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The prostate is a gland that contributes to men's fertility. It is highly responsive to androgens and is often the site of carcinogenesis,as prostate cancer is the most frequent cancer in men in over a hundred countries. To study the normal prostate,few in vitro models exist,and most of them do not express the androgen receptor (AR). To overcome this issue,prostate epithelial cells can be grown in primary culture ex vivo in 2- and 3-dimensional culture (organoids). However,methods to purify these cells often require flow cytometry,thus necessitating specialized instruments and expertise. Herein,we present a detailed protocol for the harvest,purification,and primary culture of mouse prostate epithelial cells to grow prostate organoids ex vivo. This protocol does not require flow cytometry approaches,facilitating its implementation in most research laboratories,and organoids grown with this protocol are highly responsive to androgens. In summary,we present a new simple method that can be used to grow prostate organoids that recapitulate the androgen response of this gland in vivo. View Publication

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

BACKGROUND Endometriosis is a common,complex disorder which is underrecognized and subject to prolonged delays in diagnosis. It is accompanied by significant changes in the eutopic endometrial lining. METHODS We have undertaken the first single-cell RNA-sequencing (scRNA-Seq) comparison of endometrial tissues in freshly collected menstrual effluent (ME) from 33 subjects,including confirmed endometriosis patients (cases) and controls as well as symptomatic subjects (who have chronic symptoms suggestive of endometriosis but have not been diagnosed). RESULTS We identify a unique subcluster of proliferating uterine natural killer (uNK) cells in ME-tissues from controls that is almost absent from endometriosis cases,along with a striking reduction of total uNK cells in the ME of cases (p?? View Publication

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets,neutrophils,fibrin,extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast,in vivo blockage of Fc$\gamma$RIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via Fc$\gamma$RIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and Fc$\gamma$RIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively,leading to better patient outcomes. 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

Type-2 bitter taste receptors (Tas2Rs) are a large family of G protein-coupled receptors that are expressed in the oral cavity and serve to detect substances with bitter tastes in foods and medicines. Recent evidence suggests that Tas2Rs are also expressed extraorally,including in immune cells. However,the role of Tas2Rs in immune cells remains controversial. Here,we demonstrate that Tas2R126,Tas2R135,and Tas2R143 are expressed in mouse neutrophils,but not in other immune cells such as macrophages or T and B lymphocytes. Treatment of bone marrow-derived neutrophils from wild-type mice with the Tas2R126/143 agonists arbutin and d-salicin led to enhanced C-X-C motif chemokine ligand 2 (CXCL2)-stimulated migration in vitro,but this response was not observed in neutrophils from Tas2r126/135/143-deficient mice. Enhancement of CXCL2-stimulated migration by Tas2R agonists was accompanied by increased phosphorylation of myosin light chain 2 (MLC2) and was blocked by pretreatment of neutrophils with inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK),but not by inhibitors of the small GTPase RhoA. Taken together,these results demonstrate that mouse neutrophils express functional Tas2R126/143 and suggest a role for Tas2R126/143-ROCK-MLC2-dependent signaling in the regulation of neutrophil migration. View Publication

Type 1 conventional dendritic cells (cDC1) efficiently cross-present antigens that prime cytotoxic CD8+ T cells. cDC1 therefore constitute conceivable targets in cancer vaccine development. We generated recombinant fusion cancer vaccines that aimed to concomitantly deliver tumor antigen and adjuvant to CD103+ migratory cDC1,following intranasal administration. The fusion vaccine constructs comprised a cDC1-targeting anti-CD103 single chain antibody (aCD103) and a cholera toxin A1 (CTA1) subunit adjuvant,fused with MHC class I and II- or class II-restricted tumor cell antigens to generate a CTA1-I/II-aCD103 vaccine and a CTA1-II-aCD103 vaccine. The immunostimulatory and anti-tumor efficacy of these vaccines was evaluated in murine B16F1-ovalbumin (OVA) melanoma models in C57BL/6 J mice. The CTA1-I/II-aCD103 vaccine was most efficacious and triggered robust tumor antigen-specific CD8+ T cell responses along with a Th17-polarized CD4+ T cell response. This vaccine construct reduced the local growth of implanted B16F1-OVA melanomas and efficiently prevented hematogenous lung metastasis after prophylactic and therapeutic vaccination. Anti-tumor effects of the CTA1-I/II-aCD103 vaccine were antigen-specific and long-lasting. These results imply that adjuvant-containing recombinant fusion vaccines that target and activate cDC1 trigger effective anti-tumor immunity to control tumor growth and metastasis. View Publication