Background/Purpose: The early stage transitional B cell has been shown to be the key peripheral B-cell tolerance control point defect in SLE patients.  Anti-RNP Abs can occur before the onset of SLE, but the mechanism underlying tolerance loss to RNPs is unclear. Our previous B cell RNP Ag tetramer analysis indicates abnormal maturation of La_13-27 epitope reactive transitional T1 B cells in autoimmune BXD2 mice.  As B-cell signaling strength and NF-kB p-p65 signaling play an important role in T1 B cell maturation, the purpose of this study was to determine the contribution of elevated IL-17R mediated NF-kp-p65 signaling in autoreactive transitional B cell development. 

Methods: Using a La-peptide specific tetramer, La_13-27 autoreactive B cells from the spleens of B6, autoimmune WT BXD2, and BXD2-Il17r^‒⁄‒ mice were analyzed by FACS for the development of CD93⁺ transitional B cell subsets.  T1 B cells were co-transferred into WT vs. BXD2-Il17r^‒⁄‒.  B cell proliferative responses to anti-IgM and PMA/Ionomycin were determined by the Dojindo assay, and NF-kB p-p65 and p-Tyr levels were determined by phospho-flow. 

Results: Tetramer analysis of CD93⁺ transitional B cells revealed a 2-fold accelerated development of La_13-27⁺ T3 transitional spleen B cells (CD23^–IgM^hi) in BXD2 (38%±5%) compared to that in B6 (18%±3%) mice (n=8, p≤0.01).  Interestingly, we have identified that there was mislocalization of IgM^hiCD93⁺ T1 B cells in the follicles of BXD2 mice.  However, in normal B6 mice, T1 B cells are located in the peri-follicular region.  IL-17R signaling was previously shown to stabilize B-cell retention in the germinal center light zone.  Follicular mislocation of T1 B cells was normalized in BXD2-Il17r^‒⁄‒ mice. Interestingly, a deficiency in IL17R signaling in BXD2-Il17r^‒⁄‒ mice prevented the abnormal maturation of T1 to T3 La_13-27⁺ B cells (T3=14±2% vs 38±5%, n=8, p≤0.01). In vivo co-transfer of WT and BXD2-Il17r^‒⁄‒ into WT or BXD2-Il17r^‒⁄‒recipients support a role for IL-17R signaling in transitional B cell development in BXD2 mice.  Phospho-flow experiments suggest IL-17R mediated elevated NF-kB p-p65 activation at the T1 stage in BXD2 compared to B6 mice.  This elevation was associated with increased protein kinase C (PKC) activation and proliferation in BXD2 B cells stimulated in vitro with anti-IgM and PMA/ionomycin (p≤0.01).  A deficiency of IL-17R normalized these responses in BXD2-Il17r^‒⁄‒ mice. 

Conclusion:   The present data suggest that follicular exclusion of T1 B cells promotes peripheral tolerance, whereas follicular translocation and retention of T1 B cells promotes maturation.  Furthermore, IL-17 can promote NF-kB signaling in T1 B cells that have entered the follicle, bypassing tolerance checkpoint two in BXD2 mice.  This provides a mechanistic basis for checkpoint two defects.  As anti-La autoAbs can occur before the onset of SLE, these mechanisms may play a key role in initiation of disease.

This work was supported by the NIH (RO1-AI-071110 to JDM, RO1-AI-083705 to HCH, P30-AR-048311 to the UAB Comp. Flow Cyt. Core and the Analytic Imaging & Immunoreagent Core and P30-AI-027767 to the UAB Center for AIDS Research Comp. Flow Cyt. Core) and the VA (Merit Review grant 1I01BX000600-01 to JDM).  IL17R -/- mouse is a generous gift from Amgen Inc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il17-promotes-development-of-autoreactive-early-stage-b-cells-in-distinct-regions-of-the-spleen-follicle/