CD5 costimulation enhances prolonged STAT3 activation and ROR-γt transcription. (A) Naive T cells were stimulated by plate-bound antibodies directed against CD3/CD28 or CD3/CD5 and cultured with Th17-inducing conditions. Phospho-STAT3 levels were measured by FACS. Data shown are means from 8 individual experiments with different donors. (B-C) Phospho-STAT3 was measured after 18 hours (B) or 72 hours (C) of naive T cells stimulated as described under panel A. Data shown are means ± SD from 8 individual experiments using different donors. (D) Real-time semiquantitative PCR of mRNA expression of RORC of naive T cells stimulated via coated antibodies directed against CD3/CD28 or CD3/CD5 in Th17-polarizing conditions. Samples were measured after 3 days, 6 days, and 11 days of culture and the expression is relative to CD3/CD28 stimulated naive cells in medium, at day 3. Data shown are mean ± SD of triplo measurement from one representative experiment of 4 independent experiments using different donors. (E) Naive T cells were stimulated via CD3/CD28, CD3/CD5, or CD3/CD5/CD28 in Th17-inducing conditions. Phospho-STAT3 levels were measured by FACS. (F) Phospho-STAT3 levels at 72 hours, of cells stimulated as described under panel E. Data shown are from 3 individual experiments with different donors. (G) Naive T cells were stimulated via CD3/CD28, CD3/CD5, or CD3/CD5/CD28 in Th17-inducing conditions. IL-23R expression was measured by FACS at day 3 and day 6. Data shown are from 1 representative experiment of 4 independent experiments using different donors.