Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates

Abstract

Transplant tolerance, defined as indefinite allograft survival without immunosuppression, has been regularly achieved in laboratory mice but not in nonhuman primates or humans. In contrast to laboratory mice, primates regularly have high frequencies of alloreactive memory T cells (TMEMs) before transplantation. These TMEMs are poorly sensitive to conventional immunosuppression and costimulation blockade, and the presence of donor-reactive TMEMs in primates may account for their resistance to transplant tolerance protocols that have proven consistently effective in mice. We measured the frequencies of anti-donor TMEMs before and after transplantation in a series of rejecting and tolerant monkeys that underwent nonmyeloablative conditioning, short-term immunosuppression, and combined allogeneic kidney/cell transplantation. Transplants were acutely rejected in all the monkeys with high numbers of donor-specific TMEMs before transplantation. In contrast, long-term survival was observed in the recipients harboring lower frequencies of anti-donor TMEMs before transplantation. Similar amounts of TMEM homeostatic expansion were recorded in all transplanted monkeys upon hematopoietic reconstitution; however, only the tolerant monkeys had no expansion or activation of donor-reactive TMEMs after transplantation. These results indicate that the presence of high frequencies of host donor-reactive TMEMs before transplantation impairs tolerance induction to kidney allografts in this nonhuman primate model. Indeed, recipients harboring a low anamnestic reactivity to their donor before transplantation were successfully rendered tolerant via infusion of donor cells and short-term immunosuppression. This suggests that selection of allogeneic donors with low memory responses in recipients may be essential to successful transplant tolerance induction in patients.

Fig. 1

Memory T cell (TMEM) responses before transplantation in cynomolgus monkeys. (A and B) Pretransplant frequencies of donor-reactive TMEMs in recipients. (A) The peripheral blood TMEMs from a panel of individual monkeys were isolated by FACS using CD95 and C28 markers and stimulated in vitro with irradiated allogeneic stimulator cells (direct allorecognition). The frequencies of IFN-γ−producing TMEMs were determined by ELISPOT in each of 251 responder/stimulator combinations. The arrows correspond to the eight monkey combinations selected for combined bone marrow/kidney transplantation. (B) Frequencies of IFN-γ–secreting TMEMs measured in the eight monkeys selected as recipients after stimulation with allogeneic APCs (from the allogeneic monkeys selected as donors) or control syngeneic (autologous) APCs or in the absence of APCs (medium).

Fig. 2

Conditioning and combined donor cell/kidney transplantation. The monkeys underwent total body irradiation (TBI) (1.5 Gy) at days −6 and −5 and thymic irradiation (TI) (7 Gy) at day −1. They also received ATG at days −2, −1, and 0 (50 mg/kg given intravenously). At day 0, they were transplanted with an allogeneic kidney and injected with bone marrow cells (BMCs) or spleen cells (3 × 10⁸ to 4 × 10⁸ cells/kg and 0.1 × 10⁸ to 0.4 × 10⁸ cells/kg, respectively) from the same donor, followed by a 28-day course of cyclosporine A (CYA) (15 mg/kg per day).

Fig. 3

Expansion of TMEMs after conditioning and transplantation. (A) Kinetics of posttransplant expansion of TMEMs measured in the peripheral blood of two recipients that underwent acute rejection (Rej) (dotted lines, open symbols) and three tolerant (Tol) monkeys (solid lines and symbols). (B) T cells from the peripheral blood were isolated and stained with anti-CD95 APC (DX2), anti-CD28 PE (CD28.2), and anti-CD3 PerCP (SP34-2) mAbs and analyzed via cytofluorometry. This plot shows the distribution of CD95⁻CD28⁺ (lower right: naïve T cells), CD95⁺CD28⁺ (upper right: central TMEMs), and CD95⁺CD28⁻ (upper left: effector TMEMs), gated on CD3⁺ T cells. This plot is representative of the six monkeys tested individually before transplantation (Pre-Tx, top panel) and 50 days after transplantation (Post-Tx, lower panel). The variation of the percentages of T cell subsets between monkeys is shown in fig. S1. The numbers shown in each quadrant indicate the percentages of each T cell subset among T cells.

Fig. 4

Posttransplant activation/expansion of donor-specific TMEMs. TMEMs were collected from the peripheral blood of recipients before transplantation and at different time points after combined bone marrow/kidney transplantation. The frequencies of IFN-γ–producing TMEMs recognizing donor MHC antigens directly were determined by ELISPOT. The results are expressed as numbers of cytokine-producing cells per million TMEMs. Direct memory alloresponses were assessed in four tolerant recipients (solid lines and dots) and two acutely rejecting monkeys (dotted lines and open symbols). In addition, memory alloreactivity was monitored in a control recipient, which received no immunosuppressive and conditioning treatment before transplantation and underwent acute rejection within 6 days after the placement of an allogeneic kidney (gray line and symbols).