Introduction: ABO-incompatible heart transplantation (ABOi HTx) is safe during infancy and allows increased donor access. B cell tolerance develops to donor A/B-antigen(s) after ABOi HTx by mechanisms not well defined. We developed A-transgenic (A-Tg) mice constitutively expressing human A-antigen on vascular endothelium and erythrocytes (RBC) to study B cell immunity and tolerance. Here we studied A-incompatibility in the context of syngeneic, allogeneic and xenogeneic stimulation.
Methods:Part I: Adult wild-type (WT) C57BL/6 (B6; H-2b), BALB/c (BALB; H-2d), or C3H/He mice (C3H; H-2k) received i.p. injection weekly x3 of B6 or BALB A-Tg RBC membrane (100 μl of 10% vol/vol); or human RBC membranes (100 μl of 10% vol/vol) from blood group A or O; or A-incompatible heart allografts (Table). Serum anti-A antibody was measured by hemagglutination and ELISA (IgG and IgM); graft survival was assessed by palpation. Part II: a) to assess requirement for ‘linkage’ of the non-self carbohydrate A-antigen with foreign protein to stimulate anti-A, human O-RBC were co-injected in B6 WT mice with syngeneic A-Tg cells; b) to assess T cell dependence of anti-A response, CD4+ T cells were depleted (mAb GK1.5) from B6 WT mice before human A RBC injection.
Results:Part I: Exposure to incompatible A-antigen in the context of allogeneic stimulation (MHC-mismatched A-Tg blood or heart graft) or xenogeneic stimulation (human A-RBC) induced abundant anti-A production (Table), whereas A-Tg syngeneic (MHC-matched) cells did not. Part II: a) a mixture of syngeneic A-Tg and xenogeneic human O-RBC did not induce anti-A; b) after CD4+ T cell depletion, xenogeneic cells failed to elicit anti-A.
Conclusion: Exposure to non-self A-antigen in syngeneic cells is insufficient to induce anti-A production; stimulation of antibody depends not only on exposure to the foreign carbohydrate but co-engagement with allogeneic or xenogeneic protein, and requires CD4 T cell help. These results are consistent with our previous data showing a requirement for chemical/physical linkage of non-self A-antigen and foreign protein.
This research was supported by the Heart and Stroke Foundation of Canada, Canadian Institutes of Health Research, Canadian Glycomics Network, and Stollery Children's Hospital Foundation through the Women and Children's Health Research Institute.