Cognitive behavior therapy for autistic adolescents, awareness and care for my autistic traits program: a multicenter randomized controlled trial.

BACKGROUND: Autistic people demonstrate focused interests, sensitivity to sensory stimulation, and, compared with the general population, differences in social communication and interaction. We examined whether a combination of the Awareness and Care for My Autistic Traits (ACAT) program and treatment-as-usual is more effective than only treatment-as-usual in increasing the understanding of autistic attributes, reducing treatment stigma, and improving mental health and social adaptation among autistic adolescents and their parents/guardians. METHODS: Forty-nine adolescents and their parents/guardians were randomly assigned to either a combination of ACAT and treatment-as-usual or only treatment-as-usual. The combined group received six weekly 100-minute ACAT sessions, while the treatment-as-usual group received no additional intervention. The primary outcome was the change in understanding of autistic attributes (Autism Knowledge Quiz-Child), administered from pre- to post-intervention. The secondary outcomes included the change in Autism Knowledge Quiz-Parent, reduced treatment stigma, and improved mental health and social adaptation among autistic adolescents and their parents/guardians. A primary outcome measure scale was scored by assessors who were blind to the group assignment. RESULTS: The combined group (both autistic adolescents and their parents/guardians) showed an increase in Autism Knowledge Quiz scores compared to those in the treatment-as-usual group. Autistic adolescents in the combined group also demonstrated a decrease in treatment-related stigma and an improvement in general mental health compared to those in the treatment-as-usual group, while there were no group differences in the change in social adaptation. For parents/guardians, there were no group differences in the change in treatment-related stigma, general mental health, adaptive skills, or attitudes toward their children. CONCLUSIONS: The ACAT program could be an effective treatment modality to increase the understanding of autistic attributes among both autistic adolescents and their parents/guardians. The ACAT program positively affects self-understanding, reduces treatment stigma, and stabilizes behavioral issues for autistic adolescents as a part of mental health measures, but it does not effectively reduce treatment barriers or improve mental health for parents/guardians. Further research should consider whether additional support for parents/guardians could be beneficial. TRIAL REGISTRATION: The study was registered in UMIN (UMIN000029851, 06/01/2018).

Upstream Position of Proline Defines Peptide-HLA Class I Repertoire Formation and CD8+ T Cell Responses.

Cytotoxic CD8+ T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.

Osteosarcoma-initiating cells show high aerobic glycolysis and attenuation of oxidative phosphorylation mediated by LIN28B.

Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non-responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma-initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC-related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH ) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA-sequencing, we identified LIN28B as a SIC-related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low-dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so-called cancer/testis antigen, might be a good approach.

Development of an artificial antibody specific for HLA/peptide complex derived from cancer stem-like cell/cancer-initiating cell antigen DNAJB8.

BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

Peptide vaccinations elicited strong immune responses that were reboosted by anti-PD1 therapy in a patient with myxofibrosarcoma.

Peptide-based immunotherapy does not usually elicit strong immunological and clinical responses in patients with end-stage cancer, including sarcoma. Here we report a myxofibrosarcoma patient who showed a strong clinical response to peptide vaccinations and whose immune responses were reboosted by anti-PD1 therapy combined with peptide vaccinations. The 46-year-old man showed a strong response to the peptide vaccinations (papillomavirus binding factor peptide, survivin-2B peptide, incomplete Freund's adjuvant, and polyethylene glycol-conjugated interferon-alpha 2a) and subsequent wide necrosis and massive infiltration of CD8+ T cells in a recurrent tumor. The patient's immune responses weakened after surgical resection; however, they were reboosted following the administration of nivolumab combined with peptide vaccinations. Thus, anti-PD1 therapy combined with peptide vaccinations might be beneficial, as suggested by the observations in this sarcoma patient.

Development of a T-cell receptor multimer with high avidity for detecting a naturally presented tumor-associated antigen on osteosarcoma cells.

For efficacy of peptide vaccination immunotherapy for patients with cancer, endogenous expression of the target peptide/human leukocyte antigen (HLA) on cancer cells is required. However, it is difficult to evaluate the expression status of a peptide/HLA complex because of the lack of a soluble T-cell receptor (TCR) that reacts with tumor-associated antigen (TAA) with high avidity. In the present study, we developed two soluble TCR-multimers that were each directed to TAA, survivin-2B (SVN-2B) and PBF in the context of HLA-A24 (SVN-2B TCR-multimer and PBF TCR-multimer, respectively), from CTL clones that were established from peptide-vaccinated patients. Both TCR multimers could recognize cognate peptide-pulsed antigen-presenting cells, C1R-A24 cells, in a CD8-independent method. Moreover, the PBF TCR-multimer successfully recognized a PBF peptide naturally presented on HLA-A24+ PBF+ osteosarcoma cells. Taken together, the results indicated that a TCR-multimer might be useful for detection of a TAA-derived peptide presented by HLA in patients receiving immunotherapy.

Immunohistological analysis of pancreatic carcinoma after vaccination with survivin 2B peptide: Analysis of an autopsy series.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer by providing new options in addition to existing therapies. However, peptide vaccination therapies still represent an attractive approach, because of the antigen specificity. We identified survivin 2B peptide (SVN-2B), a 9-mer antigenic peptide encoded by survivin, and an SVN-2B peptide vaccine-based phase II randomized clinical trial targeting unresectable and refractory pancreatic carcinoma was undertaken. The SVN-2B peptide vaccine did not have any statistically significant clinical benefits in that study. Therefore, we undertook an autopsy study to analyze the immune status of the pancreatic cancer lesions at the histological level. Autopsies were carried out in 13 patients who had died of pancreatic cancer, including 7 who had received SVN-2B peptide vaccination and 6 who had not, as negative controls. The expression of immune-related molecules was analyzed by immunohistochemical staining. Cytotoxic T lymphocytes were analyzed by tetramer staining and enzyme-linked immunospot assay. Histological analysis revealed dense infiltration of CD8+ T cells in some lesions in patients who had received the SVN-2B peptide vaccine. A high rate of programmed cell death ligand 1 expression in cancer cells was observed in these cases, indicating that CTLs were induced by SVN-2B peptide vaccination and had infiltrated the lesions. The lack of a significant antitumor effect was most likely attributable to the expression of immune checkpoint molecules. These findings suggest that the combination of a tumor-specific peptide vaccine and an ICI might be a promising approach to the treatment of pancreatic carcinoma in the future.

Randomized phase II trial of survivin 2B peptide vaccination for patients with HLA-A24-positive pancreatic adenocarcinoma.

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-ß (IFNß); (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 IFNß. 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 IFNß (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 IFNß group by tetramer assay. Among patients who participated in Step 2, those who had received SVN-2B plus IFNß in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN-2B plus IFNß did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN-2B plus IFNß vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).

Clinical factors associated with the therapeutic outcome of chemotherapy in very elderly cancer patients.

BACKGROUND: The purpose of this study was to detect background factors that might be associated with the therapeutic and curative outcome of chemotherapy in elderly cancer patients aged over 75 years. METHODS: A retrospective study was conducted for elderly cancer patients aged over 75 years who had received more than 2 courses of chemotherapy at our hospital. We analyzed the relationships between RECIST outcome and background factors, such as age, sex, clinical TNM stage, pre-treatment history, ECOG performance status, serum albumin, and Charlson comorbidity index using logistic regression analysis. RESULTS: A total of 103 cancer patients aged over 75 years were analyzed in this study, including 28 with hematological neoplasia, 36 with gastrointestinal tract cancers, 25 with breast cancers, and 14 with other malignancies originating in various tissues. Seventy-one patients (69.1%) had a positive clinical outcome including RECIST CR (complete response), PR (partial response) and SD (stable disease). Multivariate analysis showed that a high serum albumin level of more than 3.5 g/dl and a Charlson comorbidity index score of less than 2 points were positively correlated with a favorable therapeutic outcome. CONCLUSIONS: The results of the current study suggested that serum albumin level and comorbidity index are the principal clinical factors affecting therapeutic outcomes in elderly cancer patients receiving chemotherapy. In the future, these factors may make chemotherapy adaptations, continuity, and effectiveness easier to predict than GA screening.

Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

Case report: Long-term survival of a pancreatic cancer patient immunized with an SVN-2B peptide vaccine.

A 62-year-old woman who underwent surgery to treat pancreatic cancer provided written, informed consent to undergo adjuvant therapy with gemcitabine, tegafur, and uracil. However, this was stopped after only 14 days due to Grade 4 neutropenia. She was then started on vaccine therapy with Survivin 2B peptide (SVN-2B) including IFA and INF-α. Although metastatic lung tumors were identified and resected at 82 months after surgery, the patient has remained free of new or relapsed disease for 12 years thereafter. Tetramer and ELISPOT assays revealed the continuous circulation of SVN-2B-restricted cytotoxic T-lymphocytes (CTLs) in her peripheral blood, and CTL clones had specific activity for SVN-2B at 12 years after surgery. The adverse effects of the peptide vaccination were tolerable and comprised low-grade headache, nausea, and fatigue. A prognosis beyond 10 years in the face of pancreatic cancer with distant metastasis is extremely rare. This experience might indicate the value of cancer vaccination therapy.

Loss of sorting nexin 5 stabilizes internalized growth factor receptors to promote thyroid cancer progression.

Thyroid carcinoma is the most common endocrine malignancy and its prevalence has recently been increasing worldwide. We previously reported that the level of sorting nexin 5 (Snx5), an endosomal translocator, is preferentially decreased during the progression of well-differentiated thyroid carcinoma into poorly differentiated carcinoma. To address the functional role of Snx5 in the development and progression of thyroid carcinoma, we established Snx5-deficient (Snx5-/- ) mice. In comparison to wild-type (Snx5+/+ ) mice, Snx5-/- mice showed enlarged thyroid glands that consisted of thyrocytes with large irregular-shaped vacuoles. Snx5-/- thyrocytes exhibited a higher growth potential and higher sensitivity to thyroid-stimulating hormone (TSH). A high content of early endosomes enriched with TSH receptors was found in Snx5-/- thyrocytes, suggesting that loss of Snx5 caused retention of the TSH receptor (TSHR) in response to TSH. Similar data were found for internalized EGF in primary thyrocytes. The increased TSH sensitivities in Snx5-/- thyrocytes were also confirmed by results showing that Snx5-/- mice steadily developed thyroid tumors with high metastatic potential under high TSH. Furthermore, a thyroid cancer model using carcinogen and an anti-thyroidal agent revealed that Snx5-/- mice developed metastasizing thyroid tumors with activation of MAP kinase and AKT pathways, which are postulated to be major pathways of malignant progression of human thyroid carcinoma. Our results suggest that thyrocytes require Snx5 to lessen tumorigenic signaling driven by TSH, which is a major risk factor for thyroid carcinoma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster.

During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.

Implication of chemo-resistant memory T cells for immune surveillance in patients with sarcoma receiving chemotherapy.

Chemotherapy has improved the prognosis of patients with sarcomas. However, it may suppress anti-tumor immunity. Recently, we reported a novel CD8+ memory T cell population with a chemo-resistance property, "young memory" T (TYM ) cells. In this study, we investigated the proportion and function of TYM cells in peripheral blood of healthy donors and sarcoma patients who received chemotherapy and those who did not. The proportion of TYM cells was significantly decreased in patients compared with that in healthy donors. In healthy donors, anti-EBV CTLs were induced using mixed lymphocyte peptide culture, from not only TYM cells but also TCM and TEM cells. No CTLs directed to tumor-associated antigens were induced. In sarcoma patients who did not receive chemotherapy, in addition to anti-EBV CTLs, CTLs directed to the tumor-associated antigen PBF were induced from TYM , TCM and TEM cells. In sarcoma patients who received chemotherapy, EBV-specific CTLs were induced from TYM cells but were hardly induced from TEM cells. Interestingly, CTLs directed to the anti-tumor-associated antigen PBF were induced from TYM cells but not from the TCM and TEM cells in sarcoma patients who received chemotherapy. The findings suggest that TYM cells are resistant to chemotherapy and can firstly recover from the nadir. TYM cells might be important for immunological memory, especially in sarcoma patients receiving chemotherapy.

Microenvironmental stresses induce HLA-E/Qa-1 surface expression and thereby reduce CD8(+) T-cell recognition of stressed cells.

Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8(+) T-cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA-E in human and Qa-1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa-1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8(+) T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8(+) T-cell recognition.

Hypoxia augments MHC class I antigen presentation via facilitation of ERO1-α-mediated oxidative folding in murine tumor cells.

To establish an effective cancer immunotherapy, it is crucial that cancer cells present a cancer-specific antigen in a hypoxic area, a hallmark of the tumor microenvironment. Here, we show the impact of hypoxia on MHC class I antigen presentation in vitro and in vivo in murine tumors. Activation of antigen-specific CTLs by tumor cells that had been pre-incubated under a condition of hypoxia was enhanced compared with that by tumor cells pre-incubated under a condition of normoxia. Cell surface expression of MHC class I-peptide complex on the tumor cells was increased under a condition of hypoxia, thereby leading to higher susceptibility to specific CTLs. We show that the hypoxia-inducible ER-resident oxidase ERO1-α plays an important role in the hypoxia-induced augmentation of MHC class I-peptide complex expression. ERO1-α facilitated oxidative folding of MHC class I heavy chains, thereby resulting in the augmentation of cell surface expression of MHC class I-peptide complex under hypoxic conditions. These results suggest that since the expression of MHC class I-peptide complex is augmented in a hypoxic tumor microenvironment, strategies for inhibiting the function of regulatory T cells and myeloid-derived suppressor cells and/or immunotherapy with immune checkpoint inhibitors are promising for improving cancer immunotherapy.

LY6/PLAUR domain containing 3 has a role in the maintenance of colorectal cancer stem-like cells.

Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) is preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs.

Identification and functional analysis of variants of a cancer/testis antigen LEMD1 in colorectal cancer stem-like cells.

Colorectal cancer (CRC) is one of the most common malignancy, and the prognosis is not still satisfactory due to treatment resistance, recurrence and distant metastasis. Cancer stem cells (CSCs)/cancer-initiating cells (CICs) is endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability, and CSCs/CICs are resistant to treatments. Thus, CSCs/CICs are thought to be responsible for recurrence and distant metastasis, and eradication of CSCs/CICs is essential to cure CRCs. However, the molecular mechanisms of CSCs/CICs are remain unknown, and we aimed to elucidate molecular aspects of CR-CSCs/CICs in this study. We screened the transcriptome data of primary human CR-CSCs/CICs that we previously established, and found that LEM domain containing 1 (LEMD1) is preferentially expressed in CR-CSCs/CICs. LEMD1 belongs to cancer-testis (CT) antigen, and has five transcript variants (variant 1 [V1] - variant 5 [V5]). We found that LEMD1 V1, V2 and V3 is expressed in testis and CR-CSCs/CICs, whereas LEMD1 V4 and V5 is ubiquitously expressed. LEMD1 gene knockdown experiments using siRNAs and gene overexpression experiments revealed that LEMD1 has a role in the maintenance of CR-CSCs/CICs. These observations indicate that CR-CSC/CIC-specific LEMD1 variants are reasonable target of CR-CSC/CIC-targeted therapy.

Cancer-associated oxidoreductase ERO1-α drives the production of tumor-promoting myeloid-derived suppressor cells via oxidative protein folding.

Endoplasmic reticulum disulfide oxidase ERO1-α plays a role in the formation of disulfide bonds in collaboration with protein disulfide isomerase. Disulfide bond formation is required for the proper conformation and function of secreted and cell surface proteins. We found that ERO1-α was overexpressed in a variety of tumor types; therefore, we examined its role in tumor growth. In BALB/c mice, knockdown of ERO1-α within 4T1 mouse mammary gland cancer (KD) cells caused retardation of in vivo tumor growth compared with tumor growth of scrambled control (SCR) cells. In contrast, when ERO1-α-overexpressed 4T1 (OE) cells were compared with mock control cells, OE cells showed augmented tumor growth. However, differences in tumor growth were not observed among four groups of nude mice, suggesting that expression of ERO1-α diminished antitumor immunity. We observed dense peritumoral granulocytic infiltrates in tumors of wild-type 4T1 and SCR cells but not KD cells, and these cells were identified as polymorphonuclear myeloid-derived suppressor cells (MDSCs). In addition, production of G-CSF and CXCL1/2, which have intramolecular disulfide bonds, from KD cells was significantly decreased compared with that from SCR cells. In contrast, OE cells produced a larger amount of these molecules than did mock cells. These changes were regulated at the posttranscriptional level. These results suggest that overexpression of ERO1-α in the tumor inhibits the T cell response by recruiting polymorphonuclear MDSCs via regulation of MDSC-prone cytokines and chemokines.

Cancer-Associated Oxidase ERO1-α Regulates the Expression of MHC Class I Molecule via Oxidative Folding.

ERO1-α is an oxidizing enzyme that exists in the endoplasmic reticulum and is induced under hypoxia. It reoxidizes the reduced form of protein disulfide isomerase that has oxidized target proteins. We found that ERO1-α is overexpressed in a variety of tumor types. MHC class I H chain (HC) has two disulfide bonds in the α2 and α3 domains. MHC class I HC folding is linked to the assembly of MHC class I molecules because only fully disulfide-bonded class I HCs efficiently assemble with ß2-microglobulin. In this study, we show that ERO1-α associates with protein disulfide isomerase, calnexin, and immature MHC class I before being incorporated into the TAP-1-associated peptide-loading complex. Importantly, ERO1-α regulates the redox state as well as cell surface expression of MHC class I, leading to alteration of susceptibility by CD8(+) T cells. Similarly, the ERO1-α expression within cancer cells was associated with the expression level of MHC class I in colon cancer tissues. Thus, the cancer-associated ERO1-α regulates the expression of the MHC class I molecule via oxidative folding.