Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig

Abstract

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (β-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

Figure 1.. Xenokidney transplantation in allosensitized rhesus recipients.

(A) A schematic representation of allosensitization. Six rhesus recipients were sensitized by skin transplantation from maximally MHC-mismatched donors (2 skin grafts from the same allo-donor placed at 8-week intervals). (B) Allo- and xeno-reactive antibodies after skin sensitization. Allosensitization was confirmed by elevation of DSA. Anti-pig antibodies in naïve, first sensitization (peak 1), second sensitization (peak 2), and before xenotransplant were measured. (C) A schematic representation of experimental design and immunosuppression regimen. Six sensitized rhesus monkeys received kidney transplantation from GGTA1KO.hCD55 transgenic pigs. Induction immunosuppression consisted of anti-CD4 mAb and anti-CD8 mAb (50 mg/kg). Maintenance immunosuppression consisted of costimulation blockade with anti-CD154 mAb (20 mg/kg), MMF, and steroids. (D) Kaplan-Meier curve of survival of xenograft in sensitized recipients (blue solid line, n=6, one censored) and allograft in sensitized recipients (red dotted line, n=4).. (E) Post-transplant serum creatinine (sCr) changes (left) and representative images of xenokidney (right) at time of rejection. A rise in serum creatinine corresponds with xenograft rejection. (F) Flow crossmatch of circulating anti-pig IgG after xenokidney transplantation. (G) Representative images of histology (H&E staining, left) and C4d immunostaining (right) at the time of rejection (scale bar: 200μm). (H) Quantification of allo- and xeno-DSA after xenokidney transplantation (n=5). Error bars represent SD. All data points indicate biologically independent animals; *p<0.05; **P<0.01; ***p<0.001 using two-tailed parametric paired t test; NS indicates no statistical significance.

Figure 2.. Induction of germinal center response after xenotransplantation.

(A) Representative flow cytometry plots for ICOS⁺PD-1⁺ and ICOS⁺PD-1^hi CD4⁺ T cells (left panels) and quantification (right panels) for lymph node ICOS⁺PD-1⁺ and ICOS⁺PD-1^hi CD4⁺ T cell populations at pre-transplant and rejection (n=5). (B) Representative flow cytometry plots (left panels) and quantification (right panels) for PD-1^hiBCL-6⁺ Tfh cell (n=5). (C) Representative ELIspot images (left panels) and compiled data (right panel) for antibody-secreting cells (ASCs) in lymph node (LN) at pre-transplantation and rejection (n=5). Error bars represent SD. Each dot indicates biologically independent animals; Data were analyzed with all samples (black, n=5) and with samples collected at least 3 weeks after transplantation (red, n=3); p-values were calculated using two-tailed parametric paired t test.

Figure 3.. Aberrant expansion of circulating ICOS⁺PD-1⁺CD4⁺ T cells after xenokidney transplantation.

(A) Representative histogram (upper) and quantitation (lower) of PD-1 (left) and ICOS (right) expression on circulating CD4⁺ T cells at pre-transplant (red) and rejection (blue). (B) Representative flow cytometry plots for ICOS⁺PD-1⁺ and ICOS⁺PD-1^hi cells in gated CD4⁺ cells during T cell repopulation. (C) Kinetics (left) and frequency (right) of post-transplant circulating ICOS⁺PD-1⁺ (top) and ICOS⁺PD-1^hi (bottom) CD4⁺ T cells. Error bars represent SD. Each dot indicates a biologically independent animal; Data were analyzed with all samples (black, n=5) and with samples collected at least 3 weeks after transplantation (red, n=3); p-values were provided using two-tailed parametric paired t test.

Figure 4.. Transplantation of 3KO.7TG xenokidneys results in prolonged graft survival and reduced antibody-mediated rejection.

(A). Kaplan-Meier curve of survival of xenograft in sensitized recipients with 3KO.7TG (n=3) and 1KO.1TG (N=5) pig donor. (B) Individual (left panel) and quantitated (right panel) post-xenotransplant serum creatinine (sCr). (C) Individual (left panel) and quantitated (right panel) post-xenotransplant DSA (anti-pig donor antibodies). (D) Representative PAS staining of 1KO.1TG (top left) and 3KO.7TG (top right) grafts at one-month post-transplantation and collated clustered Banff pathology gradings (bottom) for antibody-mediated rejection (g+ptc), microvascular injury (mm+cg), T-cell mediated rejection (t+v+i) at one-month post-transplantation in 1KO.1TG (blue) or 3KO.7TG (green) xenokidneys. (E) Representative C3d immunostaining of 1KO.1TG (top left) and 3KO.7TG (top right) grafts at one-month post-transplantation and collated grading (bottom) for C4d at peritubular capillaries (PTC), C3d at peritubular capillaries (PTC), and C3d at glomeruli (C3d_G) at one-month post-transplantation with 1KO.1TG (blue) or 3KO.7TG (green) xenokidneys. (F) Volcano plots (left) and hierarchical clustering (right) of differentially expressed genes at one month after xenotransplantation of 1KO.1TG (n=3) or 3KO.7TG (n=3) using human NanoString platform. (G) Differentially expressed genes between 1KO.1TG and 3KO.7TG xenografts at one-month post-transplantation. The Purple background indicates 31 downregulated genes, while the green background indicates 18 upregulated genes (including human transgenes) in 3KO.7TG (n=3) vs. 1KO.1TG (n=3) xenokidney transplantation. g, glomerulitis; ptc, peritubular capilaritis; cg, glomerular basement membrane double contours; mm, mesangial matrix expansion; t, tubulitis; v, intimal arteritis; i, interstitial inflammation. Error bars represent SD. N number indicates biologically independent animals; *p<0.05 using two-tailed parametric unpaired t test; NS indicates no statistical significance.

Figure 5.. Transplantation of 3KO.7TG xenokidneys results in reduced innate and adaptive immune cell responses.

(A) Absolute number of circulating lymphocytes, monocytes, and neutrophils following anti-CD4/CD8 induction in both the 1KO.1TG (n=6) and 3KO.7TG (n=4) groups. (B) Absolute number of circulating T cells, B cells, and plasmablasts following anti-CD4/CD8 induction in both the 1KO.1TG (n=6) and 3KO.7TG (n=4) groups. (C) Representative ELIspot images (top) and quantification (bottom) for ASCs from LN from 1KO.1TG (n=5) vs. 3KO.7TG (n=3). (D) Representative CD4⁺ (top) and CD8⁺ (bottom) T cell subset analysis based on the expression of CCR7 and CD45RA in 1KO.1TG (left, n=3) and 3KO.7TG (right, n=3) in the lymph node at one-month post-transplantation. Quantitated CD4⁺ (top) and CD8⁺ (bottom) T cell subsets are depicted on the right. (E) Representative histology (H&E) images showing lymph node and germinal center architectures (white dotted line) before and after xenokidney transplantation (one-month) in the 3KO.7TG group. (F) Lymph node GC (CXCR5⁺PD-1⁺ and BCL6⁺) CD4⁺ T and (BCL6⁺) B cells were analyzed in the 1KO.1TG (n=3) and 3KO.7TG (n=3) group at 1-month post-transplant. Error bars represent SD. Each dot indicates biologically independent animals; *p<0.05 using two-tailed parametric un-paired t test; NS indicates no statistical significance.

Figure 6.. Rejection by AMR after discontinuation of immunosuppression in 3KO.7TG xenokidney grafts.

A. The number of circulating lymphocytes, monocytes, neutrophils, and T (total, CD4⁺, and CD8⁺) cells before and after withdrawal of anti-CD154-based immunosuppression. B. CD4⁺ (left) and CD8⁺ (right) T cell subsets analysis based on the expression of CCR7 and CD45RA following withdrawal of immunosuppression in 3KO.7TG (right, n=3) C. Representative PAS images of 3KO.7TG graft after withdrawal of immunosuppression (left) and at rejection (right). Collated clustered Banff pathology gradings for AMR (g+ptc), microvascular injury (mm+cg), ACR (t+v+i), and glomerular C3d deposition (C3d_G) at one-month post-transplantation (n=3), before withdrawal of immunosuppression (n=3), and at rejection (n=3) are also shown. D. Differential gene expression in xenografts pre- vs. post-withdrawal of immunosuppression. E. Representative flow plot (left) and quantification (right) of circulating plasmablasts at one month after xenotransplantation (n=3), after withdrawal of anti-CD154mAb-based immunosuppression, and at rejection (n=3). Error bars represent SD. N number indicates biologically independent animals; *p<0.05; **P<0.01; ***p<0.001 using two-tailed parametric paired t test; NS indicates no statistical significance.