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Methods and Results: Tregs from normal donors were isolated by CD25 immunoabsorption and cultured with dendritic cells (DC) from HLA-matched siblings in the presence of IL-2, IL-15 and rapamycin. We detected a 15-fold increase in 3H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical, mHA incompatible sibling DC compared to self-DC. The median precursor frequency of mHA-specific Treg is 1.3 (range, 0.1-13.5) cells per million total blood CD4 T cells, while the median frequency of mHA-specific conventional CD4 (Tconv) is 25 (range, 3-141) per million total blood CD4 T cells. Among 13 HLA-identical sibling pair tested in any gender combination, there was a 26 (range, 8 to 39)-fold excess mHA-specific Tconv over mHA-specific Treg in the donor blood. It is the predominance of alloreactive Tconv over Tregs that drives the immune response causing GVHD. Purified Tregs were expanded in culture with for 12 days with DC from a HLA-identical sibling, IL-2, IL-15 and rapamycin (n=5), and the frequency of mHA-specific Tregs expanded 147 to 394-folds. Tregs responded to re-stimulation with DC from the original HLA-identical sibling, but not self-DC. The mHA-specific Tregs exhibited antigen specific suppression. When Tregs were cultured at limiting dilution, we obtained 4 mHA-specific Treg clones that retained TGFb secretion in response to the sibling’s mHA-disparate DC but not self-DC. One of these clones from a female donor responded to HY-antigen DBY expressed on self-APC after gene transduction and not UTY or DC alone, while another clone responded to UTY not DBY or self-DC alone. We adapted our protocol to GMP-compatible, large scale expansion of Treg from apheresis of 3.7 x 1010cells. Cultured Tregs maintained high levels of Foxp3 expression and demethylation of the Treg-specific Foxp3 gene promoter of the end-culture. mHA-specific Tregs were enriched 324-folds during the 12-day culture, indicating that we will be able to produce enough cells to exceed the numbers of mHA-specific CD4 Tconv (CD4 Tconv/Treg ratio, range 8-29) using a leukapheresis. We could not detect alloreactive T cells in this culture, besides TGFb-producing Tregs. The end-of-culture Tregs produced allospecific suppression with an IC50 between 1:1,000 and 1:10,000.
Conclusions: 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 against ubiquitous mHAs may prevent GVHD.