Increased Pretransplant Frequency of CD28+ CD4+ TEM Predicts Belatacept-Resistant Rejection in Human Renal Transplant Recipients

Abstract

While most human T cells express the CD28 costimulatory molecule constitutively, it is well known that age, inflammation, and viral infection can drive the generation of CD28^null T cells. In vitro studies have demonstrated that CD28^null cell effector function is not impacted by the presence of the CD28 costimulation blocker belatacept. As such, a prevailing hypothesis suggests that CD28^null cells may precipitate costimulation blockade-resistant rejection. However, CD28⁺ cells possess more proliferative and multifunctional capacity, factors that may increase their ability to successfully mediate rejection. Here, we performed a retrospective immunophenotypic analysis of adult renal transplant recipients who experienced acute rejection on belatacept treatment as compared to those who did not. Intriguingly, our findings suggest that patients possessing higher frequency of CD28⁺ CD4⁺ T_EM prior to transplant were more likely to experience acute rejection following treatment with a belatacept-based immunosuppressive regimen. Mechanistically, CD28⁺ CD4⁺ T_EM contained significantly more IL-2 producers. In contrast, CD28^null CD4⁺ T_EM isolated from stable belatacept-treated patients exhibited higher expression of the 2B4 coinhibitory molecule as compared to those isolated from patients who rejected. These data raise the possibility that pretransplant frequencies of CD28⁺ CD4⁺ T_EM could be used as a biomarker to predict risk of rejection following treatment with belatacept.

Keywords: T cell biology; basic (laboratory) research/science; biomarker; costimulation; fusion proteins and monoclonal antibodies: belatacept; immune regulation; immunobiology; immunosuppressant; immunosuppression/immune modulation; kidney transplantation/nephrology.

Figure 1. Pre-transplant CD28 expression within the CD4⁺ and CD8⁺ T cell compartments of patients that remained stable on belatacept-based immunosuppression vs. those that went on to reject

(A) Representative flow plots of the gating strategy to identify CD28^null CD4⁺ and CD8⁺ T cells. (B) Representative flow plot of pre-transplant CD28^null CD4⁺ expression in patients that remained stable while on belatacept-based therapy and those that rejected. (C) Patients who went on to experience acute rejection while on belatacept exhibited decreased frequencies of CD28^null CD4⁺ T cells as compared to those that remained stable while on belatacept-based therapy (p=0.03), and the frequency of CD28^null CD4⁺ T cells in stable patients was actually higher than that observed in normal healthy controls (p=0.01). Patients who went on to experience acute rejection possessed increased frequencies of CD28⁺ CD4⁺ T cells compared to those that remained stable (p=0.03) D. Representative flow plots of pre-transplant CD28^null CD8⁺ expression in patients that remained stable while on belatacept-based therapy and those that rejected. E. No differences in the frequencies of CD28^null cells and CD28⁺ cells within the CD8⁺ T cell compartments of stables vs. rejectors were observed.

Figure 2. Pre-transplant frequencies of CD28⁺ CD4⁺ T_EM, CD28⁺ CD4⁺ T_EMRA, and CD28⁺ CD8⁺ T_EM are increased in patients who experienced belatacept-resistant rejection

(A) Representative flow plots and (B) summary data of frequencies of CD28⁺ cells within naïve (CCR7⁺ CD45RA⁺), T_CM (CCR7⁺ CD45RA-), T_EM (CCR7- CD45RA-), and T_EMRA (CCR7- CD45RA⁺) subsets of CD4⁺ T cells in patients that experienced belatacept-resistant rejection (n=10) vs. those that remained stable (n=13). Pre-transplant frequency of CD28⁺ cells among CD4⁺ T_EM and CD4⁺ T_EMRA cells were significantly increased in patients who went on to reject vs. those that did not (p<0.0001 and p=0.01, respectively). (C) Representative flow plots and (D) summary data of frequencies of CD28⁺ cells within naïve (CCR7⁺ CD45RA⁺), T_CM (CCR7⁺ CD45RA-), T_EM (CCR7- CD45RA-), and T_EMRA (CCR7- CD45RA⁺) subsets of CD8⁺ T cells in patients that experienced belatacept-resistant rejection (n=10) vs. those that remained stable (n=13). Pre-transplant frequency of CD28⁺ cells among CD8⁺ T_EM were significantly increased in patients who went on to reject vs. those that did not (p=0.05).

Figure 3. Pre-transplant frequencies of CD28⁺ CD4⁺ or CD8⁺ T cells are not increased in patients who experienced rejection following treatment with tacrolimus-based immunosuppression vs. those that remained stable

(A and B) Summary data of frequencies of CD28⁺ cells within the total CD4⁺ compartment (A) and T_EM subset (CCR7- CD45RA-) (B) of CD4⁺ T cells from PBMC of patients that experienced rejection during treatment with tacrolimus (n=7) vs. those that remained stable (n=8). (C and D) Summary data of frequencies of CD28⁺ cells within the total CD8⁺ compartment (A) and T_EM subset (CCR7- CD45RA-) (B) of CD8⁺ T cells from PBMC of patients that experienced rejection during treatment with tacrolimus (n=7) vs. those that remained stable (n=8). p=ns for all pairs.

Figure 4. CD28⁺ CD4⁺ T_EM produce more IL-2 and rapidly downregulate CD28 following ex vivo restimulation

(A–C) PBMC from healthy controls were stimulated ex vivo with PMA/ionomycin for 4 hours and intracellular cytokine staining was performed. CD4⁺ cells were gated on CD28⁺ vs. CD28⁻ and analyzed for IFN-γ and IL-2 production. Representative flow plots are shown in A. B–C, Significantly higher frequencies of CD28⁺ CD4⁺ T cells secreted IL-2 (but not IFN-γ) relative to CD28^null CD4⁺ T cells (n=3, p<0.05). (D) PBMC isolated from belatacept-treated stables and rejectors did not differ with regard to the frequencies of IFN-γ-secreting cells within their CD4⁺ T cell compartments following ex vivo restimulation. (E) Belatacept-treated patients who rejected exhibited a statistically significantly higher frequency of IL-2-secreting CD4⁺ T cells as compared to patients that were rejection-free (p<0.05). (F–G) PBMC from belatacept-treated rejectors vs. stables were stimulated ex vivo for 4 hours and the frequencies of CD28^null cells following ex vivo stimulation were quantified. F, The frequencies of CD28⁻CD4⁺ T cells were not different after ex vivo stimulation in patients that were stable. (G) The frequencies of CD28⁻CD4⁺ T cells significantly increased after ex vivo stimulation in patients that experienced belatacept-resistant rejection.

Figure 5. 2B4 coinhibitory molecule expression was increased on CD28^null cells in belatacept-treated stables relative to rejectors

(A) Representative flow plots demonstrating that 2B4 is highly enriched on CD28^null cells relative to CD28⁺ CD4⁺ T cells (B) No statistical difference was observed in 2B4 expression on CD28⁺ CD4⁺ T_EM in PBMC isolated from belatacept-treated patients that were stable versus those that experienced rejection. (C) 2B4 expression was significantly higher in CD28^null CD4⁺ T_EM in PBMC isolated from belatacept-treated patients that were stable versus either those that experienced rejection or healthy controls (p=0.05).

Figure 6. Assessment of CD28 expression in patients with ESRD compared to patients with ESLD and healthy controls

(A) PBMC isolated from healthy controls (n=8), patients with ESRD (n=37) and ESLD (n=9) were assessed for frequencies of CD28⁺ cells within both the CD4⁺ (A) and CD8⁺ (B) T cell compartments. Both ESRD and ESLD presented a statistically significant increase in CD28^null cells as compared to normal HC within the CD4⁺ T cell compartment (p=0.03 and p=0.04 respectively). (B) No difference in the frequencies of CD28^null cells in the CD8⁺ T cell compartment was observed between patients with ESRD, ESLD and HC. (C) PBMC isolated from patients with ESLD were gated on CD4⁺ T_EM (CCR7- CD45RA-) and frequencies of CD28^null cells were assessed.