NIV Congres

In addition to thymically derived natural regulatory T cells (nTreg), Treg can be induced in the periphery (iTreg) by antigen exposure and cytokines. It is currently unknown whether iTreg are induced during alloreactivity and whether these iTreg specifically suppress alloreactive T cells. Therefore, we studied the generation of iTregs and examined their immunosuppressive capacity and specificity in an alloreactive setting.

Sorted CD4+CD25-FOXP3- T cells from healthy blood bank donors (n=7) were stimulated with alloantigen and the induced CD4+CD25+FOXP3+ Treg were subdivided into CD127- iTreg and CD127+ iTreg by FACS sorting. Their suppressive capacity and specificity was examined in a secondary MLR. To validate the induced character of the iTreg, the methylation status of the TSDR (Treg Specific Demethylated Region) in the FOXP3 gene was measured.

After stimulation of CD4+CD25-FOXP3- T cells with alloantigen for 1 week, 78 ± 9% (mean ± SD) of the CD4+ cells became CD25+FOXP3+ (iTreg). Both CD127+ iTreg and CD127- iTreg robustly inhibited the proliferation of CD25- T cells in the secondary MLR (CD127+ iTreg: 93 ± 5%; CD127- iTreg: 95 ± 3%; ratio iTreg : CD25- T cells = 1:5) by restimulation with the same alloantigen. By stimulation with a third party alloantigen (fully mismatched at HLA-DR) in the secondary MLR, the proliferation of CD25- T cells was significantly less inhibited (iCD127+ iTreg: 68 ± 17%, p=0.004; iCD127- iTreg: 68 ± 13%, p=0.0003). By stimulation with a comparable alloantigen (1 HLA-DR mismatch) in the secondary MLR, the proliferation of CD25- T cells was inhibited with 96 ± 1% by CD127+ iTreg and 93 ± 2% by CD127- iTreg, which was comparable to the inhibition found by restimulation with the same alloantigen (p=0.4 and p=0.2). Non-activated CD3+CD4+CD25- T cells were unable to inhibit the proliferation of CD25- T cells. Both iTreg subsets were fully methylated at the TSDR in the FOXP3 gene, 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.