In primed allo-tolerance, TIM-3-Ig rapidly suppresses TGFbeta, but has no immediate effect on Foxp3.

T-cell immunoglobulin mucin-3 (TIM-3) is only expressed by differentiated TH1 cells following their proliferative response to antigen, functioning to terminate TH1-mediated immunity upon binding to the TIM-3 ligand, galectin-9. This critical regulatory process involves Treg cells via their stable expression of galectin-9. Soluble TIM-3-Ig blocks galectin-9 and prevents induction of peripheral tolerance. Here we have looked for evidence that TIM-3-Ig might also break established regulatory tolerance. Using allo-primed spleen cells cultured ex vivo and challenged with irradiated donor-type stimulator cells either alone or together with 20 microg/ml TIM-3-Ig, we measured daily cytokine release [IL2, inferon gamma (INFgamma), transforming growth factor beta (TGFbeta), IL6, IL10] and cellular Foxp3 protein. In allo-tolerance, a specific effect of TIM-3-Ig was some fourfold reduction in TGFbeta. Foxp3 was induced in the allo-tolerant response to donor and this was not altered by TIM-3-Ig over the 5-day culture period. No Foxp3 was detected in either rejected or donor stimulator cells at any time. Thus, in an ex vivo model of in vivo tolerance to heart allografts, TIM-3-Ig therapy appears to reduce the stable tolerogenic environment by a rapid and specific repression of TGFbeta release.