Scientific Resources

Interactions with Ag-specific T cells drive B cell activation and fate choices that ultimately determine the quality of high-affinity Ab responses. As such,these interactions,and especially the long-lived interactions that occur before germinal center formation,may be important checkpoints to regulate undesirable responses. Using mouse model Ag systems,we directly observed interactions between T and B cells responding to the self-antigen myelin oligodendrocyte glycoprotein (MOG) and found that they are of lower quality compared with interactions between cells responding to the model foreign Ag nitrophenyl-haptenated OVA. This was associated with reduced expression of molecules that facilitate these interactions on the B cells,but not on T cells. B cell expression of these molecules was not dictated by the T cell partner,nor could the relative lack of expression on MOG-specific (MOG-sp.) B cells be reversed by a multivalent Ag. Instead,MOG-sp. B cells were inherently less responsive to BCR stimulation than MOG-non-sp. cells. However,the phenotype of MOG-sp. B cells was not consistent with previous descriptions of autoimmune B cells that had been tolerized via regular exposure to systemically expressed self-antigen. This suggests that alternate anergy pathways may exist to limit B cell responses to tissue-restricted self-antigens. View Publication

Ov-ASP-1 (rASP-1),a parasite-derived protein secreted by the helminth Onchocerca volvulus,is an adjuvant which enhances the potency of the influenza trivalent vaccine (IIV3),even when used with 40-fold less IIV3. This study is aimed to provide a deeper insight into the molecular networks that underline the adjuvanticity of rASP-1. Here we show that rASP-1 stimulates mouse CD11c+ bone marrow-derived dendritic (BMDCs) to secrete elevated levels of IL-12p40,TNF-?,IP-10 and IFN-? in a TRIF-dependent but MyD88-independent manner. rASP-1-activated BMDCs promoted the differentiation of na?ve CD4+ T cells into Th1 cells (IFN-?+) that was TRIF- and type I interferon receptor (IFNAR)-dependent,and into Tfh-like cells (IL21+) and Tfh1 (IFN-?+ IL21+) that were TRIF-,MyD88- and IFNAR-dependent. rASP-1-activated BMDCs promoted the differentiation of na?ve CD4+ T cells into Th17 (IL-17+) cells only when the MyD88 pathway was inhibited. Importantly,rASP-1-activated human blood cDCs expressed upregulated genes that are associated with DC maturation,type I IFN and type II IFN signaling,as well as TLR4-TRIF dependent signaling. These activated cDCs promoted the differentiation of na?ve human CD4+ T cells into Th1,Tfh-like and Th17 cells. Our data thus confirms that the rASP-1 is a potent innate adjuvant that polarizes the adaptive T cell responses to Th1/Tfh1 in both mouse and human DCs. Notably,the rASP-1-adjuvanted IIV3 vaccine elicited protection of mice from a lethal H1N1 infection that is also dependent on the TLR4-TRIF axis and IFNAR signaling pathway,as well as on its ability to induce anti-IIV3 antibody production. View Publication

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example,ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However,additional antitumor benefits from targeting the DDR pathways,which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal,inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here,we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However,STING deficiency in tumor cells does not prevent DC activation,suggesting that transactivation of the STING pathway occurs within DCs. Furthermore,depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells,indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary,in this study,we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation,which is enhanced in ATM-deficient cells. View Publication

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection,but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model,we evaluated allografts from BALB/c †’ C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet,IFN-$\gamma$,and TRAF proteins 1-5 compared with isografts. In the acute model,treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3,preserved FBXL2,and substantially reduced T-bet,IFN-$\gamma$,and TRAFs 1-5,consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model,DBA/2J/C57BL/6F1 > DBA/2J (day 28),we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-$\gamma$ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines,TNF-$\alpha$ was found to negatively regulate FBXL2 protein and mRNA levels. Together,our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2,with negative cross-regulation of TNF-$\alpha$ on FBXL2 during lung allograft rejection. View Publication

BACKGROUND Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has become an increasingly vital tool for modifying gene expression in a variety of cell types. Lentiviral transduction and electroporation are the two main approaches used to deliver CRISPR/Cas9 into cells. However,the application of CRISPR/Cas9 in primary hematopoietic cells has been limited due to either low transduction efficiency in terms of viral-based delivery or difficult selection and enrichment of transfected and edited cells with respect to electroporation of CRISPR/Cas9 ribonucleoprotein (RNP). METHODS In this study in vitro transcription was used to synthesize the guide RNA (gRNA),and plasmid pL-CRISPR.EFS.GFP was used as its DNA template. Then the in vitro transcribed gRNA was labeled with pCp-Cy5 via T4 ligase before incubating with Cas9 protein. Furthermore,CRISPR/Cas9 RNP was electroporated into primary CD34+ cells isolated from cord blood,and cell survival rate and transfection efficiency were calculated and compared to that of lentiviral transduction. RESULTS Here,we show that electroporation of CRISPR/Cas9 RNP resulted in higher cell viability compared to electroporation of CRISPR/Cas9 all-in-one plasmid,providing important findings for further studies in hematology via CRISPR/Cas9 technology. Moreover,we established a method for labeling in vitro-transcribed gRNA with fluorophore and the sorted fluorescent cells displayed higher knockout efficiency than nonsorted transfected cells. CONCLUSIONS Electroporation of fluorescence labeled CRISPR/Cas9 RNP is a perspective approach of gene editing. Our study provides an efficient and time-saving approach for genome-editing in hematopoietic cells. View Publication

The biological macromolecule Nocardia rubra cell-wall skeleton (Nr-CWS) has well-established immune-stimulating and anti-tumor activities. However,the role of Nr-CWS on natural killer (NK) cells remains unclear. Here,we explore the function and related mechanisms of Nr-CWS on NK cells. Using a tumor-bearing model,we show that Nr-CWS has slightly effect on solid tumor. In addition,using a tumor metastasis model,we show that Nr-CWS suppresses the lung metastasis induced by B16F10 melanoma cells in mice,which indicates that Nr-CWS may up-regulate the function of NK cells. Further investigation demonstrated that Nr-CWS can increase the expression of TRAIL and FasL on spleen NK cells from Nr-CWS treated B16F10 tumor metastasis mice. The spleen index and serum levels of TNF-$\alpha$,IFN-$\gamma$,and IL-2 in B16F10 tumor metastasis mice treated with Nr-CWS were significantly increased. In vitro,the studies using purified or sorted NK cells revealed that Nr-CWS increases the expression of CD69,TRAIL,and FasL,decreases the expression of CD27,and enhances NK cell cytotoxicity. The intracellular expression of IFN-$\gamma$,TNF-$\alpha$,perforin (prf),granzyme-B (GrzB),and secreted TNF-$\alpha$,IFN-$\gamma$,IL-6 of the cultured NK cells were significantly increased after treatment with Nr-CWS. Overall,the findings indicate that Nr-CWS could suppress the lung metastasis induced by B16F10 melanoma cells,which may be exerted through its effect on NK cells by promoting NK cell terminal differentiation (CD27lowCD11bhigh),and up-regulating the production of cytokines and cytotoxic molecules. View Publication

Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However,it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4R$\alpha$-/- ) and mice with a myeloid-specific deletion of IL-4R$\alpha$ (IL-4R$\alpha$f/f LysMcre ),we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4R$\alpha$,murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4R$\alpha$-/- knockout animals. Furthermore,assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4R$\alpha$-/- mice,whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore,IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway,including the phosphorylation of the NF$\kappa$Bp65 protein. In a cohort of human patients,serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model,reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4R$\alpha$f/f LysMcre mice without changes in overall migratory function. Thus,we identified a homeostatic role of IL-4R$\alpha$ in regulating the lifespan of monocytes in vivo. View Publication

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