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Michael C. Carroll 
Harvard Medical School
Boston, MA

A Critical Role for Complement in Clonal Selection of B Lymphocytes

Participation of the complement system in clonal selection of B cells and the memory response is well supported by studies both in vitro and in vivo. For example, mice deficient in complement C3 (C3null) or C4 (C4null) or complement receptors CD21 and CD35 (Cr2null) have a severely impaired primary and secondary response to T-dependent antigens. Regulation of the B cell response is mediated by CD21 and CD35 which are expressed on B cells and follicular dendritic cells (FDC). On B cells, the receptors form a signaling complex with CD19 and Tapa-1, which provides coreceptor function in conjunction with the B cell receptor. CD21/CD35 expression on FDC is important for trapping and retention of C3d-antigen complexes. To identify the cellular defect in Cr2null mice, they were crossed with mice bearing the high affinity hen egg lysozyme specific immunoglobulin (Ig) transgene (tg). WT mice were immunized with different forms of lysozyme and then 7 days later injected with either Cr2null or Cr2+ HEL-binding B cells. Analysis of splenocytes harvested from the different groups of recipients 5 days after transfer by flow cytometry and immunohistology, revealed that expression of the CD21/CD19 coreceptor was essential for B cell survival within the splenic follicle and germinal center in response to both intermediate and very high affinity binding antigens, respectively. These results demonstrate that the B cell coreceptor is essential in both initial activation of B cells and providing a survival signal within the germinal center. Furthermore, germinal center survival is dependent on contact with C3d-antigen complexes retained on the FDC.

Given the importance of complement in the B cell response to foreign antigens, it was reasoned that it might be equally important in the response to self antigens. To test this hypothesis, Cr2+ or Cr2null HEL+ Ig transgenic mice were crossed with mice bearing a transgene encoding soluble HEL (sHEL). In this well-defined double transgenic model, self-reactive B cells are anergized in the bone marrow but migrate into the periphery, where they have a dramatically reduced half-life and do not respond to antigen stimulation in vitro. By contrast, self-reactive B cells deficient in CD21/CD35 were not anergized by soluble self antigen, as they appeared to accumulate at normal frequencies and were responsive to self-antigen in in vitro stimulation assays. Thus, the complement receptors CD21/CD35 appear to be involved in negative selection of self-reactive B cells.

To further test a role for complement in maintenance of self-tolerance, a murine model of lupus – the fas-deficient lpr strain of mouse – was crossed with mice deficient in either CD21/CD35 or complement C4. Severity of lupus-like disease in these mice is strain-dependent, and on the mixed 129 X C57BL/6 background the lpr mice have a mild form of the disease. By contrast, lpr mice bearing deficiency in either CD21/CD35 or C4 develop a more severe form of lupus, characterized by increased lymphadenopathy and high titers of anti-nuclear and -dsDNA antibodies. Examination of kidneys by histology revealed a significant increased frequency of deposition of immune complexes and glomerulonephritis in the complement-deficient lpr mice in comparison with the complement-sufficient controls. Thus, in the absence of early classical pathway complement or receptors CD21/CD35, self-reactive B cells accumulate and in the presence of self-reactive T cells, expand and release autoantibodies leading to glomerulonephritis in the lpr mice. These results provide a novel explanation for the long-standing paradox of genetic association of complement deficiency with susceptibility to systemic lupus erythematosus in humans.