Postdepletion Lymphocyte Reconstitution During Belatacept and Rapamycin Treatment in Kidney Transplant Recipients

Abstract

Belatacept is used to prevent allograft rejection but fails to do so in a sizable minority of patients due to inadequate control of costimulation-resistant T cells. In this study, we report control of costimulation-resistant rejection when belatacept was combined with perioperative alemtuzumab-mediated lymphocyte depletion and rapamycin. To assess the means by which the alemtuzumab, belatacept and rapamycin (ABR) regimen controls belatacept-resistant rejection, we studied 20 ABR-treated patients and characterized peripheral lymphocyte phenotype and functional responses to donor, third-party and viral antigens using flow cytometry, intracellular cytokine staining and carboxyfluorescein succinimidyl ester-based lymphocyte proliferation. Compared with conventional immunosuppression in 10 patients, lymphocyte depletion evoked substantial homeostatic lymphocyte activation balanced by regulatory T and B cell phenotypes. The reconstituted T cell repertoire was enriched for CD28(+) naïve cells, notably diminished in belatacept-resistant CD28(-) memory subsets and depleted of polyfunctional donor-specific T cells but able to respond to third-party and latent herpes viruses. B cell responses were similarly favorable, without alloantibody development and a reduction in memory subsets-changes not seen in conventionally treated patients. The ABR regimen uniquely altered the immune profile, producing a repertoire enriched for CD28(+) T cells, hyporesponsive to donor alloantigen and competent in its protective immune capabilities. The resulting repertoire was permissive for control of rejection with belatacept monotherapy.

Trial registration: ClinicalTrials.gov NCT00565773.

Keywords: basic (laboratory) research / science; clinical research / practice; fusion proteins and monoclonal antibodies: alemtuzumab; fusion proteins and monoclonal antibodies: belatacept; immune regulation; immunobiology; immunosuppressant; immunosuppression / immune modulation; kidney transplantation / nephrology; lymphocyte biology: differentiation / maturation; mechanistic target of rapamycin (mTOR).

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1. Repopulation of peripheral blood mononuclear cells after renal allograft transplantation

The absolute cell counts and subsets were analyzed by polychromatic flow cytometry. Leukocytes and granulocyte absolute numbers remain unchanged post-transplantation. Monocyte absolute counts decrease transiently within first month post-transplantation. In contrast, profound T cell (CD3+) and B cell (CD3-CD20+) depletion is achieved following alemtuzumab induction. T cell repopulation remained lethargic; however, B cells show rapid repopulation, with absolute numbers exceeding baseline values at 6 months post-transplantation. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 2. Post-depletional homeostatic proliferation and activation of CD4+ and CD8+ cells after renal allograft transplantation

(A) The dynamics of homeostatic proliferation for both CD4+ and CD8+ cells post–alemtuzumab induction. Significantly higher percentages of Ki-67-expressing cells are seen in belatacept/rapamycin maintenance when compared with the standard care controls. (B) CD4+ Ki-67+ and CD8+ Ki-67+ cells which express the anti-apoptosis marker Bcl-2 are segregated by CD45RA and CD197 expression to determine naïve versus memory phenotypes. After depletion, both CD4+ and CD8+ Ki-67+Bcl-2+ double positive cells are more pronounced in the TNaïve population compared to baseline, with the reverse phenomenon observed in the TEM population. (C) The activation of T cells post-depletion is detected by up-regulation of CD69 and CD38/HLA-DR expression. Significant up-regulation for CD69 and CD38/HLA-DR is seen during early phase of both CD4+ and CD8+ T cell repopulation when compared with baseline. (D) CD69 and CD38/HLA-DR expressing cells are subdivided to memory and naïve subsets based on CD45RA and CD197 expression. TNaïve cells demonstrated a higher frequency of activation post-depletion, with decreased activation frequency in TEM cells. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 2. Post-depletional homeostatic proliferation and activation of CD4+ and CD8+ cells after renal allograft transplantation

(A) The dynamics of homeostatic proliferation for both CD4+ and CD8+ cells post–alemtuzumab induction. Significantly higher percentages of Ki-67-expressing cells are seen in belatacept/rapamycin maintenance when compared with the standard care controls. (B) CD4+ Ki-67+ and CD8+ Ki-67+ cells which express the anti-apoptosis marker Bcl-2 are segregated by CD45RA and CD197 expression to determine naïve versus memory phenotypes. After depletion, both CD4+ and CD8+ Ki-67+Bcl-2+ double positive cells are more pronounced in the TNaïve population compared to baseline, with the reverse phenomenon observed in the TEM population. (C) The activation of T cells post-depletion is detected by up-regulation of CD69 and CD38/HLA-DR expression. Significant up-regulation for CD69 and CD38/HLA-DR is seen during early phase of both CD4+ and CD8+ T cell repopulation when compared with baseline. (D) CD69 and CD38/HLA-DR expressing cells are subdivided to memory and naïve subsets based on CD45RA and CD197 expression. TNaïve cells demonstrated a higher frequency of activation post-depletion, with decreased activation frequency in TEM cells. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 3. Dynamics of repopulating T cell subsets post-transplantation

(A) CD4+ T cells to increase frequency of TNaïve cells and reduce the frequency of TEM cells during repopulation; however, this did not reach statistical significance when compared with baseline proportions. In contrast, CD8+ reconstitution contained increased in TNaïve and decreased in TEM and TEMRA cell percentages. (B) Patients treated with alemtuzumab induction followed by belatacept/rapamycin maintenance regimen show significantly higher percentages of TNaïve cells in both CD4+ and CD8+ compartments, with a significantly lower frequency of CD8+ TEM cells when compared with a cohort treated with basiliximab induction and standard care maintenance regimen. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 3. Dynamics of repopulating T cell subsets post-transplantation

(A) CD4+ T cells to increase frequency of TNaïve cells and reduce the frequency of TEM cells during repopulation; however, this did not reach statistical significance when compared with baseline proportions. In contrast, CD8+ reconstitution contained increased in TNaïve and decreased in TEM and TEMRA cell percentages. (B) Patients treated with alemtuzumab induction followed by belatacept/rapamycin maintenance regimen show significantly higher percentages of TNaïve cells in both CD4+ and CD8+ compartments, with a significantly lower frequency of CD8+ TEM cells when compared with a cohort treated with basiliximab induction and standard care maintenance regimen. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 4. Dynamics and memory phenotypes of CD28 expressing T cells during post-depletion repopulation

(A) Ratio of CD28 expression among repopulating CD4+ and CD8+ cells. The CD8+CD28- cells show a transient increase in frequency above the baseline within first two months. There is no change in frequency of CD28 expression observed in repopulating CD4+ cells. (B) CD8+ cells are segregated by CD2 and CD28 expression, demonstrating a significant reduction of CD8+CD2hiCD28- frequency with a concomitant increase in CD8+CD2loCD28+ frequency when compared and a standard care control group. (C) The CD8+CD2loCD28+ cells are subdivided based on CD45RA and CD197 expression, with a reduction in TEM frequency and increase in TNaïve frequency during reconstitution of the T cell population. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 5. B cell repopulation and dynamic changes of phenotypes post-depletion

(A) Representative dot plots of gating on singlet of CD19+ B cells are segregated by the expression of CD27 and IgD into switched memory (CD27+IgD-), un-switched memory (CD27+IgD+), naïve (CD27-IgD+), and exhausted memory (CD27-IgD-) subsets. (B) Frequencies over time of memory and naïve B cells showing rapid repopulation of naïve cells compared to memory subsets post–alemtuzumab induction. (C) Comparisons of B cell subsets between patients receiving alemtuzumab induction followed by belatacept/rapamycin maintenance and patients with standard care immunosuppression. (D) CD19+ naïve and memory B cell subsets are defined by Bm1-Bm5 Classification based on CD38/IgD expression showing rapid repopulation of naïve Bm-2 subset. (E) Comparisons of B cell subsets defined by Bm1–Bm5 classification between study immunosuppression and standard care immunosuppression. (F) Serum BAFF and APRIL concentrations before and after transplantation measured by ELISA. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 5. B cell repopulation and dynamic changes of phenotypes post-depletion

(A) Representative dot plots of gating on singlet of CD19+ B cells are segregated by the expression of CD27 and IgD into switched memory (CD27+IgD-), un-switched memory (CD27+IgD+), naïve (CD27-IgD+), and exhausted memory (CD27-IgD-) subsets. (B) Frequencies over time of memory and naïve B cells showing rapid repopulation of naïve cells compared to memory subsets post–alemtuzumab induction. (C) Comparisons of B cell subsets between patients receiving alemtuzumab induction followed by belatacept/rapamycin maintenance and patients with standard care immunosuppression. (D) CD19+ naïve and memory B cell subsets are defined by Bm1-Bm5 Classification based on CD38/IgD expression showing rapid repopulation of naïve Bm-2 subset. (E) Comparisons of B cell subsets defined by Bm1–Bm5 classification between study immunosuppression and standard care immunosuppression. (F) Serum BAFF and APRIL concentrations before and after transplantation measured by ELISA. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 5. B cell repopulation and dynamic changes of phenotypes post-depletion

(A) Representative dot plots of gating on singlet of CD19+ B cells are segregated by the expression of CD27 and IgD into switched memory (CD27+IgD-), un-switched memory (CD27+IgD+), naïve (CD27-IgD+), and exhausted memory (CD27-IgD-) subsets. (B) Frequencies over time of memory and naïve B cells showing rapid repopulation of naïve cells compared to memory subsets post–alemtuzumab induction. (C) Comparisons of B cell subsets between patients receiving alemtuzumab induction followed by belatacept/rapamycin maintenance and patients with standard care immunosuppression. (D) CD19+ naïve and memory B cell subsets are defined by Bm1-Bm5 Classification based on CD38/IgD expression showing rapid repopulation of naïve Bm-2 subset. (E) Comparisons of B cell subsets defined by Bm1–Bm5 classification between study immunosuppression and standard care immunosuppression. (F) Serum BAFF and APRIL concentrations before and after transplantation measured by ELISA. (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Figure 6. Intracellular cytokine production and CD28 expression by allo- and viral-reactive TEM cells

(A) PBMCs collected from patients are stimulated with CD3-depleted donor and third-party PBMCs followed by ICCS. Activating allo-specific CD4+ and CD8+ T cells are identified as TNF-α/IFN-γ dual producers, and the IL-2/TNF-α/IFN-γ triple producers are determined by gating on dual cytokine producers. Representative results from one individual are shown in top panel. The frequency of CD4+ and CD8+ triple producers in response to donor-specific antigens is decreased within 36 months after renal transplantation with this novel regimen. In contrast, T cell responses to HLA-mismatched third-party remain intact. (B) PBMCs from study patients are stimulated with CMV-pp65 peptides followed by ICCS to detect IL-2, TNF-α, and IFN-γ. Representative results from one individual are shown in top panel. Activation of CMV-specific is defined as triple cytokine producers, and both CMV-specific CD4+ and CD8+ T cell responses remain intact within 36 months post-transplantation. Activation of EBV-specific CD8+ T cells remains unchanged through the timeframe of the study with consistent phenotypes of IL-2/TNF-α/IFN-γ triple producers following stimulation with EBV proteins. (C) TNF-α and IFN-γ dual producers after allo- and viral-stimulation are segregated into CD28+ and CD28- subsets. The frequency of CD8+CD28- subset in allo-specific responders reduced significantly (p ≤ 0.0261) with an expected significant increase of the CD8+CD28+ subset (p ≤ 0.0261) within 36 months post-transplantation. In contrast, CD28 expression on viral-specific T cells after viral-stimulation remains unchanged post-depletion and transplant. The CD28 expression on CD4+ allo-responding cells remains unchanged post-depletion and transplant.

Figure 6. Intracellular cytokine production and CD28 expression by allo- and viral-reactive TEM cells

(A) PBMCs collected from patients are stimulated with CD3-depleted donor and third-party PBMCs followed by ICCS. Activating allo-specific CD4+ and CD8+ T cells are identified as TNF-α/IFN-γ dual producers, and the IL-2/TNF-α/IFN-γ triple producers are determined by gating on dual cytokine producers. Representative results from one individual are shown in top panel. The frequency of CD4+ and CD8+ triple producers in response to donor-specific antigens is decreased within 36 months after renal transplantation with this novel regimen. In contrast, T cell responses to HLA-mismatched third-party remain intact. (B) PBMCs from study patients are stimulated with CMV-pp65 peptides followed by ICCS to detect IL-2, TNF-α, and IFN-γ. Representative results from one individual are shown in top panel. Activation of CMV-specific is defined as triple cytokine producers, and both CMV-specific CD4+ and CD8+ T cell responses remain intact within 36 months post-transplantation. Activation of EBV-specific CD8+ T cells remains unchanged through the timeframe of the study with consistent phenotypes of IL-2/TNF-α/IFN-γ triple producers following stimulation with EBV proteins. (C) TNF-α and IFN-γ dual producers after allo- and viral-stimulation are segregated into CD28+ and CD28- subsets. The frequency of CD8+CD28- subset in allo-specific responders reduced significantly (p ≤ 0.0261) with an expected significant increase of the CD8+CD28+ subset (p ≤ 0.0261) within 36 months post-transplantation. In contrast, CD28 expression on viral-specific T cells after viral-stimulation remains unchanged post-depletion and transplant. The CD28 expression on CD4+ allo-responding cells remains unchanged post-depletion and transplant.

Figure 6. Intracellular cytokine production and CD28 expression by allo- and viral-reactive TEM cells

(A) PBMCs collected from patients are stimulated with CD3-depleted donor and third-party PBMCs followed by ICCS. Activating allo-specific CD4+ and CD8+ T cells are identified as TNF-α/IFN-γ dual producers, and the IL-2/TNF-α/IFN-γ triple producers are determined by gating on dual cytokine producers. Representative results from one individual are shown in top panel. The frequency of CD4+ and CD8+ triple producers in response to donor-specific antigens is decreased within 36 months after renal transplantation with this novel regimen. In contrast, T cell responses to HLA-mismatched third-party remain intact. (B) PBMCs from study patients are stimulated with CMV-pp65 peptides followed by ICCS to detect IL-2, TNF-α, and IFN-γ. Representative results from one individual are shown in top panel. Activation of CMV-specific is defined as triple cytokine producers, and both CMV-specific CD4+ and CD8+ T cell responses remain intact within 36 months post-transplantation. Activation of EBV-specific CD8+ T cells remains unchanged through the timeframe of the study with consistent phenotypes of IL-2/TNF-α/IFN-γ triple producers following stimulation with EBV proteins. (C) TNF-α and IFN-γ dual producers after allo- and viral-stimulation are segregated into CD28+ and CD28- subsets. The frequency of CD8+CD28- subset in allo-specific responders reduced significantly (p ≤ 0.0261) with an expected significant increase of the CD8+CD28+ subset (p ≤ 0.0261) within 36 months post-transplantation. In contrast, CD28 expression on viral-specific T cells after viral-stimulation remains unchanged post-depletion and transplant. The CD28 expression on CD4+ allo-responding cells remains unchanged post-depletion and transplant.

Figure 7. Proliferative responses of allo-responding T cells posttransplantation

(A) CFSE-labeled responder PBMCs stimulated by donor and third-party cells and CD3/CD28 beads are analyzed for proliferation after 5 days through assessment of CFSE dilution. The proliferative responses to both specific donor and third-party allo-stimulation post-transplantation are comparable to baseline throughout the study period and predominated by CD28+ cells (B).