T regulatory cells are a specialised subset of CD4⁺ T cells that regulate immune responses and are identified as CD4⁺CD25⁺CD127^loFoxp3⁺. Treg population itself is heterogenous and comprise of three populations. These three populations constitute population (Pop) I-III of 5 populations based on CD25/Foxp3 and CD45RA expression within CD4⁺ T cells.  Within CD4⁺ cells, Pop I are naïve Treg (CD25⁺FOXP3⁺CD45RA⁺); Pop II activated Treg (CD25^hiFOXP3^hiCD45RA^-); Pop III as CD25⁺FOXP3⁺CD45RA^-, Pop IV activated effectors (CD25^-FOXP3^-CD45RA^-); Pop V naive T cells (CD25^-FOXP3^-CD45RA⁺). Changes in these populations may indicate transplant tolerance and identify patients who could reduce immunosuppression. We examined 5 populations of CD4+ T cells in patients with long surviving kidney allograft and compared them with healthy volunteers. To identify activated cells, expression of chemokine receptors and Treg effector molecules CD39, Class II MHC and PD-1 were assayed.
Blood from healthy volunteers (HV) (n=44) and stable renal transplant patients with grafts surviving >10yrs (RT) (n=25) was examined for T, B, and NK cells using CD3/CD4/CD8/CD45/CD19/CD16&CD56 (TBNK reagent, BD). Panels of mAb for CD4⁺ populations included CD4/CD25/CD127/CD45RA/Foxp3. Expression of chemokines (CCR4/CXCR3/CCR6) and activation markers (CD39/HLA-DR/PD-1) was analysed for each population. Data was acquired on BD FACSCanto II using FACS DIVA 8.0 and analysed using FlowJo (BD) to examine Treg and effector T cell populations and Treg/B cells ratio.
Lymphocyte and CD4+ T cell numbers in HV and RT were similar. Total Treg proportion within CD4 cells was lower (p<0.01) in RT than HV. RT had lower naïve Treg (Pop I) (p<0.01) and naïve CD4⁺ T cells (Pop V) (p<0.001), but higher activated CD4⁺ effectors (pop IV) than HV (p<0.001). Pop II and III were not different in two groups. Expression of CXCR3 or CCR6 was less in Pop I and V whereas Pop II had ~50% CXCR3⁺ or CCR6⁺ cells. RT had significantly lower CXCR3⁺ (p<0.0001) or CCR6⁺ (p<0.01) cells in Pop II compared to HV. Both Pop II and III had significantly higher (p<0.01) CXCR3^-CCR6^- cells and lower CXCR3⁺CCR6⁺ cells (p<0.001) in RT than HV. Examining CCR4⁺ first, then for CXCR3 or CCR6 identifies Th-like Treg. Th2-like Treg (CCR4⁺CXCR3^-CCR6^-) were higher in RT in Pop II (p<0.01) and Pop III (p<0.05). Th1/Th17-like Treg (CCR4⁺CXCR3⁺CCR6⁺) in Pop II (p<0.001) and Pop III (p<0.01) were lower in RT than HV. Treg effector molecules analysis revealed no differences in CD39, HLA-DR and PD-1 expression. Ratio of activated Treg (Pop II & III) to B cells was markedly greater in RT (p<0.05).
Long surviving RT patients had reduced naïve Treg, but increased proportions of effector cells. Unanticipated, activated Treg had a lower proportion of Th1- and Th17-like Treg cells and an increased proportion of Th2-like Treg.