Abrogation of renal allograft tolerance in MGH miniature swine: the role of intra-graft and peripheral factors in long-term tolerance

Abstract

We have previously demonstrated that long-term tolerance (LTT) of an MHC class-I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class-I mismatched primary kidneys were subjected to a treatment consisting of donor-specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor-MHC-matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4(+) Foxp3(+) Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class-I mismatched allotransplants.

Keywords: Animal models: porcine; basic (laboratory) research/science; immunosuppression/immune modulation; kidney transplantation/nephrology; tolerance: experimental; translational research/science.

Figure 1. Experiments of tolerance abrogation and experimental animals

A). List of Experimental Groups. DST = Donor Specific Transfusion; Retransplantation = Graftectomy of primary kidney followed by retransplantation with a Donor MHC Matched (Class I MHC mismatched, Class II MHC matched) kidney; Early Rejection = Rejection Crisis within 10 days following kidney retransplantation; Late Rejection = Rejection Crisis >90 days following kidney retransplantation; Early Peak Creatinine = Peak Creatinine Level Observed in Early Rejection Period; Late Peak Creatinine = Peak Creatinine Level Observed in Late Rejection Period. (*). n/a = not applicable. Because animal #19312 died of rejection on POD 8, late peak creatinine was not obtainable. Animals #18959 and #18956 exhibited prolonged, full rejection only after skin grafting ($). B). Timeline of experiments. Time points are relative to the day of the second transplantation (day 0). Skin grafts were performed on animals 18800, 19324, 18956, and 18959.

Figure 2. Clinical course and immune status of recipients of first and second renal transplants

A) Control animals # 20105 and # 20206 underwent DST and retransplantation, without removal of peripheral cells by leukapheresis. A-1: Serum creatinine levels for animals in group 1; A-2: Representative graft histology from animals of group 1 (animal 20206, POD 100 after retransplantation) and; A-3: Corresponding CTL reactivity representative of animals in group 1 (animal 20206, POD 100). B). Control LTT animals included pigs# 18800 and 18810 that received DST + leukapheresis without retransplantation and # 18913 which was retransplanted without treatment (Figure 1A, group 1). B-1: Serum creatinine levels for animals in group 1; B-2: Representative graft histology from animals of group 1 (animal 18800) and; B-3: Corresponding CTL reactivity representative of animals in group 1 (animal 18800). This CML assay was performed 2 weeks after DST and leukapheresis. C). Animals receiving second renal transplants after DST + leukapheresis. C-1: Serum creatinine for animals in groups 2A, B, and C. levels. C-2: Graft histology representative of group rejectors from group 2 (animal # 18959); this sample was obtained 8 days after retransplantation (ACR2-3). C-3: Corresponding cytotoxic activity for animal 18959 which was representative for animals of group 2. E/T: effector to target ratio in CML assays. ACR = Acute cellular rejection

Figure 3. Effect of leukapheresis

A). More extensive leukaphereses correlated with higher initial peak creatinine, which was used as read out of rejection. Assessment of tolerance of renal allografts with skin grafts (B-D): Two animals (18956, 18959) which experienced rejection crises (Figure 2, panel C1) that subsequently resolved were challenged with 2 consecutive donor skin grafts according to the timeline of Figure 1B (day 0 = 2^nd skin grafting). Control animals (18800 and 19324) are recipients of first kidney grafts that received (18800) or not (19324) the DST + leukapheresis treatment prior to skin grafting. B). Serum creatinine in recipients of skin transplants. C and D). CML assays, with representative data, performed 2 weeks after skin grafting. Studies for animals # 18800 and experimental animal #18956 are shown.

Figure 4. Potential involvement of regulatory T cells in tolerance to kidney grafts

A). The CML Suppression assay. Control anti-donor CML involved recipient matched PBLs stimulated with donor-type PBLs (no-suppressors, solid circles). Percent lysis observed when suppressor populations added were either naïve recipient MHC-matched (open-circles), or leukapheresis product from LTT animals (Tol. Leuka Cells, solid squares). Specificity controls for suppression involved LTT leukapheresis products added to recipient CML against 3^rd party (3^rd party-MHC-I/recipient matched Class-II). Additional negative controls included 3^rd party cells cultured independent of cells in leukapheresis product and self anti-self controls (not shown). Suppression assays were performed in duplicate. B). Monitoring of CD8+: CD4+ Foxp3+ ratios (left axis) and serum creatinine levels (right axis) in experimental animals. Error bars reflect standard deviation. C). Serum TGF(−1 levels (ng/mL) for four animals which experienced rejection of second renal transplants, but demonstrated resolution of renal function (18913, 18956, 18959, and 19381) are presented as mean TGF(−1 levels with associated standard deviations during times when animals experienced acute rejection. Increases in TGF-B1 correlated temporally with the expansion of peripheral Tregs (See 4B).

Figure 5

Accepted second transplants are selectively infiltrated by Foxp3+ Tregs. Renal graft biopsies from animal # 18958, which experienced complete rejection (Rejection) and from animal # 18959 (pre-skin graft) with resolved rejection (Tolerance) were analyzed by immunohistochemistry for their respective content in CD3+ (brown, A and D). Foxp3+ (brown, B and E) and Foxp3+/CD25+ lymphocytes (C and F). Black arrowheads in 5E highlight brown FoxP3+ cells. Double labeling for CD25-Foxp3 identifies Treg cells with surface CD25 (green) and intracellular Foxp3 (red) labeling (white arrow heads). Magnification = 200X (A, B, D, E) and 400X (C and F). These data are from animals experiencing full rejection or acceptance of second transplants.