SAT0158 IL-7 and IL-7 receptor blockade selectively inhibit TLR7-induced B cell activation in primary sjögren’s syndrome

Abstract

Background Toll-like receptors (TLRs) have been implicated in several auto-immune diseases, especially those involved in the recognition of nucleic acids (viral, bacterial, and possibly self). In primary Sjögren’s syndrome (pSS) TLR7-induced B cell activation has been implicated in the immunopathology. An increased expression of TLR7 mRNA has been found in the parotid gland of pSS patients. In addition, TLR7 specifically recognizes ssRNA from viruses, and possibly self-ssRNA, with which Ro- and/or La-proteins form complexes, facilitating anti-SSA/SSB auto-antibody production, one of the hallmark disease parameters of pSS. Interestingly, EBV-transformed B cells have been shown to express the IL-7R and IL-7. Recently, we found increased levels of IL-7 in the minor salivary gland as well. Objectives To investigate the role of IL-7/IL-7 receptor-mediated immune activation in TLR7-induced B cell activation in pSS patients. Methods Isolated CD4 T cells and CD19 B cells from HC (n=7) and pSS patients (n=5) were co-cultured with and without a TLR7 agonist (TLR7A, Gardiquimod) in the presence or absence of CD14 monocytes/macrophages. Additionally, PBMCs (HC n=5, pSS n=8) were cultured with TLR7A with and without soluble human IL-7R (shuIL7R) and fully human anti-human IL-7 mAb. Proliferation of T cells and B cells was measured using 3H-thymidine incorporation and Ki67 expression (FACS analysis). Activation markers (CD19, HLA-DR, CD25) and intracellular IL-7 and IL-7Rα expression by B cells were measured by FACS analysis. Results TLR7A-increased proliferation of T and B cell co-cultures was associated with significant and selective increases in Ki67+ CD19 B cells (HC from 1.2±0.2% to 9.3±1.3%, p<0.01 vs. pSS from 1.2±0.2% to 7.0±2.2%, p<0.05), but not CD4 T cells. Additionally, markers of activation on CD19 B cells (HC: CD25+ from 42.2±4.8% to 80.1±4.4%; CD19 MFI from 26.8±3.3% to 63.4±9.6%; HLA-DR MFI from 214±32 to 649±105) were significantly increased, equally effective in HC and pSS. TLR7-induced B cell activation was further increased in the presence of monocytes (Ki67+ B cells: HC from 0.9±0.1% to 30.2±8.9% vs. pSS from 1.0±0.1% to 11.6±2.9%, both p<0.05). IL-7(R) blockade markedly inhibited proliferation of TLR7A stimulated PBMCs from HC (from 8880±2069 cpm to 2289±624 cpm; p<0.05) and pSS patients (from 8580±2555 cpm to 4802±1526 cpm; p<0.01), associated by a selective inhibition of Ki67-proliferating B cells. The specific role of IL-7R-mediated activation was supported by an increase in intracellular IL-7Rα and IL-7 upon TLR7 triggering in B cells, and confirmed by blockade of TLR7-induced B cell activation with a fully human anti-IL-7 mAb (mean inhibition 50%, p<0.05). Conclusions Our results show that TLR7 triggering activates B cells in pSS, which is facilitated by monocytes. Interestingly, TLR7A-induced B cell activation is potently and selectively inhibited by IL-7/IL-7R blockade. Together with the up regulation of IL-7 and IL-7R upon TLR7 activation, these results suggest that shuIL-7R/anti-IL-7 mAb block...