Background/Purpose: TLR7 is required for the generation of anti-RNA antibodies and excess TLR7 confers a SLE-like phenotype in mice. Recent studies have shown that TLR7 expression in B cells is sufficient for this phenotype and that TLR7 excess enhances germinal center (GC) maturation. The goal of this study was to determine how TLR7 influences the GC derived autoreactive B cell repertoire.

Methods: TLR7 deficiency was crossed into NZW mice for 13 generations. The 3H9 IgVH transgene that confers anti-DNA and anti-cardiolipin specificity was introduced into NZW/BXSB.Yaa and NZW.TLR7+/-/BXSB.Yaa lupus prone mice. The Yaa locus confers an extra copy of TLR7 in males and an accelerated lupus phenotype and NZW.TLR7+/-/BXSB.Yaa mice retain the Yaa locus but carry only one copy of TLR7. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated 50% 3H9/50% wild type bone marrow chimeras in which transferred male 3H9. TLR7+/+ or TLR7+/-cells are GFP+ and can be easily identified.  Mice were followed clinically and sacrificed at the onset of fixed proteinuria. Spleen cells were phenotyped and GC B cells were analyzed by single cell PCR for the repertoire of Vk light chains associated with the 3H9 heavy chain. Full length Vk5-43 encoded light chains were resequenced and their mutation frequency analyzed.

Results: Disease onset occurred with the same kinetics in TLR7+/+ and TLR7+/- chimeras and spleens from both sets of chimeras were phenotypically indistinguishable. We have previously shown a preferential selection of Vk5-43 and Vk5-48 light chains into the GCs of 3H9 SLE prone mice; these light chains confer anti-chromatin activity in their germline configuration whereas anti-DNA and anti-cardiolipin activity is acquired as a result of somatic mutations.  We found that Vk5-48/Jk4 encoded light chains that have a high affinity for chromatin were preferentially selected into the GCs when 3H9 TLR7+/+ cells were transferred together with their non-3H9 counterparts but were found rarely when 3H9 TLR7+/- cells were transferred. By contrast, Vk5-43 encoded light chains were selected into the GCs in both TLR7+/+ and TLR7+/- chimeras. We therefore examined the frequency of somatic mutations in these light chains. The frequency of somatic mutations was significantly less in Vk5-43 light chains from 3H9+ GC cells from TLR7+/- chimeras than in 3H9+ GC cells from TLR7+/+ chimeras.  Finally 3H9+ B cells were significantly more expanded in the GCs of TLR7+/+ than TLR7+/- chimeras.

Conclusion: We have demonstrated that TLR7 influences selection of autoreactive B cells into the GC, as well as their expansion and the frequency of somatic mutations. Our results suggest that TLR7 influences proliferation in the dark zone of the GC since this is linked to mutation frequency. These effects of TLR7 are independent of other genes in the Yaa locus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tlr7-influences-autoreactive-b-cell-selection-in-the-germinal-center/