Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates

Abstract

Anergic T cells generated ex vivo are reported to have immunosuppressive effects in vitro and in vivo. Here, we tested this concept in nonhuman primates. Alloreactive T cells were rendered anergic ex vivo by coculture with donor alloantigen in the presence of anti-CD80/CD86 mAbs before adoptive transfer via renal allograft to rhesus monkey recipients. The recipients were briefly treated with cyclophosphamide and cyclosporine A during the preparation of the anergic cells. Thirteen days after renal transplantation, the anergic T cells were transferred to the recipient, after which no further immunosuppressive agents were administered. Rejection-free survival was prolonged in all treated recipients, and 3 of 6 animals survived long term (410-880 days at study's end). In the long-surviving recipients, proliferative responses against alloantigen were inhibited in a donor-specific manner, and donor-type, but not third-party, skin allografts were also accepted, which demonstrated that antigen-specific tolerance had been induced. We conclude that anergic T cells generated ex vivo by blocking CD28/B7 costimulation can suppress renal allograft rejection after adoptive transfer in nonhuman primates. This strategy may be applicable to the design of safe clinical trials in humans.

Figure 1

Effect of anti-human CD80/CD86 mAbs on MLR in rhesus monkeys and functional activities of the cultured cells. (A) Freshly isolated CD4⁺ T cells (fresh cells) from peripheral blood of rhesus monkeys were cocultured with irradiated allogeneic PBMCs (stimulator) in the presence or absence of anti-human CD80/CD86 mAbs (10 μg/ml each) for 5 days. (B) Peripheral blood CD4⁺ T cells (fresh cells, white bars) or the cultured cells (black bars) were stimulated with donor or third-party splenocytes (gray bars). After 3 days culture or 5 days (for fresh cells), the responder cells were evaluated for their proliferation. (C and D) Dose-dependent suppression of the alloresponses of peripheral blood CD4⁺ T cells to donor-type stimulator cells by the cultured anergic cells. Cultures were set up with recipient CD4⁺ T cells (10⁵ cells/well) and donor (C) or third-party (D) stimulators (10⁵ cells/well) for 7 days. Cultured donor splenocytes (10⁵) or different numbers of the cultured cells were also added in some wells. In all assays, cells were incubated for 6 days and then pulsed with 10 μCi of [³H]thymidine for the last 18 hours and counted. The bars represent the mean of triplicate values and the brackets indicate the SD. rIL-2, recombinant IL-2.

Figure 2

Characterization of the cultured anergic cells. (A–H) Freshly isolated PBMCs (A, C, E, and G) and the cultured anergic cells (B, D, F, and H) were analyzed for cell-surface expression of CD3, CD20, CD4, CD14, and CD25. (I) CTLA-4 (CD152) expression in cultured CD4⁺CD25⁺ T cells (open histogram) and freshly isolated T cells (filled histogram). Results are representative of 3 independent experiments.

Figure 3

Renal function was determined by serum creatinine level (mg/dl) after transplantation in group A (n = 6). Solid arrowheads indicate continued survival without rejection.

Figure 4

CsA whole blood levels (ng/ml) in recipients (group A). CsA (8 mg/kg) was injected intramuscularly daily from the day of operation to 7 days after transplantation and thereafter on days 9, 11, and 13. Solid lines, animals surviving more than 1 year (n = 3); dotted lines, animals dying within 1 year (n = 3).

Figure 5

Assessment of the responses against alloantigens in tolerance-induced recipients. (A) Proliferative response of peripheral blood CD4⁺ T cells against alloantigens. Peripheral blood CD4⁺ T cells from a recipient with a long-surviving renal allograft were cocultured with irradiated donor (black bars) or third-party splenocytes (gray bars) preoperatively (Preop.) or 60 and 320 days after transplantation. After 7 days of culture, [³H]thymidine incorporation during the final 18 hours of culture was counted. Data are expressed as mean ± SD of triplicate samples. (B) Challenge with skin allografts. A long-surviving recipient was transplanted with autologous skin (host; autograft), donor skin (allograft), or third-party skin. Forty days after skin transplantation, the third-party skin was completely rejected, although both host and donor skin grafts remained intact, with no signs of rejection.

Figure 6

Representative pathological findings of kidney allografts in groups A and E. (A and B) Histology of the surviving allograft on POD 810 in group A. (A) There was minimal cellular infiltrate in evidence around small arteries but no tubulitis, glomerulitis, or endothelialitis (H&E stain; magnification, ×200). (B) Medium and small-sized arteries with elastic fiber stain showed intact architecture without intimal hyperplasia (Movat pentachrome stain; magnification, ×400). (C and D) Histology of the rejected allograft on POD 67 in group E. Diffuse and marked cell infiltration occurred. (C) The small arteries showed acute endothelialitis with fibrinoid necrosis (double arrow) and chronic allograft vasculopathy with intimal fibrous thickening and cell infiltration (single arrow), which indicated that severe and prolonged rejection developed in the graft (H&E stain; magnification, ×200). (D) Chronic allograft vasculopathy developed in medium- and small-sized arteries (Movat pentachrome stain; magnification, ×400).

Figure 7

Schematic diagram of the regimen (group A). CsA (8 mg/kg/d) was administered intramuscularly on the days indicated by asterisks. CP (30 mg/kg) was administered intramuscularly at PODs 6, 7, and 8. During the operation, the spleen was removed from both donor and recipient. Splenic T cells from the recipient were cocultured with irradiated donor splenocytes for 13 days in the presence of anti-CD80/CD86 mAbs and injected into the recipient. No further immunosuppression was given thereafter.