Circulating cell-free DNA directly inhibits T cell function and contributes to peripheral immunosuppression in experimental GBM

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).