Figure 1
CD5 is superior to CD28 in Th17 differentiation. (A) Naive CD4+CD45RA+CD45RO− T cells were stimulated via plate bound antibodies directed against CD3/CD28 or CD3/CD5 in Th0, Th1 (+ IL-12), or Th17 polarizing conditions (IL-23, IL-1β, IL-6, TGF-β, and anti-IFN-γ). IFN-γ and IL-17 levels were measured by ELISA at day 12, after 24 hours of restimulation with a CD3-specific mAb (1 μg/mL) and PdBu (50nM). Data shown are from 1 representative experiment of 4 independent experiments with different donors. (B) ELISA of IL-17A and IFN-γ of naive T cells stimulated via CD3/CD28 or CD3/CD5 in Th17-polarising conditions as described under (A). After 11 days of culture, cells were restimulated for 24 hours with anti-CD3/PdBu. Average IL-17 production levels; CD3/CD28: 0.45 ng/mL and CD3/CD5: 6.4 ng/mL IL-17. Average IFN-γ production levels; CD3/CD28: 0.3 ng/mL and CD3/CD5: 0.7 ng/mL IFN-γ. Data are shown as mean of 19 individual experiments with different donors. (C) Naive T cells were stimulated via CD3/CD28 (left) or CD3/CD5 antibodies (right) in Th17 polarizing conditions as described under panel A. Intracellular levels of IFN-γ and IL-17A levels were measured at day 11, after 5 hours of restimulation with PMA, ionomycin, and BFA. (D) FACS plots shown are from one representative of 18 different donors, shown combined in a graph. (E) Correlation between IL-17 production measured by ELISA and IL-17 expression measured by intracellular cytokine staining. CD3/CD28 stimulated cells (red) does not show a correlation (R = 0.21, P = .3982), whereas CD3/CD5 (black) does show a significant correlation (R = 0.78, P = .0002). Data are of 17 independent experiments using different donors. (F) Real-time semiquantitative PCR of mRNA expression of IL17A and IL17F of naive T cells stimulated via coated antibodies directed against CD3/CD28 or CD3/CD5 in Th17-polarising 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 1 representative experiment of 4 independent experiments using different donors. (G) CFSE labeled naive CD4+ T cells were stimulated by plate-bound antibodies directed against CD3 (fixed at 1 μg/mL) in combination with increasing concentration of either CD28-, CD5-, or irrelevant Betv1-specific antibody. Proliferation was measured after 3 day culture in Th17-inducing conditions. Data are shown as mean ± SD for 2 different donors.

CD5 is superior to CD28 in Th17 differentiation. (A) Naive CD4+CD45RA+CD45RO− T cells were stimulated via plate bound antibodies directed against CD3/CD28 or CD3/CD5 in Th0, Th1 (+ IL-12), or Th17 polarizing conditions (IL-23, IL-1β, IL-6, TGF-β, and anti-IFN-γ). IFN-γ and IL-17 levels were measured by ELISA at day 12, after 24 hours of restimulation with a CD3-specific mAb (1 μg/mL) and PdBu (50nM). Data shown are from 1 representative experiment of 4 independent experiments with different donors. (B) ELISA of IL-17A and IFN-γ of naive T cells stimulated via CD3/CD28 or CD3/CD5 in Th17-polarising conditions as described under (A). After 11 days of culture, cells were restimulated for 24 hours with anti-CD3/PdBu. Average IL-17 production levels; CD3/CD28: 0.45 ng/mL and CD3/CD5: 6.4 ng/mL IL-17. Average IFN-γ production levels; CD3/CD28: 0.3 ng/mL and CD3/CD5: 0.7 ng/mL IFN-γ. Data are shown as mean of 19 individual experiments with different donors. (C) Naive T cells were stimulated via CD3/CD28 (left) or CD3/CD5 antibodies (right) in Th17 polarizing conditions as described under panel A. Intracellular levels of IFN-γ and IL-17A levels were measured at day 11, after 5 hours of restimulation with PMA, ionomycin, and BFA. (D) FACS plots shown are from one representative of 18 different donors, shown combined in a graph. (E) Correlation between IL-17 production measured by ELISA and IL-17 expression measured by intracellular cytokine staining. CD3/CD28 stimulated cells (red) does not show a correlation (R = 0.21, P = .3982), whereas CD3/CD5 (black) does show a significant correlation (R = 0.78, P = .0002). Data are of 17 independent experiments using different donors. (F) Real-time semiquantitative PCR of mRNA expression of IL17A and IL17F of naive T cells stimulated via coated antibodies directed against CD3/CD28 or CD3/CD5 in Th17-polarising 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 1 representative experiment of 4 independent experiments using different donors. (G) CFSE labeled naive CD4+ T cells were stimulated by plate-bound antibodies directed against CD3 (fixed at 1 μg/mL) in combination with increasing concentration of either CD28-, CD5-, or irrelevant Betv1-specific antibody. Proliferation was measured after 3 day culture in Th17-inducing conditions. Data are shown as mean ± SD for 2 different donors.

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