Selection and Characterisation of Minor Histocompatibility Antigen-Specific Regulatory T Cells in Fully HLA-Matched Setting for GVHD Therapy

Graft-versus-host disease is mediated by donor-derived T cells reactive against the recipient's broadly expressed minor histocompatibility antigens (mHA). Regulatory T cells (Treg) have been explored as a therapeutic approach for chronic GVHD (cGVHD). The promising results from polyclonal Treg trials in this setting have led us to develop a Treg product specific for mismatched minor antigens between patient and donor (mTreg), circumventing broad immune suppression risks. HLA-matched siblings of opposite sexes were used to obtain the sister's CD4+CD25hiCD127low Treg for co-culture with the respective brother's dendritic cells as a source of mismatched mHA. We have established the optimal culture conditions resulting in the highest mTreg proliferation and viability. Comprehensive phenotyping during the ex vivo selection shows PD-1, CTLA-4, CD39 and HLA-DR expression. Transcriptomic analysis revealed a switch in metabolic process, and up-regulation of functional Treg genes. Furthermore, mTreg possess specific and potent suppressive activity, in which there is a dependency on cell-to-cell contact and a role for HLA class II expression on mTreg. This protocol would allow the generation of Treg specific to an array of mHA from the recipient's healthy tissues, likely providing a directed and strong suppression of cGVHD.