Na citrate (pH 6.5) in a microwave for 15 min to retrieve antigenic determinants masked by paraffin embedding. After treatment with 1% H₂O₂ to inactivate endogenous peroxidase activity and blocking with 5% goat serum in PBS for 12 h, sections were incubated with polyclonal rabbit primary Abs for Kv1.3 (1:500 of 4.1 μg/ml IgG) or IgD (1:500; A0093; DakoCytomation) or CD27 (1:100; H-260; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. Bound primary Abs 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 with PBS 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, Hampton, NH), dehydrated, and mounted with Permount (Fisher Scientific). The polyclonal rabbit Kv1.3 Ab was previously shown to be specific for Kv1.3 (36).

[³H]Thymidine incorporation

Tonsillar IgD⁺ or IgD⁻CD27⁺ B cells (1 × 10⁵ cells per well) were incubated in complete RPMI medium in 96-well plates in the presence or absence of 10 nM PMA plus 175 nM ionomycin, and with or without ShK (Bachem Biosciences) or TRAM-34 (30). [³H]Thymidine (1 μCi/well) was added 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 for these experiments as Kv1.3 and IKCa1 blockers, because both compounds have demonstrated therapeutic efficacy in animal models of disease (16). [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 − resting cpm/maximal cpm − resting cpm (counts for IgD⁺ cells were typically ∼80,000 cpm, and for IgD⁻CD27⁺ cells ∼10,000 cpm). For anti-CD40 activation, 5 × 10⁴ IgD⁺ or IgD⁻CD27⁺ cells were incubated with 2 × 10⁴ irradiated CD32-transfected K562 cells and 1 μg/ml anti-CD40 mAb for 48 h (IgD⁻CD27⁺) or 96 h (IgD⁺) in round-bottom 96-well plates to allow sufficient contact. [³H]Thymidine was added for the last 12 h of culture (maximal counts for IgD⁺ cells were 35,000 cpm, and for IgD⁻CD27⁺ cells 7,000 cpm).

K⁺ 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 (>98% 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 K⁺ channel expression patterns were determined by whole-cell patch clamp. In the example shown in Fig. 1⇓a, the patch-clamped IgD⁻CD27⁺ memory cell is distinguished from the surrounding naive IgD⁺CD27⁻ cells by its larger size (left) and its green fluorescence (right). Both subsets expressed voltage-gated (K_V) and calcium-activated (K_Ca) K⁺ currents with the characteristic fingerprints of Kv1.3 and IKCa1 channels (Figs. 1⇓ and 2⇓; Table I⇓). The amplitude of both the Kv1.3 and the IKCa1 current was substantially larger in the IgD⁻CD27⁺ class-switched memory B cells than in IgD⁺CD27⁻ naive B cells (Figs. 1⇓ and 2⇓). Staining with the anti-IgD and the anti-CD27 Abs did not alter the properties or the expression of the Kv1.3 and IKCa1 currents (data not shown).

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FIGURE 1.

IgD⁻CD27⁺ memory B cells express much larger Kv1.3 currents than naive B cells. a, Transmitted light (left) and fluorescent image (right) of CD19⁺ B cells stained with PE-conjugated IgD and FITC-conjugated CD27 Abs. A IgD⁻CD27⁺ memory B cell is patch-clamped. b, K_V currents in both IgD⁻CD27⁺ memory (left) and IgD⁺CD27⁻ naive (right) B cells exhibit the characteristic use-dependence of Kv1.3. Currents were elicited by 200-ms pulses from −80 to 40 mV applied every 1 s. The numbers 1, 2, and 3 above the traces refer to the first, second, and third depolarizing pulse. The inset in the right panel shows the current in the IgD⁺CD27⁻ naive cell on a ×18 expanded scale. c, Normalized peak K⁺ conductance-voltage relationship for the K_V current in IgD⁻CD27⁺ memory (left) and IgD⁺CD27⁻ naive (right) B cells (n = 3). The line through the points was fitted with the Boltzmann equation. d, Blockade of K_V currents in both subsets by ShK. Currents were elicited by 200-ms depolarizing pulses from −80 to 40 mV applied every 30 s. e, The Kv1.3-specific ShK derivative ShK-F6CA completely inhibits the K_V current in both IgD⁻CD27⁺ (left) and IgD⁺CD27⁻ (right) B cells, whereas the Kv1.1 blocker DTX-I has no effect on the current at 100 nM. f, Dose-dependent block of K_V currents in both subsets by Psora-4, the most potent small-molecule inhibitor of Kv1.3. All experiments shown in b–f were conducted with a Ca²⁺-free KF-based pipette solution.

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FIGURE 2.

IKCa1 currents in IgD⁻CD27⁺ memory and IgD⁺CD27⁻ naive B cells. a, IgD⁻CD27⁻ class-switched memory B cells dialyzed with 1 μM free Ca²⁺ through the patch pipette exhibit a voltage-independent K_Ca current in voltage ramps from −120 to 40 mV. This current is not present when the pipette solution contains 50 nM free Ca²⁺. The voltage-activated current visible above −40 mV is Kv1.3. b, Effect of increasing concentrations of the selective IKCa1 inhibitor TRAM-34 on the K_Ca current in a IgD⁻CD27⁺ memory B cell. Kv1.3 was completely blocked by 1 nM ShK-Dap²² in this experiment. c, Kv1.3 and IKCa1 currents in IgD⁻CD27⁺ (left) and IgD⁺CD27⁻ (right) cells. The current seen at voltages below −40 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 IgD⁺CD27⁻ naive cell on a ×15 expanded scale.

K_V currents in both B cell subsets exhibited use-dependence, a property characteristic of Kv1.3, in which rapid repetitive depolarizing pulses cause a progressive decrease in the K_V current amplitude due to channel trapping in the inactivated state (Fig. 1⇑b). Their half-activation voltage and inactivation time-constant were also similar to Kv1.3 (Fig. 1⇑c; Table I⇑). The polypeptides ShK (Fig. 1⇑d), ShK-Dap (22), charybdotoxin, and margatoxin blocked the B cell K_V currents with the same potencies as Kv1.3 (Table I⇑). ShK-F6CA, an analog of ShK (37) that exhibits ∼80-fold selectivity for Kv1.3 over closely related K_V family channels, and Psora-4 (31), the most potent small molecule inhibitor of Kv1.3, also blocked the K_V current at concentrations that block Kv1.3 (Fig. 1⇑, e and f; Table I⇑). Dendrotoxin-1 and dendrotoxin-κ, two polypeptides that inhibit the Kv1.1 channel that has been reported in mouse thymocytes (38) and mouse T cells (39, 40) but not in human T cells (6), had no effect on the K_V currents in the B cell subsets (Fig. 1⇑e; Table I⇑). Taken together, these results show that the biophysical and pharmacological properties of the K_V channel in human B cells are identical to those of Kv1.3, and suggest that Kv1.3 is the only K_V channel expressed in human B cells (14, 15, 16, 35).

We also observed K_Ca currents in both B cell subsets when recording with a pipette solution containing 1 μM free calcium. In the ramp protocol from an IgD⁻CD27⁺ memory B cell a calcium-dependent, voltage-independent, weakly inwardly rectifying K_Ca current is seen that reverses at −80 mV (Fig. 2⇑a). This current was blocked by the IKCa1-specific inhibitor TRAM-34 (Fig. 2⇑b) and by charybdotoxin with potencies identical to those of the cloned IKCa1 channel (Table I⇑). Together, these results strongly argue that the IKCa1 channel underlies the K_Ca currents in naive and class-switched human B cells. Furthermore, the complete inhibition of all K⁺ current by 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 are shown in the flow cytometry profile in Fig. 3⇓a. The relative proportions of each subset varied in different individuals: naive IgD⁺CD27⁻ cells (45–73% in five donors), IgD⁺CD27⁺ memory cells (3–21%), class-switched IgD⁻CD27⁺ memory cells (4–20%), and IgD⁻CD27⁻ cells (2–11%). The activation markers CD86 and CD38 were not expressed or expressed only at very low levels in all four subsets (data not shown), suggesting that these 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 CD80 negative (Fig. 3⇓b). Identical results were obtained with tonsillar B cells.

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FIGURE 3.

K⁺ channel expression in B cells subsets. a, Flow cytometry profile showing IgD and CD27 expression in quiescent CD19⁺ B cells. Four subsets are distinguished: IgD⁺CD27⁻ (naive), IgD⁺CD27⁺, IgD⁻CD27⁺ (class-switched memory B cell), and IgD⁻CD27⁻. Representative Kv1.3 and IKCa1 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-based pipette solution. IKCa1 currents were elicited by 200-ms ramp pulses from −120 to 40 mV with 1 μM free Ca²⁺ 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 IgD⁺CD27⁻ (left), IgD⁺CD27⁺ (middle), and class-switched IgD⁻CD27⁺ (right) B cells.

Representative Kv1.3 and IKCa1 currents of the four major B cell subsets are shown next to the respective FACS quadrants in Fig. 3⇑a. The properties of the K_V and K_Ca currents were identical in the four B cell subsets. The amplitude of the Kv1.3 and IKCa1 currents in quiescent class-switched B cells was significantly larger than in the other three B cell subsets. We determined channel numbers per cell by dividing the whole-cell conductance for each channel in each cell with the corresponding single-channel conductance values. The Kv1.3 and IKCa1 channel numbers per cell in the four quiescent B cell subsets are shown in Fig. 4⇓a. Naive B cells expressed small numbers of Kv1.3 (∼100/cell) and IKCa1 (∼5/cell) channels. IgD⁺CD27⁺ memory B cells expressed more Kv1.3 channels (∼250/cell) than naive cells but the same number of IKCa1 channels (∼7/cell). IgD⁻CD27⁻ cells also expressed small numbers of Kv1.3 (∼70/cell) and IKCa1 (∼18/cell) channels. In contrast, class-switched IgD⁻CD27⁺ cells expressed 10–20 times more Kv1.3 channels (∼2400/cell; p < 0.00001 for all 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 expression pattern has not been seen before in any quiescent lymphoid subset. The corresponding B cell subsets in the human tonsil exhibited K⁺ channel expression patterns identical with their PB counterparts (see Fig. 4⇓a). Because class-switched memory B cells are significantly larger than the other three subsets, we normalized channel expression levels for cell size by calculating the channel density per square micrometer of surface area. Class-switched B cells had 6- to 21-fold higher Kv1.3 (p < 0.00001 in all three cases) and 2- to 8-fold higher IKCa1 channel densities (p = 0.00012–0.05663) than the other three subsets (Fig. 4⇓a).

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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 IKCa1 channel numbers per cell in the four B cell subsets from PB in the resting state (○, ▵, □, ⋄) and after activation (•, ▴, ▪, ♦). The bar shows the mean of the respective subset. Mean channel numbers per cell (±SEM), 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 ± 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 ± 1.2 IKCa1, 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 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 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.

Class-switched IgD⁻CD27⁺ memory cells express other Ig isotypes 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. 4⇑b, left) and IgG⁺CD27⁺ (middle) cells expressed large Kv1.3 currents that exhibited use-dependence. IgG⁺ cells expressed slightly higher Kv1.3 channel numbers per cell than IgA⁺ cells (p < 0.05). Both class-switched subsets expressed 10-to 20-fold more Kv1.3 channels than naive or IgD⁺CD27⁺ cells (Fig. 4⇑b, right).

In summary, differentiation of human B cells from a naive to a class-switched memory B cell stage is accompanied by a change in K⁺ channel expression. Class-switched B cells express significantly higher numbers of Kv1.3 and IKCa1 channels than the other three B cell subsets. Our flow cytometry data show that the majority of class-switched B cells express the activation marker CD80, the ligand for CD28. Interestingly, the CD27⁺CD80⁺ subset (class-switched B cells based on our results) has been reported to effectively present Ag to CD4⁺ T cells without requiring preactivation, activate at lower thresholds, and differentiate rapidly into cells that secrete large amounts of class-switched Abs (41), and has also been implicated in autoimmune pathophysiology (42, 43, 44, 45). The Kv1.3^high pattern of these cells may position them for rapid activation.

IKCa1 is up-regulated in naive and IgD⁺CD27⁺ B cells following activation, whereas class-switched memory B cells retain their Kv1.3^high expression following activation

We examined the effect of mitogenic activation on channel expression in the four human B cell subsets. PB CD19⁺ B lymphocytes were stimulated for 30–96 h with anti-CD40 Ab (presented by CD32-transfected K562 cells (34)) to mimic T cell-mediated activation through CD40 (33, 46), or with a combination of PMA plus ionomycin (32). Cells were immunostained for IgD and CD27, and then visualized by fluorescence microscopy and patch clamped. Analysis of multiple cells showed that naive, IgD⁺CD27⁺ and IgD⁻CD27⁻ B cells, up-regulated IKCa1 expression following activation, whereas Kv1.3 levels did not change (Fig. 4⇑a). When normalized for cell size, IKCa1 density in activated cells of these three subsets was ∼45-fold higher (p < 0.000001) than in the resting state, whereas Kv1.3 density was 4-fold lower (p = 0.00003). In contrast, class-switched memory B cells augmented Kv1.3 levels following activation, but this increase was proportionate to the change in size and did not alter channel density (Fig. 4⇑a). IKCa1 density in these cells did not change with activation. Similar results were obtained with tonsillar B cells (Fig. 4⇑a). Thus, activation enhances IKCa1 density and reduces Kv1.3 density in naive, IgD⁺CD27⁺ memory and IgD⁻CD27⁻ B cells, but has no significant effect on channel density in class-switched IgD⁻CD27⁺ memory B cells, which retain their Kv1.3^high pattern.

Kv1.3^high B cells reside in memory compartments of human lymphoid tissues

The number of Kv1.3 channels (∼2000 channels/cell) in quiescent class-switched memory B cells should be sufficiently high to detect Kv1.3 protein within the B cell compartments of human secondary lymphoid tissues by immunostaining with Kv1.3-specific Abs. To determine the regional distribution of K⁺ channels in intact tissue samples and to compare it with our electrophysiological data, we stained paraffin sections of human tonsil and spleen with a polyclonal Ab that has been previously demonstrated to be specific for Kv1.3 (36). In the tonsillar section shown in Fig. 5⇓a, a ring of IgD⁺ cells is seen in the mantle of the B cell follicles surrounding the germinal center (GC). Robust Kv1.3 staining was detected within the GC where class-switched memory B cells are found (Fig. 5⇓b) but not in the mantle where IgD⁺ B cells reside. The expression pattern in the GC varied—in some cases, Kv1.3⁺ cells were dispersed throughout (Fig. 5⇓b), whereas in others, they were clustered at one edge of the GC (d). Additional Kv1.3 staining was seen outside the GC (Fig. 5⇓c) in a region that has been described as marginal zone (MZ)-like, because it contains IgD⁻ class-switched memory B cells (47, 48). Serial sections stained for Kv1.3 (Fig. 5⇓d) and CD27 (e) confirmed that these markers were expressed 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 levels of resting naive, T_CM and T_EM 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 observed with rabbit control IgG. In summary, Kv1.3^high cells are found in the GC and MZ of the tonsil and spleen where class-switched memory B cells reside or traffic through.

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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). 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 ×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.

IKCa1 blockade suppresses proliferation of naive B cells and IgD⁺CD27⁺ memory B cells, whereas Kv1.3 blockade suppresses proliferation of class-switched memory B cells

The availability of highly specific Kv1.3 and IKCa1 inhibitors and the subset-specific patterns of Kv1.3 and IKCa1 expression raise the possibility of using IKCa1-specific inhibitors to suppress the proliferation of naive and IgD⁺CD27⁺ memory B cells and Kv1.3 blockers to suppress class-switched Kv1.3^high memory B cells. To test this idea, human tonsillar CD19⁺ B cells were isolated and then further separated into two subsets, an IgD⁺ subset containing both naive and IgD⁺CD27⁺ cells, and a second subset containing class-switched IgD⁻CD27⁺ memory B cells. Both subsets were activated with anti-CD40 Ab (33, 46) or with a combination of PMA and ionomycin (32, 33, 51, 52, 53, 54, 55, 56) in the presence or absence of the IKCa1 blocker TRAM-34 or the Kv1.3 blocker ShK, and [³H]thymidine incorporation was measured 24–72 h later. In keeping with our expectation, TRAM-34 suppressed the proliferation of the IgD⁺ subset (Fig. 6⇓) with EC₅₀ values of 200 nM (anti-CD40 Ab) and 100 nM (PMA plus ionomycin), and ShK had no effect at a concentration (10 nM) that completely blocks Kv1.3 channels. At a 10-fold higher ShK concentration (100 nM) that also affects IKCa1 channels, 40–50% suppression was observed. Also, as anticipated, ShK suppressed the proliferation of class-switched Kv1.3^highIgD⁻CD27⁺ memory B cells with EC₅₀ values of 1 nM (anti-CD40 Ab) and 4 nM (PMA plus ionomycin), concentrations that selectively block Kv1.3, whereas TRAM-34 was ineffective (EC₅₀ > 1 μM) (Fig. 6⇓). Similar results were obtained with Psora-4, the most potent small molecule inhibitor of Kv1.3 (31). Psora-4 suppressed the proliferation of IgD⁻CD27⁺ memory B cells with an EC₅₀ of 250 nM (data not shown) but had no effect on the proliferation of IgD⁺ B cells at concentrations up to 1 μM. These results indicate that Kv1.3 blockers preferentially suppress the proliferation of class-switched memory B cells, whereas IKCa1 blockers suppress the proliferation of naive and IgD⁺CD27⁺ B cells.

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FIGURE 6.

Suppression of B cell proliferation by K⁺ channel blockers. Effect of TRAM-34 (▪) and ShK (▦) on anti-CD40 Ab- and PMA-plus-ionomycin-stimulated [³H]thymidine incorporation by IgD⁺ cells (top) vs IgD⁻CD27⁺ B cells (bottom).