Johnson, Aaron

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.