Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target?

Regulatory T (Treg) cells suppress abnormal/excessive immune responses to self- and nonself-antigens to maintain immune homeostasis. In tumor immunity, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. There are several Treg cell immune suppressive mechanisms: inhibition of costimulatory signals by CD80 and CD86 expressed by dendritic cells through cytotoxic T-lymphocyte antigen-4, interleukin (IL)-2 consumption by high-affinity IL-2 receptors with high CD25 (IL-2 receptor α-chain) expression, secretion of inhibitory cytokines, metabolic modulation of tryptophan and adenosine, and direct killing of effector T cells. Infiltration of Treg cells into the tumor microenvironment (TME) occurs in multiple murine and human tumors. Regulatory T cells are chemoattracted to the TME by chemokine gradients such as CCR4-CCL17/22, CCR8-CCL1, CCR10-CCL28, and CXCR3-CCL9/10/11. Regulatory T cells are then activated and inhibit antitumor immune responses. A high infiltration by Treg cells is associated with poor survival in various types of cancer. Therefore, strategies to deplete Treg cells and control of Treg cell functions to increase antitumor immune responses are urgently required in the cancer immunotherapy field. Various molecules that are highly expressed by Treg cells, such as immune checkpoint molecules, chemokine receptors, and metabolites, have been targeted by Abs or small molecules, but additional strategies are needed to fine-tune and optimize for augmenting antitumor effects restricted in the TME while avoiding systemic autoimmunity. Here, we provide a brief synopsis of these cells in cancer and how they can be controlled to achieve therapeutic outcomes.

[Current Topics of Cancer Antigen].

Cancer antigens are classified into shared antigens and tumor-specific antigens based on their expression pattern or immunogenicity. Neo-antigens are defined as a subset of tumor-specific antigens generated by non-synonymous mutations, Indel gene mutation or gene fusions, presented by major histocompatibility complex molecules that were recognized by effector CD8+ T cells. Cancer cells with inherent genetic instability form abnormal proteins that have not been previously recognized by the immune system and these proteins become highly immunogenic antigens(neo-antigens)that can spontaneously trigger CD8+ T cell responses. Cancer cells that present extremely immunogenic neo-antigens are eliminated from the host through the process of carcinogenesis. Therefore, recognition of neo-antigens could be an important subject of the clinical utility of both neo-antigen cancer vaccine and adoptive TCR-T cell therapy as cancer immunotherapies. Furthermore, monitoring the quantity and quality of the neo-antigen derived in each patient could deduce the clinical response to immune checkpoint inhibitors such as anti-PD-1 therapy in various cancers. Here we review the evidence for the relevance of tumor neo-antigens and host immune response. We discuss the utility as a biomarker for cancer immunotherapy using the neoantigen expression in tumor tissues and the development of neo-antigen-targeted cancer vaccine and T cell mediated therapies.

Biomarkers for differentiation of patients with asthma and chronic obstructive pulmonary disease.

OBJECTIVE: Asthma and chronic obstructive pulmonary disease (COPD) are airflow limitation diseases with similar clinical manifestations but different pathophysiologic mechanisms. To implement the appropriate treatment, it is important to distinguish between asthma and COPD which sometimes might result difficult in clinical practice. We evaluated biomarkers to distinguish between asthma and COPD. METHODS: Blood eosinophil counts and fractional exhaled nitric oxide (FeNO) levels were analyzed. Serum periostin, interleukin-25 (IL-25), and immunoglobulin E (IgE) concentrations were compared between patients with asthma (n = 60), including atopic-asthma (n = 30) and non-atopic asthma (n = 30), and patients with COPD (n = 30). RESULTS: Significantly higher peripheral blood eosinophil counts (p < 0.001), FeNO levels (p < 0.001), and total serum IgE (P = 0.003) concentrations, but not serum periostin (p = 0.584) or serum IL-25 (p = 0.085) concentrations, were detected in patients with asthma compared to patients with COPD. Serum periostin and IgE concentrations were increased in patients with atopic-asthma compared with those with non-atopic asthma and COPD (p < 0.05). The FeNO levels were significantly correlated with the peripheral blood eosinophil counts (r = 0.430, p = 0.001) and serum IL-25 concentrations (r = 0.338, p = 0.009) in patients with asthma. The serum periostin concentrations were also correlated with the serum IgE concentrations (r = 0.375, p = 0.003)and FeNO levels (r = 0.291, p = 0.024) in patients with asthma. Asthma patients were effectively differentiated from COPD patients based on the FeNO levels (p < 0.001) and peripheral blood eosinophil counts (p < 0.001). CONCLUSIONS: FeNO levels and peripheral blood eosinophil counts were useful biomarkers for distinguishing between patients with asthma and COPD. Serum periostin and IgE concentrations could be biomarkers for atopic asthma.

Possible Role of IL-25 in Eosinophilic Lung Inflammation in Patients with Chronic Eosinophilic Pneumonia.

PURPOSE: Interleukin (IL)-25 and IL-33 induce IL-5 production by various types of cells, such as type 2 helper T (Th2) cells and type 2 innate lymphoid cells. The number of Th2 cells and concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) are increased in patients with eosinophilic pneumonia (EP). To examine the contribution of IL-25 and IL-33 to eosinophilic inflammation of the lung in humans, we evaluated IL-5, IL-25 and IL-33 levels in the BALF of patients with EP. METHODS: IL-5, IL-25, and IL-33 concentrations in the BALF were measured by enzyme-linked immunosorbent assay in patients with acute eosinophilic pneumonia (AEP), chronic eosinophilic pneumonia (CEP), idiopathic pulmonary fibrosis (IPF), and sarcoidosis. RESULTS: The absolute number of eosinophils, and IL-5 levels, but not IL-33 levels, in the BALF were significantly higher in patients with EP than in patients with IPF and sarcoidosis. IL-25 levels in the BALF were significantly higher in patients with CEP, but not in patients with AEP, than in patients with IPF and sarcoidosis. The absolute number of eosinophils was significantly correlated with the IL-5 concentration in the BALF of patients with EP. IL-5 concentrations were significantly correlated with IL-25 concentrations in the BALF of patients with CEP, but not in patients with AEP. IL-5 levels were not correlated with IL-33 levels in the BALF of patients with EP. CONCLUSIONS: Our findings suggest that IL-25 plays an important role via IL-5 in eosinophilic lung inflammation in patients with CEP.

Drug fever after cancer chemotherapy is most commonly observed on posttreatment days 3 and 4.

BACKGROUND: This study was undertaken to analyze the characteristics of fever after cancer chemotherapy in order to reduce unnecessary medical care. METHODS: Retrospectively, 1016 consecutive cycles of cancer chemotherapy were analyzed. Fever was defined as a temperature of ≥ 37.5 °C lasting for 1 h. Age, sex, tumor histology, the treatment regimen, the timing of fever onset, the number of days for which the fever persisted, the cause of the fever, the presence or absence of radiotherapy, and the use of granulocyte colony-stimulating factor (G-CSF) were examined. RESULTS: The patients included 748 males and 268 females (median age = 68, range = 29-88), of whom 949, 52, and 15 were suffering from lung cancer, malignant pleural mesothelioma, and other diseases, respectively. Fever was observed in 367 cycles (36 %), including 280 cycles (37 %) involving males and 87 cycles (32 %) involving females. Fever occurred most commonly in the first cycles and was higher than later cycles (41 vs. 30 %, p < 0.001). Fever occurred most frequently on posttreatment days 4 (8 %), 3 (7 %), and 12 (7 %), and the distribution of fever episodes exhibited two peaks on posttreatment days 3 and 4 and 10-14. Fever on posttreatment days 3 and 4 was most commonly observed in patients treated with gemcitabine (20 %) or docetaxel (18 %). The causes of fever included infection (47 %; including febrile neutropenia [24 %]), adverse drug effects (24 %), unknown causes (19 %), and tumors (7 %). Radiotherapy led to a significant increase in the frequency of fever (46 vs. 34 %, p < 0.001). Thirty-three percent of patients received G-CSF, and the incidence ratios of fever in patients who received G-CSF were higher than those who did not receive G-CSF (44 vs. 31 %, p < 0.001). CONCLUSION: The febrile episodes that occurred on posttreatment days 3 and 4 were considered to represent adverse drug reactions after cancer chemotherapy. Physicians should be aware of this feature of chemotherapy-associated fever and avoid unnecessary examination and treatments including prescribing antibiotics.

Increased Galectin-9 Concentration and Number of CD4+Foxp3high+Cells in Bronchoalveolar Lavage Fluid of Patients with Cryptogenic Organizing Pneumonia.

PURPOSE: Galectin-9 (Gal-9) is a ß-galactoside-binding protein that exhibits various biological reactions, such as chemoattraction, cell aggregation, and apoptosis. Recent studies demonstrated that Gal-9 has a role as an immunomodulator in excessive immunological reactions by expanded regulatory T cells (Tregs). We examined the role of Gal-9 in the pathogenesis of one of the major idiopathic interstitial pneumonias, cryptogenic organizing pneumonia (COP) as compared with idiopathic pulmonary fibrosis (IPF). METHODS: Gal-9, transforming growth factor-ß1, and interleukin (IL)-10 levels in the bronchoalveolar lavage fluid (BALF) of patients with COP and IPF were estimated by enzyme-linked immunosorbent assay. Forkhead box protein 3 (Foxp3) expressing Tregs were evaluated by flow cytometry. The effect of Gal-9 on interactions between human lung fibroblast cells and hyarulonan was assessed in vitro. RESULTS: Gal-9 and IL-10 levels in the BALF were significantly higher in patients with COP than in patients with IPF. The number of CD4+Foxp3high+cells was significantly higher in the BALF of patients with COP than in those with IPF. Gal-9 levels significantly correlated with the absolute number of CD4+CD25+Foxp3+cells or CD4+Foxp3high+cells, but not with the absolute number of CD4+CD25+Foxp3-cells, in the BALF of patients with COP. Gal-9 suppressed the CD44-dependent interaction of human lung fibroblast cells with hyarulonan in a dose-dependent manner. CONCLUSIONS: Our findings suggest that increased Gal-9 levels in the lung have a protective role against lung inflammation and fibrosis in patients with COP through the induction of Tregs in the lung and CD44-dependent inhibitory effects on lung fibroblast cells.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Step-down of budesonide/formoterol in early stages of asthma treatment leads to insufficient anti-inflammatory effect.

OBJECTIVE: Administration of the combination of an inhaled corticosteroid (ICS) and a long-acting beta agonist (LABA) is the main treatment strategy for bronchial asthma. The ICS/LABA dosage can be reduced (stepped down) when the patient's symptoms and lung functions are well-controlled. In this study, we obtained fractional exhaled nitric oxide (FeNO) measurements to clarify whether the anti-inflammatory effect of budesonide/formoterol is shortened by step-down. METHODS: Fifty-four patients who visited the Kawasaki Medical School Hospital with newly diagnosed asthma from November 2008 to July 2010 received budesonide/formoterol for 8 weeks or more. In 29 patients, the forced expiratory volume in 1 s% predicted increased to 80% or more, and the Asthma Control Questionnaire (ACQ) score decreased to 0.5 or less within 12 weeks. These 29 patients were randomly divided into two groups: the dosage-continued group (n = 14) and the step-down group (n = 15). Then, the impact of budesonide/formoterol step-down on ACQ score, pulmonary function and FeNO level was compared between the groups. RESULTS: In the step-down group, the dosage was stepped down from 538 mcg/day to 331 mcg/day. In both groups, pulmonary function indicators and symptoms did not change. However, the mean FeNO level decreased significantly in the dosage-continued group (from 50.9 ppb to 45.0 ppb), and increased significantly in the step-down group (from 51.0 ppb to 65.7 ppb). CONCLUSIONS: Clinicians should be more careful when stepping down budesonide/formoterol based solely on patients' symptoms and/or pulmonary function.

[Anti-CCR4 mAb and regulatory T cells].

Despite receiving multidisciplinary therapy, patients with advanced or recurrent cancer experience poor survival. Therefore, novel and effective therapies should be developed. In various malignancies, tumor cells can evade the host immune defenses, in which CD4+CD25+ regulatory Tcells (Treg) play an important role. Tregs maintain self-tolerance and homeostasis in the immune system, thereby suppressing the antitumor immune responses in cancer patients. Thus, Tregs are crucial in controlling antitumor immune responses. Several clinical studies have shown that the presence of Tregs at the tumor site was correlated with poor prognosis, and Tregs reportedly suppress the antigen-specific T-cell induction in immunotherapy. Therefore, controlling Treg function may be a promising immunotherapy. Based on the findings of adult T-cell leukemia-lymphoma research, Tregs have been shown to display high cell-surface expression of the CC chemokine receptor, CCR4. The anti-CCR4 monoclonal antibody recognizes the CCR4 molecule and induces robust ADCC activity against CCR4-positive cells such as Tregs. Thus, Treg depletion using humanized anti-CCR4 monoclonal antibodies may enhance the host immune response against tumors. The current ongoing clinical trial investigates the use of humanized anti-human-CCR4 monoclonal antibody, mogamulizumab, in the treatment of advanced solid tumors.

Spontaneous antibody, and CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a) in non-small cell lung cancer patients.

The spontaneous immune responses against XAGE-1b (GAGED2a) were analyzed in non-small cell lung cancer (NSCLC) patients. An antibody response against XAGE-1b (GAGED2a) was observed in 10% (20/200) of NSCLC patients and in 19% (13/69) of stage IIIB/IV lung adenocarcinoma patients. A CD4 T-cell response was detected in 88% (14/16) and a CD8 T-cell response in 67% (6/9) in the XAGE-1b (GAGED2a) antibody-positive patients examined. Frequent antibody responses and CD4 and CD8 T-cell responses in XAGE-1b (GAGED2a) antibody-positive patients indicate the strong immunogenicity of the XAGE-1b (GAGED2a) antigen in NSCLC patients. We established T-cell clones from PBMCs of antibody-positive patients and determined the DRB1*04:05-restricted XAGE-1b (GAGED2a) 18-31 peptide (14-mer) as a CD4 T cell epitope and the A*02:06-restricted XAGE-1b (GAGED2a) 21-29 peptide (9-mer) as a CD8 T cell epitope. As for peptide recognition, CD4 and CD8 T-cell clones responded to naturally processed antigen. The CD4 T-cell clone recognized DCs pulsed with the synthetic protein or a lysate from XAGE-1b-transfected 293T cells. The CD8 T-cell clone showed cytotoxicity against a tumor expressing XAGE-1b (GAGED2a) and the appropriate HLA class I allele. These findings establish XAGE-1b (GAGED2a) as a promising target for a lung cancer vaccine.

Enrichment of Foxp3+ CD4 regulatory T cells in migrated T cells to IL-6- and IL-8-expressing tumors through predominant induction of CXCR1 by IL-6.

Analysis of cytokine and chemokine production by tumor cell lines including five lung cancers, a malignant mesothelioma, and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma, and the malignant melanoma. We investigated the migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3(+) CD4 regulatory T cells (Tregs) in migrated T cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3(+) CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be responsible for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8R expression by IL-6 is one of the mechanisms for tumor escape.

Comparison of T-cell interferon-gamma release assays for Mycobacterium tuberculosis-specific antigens in patients with active and latent tuberculosis.

Through the use of QuantiFERON-TB Gold, a commercial IFN-gamma assay, we compared differences in quantitative T-cell responses to Mycobacterium tuberculosis (MTB)-specific antigens [QuantiFERON TB-2G (QFT-2G)] between patients with active tuberculosis (TB) disease and those with latent TB infection (LTBI). The patient group consisted of 180 patients with active TB disease (culture-positive for MTB) and 50 screening contacts with LTBI-positive response to the QFT-2G test. We prospectively performed a tuberculin skin test (TST) and a QFT-2G test for all subjects. The median IFN-gamma levels upon the application of both antigens, ESAT-6 and CFP-10, were significantly higher in patients with active TB disease than in those with LTBI. A combined positive response to both antigens occurred at a higher rate in patients with active TB disease than in those with LTBI. There were no significant relationships between the quantitative responses of IFN-gamma to both antigens and the maximum induration on TST in both patient groups. We demonstrated significant differences in the quantitative responses of IFN-gamma to MTB between patients with active TB disease and those with LTBI in this study. However, there was an overlap in the IFN-gamma levels between active TB disease and LTBI groups. Therefore, it would be difficult to use the QFT-2G test to completely discriminate active TB disease from LTBI.

False negative results of QuantiFERON TB-2G test in patients with active tuberculosis.

Ten of 140 patients (7.1%) showed negative results on a QFT-2G test. The 10 patients consisted of seven males and three females (mean age: 66.0 years old). Three of the 10 patients had no clinical symptoms and no underlying diseases. They showed negative results on the QFT-2G test repeatedly within 1 month and a negative response on a tuberculin skin test (TST). Six of the seven remaining patients were of advanced age, and all of them had underlying diseases. Although all seven patients showed negative results on the QFT-2G test repeatedly within 1 month, one of them showed a negative response on TST. The lymphocyte count was within the normal range in three patients with nutritional conditions. Although five of nine patients with pulmonary tuberculosis had cavitary lesions, six had lesions within one-third of the unilateral lung field. The clinical effect in all patients was good. We suspect that there are two patterns by which false negative results are shown on QFT-2G tests; one pattern involves the decrease of interferon-gamma production due to advanced patient age or lymphocytopenia, and in the other lymphocytes cannot produce interferon-gamma for Mycobacterium tuberculosis-specific antigen in young patients without underlying disease.

Serum Antibody Against NY-ESO-1 and XAGE1 Antigens Potentially Predicts Clinical Responses to Anti-Programmed Cell Death-1 Therapy in NSCLC.

INTRODUCTION: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti-PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits. METHODS: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti-PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing. RESULTS: In the discovery cohort (n = 13), six antibody-positive NSCLC cases responded to anti-PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8+ T-cell infiltration. In the validation cohort (n = 75), 17 antibody-positive NSCLC cases responded well to anti-PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p = 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio = 0.4, p = 0.01) and overall survival (hazard ratio = 0.2, p = 0.004). CONCLUSIONS: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti-PD-1 therapy for NSCLC and probably for other cancers.

[Asthma control test (ACT) faultiness and the remediation by peak flow (PEF) measurement at outpatient department].

BACKGROUND: To grasp the asthma patients' control level, an asthma control test (ACT), an easy questionnaire, is getting to be used as the alternative method of asthma diary recording. ACT is based on the patient's 4-week memory and the accuracy is not confirmed thoroughly METHODS: Two hundred and eighty three visits of 88 asthmatics (male 29, female 59) were subjected. The accuracy of ACT to detect "truly good control" was analyzed by comparing with the asthma diary (symptoms and PEF were recorded). RESULTS: Sensitivities, specificities (chi square values) for extraction of truly good control were 0.88, 0.42 (23.7) by the threshold of ACT > or =23, 0.77, 0.51 (18.1) by ACT > or =24, and 0.54, 0.69 (12.9) by ACT=25, significantly (p<0.001, respectively). When PEF% predicted > or =80% was added as the second factor, the chi square values improved to 29.9 by ACT > or =23 and to 30.3 by ACT > or =24 significantly (p<0.001, respectively). CONCLUSION: Addition of PEF measurement on visit on ACT may help understanding the asthmatic patient condition.

NY-ESO-1 Protein Cancer Vaccine With Poly-ICLC and OK-432: Rapid and Strong Induction of NY-ESO-1-specific Immune Responses by Poly-ICLC.

We conducted a clinical trial of a cancer vaccine using NY-ESO-1 protein with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) and/or OK-432 against solid tumors. A total of 15 patients were sequentially enrolled in 4 cohorts. Patients in cohort 1 received NY-ESO-1 protein; cohort 2a received NY-ESO-1 protein+OK-432; cohort 2b received NY-ESO-1 protein+poly-ICLC; cohort 3 received NY-ESO-1 protein+OK-432+poly-ICLC with Montanide ISA-51. The endpoints of this trial were safety, NY-ESO-1 immune responses, and clinical response. Vaccine-related adverse events observed were fever and injection-site reaction (grade 1). Two patients showed stable disease after vaccination. NY-ESO-1 antibodies were observed in 4 patients at the baseline (sero-positive) and augmented in all patients after vaccination. Eleven patients showed a conversion of negative antibody responses at baseline to positive after vaccination (seroconversion). The seroconversions were observed in all 11 sero-negative patients by the fourth immunization; in particular, it was observed by the second immunization in patients with poly-ICLC, and these induced antibody responses were stronger than those in patients immunized without poly-ICLC. The number of NY-ESO-1-specific interferon (IFN)γ-producing T cells was increased in patients immunized with poly-ICLC and/or OK-432, and furthermore, the increase of IFNγ-producing CD8 T cells in patients immunized with poly-ICLC was significantly higher than that in patients without poly-ICLC. Nonspecific activations of T-cell or antigen presenting cells were not observed. Our present study showed that poly-ICLC is a promising adjuvant for cancer vaccines.

Survival of Lung Adenocarcinoma Patients Predicted from Expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a) on Tumor Cells and Tumor-Infiltrating T Cells.

The immune status of tumors varies, and this may affect the overall survival (OS) of patients. We examined tumors from 120 patients with lung adenocarcinomas with a tissue microarray for T-cell infiltration and the expression of PD-L1 and Galectin-9 (both ligands for inhibitory receptors on T cells), and cancer/testis (CT) antigen XAGE1 (GAGED2a; a tumor antigen often found on lung tumors) expression, to determine their relevance to OS. Patients defined as pStage I-IIIA could be grouped, based on the expression profiles of PD-L1, Galectin-9, and XAGE1, into cluster A, who had prolonged survival, and cluster B, who had shorter survival. The difference in survival of the clusters was confirmed separately for pStage I and pStage II-IIIA patients. Cluster A patients who also had CD4 and CD8 T-cell infiltration showed even better survival, as expected. The findings were confirmed by examining an independent validation cohort of 68 pStage I lung adenocarcinoma patients. Our data showed that PD-L1 expression was a positive indicator, whereas Galectin-9 and XAGE1 expression was negative. In vitro analyses suggested that PD-L1 expression was upregulated by IFNγ secreted from activated T cells in the tumor and Galectin-9 expression was counteracting those T cells. Thus, use of these immune markers enables the creation of a discriminant function with which to classify tumors and predict survival. Cancer Immunol Res; 4(12); 1049-60. ©2016 AACR.

Increase in activated Treg in TIL in lung cancer and in vitro depletion of Treg by ADCC using an antihuman CCR4 mAb (KM2760).

INTRODUCTION: Tregs infiltrate tumors and inhibit immune responses against them. METHODS: We investigated subpopulations of Foxp3 CD4 T cells previously defined by Miyara et al. (Immunity 30, 899-911, 2009) in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) in lung cancer. We also showed that Tregs in healthy donors that express CCR4 could be efficiently eliminated in vitro by cotreatment with antihuman (h) CCR4 mAb (KM2760) and NK cells. RESULTS: In lung cancer, the number of activated/effector Tregs and non-Tregs, but not resting/naive Tregs, was increased in TILs compared with the number of those cells in PBMCs. The non-Treg population contained Th2 and Th17. CCR4 expression on activated/effector Tregs and non-Tregs in TILs was down-regulated compared with that on those cells in PBMCs. Chemokinetic migration of CD25 CD4 T cells containing the Treg population sorted from the PBMCs of healthy donors to CCL22/MDC was abrogated by pretreatment with anti-hCCR4 mAb (KM2760). The inhibitory activity of CD25 CD127 CD4 Tregs on the proliferative response of CD4 and CD8 T cells stimulated with anti-CD3/CD28 coated beads was abrogated by adding an anti-hCCR4 mAb (KM2760) and CD56 NK cells to the culture. CONCLUSIONS: The findings suggested the CCR4 on activated/effector Tregs and non-Tregs was functionally involved in the chemokinetic migration and accumulation of those cells to the tumor site. In vitro findings of efficient elimination of Tregs may give the basis for implementation of a clinical trial to investigate Treg depletion by administration of an anti-hCCR4 mAb to solid cancer patients.

Phase Ia Study of FoxP3+ CD4 Treg Depletion by Infusion of a Humanized Anti-CCR4 Antibody, KW-0761, in Cancer Patients.

PURPOSE: FoxP3(+) Tregs inhibit immune responses against tumors. KW-0761 is a humanized anti-human CCR4 monoclonal antibody (mAb) that has antibody-dependent cellular cytotoxicity activity. Depletion of CCR4-expressing FoxP3(+) CD4 Tregs by KW-0761 infusion was investigated in solid cancer patients. EXPERIMENTAL DESIGN: We conducted a phase Ia clinical trial of KW-0761 infusion in 7 lung and 3 esophageal cancer patients. Toxicity, clinical efficacy, changes in lymphocyte subpopulations, including Tregs, and induction of immune responses were analyzed. RESULTS: The results showed that KW-0761 infusion in a dose range between 0.1 mg/kg and 1.0 mg/kg was safe and well tolerated. No dose-limiting toxicity was observed. Four of 10 patients showed stable disease during treatment and were long survivors. The monitoring of FoxP3(+) Tregs in the peripheral blood mononuclear cells during treatment indicated efficient depletion of those cells, even at the lowest dose of 0.1 mg/kg used. The reduction in Th 1 CD4 T cells and CD8 T cells was limited, whereas a significant reduction was observed with Th 2 and Th 17 CD4 T cells. Immune responses to cancer/testis (CT) antigens and an autoantibody response to thyroid peroxidase were observed in some patients. CONCLUSIONS: The findings showed Tregs depletion and the possible occurrence of an immune response following KW-0761 infusion. Combined use of KW-0761 to deplete FoxP3(+) Tregs with other immunotherapies, such as cancer vaccines or checkpoint inhibitors, is a promising approach to augment immune responses.

Antibody response to cancer/testis (CT) antigens: A prognostic marker in cancer patients.

Immune responses to tumor antigens have been reported in cancer patients. However, the relevance of such spontaneous immune responses to the clinical course has not been studied extensively. We showed that the overall survival of patients with antibodies against NY-ESO-1 or XAGE1 (GAGED2a) antigen was prolonged in gastric or lung cancer patients, respectively.