Fetal porcine thymus engraftment, survival and CD4 reconstitution in αGal‐KO mice is impaired in the presence of high levels of antibodies against αGal

Abstract

Xenospecific T-cell tolerance can be induced among murine and human T-cells by porcine thymic grafting. However, anti-alpha 1,3-galactosyltranserase (alphaGal) (Galalpha1-3Galbeta1-4GlcNAc-R) natural antibodies (NAbs) pose a major barrier to porcine xenografts in humans. We used alphaGal knockout (KO) and muchain KO mice to explore the effect of natural anti-alphaGal and other xenoantibodies on porcine thymic engraftment and to examine the potential of thymic tissue to tolerize anti-alphaGal antibody-producing cells. Thymectomized [adult thymectomy (ATX)] non-immunized and rabbit red blood cell (RRBC) pre-transplant immunized alphaGal-KO (knockout), wild-type (WT) and mu chain KO B6 mice were treated with 3Gy total body irradiation (TBI), and T and natural killer (NK) cell depleting monoclonal antibodies (mAbs). These conditioned mice were grafted with fetal porcine thymus and liver (FP THY/LIV) tissue under the kidney capsule. Flow cytometric analysis was performed to follow CD4 reconstitution as a measure of FP THY engraftment and function. Only mice with >10% CD4+ peripheral blood lymphocytes (PBL) were considered successfully engrafted. Enzyme-linked immunosorbent assay (ELISA) was used to assess the kinetics of immunoglobulin M (IgM) and IgG anti-alphaGal antibodies. Anti-pig antibodies were monitored by flow cytometry (FCM). FP THY engrafted successfully in most of the immunoglobulin deficient mice (11 out of 12, 92%) and the outcome was similar in WT B6 controls (8 out of 12, 67%). Non-immunized alphaGal-KO mice grafted with FP THY had a similar success rate (7 out of 11) to that observed in non-immunized alphaGal-WT controls (2 out of 4). In contrast, alphaGal-KO mice immunized pre-transplant with RRBC, then grafted with FP THY/LIV, showed a significant reduction in the success of thymic grafting (2 out of 9, 22%) compared with pre-transplant immunized WT controls (4 out of 7; 57%) and non-immunized alphaGal-KO mice (7 out of 11, 64%). Anti-Gal and anti-pig antibody levels were not markedly augmented by porcine thymus grafts in mice with successful thymus grafts. FP THY engraftment is impaired in the presence of high levels of anti-alphaGal xenoantibodies. However, low levels of anti-alphaGal antibodies and other mouse anti-pig NAbs appear not to play a major role in the rejection of FP THY. Although grafting FP THY expressing the alphaGal epitope did not tolerize B cells producing anti-alphaGal antibodies in a T-cell independent manner, it prevented T-cell dependent sensitization by inducing T-cell tolerance to porcine antigens.

B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-αGAL–producing B-1 cells

“…55 In contrast, the induced anti-␣Gal IgM response is largely T-cell dependent. 54,56 Both baseline and induced anti-␣Gal levels were enhanced by nonhematopoietic CR1/CR2 expression, suggesting a role for FDC CR1/CR2 expression in both T-independent and T-dependent Ab responses.…”

“…55 In contrast, the induced anti-␣Gal IgM response is largely T-cell dependent. 54,56 Both baseline and induced anti-␣Gal levels were enhanced by nonhematopoietic CR1/CR2 expression, suggesting a role for FDC CR1/CR2 expression in both T-independent and T-dependent Ab responses.Total serum IgM levels were unaffected by the presence or absence of CR1/CR2 on hematopoietic and/or nonhematopoietic cells in our model. Our previous studies showing that anti-␣Gal-and total IgM-producing cells belong to the same B-cell subset 9 led to the expectation that total IgM levels would be reduced in mice lacking nonhematopoietic cell CR1/CR2 expression.…”

B cell tolerance and xenotransplantation

“…avoided with a single treatment by induction of mixed xenogeneic hematopoietic chimerism in a aGalT +/+ rat/ aGalT ÿ/ÿ mouse model using a high dose of bone marrow cells after non-myeloablative conditioning (Kawahara and Sykes, unpublished data) [29,35,37]. The induction of mixed xenogeneic chimerism prevents hyperacute, acute vascular and cell-mediated rejection of primarily vascularized aGal-expressing cardiac xenografts.…”

“…Thymus xenografts were previously shown to induce and maintain T-dependent but not T-independent B cell unresponsiveness [37]. Another recent study showed that skin xenografts alone induce T cell-independent B cell tolerance but sensitize the Tcell compartment when cotransplanted with thymus tissue with leflunomide treatment [67,68].…”

Mixed Xenogeneic Chimerism Induces Donor-Specific Humoral and Cellular Immune Tolerance for Cardiac Xenografts

“…Unfortunately, clinical xenotransplantation has been thwarted by the vigorous rejection response elicited against solid organs transplanted across a species barrier (2)(3)(4). Transplantation of xenografts between widely disparate species has been limited by rejection due to the presence of natural Abs (5)(6)(7)(8)(9). Even when hyperacute rejection is prevented with plasmapheresis, complement depletion, immunoaffinity columns, or the use of more closely related species, long-term xenotransplantation is still limited by robust acute humoral and cellular immune rejection (10 -16).…”