Expression of Kv1.3 on inflammatory cells in MS brain. Paraffin sections were stained by indirect immunoperoxidase for Kv1.3, CD3, CD4, CCR7, and CCR5. Areas used in sectioning were from a white matter plaque. There were many perivascular inflammatory cells that stained positively for CD3 (A), Kv1.3 (B), and CD4 (B Inset) on consecutive sections. (C) Kv1.3 was also localized on inflammatory cells in the white matter parenchyma (Inset reveals membrane polarization of Kv1.3 staining). (D) Consecutive sections through another perivascular infiltrate revealed numerous Kv1.3⁺ inflammatory cells, which were predominantly CCR7^- (E) (Inset reveals rare CCR7 positive staining), and CCR5⁺ (F). (Scale bar, 50 μmin A and B and 20 μmin C, D, E, and F.)

Tissue expression of Kv1.3 in normal appearing white and gray matter. MS brain cryostat sections that were grossly uninvolved were examined for Kv1.3 expression. (A) Kv1.3 deposits were found on perivascular and parenchymal inflammatory cells in the normal appearing white matter. (B) Many of these cells stained positively for CD68. (C) Kv1.3 staining was also seen on parenchymal cells in the gray matter. (D) Phosphorylated neurofilament staining showed axonal fragmentation and swelling in a normal appearing gray matter area, which was a consecutive section for the Kv1.3 staining seen only on inflammatory cells in C. (E) Some of the cells were branched process bearing cells with an appearance suggestive of microglia. (F) Double labeling identified some Kv1.3⁺ cells as CD68⁺ cells (arrows red-Kv1.3, dark brown CD68); not all CD68 cells expressed Kv1.3 (arrowheads). (G) Double labeling with CD3 (dark brown) and Kv1.3 (red) was also clearly seen. Not all Kv1.3-expressing cells are CD68 positive. (H) Control of the immunoperoxidase reaction. (Scale bar, 20 μmin A, B, D, G, and H; 50 μmin C and F; and 10 μm in E.)

Confocal microscopy of Kv1.3 and CD3/CD4 in MS brain tissue. Confocal microscopic images of 20-μm floating sections of postmortem MS brain tissue. (A and B) Parenchymal infiltrate with positive CD3 and punctate Kv1.3 staining in the membrane. (C) A cluster of lymphocytes stained positively for Kv1.3 and CD4, as well as occasional CD8 (Inset). (D) CD3⁺ cells showed extensive Kv1.3 and CD3 colocalization in the membrane. Immunofluorescent staining of PB-derived T cells concentrated on a slide by cytospin showed membrane patterns of staining of CD4, CD8, and Kv1.3. (E) Day 7-activated naive and T_CM are CD4⁺, CCR7⁺ (data not shown) and Kv1.3^-.(F) Chronically stimulated resting T_EM were CD4⁺, CCR7^- (data not shown) and expressed Kv1.3 in the membrane but with minimal colocalization with CD4. (G)T_EM that had been recently activated expressed increased amounts of Kv1.3 and demonstrated more colocalization with CD4 similar to the brain tissue lymphocyte in D. (H) Naive CD8⁺ T cells expressed no Kv1.3 but, when chronically stimulated and activated (I), exhibited intense Kv1.3 expression and colocalization with CD8. (J) Isotype controls using nonspecific rabbit primary antibody followed by usual secondary label and with primary rabbit anti-human and nonspecific labeled mouse anti-rabbit secondary antibody showed no background staining.

Immunofluorescence and electrophysiology of peripheral and CSF T cells. FACS plots revealed staining patterns of CCR7 vs. CD45RA. (A) CD4-gated MS-derived CSF cells revealed a predominantly T_CM phenotype immediately ex vivo, but with rapid conversion to short-lived T_EM after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads (B and C). By comparison, negatively sorted CSF derived from noninflammatory neurological controls showed CD4⁺ cells that were predominantly T_CM (D) and remained T_CM at day 7 despite identical manner of stimulation (E). (F-H) CD4 cells derived from PB were predominantly T_CM and naive phenotype (F), and maintained high levels of CCR7 even after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads (G and H). (I) Single cell patch-clamp analysis of MS-derived CSF cells revealed high Kv1.3 channel numbers per cell; mean channel number per cell was 1,082 ± 489 (mean ± SEM, n = 28). Shown for comparison on the right are mean Kv1.3 channel numbers from activated PB T cells from three MS patients (n = 32), and from 10 healthy controls (n = 33). (J and K) Family of currents. The test potential was changed from -60 to 60 mV in 10-mV increments every 30 s (V_1/2 = -28 mV). (L) The current in CSF cells exhibited the characteristic use-dependence of Kv1.3 when 200-ms pulses were applied every second to 40 mV. (M and N) Pharmacological blockade with the potent and selective Kv1.3 inhibitor ShK(L5) (14) confirmed that the current is carried by Kv1.3. (O) Representative images of CSF resting (top) or activated (bottom) T cells stained with anti-Kv1.3 and CCR7 Abs. (P) Staining intensities for Kv1.3 and CCR7 in resting and activated CSF T cells. (Q) Z-stack of confocal images through an activated CSF T cell.