The human "treg MLR": Immune monitoring for FOXP3+ T regulatory cell generation

BACKGROUND: Controversy exists about the conditions effecting the development of forkhead/winghead helix transcription factor P3 (FOXP3) expressing T cells and their relevance in transplant recipients. METHODS: We generated carboxy-fluorescein diacetate succinimidyl ester-labeled CD4CD25 FOXP3 cells in mixed lymphocyte reactions (MLRs) ("the Treg MLRg"), with varying human leukocyte antigen (HLA) disparities and cell components. Five color flow cytometry and H-thymidine uptakes were the readouts. RESULTS: (1) Despite lower stimulation indices (SIs) than two DR-mismatched MLRs, 2 DR-matched MLRs generated more than twofold higher percentages when gating on proliferating CD4CD25 FOXP3 cells; (2) Even with low numbers of proliferating cells, autologous and HLA identical MLRs generated the highest FOXP3:FOXP3 cell ratios; (3) Elimination of either non-CD3 responding cells (resulting in "direct presentationg" only) or responding CD25 (Treg generating) cells increased the SI but inhibited proliferating CD4CD25 FOXP3 cell development; (4) MLR-generated CD4CD25 FOXP3 cells added as third components specifically inhibited the same freshly set MLR SI and caused recruitment of new CD4CD25 FOXP3 cells. As an example of the "Treg MLR" immune monitoring potential, addition of third component peripheral blood mononuclear cell containing high percentages of CD4CD25 FOXP3 cells from an HLA identical kidney transplant recipient (in a tolerance protocol) caused donor-specific Treg MLR inhibition or recruitment. This was similar to the third component MLR Tregs generated entirely in vitro. CONCLUSION: In the Treg MLR, the generation of CD4CD25 FOXP3 cells is more pronounced in the context of self-recognition (HLA matching, indirect presentation). These cells can be assayed for MLR inhibitory and Treg recruitment functions, so as to immunologically monitor the allospecific regulation after transplantation.