IFN-γ plays an essential role in BBI induced Tr1 cells and its therapeutic effect on EAE.

Regulatory T cells play an important role in suppression of inflammatory and autoimmune responses. Type 1 regulatory T cells (CD4⁺Foxp3^-LAG3⁺CD49b⁺), or Tr1 cells, have a remarkable capacity for suppressing autoimmune inflammation through IL-10. We have previously shown that Bowman Birk Inhibitor (BBI) suppresses CNS autoimmunity in experimental autoimmune encephalitis model. In this study, we report that BBI acts directly on T cells and induce Tr1 cells in comparable amount as IL-27 in both mouse and human cells. Although the transcriptional factors c-Maf and Ahr contribute to the differentiation of Tr1 cells from naïve CD4⁺ T cells, it is unclear which signals trigger this differentiation pathway.

C57BL/6, Il27ra^-/-, Tbx21^-/-, Stat1^-/- were used for In-vitro and In-vivo experiments. For EAE, female C57BL/6 mice, 8–12 weeks old, were immunized in the flanks subcutaneously with MOG_35–55 in CFA. Spleens were collected from naïve mice and cells were activated with anti-CD3/CD28 in the presence or absence of BBI. Duoset ELISA  were used for quantification of cytokines in culture supernatants.

BBI directly act on CD4 T cells and induce IL-10 in comparable amount as IL-27. We closely looked at the phenotype of IL-10 producing CD4 by staining multiple transcription factors and intracellular cytokines. Our results showed that BBI did not induce Foxp3 expression in IL-10⁺CD4⁺ T cells; however, it did induce T-bet and IFN-γ expression. In addition, Co-expression of LAG3 and CD49b identifies Tr1 cells and distinguishes them from Th1 cells in human and mice.Additionally, we have shown that lack of IFN-γ abrogates Tr1 and IL-10 inducing effect of BBI In-vitro and its therapeutic effect invivo.