Characterization of the T-cell receptor beta chain repertoire in tumor-infiltrating lymphocytes

Abstract

Tumor-infiltrating lymphocytes (TILs) are direct effectors of tumor immunity, and their characterization is important for further development of immunotherapy. Recent advances in high-throughput sequencing technologies have enabled a comprehensive analysis of T-cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, which may provide information of therapeutic importance. We developed a high-fidelity target sequencing method with the ability for absolute quantitation, and performed large-scale sequencing of TCR beta chain (TCRB) CDR3 regions in TILs and peripheral blood lymphocytes (PBLs). The estimated TCRB repertoire sizes of PBLs from four healthy individuals and TILs from four colorectal cancer tissue samples were 608,664-1,003,098 and 90,228-223,757, respectively. The usage of J- and V-regions was similar in PBLs and TILs. Proportions of CDR3 amino acid (aa) sequences occupying more than 0.01% of the total molecular population were 0.33-0.43% in PBLs and 1.3-3.6% in TILs. Additional low coverage sequencing of 15 samples identified five CDR3 aa sequences that were shared by nine patients, one sequence shared by 10 patients, and one sequence shared by 12 patients. The estimated size of the TCRB repertoire in TILs was significantly smaller than that in PBLs. The proportion of abundant species (>0.01%) in TILs was larger than that in PBLs. Shared CDR3 aa sequences represent a response to common antigens, and the identification of such CDR3 sequences may be beneficial in developing clinical biomarkers.

Keywords: Barcode sequences; T-cell receptor; Tumor-infiltrating lymphocytes; colorectal cancer; next-generation sequencing.

Figure 1

The schematic representation of NOIR‐SS for TCRB. NOIR‐SS, non‐overlapping integrated read sequencing system; TCRB, T‐cell receptor beta chain.

Figure 2

Monitoring the errors in the barcode sequence tags. (A) Distribution of reads per barcode tag. Vertical axis: number of different barcode tags. Horizontal axis: number of reads per tag. (B) The mean proportion of 12‐bp barcode tags after including the 11‐ and 13‐bp tags that differed from a 12‐bp tag only by the insertion or deletion of a single base. Red line, threshold for the removal of reads.

Figure 3

Rarefaction curves. Horizontal axis: number of molecules randomly selected from each pool of sequenced molecules. Vertical axis: number of TCRB CDR3 nucleotide sequences identified. TCRB, T‐cell receptor beta chain; CDR3, complementarity‐determining region 3.

Figure 4

Usage of J‐ and V‐regions in PBLs and TILs. (A) J‐regions. (B) V‐regions. PBLs, peripheral blood lymphocytes; TILs, tumor‐infiltrating lymphocytes.

Figure 5

Frequency fractions of CDR3 amino acid sequences. A total of 100,000 molecules were selected from each pool of sequenced molecules, and the numbers of TCRB amino acid types of each frequency fraction were deduced. CDR3, complementarity‐determining region 3; TCRB, T‐cell receptor beta chain.