Tregs Specific for Minor Histocompatibility Antigens

The risk of acute GVHD after HSCT is increased in male recipients of female grafts. Disparities for the male-associated H-Y and other minor histocompatibility antigens (mHAs) have the capacity to sensitize alloreactive donor T cells and cause GVHD in HLA-matched recipients. These mHAs are polymorphic proteins that differ between donor and recipient and are presented as peptides by HLA molecules on recipient or donor antigen-presenting cells to donor immune cells. Currently, there is no evidence that minor histocompatibility antigen specific Tregs exist. Earlier in our laboratory, we have measured the frequency, growth requirements, and function of human blood Tregs specific for allo-MHC. In the present study, we sought to detect the frequency, expansion kinetics and characteristics of the minor antigens specific Tregs in the blood of HLA-matched sibling pair.

CD4⁺CD25⁺CD127⁻ Tregs were isolated by immunoabsorption from sibling donors, and cultured with HLA-matched sibling recipient antigen-presenting cells in the presence of IL-2, IL-15 and rapamycin. We detected 30–50 fold increase in H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical sibling compared to self DC. The precursor frequency of mHA-specific Tregs are between 7 and 43 (median - 13) cells per one million blood Tregs. The frequency of mHA-specific conventional CD4 T cells among total blood CD4 T cells is similar in HLA-matched sibling donors. Ex vivo expanded mHA-specific Tregs maintained higher levels of Foxp3 expression, retained the lymphoid homing receptor CD62L and a chemokine receptor, CCR7, suggesting that they are functional and are able to migrate to lymphoid tissue in vivo. Split well assay on day 12 demonstrated the mHA specificity, since Treg responded to restimulation with DC from the original HLA-identical sibling, but not self DC. The mHA-specific Tregs expanded to more than 100 fold in vitro, and exhibited antigen specific suppression. When Tregs were cultured at limiting dilution, we obtained 6 mHA-specific Treg clones that retained TGF-beta secretion in response to the sibling's mHA-disparate DC but not self DC.

We demonstrated for the first time that it is possible to detect and expand mHA specific Tregs from HLA-matched sibling pairs, immunotherapy with mHA-specific Tregs may prevent GVHD.

No relevant conflicts of interest to declare.