Background Activation of autoreactive T cells is a critical step in the pathogenesis of lupus. These T cells help autoreactive B cells, make inflammatory cytokines, and infiltrate tissues. Yet, compared to autoreactive B cells, little is known about the specificity, identity, origins, or functions of autoreactive T cells. Therefore, we sought to isolate, clone and characterise T cells that recognise peptides derived from the targets of anti-nuclear antibodies/B cells.
Materials and methods Using anti-IgG2a (“rheumatoid factor”, RF) B cells from site-directed transgenic mice as “universal APC” for the contents of dying cells that are in turn bound by IgG2a anti-nuclear antibodies (ANAs). We relied on a monoclonal IgG2a anti-chromatin to stimulate the T cells initially and then a panel of monoclonal ANAs to test them. These ANAs naturally form immune complexes (ICs) with material released from dead cells either in vitro or in vivo. We used these tools to serially stimulate primary polyclonal T cells and make autoreactive T cell hybridomas. We then cloned the TCRs of these hybridomas into retroviral vectors to make “retrogenic mice”, a source of primary T cells.
Results We isolated multiple clones of T cells that could help RF B cells make proliferate and differentiate both in vitro and in vivo and characterised two in detail. These T cells were activated by the combination of RF B cells and either IgG2a anti-chromatin or anti-RNA, indicating that the T cells recognised peptides derived from the ICs formed by the ANAs used to stimulate them. We further found that while TLR7 and 9 were required to stimulate the RF B cells in the absence of T cells, the requirement for these molecules was bypassed by the presence of autoreactive T cells. Reciprocally, the APC function of B cells for T cells also did not require TLR7/9 expression in B cells
Conclusions We used a novel method to clone and characterise autoreactive T cells that help autoreactive B cells. These T cells were isolated from normal animals without use of adjuvant or foreign Ag, confirming that normal primary repertoires contain relevant autoreactive T cells. These cells enhanced multiple modes of B cell activation and differentiation in vivo and themselves were activated and differentiated in divergent ways. Most importantly, because they could bypass in large part the need for B cell-intrinsic TLR stimulation, they support the idea that TLRs may be more important for initiation of autoimmunity rather than propagating it once it is well-established.
Acknowledgements Supported by a grant from the Lupus Research Institute.
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