#1167 Examination of subsets of CD4+ lymphocyte subpopulations in long-surviving renal transplant patients
Authors: Rakesh Prateek , Zahorowska Beata , Diep Jason , Cheung Jason , Wu Pamela , Al-Atiyah Ranje , Suranyi Michael , Spicer Tim , Wong Jeffrey , Hodgkinson Suzanne J , Hall Bruce M , Verma Nirupama D 
Published: May 2024

Abstract

Background and Aims
Subpopulations of CD4+ cells, play a major role in mediation of rejection and tolerance to a renal transplant. This study examined subsets of CD4+ T cells and CD4+CD25+CD127lo Treg in blood of renal graft recipients with grafts surviving &gt;10 years for changes in lymphocyte subpopulations that may indicate transplant tolerance and potentially identify patients who could reduce immunosuppression.
Based on CD45RA and CD25/Foxp3 expression, Miyara et al identified five populations (Pop) within CD4+ cells. Pop I, II and III are T regulatory cells (Treg); Pop I as naïve (CD45RA+Foxp3+) Treg, Pop II as activated (CD45RA-Foxp3hi) Treg, and Pop III as cytokine secreting (CD45RA-Foxp3+) cells which also includes activated effector cells. Pop IV are activated T cells (CD45RA-Foxp3−), and Pop V are naïve T cells (CD45RA+Foxp3−). Here, we examined three Treg populations (Pop I-III) within CD4+CD25+CD127lo Treg, and Pop IV and V within CD4+ T cells. To further characterise these subpopulations, we examined chemokine receptor expression (CXCR3, CCR4, CCR6, and CCR7) to identify T helper phenotype (Th-like) of Treg populations. Th1-like Treg were identified as CCR4+CXCR3+CCR6−, Th2-like as CCR4+CXCR3-CCR6−, Th17-like as CCR4+CXCR3-CCR6+, and Th1/Th17-like as CCR4+CXCR3+CCR6+. We also examined expression of activated Treg effector molecules CD39, Class II MHC, and PD-1.

Method
Peripheral blood mononuclear cells (PBMC) were isolated from fresh blood collected from healthy volunteers (HV) (n = 44) and long-term stable renal transplant patients with grafts surviving &gt;10 yrs (RT) (n = 25). Panels of monoclonal antibodies for Treg (CD4/CD25/CD127/CD45RA/Foxp3), chemokine receptors (CXCR3, CCR4, CCR6, CCR7), and Treg activation and suppression-related molecules (CD39/HLA-DR/PD-1) were also used. Data was acquired on BD FACSCanto II using BD FACSDiva software (v8.0) and analysed using FlowJo. Lymphocyte populations were examined after FSC vs SSC gating and doublets exclusion. T cell and Treg populations were analysed within CD4+ and CD4+CD25+CD127lo T cells.

Results
RT had lower lymphocyte counts than HV (p = 0.0156) but had a similar percentage of CD4+ T cells. Treg were significantly lower in RT compared to HV (p = 0.0012). RT patients had less naïve Treg (Pop I, p = 0.0083) and naïve effector T cells (Pop V, p = 0.0003), but had higher activated effector T cells (Pop IV, p = 0.0002) than HV. There were no differences in activated Treg Pop II or III between the two groups. There were significantly less cells expressing CXCR3 in RT than HV in Pop I (p = 0.0120), Pop II (p &lt; 0.0001), Pop III (p &lt; 0.0001) and Pop IV (p = 0.0047). Similarly, RT had fewer cells expressing CCR6 compared to HV in Pop II (p = 0.0019), Pop III (p = 0.0015), and Pop V (p = 0.0017). RT had lower proportions of CCR7 expressing cells in Pop I (p = 0.0035), Pop IV (p = 0.0097), and Pop V (p = 0.0002), but higher in Pop II (p = 0.0041) compared to HV. There were similar percentages of cells with Th1 and Th17-like phenotypes in both groups. However, RT had significantly more Th2-like Treg in Pop II (p = 0.0033) and Pop III (p = 0.0100), and significantly less Th1/Th17-like Treg in Pop II (p = 0.0005) and Pop III (p = 0.0017) compared to HV. There were little to no differences in the Treg activation and suppression-related molecules within subpopulations of Treg.

Conclusion
Long-surviving RT patients had less naïve Treg and naïve effector cells, but increased proportions of activated effector cells. There were little to no differences in the expression of Treg activation or suppression-related molecules in Treg subpopulations.
Unexpectedly, activated Treg in RT had significantly higher proportions with a Th2 phenotype and significantly less Treg with a Th1/Th17 phenotype. Whether this is a consequence of immunosuppression, or a biological shift associated with tolerance, remains to be resolved.
Further studies are required to investigate the relevance of these changes in identifying induction of transplant tolerance.