Characterization of tumor-infiltrating CD8+ T lymphocytes in diffuse large B cell lymphoma
Mutsunori Murahashi1, Hiroshi Hamana2, Hiroyuki Kishi2, Kotoe Katayama3, Rui Yamaguchi3, Satoru Miyano3, Hisanobu Ogata1, Shohei Miyamoto5, Kenzaburo Tani5, Hideya Onishi6, Yasushi Takamatsu4, Yoichi Nakanishi7.
1Department of Advanced Cell and Molecular Therapy, Kyushu University Hospital; 2Department of Immunology, Graduate School of Medicine and Pharmaceutical Science, Toyama University; 3Human Genome Center, Institute of Medical Science, University of Tokyo; 4Division of Medical Oncology- Hematology and Infectious Diseases, Fukuoka University; 5Project Division of ALA Advanced Medical Research, Institute of Medical Science, University of Tokyo; 6Department of Cancer Therapy and Research, Graduate School of Medical Sciences, Kyushu University; 7Institute of Diseases of Chest, Kyushu University.
Background: Adoptive immunotherapy using genetically engineered TCR-T cells is a promising next-generation tumor therapy. Recently, we developed a single-T cell analysis protocol that allows us to rapidly and easily obtain TCR cDNAs (1). In this study, we analyzed TCR repertoire of CD8+ TILs in diffuse large B cell lymphoma (DLBCL) and explored neoantigens using whole exome sequencing to investigate the possibility for application to treatment.
Material and Methods: Four patients who were pathologically diagnosed as DLBCL were evaluated. We used FACS to sort single CD137 or PD-1-positive cells in CD8+ TILs. TCRα and β cDNAs were amplified using RT-PCR to determine amino acid sequences of V region and CDR3. IFNγ production was evaluated in CD8+ TILs after culture with nivolumab. Genomic DNA was isolated from tumor specimens, and targeted exon capture and sequencing was performed at Riken Genesis, Japan.
Results: TCR repertoire analysis showed many CD8+ TILs expressing the same TCR in three patients among 4 cases. Five clones occupied 69.4% among all clones in the most significant case. Nivolumab treatment of CD8+ TILs showed an increase of IFNγ production in the case with oligoclonal TCR repertoire.
Conclusions: Our results suggest that CD8+ TILs expressing the same TCR are supposed to be clonally expanded T cells by antigen stimulation, and first demonstrating oligoclonal CD8+ TILs in DLBCL. Analysis of neoantigens are now underway and would be useful to identify the targets for clonally expanded T cells.
Reference 1. Kobayashi E, et al. Nat Med. 19:1542-6, 2013.