B Cells Alter Adaptive Autoimmunity to Protect from Ischemic Injury After Stroke

More than 800,000 people in the U.S. will suffer from stroke each year, yet there remains only one FDAapproved acute intervention. Clinical trials to minimize post-stroke inflammation failed, highlighting the need to better understand inflammatory and neuroprotective mechanisms within the CNS. Repetitive hypoxic preconditioning (RHP) naturally protects from stroke for months beyond treatment, minimizing neurovascular injury, including reductions in infarct volumes, blood-brain barrier (BBB) disruption, and leukocyte diapedesis. RHP, however, specifically recruits B cells into the injured CNS. RHP creates an immunosuppressed B cell phenotype prior to stroke, and the adoptive transfer of these RHP-treated B cells (B(RHP)cells) 6h after stroke onset reduces infarct volumes and alters the adaptive immune response in untreated, recipient wild-type (WT) mice. B(RHP)cell therapy also enhances a post-stroke autoimmune response to CNS Ag in recipient mice, a novel finding for adaptive autoimmunity that has not been studied in preclinical models of stroke except in the context of secondary infection. The goal of this New Investigator grant is to determine if B(RHP)cells, as part of a transferrable adaptive immunity to mild hypoxia, mediate endogenous protection through the production of interleukin 10 (IL10) and interferon-ã (IFN-ã) and enhance autoimmunity to promote neuronal recovery. Specific Aim 1 will test the hypothesis that RHP enhances the production of IL10 by regulatory B (B10) cells. RHP-enhanced B10 populations in the ischemic hemisphere will be quantified by flow cytometry, and the ability of B10 cells to prevent neuronal injury confirmed in an in vitro Campenot chamber assay. The post-stroke adoptive transfer of IL10-null B(RHP)cells will determine the requirement of IL10 for acute (e.g. BBB disruption, infarct volumes) protection from stroke. Specific Aim 2 will test the hypothesis that B(RHP)cell-induced neuroprotection requires CNS Ag-dependent specificity. CNS-specific targets for Ag presentation and Ab production in the ischemic CNS will be identified by flow cytometry and whole protein self-Ag array. The contribution of CNS Ag-specificity during B(RHP)cell therapy will be determined using B cells that only produce IgM specific for hen egg lysozyme. The requirement of B(RHP)cell-derived CNS Ag presentation for neuroprotection in recipient mice will be determined by transferring WT B(RHP)cells to mice that have CD4 populations specific for only ovalbumin. Both acute injury mechanisms and long-term functional recovery and plasticity will be evaluated. Specific Aim 3 will test the hypothesis that B(RHP)cells convert neuropathological T cells to regulatory T cells (Tregs) in recipient mice. IFN-ã-null B(RHP)cells will not induce a conversion to Tregs, as determined by flow cytometry and in vitro co-culture assay, a population that can suppress pro-inflammatory autoreactive responses in CD4 T cells. The transfer of Tregs exposed in vitro to B(RHP)cells will also reduce acute injury and promote long-term recovery. These studies offer the potential for neurotherapeutic interventions through investigation of previously unstudied, endogenous post-stroke autoimmune responses.