#1896 Monitoring of peripheral blood B cell subsets 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
Studies of peripheral blood B cells subsets in renal transplant patients with operational tolerance off immunosuppressive therapy have identified several differences in B cell subsets. Here we studied B cells in blood lymphocytes from long-surviving renal transplant patients on immunosuppression. Populations of CD4+ T cells, CD8+ T cells, NK cells and B cells, along with subpopulations of B cells, in long-surviving renal transplant recipients were compared to healthy volunteers. We were looking for changes in B cell subsets, which may indicate transplant tolerance and potentially identify patients who could reduce immunosuppression.

Method
Fresh blood was taken from healthy volunteers (HV) (n = 44) and stable renal transplant patients with grafts surviving &gt;10yrs (RT) (n = 25). T, B, and NK cells were examined using a 6-color TBNK reagent (BD) (CD3/CD4/CD8/CD45/CD19/CD16&CD56). Peripheral blood mononuclear cells were isolated from the remaining fresh blood and were stained with a panel of monoclonal antibodies (CD19/CD21/CD24/CD27/CD38/CD45/IgD/IgM) to identify subpopulations of B cells. 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.

Results
RT had significantly less lymphocyte counts in comparison to HV (p = 0.0156). There were no significant differences between the CD4+ T cell, CD8+ T cell, or NK cell percentages between RT and HV. B cells were markedly reduced in RT (p = 0.0001) vs HV when examined by TBNK staining. Peripheral blood mononuclear cells of HV (n = 13) and RT (n = 23) stained with a panel of B cell-related antibodies showed CD19+ cells were also significantly reduced in RT (p = 0.0006). There were less CD21hi cells (p = 0.0014) and more CD21lo (p = 0.0014) cells in RT than HV. No differences were seen between the two groups in their proportion of naïve B cells, marginal zone B cells, memory B cells, or class unswitched B cells. However, switched memory B cells were higher in RT than HV (p = 0.0181). Plasmablasts were also higher in RT compared to HV (p = 0.0491). Transitional B cells in RT appeared lower than HV on average, but this did not reach statistical significance (p = 0.1265). The ratio of Treg to B cells, RT (n = 25) showed significantly higher (p = 0.0256) proportions of activated Treg (CD4+CD25+CD127loCD45RA−Foxp3+/hi) to B cells, in comparison to HV (n = 44). There were no significant differences between the two groups in the ratio of naïve Treg (CD4+CD25+CD127loCD45RA+Foxp3+) to naïve T cells (CD4+CD45RA+Foxp3−), or highly activated Treg (CD4+CD25+CD127loCD45RA−Foxp3hi) to activated effector T cells (CD4+CD45RA−Foxp3−).

Conclusion
B cells were significantly lower in RT compared to HV. There were more plasmablasts and switched memory B cells in RT than HV. The ratio of activated Treg to B cells was higher in RT. This higher proportion of Treg to B cells may be clinically relevant and potentially demonstrate increased immunological suppression in long-surviving renal transplant recipients.