Bootcongres

In addition to thymically derived natural regulatory T cells (nTreg), Treg can be induced in the periphery (iTreg) by antigen exposure and cytokines. Here we studied the generation of iTreg during alloreactivity and examined their immunosuppressive capacity and specificity in an alloreactive setting.

Sorted CD4+CD25- T cells from healthy donors (n=9) were stimulated with HLA mismatched alloantigen to induce CD4+CD25+FOXP3+ T cells (iTreg). These CD4+CD25+ iTreg were subdivided into CD127- iTreg and CD127+ iTreg by FACS sorting and tested in a secondary MLR for their immunosuppressive capacity and specificity. 

In the secondary MLR, by using the original stimulator of the primary MLR, the proliferation of CD4+CD25- T cells was robustly inhibited by both CD127+ iTreg (94% (84-98%); ratio iTreg:CD4CD25- = 1:5) and CD127- iTreg (94% (70-97%)). Antigen specificity was confirmed by the observation that both iTreg subsets inhibited the proliferation of CD4+CD25- T cells using a stimulator sharing one HLA-DR with the original stimulator (CD127+ iTreg: 95% (84-97%) and CD127- iTreg: 92% (60-95%)) while stimulation with a fully mismatched 3P antigen resulted in significant less inhibition (CD127+ iTreg: 67% (44-84%), p=0.0008 and CD127-iTreg: 66% (52-86%), p=0.002). The immunosuppressive capacity and specificity of the iTreg subsets to alloactivated CD4+CD25- T cells was confirmed for CD8+CD25- T cells using FACS analysis. CD3+CD4+CD25- T cells, which were not activated in the primary MLR, were unable to inhibit the proliferation of either CD4CD25- T or CD8CD25- T cells. Further characterization of the iTreg subsets demonstrated that both subsets produce IFNγ (CD127+ iTreg: 52-60%; CD127- iTreg: 38-55%), but no IL10 (CD127+ iTreg: 0-0.6%; CD127-iTreg: 0-0.8%). Additionally, the TSDR in the FOXP3 gene was fully methylated (>99%) in both iTreg subsets, confirming their induced character. 

Alloantigen specific iTreg are induced during alloreactivity. Even though the mechanism by which suppression is mediated needs further investigation, these data indicate that iTreg could play an important role in controlling alloreactive T cells after transplantation.