Anani-Wolf, Delaney

Abstract Introduction Bone marrow (BM) is the primary immune organ responsible for stem cell maintenance. Neurotropic infections elicit sequestration of T cells within the BM. Changes of the BM niche during brain viral infections are unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) in C57BL/6 mouse is cleared by 30 days due to generation of an antigen-specific CD8 T cell response. Methods We used brain viral infection and injury models to test T cell responses against brain antigens in the bone marrow. Results We determined that 1-10% of CD8 T cells present in the sternal, femoral, and cranial BM were virus antigen-specific 7 days post intracranial infection. Over 70% of these CD8 T cells were tissue resident. We determined antigen-specific CD8 T cells were generated in the BM within 4 days of infection. Continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in any BM compartment. CD8 T cells in the BM established durable memory populations and were reactivated upon antigen reencounter. In the memory phase (200 days post brain TMEV infection), bone marrow resident CD8 T cell could be reactivated upon cognate peptide delivery. Such reactivation of antigen specific memory CD8 T cells caused an increase in lineage-, Sca-1+, ckit + (LSK) cells in the BM in a CD8 dependent manner. Similarly, injection of the model antigen AF488-conjugated OVA mixed with Poly IC into the brain recapitulated antigen specific CD8 T cell generation within the bone marrow and further demonstrated the antigen presenting cell involved in the response to be a CD103 expressing myeloid DC. We conclude that viral infections and inflammatory stimulation in the brain induce in situ effector and memory T cell responses within the BM which is associated with significant stem cell dysregulation. Conclusion Our data paves the way for crucial studies of BM resident antigen-specific CD8 T cells in health and disease. Funding Source NIH K99/R00 Topic Categories Viral Immunology (VIR)

Abstract Introduction Glioblastoma is an aggressive brain cancer with poor prognosis for which immunotherapies have fared poorly. In part, the failure in immunotherapies is attributed to the existence of severe local and systemic immunosuppression in GBM. We determined that majority of hallmark features of systemic immunosuppression were linked to immunosuppressive factors in serum. Our data indicate that serum of glioma-bearing mice harbors heightened levels of cell-free DNA (CF-DNA). Methods We evaluated the extent to which CF-DNA contributes to systemic T cell dysfunction in mouse models of GBM and GBM patient plasma samples. Results We determined that majority of hallmark features of systemic immunosuppression were linked to immunosuppressive factors in serum. Our data indicate that serum of glioma-bearing mice harbors heightened levels of cell-free DNA (CF-DNA). Removing CF-DNA from serum improves T cell responses while adding CF-DNA derived from serum of glioma-bearing subjects to healthy T cells directly inhibits their functions. We have also verified that CF-DNA is detectible in sera isolated from patients with GBM. When compared to healthy controls, GBM patients have higher quantities of CF-DNA is their plasma. Interestingly, our preliminary data implicates neutrophil NETosis as the likely source of CF-DNA in sera of GBM patients. Moreover, we stained GBM tumor sections with citrullinated histone 3 (cit-H3) to evaluate the extent of NETosis in GBM patients. We determined evidence of significant NETosis within patient brain tumors. Systemic immunosuppression is detrimental to the success of immune-modulating therapies and patient survival. By identifying and removing the source of systemic immunosuppression, we will enhance immune responses against the tumor likely improving patient survival. Conclusion Glioblastoma causes immune suppression outside of the brain via the released CF-DNA. Our studies in GBM patients and mouse models help develop strategies to reverse immune dysfunction and improve outcomes. Funding Source NIH K99/R00 Topic Categories Immune Mechanisms of Human Disease (HUM)

ABSTRACT Brain atrophy is a common feature of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and more recently SARS‐CoV‐2 infection for which the neuroinflammatory mechanism is not fully understood. Nevertheless, neuroinflammation and CD8 T cell accumulation is frequently observed in postmortem brain tissue of patients with neurodegeneration. We therefore developed a murine model of brain atrophy using the Theiler's murine encephalomyelitis virus (TMEV) infection model of multiple sclerosis. We employ single major histocompatibility complex (MHC) class I molecule conditional knockout mice generated by our program to analyze the contribution of immune cell infiltration and onset of atrophy. TMEV infected C57BL/6 mice that singularly express H‐2K b or H‐2D b were evaluated. TMEV infection of these mice resulted in ventricular atrophy at 14 d.p.i. However, H‐2D b expressing mice presented with significantly greater and continuous ventricular atrophy compared to H‐2K b expressing mice and MHC class I knockout control mice. Flow cytometric analysis revealed H‐2D b expressing mice had greater brain infiltrating CD8 T cell responses at 7 and 28 dpi which correlated with the extent of ventricular atrophy. Meanwhile, H‐2K b and MHC class I knockout mice also had distinct positive correlations with ventricular atrophy and global neuroinflammation in general. These findings support the role of immune cell infiltration into the CNS as a putative mechanism of ventricular atrophy following TMEV infection, with the progressive enlargement of ventricular atrophy observed in H‐2D b mice being associated with the presence of long‐lived memory CD8 T cells residing in the brain.