RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement.

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603-607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837-847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-alpha, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

DNA and RNA autoantigens as autoadjuvants.

AM14 B cells are a prototype for those low affinity autoreactive B cells that routinely mature as naïve cells in peripheral lymphoid tissues. These cells express a transgene-encoded receptor specific for IgG2a and can be effectively activated by immune complexes that incorporate either mammalian DNA or mammalian RNA that has been released from dead or dying cells. Activation depends on the ability of the B-cell receptor to deliver antigen to an internal vesicular compartment containing either Toll-like receptor-9 (TLR9) or TLR7. Since TLR9 and TLR7 are thought to recognize microbial DNA and RNA preferentially, it is important to determine under what conditions mammalian DNA and RNA become effective TLR ligands, and whether the determining factor is delivery or structure. This issue has been addressed by using IgG2a mAbs to deliver immune complexes preloaded with defined fragments of DNA or RNA, or by using modified ODNs/ORNs. The data demonstrate that only certain nucleic acid sequences or structures can induce autoreactive B-cell proliferation, even when delivery to the appropriate TLR compartment is facilitated by uptake through the B-cell receptor (BCR).

Comparison of CpG s-ODNs, chromatin immune complexes, and dsDNA fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells.

Synthetic single-stranded oligodeoxynucleotides (15-30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments ( approximately 600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN and small molecule inhibitors suggests that stimulatory CpG sODN and chromatin IC may either access TLR9 via different routes or depend on discrete activation parameters. These data have important implications regarding the therapeutic application of TLR9 inhibitors to the treatment of systemic autoimmune diseases.

Functional outcome of B cell activation by chromatin immune complex engagement of the B cell receptor and TLR9.

We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-kappaB activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.