Scientific Resources

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSCs) in cancer,inflammation,and fibrosis,with some of these cells expressing B7H3. We sought to investigate the role of MDSCs in IPF and its potential mediation via B7H3. Here we prospectively collected peripheral blood samples from IPF patients to analyze for circulating MDSCs and B7H3 expression to assess their clinical significance and potential impact on co-cultured lung fibroblasts and T-cell activation. In parallel,we assess MDSC recruitment and potential B7H3 dependence in a mouse model of pulmonary fibrosis. Expansion of MDSCs in IPF patients correlated with disease severity. Co-culture of soluble B7H3 (sB7H3)-treated mouse monocytic MDSCs (M-MDSCs),but not granulocytic MDSCs (G-MDSCs),activated lung fibroblasts and myofibroblast differentiation. Additionally,sB7H3 significantly enhanced MDSC suppression of T-cell proliferation. Activated M-MDSCs displayed elevated TGF$\beta$ and Arg1 expression relative to that in G-MDSCs. Treatment with anti-B7H3 antibodies inhibited bone marrow-derived MDSC recruitment into the bleomycin-injured lung,accompanied by reduced expression of inflammation and fibrosis markers. Selective telomerase reverse transcriptase (TERT) deficiency in myeloid cells also diminished MDSC recruitment associated with the reduced plasma level of sB7H3,lung recruitment of c-Kit+ hematopoietic progenitors,myofibroblast differentiation,and fibrosis. Lung single-cell RNA sequencing (scRNA-seq) revealed fibroblasts as a predominant potential source of sB7H3,and indeed the conditioned medium from activated mouse lung fibroblasts had a chemotactic effect on bone marrow (BM)-MDSC,which was abolished by B7H3 blocking antibody. Thus,in addition to their immunosuppressive activity,TERT and B7H3-dependent MDSC expansion/recruitment from BM could play a paracrine role to activate myofibroblast differentiation during pulmonary fibrosis with potential significance for disease progression mediated by sB7H3. View Publication

BACKGROUND Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor. Although numerous postoperative therapeutic strategies have already been developed,including radiotherapy,tumors inevitably recur after several years of treatment. The coinhibitory molecule B7-H4 negatively regulates T cell immune responses and promotes immune escape. Exosomes mediate intercellular communication and initiate immune evasion in the tumor microenvironment (TME). OBJECTIVE This study aimed to determine whether B7-H4 is upregulated by radiation and loaded into exosomes,thus contributing to immunosuppression and enhancing tumor growth. METHODS Iodixanol density-gradient centrifugation and flow cytometry were used to verify exosomal B7-H4. Na{\{i}}ve T cells were differentiated into Th1 cells with or without exosomes. T cell-secreted cytokines and markers of T cell subsets were measured. Mechanistically the roles of B7-H4 and ALIX in GBM were analyzed using databases and tissue samples. Co-immunoprecipitation and pull-down assays were used to tested the direct interactions between ATM and ALIX or STAT3. In vitro ATM kinase assays western blotting and site-directed mutation were used to assess ATM-mediated STAT3 phosphorylation. Finally the contribution of exosomal B7-H4 to immunosuppression and tumor growth was investigated in vivo. RESULTS Exosomes from irradiated GBM cells decreased the anti-tumor immune response of T cell in vitro and in vivo via delivered B7-H4. Mechanistically irradiation promoted exosome biogenesis by increasing the ATM-ALIX interaction. Furthermore the ATM-phosphorylated STAT3 was found to directly binds to the B7-H4 promoter to increase its expression. Finally the radiation-induced increase in exosomal B7-H4 induced FoxP3 expression during Th1 cell differentiation via the activated STAT1 pathway. In vivo exosomal B7-H4 decreased the radiation sensitivity of GBM cells and reduced the survival of GBM mice model. CONCLUSION This study showed that radiation-enhanced exosomal B7-H4 promoted immunosuppression and tumor growth hence defining a direct link between irradiation and anti-tumor immune responses. Our results suggest that co-administration of radiotherapy with anti-B7-H4 therapy could improve local tumor control and identify exosomal B7-H4 as a potential tumor biomarker." View Publication

Coronary heart disease (CHD) is a leading cause of death globally,while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration,and CD34+ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However,the isolation and culture of non-adherent CD34+ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34+ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment,isolation,and expansion. The culture was subsequently observed for their viability,adherence,and confluence. Day 0 observation of the culture showed a healthy CD34+ cell with a round cell shape,without any adherent cells present yet. Day 4 of observation showed that CD34+ cells within the blood plasma medium became adherent,indicated by their transformations into spindle or oval morphologies. Meanwhile,CD34+ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34+ cells in blood plasma medium,and which had 75% of confluence. In conclusion,the CD34+ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium,but not in cultures using fibronectin and vitronectin. View Publication

BACKGROUND In kidney transplantation,early allograft inflammation impairs long-term allograft function. However,precise mediators of early kidney allograft inflammation are unclear,making it challenging to design therapeutic interventions. METHODS We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. RESULTS Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period,they were also induced to express a high level of Ccl8,which,in turn,promoted recipient monocyte graft infiltration,their differentiation to resident macrophages,and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed,including CD4,CD8,and ?? T cells. Consequently,blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. CONCLUSIONS Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation. View Publication

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset,which express na{\{i}}ve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of na{\"{i}}ve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy." View Publication

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