Regulatory T cells underlies resistance to radio-immunotherapy, prevent the formation of tertiary lymphoid structures and impair CD8 T cell activation in glioblastoma

Glioblastomas (GBM) are aggressive primary brain tumors with an inherent resistance to T cell-centric immunotherapy imparted to their low mutational burden and immunosuppressive tumor microenvironment (TME). Examination of the GBM TME dynamics following fractionated RT revealed a 10-fold increase in T cell content, an enrichment also observed by spatial imaging mass cytometry in matched primary and recurrent human GBM. Implementation of a-PD-1 treatment at the peak of T cell infiltration results in a modest, albeit distinct survival benefit compared to concurrent a-PD-1 administration in GBM-bearing mice. CD103+ Tregs presenting increased cholesterol pathway activity are recruited to the GBM TME in response to a-PD-1 therapy, suggestive of their ability to restrain the response to immune checkpoint blockade. Targeting of Tregs elicits the formation of tertiary lymphoid structures, enhanced CD8 T cell content and activation and enables the therapeutic efficacy of radio-immunotherapy. These results support the rational design of therapeutic regimens limiting the induction of immunosuppressive feedback pathways in order to unleash the efficacy of T-cell centric immunotherapy in glioblastoma. Overall design: In light of recent studies underlining that not only the content of T cells but also their education profiles segregate ICB responders and non-responders, we performed in-depth investigation of the T cells transcriptional changes occurring in response to radio-immunotherapy in the concurrent or adjuvant setting. We FACS-purified CD8+ and CD4+ T cells from PDG-Ink4a GBM at day 12 post treatment initiation, a timepoint where T cell infiltration is at its peak, and subjected these cells to RNA sequencing.