Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion

Abstract

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti-pig antibodies significantly improved survival in a pig-to-rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan-T cell depletion and an anti-CD154-based maintenance regimen. Selective depletion of CD4⁺ T cells but not CD8⁺ T cells resulted in long-term survival (median survival time >400 days vs 6 days). These studies suggested that CD4⁺ T cells may have a more prominent role in xenograft rejection compared with CD8⁺ T cells. Although animals that received selective depletion of CD8⁺ T cells showed signs of early cellular rejection (marked CD4⁺ infiltrates), animals receiving selective CD4⁺ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4⁺ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti-pig antibody and CD4 depletion resulted in consistent, long-term xenograft survival.

Keywords: animal models: nonhuman primate; basic (laboratory) research/science; costimulation; immunosuppressant - fusion proteins and monoclonal antibodies: costimulation molecule specific; immunosuppression/immune modulation; kidney transplantation/nephrology; translational research/science; xenoantibody; xenoantigen; xenotransplantation.

Figure 1.. Treatment schema for pig-to-non-human primate kidney transplantation.

All animals underwent bilateral native nephrectomy and concurrent renal xenotransplantation from GGTKO/hDAF porcine donors. (A) Animals received two doses of αCD4 depletion (day −3 and day 0) and one dose of αCD8 depletion on the day 0. All animals received mycophenolate mofetil dosing twice daily and a solumedrol taper beginning on the date of surgery and continuing throughout the duration of the experiment. Anti-CD154 (5C8) was administered at the time of transplant, at 1 week after the transplant and then every two weeks thereafter. (B) In a second set of experiments, animals (n=3 in each group) were randomized to receive either only αCD4 depletion (day −3 and day 0) or only αCD8 depletion (day 0). All recipients then were treated similarly to previous xenograft recipients, solumedrol taper beginning on the date of surgery, twice daily dosing of mycophenolate mofetil, and anti-CD154 dosing for the duration of xenograft survival.

Figure 2.. Selection of recipients with low anti-pig antibody titers significantly prolongs kidney xenograft survival.

(A) Survival in the low-titer recipients (—) was significantly improved (MST 235 days) over high-titer recipients (····, MST 6 days). (B) Creatinine values of low-titer recipients remained within the normal range for longer and correlated with extended survival times compared with the high-titer recipient. (C) Flow cytometry was performed on samples of the recipients’ peripheral blood to confirm successful depletion of the CD4 and CD8 T cell subsets. (D) Urine protein measurements remained low for the first 100 days post-transplant but increased at times of late rejection. (E) Anti-pig antibodies developed in some long-term survivors.

Figure 3.. CD4⁺ T cells proliferate and express more IL-2 in response to xenogeneic stimulation.

CD4⁺ T cells were labeled in vitro with Cell Trace Violet (CTV); with every cell division, CTV fluorescence diminished. A negative control of CTV-labeled responder cells alone with no xenogeneic stimulators was used as a negative control (A) When nonhuman primate PBMCs were cultured with xenogeneic stimulators for 4 days in a mixed lymphocyte reaction, a greater percentage of CD4⁺ T cells proliferated compared to CD8⁺ T cells. (B) Similar to what is seen after alloantigen stimulation, IL-2 production is predominantly from CD4⁺ cells. The addition of anti-CD154 to the cell culture did not significantly impact proliferation or IL-2 expression in-vitro.

Figure 4.. Selective CD4 depletion is necessary for long-term renal xenograft survival.

(A) Survival of recipients that only received selective CD4 depletion (—) was significantly longer (MST 414 days) than recipients that only received CD8 depletion (····, MST 6 days). (B) Creatinine values of anti-CD4 treated recipients remained low for longer periods of time and correlated with longer survival times compared with the high-titer recipient. (C) Flow cytometry was performed on samples of the recipients’ peripheral blood to confirm successful depletion of the CD4 and CD8 T cell subsets in experimental groups that received either only αCD4 depletion () or only αCD8 depletion () (D) Urine protein measurements remained low post-transplant in long-term survivors until late. (E) Of the long-term survivors treated with αCD4 depletion and anti-CD154, none went on to develop anti-pig antibodies. (F) Representative histology from long-term survivors receiving only αCD4 depletion demonstrated little cellular infiltrate and no glomerulopathy at post-transplant day 200.

Figure 5.. Immunohistochemistry staining at the time of early xenograft rejection revealed significant CD4⁺ infiltrate.

(A) An explanted xenograft at the time of rejection was also stained for nonhuman primate αCD4 and αCD8. The xenograft stained positively for nonhuman primate αCD4 but not αCD8. (B) For a negative control comparison, a non-transplanted GGTKO/hDAF pig kidney was also stained for nonhuman primate αCD4 and αCD8. (C) Flow cytometry analysis of graft-infiltrating cells confirmed that the amount of CD4+ T cells were elevated relative to CD8+ at the time of rejection.

Figure 6.. Late xenografts from long-term surviving recipients of selective αCD4 depletion demonstrated an association of antibody-mediated injury targeted and expression of products of the β4GalNT2 enzyme.

(A) The xenografts of animals treated selectively with αCD4 depletion were noted to have evidence of chronic transplant glomerulopathy suggestive of thrombotic microangiopathy or antibody-mediated rejection on both H&E and PAS staining. Further immunohistochemistry demonstrated +IgG staining (B) and +C4d staining (C) suggestive of antibody-mediated rejection. (D) Additional analysis using fluorescently labeled DBA lectin demonstrated Sd^a antigen expression in the transplanted kidney which is produced by the β4GalNT2 enzyme; this enzyme can be silenced in future porcine donors. (E) When mixed lymphocyte reactions were performed with endothelial cell lines with SLA I, but not SLA II, expression (SLA I⁺/II^-), (F) the CD4⁺ T cell proliferative response was significantly decreased, which opens the possibility for future experiments where SLA II could be silenced in the porcine donors to avoid T cell depletion therapies prior to transplantation.