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Supplementary materials and methods

Figure S6 shows the differences in BAFF and S100 levels between immunotherapy responders and non-responders

Figure S7 shows the individual tumor growths comprising the main figures

Supplementary Table 1 provides a list of significantly changed genes between monocytes harvested from BAFF and control tumors

Figure S1 shows the in vitro characterization of the BAFF-expressing melanoma cell line

Figure S5 shows the infiltration and contribution of different T cells and cytolines to the phenotypic growth differences between BAFF-expressing and control tumors

Figure S2 shows the IgG and IgM in BAFF-expressing and control tumors

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to be an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-κB signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease.Significance:The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity.

Supplementary Table 3 shows different PD-L1 cut-offs to stratify responders and non-responders to immunotherapy

Supplementary Table 2 provides the clinical details of melanoma patients treated with immunotherapy

Figure S3 further characterizes the differences between BAFF-expressing and control tumors

Figure S4 shows the gating strategy of monocytes sorted from BAFF-expressing and control tumors

Genes differentially expressed between IgG control and anti-PD-L1 treatment.

Supplementary title page. Figure S1: CD38 on anti-PD-L1 resistant tumor cells is up-regulated, which is associated with tumor progression. Figure S2: anti-PD-L1 resistant tumors demonstrate the distinct mRNA and protein profiling for immune signature, reflecting the upregulation of CD38. Figure S3: PD-1/PD-L1 blockade results in CD38 up-regulation and acquired resistance in KP-derived lung and melanoma transplantation tumors. Figure S4: Tumor-associated PD-L1 promotes tumor growth but PD-L1 knockout cancer cells still form tumors. Figure S5: PD-L1 knockout effect on tumor growth is CD8 T cell-dependent. Figure S6: CD38 up-regulation after anti-PD-L1 treatment is associated with all-trans retinoic acid signaling. Figure S7: IFN-b, which is enriched in anti-PD-L1 treated tumors, up-regulates CD38 expression on multiple cancer cell lines. Figure S8: IRF1, which links ATRA and IFN-b, is upregulated after anti-PD-L1 treatment. Figure S9: IFN-g, TNF-a, IL-2, and IL-1b don''t regulate CD38 expression on lung cancer cells. Figure S10: The anti-PD-L1 resistant tumors demonstrate an immune suppressive microenvironment. Figure S11: CD38 substantially changes in vivo tumor formation of PD-L1KO cancer cells, but does not change in vitro cell growth rate and cell cycle. Figure S12: The elimination of PD-L1KOCD38negative cancer cells is CD8+ T cell dependent. Figure S13: CD38 on tumor cells inhibits CD8+ T cell function and protects tumor cells from CD8+ T cell killing. Figure S14: CD38-mediated CD8+TIL dysfunction is not affected by tumor growth rate/tumor size. Figure S15: CD38 expression in cancer cell lines and patient tissues, which is associated with the differentiated immune features. Figure S16: CD38 or PD-L1 expression is not correlated with overall survival in early-stage lung cancer. Figure S17: Pre-treatment levels of CD38 and PD-L1 expression in NSCLC patients who received anti-PD-1/PD-L1 therapy, as divided by clinical outcome. Figure S18: The effect of cancer immunotherapy by anti-CD38 is CD8 T cell dependent. Figure S19: Combined inhibitors of CD38 and PD-L1 inhibits tumor growth and metastases. Figure S20: The co-inhibition of PD-L1 and CD38 leads to a favorable antitumor immune microenvironment. Figure S21: Sequential treatment of anti-PD-L1 and anti-CD38 results in enhanced immune response in tumor microenvironment. Figure S22: anti-mouse CD38 antibody (NIMR-5) does not directly kill tumor cells through ADCC and CDC, but causes CD38 internalization. Table S2: Tumor immune markers with the greatest differential transcriptional levels after anti-PD-L1 treatment. Table S6: The most changed immune-related genes after anti-PD-L1 treatment. Table S9:The correlation between CD38 and suppressive immune markers in LUAD dataset. Table S10: The correlation between CD38 and suppressive immune markers in LUSC dataset. Table S11: The available NSCLC patients with CD38 and PD-L1 tumor cell IHC staining and responses to anti-PD-1 therapy. Table S12: The co-inhibition effect of PD-L1 and CD38 on tumor growth and metastasis. Supplementary Material and Methods: Reagents, Cells and Mice, CRISPR/Cas9 Editing, Antibody-mediated Cell Depletion, CD8+ T Cell Adoptive Transfer, mRNA Profiling of Murine Tumors, Flow Cytometry, Nanostring Analysis, qRT-PCR and Western Blotting, Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis, Histologic Analysis, ELISA and RPPA, ADCC/CDC and Internalization Assays, Human Samples, Statistics. Supplementary references

Background. Large scalp defects, especially those complicated by calvarial defects, titanium mesh exposure, or cerebrospinal fluid (CSF) leak, pose a challenge for the neurosurgeon and plastic surgeon. Here, we describe our experience of reconstructing the complex scalp defect with free flap transfer. Methods. From October 2012 to September 2017, 8 patients underwent free flap transfer for the reconstruction of the scalp or complicated scalp and calvarial defects. Five patients presented with scalp tumor and the other 3 patients with scalp necrosis or ulceration (2 patients with titanium plate exposure). Seven anterolateral thigh flaps and one radial forearm flap were harvested and employed. The clinical data, including defect characteristics, flap type, complications, and outcomes, were recorded and analyzed. Results. Five patients were pathologically diagnosed with malignant tumor, and 3 of them were given further radiotherapy. For the 2 patients with exposure of titanium plate, no titanium plate was removed. For the patient with scalp necrosis after decompressive craniectomy accompanied by CSF leakage, the CSF leak was stopped after reconstruction. The size of the flaps ranged from 3 to 14 cm in width and 4 to 18 cm in length. No flap failure occurred in these cases. From follow-up to the present, no ulceration or necrosis occurred. Conclusions. Free flap transfer is an ideal method for the reconstruction of large, complicated scalp defects with a one-stage operation. The anterolateral thigh flap is favored because of its durability, adjustability, water tightness, and infection prevention.

The reconstruction of peripheral nerves has lately received great attention as many patients suffer from peripheral nerve injury every year around the world. However, the damage to human nerve cells has different degrees of irreversibility due to a slow growth speed and low adhesion with the surrounding tissues. In an effort to overcome this challenge, we applied novel laminin (LN)-modified thiolated gellan gum (TGG) and loaded the nerve growth factor (NGF) as a tissue engineering scaffold for facilitating neuronal stem cell proliferation via a synergy effect for the ERK–MAPK pathway. TGG was characterized by 1H NMR spectroscopy and scanning electron microscopy, and its rheological behavior was also studied. The NGF release curve fitted the Korsmeyer–Peppas model and belonged to a Fickian diffusion-controlled release mechanism. The neuronal stem cells from newborn SD rats could adhere tightly and proliferate at a relatively rapid speed, showing excellent biocompatibility and the ability to promote growth in the modified TGG. LN and NGF could decrease the apoptosis effects of neuronal stem cells, as shown via the flow cytometry results. In a three-dimensional culture environment, LN and NGF could facilitate neuronal stem cells to differentiate into neurons, as proved by immunofluorescence, q-PCR, and western blot analyses. Therefore, the rational design of the TGG gel loaded with NGF has promising applications in the reconstruction of peripheral nerves.

Background Minimally invasive separation surgery (MISS) is a safe and effective surgical technique, the current optimal treatment for spinal metastases. However, the learning curve for this technique has not been analyzed. This study aimed to define and analyze the surgical learning curve of MISS for the treatment of spinal metastases with small incision and freehand pedicle screw fixation. Methods A continuous series of 62 patients with spinal metastases who underwent MISS were included. Each patient's operative data were accurately counted. The improvement of the patients' neurological function was followed up after surgery to evaluate the surgical treatment effect. Logarithmic curve-fit regression was used to analyze the surgical learning curve of MISS. The number of cases needed to achieve proficiency was analyzed. Based on this cut-off point, this series of cases was divided into the early phase and later phase groups. The influence of the time sequence of MISS on surgical data and surgical efficacy was analyzed. Results The operative time decreased gradually with the number of surgical cases increasing and stabilized after the 20th patient. There was no statistical difference in demographic characteristics and preoperative characteristics between the two groups. The mean operative time in the later phase group was about 39 min shorter than that in the early phase group (mean 227.95 vs. 189.02 min, P = 0.027). However, it did not affect other operative data or the surgical treatment effect. Conclusion The learning curve of MISS for spinal metastases is not steep. With the increase of surgeons' experience, the operative time drops rapidly and stabilizes within a certain range. MISS can be safely and effectively performed at the beginning of a surgeon's caree.

Background: Several clinical trials have shown that immunotherapy plays a pivotal role in the treatment of patients with metastatic synovial sarcoma. Immune-related genes (IRGs) have been demonstrated to play an important role in tumorigenesis and tumour microenvironment formation. However, the clinical significance of IRGs in patients with synovial sarcoma(SS) is still unclear, and systematic analysis is lacking. Methods: We downloaded the GSE40021 dataset from the GEO database, which included 30 cases of nonmetastatic and 28 cases of metastatic SS. Firstly,We combined the immune-related ImmPort gene set to search for SS related to metastatic and differentially expressed immune-related genes (DEIRGs). We then performed univariate Cox regression analysis from soft tissue sarcoma database in TCGA to identify DEIRGs that related to overall survival, and constructed an immune-related prognostic assessment model. We used the assessment model to evaluate the infiltration of immune cells through the ssGSEA algorithm. Finally, we collected tumour tissues in our centre to verify the RNA expression levels by real-time quantitative reverse transcription(RT-qPCR) analysis. Findings: The study screened a total of six DEIRGs which were closely related to prognosisin in metastatic SS and constructed an immune-related prognostic assessment model which was an independent prognostic factor different from other clinical features. Further analysis showed that there was no significant difference in the expression of several immune checkpoints between the two groups in the GSE40021 data. Moreover, the GREM2 and CTSS genes were significantly expressed in metastatic patients. Further verification of SS tissues from our centre by RT-qPCR analysis demonstrated reduced infiltration of activated NK cells and macrophages but increased M2-type macrophages in metastatic patients. Interpretation: The study successfully constructed an immune-related prognostic assessment model and probably explain the poor efficacy PD-1 inhibitors for SS patients. Together,the research deepens our understanding of the tumor immune microenvironment and proposed a new immune mechanism of metastatic SS. Funding Information: This study was supported by the National Natural Science Foundation of China (91959115, 81872268,and 81702665), Natural Science Foundation of Guangdong Province (2019A1515011192) and the Fundamental Research Funds for the Central Universities (19ykpy189). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Sun Yat-sen University Cancer Center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A.

Background Achieving satisfactory internal fixation for patients with Pauwels type III femoral neck fractures has become a critical problem. The purpose of this study was to compare a common standard internal reduction and fixation method for femoral neck fractures to the modified fixation methods. Methods A computed tomography scan of the femur was performed to make a Three-dimensional (3D) model, and a fracture line was simulated in the femoral neck. 3Dfinite element analysis was carried out for different insertion methods of cannulated tension screws. Six healthy femur specimens were harvested from three formalin-fixed cadavers, and Pauwels type III femoral neck fracture was artificially created in bilateral femurs. The right side was treated with the inverted triangle construct method and the left side by the modified screw fixation method. After fixation, uniaxial compression and maximum load experiments on the bilateral femoral necks were carried out using the non-contact full-field dynamic strain measurement system (VIC-3D) on a pressure testing machine. Findings Both 3D finite element analysis and biomechanical study showed that the modified screw fixation method(group D) provided better anti-shearing and anti-rotation properties for Pauwels type III femoral neck fractures, and offered better interfragmentary compression. Therefore, this modified screw fixation method can offer patients a better option for treatment of Pauwels type III femoral neck fractures. Interpretation Changing the placement of the anterosuperior screw in the inverted triangle construct as perpendicular to the fracture line has the advantages in anti-shearing, anti-rotation and increasing interfragmentary compression.

SUMMARY Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases., Graphical Abstract, In Brief Cham et al. describe a way to enhance natural immune responses to infections by blocking interactions between two molecules (CD47 and SIRPα) that normally put brakes on the immune system. Since this therapy targets the immune system, it could have broad applicability against a wide range of infectious agents.

Summary: Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor β (TGF-β). Lack of Mertk in Mertk−/− mice prevents induction of IL-10 and TGF-β, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV. : Adomati et al. show that the cytopathic virus VSV leads to innate immune cell anergy. Innate anergy is associated with apoptotic cells through activation of the TAM receptor Mertk and induction of the cytokines IL-10 and TGF-β.

Objective To determine the biomechanical effect of anterior talofibular ligament injury in Weber B lateral malleolus fractures after lateral plate fixation. Method A three-dimensional model was established based on CT images from a healthy volunteer. The simulation of lateral malleolus fracture, and the modeling and assembly of plate were completed by referring to characteristics of Weber B lateral malleolus fractures, as well as the technical characteristics of open reduction and internal fixation of lateral plate. Operating conditions were set up for groups A−D. The proximal end of the model was restrained in all four groups, 200 N of upward force and 100 N of backward force were applied at anterior of talus head in order to simulate the dorsiflexion of ankle joint. Biomechanical differences of the lateral plate were observed under various conditions of different ligament ruptures. Results The maximum stress value of group A was the smallest, approximately 78.47 N, while that of group C was the largest, approximately 238.83 N. The maximum stress value of group B was about 91.69 N; and that of group D was about 184.08N. Importantly, location of the maximum stress in group D (CUT ATaF) was displaced from the posterior edge to the anterior edge of the plate, which was different from those of the other three groups. Conclusions The anterior talofibular ligament injury may be a major contributing factor to the stress of lateral plate fixation following Weber B lateral malleolus fracture. It should be considered as an essential risk factor for evaluation of the stability in these fractures.

'NK cell-mediated regulation of antigen-specific T cells can contribute to and exacerbate chronic viral infection, but the protective mechanisms against NK cell-mediated attack on T cell immunity are poorly understood. Here, we show that progranulin (PGRN) can reduce NK cell cytotoxicity through reduction of NK cell expansion, granzyme B transcription, and NK cell-mediated lysis of target cells. Following infection with the lymphocytic choriomeningitis virus (LCMV), PGRN levels increased - a phenomenon dependent on the presence of macrophages and type I IFN signaling. Absence of PGRN in mice (Grn⁻/⁻) resulted in enhanced NK cell activity, increased NK cell-mediated killing of antiviral T cells, reduced antiviral T cell immunity, and increased viral burden, culminating in increased liver immunopathology. Depletion of NK cells restored antiviral immunity and alleviated pathology during infection in Grn⁻/⁻ mice. In turn, PGRN treatment improved antiviral T cell immunity. Taken together, we identified PGRN as a critical factor capable of reducing NK cell- mediated attack of antiviral T cells. OA platinum

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNβ in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment. Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156–75. ©2018 AACR. See related commentary by Mittal et al., p. 1066. This article is highlighted in the In This Issue feature, p. 1047

Huge efforts have been devoted to develop therapeutic monoclonal antibodies targeting human Programmed death-ligand 1 (hPD-L1) for treating various types of human cancers. However, thus far there is no suitable animal model for evaluating the anti-tumor efficacy of such antibodies against hPD-L1. Here we report the generation of a robust and effective system utilizing hPD-L1-expressing mouse tumor cells to study the therapeutic activity and mode of action of anti-human PD-L1 in mice. The model has been validated by using a clinically proven hPD-L1 blocking antibody. The anti-hPD-L1 antibody treatment resulted in potent dose-dependent rejection of the human PD-L1-expressing tumors in mice. Consistent with what have observed in autochthonous mouse tumor models and cancer patients, the hPD-L1 tumor bearing mice treated by anti-hPD-L1 antibody showed rapid activation, proliferation and reinvigoration of the cytolytic effector function of CD8+T cells inside tumor tissues. Moreover, anti-hPD-L1 treatment also led to profound inhibition of Treg expansion and shifting of myeloid cell profiles, showing bona fide induction of multilateral anti-tumor responses by anti-hPD-L1 blockade. Thus, this hPD-L1 mouse model system would facilitate the pre-clinical investigation of therapeutic efficacy and immune modulatory function of various forms of anti-hPD-L1 antibodies.

Mice are some of the widely used experimental animal models for studying human diseases. Defining the compositions of immune cell populations in various tissues from experimental mouse models is critical to understanding the involvement of immune responses in various physiological and patho-physiological conditions. However, non-lymphoid tissues are normally composed of vast and diverse cellular components, which make it difficult to quantify the relative proportions of immune cell types. Here we report the development of a computational algorithm, ImmuCC, to infer the relative compositions of 25 immune cell types in mouse tissues using microarray-based mRNA expression data. The ImmuCC algorithm showed good performance and robustness in many simulated datasets. Remarkable concordances were observed when ImmuCC was used on three public datasets, one including enriched immune cells, one with normal single positive T cells, and one with leukemia cell samples. To validate the performance of ImmuCC objectively, thorough cross-comparison of ImmuCC predicted compositions and flow cytometry results was done with in-house generated datasets collected from four distinct mouse lymphoid tissues and three different types of tumor tissues. The good correlation and biologically meaningful results demonstrate the broad utility of ImmuCC for assessing immune cell composition in diverse mouse tissues under various conditions.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population and show significant expansion under pathological conditions. microRNA plays important roles in many biological processes, whether microRNAs have a function in the expansion of MDSCs is still not very clear. In this study, miR-34a overexpression can induce the expansion of MDSCs in bone marrow chimera and transgenic mice model. The experimental results suggest that miR-34a inhibited the apoptosis of MDSCs but did not affect the proliferation of MDSCs. The distinct mRNA microarray profiles of MDSCs of wild type and miR-34a over-expressing MDSCs combined with the target prediction of miR-34a suggest that miR-34a may target genes such as p2rx7, Tia1, and plekhf1 to inhibit the apoptosis of MDSCs. Taken together, miR-34a contributes to the expansion of MDSCs by inhibiting the apoptosis of MDSCs.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous populations of immature myeloid cells with strong immunosuppressive function, and play a critical role in the immune evasion of cancer. A subset of MDSCs share many similar characteristics with tumor-associated macrophages (TAMs), but it is largely unclear whether MDSCs also have M1/M2 type polarization in tumor microenvironments. In the present study, we found that Gr-1(+)CD115(+) monocytes in tumor-bearing mice exhibited M2 characteristics with significantly lower expression of iNOS and higher expression of Arginase I. Immunofluorescence staining showed that Gr-1(+)CD115(+) monocytes in tumor sites from LPS-injected mice had a higher expression of iNOS. Similarly, in vitro experiments displayed that LPS-treated Gr-1(+)CD115(+) cells expressed higher levels of iNOS, IL-6, TNF, IL-12, and IL-10 compared with those in non-treated Gr-1(+)CD115(+) monocytes. Extensive study showed that LPS-treated Gr-1(+)CD115(+) monocytes had less ability to convert the CD4(+)CD25(-)cells into CD4(+)CD25(+) Tregs, and also had less suppressive function on CD4(+)CD25(-) conventional T cells. LLC tumors in LPS-injected mice grew significantly slower than those in non-LPS-injected mice. Further experiments suggested that LPS may function through the P38 MAPK signaling pathway to increase the expression of iNOS, and of MyD88 independently. Thus, we can get conclusion that Gr-1(+)CD115(+) monocytes in tumor-bearing mice show M2 type characteristics and LPS can skew this M2 type cells into M1 type through the P38 MAPK pathway and lead to inhibition of the suppressive function of Gr-1(+)CD115(+) monocytes. It suggests that LPS or its analogs may be potential drugs for tumor treatment, inflammation induced by LPS or other components of bacterium or virus may be benefit to the inhibition of tumor cell growth in vivo.

// Anfei Huang 1, * , Yi Yang 1, * , Si Chen 1 , Fei Xia 1 , Di Sun 1 , Deyu Fang 2 , Sidong Xiong 1 , Liping Jin 3 , Jinping Zhang 1 1 Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province 215123, People’s Republic of China 2 Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA 3 Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 200040, China * These authors have contributed equally to the work Correspondence to: Jinping Zhang, email: j_pzhang@suda.edu.cn Liping Jin, email: jinlp01@163.com Keywords: miR-34a, cDCs, development, IL-17a, T cell activation Received: July 07, 2016 Accepted: January 16, 2017 Published: February 09, 2017 ABSTRACT MicroRNAs serve important functions in numerous biological processes. Whether microRNAs also act on dendritic cell (DC) differentiation and function remains unclear. In this study, both conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were increased in miR-34a overexpressing bone marrow chimeric and transgenic (TG) mice. Further experiments showed that miR-34a promoted preDC differentiated into cDCs and pDCs without affecting the proliferation and apoptosis of DCs. Luciferase report assay and Western blot experiments demonstrated that WNT1 is the direct target of miR-34a in DCs. Interestingly, miR-34a overexpressing cDCs also produced a large amount of IL-17a and suppressed T cell activation because of the inhibition of TCF1 expression, thus increasing RORγT expression. Taken together, miR-34a promotes preDC to differentiate into cDCs and pDCs, as well as inhibits the function of cDCs on the activation of CD4 + T cells by producing IL-17a.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early myeloid progenitors, which possess strong immunosuppressive functions. MDSCs are found in increased numbers in infectious and inflammatory pathological conditions. However, whether microRNAs have a role in the expansion remains unclear. Here in our study, we found that overexpression of miR-34a could induce the expansion of MDSCs in the bone marrow and spleen both in chimera and transgenic mice. And further experiments demonstrated that miR-34a inhibited the apoptosis through reduced translation of N-myc without affecting the proliferation. Luciferase assay and western blotting experiments implied that N-myc is the direct target of miR-34a in MDSCs. Overexpressed mir-34a changes the cytokine expression profile in MDSCs and skewed the MDSCs to M1 phenotype. And miR-34a-overexpressed MDSCs significantly slowed down the tumor growth. Taken together, miR-34a contributes to the expansion of MDSCs by inhibiting the apoptosis via suppressing the expression of N-myc.

BACKGROUND: Recent evidence shows that long non-coding RNA (LncRNA) play important regulatory roles in many biology process, including cell development, activation and oncogenesis. However, the roles of these LncRNAs in the development and activation of CD4+ T cells, which is an important component of immune response, remain unknown. RESULTS: To predict the function of LncRNA in the development and activation of CD4+ T cells, first, we examined the expression profiles of LncRNAs and mRNAs in CD4-CD8- (DN), CD4+CD8+ (DP), CD4+CD8-, and activated CD4+CD8- T cells in a microarray analysis and verified these results by real time PCRs (qPCR). We found that the expression of hundreds of LncRNAs significantly changed in each process of developmental transition, including DN into DP, DP into CD4+CD8-, and CD4+CD8- into activated CD4+ T cells. A Kendall distance analysis suggested that the expression of LncRNAs in DN, DP, CD4+CD8- T cells and activated CD4+ T cells were correlated with the expression of mRNAs in these T cells. The Blat algorithm and GO analysis suggested that LncRNAs may exert important roles in the development and activation of CD4+ T cells. These roles included proliferation, homeostasis, maturation, activation, migration, apoptosis and calcium ion transportation. CONCLUSION: The present study found that the expression profiles of LncRNAs in different stages of CD4+ T cells are distinguishable. LncRNAs are involved in the key biological process in CD4+ T cell development and activation.

Resveratrol, a natural polyphenol compound, has broad effects on critical events, including inflammation, oxidation, cancer and aging. However, the function and molecular mechanisms of resveratrol on T cell activation are controversial. In the present study, we found that resveratrol significantly inhibits the activation and cytokine production of T cells in vitro and in vivo. Sirt1 expression was up-regulated in resveratrol-treated T cells. Once Sirt1 was down-regulated in the T cells, the resveratrol-induced inhibition of T cell activation noticeably diminished. The acetylation of c-Jun decreased and its translocation was impeded in the resveratrol-treated T cells. The incidence and severity of collagen-induced arthritis in the resveratrol-treated mice were considerably reduced.