ABO self-tolerance in a mouse model: Evidence of tolerance at B cell but not T cell level
Ibrahim Adam1,6, Bruce Motyka2, Peter Cowan5, Lori J. West1,2,3,4,6.
1Immunology, University of Alberta, Edmonton, AB, Canada; 2Pediatrics, University of Alberta, Edmonton, AB, Canada; 3Surgery, University of Alberta, Edmonton, AB, Canada; 4Pathology/Lab Medicine, University of Alberta, Edmonton, AB, Canada; 5St. Vincent's Hospital , University of Melbourne, Melbourne, Australia; 6Canadian Donation and Transplantation Research Program, University of Alberta, Edmonton, AB, Canada
Introduction: We showed that human infants become tolerant to donor A/B-antigens (Ag) after ABO-incompatible heart transplantation (ABOi-HTx) by mechanisms not well-defined. To expand ABOi-HTx effectively beyond infancy, a clear understanding of tolerance mechanisms is needed. There are major limitations in studying mechanisms of tolerance in humans, thus we generated transgenic (A-Tg) mice (C57BL/6 [B6] background) expressing human A-Ag on vascular endothelium, erythrocytes and leukocytes. Here we investigated ABO self-tolerance in A-Tg mice.
Methods: Part 1: To test for self-tolerance to A-Ag, A-Tg mice and wild-type (WT) controls at age 6-7 weeks were challenged weekly x3 by ip injection with human A/B blood cell membranes (Hu A/B-BCM). A/B antibody (Ab) titre was determined by hemagglutination weekly using human A and B reagent erythrocytes (RBC). A-Tg native hearts were examined for C4d deposition by immunohistochemistry. Part 2: To test for the presence of B cells with specificity to A-Ag, A-Tg or WT splenocytes were either (A) cultured with IL-2 and R848 (TLR7/8-MyD88 agonist) and the supernatant assessed for anti-A Ab, or (B) adoptively transferred to B cell knock-out (BKO) mice, challenged with Hu A-BCM and assessed for anti-A Ab. Part 3: We previously showed that induced ABO Ab production requires CD4 T cell help. To test whether CD4 T cells from A-Tg mice can provide help for anti-A Ab production, we adoptively transferred CD4 T cells of A-Tg or WT mice into CD4KO mice, challenged with Hu A-BCM and assessed for anti-A Ab.
Results: Part 1: A-Tg mice produced only anti-B Ab whereas WT mice produced both anti-A and anti-B. C4d was not detected in native A-Tg hearts, indicating that absence of circulating anti-A was not due to tissue adsorption. Part 2:(A) A-Tg splenocytes did not produce anti-A Ab in culture, whereas WT splenocytes produced anti-A Ab as expected. (B) BKO mice reconstituted with A-Tg splenocytes did not produce anti-A Ab in response to Hu A-BCM stimulation whereas those reconstituted with WT splenocytes did. Part 3: CD4KO mice produce very low natural anti-A Ab, and do not respond to Hu A-BCM stimulation. However, CD4KO mice reconstituted with A-Tg CD4 T cells produced anti-A Ab in response to Hu A-BCM stimulation, comparable to anti-A production in CD4KO mice reconstituted with WT CD4 T cells.
Conclusions: B cells in A-Tg mice are tolerant to self A-Ag (Part 1). Furthermore, B cell self-tolerance in A-Tg mice cannot be broken in vitro or in vivo (Part 2). However, CD4 T cells from A-Tg mice can provide help for induction of anti-A Ab production (ie, against self A-Ag) (Part 3). How A-Tg CD4 T cells provide help for anti-A Ab production against self A-Ag is not completely clear. Some studies suggest that CD4 T cells can interact with xenogeneic proteins expressed in human RBC; others suggest that CD4 T cells can interact with carbohydrate Ags present on B cells.
