Abstract
OBJECTIVE: To investigate the molecular mechanism for biased interleukin-17 (IL-17) production by DBA/1 CD4+ T cells upon T cell receptor (TCR) and transforming growth factor β (TGFβ) stimulation. METHODS: Purified naive CD4+ T cells were stimulated with anti-CD3/CD28 under Th1, Th2, Th17, and induced T regulatory (iTreg) cell conditions. Cytokine production was assayed by intracellular staining and enzyme-linked immunosorbent assay. Expression of transcription factors was determined by reverse transcription-polymerase chain reaction, flow cytometry, and immunoblotting techniques. RESULTS: Naive CD4+ T cells from DBA/1 mice produced more IL-17 under Th17 cell polarizing conditions as compared with those from C57BL/6 or BALB/c mice. Further investigation revealed no difference among the strains in terms of CD4+ T cell survival, upstream TCR signaling, or CD69 expression or in the phosphorylation of STAT-3 and expression of suppressor of cytokine signaling 3 that positively or negatively regulate IL-17 cell production. However, DBA/1 CD4+ T cells expressed increased levels of retinoic acid-related orphan receptor γt (RORγt). Furthermore, under iTreg cell polarizing conditions, DBA/1 CD4+ T cells showed a strikingly reduced level of FoxP3 expression. When interferon-γ and IL-4 were blocked, FoxP3 expression increased but remained lower in DBA/1 CD4+ T cells following exposure to TGFβ as compared with C57BL/6 CD4+ T cells. Moreover, DBA/1 CD4+ T cells showed reduced phosphorylation of Smad2 and Smad3 under both Th17 and iTreg cell polarizing conditions. CONCLUSION: These results indicate that naive CD4+ T cells from DBA/1 mice have a dichotomous response to TGFβ: enhanced RORγt, yet reduced FoxP3, up-regulation. This observation may help to elucidate the branch point of TGFβ signaling that leads to skewed Th17, but reduced iTreg, cell differentiation.