Tianshu Zhang

BACKGROUND: The induced antibodies against Galalpha1,3Gal (Gal) and non-Gal epitopes may contribute to delayed xenograft rejection (DXR). We asked whether blockade of the CD40/CD154 and CD28/B7 co-stimulatory pathways modulates the baboon elicited antibody response to pig Gal and non-Gal antigens. METHODS: Eighteen baboons received heterotopic heart transplants from pigs transgenic for human decay-accelerating factor (n = 13) or membrane cofactor protein (n = 5). Ten reference ''conventional therapy'' animals received cyclosporin A, cyclophosphamide and mycophenolate mofetil, with (n = 4) or without (n = 6) anti-CD20. Eight ''co-stimulation blockade'' animals received anti-CD154 mAb (IDEC-131) and anti-thymocyte globulin, with (n = 4) or without (n = 4) anti-CD20; two of these animals also received CTLA4-Fc. Anti-alphaGal IgG and IgM, anti-non-Gal antibodies and graft histology were assessed serially. RESULTS: Excluding two early graft failures, median graft survival with conventional therapy was 15 days (range 6 to 36 days, n = 8). Anti-Gal IgG antibody remained low through day 6 to 10, only one graft failure was accompanied by significant rise in anti-Gal IgG, and the anti-non-Gal response was weak (n = 2) or absent (n = 7). However many recipients succumbed with infection (n = 4) or coagulopathy (n = 2); DXR and ICOS+ T cells were prevalent in long-surviving grafts. With co-stimulation blockade, excluding three early graft failures, median graft survival was 7 days (range 6 to 11 days, n = 5). This regimen was very well tolerated, but increased anti-Gal antibody titer within 14 days was associated with graft failure in four of six animals. Although an anti-non-Gal response was present in three of six animals during IDEC-131 monotherapy (one strong, two weak), it was absent in both cases with additional CTLA4-Fc treatment. CONCLUSIONS: As used here, CD154 blockade alone does not completely prevent induction of Gal and non-Gal anti-pig antibodies. Our preliminary data suggest that other co-stimulation pathways, including CD28/B7 and ICOS, are sufficient to mediate high-titer anti-non-Gal antibody to porcine antigens in baboons, and contribute significantly to the pathogenesis of DXR.

Nguyen B‐NH, Azimzadeh AM, Schroeder C, Buddensick T, Zhang T, Laaris A, Cochrane M, Schuurman H‐J, Sachs DH, Allan JS, Pierson RN. Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection. Xenotransplantation 2011; 18: 94–107. © 2011 John Wiley & Sons A/S. Abstract: Background: Galactosyl transferase gene knock‐out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild‐type and human decay‐accelerating factor‐transgenic (hDAF +/+ ) pig lungs. Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild‐type lungs (P = 0.001) and 45 ± 60 min for hDAF +/+ lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild‐type or hDAF +/+ lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild‐type or hDAF +/+ lungs. However, GalTKO lungs adsorbed cytotoxic anti‐non‐Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti‐non‐Gal antibody and complement, platelet adhesion and non‐physiologic intravascular coagulation contribute to Gal‐independent lung injury mechanisms.

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.

Rationale: It was hypothesized that the dynamics of leukocyte populations in peripheral blood (PB) or peri-graft lymph nodes (LNs) in cynomolgus monkey recipients of a heterotopic heart allotransplant, completed with the determination of graft-infiltrating lymphocyte (GIL) populations at explant, may vary in association with immune rejection mechanisms or immunomodulatory treatments. Methods: Among 15 cynomolgus monkey recipients of heterotopic heart allografts, 13 were treated with a variety of costimulation-blocking immunosuppressive (IS) agents targeting CD80/CD86, CD28, CD40, or CD154, and two were untreated (controls). Leukocyte populations were characterized using hemocytometry and flow cytometry. Results: In PB, neutrophils and monocytes increased significantly (p < 0.001) during the first 2 weeks after transplant. Eosinophils and monocytes steadily increased after transplant, peaking around the time of graft failure (p < 0.01), a trend most prominent in association with belatacept. After the initial nadir on day 1 after transplant, PB lymphocytes increased steadily, particularly in association with belatacept and hu5c8, to a peak 1 week before graft rejection (p < 0.05), like CD3 cells. In PB, the CD4/CD8 ratio consistently trended down in all treated groups, most prominently in association with 5c8. In LNs at explant, CD4 cells outnumbered CD8 cells (p < 0.001), whereas in graft-infiltrating lymphocytes (GILs), CD8 cells predominated (p < 0.001). Among GILs at the time of rejection, CD8+CD62- central and effector memory cells were prominent, along with CD4+CD8+ T cells and IgD-CD27- B cells. At explant time, analysis of CD3 CD127lowCD25highFoxp3+ cell populations identified in GILs two clusters of CD4+CD8+ and three clusters of CD8 cells, which were expanded relative to PB or LNs. Conclusions: Observations regarding CD8 T-cell subpopulations in PB, LNs, and GILs support the conventional paradigm regarding their role as key effector cells mediating graft injury. The prominence of CD4+CD8+CD127lowCD25highFoxp3+ T cells and that of IgD-CD27- B cells among GILs have not previously been described. Expansion of circulating eosinophils around the time of rejection may implicate these cells in rejection mechanisms. Comparison of graft lymphocyte subpopulations with LNs or PB highlights mechanistically plausible differences that justify further efforts to elucidate their roles in graft injury and protection as a strategy to identify new candidate approaches to prevent rejection and promote tolerance.