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We report orthotopic (life‐supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti‐CD40 monoclonal antibody‐based immunosuppression. In this current study, life‐supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti‐inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi‐gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi‐gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans. [ABSTRACT FROM AUTHOR]

We describe the incidence of early graft failure ( EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of Gal TKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (Gal TKO. hCPRP). Baboon recipients of life-supporting Gal TKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a Gal TKO. hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of Gal TKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the Gal TKO EGF incidence to 7% (1/14; P < 0.01 vs. Gal TKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a Gal TKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early Gal TKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation. [ABSTRACT FROM AUTHOR]

Singh AK, Seavey CN, Horvath KA, Mohiuddin MM. Ex-vivo expanded baboon CD4+ CD25Hi Treg cells suppress baboon anti-pig T and B cell immune response. Xenotransplantation 2012; 19: 102-111. © 2012 John Wiley & Sons A/S. Abstract: Background: CD4+ CD25+ FoxP3+ regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex-vivo expansion of naturally occurring CD4+ CD25+ T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens. Methods: Naturally occurring baboon CD4+ CD25+ regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti-CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL-2. Treg cells were also enriched directly from CD4+ T cells cultured in the presence of rapamycin (0.1-10 n m). Mixed lymphocyte culture and polyclonal B cell stimulation with ex-vivo Treg cells were performed to assess the function of ex-vivo expanded Treg cells. Results: The nTreg cells were expanded to more than 200-fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2-fold increase in enrichment of CD4+ CD25+ FoxP3+ Treg cells from CD4+ cells was observed with rapamycin compared to cultures without rapamycin. The ex-vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti-porcine xenogeneic T and B cell immune response in-vitro efficiently (more than 90% suppression at 1 : 1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1 : 256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1 : 1 ratio, and their suppressive ability was reduced to ≤50% at 1 : 16 ratio. Furthermore, we have found that ex-vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex-vivo expanded Treg cells were added to the culture at a 1 : 1 ratio. The addition of CD4+ CD25Neg cells however induced vigorous proliferation. Conclusion: Ex-vivo expanded CD4+ CD25+ FoxP3+ Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex-vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models. [ABSTRACT FROM AUTHOR]