B cells alter adaptive autoimmunity to protect from ischemic injury after stroke

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 DESCRIPTION (provided by applicant): More than 800,000 people in the U.S. will suffer from stroke each year, yet there remains only one FDA- approved acute intervention. Clinical trials to minimize post-stroke inflammation failed, highlighting the need to better understand inflammatory and neuroprotective mechanisms within the central nervous system (CNS). Repetitive hypoxic preconditioning (RHP) naturally protects from stroke for months beyond treatment, minimizing infarct volumes, blood-brain barrier (BBB) disruption, and monocyte, T cell, and neutrophil diapedesis into the ischemic brain. In contrast, RHP specifically induces an immunosuppressed B cell phenotype that is enhanced in the injured CNS of preconditioned mice following stroke. The adoptive transfer of RHP-treated B cells (B(RHP)cells) 6h after transient stroke in male mice reduced infarct volumes and improved functional recovery compared to PBS- or wild type (WT) B cell-treated controls. B(RHP)cell therapy also suppressed a post-stroke autoimmune response to CNS-derived neuronal and myelin antigen (Ag), a novel finding for adaptive autoimmunity that has only been studied in preclinical models of stroke 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), a cytokine that promotes neuronal repair, as well as interferon (IFN)-γ, a cytokine that converts effector (i.e. pro-inflammatory, Th1) T cells to regulatory T cells (Tregs) that suppress other neuropathological immune cells. Specific Aim 1 will test the hypothesis that B cells confer acute neuroprotection through the production of IL10 and direct neuronal interaction. We will confirm, using state-of-the-art 3-D serial two-photon tomography for whole brain imaging, MRI, and behavioral assays, that IL10- enhanced B(RHP)cells colocalize to a higher number of surviving neurons in the ischemic hemisphere to improve long-term functional recovery. We will also confirm the role of B(RHP)cell-derived IL10 on neuronal viability and axon outgrowth in vitro following oxygen-glucose deprivation (OGD), with B(RHP)cells harvested from IL10-null and WT mice. Specific Aim 2 will test the hypothesis that B(RHP)cells from WT but not IFN-γ-null mice will induce a novel Treg population to suppress pro-inflammatory autoreactive responses from Th1 cells. The transfer of Tregs exposed in vitro to B(RHP)cells will also promote long-term neurovascular and functional recovery (Aim 1A methods), in addition to suppressing CNS-derived autoreactivity. Finally, Glatiramer Acetate, or Copaxone(r), is an FDA-approved drug that upregulates B cell-derived IL10 and Treg conversion in patients with multiple sclerosis. Specific Aim 3 will test the hypothesis that WT B cells treated with Copaxone will upregulate IL10 and IFN-γ to phenocopy B(RHP)cell-mediated protection. We will test the efficacy of Copaxone-treated B cells on neuronal viability in vitro following OGD (Aim 1B methods). Copaxone- or control Betaseron-treated B cells will be adoptively transferred post-stroke to determine efficacy on long-term recovery (Aim 1A methods).