Immune checkpoint molecule Tim-3 promotes NKT cell apoptosis and predicts poorer prognosis in Sepsis.

BACKGROUND: Sepsis is a leading cause of death among critically ill patients, which is defined as life-threatening organ dysfunction caused by a deregulated host immune response to infection. Immune checkpoint molecule Tim-3 plays important and complex roles in regulating immune responses and in inducing immune tolerance. Although immune checkpoint blockade would be expected as a promising therapeutic strategy for sepsis, but the underlying mechanism remain unknown, especially under clinical conditions. METHODS: Tim-3 expression and apoptosis in NKT cells were compared in septic patients (27 patients with sepsis and 28 patients with septic shock). Phenotypic and functional characterization of Tim-3+ NKT cells were analysed, and then the relationship between Tim-3 + NKT cells and clinical prognosis were investigated in septic patients. α-lactose (Tim-3/Galectin-9 signalling inhibitor) and Tim-3 mutant mice (targeting mutation of the Tim-3 cytoplasmic domain) were utilized to evaluate the protective effect of Tim-3 signalling blockade following septic challenge. RESULTS: There is a close correlation between Tim-3 expression and the functional status of NKT cells in septic patients, Upregulated Tim-3 expression promoted NKT cell activation and apoptosis during the early stage of sepsis, and it was associated with worse disease severity and poorer prognosis in septic patients. Blockade of the Tim-3/Galectin-9 signal axis using α-lactose inhibited in vitro apoptosis of NKT cells isolated from septic patients. Impaired activity of Tim-3 protected mice following septic challenge. CONCLUSIONS: Overall, these findings demonstrated that immune checkpoint molecule Tim-3 in NKT cells plays a critical role in the immunopathogenesis of septic patients. Blockade of immune checkpoint molecule Tim-3 may be a promising immunomodulatory strategy in future clinical practice for the management of sepsis.

Administration of Recombinant TAPBPL Protein Ameliorates Collagen-Induced Arthritis in Mice.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease distinguished by synovial hyperplasia and a progressive destruction of joints. T cells are critical players in the pathogenesis of RA. We have previously identified a novel immune checkpoint molecule, TAPBPL, that inhibits T cell functions in vitro. As a model for human RA, we investigated the ability of the TAPBPL protein to ameliorate collagen type II (CII)-induced arthritis (CIA) in mice that were injected with recombinant TAPBPL or a control protein. The mice were analyzed for CIA development, immune cells, and their responses. We found that TAPBPL protein significantly decreased CIA incidence and reduced clinical and pathological arthritis scores, which were related to a lower number of activated CD4 T cells but a greater number of regulatory T cells (Tregs) in the spleen, and a reduction of Th1/Th17 inflammatory cytokines in the joints and serum. Importantly, TAPBPL protein inhibited CII-specific T cell growth and Th1 and Th17 cytokine expression and reduced the production of CII autoantibodies in the serum. Our results suggest that TAPBPL protein can ameliorate CIA in mice and has the potential to be used in the treatment of patients with RA.

PD-1/PD-L1 Control of Antigen-Specifically Activated CD4 T-Cells of Neonates.

Newborns are highly susceptible to infections; however, the underlying mechanisms that regulate the anti-microbial T-helper cells shortly after birth remain incompletely understood. To address neonatal antigen-specific human T-cell responses against bacteria, Staphylococcus aureus (S. aureus) was used as a model pathogen and comparatively analyzed in terms of the polyclonal staphylococcal enterotoxin B (SEB) superantigen responses. Here, we report that neonatal CD4 T-cells perform activation-induced events upon S. aureus/APC-encounter including the expression of CD40L and PD-1, as well as the production of Th1 cytokines, concomitant to T-cell proliferation. The application of a multiple regression analysis revealed that the proliferation of neonatal T-helper cells was determined by sex, IL-2 receptor expression and the impact of the PD-1/PD-L1 blockade. Indeed, the treatment of S. aureus-activated neonatal T-helper cells with PD-1 and PD-L1 blocking antibodies revealed the specific regulation of the immediate neonatal T-cell responses with respect to the proliferation and frequencies of IFNγ producers, which resembled in part the response of adults' memory T-cells. Intriguingly, the generation of multifunctional T-helper cells was regulated by the PD-1/PD-L1 axis exclusively in the neonatal CD4 T-cell lineage. Together, albeit missing memory T-cells in neonates, their unexperienced CD4 T-cells are well adapted to mount immediate and strong anti-bacterial responses that are tightly controlled by the PD-1/PD-L1 axis, thereby resembling the regulation of recalled memory T-cells of adults.

VSIG4/CRIg directly regulates early CD8+ T cell activation through its counter-receptor in a narrow window.

T-cell responses are fine-tuned by positive and negative co-signal molecules expressed on immune cells and adjacent tissues. VSIG4 is a newly identified member of the B7 family of ligands, which negatively regulates innate inflammatory and CD4+ T cell-mediated responses. However, little is known about the direct effects of VSIG4, which are exerted through an unidentified counter-receptor on CD8+ T cells. We investigated the binding of the VSIG4-Ig fusion protein during CD8+ T cell activation, and the functional involvement of VSIG4 pathway, using VSIG4-Ig and VSIG4-transfectants. VSIG4-Ig binding to CD8+ T cells was temporally observed in the CD44high phenotype during initial activation. VSIG4-Ig binding was observed earlier than the induction of PD-1, LAG3, and TIM-3, which are immune checkpoint receptors for exhausted CD8+ T cells. Immobilized VSIG4-Ig inhibited anti-CD3/CD28 mAb-induced CD8+ T cell activation, as indicated by proliferation and IFN-γ production, similar to the downregulation of T-bet and Eomesodermin transcription factors. VSIG4 on FcγR+ P815 or specific antigen-presenting E.G7 cells inhibited the generation of effector CD8+ T cells, as indicated by proliferation, IFN-γ and TNF-α expression, and granule degradation, compared to parental cells. However, the window for the regulatory function of VSIG4 was narrow and dependent on the strength of TCR (and CD28)-mediated signals. Our results suggested that VSIG4 directly delivers co-inhibitory signals via an as-yet unidentified counter-receptor on activated CD8+ T cells. VSIG4-mediated CD8+ T cell tolerance might contribute to the steady-state maintenance of homeostasis.

The mechanism of low-dose radiation-induced upregulation of immune checkpoint molecule expression in lung cancer cells.

INTRODUCTION: This study explored the effect of low-dose radiation on the expression of immune checkpoint molecules in lung cancer cells and its mechanism, as well as the antitumour effect of combined low-dose radiation and immune checkpoint inhibitors. METHODS: Western blot analysis was used to assess the expression of the immune checkpoint molecules CD47, PD-L1, FGL-1 and CD155 in lung cancer cells after radiation. Western blotting was also used to explore changes in the JAK2/STAT3 pathway. CD8+ T lymphocyte infiltration in tumour tissues were assessed by immunohistochemistry in a mouse model. The inhibitory effect of low-dose radiation combined with PD-L1 or CD47 inhibitors on tumor growth was evaluated by measuring tumor volume. RESULTS: In response to low-dose irradiation, the expression of CD47 and PD-L1 in A549 and LLC cells was increased, the expression of p-JAK2 and p-STAT3 was also increased. AG490-mediated inhibition of the JAK2/STAT3 pathway before irradiation significantly reduced the expression of p-JAK2 and p-STAT3 in lung cancer cells, in the meantime, expression of CD47 and PD-L1 was also reduced. Conventional dose exposure exhibited the same trend. PD-L1 and CD47 protein levels increased after low-dose irradiation in an LLC tumour-bearing mouse model. Low-dose irradiation combined with PD-L1 or CD47 inhibitor treatment reduced levels of PD-L1 or CD47 in tumour tissues, increased the proportion of CD8+ T lymphocytes, and significantly inhibited tumour growth. CONCLUSIONS: Both low-dose and regular-dose irradiation upregulate expression of the immune checkpoint molecules CD47 and PD-L1 in lung cancer cells, and the mechanism may be related to the JAK2/STAT3 pathway. Furthermore, low-dose irradiation combined with PD-L1 or CD47 inhibitors significantly inhibits tumour growth.

Clinicopathological analysis of immunohistochemical expression of immune checkpoint molecules in follicular lymphoma.

Follicular lymphoma (FL) is characterized by an indolent clinical course and a high relapse rate, and often exhibits a diffuse pattern beyond the follicular area. Our group previously reported that immune checkpoint (ICP) pathways, such as programmed cell death (PD-1) and programmed death ligand 1 (PD-L1), are poor prognostic factors for diffuse large B-cell lymphoma and adult T-cell leukemia/lymphoma. In this study, the association between the expression of multiple ICP molecules according to immunohistochemistry and clinicopathological features in FL was determined via immunostaining of 173 biopsy samples. Membrane and/or cytoplasm expression of CD86 (nCD86) and PD-L1 (nPD-L1) was found in tumor cells, whereas PD-1 (miPD-1), Galectin-9 (miGalectin-9), OX40 (miOX40), CTLA-4 (miCTLA-4), Tim-3 (miTim-3), OX40L (miOX40L), and LAG-3 (miLAG-3) were expressed in non-neoplastic stromal cells. MiPD-1 expression was significantly higher in the follicular area than in the diffuse area (p = 0.0450). Expression of miOX40 and miCTLA-4 was significantly higher in the diffuse area than in the follicular area (respectively, p = 0.0053 and p = 0.0092). MiTim-3 tended to be higher in the diffuse area than in the follicular area (p = 0.0616). MiTim-3 was significantly higher in relapse cases than in new-onset cases (p = 0.0440); miLAG-3 tended to be higher in relapse cases than in new-onset cases (p = 0.0622, not significant). The miOX40L-high FL group had a significantly worse overall survival than the miOX40L-low group (p = 0.0320). The expression of multiple ICP molecules on several cells reflects activated anti-tumor immunity and the unique FL microenvironment. Further studies on gene expression or genomic abnormalities will reveal the clinical and biological significance of ICP molecules in FL.

Expression of the immune checkpoint molecule V-set immunoglobulin domain-containing 4 is associated with poor prognosis in patients with advanced gastric cancer.

BACKGROUND: Recent clinical studies on immune checkpoint (IC) inhibitors in the context of advanced gastric cancer (AGC) have failed to show significant survival benefits but have suggested the possible role of IC inhibitors in anti-AGC immunity. Considering the low efficacy of targeted drugs in AGC, there is an urgent need for the discovery of new targets for the development of immunotherapeutics and prognostic markers for patient selection. This study aimed to investigate the expression of a new IC molecule, V-set Ig domain-containing 4 (VSIG4), and its clinical significance in AGC and other major cancers. METHODS: We analyzed the expression of VSIG4 and its correlation with survival in various carcinomas, including 882 surgically resected samples from patients with stage II-III AGC (two academic hospitals). RESULTS: VSIG4 positivity in AGC was significantly associated with overall survival (OS; Hazard ratio (HR) = 2.661, 95% confidence interval [CI] = 2.012-3.519, P < 0.001) and event-free survival (HR = 2.8, 95% CI = 2.18-3.72, P < 0.001). These findings were successfully validated in independent cohorts. VSIG4 expression was also significantly correlated with low intratumoral CD8 + T-cell infiltration (CD8i) (P = 0.029) and high Foxp3 + /CD8i ratio (P = 0.026), which is consistent with the previously reported immunological function of VSIG4. However, VSIG4 expression was not associated with survival in other cancers (colon, P = 0.459; lung, P = 0.275; kidney, P = 0.121; breast, P = 0.147). CONCLUSION: Our results suggest that VSIG4 is an independent prognostic factor in AGC and also implies that VSIG4 is a second-tier IC molecule in AGC, thus, providing an important basis for the development of gastric cancer-specific immunotherapeutics.

Cytokines: Signalling Improved Immunotherapy?

PURPOSE OF REVIEW: Immune checkpoint immunotherapies (ICI) are now approved for over 20 types of cancer and there are almost 6000 ongoing clinical trials investigating immuno-modulators as cancer therapies. This review investigated the effect of monoclonal antibody-based immune checkpoint immunotherapies when combined with cytokine therapy. We reviewed published clinical trial results from 2005 to 2020 for studies that used approved monoclonal antibody ICI in combination with the cytokines. Studies that met the search criteria were assessed for treatment efficacy and immunological changes associated with treatment. RECENT FINDING: ICI often fails to result in improved clinical outcomes for patients and lasting protection from cancer recurrence. The use of pro-inflammatory cytokines alongside ICI has been shown to enhance the efficacy of these therapies in vitro and in animal studies. However, the results in human clinical trials are less clear and many clinical trials do not publish results at the end of the trial. A deeper understanding of the molecular interactions between cytokines, tumors, and immune cells is needed to improve overall ICI outcomes and design combination trials. Critical examination of the design and characteristics of previous clinical trials can provide insight into the lack of effective clinical translation for many immunotherapeutic drugs.

A signature of immune-related gene pairs predicts oncologic outcomes and response to immunotherapy in lung adenocarcinoma.

In this study, we established the predictive model for lung adenocarcinoma (LUAD) depending on immune-related gene pairs (IRGPs) signature, which could not consider the technical bias of different platforms. Furthermore, we explored the predictive model with regard to the immune microenvironment and response to immunotherapy and identified specific drugs targeting the IRGPs model. Twenty-three IRGPs were identified and comprised the predictive model. When compared with the high-risk group, the low-risk group displayed a distinctly favorable prognosis and was characterized by increased immune score and decreased tumor purity. In addition, the low-risk group exhibited higher expression of immune checkpoint molecules, lower tumor stemness index, and was much more sensitive to immunotherapy. Lastly, candidate drugs that aimed at LUAD subtype differentiation were identified. The derived IRGPs model is an adverse independent biomarker for estimating oncologic outcomes in LUAD patients, and may be helpful to formulate personalized immunotherapy strategy.

Differences in the immunosurveillance pattern associated with DNA mismatch repair status between right-sided and left-sided colorectal cancer.

Tumor location and immunity play important roles in the progression of colorectal cancer (CRC). This study aimed to investigate the differences in the immunosurveillance pattern between right- and left-sided CRC and analyze their association with clinicopathologic features, including mismatch repair (MMR) status. We included surgically resected stage II/III CRC cases and evaluated the immunohistochemical findings of HLA class I, HLA class II, programmed cell death-ligand 1 (PD-L1), PD-1, CTLA-4, CD3, CD4, CD8, TIA-1, T-bet, GATA3, RORγT, Foxp3, and CD163. A total of 117 patients were included in the analyses; of these, 30 and 87 had right- and left-sided cancer, respectively. Tumor immunity varied according to the tumor location in the overall cohort. Analysis of the tumors excluding those with DNA mismatch repair (MMR) deficiency also revealed that tumor immunity differed according to the tumor location. In right-sided colon cancer (CC), high expression of Foxp3 (P = .0055) and TIA-1 (P = .0396) were associated with significantly better disease-free survival (DFS). High CD8 (P = .0808) and CD3 (P = .0863) expression tended to have better DFS. Furthermore, in left-sided CRC, only high PD-L1 expression in the stroma (P = .0426) was associated with better DFS. In multivariate analysis, high Foxp3 expression in right-sided CC was an independent prognostic factor for DFS (hazard ratio, 7.6445; 95% confidence interval, 1.2091-150.35; P = .0284). In conclusion, the immunosurveillance pattern differs between right- and left-sided CRC, even after adjusting for MMR deficiency.

Galectin-9 expression as a poor prognostic factor in patients with renal cell carcinoma.

Recently, the effectiveness of anti-programmed death 1 (PD-1) antibody therapy in the treatment of renal cell carcinoma (RCC) has been established. Nevertheless, efficacy has been reported to be limited to only 10-30% of patients. To develop more effective immunotherapy for RCC, we analyzed the immunological characteristics in RCC tissues by immunohistochemistry (IHC). We prepared a tissue microarray that consisted of tumor tissue sections (1 mm in diameter) from 83 RCC patients in Kanagawa Cancer Center between 2006 and 2015. IHC analysis was performed with antibodies specific to immune-related (CD8 and Foxp3) and immune checkpoint (programmed death ligand 1 (PD-L1) and 2 (PD-L2), B7-H4 and galectin-9) molecules. The numbers and proportions of positively stained tumor cells or immune cells were determined in each section. From multivariate analysis of all 83 patients, higher galectin-9 expression was detected as a factor associated with worse overall survival (OS) (P = 0.029) and that higher stage and higher B7-H4 expression were associated with worse progression-free survival (PFS) (P < 0.001 and P = 0.021, respectively). Similarly, in multivariate analysis of 69 patients with clear cell RCC, though not statistically significant, there was a trend for association between higher galectin-9 expression and worse OS (P = 0.067), while higher stage was associated with worse PFS (P < 0.001). This study suggests that higher galectin-9 expression is an independent adverse prognostic factor of OS in RCC patients. Therefore, to develop more effective personalized immunotherapy to treat RCC, it may be important to target not only PD-1/PD-L1, but also other immune checkpoint molecules such as galectin-9.

Spatiotemporal Changes in Checkpoint Molecule Expression.

Immune checkpoint inhibitors (ICIs), particularly PD-1/PD-L1 blockade, have led to therapeutic breakthrough in patients with advanced malignancy, covering the lung, breast, gastrointestinal, head and neck, urinary system, lymphoma, and solid tumor harboring MSI/dMMR. In certain cancer types, the expression level of immune checkpoint molecule will be required if the immune-based approaches are considered, especially the PD-L1 expression. However, in other types, survival benefit has been proven regardless of PD-L1 expression. It raises a question of how to select patients for immune therapy and whether the expression of immune checkpoint molecules will be optimal biomarkers. Before answering this question, a comprehensive map for the expression of immune checkpoint molecules is needed. In this chapter, we describe our current knowledge on the spatiotemporal changes in the expression of checkpoint molecules. We discuss the different frequencies of expression depending on tumor types and stages, the different patterns between primary and metastatic tumors, as well as the change of expression before and after treatment. The expression of PD-L1 has been most studied, but the threshold that separate "positive" and "negative" PD-L1 expressions and the consistency of testing platform remain under debate. Better understanding on the tumor microenvironment and expression of checkpoint molecules will help to identify patients who will benefit from checkpoint blockade therapy.

CD276 Promotes an Inhibitory Tumor Microenvironment in Hepatocellular Carcinoma and is Associated with Poor Prognosis.

Background: CD276 is an emerging immune checkpoint molecule that has been implicated in various cancers. However, its specific role in hepatocellular carcinoma (HCC) remains unclear. This study examined the impact of CD276 on patient prognosis and the tumor microenvironment (TME). Methods: The Cancer Genome Atlas (TCGA) database was utilized to evaluate CD276 expression in HCC and the association between CD276 and immune indicators was also analyzed. The signaling pathways correlated with CD276 expression were identified by gene set enrichment analysis (GSEA). Different algorithms were used to assess immune cell infiltration. The effect of CD276 knockdown on HCC cell phenotypes and its relationship with macrophage polarization was examined using the cell counting kit 8 (CCK-8) assay and co-culture system. Results: CD276 was upregulated in HCC and associated with unfavorable clinical outcomes. Hgh CD276 expression was associated with enrichment of the G2/M checkpoint, E2F targets, and mitotic spindles. CD276 expression was correlated with the infiltration of immune cells, including high level of tumor-associated macrophages and low levels of CD8+ T cells. Knockdown of CD276 decreased HCC cell proliferation and increased apoptosis. CD276 silencing in HCC cells and co-culture with THP-1-derived macrophages had a regulatory effect on macrophage polarization and macrophage-mediated cell proliferation and migration. Conclusion: CD276 expression in HCC is associated with unfavorable clinical outcomes and may contribute to the development of an immunosuppressive microenvironment. Specifically, CD276 was associated with alterations in immune cell infiltration, immune marker expression, and macrophage polarization during HCC progression, suggesting its potential as a prognostic indicator and promising target for immunotherapeutic intervention in HCC.

Characterizing soluble immune checkpoint molecules and TGF-ß1,2,3 in pleural effusion of malignant pleural mesothelioma.

The clinical impact of soluble molecules in pleural effusion (PE) is unclear in patients with malignant pleural mesothelioma (MPM). In this single-center, retrospective, observational study, we assessed soluble forms of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-1 ligand 1 (PD-L1) using enzyme-linked immunosorbent assays; three TGF-ß isoforms were measured via multiplex assay in PE of patients with fibrinous pleuritis (FP) or MPM, to assess relationships between the levels of six molecules, clinicopathological characteristics, and efficacy of immune checkpoint inhibitors. Soluble forms of CTLA-4, PD-L1, PD-1, TGF-ß1, TGF-ß2, and TGF-ß3 were variably produced in PE of FP (n = 34) and MPM (n = 79); we found significant relationships between the six molecules and clinicopathological features. Although none of the three soluble immune checkpoint molecules showed diagnostic or prognostic effects in patients with MPM, TGF-ß2 level in PE is a useful differential diagnostic marker between FP and MPM. Both TGF-ß1 and TGF-ß3 levels are promising prognostic markers for MPM. Moreover, we found that higher baseline levels of PD-1 soluble forms predicted the response to anti-PD1 monotherapy. Our findings identify novel diagnostic, prognostic, and predictive biomarkers for anti-PD1 therapy in patients with MPM.

Exploring Immunological Effects and Novel Immune Adjuvants in Immunotherapy for Salivary Gland Cancers.

Salivary gland cancer (SGC) is rare and comprises over 20 histological subtypes. Recently, clinical experience regarding immunotherapies for SGCs has been accumulating, yet their efficacy remains controversial. Understanding the tumor microenvironment (TME), including the expression of immune checkpoint molecules in SGC, is crucial to optimizing immunotherapy. In this review, we demonstrate that high-grade mucoepidermoid carcinoma and salivary duct carcinoma generally exhibit immune-hot TME with high immune cell infiltration, frequent genetic mutations, and robust immune checkpoint molecule expression. In contrast, adenoid cystic carcinomas exhibit an immune-cold TME. While the reported efficacy of immune checkpoint inhibitors (ICIs) for SGCs is generally poor, several studies showed promising clinical efficacy of ICIs, with an objective response rate ranging from 20.0-33.3%, indicating that ICIs might be beneficial for a specific population of SGC. Molecule-targeted therapies including anti-human epidermal growth factor receptor 2 and anti-androgen receptor therapies have shown promising clinical efficacy against SGC. Recent evidence indicates that these molecules could be targets for antigen-specific immunotherapies including chimeric antigen receptor-T therapy and cancer vaccines. This review discusses the current understanding and future directions of immunotherapies for SGCs, including ongoing clinical trials.

Moesin affects the plasma membrane expression and the immune checkpoint function of CD47 in human ovarian clear cell carcinoma.

Among major histological subtypes of epithelial ovarian cancer, a higher incidence of ovarian clear cell carcinoma (OCCC) is observed in East Asian populations, particularly in Japan. Despite recent progress in the immune checkpoint inhibitors for a wide variety of cancer cell types, patients with OCCC exhibit considerably low response rates to these drugs. Hence, urgent efforts are needed to develop a novel immunotherapeutic approach for OCCC. CD47, a transmembrane protein, is overexpressed in almost all cancer cells and disrupts macrophage phagocytic activity in cancer cells. Ezrin-Radixin-Moesin (ERM) family member of proteins serve as scaffold proteins by crosslinking certain transmembrane proteins with the actin cytoskeleton, contributing to their plasma membrane localization. Here, we examined the role of ERM family in the plasma membrane localization and functionality of CD47 in OCCC cell lines derived from Japanese women. Confocal laser scanning microscopy analysis showed colocalization of CD47 with all three ERM in the plasma membrane of OCCC cells. RNA interference-mediated gene silencing of moesin, but not others, decreased the plasma membrane expression and immune checkpoint function of CD47, as determined by flow cytometry and in vitro phagocytosis assay using human macrophage-like cells, respectively. Interestingly, clinical database analysis indicated that moesin expression in OCCC was higher than that in other histological subtypes of ovarian cancers, and the expression of CD47 and moesin increased with the cancer stage. In conclusion, moesin is overexpressed in OCCC and may be the predominant scaffold protein responsible for CD47 plasma membrane localization and function in OCCC.

[Corrigendum] HHLA2 is a novel prognostic predictor and potential therapeutic target in malignant glioma.

Following the publication of this article, an interested reader drew to the authors' attention that, in Fig. 1F on p. 2311 showing a representative high­grade glioma specimen, the data were either duplicated or overlapping with the data featured in Fig. 1D, which showed a low­grade glioma specimen. After having consulted their original data, the authors have realized that the data for Fig. 1D were inadvertently selected incorrectly. The corrected version of Fig. 1, now showing the correct data for the high­magnification high­grade glioma specimen in Fig. 1F, is shown on the next page. The authors sincerely apologize for the error that was introduced during the preparation of this figure, thank the Editor of Oncology Reports for granting them the opportunity to publish a Corrigendum, and are grateful to the reader for alerting them to this issue. The authors also regret any inconvenience that this mistake may have caused. [Oncology Reports 42: 2309-2322, 2019; DOI: 10.3892/or.2019.7343].

Dominant negative TGFß receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

BACKGROUND: The immune checkpoint molecules, Transforming growth factor beta receptor II (TGFßRII) and T cell immunoglobulin and mucin domain 3 (TIM3), have been identified as contributors to T cell immune suppression in prostate cancer. The objective of this investigation was to improve the tumor killing capability of prostate-specific membrane antigen (PSMA)-chimeric antigen receptor T (CAR-T) cells by targeting TIM3 and TGFßRII simultaneously. METHODS: To generate dnTGFßRII-trTIM3-PSMA-CAR-T (DT-PSMA-CAR-T) cells, the surface of PSMA-CAR-T cells was overexpressed with dominant negative TGFßRII (dnTGFßRII) and truncated extracellular TIM3 (trTIM3). The efficacy of DT-PSMA-CAR-T cells was assessed through in vitro killing experiments and animal experiments. RESULTS: The DT-PSMA-CAR-T cells demonstrated the ability to eradicate PSMA-positive prostate cancer cells, even in the presence of exogenous TGF-ß and/or TIM3 activating antibodies. In addition, the cells demonstrated the ability to eliminate tumor tissue in an immunodeficient mouse model transplanted with GAL9-PSMA-PC3 cells in vitro, prolonging survival without significant toxic side effects. CONCLUSIONS: This study emphasizes that upregulating dnTGFßRII and trTIM3 on the surface of T cells can potentially diminish the inhibitory effects of TGFßRII and TIM3.

LAG-3 expression in microglia regulated by IFN-γ/STAT1 pathway and metalloproteases.

Microglia are resident innate immune cells in the central nervous system (CNS) and play important roles in the development of CNS homeostasis. Excessive activation and neurotoxicity of microglia are observed in several CNS disorders, but the mechanisms regulating their activation remain unclear. Immune checkpoint molecules are expressed on activated immune cells and regulate their activation in peripheral immunity. However, the expression mechanism of immune checkpoint molecules in activated microglia is still unknown. Here, we analyzed the expression of immune checkpoint molecules in activated microglia using the mouse microglial cell line BV2 and primary cultured microglia. The expression of lymphocyte activation gene-3 (LAG-3), a type of immune checkpoint molecule, was increased in microglia activated by IFN-γ. IFN-γ-induced LAG-3 expression in microglia was suppressed by transfection of siRNA targeting STAT1. LAG-3 has two forms, membrane and soluble, and both forms were upregulated in microglia activated by IFN-γ. The production of soluble LAG-3 was suppressed by treatment with inhibitors of metalloproteinases such as ADAM10 and ADAM17. IFN-γ administration into cisterna magna of mice increased LAG-3 expression in spinal microglia. Furthermore, LAG-3 knockdown in microglia promoted nitric oxide production by IFN-γ. Our results demonstrate that LAG-3 expression in microglia is induced by the IFN-γ-STAT1 pathway and soluble LAG-3 production is regulated via cleavage of membranous LAG-3 by metalloproteinases including ADAM10 and ADAM17.

Methylation related genes are associated with prognosis of patients with head and neck squamous cell carcinoma via altering tumor immune microenvironment.

Background/purpose: Analysis of methylomes may enable prognostic stratification in patients with head and neck squamous cell carcinoma (HNSCC). This study aimed to identify methylation-related differentially expressed genes (mrDEGs), and to assess their efficacy in predicting patients' survival, tumor immune microenvironment alterations and immune checkpoints in patients with HNSCC. Materials and methods: The methylome and transcriptome data of 528 HNSCC and 50 normal samples from TCGA database were used as training cohort. We identified mrDEGs and constituted a risk score model using Kaplan-Meier analysis and multivariate Cox regression. The prognostic efficacy of the risk score was validated in GSE65858 and GSE41613. We determined the enrichment of previously defined biological processes of mrDEGs. We separated the HNSCC patients into low-risk and high-risk groups and compared their immune cell infiltration and immune checkpoints' expressions. Results: The risk score model was constituted by nine prognostic mrDEGs, including LIMD2, SYCP2, EPHX3, UCLH1, STC2, PRAME, SLC7A4, PLOD2, and ACADL. The risk score was a significant prognostic factor both in training (P < 0.001) and validation dataset (GSE65858: P = 0.008; GSE41613 = 0.015). The prognostic mrDEGs were enriched in multiple immune-associated pathways. Effector immune cells were increased in low-risk patients, including CD8+ T cells, activated CD4+ T cells, and plasma cells, whereas tumor associated M2 macrophages were recruited in the high-risk group. Expressions of immune checkpoints were generally higher in low-risk patients, including CTLA-4, PD-1 and LAG3. Conclusion: The mrDEGs can stratify HNSCC patients' prognosis, which correlates with alterations in tumor immune infiltrations and immune checkpoints.