Forston, Michael D.

Abstract Introduction The anatomical locations of generating adaptive immune responses to neurotropic viruses are still being investigated. Skull bone marrow (BM) has recently been identified as a pivotal immune site that can sense and respond to a local insult in the brain. Yet, there is a lack of understanding of the role of skull BM during viral encephalitis. We hypothesized that the skull BM is an important niche for antiviral CD8 T cells priming, expansion, and maintenance during Theiler’s murine encephalomyelitis virus infection. Methods Using peptide:MHC tetramer analysis, we determined that antiviral CD8 T cells are primed in the skull BM in early infection, at time points comparable to cervical lymph nodes. Employing MHC class I conditional knockout mice generated by our research group, we inactivated H-2Kb and H-2Db class I molecules on discrete antigen-presenting cell types. Results We determined using these transgenic mice that viral antigen presentation on dendritic cells (DCs) and endothelial cells is essential for the generation of virus-specific T cells in skull BM during acute viral encephalitis. However, antigen presentation by DCs is not required for long-lived antiviral CD8 T cells that reside in skull BM. Moreover, the use of FTY720 to block lymphocyte migration diminished skull antiviral CD8 T cell numbers, suggesting that peripheral trafficking is required for generating skull antiviral CD8 T cells. Conclusion We present compelling evidence for the importance of skull BM as a responsive site that is highly suitable for rapid generation of antiviral CD8 T cells during acute viral encephalitis. Skull BM also serves as a reservoir for memory T cells, long after the resolution of a neurotropic virus infection. This work sheds light on the skull as a potential target for further modulation of the immune response to viral infections in the brain. Funding Source NIH grants R01 NS5103212 and RF1 NS122174 Topic Categories Viral Immunology (VIR)

Abstract The impact of microglia antigen presentation on CNS infiltrating CD8 T cells responses during neurotropic virus infection remains undefined. Using Theiler’s murine encephalomyelitis virus (TMEV) infection of neurons as a model system, we generated H-2K b and H-2D b Tmem119 conditional knockout mice. During TMEV infection, we determined using Tmem119 K b cKO mice that microglia H-2K b contributes to antiviral CD8 T cell proliferation in the brain. Meanwhile, using Tmem119 D b cKO mice we determined that microglia H-2D b increased antiviral CD8 T cell perforin levels and the capacity to induce blood-brain barrier (BBB) disruption. Furthermore, microglial H-2D b was essential for antigen-specific reactivation of CD8 tissue resident memory (TRM) cell recall responses and BBB disruption long after virus had been cleared. These findings demonstrate discrete roles of individual MHC class I genes on microglia in CD8 T cell proliferation, effector functions, memory recall and neuropathology.

Abstract Skull bone marrow (BM) is a recently identified site for immune activation responsive to CNS insults. The extent to which adaptive immunity is generated through skull BM is poorly understood. In this study, we evaluated how skull BM supports the early expansion of antiviral CD8 T cells during acute neurotropic virus infection. Virus antigen-specific CD8 T cells were rapidly identified in skull BM at a rate comparable to lymphoid compartments during acute Theiler’s murine encephalomyelitis viral (TMEV) infection. We therefore employed MHC class I conditional knockout mice and demonstrated the necessity of CD11c+ antigen-presenting cells (APCs) for the generation of various subsets of TMEV-specific CD8 T cells in skull BM. Importantly, the early expansion of skull BM-derived antiviral T cells was impeded by FTY720 treatment and restored upon shared circulation with a mouse exhibiting intact antigen presentation by CD11c+ APCs. Additionally, TMEV-specific CD8 T cells were retained in skull BM during both chronic CNS infection and long after infection has been resolved. Overall, we determined that skull BM responds to acute neurotropic viral infection through the rapid recruitment of virus-specific CD8 T cells primed in the periphery before serving as a reservoir for immunologic memory.

Abstract The impact of microglia antigen presentation on CNS infiltrating CD8 T cells responses during neurotropic virus infection has been difficult to define. Using Theiler’s murine encephalomyelitis virus (TMEV) infection of neurons as a model system, our laboratory has previously determined that H-2D b restricted, but not H-2K b restricted CD8 T cells are required for viral clearance, demonstrating the role of discrete MHC class I alleles. To determine the extent microglia antigen presentation impacts brain-infiltrating CD8 T cells, our laboratory generated novel single MHC class I conditional knockout mice in which H-2K b or H-2D b can be specifically deactivated in TMEM119+ microglia with tamoxifen administration. During TMEV infection, conditional knockout of H-2K b in microglia reduced antigen-specific CD8 T cell proliferation in the brain. Meanwhile, mice with deletion of D b in microglia had reduced levels of perforin in antigen-specific CD8 T cells. Furthermore, microglia specific deletion of H-2D b reduced CD8 T cell numbers in the brain and preserved blood-brain barrier (BBB) integrity. Microglial D b restricted antigen presentation was also essential for the reactivation of CD8 tissue resident memory (TRM) cells and BBB integrity during memory recall responses. These findings further our understanding of how brain infiltrating antiviral CD8 T cell responses are impacted by microglia, as well as define how this cellular interaction contributes to BBB disruption during neuroinflammation. These findings also have high significance to our understanding of how microglia impact CD8 TRM cell populations that reside in the brain long after virus infection is cleared. Graphical abstract