Figure 6 : Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses.

From: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

Figure 6

(a) PBMC from HLA-DR4+ T1D donors (n=10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. (b) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein (n=13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student’s t-test. (c) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim’ denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 (KCNA3, d) and KCa3.1 (KCNN4, e) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). (f,g) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml−1) in the absence or presence of ShK (f) or TRAM-34 (g) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.