Background Controversy exists about the conditions effecting the development of FOXP3 expressing T cells and their relevance in transplant recipients. Methods We generated CFSE-labeled CD4+CD25highFOXP3+ cells in MLRs (‘the Treg MLR’), with varying HLA disparities and cell components. Five color flow cytometry and 3H TdR uptakes were the readouts. Results 1) Despite lower Stimulation Indices (SI) than 2 DR-mismatched MLRs, 2 DR-matched MLRs generated >2 fold higher percentages when gating on proliferating CD4+CD25highFOXP3+ 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 presentation’ only) or responding CD25+ (Treg generating) cells increased the SI but inhibited proliferating CD4+CD25HighFOXP3+ cell development; 4) MLR-generated CD4+CD25HighFOXP3+ cells added as third components specifically inhibited the same freshly set MLR SI and caused recruitment of new CD4+CD25HighFOXP3+ cells. As an example of the ‘Treg MLR’ immune monitoring potential, addition of third component PBMC containing high percentages of CD4+CD25highFOXP3+ cells from an HLA identical kidney transplant recipient (in a tolerance protocol) caused donor-specific Treg MLR inhibition/recruitment. This was similar to the third component MLR Tregs generated entirely in vitro. Conclusion In the ‘Treg MLR’, the generation of CD4+CD25High 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 allo-specific regulation after transplantation.

We compared peripheral blood immunophenotyping in 31 adult liver transplant recipients on differing long-term immunosuppressive (IS) monotherapy with and without peri-transplantation alemtuzumab (AL) induction. All patients had been stable on monotherapy with either sirolimus (SRL) (n = 10) or without SRL (tacrolimus (TAC) (n = 10), mycophenolate mofetil (MMF) (n = 11)) for more than 6 months. Five-color flow cytometry for putative “regulatory” T and dendritic cells as well as serum assays for soluble HLA-G (sHLA-G) were performed. The SRL monotherapy group had significantly higher percentages of CD4+CD25high+ Foxp3+ T cells (1.3 ± 1.0) compared with the non-SRL group (0.7 ± 0.6) (p = 0.04). The SRL effect was even higher in a subset with prior AL induction and no prior hepatitis C or rejection (1.7 ± 0.2) compared with all other subgroups (0.7 ± 0.6) (p = 0.02). TAC patients showed significantly higher “regulatory” DC2:DC1 ratios (10 ± 7.6) compared with non-TAC patients (4.1 ± 2.3) (p = 0.04). Although sHLA-G levels appeared higher in TAC patients, the differences were not statistically significant. In conclusion, IS monotherapy provides an opportunity to investigate regulatory roles of individual agents. SRL maintenance and prior AL induction in subsets of patients appeared to show a regulatory T cell immunophenotype. However, TAC patients may have other regulatory characteristics, supporting the need for larger, prospective studies to clarify differences.

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