T cells polarized by TSLP-DC possess key features of human Tfh cells. (A) ELISA for CXCL13 production by CD4 T cells differentiated for 6 d in co-culture with DCs, TSLP-DC, or LPS-DC. Cytokines secretion was measured after an additional 24 h of anti-CD3/CD28 bead stimulation. Data are mean ± SEM from 20 independent experiments. **, P < 0.01, paired Student’s t test. For the kinetic of CXCL13 expression, CD4 T cells were restimulated for 24 h with anti-CD3/CD28 beads after 3, 4, 5, or 6 d of co-culture with DCs (circles) or TSLP-DC (triangles). SEM for seven independent experiments. (B) FACS staining for ICOS, PD1, and CXCR5 in CD4 T cells after 4 d of co-culture with DCs. CXCR5^hi/ICOS^hi and CXCR5^hi/PD1^hi cells within CD4 T DAPI⁻ cells from a representative donor are shown. (C) Quantification of cell populations as indicated in B in naive CD4 T cells after 0, 2, 4, or 6 d of co-culture with DCs (circles), TSLP-DC (filled triangles), or LPS-DC (open triangles). SEM from six independent experiments. (D) CXCR5^hi/PD1^hi and CXCR5^lo/PD1^lo CD4 T cells polarized 4 d by TSLP-DC were sorted (top), and co-cultured with autologous memory B cells for 14 d. CD38 and CD27 were measured by FACS on B cells (DAPI⁻/CD3⁻/CD4⁻/CD19⁺). One representative plot is shown. (E) IgA, IgG, IgG4, and IgE were quantified in the supernatants of co-cultures, as in D, in the indicated conditions. Mean ± SEM for five donors. n.d., not detected. (F) Quantification of IgG and IgE in the supernatants of memory B cells co-cultured as in D, plus IL4R-α blocking or isotype control antibodies. SEM from five independent experiments are plotted. *, P < 0.05; **, P < 0.01, paired Student’s t test.