CD11b+ Gr1+ myeloid-derived suppressor cells (MDSCs) play critical roles in controlling the processes of tumors, infections, autoimmunity and graft rejection. Immunosuppressive drug rapamycin (RPM), targeting on the key cellular metabolism molecule mTOR, is currently used in clinics to treat patients with allo-grafts, autoimmune diseases and tumors. However, the effect of RPM on MDSCs has not been studied. RPM significantly decreases the cell number and the immunosuppressive ability on T cells of CD11b+ Ly6Chigh monocytic MDSCs (M-MDSCs) in both allo-grafts-transplanted and tumor-bearing mice respectively. Mice with a myeloid-specific deletion of mTOR have poor M-MDSCs after grafting with allo-skin tissue or a tumor. Grafting of allo-skin or tumors significantly activates glycolysis pathways in myeloid precursor cells in bone marrow, which is inhibited by RPM or mTOR deletion. 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway, inhibits M-MDSC differentiation from precursors, while enhancing glycolysis by metformin significantly rescues the RPM-caused deficiency of M-MDSCs. Therefore, we offer evidence supporting that mTOR is an intrinsic factor essential for the differentiation and immunosuppressive function of M-MDSCs and that these metabolism-relevant medicines may impact MDSCs-mediated immunosuppression or immune tolerance induction, which is of considerable clinical importance in treating graft rejection, autoimmune diseases and cancers.

Lung adenocarcinoma is heterogeneous and hierarchically organized, with a subpopulation of stem-like cells (CSCs) that reside at the apex of the hierarchy, in which exosomes act as important mediators by transporting specific molecules among different cell populations. Although there have been numerous studies on tumor exosomes, the constituents and functional properties of CSC-derived exosomes are still poorly characterized. Here we present a detail transcriptome and proteome atlas of the exosomes released by human lung adenocarcinoma stem-like cells (LSLCs). The transcriptome analysis indicates the specific patterns of exosomal constituents, including the fragmentation of transcripts and the low-level presence of circular RNAs, and identifies multiple exosomal-enriched mRNAs and lncRNAs. Integrative analysis of transcriptome and proteome data reveals the diverse functions of exosomal-enriched RNAs and proteins, many of which are associated with tumorigenesis. Importantly, several LSLC markers we identified are highly expressed in LSLC-derived exosomes and associate with poor survival, which may serve as promising liquid biopsy biomarkers for lung adenocarcinoma diagnosis. Our study provides a resource for the future elucidation of the functions of tumor-derived exosomes and their regulatory mechanisms in mediating lung cancer development.

Interleukin-35 (IL-35) has recently been implicated in tumor immunity. The aim of this study was to explore the clinical role of plasma IL-35 in patients with non-small cell lung cancer (NSCLC). Plasma collected from 106 patients with NSCLC cases and 78 healthy controls (HC) were subjected to IL-35 enzyme-linked immunosorbent assay (ELISA) and relationships between plasma IL-35 levels and clinical characteristics were evaluated. The correlation of IL-35 and overall survival was analyzed using Kaplan-Meier method. The prognostic value of IL-35 was tested using univariate and multivariate analysis. Circulating IL-35 levels were significantly higher in the NSCLC group in comparison with the HC group (21.37 ± 11.55 pg/ml vs. 10.09 ± 5.32 pg/ml, p < 0.001). Correlation analysis by subgroup indicated that plasma IL-35 correlated positively with tumor TNM stage (p < 0.001) and lymph node metastases (p < 0.0001). Using a cutoff level of 20.26 pg/ml (median value), IL-35 showed an inverse correlation with overall survival. Univariate and multivariate analysis indicated that plasma IL-35 was an independent prognostic factor for NSCLC patients. Circulating IL-35 in NSCLC patients significantly increased. IL-35 is a promising potential biomarker in prognostication of clinical outcome of NSCLC patients and the regulation of IL-35 expression may provide a new target for the treatment of NSCLC patients.

Thymic-derived CD4(+)CD25(+)Foxp3(+) natural regulatory T (nTreg) cells are essential for the maintenance of peripheral immune tolerance. Signaling pathways that drive immature thymic progenitors to differentiate into CD4(+)CD25(+)Foxp3(+) nTreg cells need to be elucidated. The precise role of the TSC1/2 complex, a critical negative regulator of mammalian target of rapamycin (mTOR), in thymic CD4(+)CD25(+)Foxp3(+) nTreg-cell development remains elusive. In the present study, we found that the percentage and cell number of thymic CD4(+)CD25(+)Foxp3(+) nTreg cells were significantly increased in T-cell-specific TSC1-knockout (TSC1KO) mice. Nevertheless, the levels of CD4(+)CD25(+)Foxp3(-) nTreg precursors in TSC1KO thymus were indistinguishable from those in wild-type mice. TSC1KO CD4(+)CD25(+)Foxp3(+) nTreg cells showed normal cell death but enhanced proliferative response to IL-2 in a STAT5-dependent manner. Rapamycin (Rapa) treatment failed to rescue but rather increased the frequency of CD4(+)CD25(+)Foxp3(+) nTreg cells in TSC1KO and RictorKO mice. The percentage and cell number of thymic CD4(+)CD25(+)Foxp3(+) nTreg cells were significantly increased in T-cell-specific RictorKO mice but not in PtenKO mice. Collectively, our studies suggest that TSC1 plays an important role in regulating thymic CD4(+)CD25(+)Foxp3(+) nTreg-cell development via a Rapa-resistant and mTORC2-dependent signaling pathway.

The frequency of CD14(+)HLA-DR-/low MDSCs could be considered as a poor prognostic predictor in SCLC and the elimination of MDSCs during medical interventions may improve the prognosis of SCLC patients.

Tumor-infiltrating T cells are highly expressive of inhibitory receptor/immune checkpoint molecules that bind to ligand expressed by tumor cells and antigen-presenting cells, and eventually lead to T cell dysfunction. It is a hot topic to restore T cell function by targeting immune checkpoint. In recent years, immunotherapy of blocking immune checkpoint and its receptor, such as PD-L1/PD-1 targeted therapy, has made effective progress, which brings hope for patients with advanced malignant tumor. However, only a few patients benefit from directly targeting these checkpoints or their receptors by small compounds or antibodies. Since the complexity of the regulation of immune checkpoints in tumor cells, further research is needed to identify the novel endogenous regulators of immune checkpoints which can help for developing effective drug target to improve the effect of immunotherapy. Here, we verified that microRNA-326 (miR-326) repressed the gene expression of immune checkpoint molecules PD-L1 and B7-H3 in lung adenocarcinoma (LUAD). We detected that the expression of miR-326 in LUAD tissue was negatively correlated with PD-L1/B7-H3. The repression of PD-L1 and B7-H3 expression through miR-326 overexpression leads to the modification the cytokine profile of CD8+ T cells and decreased migration capability of tumor cells. Meanwhile, the downregulation of miR-326 promoted tumor cell migration. Moreover, blocking PD-L1 and B7-H3 attenuated the tumor-promoting effect induced by miR-326 inhibitor. In tumor-bearing mice, the infiltration of CD8+ T cells was significantly increased and the expression of TNF-α, and IFN-γ was significantly enhanced which contributed to tumor progression after miR-326 overexpression. Collectively, miR-326 restrained tumor progression by downregulating PD-L1 and B7-H3 expression and increasing T cell cytotoxic function in LUAD. Our findings revealed a novel perspective on the complex regulation of immune checkpoint molecules. A new strategy of using miR-326 in tumor immunotherapy is proposed.

BackgroundGrowth differentiation factor (GDF) acted as a factor that regulated proliferation, apoptosis and differentiation in several tumors. However, the effects of growth differentiation factor (GDF11) in pancreatic cancer remain unclear.PurposeTo investigate the expression and significance of GDF11 in pancreatic cancer.Patients and methodsPancreatic cancer and corresponding paracancerous tissues (n=28) were collected from the Department of Hepatobiliary and Pancreatic Surgical Oncology of Chinese PLA General Hospital. Tissue microarray was obtained from Outdo Biotech Co., Ltd. (Shanghai, People’s Republic of China). GDF11 mRNA and protein expressions in pancreatic cancer samples and cell lines were detected using qRT-PCR, Western-Blot and immunohistochemistry. Overexpression and knockdown of GDF11 were performed with lentiviral transduction system and siRNA technique in PANC-1 cell line and CFPAC-1 cell line. Proliferation, migration and invasion of pancreatic cancer cell lines were examinated by MTS and transwell assay, respectively. Flow cytometry was used for cell apoptosis analysis.ResultsThe results of this study indicated that GDF11 was significantly down-regulated in pancreatic cancer tissues compared with adjacent tissues of pancreatic cancer. GDF11 was also associated with low expression in pancreatic cancer cell lines when compared with normal pancreatic cell line. In a cohort of 63 pancreatic cancer patients, high GDF11 expression levels was associated with favorable perineural invasion, T classification, N classification and overall survival (OS). Cox proportional hazards model revealed that high GDF11 expression was an independent predictor of favorable prognosis (HR: 0.496; 95% CI: 0.255–0.967; P=0.040). Overexpression of GDF11 in PANC-1 cells repressed the proliferation, migration and invasion abilities in vitro. Inhibition of GDF11 in CFPAC-1 showed inverse results. Furthermore, enhanced GDF11 expression promoted apoptosis and down-regulated GDF11 expression inhibited apoptosis in pancreatic cancer cell lines.ConclusionThese findings suggested that GDF11 acted as a tumor suppressor gene for pancreatic cancer.

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