Introduction: Previous studies of Treg using a limited number of markers have failed to establish a test of tolerance. It is now known that CD4⁺CD25⁺Treg are a heterogenous population of cells. Immunostaining for CD4/CD25/CD127/CD45RA and Foxp3 identifies five populations (Pop) of CD4⁺T cells, Treg comprising Pop I-III and effectors Pop IV-V. Naïve T cells express CD45RA, whereas activated/memory cells lack CD45RA. We examined CD4⁺CD25⁺CD127^lo Treg with gating on Foxp3 vs CD45RA. This defines Pop I as naive Treg (Foxp3⁺CD45RA⁺), Pop II activated/memory Treg (Foxp3^hiCD45RA^-) and Pop III (Foxp3⁺CD45RA^-). Also, within CD4⁺ gate, Pop IV was defined as effector/memory cells (Foxp3^-CD45RA⁺) and Pop V (CD45RA⁺Foxp3^-) as naïve effector CD4⁺T cells.

Here, we aim to examine changes in Treg and T effector cell populations to test our hypothesis that stable long-standing renal transplant patients have an increased proportion of activated CD4⁺CD25⁺CD127^loFoxp3⁺Treg consistent with development of transplant tolerance. Additionally, we looked at other lymphocyte subsets including CD8⁺T cells, B cells, NK cells and B cell subsets.

Methods: Fresh blood from healthy volunteers (HV) (n=14) and patients with kidney transplant surviving >10yrs (RT) (n=15) was on that day subjected to analysis of T, B, and NK cells using a 6-color TBNK reagent (BD) (CD3/CD4/CD8/CD45/CD19/CD16&CD56). Peripheral blood mononuclear cells (PBMC) were isolated from blood and stained with panels of monoclonal antibodies for Treg (CD4/CD25/CD127/CD45RA/Foxp3) and B cell subpopulations (CD19/CD21/CD24/CD27/CD38/CD45/IgD/IgM). Data was acquired on BD FACSCanto II using BD FACSDiva (V8.0) and analysed using FlowJo. Lymphocyte populations were examined after FSC vs SSC gating and doublets exclusion.

Results: Lymphocyte and CD4⁺T cells numbers in HV and RT were similar, but B cells were lower in RT (p<0.05). Naïve Treg (Pop I) (p<0.01) and naïve effector CD4⁺ T cells (Pop V) were lower in RT than HV. Activated Treg (combined Pop II and III) were greater in RT (p<0.05), as was activated effector T cells (Pop IV) in RT (p<0.05). Treg Pop II, which contain highly activated Treg, was not different in RT compared to HV. Total Treg (Pop I, II, III) in RT was less than in HV (p<0.05). Transitional B cells were also significantly lower in RT than HV (p<0.001). Ratio of activated Treg (Pop II & III) to B cells was markedly greater in RT (p<0.05). There were no differences in NK cells between RT and HV.

Conclusions: Long surviving RT patients had reduced naïve Treg and naïve effector cells but increased proportions of activated Treg and effector cells. The marked increase in the ratio of activated effector Treg to B cells may be associated with increased suppression. Further studies are needed to establish the relevance of such changes to induction of tolerance to transplanted tissues.