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.