CD103 and CD39 coexpression identifies neoantigen-specific cytotoxic T cells in colorectal cancers with low mutation burden

Abstract

Background: Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8⁺ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.

Experimental design: Whole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8⁺ T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103⁺CD39⁺ cytotoxic T cells in tumors.

Results: Neoantigen-directed reactivity was only encountered in bulk TIL populations and CD103⁺CD39⁺ (double positive, DP) CD8⁺ T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8⁺ T cells could be attributed to CD4⁺ T cells. CD8⁺ T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.

Conclusion: Coexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8⁺ T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients.

Keywords: Antigens; CD8-Positive T-Lymphocytes; Gastrointestinal Neoplasms; Immunotherapy; Lymphocytes, Tumor-Infiltrating.

Figure 1

Neoantigen-directed T cell reactivity assessment from bulk TIL and sorted CD8⁺ T cell subsets according to CD39 and CD103 expression. (A) Schematic workflow of the experimental setup. (B) A representative example of the IFN-γ and granzyme B ELISA measurements obtained in two independent assays performed in NIC16. Potential neoepitopes are depicted with the peptide number, for example, ‘L12.1’. (C) Representative example of a validation experiment in NIC16. The differential IFN-γ production upon coculture with the mutant peptide (yellow), the corresponding wild-type peptide (black) or a DMSO control (grey) was assessed in a peptide titration series ranging from 2.5 to 20 µg/mL. (D) Summary of the number of patient samples in which no reactivity was detected (gray), or with T cell responses derived from the DP subset (yellow), bulk TIL (green) or both the bulk TIL and DP subset (light blue). (E) IFN-γ production (left) and CD137 expression (right) on coculture of NIC4 bulk TIL (green) and DP subset (yellow) with the L29 epitope and controls. DP, double-positive; EBV-LCL, Epstein-Barr virus-transformed lymphoblastoid B cell lines; PBMC, peripheral blood mononuclear cell; TIL, tumor-infiltrating lymphocyte.

Figure 2

CD103 and CD39 detection on tumor-infiltrating T cells using imaging mass cytometry. (A, B) Representative tissue sections illustrating T cell infiltration (CD8 in red, CD103 in blue, CD39 in green) in relation to cancer cells (keratin, white). The arrows highlight DP CD8⁺ T cells. (C) Quantification of infiltration by DP populations as percentage of the total CD8⁺ T cell infiltrate. The number of cells was measured for the epithelium and stroma separately, and compared between both compartments using a paired samples Wilcoxon test. **, P ≤ 0.01. DP, double-positive.