Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases

Abstract

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2, SAP97, ZIP, p56(lck), and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

Fig. 1.

Kv1.3 channel expression in RA and OA T cells. (A) Kv1.3 number per cell in RA-SF-T cells, RA-PB-T cells, and OA-SF-T cells. Most RA-SF-T cells were CD4⁺ cells (Fig. 5B). (B) Confocal images of Kv1.3 (green) and Kvβ2 (red) staining in RA and OA T cells. (C) CCR7 expression in RA and OA T cells by FACS. (D) Synovial tissue from RA patients stained with anti-CD3, anti-Kv1.3, or anti-CCR7 Abs and counterstained with hematoxylin/eosin. Data are from five patients studied.

Fig. 2.

Kv1.3 expression in T cells specific for GAD65 (red), INS (black), or myelin antigens (blue) from patients with T1DM, type-2 diabetes mellitus, or MS or healthy controls. (A) Kv1.3 currents (Upper) and channel number per cell (Lower) in activated INS-, GAD65-, and myelin-specific CD4⁺ TCLs from new-onset T1DM patients, healthy controls, and MS patients (10). Each data point represents the mean ± SEM from 20–50 cells from two to four TCLs from a single donor. (B) Immunostaining for Kv1.3. (C) CCR7 expression in TCLs by FACS. (D) Kv1.3 number per cell in TCLs from a patient with both T1DM and MS and from patients with longstanding T1DM or type-2 diabetes mellitus. (E) Kv1.3 channel numbers per cell and CCR7, CD45R, and Kv1.3 protein expression in CD4⁺ T cells FACS-sorted with MHC class II tetramers loaded with GAD65 557I peptide or HA 306–318 peptide from T1DM patients.

Fig. 3.

Specific Kv1.3 blockers preferentially suppress human T_EM cells. (A) Kv1.3-containing signaling complex: Kv1.3, Kvβ2, SAP97 (synapse-associated protein 97), ZIP (PKC ζ-interacting protein, p56^lck-associated p62 protein), p56^lck, and CD4 (37). (B) Cocapping of Kv1.3 (green) with CD4 (red) in human T_EM cells. (C and D) CD4 (red) and Kv1.3 (green) staining in human GAD65-specific T_EM cells exposed to APCs loaded with GAD65 557I (C) or MBP (D). (E) SL5 100 nM does not prevent IS formation. (F) Ca²⁺ signaling in GAD-specific CD4⁺ T_EM clones triggered by anti-CD3 plus cross-linking secondary Ab (arrow) in the absence (black) or presence of SL5 at 0.1 nM (blue), 1 nM (green), or 100 nM (red). (G) SL5 suppression of cytokine production by RA-SF-T cells, RA-PB-T cells, and tetramer-sorted GAD65-specific T_EM clones from T1DM patients. Amounts of cytokines produced are in Fig. 9. (H Left) Anti-CD3 Ab-stimulated [³H]thymidine incorporation by RA-PB-T cells versus RA-SF-T cells from three RA patients. (H Right) [³H]Thymidine incorporation by same two populations after they were stimulated for 48 h with anti-CD3 Ab, rested overnight in medium, and then rechallenged with anti-CD3-Ab. (I) GAD65-specific T_EM cells escape from Kv1.3 blockade as the amount of GAD65 557I peptide increases from 10 (green) to 30 (red) to 90 (blue) μg/ml. (J) Effect of 4-AP on T_EM proliferation induced by anti-CD3 Ab. Each point represents mean ± SD of triplicates. Dotted line shows previously published data (17) on PB-T cells from healthy donors.

Fig. 4.

Kv1.3 blockers ameliorate PIA and EAD in rats. (A) SL5 treatment of PIA in which vehicle-treated animals have periostitis and joint deformation. (B) X-rays of paws. (C) Staining of joints from vehicle-treated (n = 5) and SL5-treated (n = 5) rats with PIA. (D) Cumulative incidence of EAD. (E) Islets immunostained for INS (first column), CD3 (second column), CD8 (third column), and CD68 (fourth column) in vehicle-treated (Upper) and PAP1-treated (Lower) rats. (F) PAP1 reduces β cell destruction and intraislet infiltration by T cells and macrophages on day 70. Destruction/infiltration: none, white; moderate, gray; severe, black.