Abstract 4694: Single-cell analysis of glioblastoma immune contexture identifies a subset of activated and memory tumor-reactive CD8+ TILs and a Treg signature contributing to TIL irreversible dysfunction
Authors: Sambruni Irene , Musio Silvia , Ianni Natalia Di , Maffezzini Martina , Patanè Monica , Eoli Marica , Silvani Antonio , Pollo Bianca , DiMeco Francesco , Pellegatta Serena 
Published: April 2023

Abstract
Despite the first classification of glioblastoma (GBM) as an immunologically “cold” tumor, evidence suggests that this cancer type is susceptible to T-cell infiltration, providing a glimmer of hope for developing effective immunotherapy approaches. However, whether the “exhausted” phenotype of tumor-infiltrating lymphocytes (TILs) and the immunosuppressive signaling in the GBM microenvironment (ME) can be therapeutically reprogrammed to unlock the anticancer potential of the immune system remains to be elucidated. We performed a histological analysis of GBM specimens collected pre-therapy from 24 newly-diagnosed GBM patients treated with dendritic cell (DC)-immunotherapy as part of the DENDR1 clinical trial (NCT04801147). Our preliminary results suggest an association between the distribution and density of CD8+ TIL subsets and patients’ responses to immunotherapy. TIL spatial patterns revealed either an immune-excluded or an immune-infiltrated scenario with either scattered or widespread TILs. A widespread CD8+ TIL pattern was significantly correlated with a better response to immunotherapy. It was also observed that the frequency of CD8+ TILs was significantly increased at recurrence compared to their primary tumors, suggesting that these patients are more likely to benefit from CD8+ T cell antitumor immunity. In light of these observations, we have hypothesized that tumor-reactive (TR) TILs isolated from GBM can be used as direct personalized treatment mediators exerting an effective antitumor activity with low side effects per se. TR-TILs were isolated from GBM surgical material using CD137 as a key marker, and successfully expanded from 50% of patients’ GBM showing specific reactivity against autologous tumor cells. For the remaining cases, TR-TILs failed to expand, suggesting terminal dysfunction. Single-cell sequencing of the transcriptome was performed on the immune contexture after CD137+ TILs enrichment revealing the presence of a specific signature related to exhausted, cytotoxic, and memory state. A gene signature highly upregulated in regulatory T cells (Treg) was related to a more immune-suppressive ME. Flow cytometry analysis on immune contexture confirmed the presence of FoxP3+ CD4+ T cells, expressing CD137 can be enriched and persist, dysregulating the expansion of TR-TILs during the early stage in culture. Our preliminary findings indicate that a high frequency of Treg can be investigated as a negative predictive indicator of ex-vivo expansion of TR-TILs, which should be taken into consideration when planning a personalized TIL therapy.
Citation Format: Irene Sambruni, Silvia Musio, Natalia Di Ianni, Martina Maffezzini, Monica Patanè, Marica Eoli, Antonio Silvani, Bianca Pollo, Francesco DiMeco, Serena Pellegatta. Single-cell analysis of glioblastoma immune contexture identifies a subset of activated and memory tumor-reactive CD8+ TILs and a Treg signature contributing to TIL irreversible dysfunction. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4694.