(A). TCR response. Sorted CD4⁺CD25⁻, effector T cells and iTregs were rested for 2 hours on ice and activated (+) or not (−) by anti-CD3 and anti-mouse IgG crosslinking for 3 min. Lysates of 5×10⁵ cells were analyzed by Western blot to detect activated (phosphorylated) forms and total protein expression of Lck/Fyn (anti-pY416-Src recognizes both pY394-Lck and pY417-Fyn), AKT, LAT and ERK. GAPDH expression demonstrated equivalent loading. Protein band densities were quantified by densitometry. Ratio of phosphorylated: total protein was normalized to that of mock-treated CD25⁻ cells and depicted as mock-treated (empty bars) or CD3 crosslinked (black bars) cells. iTregs exhibited intense phosphorylation of Lck/Fyn in mock cells and impaired capacity to activate AKT upon TCR engagement. (B). Comparative expression of proteins. Lysates from 5×10⁵ cells were analyzed by Western blot to detect the pattern of expression of proteins involved in early events of TCR signaling. Protein band densities were quantified and normalized to that of CD25⁻ cells as indicated below each blot. No substantial differences were noted between effector and iTregs. (C) Regulation of gene expression. Transcription of the LCK gene is not differentially regulated in iTregs. FOXP3 (as a positive control) and GAPDH (housekeeping gene) were assessed as control. Gene expression was quantified from CD25⁻, effector T cells and iTregs. Briefly, total RNA was extracted from 5–7×10⁶ sorted cells. cDNA was obtained by reverse transcription and used as a template in a microarray analysis with the Affymetrix GeneChip System (Human Genome U133 Plus 2.0 Array microchip). The results represent the comparative mean value of triplicates from three different donors relative to CD25⁻ samples; bars represent the experimental range of the triplicates. Results were validated with Real Time-PCR (not shown).

(A). Physical association between Lck and IL2-R complex in iTregs. Lysates from 10⁷ effector T cells and iTregs were incubated with Lck antibody-loaded agarose beads or Ig-isotype control (IgC). Cell extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blots using CD122 (IL2-Rβ) and Lck antibodies. The results show that Lck co-immunoprecipitates with IL2-Rβ subunit in iTregs but not in effector cells. Lck blot verified equivalent yield of immunoprecipitate. Blotting total cell lysates with anti-CD122 demonstrated equal expression of IL2-Rβ in both cell types. Quantification of co-immunoprecipitates was determined by the relative ratio of IL2-Rβ bound to Lck and normalized at 1.0 in effector cells. (B). Functional association of Lck and IL2-R complex in iTregs. Sorted iTregs were rested for 6–7 hours on ice and mock-activated, anti-CD3 crosslinked or IL2 treated. Lysates of 5×10⁵ cells were subjected to immunoblotting using the indicated antibodies. Phosphorylation of STAT5 and Lck were detected upon IL2 treatment; phosphorylation of LAT demonstrated an effective response to CD3 crosslink. LAT expression verified equal loading. Quantification of protein expression was normalized on the basis of mock-treated iTregs for each individual blot. (C). IL2 treatment supports Kv1.3 current in iTregs. Sorted iTregs were cultured for 60 h in AIMV with or without 5 ng/mL of IL2, IL6 or IL10. Depiction, measurement of Kv1.3 currents and statistical analysis were performed as described in Fig. 2. The results demonstrate that iTregs cultured with IL2 displayed the largest Kv1.3 currents compared to IL6 and IL10. Profiles of Kv1.3 currents represent the combined values of two independent experiments.

(A). Electrophysiology. CD4⁺CD25⁻ T cells and nTregs isolated from same donors were cultured in iTreg medium (see Supporting information for details). After 6 days, CD4⁺CD25⁻, effector T cells and iTregs were sorted and maintained for 60 h in serum-free AIMV medium (Invitrogen) with or without 0.15 µg/mL of soluble anti-CD3 antibody. Kv1.3 current intensity in sorted cells and nTregs was measured with high-throughput patch-clamping screening (see Supporting Information and ^[5]). Experimental values represent the combined distribution of Kv1.3 measurements on individual cells isolated from three independent experiments. N=255 CD4⁺CD25⁻ cells; N=186 effector cells; N= 88 natural Tregs; N=129 iTregs. Treg cells (“natural” or “induced”) did not increase Kv1.3 specific current intensity in response to TCR engagement. The distribution of Kv1.3 values are depicted in box plots with whiskers representing the 10^th and 90^th percentile of experimental values; boxes are limited by the 25^th and the 75^th percentiles; solid lines are the medians, and dark squares represent the mean values. A two-sample Kolmogorov-Smirnov test was used to compare paired distributions of Kv1.3 currents. (B). Expression. Sorted CD4⁺CD25⁻, effector T cells and iTregs were analyzed by Western blot to compare the expression of Kv1.3. The double banded pattern depicted in effector and iTreg Kv1.3 blots may likely correspond to different glycosylation forms of Kv1.3^(22,23). Quantification of protein expression was normalized to the band density of CD25⁻ cells. GAPDH levels demonstrate equal protein loading. Gene expression was determined as detailed in Fig. 1C.