Occurrence of specific humoral non-responsiveness to swine antigens following administration of GalT-KO bone marrow to baboons

Abstract

Background: Hematopoietic chimerism induces transplantation tolerance across allogeneic and xenogeneic barriers, but has been difficult to achieve in the pig-to-primate model. We have now utilized swine with knockout of the gene coding for alpha-1,3-galactosyltransferase (GalT-KO pigs) as bone marrow donors in an attempt to achieve chimerism and tolerance by avoiding the effects of natural antibodies to Gal determinants on pig hematopoietic cells.

Methods: Baboons (n = 4; Baboons 1 to 4 = B156, B158, B167, and B175, respectively) were splenectomized and conditioned with TBI (150 cGy), thymic irradiation (700 cGy), T cell depletion with rabbit anti-thymocyte globulin (rATG) and rat anti-primate CD2 (LoCD2b), and received FK506 and supportive therapy for 28 days. All animals received GalT-KO bone marrow (1 to 2 x 10(9) cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells by PCR of BM colony-forming units, and for cellular reactivity to pig cells by mixed lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT-KO pig cells was tested by flow cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential impact of the protocol on renal transplantation.

Results: None of the animals showed detectable pig cells by flow cytometry for more than 12 h post-BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig-derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was detected by PCR in baboons 1 and 2 for at least 28 days post-transplant. ELISA results confirmed humoral immunocompetence at time of transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) increased within 2 weeks. However, no induced antibodies to GalT-KO pig cells or increased donor specific cytotoxicity was detectable by flow cytometry. In contrast, baboons 3 and 4 developed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day 14. Retrospective analysis revealed that although all four baboons possessed low levels of antibody-mediated cytotoxicity to GalT-KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and rejected pig kidneys) had relatively high pre-transplantation titers of anti-non-Gal IgG detectable by flow cytometry, whereas baboons 1 and 2 had undetectable titers.

Conclusions: Engraftment and specific non-responsiveness to pig cells has been achieved in two of four baboons following GalT-KO pig-to-baboon BMT. Engraftment correlated with absence of preformed anti-non-Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier.

Fig. 1

Cellular recovery: (A) WBC showed recovery within a week of transplant for all animals. B158 (Baboon 1) had a suspected low grade infection following transplant, with a relative leukocytosis, that resolved with empiric antibiotic coverage; (B) T cells were depleted by irradiation, ATG and LoCD2b, which began 1 week before BM transplant, and reached maximal depletion by day -1. All animals maintained CD3 + T cell counts < 100/μl for 10 days post-transplant, except for B158 (Baboon 2), which manifested an accelerated recovery and subsequently received a second dose of ATG on day 10. Baboon 1 received a third dose of LoCD2b on D27 in preparation for an additional infusion of GalT-KO bone marrow (presumably the cause of its anaphylactic death).

Fig. 2

(A) Blood and platelet recovery: Despite frequent blood draws for in vitro assays, CBC, chemistries and coagulation panels, both animals that received only a BM transplant maintained a stable HCT above 20 without pRBC transfusions; (B) Platelet levels decreased following TBI, and reached the lowest counts 1 week following irradiation. Baboon 4 (B175) required extensive blood product support due to persistent anemia and thrombocytopenia. B156 experienced a sudden decrease in platelets on day 10, which was attributed to menses.

Fig. 3

Baboon anti-pig antibodies as measured by antibody-mediated cytotoxicity: All animals in this study had baseline low cytotoxicity that was indistinguishable from the negative control (fetal pig sera). (A) B156 (Baboon 1) and 158 (Baboon 2) maintained low levels of cytotoxicity during the study period with approximately 14% at a sera dilution of 1:2; (B) B167 (Baboon 3) and B175 (Baboon 4) in contrast, showed increasing cytotoxicity within 15 days following transplant. While B175 (Baboon 4) received a kidney transplant on day 2, which may have sensitized the animal, B165 (Baboon 3) demonstrated increasing cytotoxic antibody titers on D15, prior to its kidney transplant on day 17. Note: B158 was subsequently immunized with GalT-KO tissues to produce the positive control serum used in panel B.

Fig. 4

Mixed lymphocyte reaction (MLR): Pre-transplant MLR of B158 (Baboon 2) showed a robust proliferative response to both DD pig and allogeneic cell stimulators. By day 40, this animal showed hyporesponsiveness to allogeneic stimulators and a notable lack of reactivity to DD pig cells. By D57, the allogeneic response began to recover while stimulation by DD pig cells remained very low.

Fig. 5

LoCD2b antibody titers in B156 (Baboon 1). This animal had low levels of IgG and IgM anti-LoCD2b antibody prior to BM transplant, but developed elevated IgG titers by day 12 following transplant. High IgG titers persisted until day 28, when the animal expired following a third dose of LoCD2b. Suspicion of an anaphylactic reaction to LoCD2b prompted the retrospective analysis of anti-ATG and anti-LoCD2b serum antibody titers.

Fig. 6

Pre-transplant anti-;non-Gal IgG antibody titers. (A, B) Both B56 (Baboon 1) and B158 (Baboon 2) had low levels of anti-non-Gal IgG prior to transplant, with a mode fluorescence intensity (MFI) comparable to the negative control. No significant change in MFI was noted in the weeks following transplantation. (C, D) In contrast, B167 (Baboon 3) and B175 (Baboon 4) had higher pre-existing levels of natural anti-non-Gal IgG, and demonstrated a clear increase in titers after BM transplantation.