Benefits of Using CD45RA and CD28 to Investigate CD8 Subsets in Kidney Transplant Recipients

Abstract

The deleterious role of CD8 T cells in kidney graft outcome has regained interest over the years, and memory T cells are considered as one of the main hurdles to achieve transplantation success. Monitoring the CD8 immune response in transplant recipients involved a heterogeneous combination of markers, but the justification of their choice is rarely stated. Whereas the number of parameters is not an issue in phenotypic analysis, functional assays have to accommodate the cell number with the narrowing of the subset. The aim of the study was to investigate the similarities and differences of the subsets identified using three nomenclatures (CD45RA and CCR7/CD27/ CD28) in kidney transplant recipients with stable graft function. We found that all three nomenclatures can identify na€ ıve and effector memory (EM) rheumatoid arthritis T cell CD8 with similar features. Whereas CM CD8 could only be documented using CCR7 and CD45RA, the characteristics of EM CD8 will differ according to the nomenclature. We found that the use of the CD45RA and CD28 gives the benefit of examining two EM populations at early and late differentiation states. This systematic comparison provides a cohesive layout of the advantages of using these nomenclature strategies in kidney transplant recipients to guide the choice of their use.

Belatacept-Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells

“…For a more granular analysis of the heterogeneous and functionally distinct memory and effector populations within CD3 + T cell compartment, we included additional conventional memory markers CD45RA and CCR7 (19, 25–27). Utilizing a gating strategy involving these four memory markers (Supplemental Figure 6) revealed belatacept resistant rejection was highly associated with elevated pre-transplant frequencies of CD28 + CD95 + CD45RA + CCR7 − CD8 T cells, so-called CD28 + CD8 + T EMRA (Figure 4c–d, P<0.0001).…”

“…The use of CD28 and CD95 is a standard approach for identifying na€ ıve (CD28 + CD95 À ), central memory (CD28 + CD95 + ), and effector/effector memory (CD28 À CD95 + ) T cells when phenotyping subsets in primates (21,22,25). For a more granular analysis of the heterogeneous and functionally distinct memory and effector populations within CD3 + T cell compartment, we included additional conventional memory markers CD45RA and CCR7 (19,(25)(26)(27). Utilizing a gating strategy involving these four memory markers ( Figure S6) revealed that belatacept-resistant rejection was highly associated with elevated pretransplant frequencies of CD28 + CD95 + CD45RA + CCR7 À CD8 T cells, so-called CD28 + CD8 + T EMRA ( Figure 4C Belatacept-and tacrolimus-based immunosuppression gave rise to two distinct study populations: those animals that were "susceptible" to therapy (gray lines) and those that were "resistant" to treatment (black lines).…”

“…Classic models of memory classification define CD4 and CD8 T cells by CCR7 and CD45RA or by CD28 and CD95 (20)(21)(22). Multiparametric flow cytometry has revealed the phenotypic and functional heterogeneity of T cell subsets (23)(24)(25)(26)(27). These studies reveal functional differences between subsets defined by four or more phenotypic markers at a time, with a potentially critical transition marked by CD28 loss (28).…”

IL-15 Harnesses Pro-inflammatory Function of TEMRA CD8 in Kidney-Transplant Recipients

“…Given the low expression of costimulatory molecules CD28 and CD27 by TEMRA CD8 T cells (25), we hypothesize that the effector functions of TEMRA CD8 T cells are triggered by pro-inflammatory cytokines such as IL-15, which are expressed and transpresented by conventional dendritic cells and macrophages (26, 27). Of note, CD45RA and CD28 markers were used to identify NAÏVE (CD45RA + CD28 + ), EM (CD45RA − CD28 + ), and TEMRA (CD45RA + CD28 − ) CD8 subsets (Figure S1 in Supplementary Material) (25). Stimulation with plate-bound anti-CD3 and IL-15 results in the upregulation of the activation marker CD25 and early proliferation marker CD69 by TEMRA CD8 from KT recipients (mean ± SEM 79.0 ± 5.4 and 67.8 ± 4.6%, respectively; p  < 0.0001 vs. no stimulation; Figure 1A).…”

“…IL-15 favors the migration of memory CD8 T cells toward inflamed tissues (11) and supports the proliferation and the survival of CCR7 − memory CD8 T cell pool (30). The CCR7 − memory CD8 include EM and TEMRA with a heterogeneous frequency across HV and in patients with strong immune stimulation such as kidney transplantation (25). By investigating the reactivity of TEMRA and EM CD8 and by characterizing their metabolic profiles, we aimed to assess whether TEMRA and EM CD8 respond in similar manner to IL-15 stimulation.…”