IL-7 receptor heterogeneity as a mechanism for repertoire change during postdepletional homeostatic proliferation and its relation to costimulation blockade-resistant rejection

Abstract

Kidney transplant patients treated with belatacept without depletional induction experience higher rates of acute rejection compared to patients treated with conventional immunosuppression. Costimulation blockade-resistant rejection (CoBRR) is associated with terminally differentiated T cells. Alemtuzumab induction and belatacept/sirolimus immunotherapy effectively prevent CoBRR. We hypothesized that cells in late phases of differentiation would be selectively less capable than more naive phenotypes of repopulating postdepletion, providing a potential mechanism by which lymphocyte depletion and repopulation could reduce the risk of CoBRR. Lymphocytes from 20 recipients undergoing alemtuzumab-induced depletion and belatacept/sirolimus immunosuppression were studied longitudinally for markers of maturation (CCR7, CD45RA, CD57, PD1), recent thymic emigration (CD31), and the IL-7 receptor-α (IL-7Rα). Serum was analyzed for IL-7. Alemtuzumab induction produced profound lymphopenia followed by repopulation, during which naive IL-7Rα⁺ CD57^- PD1^- cells progressively became the predominant subset. This did not occur in a comparator group of 10 patients treated with conventional immunosuppression. Serum from depleted patients showed markedly elevated IL-7 levels posttransplantation. Sorted CD57^- PD1^- cells demonstrated robust proliferation in response to IL-7, whereas more differentiated cells proliferated poorly. These data suggest that differences in IL-7-dependent proliferation is one exploitable mechanism that distinguishes CoB-sensitive and CoB-resistant T cell populations to reduce the risk of CoBRR. (ClinicalTrials.gov - NCT00565773.).

Keywords: basic (laboratory) research/science; clinical research/practice; cytokines/cytokine receptors; immunobiology; immunosuppressant - fusion proteins and monoclonal antibodies: belatacept; immunosuppressant - mechanistic target of rapamycin: sirolimus; immunosuppression/immune modulation; immunosuppressive regimens - induction; kidney transplantation/nephrology; lymphocyte biology: differentiation/maturation.

Figure 1. Post-depletional homeostatic reconstitution of CD4⁺ and CD8⁺ CD57⁻PD1⁻ cells after kidney allograft transplantation

(A) CD57⁻PD1⁻ subset in both CD4⁺ and CD8⁺ populations demonstrated a transit reduction post-alemtuzumab induction, and increased constantly above baseline levels after 12 months post-transplantation. The absolute numbers of CD4⁺CD57⁻PD1⁻ cells remained below baseline levels (p<0.0001). In contrast, absolute numbers of CD8⁺CD57⁻PD1⁻ cells returned to baseline levels between 12 and 18 months post-transplantation, and continually increased above baseline levels therefore (p<0.0018). (B) CD57⁻PD1⁻ cells prior to depletional induction contained large fractions of T_CM, T_EM, and T_EMRA subsets. The frequency of CD4⁺ T_EMRA cells increased slightly post-depletion and then returned to normal levels 3 months post-depletion induction. The frequency of CD8⁺ T_EMRA cells demonstrated a transient increase within 4 months post-alemtuzumab induction, and returned to baseline levels thereafter. A significant reduction of T_EM subset in CD57⁻PD1⁻ cells was observed thereafter, and the CD57⁻PD1⁻ subset significantly skewed toward naïve cells during homeostatic reconstitution. *p<0.05, *** p<0.001, **** p<0.0001

Figure 1. Post-depletional homeostatic reconstitution of CD4⁺ and CD8⁺ CD57⁻PD1⁻ cells after kidney allograft transplantation

(A) CD57⁻PD1⁻ subset in both CD4⁺ and CD8⁺ populations demonstrated a transit reduction post-alemtuzumab induction, and increased constantly above baseline levels after 12 months post-transplantation. The absolute numbers of CD4⁺CD57⁻PD1⁻ cells remained below baseline levels (p<0.0001). In contrast, absolute numbers of CD8⁺CD57⁻PD1⁻ cells returned to baseline levels between 12 and 18 months post-transplantation, and continually increased above baseline levels therefore (p<0.0018). (B) CD57⁻PD1⁻ cells prior to depletional induction contained large fractions of T_CM, T_EM, and T_EMRA subsets. The frequency of CD4⁺ T_EMRA cells increased slightly post-depletion and then returned to normal levels 3 months post-depletion induction. The frequency of CD8⁺ T_EMRA cells demonstrated a transient increase within 4 months post-alemtuzumab induction, and returned to baseline levels thereafter. A significant reduction of T_EM subset in CD57⁻PD1⁻ cells was observed thereafter, and the CD57⁻PD1⁻ subset significantly skewed toward naïve cells during homeostatic reconstitution. *p<0.05, *** p<0.001, **** p<0.0001

Figure 2. Post-depletional homeostatic reconstitution of CD4⁺ and CD8⁺ CD57⁺ and/or PD1⁺ cells after kidney allograft transplantation

(A) The dynamics of homeostatic repopulation of CD4⁺CD57⁺PD1⁻, CD4⁺CD57⁺PD1⁺, and CD4⁺CD57⁻PD1⁺ subsets post-alemtuzumab induction. The frequency of PD1⁺ subsets with or without CD57 expression increased transiently during early homeostatic repopulation post-depletion and repopulated to below baseline levels thereafter. In contrast, the absolute number of these subsets remained below baseline levels post-transplantation. The large fractions of these subsets were characterized as T_CM, T_EMRA, and T_EM cells. PD1⁺ subsets demonstrated a significant reduction of T_EM frequency during repopulation. (B) The dynamics of homeostatic repopulation of CD8⁺CD57⁺PD1⁻, CD8⁺CD57⁺PD1⁺, and CD8⁺CD57⁻PD1⁺ subsets post-alemtuzumab induction. The frequency and absolute number of these subsets significantly decreased below baseline levels significantly after transient elevation during early reconstitution, and were phenotypically T_EM and T_EMRA cells prior to transplantation. A significant reduction of T_EM subset and increased frequency for T_Naïve subset during CD57⁻PD1⁺ cell repopulation were observed.

Figure 2. Post-depletional homeostatic reconstitution of CD4⁺ and CD8⁺ CD57⁺ and/or PD1⁺ cells after kidney allograft transplantation

(A) The dynamics of homeostatic repopulation of CD4⁺CD57⁺PD1⁻, CD4⁺CD57⁺PD1⁺, and CD4⁺CD57⁻PD1⁺ subsets post-alemtuzumab induction. The frequency of PD1⁺ subsets with or without CD57 expression increased transiently during early homeostatic repopulation post-depletion and repopulated to below baseline levels thereafter. In contrast, the absolute number of these subsets remained below baseline levels post-transplantation. The large fractions of these subsets were characterized as T_CM, T_EMRA, and T_EM cells. PD1⁺ subsets demonstrated a significant reduction of T_EM frequency during repopulation. (B) The dynamics of homeostatic repopulation of CD8⁺CD57⁺PD1⁻, CD8⁺CD57⁺PD1⁺, and CD8⁺CD57⁻PD1⁺ subsets post-alemtuzumab induction. The frequency and absolute number of these subsets significantly decreased below baseline levels significantly after transient elevation during early reconstitution, and were phenotypically T_EM and T_EMRA cells prior to transplantation. A significant reduction of T_EM subset and increased frequency for T_Naïve subset during CD57⁻PD1⁺ cell repopulation were observed.

Figure 3. Dynamics and phenotype of CD57⁻PD1⁻ and CD57⁺ and/or PD1⁺ cells in patients treated with non-depletional induction and CNI-based regimen

(A) The phenotype of T cell subsets based on CD57 and PD1 expression and the frequency of CD57 and PD1 expressing of 10 patients were longitudinally assessed. (B) Pretransplant naïve and memory phenotypes of these subsets were similar to post-transplant phenotypes.

Figure 4. Dynamics of IL-7Rα (CD127) expressing T cells post-depletional induction

CD4⁺ CD57⁺ cells prior to alemtuzumab induction contained a large fraction of IL-7Rα⁺ cells. The CD57⁻ cells are predominantly IL-7Rα⁺ subset. The CD4⁺ cells lacking IL-7Rα expression were resistant to depletional induction during early T cell reconstitution. CD8⁺CD57⁺ cells were predominantly IL-7Rα–negative cells and remained so after depletional induction. A vast majority of CD57⁻ CD8 cells expressed IL-7Rα prior to alemtuzumab induction, followed by significant reduction during early repopulation. The repopulating CD57⁻ IL-7Rα⁺ cells returned to baseline levels at 12 months post-transplantation.

Figure 5. Homeostatic repopulation of CD31 expressing CD4⁺ and CD8⁺ cells post-depletional induction

CD4⁺ and CD8⁺ cells demonstrated a significant increase of CD31 expressing cells and a reduction of CD31⁻ cells post-transplantation.

Figure 6. Measurement of serum IL-7 concentrations before and after transplantation

(A) Patients treated with alemtuzumab induction and belatacept-based immunosuppressive regimens were longitudinally analyzed for serum IL-7 as measured by ELISA. IL-7 production increased after depletional induction. (B) Patients treated with conventional immunosuppression without depletion induction did not show increased IL-7 production post-transplantation.

Figure 7. Proliferation of purified CD57⁻PD1⁻ and CD57⁺ and/or PD1⁺ cells in response to IL-7

Purified CD57⁻PD1⁻ and CD57⁺ and/or PD1⁺ cells were verified by flow cytometry and labeled with BD VPD-450. Following incubation with or without IL-7, the proliferation of T cell subsets was analyzed by flow cytometry. (A) CD57⁻PD1⁻ cells demonstrated significant higher frequency of IL-7Rα (CD127)-expressing cells than purified CD57⁺ and/or PD1⁺ cells. (B) The purified CD57⁺ and/or PD1⁺ cells demonstrated barely detectable proliferation in the presence of IL-7. In contrast, CD4⁺ and CD8⁺ CD57⁻PD1⁻ cells demonstrated robust proliferation in response to IL-7.