CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates

Abstract

Selective inhibition of T cell costimulation using the B7-specific fusion protein CTLA4-Ig has been shown to induce long-term allograft survival in rodents. Antibodies preventing the interaction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5-8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20-98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.

Figure 1

The effect of CTLA4-Ig and 5C8 alone and in combination on unidirectional rhesus monkey mixed lymphocyte reactions. Increasing concentrations of CTLA4-Ig result in progressive suppression, whereas the effects of 5C8 are more modest. The combination is more effective than either drug alone at 100-fold or greater concentrations. Results shown were reproduced in three independent experiments. c.p.m., counts per minute from incorporated [³H]thymidine.

Figure 2

(A) Survival and renal function as determined by serum creatinine following unmodified allogeneic renal transplantation (–) or transplantation following induction with CTLA4-Ig alone (▪) or 5C8 alone (⧫). Open arrows indicate re-treatment during biopsy-proven rejection. Solid arrows indicate continued survival. (B) Survival and renal function as determined by serum creatinine following unmodified allogeneic renal transplantation (–) or transplantation following induction with both CTLA4-Ig and 5C8. ○ indicate treatment on days 0, 2, 4, 6, 8, 10, and 12 posttransplant. • indicate treatment on days 0, 2, 4, 6, 8, 12, 16, and 28 posttransplant. Open arrows indicate re-treatment during biopsy-proven rejection for the animal depicted in ○. Solid arrows indicate continued survival free of rejection since transplantation.

Figure 3

(A) Renal allograft histology showing acute cellular rejection following unmodified renal allotransplantation in rhesus monkeys. (B) Renal allograft histology showing acute cellular rejection prior to reversal with 5C8. (C) Normal renal allograft histology from an animal with normal renal function 163 days after transplantation and induction with CTLA4-Ig and 5C8. (D) A perivascular lymphoid aggregate within the allograft shown in C. These nests of lymphocytes exist in the allograft despite normal function and the absence of immunosuppression. All micrographs are ×250.

Figure 4

Mixed lymphocyte responses against donor lymphocytes and third-party lymphocytes for two rhesus monkeys 150 days after allotransplantation with rejection-free survival and normal renal function and without any chronic therapy. Both donor and third-party responsiveness are maintained.