Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model

Abstract

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Keywords: Kidney (allograft) function/dysfunction; tolerance: experimental.

Figure 1. Schematic of the adoptive transfer experimental protocol

On day −110, adoptive transfer donors (highly-inbred SLA^dd) underwent bilateral nephrectomy and received a class I-mismatched (SLA^gg) kidney graft, followed by 12 days of high-dose CyA. After 100 days with stable renal function and in vitro unresponsiveness, these animals were deemed LTT, and underwent a DST (day −9), and an extensive leukapheresis to remove PBMCs (day −1). These cells were immediately transferred to a syngeneic recipient (highly-inbred SLA^dd), who underwent 150 cGy WBI just prior transfusion. On day 0, the donor animal was sacrificed and the LTT kidney transferred to the adoptive transfer recipient after bilateral native kidney nephrectomy. Recipients did not receive immunosuppression after transplant, and were followed for at least 100 days with serial creatinine measurements and biopsies on PODs 30, 60 and 90. Accepted LTT kidneys were removed after day 100 and replaced with naïve SLA^gg kidney grafts. DD: SLA^dd, GG: SLA^gg, DST: donor-specific transfusion, KTx: kidney transplant, Leukaph: leukapheresis, LTT: long-term tolerant, mm: mismatched, PBMC: peripheral blood mononuclear cells, WBI: whole-body irradiation.

Figure 2. Histology of the adoptively transferred LTT kidneys after transplantation

A: POD 118 biopsy from animal 20314, showing no interstitial infiltrate or cellulitis. B: POD 125 biopsy from animal 20319, showing mild glomerulitis without cellular rejection. C: POD 105 biopsy from animal 21713, consistent with ACR1, signs of glomerulopathy, no cellular rejection. D: POD 14 biopsy from animal 21586 showing massive lymphocyte infiltration and severe hemorrhagic changes. POD: Post-operative day; ACR: acute cellular rejection.

Figure 3. In vitro responsiveness after adoptive transfer

Representative day ~100 CMLs for animals in group A (19678), B (20319), and C (21713) showed hypo-/unresponsiveness to donor-type cell targets. Panel D shows responsiveness for animal 21666 in group D, who failed to achieve tolerance and rejected the adoptively transferred kidney (day 14 CML, at the time of sacrifice). PBMCs from a naïve haploidentical animal (SLA^dd) were used as control in all assays (naïve DD); all animals maintained in vitro reactivity to third party (class I-mismatched) PBMCs (data not shown), suggesting that unresponsiveness was donor-specific. CML: cellular mediated lympholysis.

Figure 4. Outcomes of donor-type challenge grafting

Panels A and B show histology and in vitro unresponsiveness on day 121 after re-transplantation from animal 20319, one of the three who accepted the re-transplanted challenge graft. Panel C shows arteritis and acute cellular rejection in the challenge graft of swine 20791 at the time of sacrifice (day 53 after re-transplantation). At that time the animal also regained responsiveness in vitro (panel D). PBMCs from a naïve haploidentical animal (SLA^dd) were used as control in all assays (naïve DD).