Fig 1.
OsK1 peptide inhibited voltage-gated Kv1.3 currents in human CD4 T cells and CHO cells transfected with recombinant human Kv1.3.
OsK1 peptide inhibited human Kv1.3 mediated whole cell currents in a concentration dependent manner in human CD4 T cells (A). Potency of OsK1 peptide on Kv1.3 currents in purified human CD4 T cells and Kv1.3 transfected CHO cells were determined and IC50 curves were shown (B). Whole cell voltage-gated currents were measured in manual patch clamp assays. All recordings were made at room temperature using Multiclamp 700A amplifier and pClamp 9 software as described in Methods and Materials.
Table 1.
Potency of different Kv1.3 blockers on human Kv1.3 channels on transfected CHO cells.
Fig 2.
Efficacy of Kv1.3 blocker OsK1peptide in inhibiting T cell activation is partial and dependent on stimulation strength.
Effect of OsK1 peptide (filled circles) or cyclosporine A (open circles) on proliferation or IL-2 production of human primary CD4 (A—F) and CD8 T cells (G—L) activated with anti-CD3/CD28 coated beads under strong stimulation (2:1 bead:cell ratio; left panels), intermediate stimulation (1:1 bead:cell ratio; middle panels), or weak stimulation (1:2 bead:cell ratio; right panels) conditions. Cyclosporine A was not tested in CD8 T cells with weak stimulation due to insufficient purified T cells. The assays were performed in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented.
Table 2.
Potency and efficacy of Kv1.3 blockers on human T cells under different stimulation conditions.
Fig 3.
Effect of Kv1.3 blockers effect on cytokine production from thapsigargin stimulated human blood and purified CD4 T cells.
Effects of OsK1 and Kv261 on IL-2 production of thapsigargin stimulated CD4 T cells (A) were shown. OsK1, Kv261 peptide and Kv261-HSA-34 were tested in thapsigargin stimulated blood (B) from healthy donors and IL-2 was measured. BTP2 and cyclosporine A were used as reference compounds. The assays were performed twice in CD4 T cells and 3 times in human blood with samples in duplicates. Data from representative experiments were presented.
Fig 4.
Effect of Kv1.3 blocker on calcium flux in T cells.
ShK peptide (1nM) reduced calcium flux induced by either anti-CD3/CD28 (A) or thapsigargin (B) in human CD4 T cells. Kinetics of calcium signal change in different samples are presented as curves over time (A, B). CD4 T cell populations with different levels of calcium signals are presented in dot plots and their Fluo4 intensities in geometric means as well as population percentages are indicated (C). The assays were repeated in 3 independent experiments and data from one representative experiment were presented.
Fig 5.
Effect of OsK1 on human CD4 T cell subsets.
Human CD45RO- CCR7+ naïve T cells, CD45RO+ CCR7+ central memory (Tcm) and CD45RO+ CCR7- effector memory (Tem) T cells were purified from human blood (A). OsK1 peptide blocked proliferation of the purified human CD4 T cell subsets induced by 2 day stimulation with anti-CD3 in the presence of PBMC (B). BTP2 and cyclosporine A were used as reference compounds in T cell assays. The assays were performed in 8 similar experiments with samples in duplicates. Data from one representative experiment were presented.
Fig 6.
Kv1.3 expression in human tissues and T cells.
Kv1.3 mRNA expression in different human tissues was profiled in Affymetrix microarrays (A). Cell surface Kv1.3 channels were measured with patch express assays on activated or resting human CD4 T cell subsets including naïve, central memory (Tcm) and effector memory (Tem) T cells, as well as antigen-specific T cells from house dust mite (HDM) stimulation. Data shown are either channels/cell (B) or number of channels normalized to cell surface area (channels/pF) (C). Time course of Kv1.3 channel activity and expression of the activation markers CD25 and CD69 are shown on effector memory CD4 T cells activated with anti-CD3/CD28 coated beads (D). Expression of Kv1.3 mRNA levels in naïve, Tcm and Tem CD4 T cells at different time points post activation with anti-CD3/CD28 coated beads are presented (E). Patch express measurement of Kv1.3 channels was performed on T cell subsets purified from 6 healthy blood donors. Statistical significance on the difference in Kv1.3 channel levels between activated and resting T cell subsets was analyzed by 2-tailed Student’s t test and P values were determined and indicated as *P < 0.05, ***P < 0.001, ****P < 0.0001.
Table 3.
Kv1.3 expression on human primary T cell subsets.
Fig 7.
Effect of Kv1.3 blockers on antigen-specific T cell activation.
Kv1.3 blocker OsK1, Kv261 peptides and Kv261-HSA-34 fusion protein blocked proliferation of CD4 T cells from house dust mite allergic patients following 5 day stimulation with house dust mite extracts (A, B). IL-5 production from house dust mite-specific T cells was inhibited by Kv1.3 blockers as shown with OsK1 and Kv261-HSA-34 (C). OsK1 peptide at 0.3μM inhibited partially the proliferation of PBMC from patients allergic to ragweed after 3 day stimulation with ragweed extracts (D). Decreased proliferative response of individual donor PBMC samples by OsK1 peptide was shown in a separated graph. CTLA4-Ig or cyclosporine A (1μM) were used as reference compounds. The assays in panels A-C were repeated in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented. The study in panel D was done in an experiment with 29 subjects per group. Statistical significance on the difference in proliferation between untreated and OsK1 treated groups were analyzed by 2-way ANOVA and the P value was determined and indicated as *P < 0.05. Readouts in (A) and (C) were performed on CD4 T cells from the same donor whereas those in (B) were from a separate donor. Data from one representative experiment were presented.