Jin, Fang

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti—programmed cell death protein 1 (anti–PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti–PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I–restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I–independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

Abstract Immune organ innervation by the peripheral nervous system enables autonomic function in the development and maintenance of immunity. Specifically, the role of thymic innervation in health and following neurological insults is unknown. An in depth identification of the connectome within the thymus and the extent to which nervous impulses delivered to the thymus affect T cell development and other biological processes within the thymus is needed. We sought to determine the exact nature of innervation within the thymus. Using both flow cytometry and confocal microscopy, we determined that the thymus contains both extrinsic neuronal processes (cell bodies outside of the thymus), and surprisingly, intrinsic neurons (cells bodies within the thymus). We further verified that subsets of neurons within the thymus express tyrosine hydroxylase, BIII tubulin, and/or NeuN, and are distinct from epithelial cells (EPCAM+, UEA1+, Ly51+) and tuft cells (DLCK1+). Furthermore, neuronal cell bodies found within the thymus did not express AIRE, consistent with their identity as neurons. Subsets of intrinsic neurons within the thymus express MHCII implying a potential role in T cell development. Importantly, using rabies virus retrograde and anterograde neurotracers, we mapped a novel, previously undescribed, connectome of neurons within the thymus. Finally, we determined that both epithelial cells and neurons are significantly reduced in RAG deficient mice, indicating a possible co-regulation between thymic neurons and epithelial cells. In summary, we describe a novel network of neurons within the thymus and put forward implications of the thymic connectome’s role in T cell development and establishment of thymic architecture.

Abstract Immune-mediated blood brain barrier (BBB) disruption is a prominent feature of various neurological conditions for which an emerging role of CD8 T cells is being realized. Our group has developed a unique model of CD8 T cell-mediated BBB disruption which employs Theiler’s murine encephalomyelitis virus (TMEV) infection. Upon intravenous administration of VP2121–130 viral peptide during the peak antiviral adaptive response, Db:VP2121–130 epitope specific CD8 T cells induce BBB disruption in a perforin dependent manner. In this model, we addressed the role of microglia in relation to antigen specific CD8+ T cells and permeable cerebral vessels. Using real time two-photon in vivo imaging, we demonstrate that microglia adopt distinct morphological features, including enlarged cell body and fewer ramified processes as early as 6 hours post administration of VP2 peptide. Notably, microglial population exhibit robust down-regulation of homeostatic markers including CX3CR1 during BBB disruption. This study demonstrates CD8 T cells can promote microglia activation in a perforin-dependent manner during BBB disruption. Importantly, microglial activation occurs concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.

Abstract Cytotoxic T cell infiltration into the central nervous system (CNS) is required for clearance of pathogen or tumor. Whether microglia, the resident innate immune cells of the brain, are involved in orchestrating CD8 T cell infiltration of the brain in an antigen dependent manner remains to be elucidated. We hypothesized that antigen presentation by major histocompatibility complex (MHC) class I molecules expressed by microglia is required to orchestrate antigen specific CD8 T cell infiltration into the brain parenchyma. To test this hypothesis, we generated a novel tamoxifen-inducible microglial MHC class I conditional knockout mouse (CX3CR1-creERT2H-2Kb cKO). In these mice, administration of tamoxifen mediates deletion of the H-2Kbmolecule in cells expressing CX3CR1. Short-lived peripheral CX3CR1+ cells repopulate within 4–6 weeks after tamoxifen treatment and restore expression of H-2Kb, leaving only long-lived microglia deficient for MHC class I. We challenged these mice with intracranial infection of Theiler’s murine encephalomyelitis virus expressing the model antigen ovalbumin (TMEV-OVA) to assess antigen specific CD8 T cell responses within the brain parenchyma. In microglia MHC I cKO mice, the frequency of brain infiltrating OVA-specific CD8 T cells was attenuated while the frequency of intravascular OVA-specific CD8 T cells was increased when compared to cre− littermate controls. These results suggest that microglial antigen presentation is important for antigen specific CD8 T cell infiltration into the brain parenchyma. These studies directly address the critical role of microglial MHC class I on antigen specific CD8 T cell responses to an in vivo neuroinflammatory insult.

Abstract Brain tissue resident memory T cells (TRM) are an emerging lymphocyte of interest. While the phenotype and gene expression pattern of brain TRMs have recently been discussed, their function and response to neurologic insults is not yet understood. Using the described phenotype of TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we analyzed the reaction of this lymphocyte in the brain to various neurological insults. We found that physical insult induced by an intracranial(ic) injection of PBS resulted in increased TRM numbers within 24 hours post insult. This TRM population later decreased yet remained above baseline by 50 days post insult. Interestingly, TRM populations also increased in the brains of animals as a function of age, suggesting natural insults experienced by mice leads to an ever increasing TRM population in the brain. We then assessed the reaction of TRMs to virus in the brain. Mice infected ic with Theiler’s Murine Encephalomyelitis virus (TMEV) had a marked increase in brain TRMs one day later, which is 4 days prior to detection of traditional TMEV antigenspecific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. Finally, we investigated the role of dendritic cell (DC) antigen presentation in generation of TRMs using our novel MHC class I conditional knockout mice. Conditional ablation of H-2Kb and H-2Db did not disrupt TRM populations in the brain. Our data is the first study to show: 1. Activation of TRMs in the brain preceded antigen specific cell infiltration and 2. MHC class I molecules on DCs are not required to generate brain resident TRMS. Understanding brain TRMs is crucial in elucidating their role in neurodegeneration and/or targeting them in CNS cancers.

Background— A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects. Methods and Results— Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in Ca V 1.2 and revealed a complex phenotype, including loss of current density and inactivation in combination with increased window and late current. Conclusions— Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype.