OR42 Expanded thymic regulatory T cells (Tregs) have low class I HLA expression levels and are HLA-DR negative – Potential for ‘Off-The-Shelf’ Cellular Therapy?

Aim Tregs modulate immune responses against alloantigens and are a promising therapeutic tool in organ transplantation. We previously showed that abundant CD25 + FOXP3 + Tregs can be isolated and expanded from discarded human thymuses, routinely removed during pediatric cardiac surgery, indicating potential use of thymic Tregs as an ‘off-the-shelf’ tolerogenic therapy. Immunogenicity of allogenic therapeutic Tregs, however, is unclear. Here we studied HLA-ABC and -DR expression on expanded thymic Tregs compared to blood Tregs. Methods Thymocytes were obtained from infant thymuses (age n  = 3) by mechanical dissociation; FOXP3 + CD25 + Tregs were isolated by magnetic-bead cell separation. Tregs were also isolated from infant peripheral blood mononuclear cells (PBMCs; n  = 3) autologous to the thymus and PBMCs from healthy adults ( n  = 3). After expansion with artificial antigen-presenting cells loaded with anti-CD3, IL-2, and rapamycin, Treg characteristics were confirmed by analyzing FOXP3 expression and suppressive capacity of proliferation of anti-CD3/28-stimulated T cells. HLA-ABC (clone W6/32) and HLA-DR (clone LN3) expression before and after expansion was assessed by flow cytometry. Results Before expansion, HLA-ABC expression levels were similar between thymic, infant blood and adult blood Tregs (Median Fluorescence Intensity (MFI) range: 3673–6059 vs. 3841–3830 vs. 6355–7952, respectively). Few thymic Tregs or infant blood Tregs expressed HLA-DR ( Conclusions Expanded thymic Tregs had lower class I HLA expression levels and contained fewer HLA-DR + cells compared to expanded blood Tregs, suggesting that thymic Tregs may be less immunogenic than blood Tregs. These findings indicate that discarded human thymus is potentially an excellent source of Tregs for ‘off-the-shelf’ tolerogenic therapy.