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Yuji Yamamoto,1 Haruhiko Hirata,1 Takayuki Shiroyama,1 Tomoki Kuge,1 Kinnosuke Matsumoto,1 Midori Yoneda,1 Makoto Yamamoto,1 Yujiro Naito,1 Yasuhiko Suga,1 Kiyoharu Fukushima,1 Kotaro Miyake,1 Shohei Koyama,1 Kota Iwahori,1 Izumi Nagatomo,1 Yoshito Takeda,1 Atsushi Kumanogoh1– 4 1Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Department of Immunopathology, WPI, Immunology Frontier Research Center (iFReC), Osaka University, Suita, Osaka, Japan; 3Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka, Japan; 4Center for Infectious Diseases for Education and Research (CiDER), Osaka University, Suita, Osaka, JapanCorrespondence: Yuji Yamamoto, Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan, Tel +81 6-36879-3833, Fax +81 6-6879-3839, Email cyyamamoto1110@gmail.comPurpose: Pulmonary fibrosis and emphysema result in relatively maintained ventilation and reduced diffusing capacity. This pulmonary functional impairment complicates the evaluation of pulmonary function in patients with combined pulmonary fibrosis and emphysema (CPFE). Therefore, a single and easy-to-use pulmonary function index to evaluate patients with CPFE warrants further studies. Respiratory impedance can easily be provided by oscillometry and might be a candidate index to evaluate pulmonary function in patients with CPFE. As a preliminary study to assess the utility of respiratory impedance, we investigated the associations of physiological indices, including respiratory impedance, in patients with idiopathic pulmonary fibrosis (IPF) with and without emphysema.Patients and Methods: This retrospective study evaluated patients with IPF who did and did not satisfy the diagnostic criteria of CPFE. All patients underwent oscillometry, spirometry, and diffusing capacity for carbon monoxide (DLCO). Correlations of the obtained physiological indices were analyzed.Results: In total, 47 patients were included (18 and 29 patients with CPFE and IPF, respectively). Respiratory reactance (Xrs) at 5 Hz (X5) in the inspiratory phase was associated with forced vital capacity (FVC) % predicted in patients with CPFE (rS=0.576, P=0.012) and IPF (rS=0.539, P=0.003). Inspiratory X5 positively correlated with DLCO % predicted only in patients CPFE (rS=0.637, P=0.004).Conclusion: Emphysema might associate Xrs with ventilation and diffusing capacity in patients with IPF and emphysema. Given the multiple correlations of Xrs with FVC and DLCO, this study warrants further studies to verify the utility of oscillometry in a large-scale study for patients with CPFE.Keywords: chronic obstructive pulmonary disease, forced oscillation technique, gas exchange, idiopathic pulmonary fibrosis, ventilation

Background/aim: We herein examined T cell immunity in esophageal cancer patients with and without Helicobacter pylori infection to establish a foundation for immunotherapeutic strategies targeting esophageal cancer in the presence of H. pylori infection., Materials and Methods: Twenty-six patients with esophageal squamous cell carcinoma between 2015 and 2017 were enrolled in the present study. Serum antibodies against H. pylori were measured. Fresh tumor tissues were obtained by endoscopic biopsy or from surgical resection. A cell suspension of these tissues was subjected to a flow cytometric analysis., Results: Among the 26 patients analyzed, 10 (38.5%) were seropositive for H. pylori. The flow cytometric analysis of tumor-infiltrating lymphocytes revealed that the percentage of CD103 + CD4 + T cells in esophageal tumors was significantly lower in H. pylori-positive patients than in H. pylori-negative patients (p=0.0105). Conversely, the percentage of CD45RA-CD25hi effector Treg cells in esophageal tumors was significantly higher in H. pylori-positive patients than in H. pylori-negative patients (p=0.0022), indicating an immunosuppressive tumor microenvironment in the former. Following neoadjuvant chemotherapy, the number of CD45RA-CD25hi effector Treg cells decreased (p=0.0248)., Conclusion: The tumor immune microenvironment of esophageal cancer patients with H. pylori infection exhibited an immunosuppressive phenotype. The targeting of Treg cells has potential in immunotherapy for this patient population., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

Background: CCR8-expressing regulatory T cells (Tregs) are selectively localized within tumors and have gained attention as potent suppressors of anti-tumor immunity. This study focused on CCR8 + Tregs and their interaction with CD8 + T cells in the tumor microenvironment of human lung cancer. We evaluated their spatial distribution impact on CD8 + T cell effector function, specifically granzyme B (GzmB) expression, and clinical outcomes., Methods: A total of 81 patients with lung squamous cell carcinoma (LSCC) who underwent radical surgical resection without preoperative treatment were enrolled. Histological analyses were performed, utilizing an automated image analysis system for double-stained immunohistochemistry assays of CCR8/Foxp3 and GzmB/CD8. We investigated the association of CCR8 + Tregs and GzmB + CD8 + T cells in tumor tissues and further evaluated the prognostic impact of their distribution profiles., Results: Histological evaluation using the region of interest (ROI) protocol showed that GzmB expression levels in CD8 + T cells were decreased in areas with high infiltration of CCR8 + Tregs, suggesting a suppressive effect of CCR8 + Tregs on T cell cytotoxicity in the local tumor microenvironment. Analysis of the association with clinical outcomes showed that patients with more CCR8 + Tregs and lower GzmB expression, represented by a low GzmB/CCR8 ratio, had worse progression-free survival., Conclusions: Our data suggest that local CCR8 + Treg accumulation is associated with reduced CD8 + T cell cytotoxic activity and poor prognosis in LSCC patients, highlighting the biological role and clinical significance of CCR8 + Tregs in the tumor microenvironment. The GzmB/CCR8 ratio may be a useful prognostic factor for future clinical applications in LSCC., (© 2024. The Author(s).)

Background: Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes., Objective: We sought to identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs)., Methods: We performed data-independent acquisition of serum EVs from 4 healthy controls, 4 noneosinophilic asthma (NEA) patients, and 4 eosinophilic asthma (EA) patients to identify novel BMs for BA. We confirmed EA-specific BMs via data-independent acquisition validation in 61 BA patients and 23 controls. To further validate these findings, we performed data-independent acquisition for 6 patients with chronic rhinosinusitis without nasal polyps and 7 patients with chronic rhinosinusitis with nasal polyps., Results: We identified 3032 proteins, 23 of which exhibited differential expression in EA. Ingenuity pathway analysis revealed that protein signatures from each phenotype reflected disease characteristics. Validation revealed 5 EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase, major basic protein, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. The potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnostic capability and detection of airway obstruction. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified in EVs and tissues from patients with chronic rhinosinusitis with nasal polyps, 5 (including Gal10 and eosinophil peroxidase) showed significant correlations between EVs and tissues. Gal10 release from EVs was implicated in eosinophil extracellular trapped cell death in vitro and in vivo., Conclusion: Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target for liquid biopsy approaches., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells., Methods: The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors., Results: The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4 + and CD8 + T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors., Conclusions: In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy., Competing Interests: Competing interests: There are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tetracyclines exhibit anti-viral, anti-inflammatory, and immunomodulatory activities via various mechanisms. The present study investigated the efficacy and safety of demeclocycline in patients hospitalized with mild-to-moderate COVID-19 via an open-label, multicenter, parallel-group, randomized controlled phase 2 trial. Primary and secondary outcomes included changes from baseline (day 1, before the study treatment) in lymphocytes, cytokines, and SARS-CoV-2 RNA on day 8. Seven, seven, and six patients in the control, demeclocycline 150 mg daily, and demeclocycline 300 mg daily groups, respectively, were included in the modified intention-to-treat population that was followed until day 29. A significant change of 191.3/μL in the number of CD4 +  T cells from day 1 to day 8 was observed in the demeclocycline 150 mg group (95% CI 5.1/μL-377.6/μL) (p = 0.023), whereas that in the control group was 47.8/μL (95% CI - 151.2/μL to 246.8/μL), which was not significant (p = 0.271). The change rates of CD4 +  T cells negatively correlated with those of IL-6 in the demeclocycline-treated groups (R = - 0.807, p = 0.009). All treatment-emergent adverse events were of mild-to-moderate severity. The present results indicate that the treatment of mild-to-moderate COVID-19 patients with demeclocycline elicits immune responses conducive to recovery from COVID-19 with good tolerability.Trial registration: This study was registered with the Japan Registry of Clinical Trials (Trial registration number: jRCTs051200049; Date of the first registration: 26/08/2020)., (© 2023. Springer Nature Limited.)

Objectives: Limited treatment options are available for non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD). The rationale for immunotherapy and its adverse events for NSCLC with ILD remains unclear. In this study, we examined T cell profiles and functions in the lung tissues of NSCLC patients with or without ILD to provide evidence for the potential mechanism of immune checkpoint inhibitor (ICI)-related pneumonitis in NSCLC patients with ILD., Material and Methods: We investigated T cell immunity in the lung tissues of NSCLC patients with ILD to support the application of immunotherapy for these patients. We analyzed T cell profiles and functions in surgically resected lung tissues from NSCLC patients with and without ILD. The T cell profiles of infiltrating cells in lung tissues were analyzed by flow cytometry. T cell functions were measured based on cytokine production by T cells stimulated with phorbol 12-myristate 13-acetate and ionomycin., Results: The percentages of CD4 +  T cells expressing immune checkpoint molecules (Tim-3, ICOS, and 4-1BB), CD103 + CD8 +  T cells, and regulatory T (Treg) cells were higher in NSCLC patients with than in those without ILD. A functional analysis of T cells in lung tissues indicated that CD103 + CD8 +  T cells positively correlated with IFNγ production, whereas Treg cells negatively correlated with IFNγ and TNFα production. Cytokine production by CD4 +  and CD8 +  T cells did not significantly differ between NSCLC patients with and without ILD, except for TNFα production by CD4 +  T cells being lower in the former than in the latter., Conclusion: In NSCLC patients with ILD stable for surgery, T cells were active participants and balanced in part by Treg cells in lung tissues, suggesting the potential development of ICI-related pneumonitis in NSCLC patients with ILD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Minocycline is often administered prophylactically or therapeutically to non-small cell lung cancer (NSCLC) patients receiving epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) for skin rash as an adverse event. We examined the effects of minocycline on the outcomes of EGFR-mutant NSCLC treated with first-line EGFR-TKIs based on a single-center retrospective analysis. In this retrospective cohort study, data were collected on NSCLC patients treated with first-line EGFR-TKIs between January 2010 and June 2021. The treatment efficacy of first-line EGFR-TKIs was compared between patients who received minocycline and those who did not. Median progression-free survival (PFS) with first-line EGFR-TKIs was significantly longer in the minocycline group (N = 32) than in the control group (N = 106); 714 (95% confidence interval CI 411-1247) days vs. 420 (95% CI 343-626) days, p = 0.019. A multivariate analysis including skin rash as a variable confirmed that the administration of minocycline for 30 days or longer correlated with good PFS and overall survival (OS) with first-line EGFR-TKIs (HR 0.44 [95% CI 0.27-0.73], p = 0.0014 and HR 0.50 [95% CI 0.27-0.92], p = 0.027, respectively). The administration of minocycline influenced good treatment efficacy with first-line EGFR-TKIs independently of skin rash., (© 2023. The Author(s).)

Immune checkpoint inhibitors (ICIs) have caused revolutionary changes in cancer treatment, but low response rates remain a challenge. Semaphorin 4A (Sema4A) modulates the immune system through multiple mechanisms in mice, although the role of human Sema4A in the tumor microenvironment remains unclear. This study demonstrates that histologically Sema4A-positive non-small cell lung cancer (NSCLC) responded significantly better to anti-programmed cell death 1 (PD-1) antibody than Sema4A-negative NSCLC. Intriguingly, SEMA4A expression in human NSCLC was mainly derived from tumor cells and was associated with T cell activation. Sema4A promoted cytotoxicity and proliferation of tumor-specific CD8 + T cells without terminal exhaustion by enhancing mammalian target of rapamycin complex 1 and polyamine synthesis, which led to improved efficacy of PD-1 inhibitors in murine models. Improved T cell activation by recombinant Sema4A was also confirmed using isolated tumor-infiltrating T cells from patients with cancer. Thus, Sema4A might be a promising therapeutic target and biomarker for predicting and promoting ICI efficacy.