Single-Cell Transcriptome Profiling of Pancreatic Islets From Early Diabetic Mice Identifies Anxa10 for Ca2+ Allostasis Toward ß-Cell Failure.

Type 2 diabetes is a progressive disorder denoted by hyperglycemia and impaired insulin secretion. Although a decrease in ß-cell function and mass is a well-known trigger for diabetes, the comprehensive mechanism is still unidentified. Here, we performed single-cell RNA sequencing of pancreatic islets from prediabetic and diabetic db/db mice, an animal model of type 2 diabetes. We discovered a diabetes-specific transcriptome landscape of endocrine and nonendocrine cell types with subpopulations of ß- and α-cells. We recognized a new prediabetic gene, Anxa10, that was induced by and regulated Ca2+ influx from metabolic stresses. Anxa10-overexpressed ß-cells displayed suppression of glucose-stimulated intracellular Ca2+ elevation and potassium-induced insulin secretion. Pseudotime analysis of ß-cells predicted that this Ca2+-surge responder cluster would proceed to mitochondria dysfunction and endoplasmic reticulum stress. Other trajectories comprised dedifferentiation and transdifferentiation, emphasizing acinar-like cells in diabetic islets. Altogether, our data provide a new insight into Ca2+ allostasis and ß-cell failure processes. ARTICLE HIGHLIGHTS: The transcriptome of single-islet cells from healthy, prediabetic, and diabetic mice was studied. Distinct ß-cell heterogeneity and islet cell-cell network in prediabetes and diabetes were found. A new prediabetic ß-cell marker, Anxa10, regulates intracellular Ca2+ and insulin secretion. Diabetes triggers ß-cell to acinar cell transdifferentiation.

The Association of Short Physical Performance Battery With Mortality and Hospitalization in Patients Receiving Hemodialysis.

OBJECTIVE: The Short Physical Performance Battery (SPPB) is recommended for evaluating physical performance in patients on hemodialysis (HD). However, the association between SPPB score and long-term health outcomes in these patients remains unclear. We examined the association of SPPB score with all-cause mortality, all-cause hospitalization, and cardiovascular hospitalization in patients on HD. DESIGN AND METHODS: This retrospective cohort study included 326 patients (median age, 68 years; 62% men) who received outpatient HD therapy. These patients were divided into 2 SPPB groups: low (SPPB ≤9) and high (SPPB >9). We investigated the association of SPPB score and their change over time with health outcomes using Cox regression analysis. RESULTS: Low SPPB score was associated with a higher risk for all-cause mortality (hazard ratio [HR]: 3.19, 95% confidence interval [95% CI]: 1.89-5.38), all-cause hospitalization (HR: 2.01, 95% CI: 1.44-2.82), and cardiovascular hospitalization (HR: 2.20, 95% CI: 1.45-3.35). Additionally, change in SPPB score over 1 year was associated with health outcomes. CONCLUSIONS: Lower SPPB score was significantly associated with a higher risk for all-cause mortality, all-cause hospitalization, and cardiovascular hospitalization. The SPPB may be a valuable indicator for risk stratification in patients on HD. Additionally, preventive treatments may be an effective management strategy in limiting the high mortality and hospitalization rates in patients with decreased SPPB score.

Changes in body composition of patients undergoing hemodialysis during the coronavirus disease 2019 pandemic: a retrospective longitudinal study.

Background: The spread of coronavirus disease 2019 (COVID-19) has dramatically altered the lifestyles of many people worldwide. Several studies reported that body weight of young adults increased during the COVID-19 pandemic; however, weight loss has been observed in the elderly population. Therefore, trends in body composition due to the COVID-19 pandemic may vary depending on the characteristics of the population. This study aimed to investigate the changes in body mass index (BMI), muscle mass, and fat mass before and during the COVID-19 pandemic among patients undergoing hemodialysis. Methods: In this retrospective longitudinal study, we enrolled 115 clinically stable outpatients (mean age: 65.7 ± 11.2 years, 62.6% men) who underwent hemodialysis thrice a week. Baseline data were collected between April 2019 and March 2020, before the declaration of the COVID-19 emergency by the Japanese government. The follow-up measurements were performed between July 2020 and March 2021 during the COVID-19 pandemic. Patient characteristics, laboratory data, and BMI measurements were collected from the medical records. Muscle mass and fat mass were measured using bioelectrical impedance analysis. Results: BMI and fat mass among the study participants were significantly higher during the COVID-19 pandemic than before the pandemic (p < 0.01), but no significant change in muscle mass was observed. A restricted cubic spline function showed that the increase in BMI appeared to correlate well with fat mass, but not with muscle mass. Conclusions: BMI and fat mass of patients on hemodialysis significantly increased due to preventive measures against the COVID-19 pandemic in Japan. These findings may provide useful information in making nutritional management decisions for patients undergoing hemodialysis during and after the COVID-19 pandemic.

[One Case of Accessory Breast Cancer Complicated by Contralateral Breast Cancer].

We experienced a case of right sided accessory breast cancer complicated by contralateral breast cancer. A 50-year-old woman came to us for an examination because a tumor in her left breast was pointed out at breast cancer screening. A breast MRI confirmed a tumor in her left breast and a tumor continuing from the skin to the subcutis of the right axilla. A skin biopsy for the tumor in the right axilla and a core needle biopsy(CNB)for the tumor in the left breast were performed. The pathological result of the CNB for the left breast indicated an invasive ductal carcinoma of the tubular formative scirrhous type. Although the tumor of the right axilla was poorly differentiated adenocarcinoma demonstrating cord-like arrays, it was examined by skin biopsy and therefore no deep part of the tissue was included. We conducted immunostaining, in consideration of the possibility of metastasis from the left sided breast cancer. ER, PgR, mammaglobin, GATA 3 were positive, strongly suggesting that the tumor in the right axilla was also derived from a mammary gland. We also performed a wide local excision of the right axilla plus axillary dissection(level Ⅰ)in addition to conducting a left mastectomy plus sentinel lymph node biopsy, in consideration of the possibility of primary right sided accessory breast cancer. The pathological result following surgery confirmed a difference in the histologic features between both sides, residual normal accessory mammary glands around the tumor on the right side, and the presence of rich DCIS and a lobular replacement image, leading to a definitive diagnosis of primary invasive ductal carcinoma of the accessory breast on the right side.

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16high Cells In Vivo.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-CreERT2-tdTomato mouse model to analyze the in vivo characteristics of p16high cells at a single-cell level. We found tdTomato-positive p16high cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16high cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16high cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.

The increased frequency of methicillin-resistant Staphylococcus aureus with low MIC of beta-lactam antibiotics isolated from hospitalized patients.

Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infectious diseases and can be life-threatening in healthcare-settings. MRSA is classified into health-care associated (HA)-MRSA strains and community acquired (CA)-MRSA strains based on genotype and phenotype. CA-MRSA has been reported to show the lower minimal inhibitory concentration (MIC) of some antibiotics as compared to HA-MRSA. Recently, the prevalence of CA-MRSA has been increased in worldwide. CA-MRSA is isolated not only from the healthy individuals in a community but also from the patients in healthcare settings. However, the changing trend in frequency of HA-MRSA and CA-MRSA in the hospital setting is not clear. Therefore, we analyzed the trend of MIC to speculate the frequency of HA-MRSA and CA-MRSA in the facility. Moreover, gene mutations were evaluated on resistant gene loci with next generation sequencer. The frequency of strains with low MIC of beta-lactam antibiotics was gradually increased in isolated MRSA strains from the hospitalized patients. Whole genome analysis revealed the frequency of gene mutation was also decreased in some resistant loci, such as blaZ and blaR1. These findings highlight the changing trend of MRSA strains isolated from hospitalized patients.

Collagen adhesion gene is associated with bloodstream infections caused by methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. METHODS: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. RESULTS: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. CONCLUSIONS: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

In vitro expansion of endogenous human alveolar epithelial type II cells in fibroblast-free spheroid culture.

Alveolar epithelial type II cells (AEC2) are stem cells of the alveoli and play crucial roles in maintaining lung homeostasis and the pathogenesis of lung diseases. We recently reported on an organoid culture system for endogenous murine AEC2. Despite advances in generation of human induced pluripotent stem cell-derived AEC2, in vitro expansion of endogenous human AEC2 has not been reported and genetic manipulation of human AEC2 has been difficult. Here, we show that endogenous human AEC2 could be cultured and passaged using a three-dimensional culture system with a specific combination of signal ligands and inhibitors. The culture system was suitable for retroviral gene transduction into AEC2. Transduction of pulmonary fibrosis-associated mutant surfactant protein C (SFPTCΔexon4) into AEC2 revealed characteristic transcriptional traits similar to those of patients with idiopathic pulmonary fibrosis. Our culture system will be a useful tool for investigating human AEC2 functions in vitro.

Gli signaling pathway modulates fibroblast activation and facilitates scar formation in pulmonary fibrosis.

Pulmonary fibrosis is characterized by progressive and irreversible scarring of alveoli, which causes reduction of surface epithelial area and eventually respiratory failure. The precise mechanism of alveolar scarring is poorly understood. In this study, we explored transcriptional signatures of activated fibroblasts in alveolar airspaces by using intratracheal transfer in bleomycin-induced lung fibrosis. Lung fibroblasts transferred into injured alveoli upregulated genes related to translation and metabolism in the first two days, and upregulated genes related to extracellular matrix (ECM) production between day 2 and 7. Upstream analysis of these upregulated genes suggested possible contribution of hypoxia-inducible factors 1a (Hif1a) to fibroblast activation in the first two days, and possible contribution of kruppel-like factor 4 (Klf4) and glioma-associated oncogene (Gli) transcription factors to fibroblast activation in the following profibrotic phase. Fibroblasts purified based on high Acta2 expression after intratracheal transfer were also characterized by ECM production and upstream regulation by Klf4 and Gli proteins. Pharmacological inhibition of Gli proteins by GANT61 in bleomycin-induced lung fibrosis altered the pattern of scarring characterized by dilated airspaces and smaller fibroblast clusters. Activated fibroblasts isolated from GANT61-treated mice showed decreased migration capacity, suggesting that Gli signaling inhibition attenuated fibroblast activation. In conclusion, we revealed transcriptional signatures and possible upstream regulators of activated fibroblasts in injured alveolar airspaces. The altered scar formation by Gli signaling inhibition supports that activated fibroblasts in alveolar airspaces may play a critical role in scar formation.

Transcriptome network analysis identifies protective role of the LXR/SREBP-1c axis in murine pulmonary fibrosis.

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Although lung fibroblasts play a central role in PF, the key regulatory molecules involved in this process remain unknown. To address this issue, we performed a time-course transcriptome analysis on lung fibroblasts of bleomycin- and silica-treated murine lungs. We found gene modules whose expression kinetics were associated with the progression of PF and human idiopathic PF (IPF). Upstream analysis of a transcriptome network helped in identifying 55 hub transcription factors that were highly connected with PF-associated gene modules. Of these hubs, the expression of Srebf1 decreased in line with progression of PF and human IPF, suggesting its suppressive role in fibroblast activation. Consistently, adoptive transfer and genetic modification studies revealed that the hub transcription factor SREBP-1c suppressed PF-associated gene expression changes in lung fibroblasts and PF pathology in vivo. Moreover, therapeutic pharmacological activation of LXR, an SREBP-1c activator, suppressed the Srebf1-dependent activation of fibroblasts and progression of PF. Thus, SREBP-1c acts as a protective hub of lung fibroblast activation in PF. Collectively, the findings of the current study may prove to be valuable in the development of effective therapeutic strategies for PF.

Mesenchymal-Epithelial Interactome Analysis Reveals Essential Factors Required for Fibroblast-Free Alveolosphere Formation.

Lung epithelial cells and fibroblasts are key cell populations in lung development. Fibroblasts support type 2 alveolar epithelial cells (AEC2) in the developing and mature lung. However, fibroblast-AEC2 interactions have not been clearly described. We addressed this in the present study by time course serial analysis of gene expression sequencing (SAGE-seq) of epithelial cells and fibroblasts of developing and mature murine lungs. We identified lung fibroblast-epithelial interactions that potentially regulate alveologenesis and are mediated by fibroblast-expressed ligands and epithelial cell surface receptors. In the epithelial-fibroblast co-culture alveolosphere formation assay, single intervention against fibroblast-expressed ligand or associated signaling cascades promoted or inhibited alveolosphere growth. Adding the ligand-associated molecules fibroblast growth factor 7 and Notch ligand and inhibitors of bone morphogenetic protein 4, transforming growth factor ß, and glycogen synthase kinase-3ß to the culture medium enabled fibroblast-free alveolosphere formation. The results revealed the essential factors regulating fibroblast-AEC2 interactions.

Lung fibroblasts express a miR-19a-19b-20a sub-cluster to suppress TGF-ß-associated fibroblast activation in murine pulmonary fibrosis.

Lung fibroblasts play a pivotal role in pulmonary fibrosis, a devastating lung disease, by producing extracellular matrix. MicroRNAs (miRNAs) suppress numerous genes post-transcriptionally; however, the roles of miRNAs in activated fibroblasts in fibrotic lungs remain poorly understood. To elucidate these roles, we performed global miRNA-expression profiling of fibroblasts from bleomycin- and silica-induced fibrotic lungs and investigated the functions of miRNAs in activated lung fibroblasts. Clustering analysis of global miRNA-expression data identified miRNA signatures exhibiting increased expression during fibrosis progression. Among these signatures, we found that a miR-19a-19b-20a sub-cluster suppressed TGF-ß-induced activation of fibroblasts in vitro. Moreover, to elucidate whether fibroblast-specific intervention against the sub-cluster modulates pathogenic activation of fibroblasts in fibrotic lungs, we intratracheally transferred the sub-cluster-overexpressing fibroblasts into bleomycin-treated lungs. Global transcriptome analysis of the intratracheally transferred fibroblasts revealed that the sub-cluster not only downregulated expression of TGF-ß-associated pro-fibrotic genes, including Acta2, Col1a1, Ctgf, and Serpine1, but also upregulated expression of the anti-fibrotic genes Dcn, Igfbp5, and Mmp3 in activated lung fibroblasts. Collectively, these findings indicated that upregulation of the miR-19a-19b-20a sub-cluster expression in lung fibroblasts counteracted TGF-ß-associated pathogenic activation of fibroblasts in murine pulmonary fibrosis.

Chemical synthesis and tyrosinase-inhibitory activity of isotachioside and its related glycosides.

Isotachioside (1) and its related natural product 2 are isolated from Isotachis japonica and Protea neriifolia, respectively, and are categorized as analogs of arbutin (3), a tyrosinase inhibitor for practical use. Both of the natural products and several derivatives such as glucoside 4, xyloside 5, cellobioside 6, and maltoside 7 were synthesized via Schmidt glycosylation as a key step, and their tyrosinase inhibitory activity was evaluated. The half maximal inhibitory concentration (IC50) of 1-3 could not be determined even when the concentration was increased to 1000 µM. Contrastingly, glycosides 4-7, missing methyl and benzoyl groups, acted as tyrosinase inhibitors with IC50s of 417 µM, 852 µM, 623 µM, and 657 µM, respectively. Among these novel inhibitors, derivative 4 was the most potent, indicating that the structural combination of resorcinol and glucose was significant for inducing the inhibitory effect.

Prognostic Effect of Lymphovascular Invasion on TNM Staging in Stage I Non-Small-cell Lung Cancer.

INTRODUCTION: Lymphovascular invasion (LVI) is a known adverse prognostic factor for early-stage non-small-cell lung cancer (NSCLC). Nonetheless, the prognostic effect of LVI on TNM staging of stage I NSCLC remains inconclusive. We thus hypothesized that it might be better to upstage pathologic stage IA NSCLC with LVI to pathologic stage IB NSCLC. PATIENTS AND METHODS: Using a Cox proportional hazards model, we examined the effect of LVI on disease-specific survival (DSS) in stage IA versus stage IB disease in 660 consecutive patients with stage I NSCLC (598 with adenocarcinoma, 62 with squamous cell carcinoma) who had undergone complete resection. RESULTS: On univariable analysis of stage IA cases, vascular invasion (VI) was significantly associated with inferior DSS (univariable hazard ratio [HR], 3.39; 95% confidence interval [CI], 1.46-7.89; P = .005). In contrast, lymphatic invasion exhibited a tendency toward inferior DSS (univariable HR, 2.90; 95% CI, 0.97-8.66; P = .056). Multivariable analysis of DSS in stage IA cases identified VI as an independent significant prognostic factor (multivariable HR, 2.86; 95% CI, 1.58-5.18; P = .007). With VI, DSS was significantly poorer for stage IB than for stage IA patients without VI (univariable HR, 3.44; 95% CI, 1.67-7.09; P < .001). In contrast, no difference was observed between patients with stage IA and VI and stage IB patients (P = .97). CONCLUSION: The presence of VI independently and significantly affects DSS in patients with stage IA NSCLC. We found that stage IA with VI and stage IB disease had equivalent prognostic outcomes. Our results suggest that stage IA with VI should be upstaged to IB in the TNM classification of NSCLC.

Photocatalytic Reduction of Low Concentration of CO2.

A novel molecular photocatalytic system with not only high reduction ability of CO2 but also high capture ability of CO2 has been developed using a Ru(II)-Re(I) dinuclear complex as a photocatalyst. By using this photocatalytic system, CO2 of 10% concentration could be selectively converted to CO with almost same photocatalysis to that under a pure CO2 atmosphere (TONCO > 1000, ΦCO > 0.4). Even 0.5% concentration of CO2 was reduced with 60% initial efficiency of CO formation by using the same system compared to that using pure CO2 (TONCO > 200). The Re(I) catalyst unit in the photocatalyst can efficiently capture CO2, which proceeds CO2 insertion to the Re-O bond, and then reduce the captured CO2 by using an electron supplied from the photochemically reduced Ru photosensitizer unit.

EFFECT OF INTERNAL LIMITING MEMBRANE PEELING DURING VITRECTOMY FOR DIABETIC MACULAR EDEMA: Systematic Review and Meta-analysis.

PURPOSE: To evaluate the effect of internal limiting membrane (ILM) peeling during vitrectomy for diabetic macular edema. METHODS: MEDLINE, EMBASE, and CENTRAL were systematically reviewed. Eligible studies included randomized or nonrandomized studies that compared surgical outcomes of vitrectomy with or without ILM peeling for diabetic macular edema. The primary and secondary outcome measures were postoperative best-corrected visual acuity and central macular thickness. Meta-analysis on mean differences between vitrectomy with and without ILM peeling was performed using inverse variance method in random effects. RESULTS: Five studies (7 articles) with 741 patients were eligible for analysis. Superiority (95% confidence interval) in postoperative best-corrected visual acuity in ILM peeling group compared with nonpeeling group was 0.04 (-0.05 to 0.13) logMAR (equivalent to 2.0 ETDRS letters, P = 0.37), and superiority in best-corrected visual acuity change in ILM peeling group was 0.04 (-0.02 to 0.09) logMAR (equivalent to 2.0 ETDRS letters, P = 0.16). There was no significant difference in postoperative central macular thickness and central macular thickness reduction between the two groups. CONCLUSION: The visual acuity outcomes using pars plana vitrectomy with ILM peeling versus no ILM peeling were not significantly different. A larger randomized prospective study would be necessary to adequately address the effectiveness of ILM peeling on visual acuity outcomes.

Development of a novel molecular detection method for clustered regularly interspaced short palindromic repeats (CRISPRs) in Taylorella organisms.

Contagious equine metritis is a bacterial infectious disease of horses caused by Taylorella equigenitalis, a Gram-negative eubacterium. The disease has been described in several continents, including Europe, North America and Asia. A novel molecular method was developed to detect clustered regularly interspaced short palindromic repeats (CRISPRs), which were separated by non-repetitive unique spacer regions (NRUSRs) of similar length, in the Taylorella equigenitalis EQ59 strain using a primer pair, f-/r-TeCRISPR-ladder, by PCR amplification. In total, 31 Taylorella isolates (17 T. equigenitalis and 14 Taylorella asinigenitalis) were examined. The T. equigenitalis isolates came from thoroughbred and cold-blooded horses from nine countries during 1980-1996, whilst the T. asinigenitalis isolates all originated from donkey jacks in France and the USA during 1997-2006. PAGE fractionated all of the 13 CRISPRs separated by 12 NRUSRs in T. equigenitalis EQ59. Permutation examples of CRISPRs, which were separated by NRUSRs for small-sized ladders, consisting of two doublet bands were shown. Putative CRISPRs separated by NRUSRs were amplified with 14/17 (82.4 %) geographically disparate T. equigenitalis isolates using the newly designed primer pair. Approximately 82.4 % of the T. equigenitalis isolates had CRISPRs separated by NRUSRs. The CRISPR locus was also found in the French T. asinigenitalis strain MCE3. Putative CRISPRs separated by NRUSRs were detected similarly in 4/14 (28.6 %) T. asinigenitalis isolates. Overall, a more detailed understanding of the molecular biology of CRISPRs within Taylorella organisms may help elucidate the pathogenic virulence and transmission mechanisms associated with this important equine pathogen.

Molecular analysis of the tlyA gene in Campylobacter lari.

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.

Robust Antitumor Effects of Combined Anti-CD4-Depleting Antibody and Anti-PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice.

Depletion of CD4(+) cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4(+) cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti-CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25(+) Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8(+) cell depletion. CD4(+) cell depletion led to the proliferation of tumor-specific CD8(+) T cells in the draining lymph node and increased infiltration of PD-1(+)CD8(+) T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies.