Figure 2
CD5-mediated Th17 development is attenuated by CD28 costimulation. (A) Naive T cells were stimulated with plate bound antibodies directed against CD3/CD5, CD3/CD5/CD28 (1 μg/mL) in Th17-polarizing conditions (IL-23, IL-1β, IL-6, TGF-β, and anti–IFN-γ). At day 12, IL-17 levels were measured by ELISA after 24 hours of restimulation with anti-CD3/PdBu. Data shown are of 9 independent experiments of individual donors. (B) Naive T cells were stimulated via CD3/CD5, CD3/CD5/CD28, or CD3/CD5/Betv1 (1 μg/mL) in Th17-polarizing conditions as described under (A). At day 12, IL-17 levels were measured by ELISA after 24 hours of restimulation with anti-CD3/PdBu. Data shown are from one representative experiment of 4 independent experiments using different donors. (C) Naive T cells were stimulated via plate bound antibodies directed against CD3/CD5 in combinations with various concentrations of coated CD28-specific antibody. After culturing with Th17-polarizing conditions for 11 days, cells were restimulated with anti-CD3/PdBU for 24 hours and IL-17 levels were measured by ELISA. One representative experiment is shown of 2 individual experiments using different donors. (D) CFSE labeled naive T cells were stimulated via CD3- specific antibodies (left) or CD3/CD5 in combination with various concentrations of CD28 specific antibody as described under (C). Proliferation was measured at day 4. Data shown are from one representative experiment of 3 independent experiments using different donors. (E) Real-time semiquantitative PCR of mRNA expression of IL2 of naive T cells stimulated via coated antibodies directed against CD3/CD28 or CD3/CD5 in Th17-polarizing conditions. Samples were measured after 18 and 72 hours of culture. Data shown are mean ± SD of triplo measurement from 1 representative experiment of 2 independent experiments using different donors. (F) Intracellular IL-2 was measured by FACS staining of naive T cells stimulated via CD3/CD28 or CD3/CD5 for 18 and 72 hours. Data shown are from one representative experiment of 4 independent experiments using different donors. (G) Naive T cells were stimulated via CD3/CD28 or CD3/CD5 in Th17 inducing conditions, in addition of extra IL-2 (100 U/mL) or IL-21 (10 ng/mL). IL-17 expression was measured at day 11 by intracellular cytokine staining. (H) Real-time semiquantitative PCR of mRNA expression of IL21 of naive T cells stimulated as described under (E). Samples were measured after 3 days, 6 days, and 11 days of culture. Data shown are mean ± SD of triplo measurement from one representative experiment of 2 independent experiments using different donors.

CD5-mediated Th17 development is attenuated by CD28 costimulation. (A) Naive T cells were stimulated with plate bound antibodies directed against CD3/CD5, CD3/CD5/CD28 (1 μg/mL) in Th17-polarizing conditions (IL-23, IL-1β, IL-6, TGF-β, and anti–IFN-γ). At day 12, IL-17 levels were measured by ELISA after 24 hours of restimulation with anti-CD3/PdBu. Data shown are of 9 independent experiments of individual donors. (B) Naive T cells were stimulated via CD3/CD5, CD3/CD5/CD28, or CD3/CD5/Betv1 (1 μg/mL) in Th17-polarizing conditions as described under (A). At day 12, IL-17 levels were measured by ELISA after 24 hours of restimulation with anti-CD3/PdBu. Data shown are from one representative experiment of 4 independent experiments using different donors. (C) Naive T cells were stimulated via plate bound antibodies directed against CD3/CD5 in combinations with various concentrations of coated CD28-specific antibody. After culturing with Th17-polarizing conditions for 11 days, cells were restimulated with anti-CD3/PdBU for 24 hours and IL-17 levels were measured by ELISA. One representative experiment is shown of 2 individual experiments using different donors. (D) CFSE labeled naive T cells were stimulated via CD3- specific antibodies (left) or CD3/CD5 in combination with various concentrations of CD28 specific antibody as described under (C). Proliferation was measured at day 4. Data shown are from one representative experiment of 3 independent experiments using different donors. (E) Real-time semiquantitative PCR of mRNA expression of IL2 of naive T cells stimulated via coated antibodies directed against CD3/CD28 or CD3/CD5 in Th17-polarizing conditions. Samples were measured after 18 and 72 hours of culture. Data shown are mean ± SD of triplo measurement from 1 representative experiment of 2 independent experiments using different donors. (F) Intracellular IL-2 was measured by FACS staining of naive T cells stimulated via CD3/CD28 or CD3/CD5 for 18 and 72 hours. Data shown are from one representative experiment of 4 independent experiments using different donors. (G) Naive T cells were stimulated via CD3/CD28 or CD3/CD5 in Th17 inducing conditions, in addition of extra IL-2 (100 U/mL) or IL-21 (10 ng/mL). IL-17 expression was measured at day 11 by intracellular cytokine staining. (H) Real-time semiquantitative PCR of mRNA expression of IL21 of naive T cells stimulated as described under (E). Samples were measured after 3 days, 6 days, and 11 days of culture. Data shown are mean ± SD of triplo measurement from one representative experiment of 2 independent experiments using different donors.

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