Cytotoxic T-lymphocytes (CTLs) lyse target cells after recognizing the complexes of peptides and MHC class I molecules (pMHC I) on cell surfaces. Tapasin is an essential component of the peptide-loading complex (PLC) and its absence influences the surface repertoire of MHC class I peptides. In the present study, we assessed tapasin expression in 85 primary tumor lesions of non-small cell lung cancer (NSCLC) patients, demonstrating that tapasin expression positively correlated with patient survival. CD8C T-cell infiltration of tumor lesions was synergistically observed with tapasin expression and correlated positively with survival. To establish a direct link between loss of tapasin and CTL recognition in human cancer models, we targeted the tapasin gene by CRISPR/Cas9 system and generated tapasin-deficient variants of human lung as well as colon cancer cells. We induced the CTLs recognizing endogenous tumor-associated antigens (TAA), survivin or cep55, and they responded to each tapasin-proficient wild type. In contrast, both CTL lines ignored the tapasin-deficient variants despite their antigen expression. Moreover, the adoptive transfer of the cep55-specific CTL line failed to prevent tumor growth in mice bearing the tapasindeficient variant. Loss of tapasin most likely limited antigen processing of TAAs and led to escape from TAA-specific CTL recognition. Tapasin expression is thus a key for CTL surveillance against human cancers.

The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2‐step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN‐2B) plus interferon‐β (IFNβ); (ii) SVN‐2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN‐2B plus IFNβ. The primary endpoint was progression‐free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty‐three patients were randomly assigned to receive SVN‐2B plus IFNβ (n = 30), SVN‐2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B‐specific CTLs were found to be increased in the SVN‐2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN‐2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN‐2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN‐2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).

Colorectal cancer consists of a small number of cancer stem cells (CSC) and many non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because they survive conventional chemo-and radiotherapies and perpetuate tumor formation in vivo. In this study, we conducted an HLA ligandome analysis to survey HLA-A24 peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified an antigen, ASB4, which was processed and presented by a CSC subset but not by nonCSCs. The ASB4 gene was expressed in CSCs of colorectal cancer, but not in cells that had differentiated into non-CSCs. Because ASB4 was not expressed by normal tissues, its peptide epitope elicited CD8 þ cytotoxic T-cell (CTL) responses, which lysed CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-associated antigen that can elicit CTL responses specific to CSCs and can discriminate between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and effectively prevented tumor growth in a mouse model. As the cancer cells implanted in these mice contained very few CSCs, the elimination of a CSC subset could be the condition necessary and sufficient to control tumor formation in vivo. These results suggest that CTL-based immunotherapies against colorectal CSCs might be useful for preventing relapses.

This study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC-MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis). The SUV39H2 peptide is immunogenic and elicits cytotoxic CD8 T-cell (CTL) responses against cancer cells and is thus a novel cancer-testis antigen. Moreover, we found that microsatellite instability (MSI)-colon cancer cells displayed a neoepitope with an amino-acid substitution, while microsatellite stable (MSS)-colon and lung cancer cells displayed its counterpart peptide without the substitution. Structure modeling of peptide-HLA-A24 complexes predicted that the mutated residue at P8 was accessible to T-cell receptors. The neoepitope readily elicited CTL responses, which discriminated it from its wild-type counterpart, and the CTLs exhibited considerably high cytotoxicity against MSS-colon cancer cells carrying the responsible gene mutation. The specific and strong CTL lysis observed in this study fosters our understanding of immune surveillance against neoantigens.

Although CD8 + T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometrybased proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch-repair deficient (dMMR)-CRC tissue sample carrying 3,967 nonsynonymous mutations, where abundant tumorinfiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of non-mutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8 + TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8 + TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.