Identification of Tumoricidal TCRs from Tumor-Infiltrating Lymphocytes by Single-Cell Analysis.

Kiyomi Shitaoka,Kenzaburo Tani,Hiroyuki Kishi,Xiuhong Piao,Mutsunori Murahashi,Fulian Lyu,Atsushi Tanemura,Yoshihiro Hayakawa,Eiji Kobayashi,Ichiro Katayama,Hiroyoshi Nishikawa,Hiroshi Hamana,Kenta Sukegawa,Takuya Nagata,Atsushi Muraguchi,Daisuke Sugiyama,Yasushi Takamatsu,Tatsuhiko Ozawa

doi:10.1158/2326-6066.cir-17-0489

Abstract

T-cell receptor (TCR) gene therapy is a promising next-generation antitumor treatment. We previously developed a single-T-cell analysis protocol that allows the rapid capture of paired TCRα and β cDNAs. Here, we applied the protocol to analyze the TCR repertoire of tumor-infiltrating lymphocytes (TIL) of various cancer patients. We found clonally expanded populations of T cells that expressed the same clonotypic TCR in 50% to 70% of CD137+CD8+ TILs, indicating that they responded to certain antigens in the tumor environment. To assess the tumor reactivity of the TCRs derived from those clonally expanded TILs in detail, we then analyzed the CD137+CD8+ TILs from the tumor of B16F10 melanoma cells in six C57BL/6 mice and analyzed their TCR repertoire. We also found clonally expanded T cells in 60% to 90% of CD137+CD8+ TILs. When the tumor reactivity of dominant clonotypic TCRs in each mouse was analyzed, 9 of 13 TCRs induced the secretion of IFNγ in response to, and showed killing of, B16F10 cells in vitro, and 2 of them showed strong antitumor activity in vivo Concerning their antigen specificity, 7 of them reacted to p15E peptide of endogenous murine leukemia virus-derived envelope glycoprotein 70, and the rest reacted to tumor-associated antigens expressed on EL4 lymphoma as well as B16 melanoma cells. These results show that our strategy enables us to simply and rapidly obtain the tumor-specific TCR repertoire with high fidelity in an antigen- and MHC haplotype-independent manner from primary TILs. Cancer Immunol Res; 6(4); 378-88. ©2018 AACR.

Highlights

Since the advent of immune checkpoint blockade therapies, tumor immunotherapy has become a promising antitumor approach [1]
The results showed that the CD137þPD-1þCD8þ tumor-infiltrating lymphocytes (TIL) could be grouped into populations of T cells expressing the same clonotypic TCRa and b pair (Fig. 1B)
Because our cloned TIL-derived T-cell receptor (TCR) were restricted to H-2Kb molecules (Supplementary Fig. S9), we examined two antigenic peptides: one is tyrosine-related protein 2-drived peptide (TRP2p; ref. 33) and the other is p15E peptide (p15Ep) from envelope protein 70 of endogenous murine-leukemia virus [34], both of which bound to H-2Kb molecules

Summary

Introduction

Since the advent of immune checkpoint blockade therapies, tumor immunotherapy has become a promising antitumor approach [1]. In this context, T-cell receptor (TCR) gene therapy and T-cell adoptive therapy are promising next-generation tumor therapies [2, 3]. T-cell receptor (TCR) gene therapy and T-cell adoptive therapy are promising next-generation tumor therapies [2, 3] To develop these T cell–related therapies, tumor-specific T cells or TCR genes are required. Treating patients using tumor-specific T cells or TCRs derived from the patients themselves is difficult because their cancer may progress rapidly. The subjects of most studies on tumor immunotherapies were skewed toward patients with major human leukocyte antigen (HLA) haplotypes (i.e., HLA-AÃ2402 for Asian patients and HLA-AÃ0201 for Caucasian patients; see The Allele Frequency Net Database, http:// allelefrequencies.net/default.asp; ref. 4), and those patients with minor HLA haplotypes cannot benefit from results from these studies

Methods

Results

Conclusion