Interleukin-4 induced 1-mediated resistance to an immune checkpoint inhibitor through suppression of CD8+ T cell infiltration in melanoma.

Cancer cells adopt multiple strategies to escape tumor surveillance by the host immune system and aberrant amino acid metabolism in the tumor microenvironment suppresses the immune system. Among the amino acid-metabolizing enzymes is an L-amino-acid oxidase called interleukin-4 induced 1 (IL4I1), which depletes essential amino acids in immune cells and is associated with a poor prognosis in various cancer types. Although IL4I1 is involved in immune metabolism abnormalities, its effect on the therapeutic efficacy of immune checkpoint inhibitors is unknown. In this study, we established murine melanoma cells overexpressing IL4I1 and investigated their effects on the intratumor immune microenvironment and the antitumor efficacy of anti-programmed death-ligand 1 (PD-L1) antibodies (Abs) in a syngeneic mouse model. As a result, we found that IL4I1-overexpressing B16-F10-derived tumors showed resistance to anti-PD-L1 Ab therapy. Transcriptome analysis revealed that immunosuppressive genes were globally upregulated in the IL4I1-overexpressing tumors. Consistently, we showed that IL4I1-overexpressing tumors exhibited an altered subset of lymphoid cells and particularly significant suppression of cytotoxic T cell infiltration compared to mock-infected B16-F10-derived tumors. After treatment with anti-PD-L1 Abs, we also found a more prominent elevation of tumor-associated macrophage (TAM) marker, CD68, in the IL4I1-overexpressing tumors than in the mock tumors. Consistently, we confirmed an enhanced TAM infiltration in the IL4I1-overexpressing tumors and a functional involvement of TAMs in the tumor growth. These observations indicate that IL4I1 reprograms the tumor microenvironment into an immunosuppressive state and thereby confers resistance to anti-PD-L1 Abs.

Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment.

BACKGROUND: Alpha-2-glycoprotein 1, zinc-binding (ZAG), a secreted protein encoded by the AZGP1 gene, is structurally similar to HLA class I. Despite its presumed immunological function, little is known about its role in tumor immunity. In this study, we thus aimed to determine the relationship between the expression of AZGP1/ZAG and the immunological profiles of breast cancer tissues at both the gene and protein level. METHODS: Using a publicly available gene expression dataset from a large-scale breast cancer cohort, we conducted gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1. We analyzed the correlation between AZGP1 expression and immune cell composition in breast cancer tissues, estimated using CIBERSORTx. Previously, we evaluated the infiltration of 11 types of immune cells for 45 breast cancer tissues using flow cytometry (FCM). ZAG expression was evaluated by immunohistochemistry on these specimens and analyzed for its relationship with immune cell infiltration. The action of ZAG in M1/M2 polarization models using primary cultures of human peripheral blood mononuclear cells (PBMC)-derived macrophage (Mφ) was analyzed based on the expression of M1/M2 markers (CD86, CD80/CD163, MRC1) and HLA class I/II by FCM. RESULTS: AZGP1 expression was negatively correlated with multiple immunological processes and specific immune cell infiltration including Mφ M1 using GSEA and CIBERSORTx. ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in tumor tissues assessed using FCM. In in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I/II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. CONCLUSION: ZAG is suggested to be a novel immunoregulatory factor affecting the Mφ phenotype in breast cancer tissues.

Expression of hormone receptors is associated with specific immunological profiles of the breast cancer microenvironment.

BACKGROUND: Elucidating the unique immunoregulatory mechanisms in breast cancer microenvironment may help develop new therapeutic strategies. Some studies have suggested that hormone receptors also have immune regulatory functions, but their mechanisms are not fully understood. In this study, we have comprehensively analyzed the relationship between the expressions of estrogen (ER), progesterone (PgR), and androgen receptors (AR), and the immunological profile in breast cancer. METHODS: Using publicly available gene expression profile datasets, METABRIC and SCAN-B, the associations between the expressions of hormone receptors and the immune cell compositions in breast cancer tissue, estimated by CIBERSORTx algorithm, were analyzed. We histologically evaluated tumor-infiltrating lymphocytes (hTIL), PD-L1 (hPD-L1) expression, and the infiltration of 11 types of immune cells by flow cytometry (FCM) for 45 breast cancer tissue samples. The relationships between them and the expressions of ER, PgR, and AR of tumor tissues, evaluated immunohistochemically, were analyzed. RESULTS: Expressions of ESR1, PGR, and AR were negatively correlated with overall immune composition. Expressions of ER and AR, but not that of PgR, were inversely associated with hTIL and hPD-L1 expression. FCM analysis showed that the expressions of ER and AR, but not that of PgR, were associated with decreased total leukocyte infiltration. Both CIBERSORTx and FCM analysis showed that ER expression was associated with reduced infiltration of macrophages and CD4+ T cells and that of AR with reduced macrophage infiltration. CONCLUSION: Hormone receptor expression correlates with specific immunological profiles in the breast cancer microenvironment both at the gene and protein expression levels.

Pretreatment tumour immune microenvironment predicts clinical response and prognosis of muscle-invasive bladder cancer in the neoadjuvant chemotherapy setting.

BACKGROUND: We examined the relationship between the tumour microenvironment and the clinical efficacy of neoadjuvant chemotherapy in patients with cT2-4aN0M0 bladder cancer using multiplex fluorescence immunohistochemistry. METHODS: The study retrospectively evaluated 51 patients who underwent radical cystectomy following neoadjuvant chemotherapy for cT2-4aN0M0 muscle-invasive bladder cancer. Patients were divided into responders (

Immune microenvironment, homologous recombination deficiency, and therapeutic response to neoadjuvant chemotherapy in triple-negative breast cancer: Japan Breast Cancer Research Group (JBCRG)22 TR.

BACKGROUND: Triple-negative breast cancer (TNBC) is a biologically diverse disease, with characteristics such as homologous recombination deficiency (HRD), gene mutation, and immune reactions. Japan Breast Cancer Research Group 22 is a multicenter trial examining TNBC's response to neoadjuvant chemotherapy (NAC) according to the HRD status. This translational research investigated the clinical significance of the immune microenvironment of TNBC in association with HRD, tumor BRCA1/2 (tBRCA1/2) mutation, and response to NAC. METHODS: Patients aged below 65 years with high HRD or germline BRCA1/2 (gBRCA1/2) mutation randomly received paclitaxel + carboplatin (group A1) or eribulin + carboplatin (A2), followed by anthracycline. Patients aged below 65 years with low HRD or those aged 65 years or older without gBRCA1/2 mutation randomly received eribulin + cyclophosphamide (B1) or eribulin + capecitabine (B2); nonresponders to the first four cycles of the therapy received anthracycline. A pathological complete response (pCR) was defined as the absence of residual cancer cells in the tissues. Pretreatment biopsy specimens were stained by multiplexed fluorescent immunohistochemistry using antibodies against CD3, CD4, CD8, Foxp3, CD204, and pan-cytokeratin. Immune cells with specific phenotypes were counted per mm2 in cancer cell nests (intratumor) and stromal regions. The immune cell densities were compared with clinicopathological and genetic factors including tumor response. RESULTS: This study analyzed 66 samples. T1 tumors had a significantly higher density of intratumoral CD8+ T cells than T2 or larger tumors. The tBRCA1/2 mutation or HRD status was not associated with the density of any immune cell. The density of intratumoral and stromal CD4+ T cells was higher in patients showing pCR than in those without pCR. In a multivariate analysis, intratumoral and stromal CD4+ T cell density significantly predicted pCR independent of age, chemotherapy dose, HRD status, and treatment groups (P = 0.009 and 0.0057, respectively). In a subgroup analysis, the predictive value of intratumoral and stromal CD4+ T cell density persisted in the platinum-containing chemotherapy group (A1+A2) but not in the non-platinum-containing group (B1+B2). CONCLUSIONS: Intratumoral and stromal CD4+ T cell density was an independent predictor of pCR in patients with TNBC. A larger study is warranted to confirm the results. TRIAL REGISTRATION: UMIN000023162.

[Dietary approach to improving the nutritional status in institutionalized elderly hemodialysis patients with a poor dietary intake: a single-arm pilot study].

OBJECTIVE: The hemodialysis (HD) diet, which is a high-calorie and high-fat regimen, may inadvertently lead to an inadequate dietary intake, resulting in undernutrition among elderly HD patients. Therefore, an attempt was made to improve the dietary intake by implementing a modified diet regimen in eligible elderly HD patients. SUBJECTS: Elderly HD patients who had ingested < 50% of the meals provided and were diagnosed with undernutrition among all elderly patients institutionalized at the special elderly nursing home annexed to Nagasaki Kidney Hospital between June and November 2012. RESULTS: Of the elderly HD patients in the nursing home (n = 27), the study included a total of 7 consecutive patients (male/female, 1/6; mean age, 84.1±6.4 years old; duration of HD, 4.3±3.8 years; geriatric nutritional index [GNRI], 83.5±8.3; normalized protein catabolic ratio [nPCR], 0.78±0.14). The modified diet regimen, which involved reducing food portion sizes and incorporating a liquid diet, led to a significant increase in their dietary intake from 48.1% at baseline to 97.1% of the meals provided 3 months after the start of the modified HD diet regimen. Their GNRI also significantly increased from 83.5±8.3 to 86.1±10.2, and their serum albumin levels significantly increased from 3.2±0.2 g/dL to 3.4±0.4 g/dL, suggesting improvements in their nutritional status. CONCLUSIONS: The attempted dietary approach for elderly HD patients was shown to potentially increase their dietary intake and improve their nutritional status without affecting the efficiency of HD being implemented.

Distinct pathways leading to TDP-43-induced cellular dysfunctions.

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component protein of inclusions found in brains of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the molecular mechanisms by which TDP-43 causes neuronal dysfunction and death remain unknown. Here, we report distinct cytotoxic effects of full-length TDP-43 (FL-TDP) and its C-terminal fragment (CTF) in SH-SY5Y cells. When FL-TDP was overexpressed in the cells using a lentiviral system, exogenous TDP-43, like endogenous TDP-43, was expressed mainly in nuclei of cells without any intracellular inclusions. However, these cells showed striking cell death, caspase activation and growth arrest at G2/M phase, indicating that even simple overexpression of TDP-43 induces cellular dysfunctions leading to apoptosis. On the other hand, cells expressing TDP-43 CTF showed cytoplasmic aggregates but without significant cell death, compared with cells expressing FL-TDP. Confocal microscopic analyses revealed that RNA polymerase II (RNA pol II) and several transcription factors, such as specificity protein 1 and cAMP-response-element-binding protein, were co-localized with the aggregates of TDP-43 CTF, suggesting that sequestration of these factors into TDP-43 aggregates caused transcriptional dysregulation. Indeed, accumulation of RNA pol II at TDP-43 inclusions was detected in brains of patients with FTLD-TDP. Furthermore, apoptosis was not observed in affected neurons of FTLD-TDP brains containing phosphorylated and aggregated TDP-43 pathology. Our results suggest that different pathways of TDP-43-induced cellular dysfunction may contribute to the degeneration cascades involved in the onset of ALS and FTLD-TDP.

Differential diagnosis of amyotrophic lateral sclerosis from Guillain-Barré syndrome by quantitative determination of TDP-43 in cerebrospinal fluid.

The aim of this study was to investigate whether an increased level of TAR DNA-binding protein 43 (TDP-43) in the cerebrospinal fluid (CSF) could be a biomarker for amyotrophic lateral sclerosis (ALS) and facilitate differential diagnosis of ALS from peripheral motor neuropathy. TDP-43 is the major constituent of neuronal and glial inclusions that neuropathologically characterize both ALS and tau-negative frontotemporal lobar degeneration. Recent discoveries of various missense mutations in the TDP-43 gene in familial ALS indicate a pivotal role of the aberrant accumulation of TDP-43 in neurodegeneration. Increased TDP-43 in the CSF could be a hallmark of ALS and other TDP-43 proteinopathy. Sandwich enzyme-linked immunosorbent assay (ELISA) was established to measure the concentration of TDP-43 in biological fluids. Culture supernatants of cells transfected with various TDP-43 constructs were used to confirm that the ELISA detected TDP-43. TDP-43 in the culture supernatant of TDP-43 transfected cells was detected by immunoprecipitation with subsequent immunoblotting and concentrations were successfully measured by sandwich ELISA. We then measured TDP-43 concentrations in the CSF of patients with ALS and Guillain-Barré syndrome (GBS). TDP-43 concentrations in CSF were significantly higher in ALS than in GBS (p = 0.016). The sensitivity of the diagnostic test was 71.4% and the specificity was 84.6%. Quantitative determination of TDP-43 concentrations in the CSF by sandwich ELISA is a potential laboratory test for differentiating ALS from peripheral motor neuropathies such as GBS.

Pathological complete response to neoadjuvant chemotherapy may improve antitumor immune response via reduction of regulatory T cells in muscle-invasive bladder cancer.

The prognosis for patients who achieve a pathologic complete response in bladder cancer is excellent, but the association between their prognosis and the tumor microenvironment is unclear. We investigated the tumor immune microenvironment of those with pathological complete response after platinum-based neoadjuvant chemotherapy for cT2-4aN0M0 bladder cancer using multiplex fluorescence immunohistochemistry. Our retrospective study included 12 patients with pathological complete response who underwent radical cystectomy following neoadjuvant chemotherapy for cT2-4aN0M0 muscle-invasive bladder cancer. We assessed the density of several immune cell types in pretreatment and posttreatment tissues via multiplex fluorescence immunohistochemical analysis. The median age was 67 years; 10 patients were male. Nine (75%) and 3 (25%) patients were cT2 and cT3, respectively. The 5-year progression-free and overall survivals were 90% and 100%, respectively. The densities of regulatory T cells (Treg; CD3+CD4+FoxP3+ cell) were significantly decreased and almost disappeared in the tumor microenvironment of posttreatment tissue compared with pretreatment tissue. Other immune cells, such as effector T cells or M2 macrophages, were not significantly changed between posttreatment and pretreatment tissues. In pathological complete response, Tregs in the tumor immune microenvironment were significantly decreased after platinum-based chemotherapy for muscle-invasive bladder cancer. The temporary arresting of immune response in the tumor microenvironment may reflect a favorable prognosis due to the decrease of Tregs with tumor shrinkage and improve the host tumor immune response.

Characterization of double-negative T cells in colorectal cancers and their corresponding lymph nodes.

TCRαß+ CD4- CD8- double-negative T (DNT) cells are minor populations in peripheral blood, and their roles have mostly been discussed in inflammation and autoimmunity. However, the functions of DNT cells in tumor microenvironment remain to be elucidated. We investigated their characteristics, possible origins and functions in colorectal cancer tissues as well as their corresponding tumor-draining lymph nodes. We found a significant enrichment of DNT cells in tumor tissues compared with their corresponding lymph nodes, especially in tumors with lower T cell infiltration. T cell receptor (TCR) sequence analysis of CD4+ T, CD8+ T and DNT cells indicated that TCR sequences detected in DNT cells were found in CD8+ T cells, but rarely in CD4+ T cells, suggesting that a part of DNT cells was likely to be originated from CD8+ T cells. Through a single-cell transcriptomic analysis of DNT cells, we found that a DNT cell cluster, which showed similar phenotypes to central memory CD8+ T cells with low expression of effector and exhaustion markers, revealed some specific gene expression patterns, including higher GZMK expression. Moreover, in flow cytometry analysis, we found that DNT cells lost production of cytotoxic mediators. These findings imply that DNT cells might function as negative regulators of anti-tumor immune responses in tumor microenvironment.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

Molecular analysis and biochemical classification of TDP-43 proteinopathy.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kDa pathology are progressive neurodegenerative diseases that are characterized by intracytoplasmic aggregates of hyperphosphorylated TAR DNA-binding protein of 43 kDa. These TAR DNA-binding protein 43 proteinopathies can be classified into subtypes, which are closely correlated with clinicopathological phenotypes, although the differences in the molecular species of TAR DNA-binding protein 43 in these diseases and the biological significance thereof, remain to be clarified. Here, we have shown that although the banding patterns of abnormally phosphorylated C-terminal fragments of TAR DNA-binding protein 43 differ between the neuropathological subtypes, these are indistinguishable between multiple brain regions and spinal cord in individual patients. Immunoblot analysis of protease-resistant TAR DNA-binding protein 43 demonstrated that the fragment patterns represent different conformations of TAR DNA-binding protein 43 molecular species in the diseases. These results suggest a new clinicopathological classification of TAR DNA-binding protein 43 proteinopathies based on their molecular properties.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

Epitope mapping of antibodies against TDP-43 and detection of protease-resistant fragments of pathological TDP-43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component of the intracellular inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we show that both monoclonal (60019-2-Ig) and polyclonal (10782-2-AP) anti-TDP-43 antibodies recognize amino acids 203-209 of human TDP-43. The monoclonal antibody labeled human TDP-43 by recognizing Glu204, Asp205 and Arg208, but failed to react with mouse TDP-43. The antibodies stained the abnormally phosphorylated C-terminal fragments of 24-26 kDa in addition to normal TDP-43 in ALS and FTLD brains. Immunoblot analysis after protease treatment demonstrated that the epitope of the antibodies (residues 203-209) constitutes part of the protease-resistant domain of TDP-43 aggregates which determine a common characteristic of the pathological TDP-43 in both ALS and FTLD-TDP. The antibodies and methods used in this study will be useful for the characterization of abnormal TDP-43 in human materials, as well as in vitro and animal models for TDP-43 proteinopathies.

Urine temperature as an index for the core temperature of industrial workers in hot or cold environments.

Workers working in hot or cold environments are at risk for heat stroke and hypothermia. In Japan, 1718 people including 47 workers died of heat stroke in 2010 (Ministry of Health Labour and Welfare, Japan 2011). While the American Conference of Governmental Industrial Hygienists (ACGIH) recommendation lists the abnormal core temperature of workers as a criterion for halting work, no method has been established for reliably measuring core temperatures at workplaces. ISO 9886 (Ergonomics-evaluation of thermal strain by physiological measurements. ISO copyright office, Geneva, pp 3-14; 2004) recognizes urine temperature as an index of core temperature only at normal temperature. In this study we ascertained whether or not urine temperature could serve as an index for core temperature at temperatures above and below the ISO range. We measured urine temperature of 31 subjects (29.8 ± 11.9 years) using a thermocouple sensor placed in the toilet bowl at ambient temperature settings of 40, 20, and 5˚C, and compared them with rectal temperature. At all ambient temperature settings, urine temperature correlated closely with rectal temperature exhibiting small mean bias. Urine temperature changed in a synchronized manner with rectal temperature at 40˚C. A Bland and Altman analysis showed that the limits of agreement (mean bias ± 2SD) between rectal and urine temperatures were -0.39 to +0.15˚C at 40˚C (95%CI -0.44 to +0.20˚C) and -0.79 to +0.29˚C at 5˚C (-0.89 to +0.39˚C). Hence, urine temperature as measured by the present method is a practical surrogate index for rectal temperature and represents a highly reliable biological monitoring index for assessing hot and cold stresses of workers at actual workplaces.

Immunological profiles of the breast cancer microenvironment represented by tumor-infiltrating lymphocytes and PD-L1 expression.

Tumor-infiltrating lymphocytes (TILs) and programmed cell death 1 ligand 1 (PD-L1) are established prognostic and predictive biomarkers for certain breast cancer subsets. However, their association with the immune response complexity is not fully understood. Therefore, we analyzed the association between the immune cell fractions in breast cancer tissues and histologically assessed TIL (hTIL) and PD-L1 (hPD-L1). Forty-five tumor and eighteen blood samples were collected from patients with breast cancer. Total leukocyte counts, frequency of 11 immune cell populations, and PD-L1 expression in each cell fraction were evaluated by flow cytometry. TILs and PD-L1 were assessed by hematoxylin and eosin staining and immunohistochemistry, respectively. A higher hTIL score showed association with increased leukocyte infiltration, higher CD4+ and CD8+ T cell proportions, and lower natural killer and natural killer T cell proportions. PD-L1 was highly expressed in nonclassical monocytes, monocyte/macrophages, myeloid-derived suppressor cells, myeloid dendritic cells, dendritic cells, and other lineages in tumors. hPD-L1 positivity reflected PD-L1 expression accurately in these fractions, as well as increased leukocyte infiltration in tumors. These results indicate that hTILs reflect differences in the immune responses in the tumor microenvironment, and certain immune cell fractions are favorably expressed in the PD-L1 pathway in breast cancer microenvironments.

Comprehensive biomarker analysis from phase II study of nivolumab in patients with thymic carcinoma.

In a phase II trial of nivolumab in advanced thymic carcinoma (UMIN000022007), long SD (SD for more than 24 weeks) was seen in three patients and irAE (Gr2 or higher) was seen in four patients among 15 patients. Here, we report preplanned comprehensive biomarker analyses. We obtained tumor samples for immunohistochemistry, peripheral blood mononuclear cells (PBMCs), plasma and serum for pharmacokinetic analysis of nivolumab and cytokine evaluations, and whole blood for immuno pharmacogenomic (PGx) analysis. PD-L1 expression on tumor cells were not associated with therapeutic efficacy, but FOXP3 expression in tumor area and stroma, CD204 expression in stroma, and MHC class I in tumor area were all low among long SD patients. PBMC of long SD patients presented with larger number of naïve/memory cells prior to treatment, suggesting priming after nivolumab administration. Immuno-PGx analysis showed non-synonymous SNVs in ITGAX and PDCD1 had some correlation with PFS. Concentration of nivolumab in blood during the treatment was not related to PFS, with their overall trend towards decreased nivolumab concentration in patients with irAEs. Low immunogenicity of thymic carcinoma demonstrated in our study may require the activation of immune systems via a combination of immune checkpoint blockades.

NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene.

We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between -200 and -179bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.