搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here,we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1–2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients,with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states,including a naive-like state,but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively,these data support the feasibility,safety,and immunogenicity of this treatment in resected NSCLC. View Publication

The molecular mechanisms regulating CD8 + cytotoxic T lymphocytes (CTL) are not fully understood. Here,we show that the peroxisome proliferator–activated receptor δ (PPARδ) suppresses CTL cytotoxicity by inhibiting RelA DNA binding. Treatment of Apc Min/+ mice with the PPARδ agonist GW501516 reduced the activation of normal and tumor-associated intestinal CD8 + T cells and increased intestinal adenoma burden. PPARδ knockout or knockdown in CTLs increased their cytotoxicity against colorectal cancer cells,whereas overexpression of PPARδ or agonist treatment decreased it. Correspondingly,perforin,granzyme B,and IFNγ protein and mRNA levels were higher in PPARδ knockout or knockdown CTLs and lower in PPARδ overexpressing or agonist-treated CTLs. Mechanistically,we found that PPARδ binds to RelA,interfering with RelA–p50 heterodimer formation in the nucleus,thereby inhibiting its DNA binding in CTLs. Thus,PPARδ is a critical regulator of CTL effector function. Significance: Here,we provide the first direct evidence that PPARδ plays a critical role in suppressing the immune response against tumors by downregulating RelA DNA-binding activity. This results in decreased expression of perforin,granzyme B,and IFNγ. Thus,PPARδ may serve as a valuable target for developing future cancer immunotherapies. View Publication

Chordoma is a slow-growing,primary malignant bone tumor that arises from notochordal tissue in the midline of the axial skeleton. Surgical excision with negative margins is the mainstay of treatment,but high local recurrence rates are reported even with negative margins. High-dose radiation therapy (RT),such as with proton or carbon ions,has been used as an alternative to surgery,but late local failure remains a problem. B7-H3 is an immune checkpoint,transmembrane protein that is dysregulated in many cancers,including chordoma. This study explores the efficacy of B7-H3 chimeric antigen receptor T (CAR-T) therapy in vitro and in vivo. Chordoma cancer stem cells (CCSCs) were identified using flow cytometry,sphere formation,and western blot analysis. The expression of B7-H3 in paraffin-embedded chordoma tissue was determined by immunohistochemical staining,and the expression of B7-H3 in chordoma cells was measured by flow cytometry. Retroviral particles containing either B7-H3 or CD19 CAR-expressing virus were transduced into T cells derived from peripheral blood mononuclear cells isolated from healthy human donor blood to prepare CAR-T cells. Animal bioluminescent imaging was used to evaluate the killing effect of CAR-T cells on chordoma cells in vivo. An irradiator was used for all irradiation (IR) experiments. The combination of B7-H3 CAR-T cell therapy and IR has a greater killing effect on killing radiation-resistant CCSCs and bulk chordoma cells compared with CAR-T cell or IR monotherapy. Additionally,increased expression of B7-H3 antigens on CCSCs and bulk tumor cells is associated with enhanced CAR-T cell killing in vitro and in vivo xenograft mouse models. Upregulation of B7-H3 expression by IR increases CCSCs sensitivity to B7-H3 CAR-T cell-mediated killing. Our preliminary data show that IR and B7-H3 CAR-T cell therapy is synergistically more effective than either IR or CAR-T cell monotherapy in killing chordoma cells in vitro and in a xenograft mouse model. These results provide preclinical evidence for further developing this combinatorial RT and B7-H3 CAR-T cell therapy model in chordoma View Publication

The airway epithelium provides a first line of defense against pathogens by release of antimicrobial factors and neutrophil-attracting chemokines. Pseudomonas (P.) aeruginosa,a Gram-negative bacterium that expresses flagellin as an important virulence factor,is a common cause of injurious airway inflammation. The aim of our study was to determine the contribution of flagellin to the inflammatory,antimicrobial,and metabolic responses of the airway epithelium to P. aeruginosa . Furthermore,as we previously showed that targeting mTOR limited the glycolytic and inflammatory response induced by flagellin,we assessed the effect of rapamycin on human bronchial epithelial (HBE) cells stimulated with flagellated and non-flagellated P. aeruginosa. Primary pseudostratified HBE cells,cultured on an air-liquid-interface,were treated on the basolateral side with medium,vehicle or rapamycin,exposed on the apical side with flagellated or flagellin-deficient P. aeruginosa,and analyzed for their inflammatory,antimicrobial,and glycolytic responses. Flagellin augmented the P. aeruginosa -induced expression of antimicrobial factors and secretion of chemokines by HBE cells but did not further increase the glycolytic response. Treatment of HBE cells with rapamycin inhibited mTOR activation in general and flagellin-augmented mTOR activation in particular,but did not affect the glycolytic response. Rapamycin,however,diminished the flagellin-augmented inflammatory and antimicrobial response induced by Pseudomonas . These results demonstrate that flagellin is a significant factor that augments the inflammatory and antimicrobial response of human airway epithelial cells upon exposure to P. aeruginosa and suggest that mTOR inhibition by rapamycin in the airway epithelium diminishes these exaggerated responses. View Publication

PURPOSE Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy,temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN ZR2002 is a combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR downstream Erk1/2 phosphorylation increased DNA strand breaks and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line decreased EGFR Erk1/2 and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability blood-brain barrier permeability and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM." View Publication

Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations. AbstractFc engineering to enhance antibody effector functions harbors the potential to improve therapeutic effects. Understanding FcγR expression and distribution in the tumor microenvironment prior to and following treatment may help guide immune-engaging antibody design and patient stratification. In this study,we investigated FcR-expressing immune effector cells in HER2+ and triple-negative breast cancers (TNBC),including neoadjuvant chemotherapy–resistant disease. FcγRIIIa expression,FcγRIIIa+ NK cells,and classically activated (M1-like) macrophages correlated with improved anti-HER2 antibody efficacy. FcγRIIIa protein and FcγRIIIa+ NK cells and macrophages were present in primary TNBC and retained in treatment-resistant tumors. FcγRIIIa was spatially associated with folate receptor alpha–positive (FRα+) tumor areas at baseline and in residual tumors following neoadjuvant chemotherapy. Wild-type and Fc-engineered antibodies recognizing two breast cancer–associated antigens,HER2 and the emerging TNBC target FRα,were designed and generated to have increased FcγRIIIa-expressing effector cell engagement. The combination of glycoengineering,including fucose removal from the N-linked Fc glycan,and Fc point mutations greatly increased antibody affinity for and retention on FcγRIIIa. The Fc-engineered antibodies enhanced immune effector activity against HER2+ breast cancer and TNBC,altering proinflammatory cytokine production by NK cells and tumor-conditioned macrophages and skewing macrophages toward proinflammatory states. Furthermore,the Fc-engineered antibodies restricted orthotopic HER2+ and FRα+ breast cancer xenograft growth at doses suboptimal for equivalent wild-type antibodies and recruited FcγRIIIa-expressing cells into tumors. Antibody design through combined glycoengineering and Fc point mutations to enhance FcγRIIIa engagement of tumor-infiltrating effector cells may be a promising strategy for developing therapies for patients with aggressive and treatment-resistant breast cancers.Significance:Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication

BACKGROUND Patient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However,the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research,and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study,our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer. METHODS We employed organoid culture methods and flow cytometric,cytologic,immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment. RESULTS We describe the culture and characterization of human pancreatic cancer organoids from resection,ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology,tissue architecture and polarity in contrast to cell line spheroids,which formed homogenous,non-lumen forming spheres. Importantly,we demonstrate the construction of complex organotypic models of tumor,stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models. CONCLUSIONS These studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor,stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration. View Publication

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However,the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular,interferon-gamma$ (IFN-gamma$)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-gamma$ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation,the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore,the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-gamma$ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-gamma$ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture,whereas IDO expression by IFN-gamma$-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore,sustained IDO expression induced by IFN-gamma$-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-gamma$-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether,microparticle delivery of IFN-gamma$ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237. View Publication

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells,including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM,we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients,we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However,we discovered,next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC,a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients),as well as CDC (56 patients). Similarly,experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly,all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. View Publication

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However,we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study,25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1193±631days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells. View Publication