CD154 blockade effectively controls antibody-mediated rejection in highly sensitized nonhuman primate kidney transplant recipients

Abstract

Current desensitization and maintenance immunosuppression regimens for kidney transplantation in sensitized individuals show limited ability to control the posttransplant humoral response, resulting in high rates of antibody-mediated rejection (ABMR) and graft failure. Here, we showed that anti-CD154 monoclonal antibody (mAb)-based immunosuppression more effectively controlled allograft rejection and humoral rebound in a highly sensitized nonhuman primate kidney transplantation model compared with tacrolimus-based standard-of-care (SOC) immunosuppression. Desensitization with an anti-CD154 mAb (5C8) and a proteasome inhibitor led to decreased donor-specific antibodies (DSAs) and disruption of lymph node germinal centers with reduction of proliferating, memory, and class-switched B cells as well as T follicular helper cells. After transplant, the nonhuman primates maintained on 5C8-based immunosuppression had significantly better survival compared with those maintained on SOC immunosuppression (135.2 days versus 32.8 days, P = 0.013). The 5C8-treated group demonstrated better suppression of DSAs after transplant, more robust suppression of B cell populations, and better induction of regulatory T cells. Fewer infectious and welfare complications, including viral reactivation and weight loss, were also observed with 5C8-based immunosuppression compared with SOC immunosuppression. Therefore, anti-CD154 mAbs may improve kidney transplant outcomes through better control of posttransplant immune responses. The superior efficacy of anti-CD154 mAb-based immunosuppression over tacrolimus-based SOC seen in this highly sensitized NHP transplant model suggests that anti-CD154 mAbs could potentially be used to desensitize and treat highly sensitized patients receiving kidney transplantation.

Figure 1.. Desensitization with anti-CD154 mAb and proteasome inhibition reduces preformed donor-specific antibodies in sensitized NHPs.

(A) A schematic representation of experimental timeline for sensitization and desensitization prior to kidney transplantation. (B) Circulating DSA before and after desensitization as measured by T cell and B cell flow crossmatch (TFXM and BFXM, respectively). (C) Representative histology images of lymph nodes stained with hematoxylin and eosin (H&E) (upper panels) and immunohistochemistry (lower panels; red–CD20, green–Ki-67) before and after desensitization. Asterisks (*, upper panels) indicate the germinal center selected for higher magnification in the insets. Scale bars: 500μm in upper panels (100μm for the insets); 100μm in lower panels. (D) Flow cytometry analysis for circulating (left) and LN (right) Tfh (ICOS⁺PD-1⁺ CD4⁺ and ICOS⁺PD-1^hiCD4⁺) cells pre- and post-desensitization treatment. (E to G) Circulating (left) and LN (right) proliferating (Ki67⁺CD20⁺) B cells, isotype switched (IgG⁺IgD⁻CD20⁺) B cells, and regulatory (CD25⁺FoxP3⁺CD4⁺) T cells. N number indicates biologically independent animals (n=12). *p < 0.05; **p < 0.01, ***p<0.001 using two-tailed parametric paired t test; ns indicates no statistical significance.

Figure 2.. Allograft survival after desensitization with anti-CD154mAb and CFZ.

(A) A schematic representation of experimental groups based on the immunosuppression regimen. Following anti-CD154 mAb/CFZ desensitization, NHPs received either SOC IS (tacrolimus/MMF/Steroid) or anti-CD154 mAb-based IS (5C8/MMF/steroid). (B) Kaplan-Meier curves of rejection-free allograft survival in Tac/MMF/Steroids (cyan line, n=5) and 5C8/MMF/Steroids (orange line, n=5) groups. (C) Graph showing post-transplant serum creatinine (sCr) concentrations over time. Rises in sCr correspond with allograft rejection and DSA production. (D) Circulating serum DSA after transplantation measured with TFXM (left) and BFXM (right). N number indicates biologically independent animals.

Figure 3.. Antibody-mediated rejection and acute cellular rejection in kidney allografts of highly sensitized NHPs.

(A) Representative PAS-stained histology (upper panels) and C4d-stained immunohistochemistry (lower panels) kidney micrographs from biopsy samples obtained during the first month post-transplant. Scale bars defined in the figure. (B) A clustered BANFF score for acute cellular rejection (ACR; t+v+i, p=0.14). (C) A clustered BANFF score for acute antibody-mediated rejection (ABMR; g+ptc+C4d, p=0.04*p < 0.05 using two-tailed parametric paired t test. (D) Representative H&E-stained histology (left) and C4d-stained immunohistochemistry (right) kidney micrographs from biopsy samples obtained at primary endpoint (POD180). Scale bars: 50μm. (E) Post-transplant ABMR scores in the SOC group vs. anti-CD154mAb-based IS group. p=0.04, assessed by a non-parametric Mann-Whitney test. N number indicates biologically independent animals.

Figure 4.. Post-transplant T and B cell subpopulations in peripheral blood and lymph nodes in animals receiving SOC or anti-CD154 mAb-based immunosuppression.

Flow cytometric analysis of post-transplant T and B cells related to the humoral response in circulation and LN was performed. A. The frequency of class-switched (IgD⁻IgG⁺) B cells in circulation and lymph nodes. B. The frequency of proliferated (Ki67⁺) B cells in circulation and lymph nodes. C. The frequency of both circulating (ICOS⁺PD-1⁺) and LN (ICOS⁺PD-1^hi) Tfh cell populations. D. The frequency of Treg (CD25⁺FoxP3⁺) cells in the periphery and lymph nodes. Mean ± S.D. is presented. N number indicates biologically independent animals. *p < 0.05; **p < 0.01; ***p<0.001 using two-tailed parametric unpaired t test; NS indicates no statistical significance.

Figure 5.. Post-transplant clinical events, infection, and immunity in anti-CD154 mAb-based IS and SOC-treated NHP transplant recipients.

A. A graphical representation of the immunosuppression protocols and time courses (top) as well as the post-transplant clinical outcomes of individual animals’ graft dysfunction events, DSA elevation, positive SPV tests, and blood transfusions in all NHPs (n=10). B. Post-transplant platelet concentrations. C. Rhesus Cytomegalovirus (RhCMV) titers for animals treated with SOC and anti-CD154mAb-based IS for the duration of study (POD180). D. Hemoglobin concentrations for animals treated with SOC and anti-CD154mAb-based IS for the duration of study (POD180). E. Post-transplant Rhesus parvo virus (RhPV) titers for animals treated with SOC and anti-CD154mAb-based IS for the duration of study (POD180). F. SARS-CoV-2 vaccination response among NHPs in the anti-CD154 mAb/MMF/methylprednisolone treatment group. Spike- (left) and RBD-specific (right) serum IgG responses shown as mean BAU/mL.

Figure 6.. ABMR following conversion from anti-CD154 mAb-based immunosuppression to tacrolimus-based immunosuppression and subsequent ABMR treatment.

A. A schematic representation of conversion to tacrolimus-based immunosuppression after primary endpoint (POD180). Three NHPs in the anti-CD154mAb group, L560, M131, M518, were switched to tacrolimus-based immunosuppression at POD 246, 224, and 224, respectively. Four months after conversion all animals began ABMR treatment in the form of weekly CFZ and anti-CD154mAb for one month then tacrolimus and CFZ were stopped and anti-CD154 mAb-based immunosuppression was continued. B. Serum Cr concentrations after conversion to SOC immunosuppression. C. Serum DSA after conversion to SOC immunosuppression. D. DSA measured by TFXM (left) and BFXM (right) at the conversion to SOC immunosuppression and at initiation of ABMR. E. DSA measured by TFXM (left) and BFXM (right) following initiation of ABMR treatment with CFZ and anti-CD154 mAb.. F. DSA measured by TFXM (left) and BFXM (right) before and after ABMR treatment. All data are presented as individual value or mean ± S.D. N number indicates biologically independent animals. *p < 0.05 using two-tailed parametric unpaired t test; NS indicates no statistical significance.