ABSTRACT
Background: Glioblastoma (GBM) is characterized by low numbers of glioma-infiltrating lymphocytes (GIL) with a dysfunctional phenotype. Whether this dysfunctional phenotype is fixed or can be reversed upon ex vivo culturing is poorly understood. The aim of this study was to assess T cell receptor (TCR)-dynamics and -specificities as well as determinants of in vitro GIL expansion by sequencing-based technologies and functional assays to explore the use of GIL for cell therapy. Methods: By means of flow cytometry, T cell functionality in GIL cultures was assessed from 9 GBM patients. TCR beta sequencing (TCRB-seq) was used for TCR repertoire profiling before and after in vitro expansion. Microarrays or RNA sequencing (RNA-seq) were performed from 6 micro-dissected GBM tissues and healthy brain RNA to assess the individual expression of GBM-associated antigens (GAA). GIL reactivity against in silico predicted tumor-associated antigens (TAA) and patient-individual GAA was assessed by ELISpot assay. Combined ex vivo single cell (sc)TCR-/RNA-seq and post-expansion TCRB-seq were used to evaluate transcriptional signatures that determine GIL expansion. Results: Human GIL regains cellular fitness upon in vitro expansion. Profound TCR dynamics were observed during in vitro expansion and only in one of six GIL cultures, reactivity against GAA was observed. Paired ex vivo scTCR/RNA-seq and TCRB-seq revealed predictive transcriptional signatures that determine GIL expansion. Conclusions: Profound TCR repertoire dynamics occur during GIL expansion. Ex vivo transcriptional T cell states determine expansion capacity in gliomas. Our observation has important implications for the use of GIL for cell therapy including genetic manipulation to maintain both antigen specificity and expansion capacity.
ABSTRACT
0.05).Activity of GIL in the culture in IL-2 of killing autologous glioma cells were significant higher than that of killing allogeneic glioma cells(P
ABSTRACT
Glioma-infiltrating lymphocytes (GIL) were isolated from 9 surgical biopsy specimens of primary brain gliomas using mechanical and enzymatic digestion and discontinuous density gtadient centrifugation. During cultured in the presence of interieukin-2 (IL-2) for a period of four weeks, GIL were expanded 48.4-fold on the averags, even up to 118-fold. GIL activated by IL-2 had specific cytorytic activity against autologous glioma cells. Analysis of T subsets of GIL freshly isolated showed that CD3+ cells were 71.0?11.9%, CD4+ cells 34.2?6.1% and CD8+ cells 37.0?7.6%. Ability of activated GIL to secrete ?-interferon (?-IFN) was significantly higher than that of freshly isolated GIL and autologous peripheral blood lymphocytes (PBL). The results suggest that GIL have many advantages for an adoptive immunotherapy of patients with brain gliomas and is a new type of antitumor immune effector.
