Donor-specific immunomodulation is mediated by mechanisms beyond CD4⁺ regulatory T cells

Donor-specific immunomodulatory cells (DSIMC) have successfully induced donor-specific tolerance in clinical living donor liver transplantation. These cells are generated from recipient peripheral blood mononuclear cells (PBMCs) stimulated with irradiated donor PBMC in the presence of costimulatory blockade, yielding a heterogeneous population of recipient-derived immune cells including T cells, B cells, and NK cells, in which CD4⁺ regulatory T cells (Tregs) are thought to play a central role. Here, we examined the cell populations and functions crucial for donor-specific immunomodulation within the DSIMC product. DSIMC were generated from PBMC of healthy volunteers using belatacept to achieve costimulatory blockade. Tregs were enriched by CD4⁺CD25⁺ magnetic-cell activated cell sorting (MACS) or CD25⁺CD127^-/low fluorescence-activated cell sorting (FACS). CD4⁺, CD8⁺, CD19⁺, and CD25⁺CD127^-/low fractions were also individually depleted from DSIMC. Immunomodulatory function of each resulting population was assessed by tritium-labelled thymidine incorporation in mixed lymphocyte cultures (MLC), and cytokine-producing capacity was evaluated by ELISA/ELISpot assays. Unsorted DSIMC exhibited donor-specific suppression at lower cell numbers than CD4⁺CD25⁺ enriched cells, while CD4⁺CD25^- T cells showed no immunosuppressive effect. Depletion of CD4⁺, CD8⁺, or CD19⁺ cells did not substantially impair immunomodulatory function. In contrast, depletion of the CD25⁺CD127^-/low fraction reduced inhibitory function and abolished donor specificity. Depletion of CD4⁺ or CD25⁺CD127^-/low reduced IFN-γ and IL-10 production in response to donor stimulation compared with unsorted DSIMC, implicating CD4⁺ Tregs as important contributors to cytokine production under allogeneic stimulation. Furthermore, Treg expansion following donor antigen restimulation was observed in unsorted DSIMC but not in CD4⁺CD25⁺-enriched cells, suggesting that Tregs can be induced from non-Treg CD4⁺ populations within the DSIMC. In conclusion, CD4⁺CD25⁺CD127^-/lowFoxP3⁺-associated cells appear to contribute to donor-specific immunomodulation; however, the enrichment of CD4⁺CD25⁺ T cell diminished DSIMC efficacy. Since the unsorted DSIMC showed stronger activity, we suggest that multiple cell populations may act together to mediate the DSIMC effect.