Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87.

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells.

Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3+ T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions.

Anti-CTLA-4 Antibody Might Be Effective Against Non-small Cell Lung Cancer With Large Size Tumor.

BACKGROUND/AIM: Immunotherapy using immune checkpoint inhibitors (ICIs) has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC). Although several ICI options are available, the treatment regimen for NSCLC with large size tumors (large NSCLC) is controversial and the efficacy of anti-CTLA-4 antibody is unclear. This study thus investigated potential biomarkers for CTLA-4 blockade. PATIENTS AND METHODS: The correlation between tumor diameter and treatment duration was examined in patients with advanced NSCLC treated with anti-PD-1 antibody monotherapy in our institution. In addition, the ratio of tumor-infiltrating CD8+ T cells and regulatory T (Treg) cells in small and large size NSCLC was also evaluated using immunohistochemical staining. Finally, the efficacy of treatment with anti-CTLA-4 antibody against large NSCLC was investigated. RESULTS: A negative correlation was found between tumor diameter and treatment duration in patients treated with anti-PD-1 antibody monotherapy. Immuno-histochemical staining revealed that Treg cell infiltration was significantly higher in large NSCLC tumors than in small tumors. Among the patients with large NSCLC, the ICI regimen including anti-CTLA-4 antibody showed significant efficacies. CONCLUSION: Anti-PD-1 antibody monotherapy might be less effective against large NSCLC due to the infiltration of Treg cells. Therefore, it might be appropriate for large NSCLC to select a treatment including an anti-CTLA-4 antibody, which can target Treg cells.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma.

HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.

The immune system is the body's way of defending itself, offering protection against diseases such as cancer. But to remove the cancer cells, the immune system must be able to identify them as different from the rest of the body. All cells break down proteins into shorter fragments, known as peptides, that are displayed on the cell surface by a protein called human leukocyte antigen, HLA for short. Cancer cells display distinctive peptides on their surface as they generate different proteins to those of healthy cells. Immune cells called T cells use these abnormal peptides to identify the cancer so that it can be destroyed. Sometimes T cells can lack the right equipment to detect abnormal peptides, allowing cancer cells to hide from the immune system. However, T cells can be trained through a treatment called immunotherapy, which provides T cells with new tools so that they can spot the peptides displayed by HLA on the previously 'hidden' cancer cells. There are many different forms of HLA, each of which can display different peptides. Current research in immunotherapy commonly targets only a subset of HLA forms, and not all cancer patients have these types. This means that immunotherapy research is only likely to be of most benefit to a limited number of patients. Immunotherapy could be made effective for more people if new cancer peptides that are displayed by the other 'under-represented' forms of HLA were identified. Murata, Nakatsugawa et al. have now used T cells that were taken from tumors in eight patients with melanoma, which is a type of skin cancer. A library of fluorescent HLA-peptides was generated ­ using a new, simplified methodology ­ with 25 forms of HLA that displayed over 800 peptides. T cells were then mixed with the library to identify which HLA-peptides they can target. As a result, Murata, Nakatsugawa et al. found the cancer targets of around 12% of the tumor-infiltrating T cells tested, including those from under-represented forms of HLA. Consequently, these findings could be used to develop new immunotherapies that can treat more patients.

A new ENG mutation in a Japanese family with hereditary hemorrhagic telangiectasia and pulmonary arteriovenous malformations.

We present a case series of four siblings with hereditary hemorrhagic telangiectasia (HHT) and pulmonary arteriovenous malformations (PAVM). The patients' mother has HHT. Case 1: A 22-year-old man developed dyspnea and epistaxis. CT revealed a large PAVM, treated by segmentectomy. Case 2: A 27-year-old woman developed epistaxis and dyspnea. CT revealed three PAVMs, treated by partial resection. Case 3: A 20-year-old woman developed dyspnea. CT revealed multiple PAVMs, treated with endovascular occlusion of the largest one. Case 4: A 12-year-old woman developed epistaxis. CT revealed multiple PAVMs, observed without treatment. Genetic testing identified a new mutation, ENG c.1517T>C (p.Leu506Pro), in all patients and their mother. We suspect that HHT in these patients may be associated with this ENG mutation.

Occult Thyroid Follicular Carcinoma Diagnosed as Metastasis to the Chest Wall.

Chest wall tumors are relatively rare, and hematogenous metastasis to the chest wall is very rare. We herein describe a rare case of occult thyroid carcinoma as metastasis to the chest wall in an 80-year-old woman. The patient received detailed examinations of the chest wall tumor, and the results suggested that she had occult thyroid carcinoma. Surgery was then performed to remove all of her thyroid. As a result, she was diagnosed with follicular carcinoma of the thyroid. We report an extremely rare case of occult thyroid carcinoma diagnosed as hematogenous metastasis to the chest wall.

Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigen-specific CTL recognition.

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. CD8+ 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 tapasin-deficient 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.

Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is a novel target of lung cancer stem-like cell immunotherapy.

Lung cancer is one of the most common malignancies with a high rate of mortality. Lung cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) play major role in resistance to treatments, recurrence and distant metastasis and eradication of CSCs/CICs is crucial to improve recent therapy. Cytotoxic T lymphocytes (CTLs) are major effectors of cancer immunotherapy, and CTLs recognize antigenic peptides derived from antigens that are presented by major histocompatibility complex (MHC) class I molecules. In this study, we analyzed the potency of a cancer-testis (CT) antigen, brother of the regulator of the imprinted site variant subfamily 6 (BORIS sf6), in lung CSC/CIC immunotherapy. BORIS sf6 mRNA was expressed in lung carcinoma cells (9/19), especially in sphere-cultured lung cancer stem-like cells, and in primary lung carcinoma tissues (4/9) by RT-PCR. Immunohistochemical staining using BORIS sf6-specific antibody revealed that high expression of BORIS sf6 is related to poorer prognosis. CTLs could be induced by using a human leukocyte antigen, (HLA)-A2 restricted antigenic peptide (BORIS C34_24(9)), from all of 3 HLA-A2-positive individuals, and CTL clone cells specific for BORIS C34_24(9) peptide could recognize BORIS sf6-positive, HLA-A2-positive lung carcinoma cells. These results indicate that BORIS sf6 is a novel target of lung cancer immunotherapy that might be useful for targeting treatment-resistant lung cancer stem-like cells.

Matrix metalloproteinase-10 regulates stemness of ovarian cancer stem-like cells by activation of canonical Wnt signaling and can be a target of chemotherapy-resistant ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the most lethal cancers in females. Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) have been reported to be origin of primary and recurrent cancers and to be resistant to several treatments. In this study, we identified matrix metalloproteinase-10 (MMP10) is expressed in CSCs/CICs of EOC. An immunohistochemical study revealed that a high expression level of MMP10 is a marker for poor prognosis and platinum resistance in multivariate analysis. MMP10 gene overexpression experiments and MMP10 gene knockdown experiments using siRNAs revealed that MMP10 has a role in the maintenance of CSCs/CICs in EOC and resistance to platinum reagent. Furthermore, MMP10 activate canonical Wnt signaling by inhibiting noncanonical Wnt signaling ligand Wnt5a. Therefore, MMP10 is a novel marker for CSCs/CICs in EOC and that targeting MMP10 is a novel promising approach for chemotherapy-resistant CSCs/CICs in EOC.

Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is involved in cervical cancer stemness and can be a target of immunotherapy.

Cervical cancer is a major cause of cancer death in females worldwide. Cervical cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are resistant to conventional radiotherapy and chemotherapy, and CSCs/CICs are thought to be responsible for recurrence. Eradication of CSCs/CICs is thus essential to cure cervical cancer. In this study, we isolated cervical CSCs/CICs by sphere culture, and we identified a cancer testis (CT) antigen, CTCFL/BORIS, that is expressed in cervical CSCs/CICs. BORIS has 23 mRNA isoform variants classified by 6 subfamilies (sfs), and they encode 17 different BORIS peptides. BORIS sf1 and sf4 are expressed in both CSCs/CICs and non-CSCs/CICs, whereas BORIS sf6 is expressed only in CSCs/CICs. Overexpression of BORIS sf6 in cervical cancer cells increased sphere formation and tumor-initiating ability compared with those in control cells, whereas overexpression of BORIS sf1 and BORIS sf4 resulted in only slight increases. Thus, BORIS sf6 is a cervical CSC/CIC-specific subfamily and has a role in the maintenance of cervical CSCs/CICs. BORIS sf6 contains a specific c-terminal domain (C34), and we identified a human leukocyte antigen (HLA)-A2-restricted antigenic peptide, BORIS C34_24(9) encoded by BORIS sf6. A BORIS C34_24(9)-specific cytotoxic T cell (CTL) clone showed cytotoxicity for BORIS sf6-overexpressing cervical cancer cells. Furthermore, the CTL clone significantly suppressed sphere formation of CaSki cells. Taken together, the results indicate that the CT antigen BORIS sf6 is specifically expressed in cervical CSCs/CICs, that BORIS sf6 has a role in the maintenance of CSCs/CICs, and that BORIS C34_24(9) peptide is a promising candidate for cervical CSC/CIC-targeting immunotherapy.

Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

Plasticity of lung cancer stem-like cells is regulated by the transcription factor HOXA5 that is induced by oxidative stress.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are reasonable targets for cancer therapy. However, recent studies have revealed that some non-CSCs/CICs have plastic ability and can dedifferentiate into CSCs/CICs. Therefore, an understanding of the molecular mechanisms that control the plasticity is essential to achieve CSC/CIC-targeting therapy. In this study, we analyzed the plasticity of lung cancer cells and found that lung non-CSCs/CICs can dedifferentiate into CSCs/CICs in accordance with the expression of stem cell transcription factor SOX2. SOX2 expression was induced by the transcription factor HOXA5. Oxidative stress repressed the expression of HDAC8 and then induced histone 3 acetylation and increased the expression of HOXA5 and SOX2. These findings indicate that lung cancer cells have plasticity under a condition of oxidative stress and that HOAX5 has a critical role in dedifferentiation.

Ectopically expressed variant form of sperm mitochondria-associated cysteine-rich protein augments tumorigenicity of the stem cell population of lung adenocarcinoma cells.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have self-renewal ability, differentiation ability and high tumor-initiating ability. CSCs/CICs are resistant to cancer therapies including chemotherapy and radiotherapy. Therefore, CSCs/CICs are thought to be responsible for cancer recurrence and distant metastasis after treatment. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we isolated CSCs/CICs as side population (SP) cells from lung carcinoma, colon carcinoma and breast carcinoma cells and analyzed the molecular mechanisms of CSCs/CICs. cDNA micro-array screening and RT-PCR analysis revealed that sperm mitochondria-associated cysteine-rich protein (SMCP) is ectopically expressed in SP cells. 5'-Rapid amplification of cDNA end (RACE) analysis revealed that the SMCP transcript in SP cells was a variant form (termed vt2) which is composed from only one exon. SMCP vt2 was detected in only cancer cells, whereas the wild-type (vt1) form of SMCP was expressed in the testis. SMCP was shown to have a role in tumor initiation by SMCP overexpression and SMCP knockdown using siRNAs in lung cancer cells. Taken together, the initiation results indicate that an ectopically expressed variant form of SMCP has a role in tumor initiation of CSCs/CICs and that the variant form of SMCP might be a novel CSC/CIC marker and a potential and promising target of CSC/CIC-targeting therapy.