Allograft outcomes in outbred mice

Abstract

Inbreeding depression and lack of genetic diversity in inbred mice could mask unappreciated causes of graft failure or remove barriers to tolerance induction. To test these possibilities, we performed heart transplantation between outbred or inbred mice. Unlike untreated inbred mice in which all allografts were rejected acutely (6-16 days posttransplantation), untreated outbred mice had heterogeneous outcomes, with grafts failing early (<4 days posttransplantation), acutely (6-24 days) or undergoing chronic rejection (>75 days). Blocking T cell costimulation induced long-term graft acceptance in both inbred and outbred mice, but did not prevent the early graft failure observed in the latter. Further investigation of this early phenotype established that it is dependent on the donor, and not the recipient, being outbred and that it is characterized by hemorrhagic necrosis and neutrophilic vasculitis in the graft without preformed, high titer antidonor antibodies in the recipient. Complement or neutrophil depletion prevented early failure of outbred grafts, whereas transplanting CD73-deficient inbred hearts, which are highly susceptible to ischemia-reperfusion injury, recapitulated the early phenotype. Therefore, outbred mice could provide broader insight into donor and recipient determinants of allograft outcomes but their hybrid vigor and genetic diversity do not constitute a uniform barrier to tolerance induction.

Fig 1. Effect of costimulation blockade on allograft survival in inbred and outbred mouse groups

Survival of (a) inbred Balb/c allografts transplanted to inbred B6 recipients (I to I) and (b) outbred (CD-1 or CF-1) allografts transplanted to outbred CD-1 recipients (O to O) was assessed in the presence or absence of recipient treatment with costimulation blockade (MR-1 + CTLA4-Ig). (c) Histopathology (H&E) of allografts that survived > 75 days in treated I to I group (left panel) and either treated or untreated O to O groups (right panels). Magnification = 2×. Insets show evidence of chronic allograft vasculopathy in all groups (magnification = 30×).

Fig 2. Histopathology of early graft failure

Representative pathology of cardiac allografts transplanted between untreated outbred mice that failed < 4 days (left panels) or between 6 and 24 days (right panels) after transplantation is shown. Gross appearance of allografts is shown in the top panels, H&E stained tissue sections in the middle panels (magnification = 4×), and immunofluorescent stained tissue section in the bottom panel. Insets in left middle panel highlight areas of neutrophil clot and neutrophilic vasculitis, while inset in right middle panel demonstrates lymphocytic arteritis typical of untreated acute cellular rejection. Note large area of hemorrhagic necrosis (*) in the early graft failure but not acute cellular rejection phenotype. Ly6G, CD31 and DAPI identify neutrophils, endothelial cells, and nuclei, respectively (bottom panel; magnification = 20×).

Fig 3. Early graft failure is dependent on donor outbred status

Survival of cardiac allografts transplanted from inbred to outbred (BALB/c to CD-1; I to O) or outbred to inbred (CD-1 to BALB/c; O to I) mice in the absence (a) or presence of costimulatory blockade (b). Note complete absence of early graft failure phenotype in recipients of allografts from inbred donors. (c) Degree of MHC II mismatch between all donor-recipient pairs used in this study as well as contemporaneous experiments involving outbred mice. Note that the degree of MHC II haplotype disparity between donor and recipient does not correlate with graft loss or rejection phenotype.

Fig 4. Lack of significant, preformed anti-donor antibodies in recipients that exhibited early graft failure

(a) Pre-formed, anti-donor thymocyte IgG (left panel) and IgM (right panel) antibodies in serum of recipients of outbred allografts that went on to develop early graft failure. Serum was obtained prior to transplantation in all mice except the sensitized group. MFI of isotype control antibody was subtracted in each case to determine net binding of IgG or IgM antibodies to donor thymocytes. (b) IgG and IgM deposits in cardiac allografts harvested at indicated time points after transplantation. Top panels (positive control) show IgG and IgM deposits in BALB/c cardiac allografts 12 hrs after transplantation to sensitized C57Bl/6 mice. Middle and bottom panels show lack of IgG and IgM deposits in outbred allografts (CD-1 to CD-1) harvested at 6 and 12 hrs after transplantation. These grafts had evidence of interstitial congestion, focal neutrophilic margination, and platelet fibrin thrombi at 6 hrs, and hemorrhagic necrosis with moderate neutrophilic margination at 12 hrs (H&E micrographs), consistent with early graft failure phenotype (magnification = 2×, inset magnification = 30×).

Fig 5. Early graft failure is dependent on complement and neutrophils

Survival of cardiac allografts after complement depletion in both donors and recipients (CD-1 to CD-1; O to O) (a) or after neutrophil depletion in the recipients (CD-1 to BALB/c; O to I) (b). Transplantation was performed on day 0. Average C3 levels in recipient blood are depicted as % of baseline level (right-hand y-axis in (a)). *Grafts that exhibited early graft failure phenotype by gross morphology and/or histopathology. H&E micrographs confirmed hemorrhagic necrosis and neutrophilic vasculitis in these grafts (magnification = 2×, inset magnification = 30×).