P04.04 Ex vivo expansion of human glioma-infiltrating lymphocytes alters the exhaustion phenotype of T cells

摘要

Immunotherapy for brain tumors is entering the clinical arena with novel immunotherapeutic strategies which potentiate T cell responses targeting tumor antigens (TA). Consequently, there is increasing interest in identifying and characterizing tumor antigen-specific tumor-infiltrating T lymphocytes (TILs) reactive to tumor antigens for further tumor immunotherapy such as adoptive T cell therapy. T cells typically upregulate exhaustion markers such as PD-1 after antigen-experience. Protocols for selecting TA-specific T cells from the periphery hence make use of these exhaustion markers to select for relevant antigen-experienced T cells. Commonly used protocols for the isolation of TILs make use of an ex vivo expansion of TILs in order to achieve reasonable T cell numbers for further analysis. Whether this expansion affects the phenotype of TILs, however, remains unclear. In this study, we investigated the influence of a commonly used ex vivo expansion protocol for the isolation of human glioblastoma (GBM)-infiltrating T lymphocytes on their phenotype. This protocol involved ex vivo expansion of TILs by culturing tumor fragments with complete medium supplemented with IL-2 and CD3 ligand, whereby TILs were allowed to migrate out of the tumor tissue and further expanded using IL-2 for two weeks. We demonstrate that the CD8/CD4 T cell ratio shifts dramatically after ex vivo expansion of GBM TILs towards a CD4 dominance, when compared to freshly isolated unperturbed TILs. Furthermore, ex vivo expansion of GBM TILs resulted in an increase of LAG-3+ and TIM-3+ CD8 and CD4 T cells, and the frequency of PD-1+ CD8 T cells decreased considerably after ex vivo expansion, whereas a greater subset of the PD-1+eomes+ CD8 T cell population indicated expression of the proliferation marker Ki67 as well as the cytotoxic serine protease granzyme B, indicating reinvigoration of exhausted T cells. In conclusion, our results show that a commonly used ex vivo expansion of patient TILs results in a profound skewing of the TIL phenotype, particularly markers of exhaustion and reinvigoration. Hence, direct analysis of freshly isolated unperturbed TILs may be necessary to allow a faithful assessment of the relevant TIL profile for further identification of TA-specific TILs.