B cell subsets with phenotypes characteristic of naive, non-isotypeswitched, memory (B mem ) cells and antibody-secreting cells (ASC) accumulate in various models of central nervous system (CNS) inflammation, including viral encephalomyelitis. During neurotropic coronavirus JHMV infection, infiltration of protective ASC occurs after T cell-mediated viral control and is preceded by accumulation of non-isotype-switched IgD ϩ and IgM ϩ B cells. However, the contribution of peripheral activation events in cervical lymph nodes (CLN) to driving humoral immune responses in the infected CNS is poorly defined. CD19, a signaling component of the B cell receptor complex, is one of multiple regulators driving B cell differentiation and germinal center (GC) formation by lowering the threshold of antigen-driven activation. JHMV-infected CD19 Ϫ/Ϫ mice were thus used to determine how CD19 affects CNS recruitment of B cell subsets. Early polyclonal ASC expansion, GC formation, and virus-specific ASC were all significantly impaired in CLN of CD19 Ϫ/Ϫ mice compared to wild-type (WT) mice, consistent with lower and unsustained virus-specific serum antibody (Ab). ASC were also significantly reduced in the CNS, resulting in increased infectious virus during persistence. Nevertheless, CD19 deficiency did not affect early CNS IgD ϩ B cell accumulation. The results support the notion that CD19-independent factors drive early B cell mobilization and recruitment to the infected CNS, while delayed accumulation of virus-specific, isotype-switched ASC requires CD19-dependent GC formation in CLN. CD19 is thus essential for both sustained serum Ab and protective local Ab within the CNS following JHMV encephalomyelitis. IMPORTANCE CD19 activation is known to promote GC formation and to sustain serum Ab responses following antigen immunization and viral infections. However, the contribution of CD19 in the context of CNS infections has not been evaluated. This study demonstrates that antiviral protective ASC in the CNS are dependent on CD19 activation and peripheral GC formation, while accumulation of early-recruited IgD ϩ B cells is CD19 independent. This indicates that IgD ϩ B cells commonly found early in the CNS do not give rise to local ASC differentiation and that only antigen-primed, peripheral GC-derived ASC infiltrate the CNS, thereby limiting potentially harmful nonspecific Ab secretion. Expanding our understanding of activation signals driving CNS migration of distinct B cell subsets during neuroinflammatory insults is critical for preventing and managing acute encephalitic infections, as well as preempting reactivation of persistent viruses during immune-suppressive therapies targeting B cells in multiple sclerosis (MS), such as rituximab and ocrelizumab.

Recent studies using advanced techniques such as single cell RNA sequencing (scRNAseq), high parameter flow cytometry, and proteomics reveal that neutrophils are more heterogeneous than previously appreciated. Unique subsets have been identified in the context of bacterial and parasitic infections, cancer, and tissue injury and repair. The characteristics of infiltrating neutrophils differ depending on the nature of the inflammation-inciting stimulus, the stage of the inflammatory response, as well as the tissue microenvironment in which they accumulate. We previously described a new subpopulation of immature Ly6Glow neutrophils that accumulate in the peritoneal cavity 3 days following intraperitoneal (i.p.) administration of the fungal cell wall extract, zymosan. These neutrophils express markers of alternative activation and possess neuroprotective/regenerative properties. In addition to inducing neurite outgrowth of explanted neurons, they enhance neuronal survival and axon regeneration in vivo following traumatic injury to the optic nerve or spinal cord. In contrast, the majority of neutrophils that accumulate in the peritoneal fluid 4 hours following i.p. zymosan injection (4h NΦ) have features of conventional, mature Ly6Ghi neutrophils and lack neuroprotective or neuroregenerative properties. In the current study, we expand upon on our previously published observations by performing a granular, in-depth analysis of these i.p. zymosan-modulated neutrophil populations using scRNAseq and high parameter flow cytometry. We also analyze cell lysates of each neutrophil population by liquid chromatography/mass spectrometry. Circulating blood neutrophils, harvested from naive mice, are analyzed in parallel as a control. When samples were pooled from all three groups, scRNAseq revealed 11 distinct neutrophil clusters. Pathway analyses demonstrated that 3d NΦ upregulate genes involved in tissue development and wound healing, while 4h NΦ upregulate genes involved in cytokine production and perpetuation of the immune response. Proteomics analysis revealed that 3d NΦ and 4h NΦ also express distinct protein signatures. Adding to our earlier findings, 3d NΦ expressed a number of neuroprotective/neuroregenerative candidate proteins that may contribute to their biological functions. Collectively, the data generated by the current study add to the growing literature on neutrophil heterogeneity and functional sub-specialization and might provide new insights in elucidating the mechanisms of action of pro-regenerative, neuroprotective neutrophil subsets.

Multiple sclerosis (MS), a neuroinflammatory disease that affects millions worldwide, is widely thought to be autoimmune in etiology. Historically, research into MS pathogenesis has focused on autoreactive CD4 T cells because of their critical role in the animal model, experimental autoimmune encephalomyelitis, and the association between MS susceptibility and single-nucleotide polymorphisms in the MHC class II region. However, recent studies have revealed prominent clonal expansions of CD8 T cells within the CNS during MS. In this paper, we review the literature on CD8 T cells in MS, with an emphasis on their potential effector and regulatory properties. We discuss the impact of disease modifying therapies, currently prescribed to reduce MS relapse rates, on CD8 T cell frequency and function. A deeper understanding of the role of CD8 T cells in MS may lead to the development of more effective and selective immunomodulatory drugs for particular subsets of patients.

The prevalence and role of antigen-specific Bmem in the CNS during viral encephalomyelitis is largely undefined. A lack of reliable markers identifying murine Bmem has made it difficult to assess their contribution to local antiviral protection via antigen presentation or conversion to ASC. Using reporter mice infected with neurotropic coronavirus to track virus-specific Bmem and ASC, this report demonstrates that both subsets only emerge in the CNS following peripheral GC formation and subsequently prevail. While early GC reactions supported preferential Bmem accumulation in the CNS, late GC reactions favored ASC accumulation, although Bmem outnumbered ASC in draining lymph nodes throughout infection. Importantly, virus-specific B cells undergoing sustained GC selection were continually recruited to the persistently infected CNS. Elucidating the factors governing temporal events within GCs, as well as regional CNS cues during viral persistence, will aid intervention to modulate CNS humoral responses in the context of infection and associated autoimmune pathologies.