B CELLS AND ANTIBODY RESPONSE

I.G. Harper¹, T. Conlon¹, C.J. Callaghan², K. Saeb-parsy¹, E.M. Bolton¹, J.A. Bradley¹, G.J. Pettigrew^1
1Department Of Surgery, University of Cambridge, Cambridge/UNITED KINGDOM, ²Department Of Surgery, Addenbrooke's Hospital, University of Cambridge, Cambridge/UNITED KINGDOM

Body: Introduction
We have recently shown that donor CD4 T cells provide help to recipient B cells through graft-versus-host (GVH) allorecognition of surface MHC II (Win TS et al 2009). Only autoantibody was produced, yet theoretically all B cells, irrespective of BCR specificity receive equivalent help, and we hypothesise that simultaneous BCR ligation by constitutively-expressed autoantigen provides an essential additional signal for plasma cell differentiation. If so, antibody responses should also develop against non-self antigens (including alloantigens) that are encountered concurrently.

Methods
The role of donor CD4 T cells in providing help for alloantibody production was examined by adoptive transfer into T cell deficient B6.TCR-KO animals. NK cells were depleted using anti-NK1.1 antibody (0.5mg i.p on D-2,0,2). IgG autoantibody responses were assayed by indirect immunofluorescent staining of nuclear-antigen-expressing Hep2 cell and alloantibody by H-2K^d-specific ELISA.

Results
Upon injection of bm12 CD4 T cells into B6.TCR-KO mice, GVH allorecognition of the disparate I-A^b MHC II on recipient B cells provoked strong autoantibody responses, but anti-OVA responses did not occur unless mice were immunized simultaneously with OVA. Similarly, injection with bm12 CD4 T cells that expressed transgenic H-2K^d (bm12K^d) prompted strong auto- and anti-K^d IgG alloantibody responses; in contrast, injection with WT bm12 cells generated autoantibody only. Autoantibody and anti-K^d IgG alloantibody responses were also detected following bm12K^d heart transplantation into TCR-KO recipients. This ability of bm12K^d CD4 T cells to provide help for anti-K^d humoral immunity is not due to recognition of K^d-peptide, but instead to GVH recognition of recipient B cell MHC II, because these cells are tolerant to self; bm12K^d mice challenged with H-2^d heart allografts do not produce anti-K^d antibody. Self tolerance does not, nevertheless, prevent alloantibody-mediated destruction, because whereas bm12 CD4 T cells survived indefinitely in B6.TCR-KO hosts, the development of anti-K^d alloantibody coincided with disappearance of bm12K^d CD4 T cells.
Injection of BALB/c x bm12 F1 CD4 T cells into B6.TCR-KO mice generated neither auto- nor alloantibody. This likely reflects NK cell-mediated killing of the more disparate F1 cells, because whereas bm12 and bm12K^d cells survived indefinitely upon transfer into B6 RAG^-/- mice, the F1 cells were detectable after the first week only if NK cells were simultaneously depleted. Similar depletion of NK cells in TCR-KO mice resulted in strong, rapid anti-K^d alloantibody responses upon injection with BALB/c or BALB/c x bm12 F1 CD4 T, confirming that disparate donor CD4 T cells can also provide help for alloantibody generation, provided that NK cell recognition is circumvented. The development of alloantibody in these animals again coincided with disappearance of the donor CD4 T cell population.
Conclusions
Our results demonstrate a novel, donor CD4 T cell dependent mechanism for production of class-switched effector alloantibody. Although normally prevented by NK cell recognition of donor lymphocytes, this mechanism may be particularly relevant to clinical transplant tolerance achieved through formation of bone marrow chimerism that results in co-existent populations of donor and recipient lymphocytes; allo- and auto-antibody responses have been described recently in such patients.

Disclosure: All authors have declared no conflicts of interest.