Gulbicki, Lauren

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 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 Glioblastoma (GBM) is an incurable brain cancer which is associated with severe peripheral immune suppression. Using experimental GBM, we recently determined that peripheral immunosuppression involves thymic atrophy, stunted T cell proliferation, and release of circulating factors that inhibit immune responses. RNA sequencing and proteomic studies have recently determined that transcripts and protein levels of Serpina3n are upregulated in the brain and serum of GBM patients and correlates with poor prognosis. We determined Serpina3n is a highly upregulated gene in the brain and the thymus of glioma-bearing mice using RNA sequencing technologies. We therefore sought to determine the role of Serpina3n in peripheral immunosuppression in GL261 glioma harboring wild type and Serpina3n knockout mice. Glioma implantation in Serpina3n knockout mice resulted in similar survival and peripheral immunosuppression compared to controls. Western blotting experiments confirmed that Serpina3n is expressed and secreted by GL261 gliomas isolated from WT and Serpina3n knockout mouse brains. Together this data suggests that tumor-derived, but not host derived, Serpina3n needs to be evaluated as a putative factor associated with peripheral immunosuppression. Supported by Brains Together for a Cure grant

Abstract The contribution of antiviral resident memory T cells (TRM) to blood-brain barrier disruption has not been defined despite the implication of this lymphocyte in clinical encephalitis. We therefore evaluated the capacity of TRM cells to induce CNS vascular permeability in mice which resolved Theiler’s murine encephalomyelitis (TMEV) infection. Following TMEV infection of the brain, persistent populations of virus antigen specific resident memory T cells are induced within the brain. This population of resident memory T cells can be further reactivated at 60 dpi through administration of the cognate immunodominant virus peptide antigen, VP2, inducing blood brain barrier disruption in the process. We determined that brain resident memory T cells expand following reactivation with administered VP2 peptide. This reactivation is associated with infiltration of T cells, and inflammatory myeloid cells into the brain. Importantly, using depletion and T cell sequestrating strategies with low dose anti CD8 antibodies and FTY720, we determined that TRM induced blood-brain barrier disruption is independent of peripheral T cells. We conclude that reactivation of brain TRM cells during peptide administration occurs without dependency on peripheral responses, highlighting the importance of this cell type in inducing neuroinflammation and underlying neuropathology.

Abstract Glioblastoma (GBM) is an incurable and 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 immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including T cell lymphopenia. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. These high molecular weight species potently inhibits T cell proliferation in vitro despite the presence of strong T cell stimulation. Our goal was to determine the identity of the immunosuppressive species in serum of glioma-bearing mice. We performed state-of-the-art comparative high throughput mass spectrometry on fractionated and multiplexed sera isolated from control and glioma-bearing mice. This proteomics analysis demonstrated histones as a top highly abundant protein in serum of glioma-bearing mice compared to naïve controls. Cell-free DNA has been recently identified as an immune suppressive factor in circulation and can be bound to histones. We, therefore, assessed cell-free DNA levels in the serum of glioma-bearing mice. We determined that sera isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Reducing the DNA content in serum, using DNAse treatment, partially improved the defect in T cell proliferation observed when T cells are exposed to serum isolated from glioma-bearing mice. These data indicate that heighted levels of circulating DNA in serum contributes to peripheral immunosuppression in GBM. These findings provide a tractable approach to investigate the cellular source and immunosuppressive effects of cell-free DNA in murine models of GBM and to devise strategies to reverse immunosuppression with the hope of improving outcomes in patients.