(A) Human naïve CD4⁺ T cells cultured with allogeneic DCs stimulated with LPS or CD40L were re-stimulated at day 7 with PMA + ionomycin to test intracellular IL-21 expression. Gated to FSC^hi activated CD4⁺ T cells. A representative out of three experiments.
(B) Activated CD4⁺ T cells sorted at day 7 were re-stimulated with CD3 + CD28 mAbs 24 h to measure IL-21 secretion.
(C) Naïve CD4⁺ T cells were stimulated by CD3 + CD28 mAbs with the supernatants of DCs activated by CD40L or TLR-ligands. IL-21 production from activated CD4⁺ T cells as in (B).
(D) Naïve CD4⁺ T cells stimulated with CD3 + CD28 mAbs in the presence of the indicated cytokines and re-stimulated at day 7 with PMA + ionomycin for intracellular IL-21 expression. A representative out of three experiments.
(E, F) IL-21 secretion from activated CD4⁺ T cells. Experiments performed with cells from five different donors are shown in (F). Paired t-test.
(G) Expression of IL-21 in naïve CD4⁺ T cells primed with IL-21 (30 ng/ml) as in (D) (left). IL-21 secretion from the activated CD4⁺ T cells (right). A representative out of three experiments.

(A) Naive B cells pre-activated with anti-IgM were cultured in the presence of CpG and SEB with autologous CD4⁺ T cells primed with the indicated cytokines. Ig titers in the supernatants were measured at day 14. Mean ± s.e.m. n=6. Unpaired two-tailed t-test. A representative out of three experiments.
(B) Memory B cells were cultured in the presence of SEB with autologous CD4⁺ T cells primed with the indicated cytokines. Ig levels at day 14. Mean ± s.e.m. n=6. Unpaired two-tailed t-test. A representative out of three experiments.
(C) Ig secretion from memory B cells cultured with CD4⁺ T cells primed with titrated amounts of IL-12. A representative out of two experiments.
(D) IL-21R-Fc was added to block IL-21 to the co-cultures of memory B cells and CD4⁺ T cells primed with IL-12. Ig levels at day 14. Mean ± s.e.m. n=6. Unpaired two-tailed t-test. A representative out of three experiments.
(E) Blocking of ICOS by ICOS-L-mIgFc during T-B cultures. Ig levels at day 14. Mean ± s.e.m. n=6. Unpaired two-tailed t-test. A representative out of three experiments.

(A, B) Fresh PBMCs were stimulated with SEB in the presence of anti IL-12p40 or IL-12p70 mAb. IL-21 (A) or the indicated cytokines (B) secreted cytokines during 48 h cultures were measured. Symbols represent results from seven different donors.
(C) Memory CD4⁺ T cells were stimulated with CD3 + CD28 mAbs in the presence of the indicated cytokines. IL-21 secretion in the supernatants was measured at 24 and 48 h of culture.
(D) Naive CD4⁺ T cells were stimulated with CD3 + CD28 mAbs in the presence IL-12. IL-21 secretion in the supernatants was measured at the indicated time points.

(A) Naïve CD4⁺ T cells were stimulated with CD3 + CD28 mAbs for 7 d in the presence of titrated amounts of IL-12 or IL-23. IL-21 and IFN-γ secretion upon 24 h CD3 + CD28 restimulation. A representative out of two experiments.
(B) Naïve CD4⁺ T cells stimulated with CD3 + CD28 mAbs in the presence of 10 ng/ml IL-12 or IL-23 were re-stimulated at day 7 with PMA + ionomycin to test intracellular cytokine expression. Gated to FSC^hi activated CD4⁺ T cells. A representative out of five experiments.
(C) Intracellular cytokine expression in CD4⁺ T cells primed with titrated amounts of IL-12. After re-stimulation with PMA + ionomycin. A representative out of two experiments.
(D) Highly purified cord blood naïve CD4⁺ T cells were stimulated for 7 d with CD3 + CD28 mAbs ± IL-12. Intracellular IL-21 and IFN-γ expression upon stimulation with PMA and ionomycin. A representative out of three experiments.
(E) Expression of T-bet and intracytoplasmic cytokines was analyzed in CD4⁺ T cells primed with IL-12, after re-stimulation with PMA + ionomycin. The levels of T-bet expression in IL-21⁺IFN-γ^- (R1), IL-21^-IFN-γ⁺ (R2) and IL-21⁺IFN-γ⁺ (R3) cells were compared to that in IL-21^-IFN-γ^- cells (R0).
(F) Expression of T-bet and intracytoplasmic cytokines was analyzed in tonsillar CD4⁺ T cells stimulated with PMA + ionomycin.
(G) Naïve CD4⁺ T cells were cultured for 7 d with CD3 + CD28 mAbs under Th0, Th1, Th2, or Th17-skewing conditions. IL-21 secretion upon 24 h CD3 + CD28 restimulation from activated CD4⁺ T cells.

(A) IL-21 secretion from naïve CD4⁺ T cells cultured with allogeneic DCs exposed to heat-killed bacteria. A representative out of five experiments.
(B) IL-12 and IL-23 secretion from DCs exposed to heat-killed bacteria. A representative out of five experiments.
(C) IL-21 secretion of CD4⁺ T cells primed with bacteria-exposed DCs in the presence of anti IL-12p40 or IL-12p70 mAb. A representative out of five experiments.
(D) IL-21 secretion of CD4⁺ T cells primed with CD40L- or LPS-activated DCs in the presence of anti IL-12p70 mAb. A representative out of three experiments.
(E) IL-12 secretion from DCs exposed to CD40L or TLR-ligands. A representative out of three experiments.
(F) Naïve CD4⁺ T cells were cultured for 7 d with allogeneic bacteria-exposed DCs in the presence of anti IL-12p40 or IL-12p70 mAbs. Activated CD4⁺ T cells were sorted and cultured with autologous memory B cells, and produced Igs were analyzed. Mean ± s.e.m. n=6. Unpaired two-tailed t-test. A representative out of four experiments.

(A) Naïve CD4⁺ T cells stimulated with CD3 + CD28 mAbs for 5-7 d were transfected with siRNA specific for STAT3 or STAT4. The levels of STAT3 and STAT4 expression at 24 h post-transfection were analyzed by flow cytometry. Cells transfected with control siRNA (gray), STAT3 siRNA (blue), and STAT4 siRNA (red) are shown. Black histogram represents staining with control isotype. Gated to AQUA^-FSC^hi activated CD4⁺ T cells. A representative out of three experiments.
(B) Transfected CD4⁺ T cells were cultured for 48 h in the presence of IL-12, IL-23 or IL-21. Activated CD4⁺ T cells were then re-stimulated with CD3 + CD28 mAbs for 24 h to measure IL-21 secretion. A representative out of three experiments.
(C) After 48 h culture with IL-12, IL-23, or IL-21, the expression of IL-21 was analyzed after 6h restimulation with PMA + ionomycin. Gated to AQUA^-FSC^hi activated CD4⁺ T cells. A representative out of three experiments.
(D) Percentages of IL-21-expressing CD4⁺ T cells in three independent experiments.
(E) Percentages of IFNγ⁺IL-21⁺ and IFNγ^-IL-21⁺ cells in STAT4 siRNA-transfected CD4⁺ T cells cultured with IL-12.