This year, the Animal Models of Autoimmunity Group has turned its attention solely to investigate the role of IL-21 in B cell biology. IL-21 is a type I cytokine that influences the function of T cells, NK cells, and B cells, and is produced by T cells. IL-21 signals through a receptor composing of the IL-21R and the common cytokine receptor gamma-chain which are both expressed by T, B, and NK cells. Last year we discovered that IL-21 is a powerful stimulus to human peripheral blood and tonsilar B cells with the capability to drive 100% of poorly responsive naive cord blood B cells to terminally differentiated plasma cells! When highly purified naive B cells were stimulated with anti-CD40, IL-21 dramatically increased proliferation and plasma cell differentiation as well as robust immunoglobulin secretion. In marked contrast, when B cells were stimulated with anti-IgM, the addition of IL-21 resulted in death of activated proliferating naive B cells. However, when B cells were activated with both CD40 and IgM antibodies in the presence of IL-21, rapid differentiation into IgG secreting plasma cells was observed. These results indicate that IL-21 plays a critical role in determining the outcome of B cell stimulation, with the potential to foster the differentiation of large numbers of immunoglobulin secreting plasma cells. Last year we also focused on TNF and it?s role in autoimmunity. Previously we showed that mice deficient in TNF develop anti-nuclear autoantibodies (ANA) and double-stranded DNA antibodies. Rachel Robbins, a post-bac in the group, showed that in T cell transfer studies, OVA-specific OT-2 transgenic T cells alone were not able to confer autoimmunity in TNF/T cell-deficient mice. However, when these mice were immunized with specific antigen, and TNF was neutralized, high titers of ANA rapidly developed. These data suggest that in TNF-deficient mice, activated autoreactive B cells become trapped within the splenic T cell areas and are induced to secrete autoantibodies via CD4+ T cell help provided by activated bystander T cells. TNF, therefore, appears to play a role in maintaining tolerance of both T and B cells, the loss of which results in autoimmunity. This year we have extended our understanding of IL-21 to physiologic mechanisms that occur in vivo. T cell-dependent B cell activation involves numerous molecular interactions that ultimately lead to B cell differentiation. When activated T cells are cultured with purified B cells, many T cell derived factors including CD40L, induce plasma cell differentiation and Ig production, via a germinal center reaction. We wanted to address if IL-21 played a role in ongoing humoral immune responses. To address this Rachel Robbins and a summer student Ryan Novice undertook two approaches. First, we examined IL-21R-deficeint mice following primary and secondary immune responses and found that although (as known previously) IL-21R mice mount a weak humoral response as evidenced by low IgG titers to the immunized antigen, these mice were able to mount strong germinal center reactions as determined by immunohistology. Interestingly, we found that these mice had many IgG1, but not IgG2a memory B cells in their splenic primary follicles. However, upon challenge with antigen two months later, we found that mice deficient in IL-21R expression could not mount a secondary immune responses as evidence by no splenic germinal centers, although, these mice had many splenic IgM plasma cells. These data suggest that loss of IL-21R signaling results in a block of IgG plasma cell differentiation, the consequence of which is a ?pile up? of post-switched IgG1+ memory B cells and low IgG titers in the blood. It appears that these IgG1 memory B cells go on to die as they do not respond (or even are present) following a secondary immunization with antigen. The second approach was to examine T cell-dependent B cell activation by culturing activated human T cells with purified B cells. Normally, these type of culture conditions lead to non-specific B cell activation , differentiation and Ig production. However, we found that in the presence of neutralizing IL-21R-Fc fusion protein this response was remarkably inhibited. This included marked reduction in B cell proliferation, total B cell and plasma cell numbers, as well as low Ig production. These data demonstrate that what we showed previously with recombinant IL-21 and purified B cells is physiologically relevant to humoral immune responses in vivo. Our group made great headway in understanding the underlying mechanisms of IL-21 actions. In the near future we hope to discover how (or if) this cytokine is contributing to the development of systemic lupus erythematosus and rheumatic diseases. We feel that our research will lead to new therapeutics as well as to understand T cell-dependent humoral immune responses.
