B CELLS AND ANTIBODY RESPONSE

T.M. Conlon, R. Motallebzadeh, K. Saeb-parsy, C.J. Callaghan, S. Sivaganesh, E.M. Bolton, J.A. Bradley, G.J. Pettigrew
Department Of Surgery, University of Cambridge, Cambridge/UNITED KINGDOM

Body: Introduction: Antibody specificity is restricted by the requirement for T cell help delivered through ‘linked cognate’ recognition of processed target antigen that is presented following BCR internalisation. Here we examine whether allo-MHC class I-specific B cells can receive help for generating anti-MHC class I alloantibody through acquisition and presentation of additional mismatched graft alloantigen. Methods and Results: As expected, T cell deficient B6 (TCR KO) mice, when reconstituted with K^d-peptide-specific TCR Tg CD4 T cells, mounted strong anti-K^d alloantibody responses to a BALB/c heart graft. Surprisingly, anti-K^d antibody responses also developed when TCR KO mice were reconstituted with either B6 TEa CD4 T cells (that recognise donor IE MHC II peptide) or B6 Mar CD4 T cells (specific for H-Y peptide) and challenged with male BALB/c hearts. No alloantibody was generated when Mar-reconstituted mice received female BALB/c hearts, even when Mar CD4 T cells were activated by simultaneous challenge with male B6 APC, suggesting that help for anti-class I alloantibody responses provided through T cell recognition of an additional alloantigen requires co-expression of both antigens on the same graft cell. To investigate the hypothesis that alloantigen-specific B cells capture neighbouring donor proteins when internalising target alloantigen and process this for presentation to helper T cells, bone marrow chimeric Mar mice were created that lacked MHC II expression only on B cells. These mice did not develop anti-K^d alloantibody responses to male BALB/c hearts; in contrast strong responses developed in MHC II^+ve control mice, confirming that provision of help by T cells that recognise additional alloantigen still requires cognate interaction with B cell MHC II. Next, donor hearts from mosaic B6.K^d/B6.IE mice (created by embryo aggregation of two transgenic strains to contain cells expressing K^d or I-E, but not both) were transplanted into TEa-reconstituted TCRKO mice. Compared to control K^d+ve/IE^+ve grafts, anti-K^d IgG alloantibody responses were reduced significantly. The residual antibody that developed probably reflects transfer, as demonstrable on flow cytometry, of small amounts of IE and K^d MHC antigens between chimeric cells in mosaic animals. Finally, to examine whether this unusual mechanism of help for class-switched alloantibody contributes to graft damage, heart grafts were excised and analysed at day 50. Marked areas of scarring and significant vasculopathy (mean luminal stenosis 51%) were present in male BALB/c hearts transplanted into Mar-reconstituted TCR KO recipients, whereas female BALB/c hearts from Mar-reconstituted mice that were additionally challenged with male B6 APC had minimal parenchymal damage and only slight vasculopathy (9%). Conclusion: Our demonstration, that help for anti-MHC class I effector alloantibody responses can be provided by CD4 T cells that recognise additional mismatched alloantigen, challenges the tenet of 'linked' antigen recognition between the BCR and helper TCR as a critical requirement for T-dependent antibody responses and provides a mechanism to explain how alloantibody specificities diversify after transplantation.

Disclosure: All authors have declared no conflicts of interest.