Scientific Resources

As a critical immune checkpoint molecule,PD-L1 is expressed at significantly higher levels in multiple neoplastic tissues compared to normal ones. PD-L1/PD-1 axis is a critical target for tumor immunotherapy,blocking the PD-L1/PD-1 axis is recognized and has achieved unprecedented success in clinical applications. However,the clinical efficacy of therapies targeting the PD-1/PD-L1 pathway remains limited,emphasizing the need for the mechanistic elucidation of PD-1/PD-L1 expression. In this study,we found that RNF125 interacted with PD-L1 and regulated PD-L1 protein expression. Mechanistically,RNF125 promoted K48-linked polyubiquitination of PD-L1 and mediated its degradation. Notably,MC-38 and H22 cell lines with RNF125 knockout,transplanted in C57BL/6 mice,exhibited a higher PD-L1 level and faster tumor growth than their parental cell lines. In contrast,overexpression of RNF125 in MC-38 and H22 cells had the opposite effect,resulting in lower PD-L1 levels and delayed tumor growth compared with parental cell lines. In addition,immunohistochemical analysis of MC-38 tumors with RNF125 overexpression showed significantly increased infiltration of CD4+,CD8+ T cells and macrophages. Consistent with these findings,analyses using The Cancer Genome Atlas (TCGA) public database revealed a positive correlation of RNF125 expression with CD4+,CD8+ T cell and macrophage tumor infiltration. Moreover,RNF125 expression was significantly downregulated in several human cancer tissues,and was negatively correlated with the clinical stage of these tumors,and patients with higher RNF125 expression had better clinical outcomes. Our findings identify a novel mechanism for regulating PD-L1 expression and may provide a new strategy to increase the efficacy of immunotherapy. View Publication

Lymphopenia is a common observation in patients with COVID-19. To explore the cause of T cell lymphopenia in the disease,laboratory results of 64 hospitalized COVID-19 patients were retrospectively analyzed and six patients were randomly selected to trace their changes of T lymphocytes and plasma concentration of IL-6 for the course of disease. Results confirmed that the T-cell lymphopenia,especially CD4+ T cell reduction in COVID-19 patients,was a reliable indicator of severity and hospitalization in infected patients. And CD4+ T cell count below 200 cells/$\mu$L predicts critical illness in COVID-19 patients. In vitro assay supported that exposure to key contributors (IL-1$\beta$,IL-6,TNF-$\alpha$ and IFN-$\gamma$) of COVID-19 cytokine storm caused substantial death of activated T cells. Among these contributors,IL-6 level was found to probably reversely correlate with T cell counts in patients. And IL-6 alone was potent to induce T cell reduction by gasderminE-mediated pyroptosis,inferring IL-6 took a part in affecting the function and status of T cells in COVID-19 patients. Intervention of IL-6 mediated T cell pryprotosis may effectively delay disease progression,maintain normal immune status at an early stage of infection. View Publication

Oncolytic viruses have been emerging as a promising therapeutic option for cancer patients,including lung cancer. Orf virus (ORFV),a DNA parapoxvirus,can infect its natural ungulate hosts and transmit into humans. Moreover,the ORFV has advantages of low toxicity,high targeted,self-amplification and can induce potent Th1-like immunity. This study explored the therapeutic potential of ORFV infection for human lung cancer therapy and investigated the molecular mechanisms. We used a previously described ORFV NA1/11 strain and tested the oncolysis of ORFV NA1/11 in two lines of lung cancer cells in vitro and in vivo. Treatment of both cell lines with ORFV NA1/11 resulted in a decrease in cell viability by inducing cell cycle arrest in G2/M phase,suppressing cyclin B1 expression and increasing their apoptosis in a caspase-dependent manner. The ORFV NA1/11-infected lung cancer cells were highly immunogenic. Evidently,ORFV NA1/11 infection of lung cancer cells induced oncolysis of tumor cells to release danger-associated molecular patterns,and promoted dendritic cell maturation,and CD8 T cell infiltration in the tumors by enhancing CXCL16 secretion. These findings may help to understand the molecular mechanisms of ORFV oncolysis and aid in the development of novel therapies for lung cancer. View Publication

The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients,many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus,overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example,with rare,propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore,it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms,and their key pathways,so that CML LSCs can be selectively targeted or sensitized to TKIs. Here,we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI),which can be further explored as therapeutic modalities against LSCs in the context of human leukemia. View Publication

In response to an intracellular infectious agent,the immune system produces a specific cellular response as well as a T cell-dependent Ab response. Precursor T cells differentiate into effector T cells,including Th1 cells,and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study,we demonstrated that the Bcl6 gene is regulated by FOXO1-binding,cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells,deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity. View Publication

Pancreatic cancer is one of the deadliest cancers,against which current immunotherapy strategies are not effective. Herein,we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically,NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However,tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression,but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer,its combination with radiation therapy hold greater therapeutic benefit. View Publication

The mechanisms that control the inflammatory-immune response play a key role in tissue remodelling in cardiovascular diseases. T cell activation receptor CD69 binds to oxidized low-density lipoprotein (oxLDL),inducing the expression of anti-inflammatory NR4A nuclear receptors and modulating inflammation in atherosclerosis. To understand the downstream T cell responses triggered by the CD69-oxLDL binding,we incubated CD69-expressing Jurkat T cells with oxLDL. RNA sequencing revealed a differential gene expression profile dependent on the presence of CD69 and the degree of LDL oxidation. CD69-oxLDL binding induced the expression of NR4A receptors (NR4A1 and NR4A3),but also of PD-1. These results were confirmed using oxLDL and a monoclonal antibody against CD69 in CD69-expressing Jurkat and primary CD4??+??lymphocytes. CD69-mediated induction of PD-1 and NR4A3 was dependent on NFAT activation. Silencing NR4A3 slightly increased PD-1 levels,suggesting a potential regulation of PD-1 by this receptor. Moreover,expression of PD-1,CD69 and NR4A3 was increased in human arteries with chronic inflammation compared to healthy controls,with a strong correlation between PD-1 and CD69 mRNA expression (r??=??0.655 P?? View Publication

BACKGROUND Several miRNAs are now known to have clear connections to the pathogenesis of asthma. The present study focused on the potential role of miR-3934 during asthma development. METHODS miR-3934 was detected as a down-regulated miRNA in basophils by sequencing analysis. Next,the expression levels of miR-3934 in peripheral blood mononuclear cells of 50 asthma patients and 50 healthy volunteers were examined by RT-qPCR methods. The basophils were then treated with AGEs and transfected with miR-3934 mimics. The apoptosis levels were examined by flow cytometry assay; and the expression levels of cytokines were detected using the ELISA kits. Finally,the Western blot was performed to examined the expression of key molecules in the TGF-$\beta$/Smad signaling pathway. RESULTS miR-3934 was down-regulated in the basophils of asthmatic patients. The expression of the pro-inflammatory cytokines IL-6,IL-8 and IL-33 was enhanced in basophils from asthmatic patients,and this effect was partially reversed by transfection of miR-3934 mimics. Furthermore,receiver operating characteristics analysis showed that miR-3934 levels can be used to distinguish asthma patients from healthy individuals. miR-3934 partially inhibited advanced glycation end products-induced increases in basophil apoptosis by suppressing expression of RAGE. CONCLUSION Our results indicate that miR-3934 acts to mitigate the pathogenesis of asthma by targeting RAGE and suppressing TGF-$\beta$/Smad signaling. View Publication

Immune-mediated red blood cell (RBC) destruction due to antibodies is an ongoing problem in transfusion medicine for the selection of the safest blood. Serological testing often revealed incompatibility with donors' RBCs. When this incompatible blood was transfused,destruction was due mostly to extravascular-mediated phagocytosis of the antibody-opsonized RBCs; however,intravascular hemolysis was sometimes observed without explanation. Based on serology,antibodies with potential for clinical sequalae could not be ascertained; thus,antigen-negative blood was usually selected for transfusion to avoid problems. Antibodies to antigens having very high frequency in the general population (>95%),however,made selection of antigen-negative blood difficult and sometimes impossible. Some patients,who were sensitized by previous transfusions or by pregnancy,developed multiple antibodies,again creating a problem for finding compatible blood for transfusion,without the ability to discern which of the antibodies may be clinically irrelevant and ignored. Transfusion medicine scientists began searching for an in vitro means to determine the in vivo outcome of transfusion of blood that was serologically incompatible. Methods such as chemiluminescence,monocyte-macrophage phagocytosis,and antibody-dependent cellular cytotoxicity (ADCC) were described. Over the years,the monocyte monolayer assay (MMA) has emerged as the most reliable in vitro assay for the prediction of the clinical relevance of a given antibody. ADCC has not been fully studied but has the potential to be useful for predicting which antibodies may result in intravascular hemolysis. This article captures the protocols for the implementation and readout of the MMA and ADCC assays for use in predicting the clinical significance of antibodies in a transfusion setting. {\textcopyright} 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Monocyte monolayer assay (MMA) Basic Protocol 2: Antibody-dependent cellular cytotoxicity assay (ADCC). View Publication

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2$\alpha$ and/or CK2$\alpha$') and two regulatory subunits (CK2$\beta$). CK2 promotes cancer progression by activating the NF-$\kappa$B,PI3K/AKT/mTOR,and JAK/STAT pathways,and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2$\alpha$ deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore,CK2$\alpha$ is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly,using a mouse Listeria monocytogenes infection model,we demonstrate that CK2$\alpha$ is required for CD8+ T cell expansion,maintenance,and effector function in both primary and memory immune responses. Collectively,our study implicates CK2$\alpha$ as an important regulator of mouse CD8+ T cell activation,metabolic reprogramming,and differentiation both in vitro and in vivo. View Publication

BACKGROUND Adoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However,even for the best-studied and clinically validated CD19-targeted chimeric antigen receptor (CAR) T-cell therapy,many patients face the challenge of lack of response or occurrence of relapse. There is increasing need to improve the efficacy of ACT so that durable,curative outcomes can be achieved in a broad patient population. METHODS Here,we investigated the impact of indomethacin (indo),a non-steroidal anti-inflammatory drug (NSAID),on the efficacy of ACT in multiple preclinical models. Mice with established B-cell lymphoma received various combinations of preconditioning chemotherapy,infusion of suboptimal dose of tumor-reactive T cells,and indo administration. Donor T cells used in the ACT models included CD4+ T cells expressing a tumor-specific T cell receptor (TCR) and T cells engineered to express CD19CAR. Mice were monitored for tumor growth and survival. The effects of indo on donor T cell phenotype and function were evaluated. The molecular mechanisms by which indo may influence the outcome of ACT were investigated. RESULTS ACT coupled with indo administration led to improved tumor growth control and prolonged mouse survival. Indo did not affect the activation status and tumor infiltration of the donor T cells. Moreover,the beneficial effect of indo in ACT did not rely on its inhibitory effect on the immunosuppressive cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis. Instead,indo-induced oxidative stress boosted the expression of death receptor 5 (DR5) in tumor cells,rendering them susceptible to donor T cells expressing TNF-related apoptosis-inducing ligand (TRAIL). Furthermore,the ACT-potentiating effect of indo was diminished against DR5-deficient tumors,but was amplified by donor T cells engineered to overexpress TRAIL. CONCLUSION Our results demonstrate that the pro-oxidative property of indo can be exploited to enhance death receptor signaling in cancer cells,providing rationale for combining indo with genetically modified T cells to intensify tumor cell killing through the TRAIL-DR5 axis. These findings implicate indo administration,and potentially similar use of other NSAIDs,as a readily applicable and cost-effective approach to augment the efficacy of ACT. View Publication

T cell antigen-receptor (TCR) signaling controls the development,activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing,translation and stability of transcripts. In the present study,we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes,control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut ROR?t+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells. View Publication