Fig. 7. Anergy induction by Tregs involves formation of gap junctions.

a The expression of connexin 43 (Cx43) impacts the number of anergic CD4⁺ cells in the spleen. Each animal is depicted by an individual symbol (n = 8 of each type). b Relative levels of Cx43 transcription in naïve (Tn, CD44⁻Foxp3^GFP−), activated/effector (Te, CD44⁺Foxp3^GFP−), anergic (Tan, CD44⁺Foxp3^GFP−FR4⁺CD73⁺), and Treg (Foxp3^GFP+) CD4⁺ cells examined by real time PCR analysis. Each cell type was analyzed in triplicate. Data from one of two experiments are shown. c Ex vivo induction of Cx43 expression by αCT-1 peptide improves Treg-mediated suppression (left y-axis) and increases the number of Tan cells (dotted box and right y-axis). Representative dot plots and graph of one of two experiments is shown. d Relative expression of Cx43 by Tregs transfected with Cx43-targeting or scrambled control siRNA. e Implemented Cx43 silencing reduces the transfer of a calcein violet between Tregs and suppressed Tn cells via gap junctions. Box within dot plots show frequencies of Tn cells receiving a calcein. The graph shows data from individual samples. f Ex vivo CD4⁺ Tn cells proliferation and FR4 expression upon inhibition by Tregs transfected with Cx43-specific or control siRNA. Tregs abbreviated to “Tr”. The graph on right (g) summarizes these data (dotted box shows increase in Tan in response to Tregs). e–g One experiment of two is shown. Statistical significance was calculated with paired Student t test (a, c, d, g) or ANOVA with Bonferroni correction (b, e). *p < 0.05, **p < 0.01, ***p < 0.001.