Citations

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with poor survival despite standard of care. Checkpoint inhibitors aimed to revive dysfunctional T cells have significantly improved outcomes in other cancers, but have failed GBM. GBM patients have severe peripheral immunosuppression, including severe lymphopenia, immune organ atrophy, and defective T cell responses. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Using parabiosis, we demonstrated that serum-derived factors were sufficient to drive hallmark features of immunosuppression, including inhibition of ex vivo T cell proliferation. This factor was nonsteroidal in nature and had molecular weights greater than 100kDa. Mass-spectrometry and pathway analysis of the suppressive serum implicated proteins involved in DNA response elements, cell death, and DNA-histone complexes. We hypothesized that the factor is cell-free DNA (cfDNA). Confirming this, serum levels of cfDNA are increased in glioma-bearing mice. Moreover, exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation ex vivo. Interestingly, exposure to genomic DNA was not sufficient to induce proliferation defects in T cells, highlighting the unique features of cfDNA. Importantly, reducing cfDNA content using DNaseI partially restored proliferation capacity. We determined that the T cell proliferation defects were independent of DNA sensing mechanisms through AIM2, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we employed methyl-seq. The chromatin signature most closely represented neutrophils. This highlights a potential role for neutrophils-derived cfDNA as a targetable strategy in GBM. Together, we contend that cfDNA induces immunosuppressive effects in GBM and devising strategies to reduce circulating cfDNA could be a therapeutic approach to improve T cell functions and outcomes in GBM patients.

Abstract Although the functions of tyrosine phosphatases in T‐cell biology have been extensively studied, our knowledge on the contribution of serine/threonine phosphatases in T cells remains poor. Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine phosphatases. It is important in thymocyte development and CD4 + T‐cell differentiation. Utilizing a genetic model in which its catalytic subunit alpha isoform (PP2A Cα) is deleted in T cells, we investigated its contribution to CD8 + T‐cell homeostasis and effector functions. Our results demonstrate that T‐cell intrinsic PP2A Cα is critically required for CD8 + T‐cell homeostasis in secondary lymphoid organs and intestinal mucosal site. Importantly, PP2A Cα–deficient CD8 + T cells exhibit reduced proliferation and survival. CD8 + T‐cell antibacterial response is strictly dependent on PP2A Cα. Expression of Bcl2 transgene rescues CD8 + T‐cell homeostasis in spleens, but not in intestinal mucosal site, nor does it restore defective antibacterial responses. Finally, proteomics and phosphoproteomics analyses reveal potential targets dependent on PP2A Cα, including mTORC1 and AKT. Thus, PP2A Cα is a key modulator of CD8 + T‐cell homeostasis and effector functions.

Abstract Glioblastoma (GBM) is aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Immunocompetent mice harboring intracranial gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. Using parabiosis, we determined that T cell lymphopenia can be transferred from glioma-bearing to healthy mice via circulation This led to the identification of a high molecular weight immunosuppressive factor in sera of glioma-bearing mice that inhibits T cell proliferation ex vivo. Proteomics analysis identified 119 proteins uniquely upregulated in the sera of glioma-bearing mice compared to controls. These proteins are associated with pathways involved in DNA response elements, cell death, and DNA-histone complexes which imply DNA release. Additional analysis revealed serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded cell free DNA (cfDNA) compared to controls. Reducing DNA content using DNase partially reverses the proliferation defects observed in T cells when exposed to serum of glioma-bearing mice. This is while, addition of cfDNA purified from serum of glioma-bearing mice, but not genomic DNA, potently inhibited T cell function. These results were AIM2 independent implicating a novel pathway of cfDNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reduce circulating cfDNA is therefore a therapeutic approach that could improve outcomes in GBM patients.

Abstract Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood–brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood–brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Abstract Bone marrow is a primary immune organ responsible for stem cell maintenance and hematopoiesis. While the bone marrow niche is mainly studied in the context of hematopoiesis, aging, and mechanisms underlying stem cell and plasma cell survival, changes of the niche during brain viral infections and primary T cell responses is unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared 30 days post infection due to generation of an antigen specific CD8 T cell response raised against the immunodominant epitope VP2121–130. We determined that 7–10% of CD8 T cells present in the sternal and femoral bone marrows and 15–20% in the cranial bone marrow were antigen specific 7 days post intracranial TMEV infection. Over 70% of these CD8 T cells were located outside of the vasculature as determined by IV labeling. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen specific CD8 T cells in any bone marrow compartment. CD8 T cells in the bone marrow established durable memory following viral clearance and were reactivated upon cognate antigen reencounter. Both acute TMEV infection and antigen specific reactivation caused an increase in lineage−, Sca-1+, ckit+ (LSK) cells in the bone marrow. This LSK dysregulation was abrogated upon CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Both acute and memory recall CD8 T cell responses cause stem cell dysregulation. Our data hence paves the way for crucial studies of bone marrow resident antigen specific CD8 T cells in health and disease. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Abstract Cerebral malaria (CM) is a severe complication of P. falciparum infection and a leading cause of death, globally. Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice serves as a murine model of CM called experimental cerebral malaria (ECM) due to the neurological and neuropathological features that simulate the infection of human CM. A putative mechanism of disease in human CM requires CD8 T cell to infiltration into the brain and blood-brain barrier disruption. The specific antigen-presenting cell type(s) that prime this CNS infiltrating CD8 T cell response capable of inducing lethal blood-brain barrier disruption has not been defined. To answer this, single class I expressing Kb loxP transgenic mice were generated. Crossing these mice to class I deficient CD11c-cre or LysM-cre mice created the LysM-cre Kb cKO and CD11c-cre Kb cKO mice, respectively. PbA infected Kb loxP and LysM-cre Kb cKO were susceptible to ECM. In contrast, CD11c-cre Kb cKO mice survived acute PbA infection and avoided neurological manifestations. We therefore employed spectral flow cytometry to analyze splenocytes and brain infiltrating immune cells during acute PbA infection in these genotypes. We observed a reduction in activated CD8 T cells in the spleens of PbA infected CD11c-cre Kb cKO mice versus the Kb loxP mice and the LysM-cre Kb cKO. This reduced peripheral CD8 T cell activation in PbA infected CD11c-cre Kb cKO mice also corresponded with reduced brain infiltration of CD8 T cells. We conclude a CD11c+ APC is therefore required for priming and potentially recruitment of CD8 T cells to the brain during lethal ECM pathology. 5R01NS103212-06

Abstract Glioblastoma (GBM) is an incurable brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. C57BL/6 mice harboring GL261 gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. We determined that peripheral immunosuppression involves release of non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. Sera of glioma-bearing mice inhibits T cell proliferation ex vivo. To determine the identity of the immunosuppressive molecules in serum, we used two parallel approaches: 1.) evaluation of abundant macromolecules and 2.) proteomics analysis using tandem mass tag mass-spectrometry (TMT-MS). We determined that serum of glioma-bearing mice contains significantly higher levels of cell free DNA (cfDNA) compared to controls. TMT-MS identified histones as a highly abundant protein in serum, further implicating DNA-histone complexes as top candidates. DNAse treatment of sera isolated from GL261 glioma-bearing mice reduced the suppressive effect on T cell proliferation. We next performed T cell proliferation assays in the presence of pure cfDNA isolated from sera of glioma-bearing mice while genomic DNA was used as control. cfDNA from serum potently inhibited T cell proliferation while control genomic DNA did not. These results were AIM2 independent implicating a novel pathway of DNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reverse immunosuppression through targeting cfDNA could improve outcomes in GBM patients. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ) and Brains Together for a Cure Foundation (KA).

Abstract Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, multiple sclerosis, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy using mice with a C57BL/6 background. The H-2DbClass I molecule not only contributed to significantly more ventricular enlargement in a T2-weighted MRI at 45 d.p.i., but this ventricular enlargement correlated to CD8 T cell infiltration. To further define the cellular and molecular mechanisms of MHC class I molecules in TMEV induced brain atrophy we investigated the cell specific contribution of brain resident antigen presenting cells (APCs) in ventricular atrophy. We developed novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells. Cx3CR1cre+/Db Lox P mice presented with significantly less ventricular atrophy at 45 d.p.i., compared to cre- littermates along with a decrease in CD8 T cell infiltration. These results strongly imply that antigen presentation by H-2Db on myeloid cells, at least in part is responsible for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. NS103212, NS122174

Abstract Intracranial infection with neurotropic Theiler’s Murine Encephalomyelitis Virus induces robust generation of brain TRMs against the immunodominant antigen, VP2121–130, following viral clearance in C57BL/6 mice. While the presence of TRMs in the brain is documented, neurological consequences of TRM reactivation and their causal role in neuropathology remain understudied. We disabled peripheral CD8 T cells via anti CD8 depleting strategies and FTY720 treatment to determine the causal link between brain TRM reactivation and neuropathology. We depleted peripheral T cells and then administrated VP2 peptide IV to reactivate TRMs 60–200 days post infection as this elegant technique isolates responses of brain TRMs away from other branches of memory. We used state-of-the-art flow cytometry and MRI analysis to evaluate neurological consequences of TRM reactivation. TRM reactivation induced severe neuroinflammation in the brain as evident by infiltration of myeloid cells and blasting of T cells. MRI detected gadolinium leakage marking severe blood-brain-barrier (BBB) disruption when brain TRMs were reactivated. Depletion of peripheral CD8 T cells with low-dose anti CD8 antibodies or their sequestration via FTY720 did not alter neuroinflammation indicating brain TRM reactivation causally initiates neuropathology. Confirming this, tissue depletion of all TRMs in the brain using high-dose anti CD8 antibodies prevented both neuroinflammation and neuropathology. Hence, TRM reactivation causes severe neuroinflammation and BBB disruption from within the brain independently of peripheral T cells. Targeting brain TRM reactivation is a novel potential therapeutic for patients with many acute and chronic neurological diseases. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Abstract The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)–cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)–cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.