Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane) (PDMS), a biocompatible silicone elastomer. We show that softer (Young’s Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4+ and CD8+ T cells compared with stiffer substrates (E >2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (non-significant) towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Naïve CD4+ T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ producing TH1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation and TH differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.
Traditionally, the identification and quantification of eosinophils in inflammatory tissues and exudates has been primarily based upon morphologic criteria and manual counting. In this study, we describe a new flow cytometry-based assay to enumerate eosinophils present in murine bronchoalveolar lavage fluid (BAL) and lung parenchyma obtained from the normal/non-inflamed respiratory tract, following experimentally-induced allergic pulmonary inflammation, and during experimental infection with respiratory syncytial virus (RSV). By using a murine Siglec-F-specific antibody in combination with antibodies directed to CD45 and CD11c, we demonstrate that eosinophils can be distinguished from other cell types in the BAL fluid and lung parenchyma based upon their distinct CD45 + Siglec-F + and CD11c low/− staining profile. In the BAL fluid, this flow cytometry-based method of eosinophil identification/quantitation yields results comparable to the standard morphology-based method without the potential observer bias or staining artifacts inherent in morphology-based quantitation. Furthermore, this flow cytometry-based method can be directly adapted to enumerate eosinophils infiltrating the inflamed lung parenchyma, thereby obviating the need for quantitative morphometry of tissue sections.
Dendritic cells (DC) are believed to play an important role in the initiation of innate and adaptive immune responses to infection, including respiratory tract infections, where respiratory DC (RDC) perform this role.In this report, we examined the susceptibilities of isolated murine RDC to influenza virus infection in vitro and the effect of the multiplicity of infection (MOI) on costimulatory ligand upregulation and inflammatory cytokine/chemokine production after infection. We found that the efficiency of influenza virus infection of RDC increased with increasing MOIs. Furthermore, distinct subpopulations of RDC differed in their susceptibilities to influenza virus infection and in the magnitude/tempo of costimulatory ligand expression. Additional characterization of the CD11c-positive (CD11c DC produced elevated levels of the murine chemokine CXCL1 (KC), interleukin 12p40, and RANTES in response to influenza virus infection. Our results indicate that RDC are targets of influenza virus infection and that distinct RDC subsets differ in their susceptibilities and responses to infection.Dendritic cells (DC) are a distinct lineage of hematopoietic mononuclear cells that are believed to play a critical role in the induction of adaptive immune responses and may also serve a direct role as effector cells in the innate immune responses to infection (4,15,30). DC are found both within secondary lymphoid organs (e.g., the spleen and lymph nodes) and at peripheral sites/body surfaces (e.g., the gastrointestinal tract, the genitourinary tract, and the respiratory tract). DC localized to body surfaces act as sentinels, monitoring these sites for the presence of foreign antigen (in particular, infectious microorganisms). These peripheral DC capture antigen (by direct antigen uptake and/or infection of the DC) and, as a result of this encounter, undergo an activation/maturation process. The activated DC then transport the antigen from the body surface to secondary lymphoid organs, where the induction of the adaptive immune responses occurs (3, 16).DC isolated from secondary lymphoid organs or peripheral sites demonstrate considerable heterogeneity in both phenotype (i.e., cell surface marker expression) and function and can be categorized into distinct subsets. Both the number of distinct DC subsets and the distribution of these DC subsets can differ dramatically depending on the sites or tissues of origin (2,12,34).The respiratory tract (RT) is a major site of antigen encounters with DC. Respiratory DC (RDC) are distributed throughout the upper and lower RT. In the RT, RDC are localized to both large and small airways, as well as to the lung parenchyma (21, 33). Airway-resident RDC are intimately associated with the airway epithelium, are uniformly distributed along the airways, and have a relatively high turnover rate. By contrast, RDC found in alveoli and the lung parenchyma are less uniformly distributed and have a lower turnover rate than airwayassociated RDC (32). RDC have been divided into distinct subsets based on ...
BackgroundSmall cell carcinoma of the bladder (SCCB) is a rare and aggressive neuroendocrine tumor with a dismal prognosis and limited treatment options. As SCCB is histologically indistinguishable from small cell lung cancer, a shared pathogenesis and cell of origin has been proposed. The aim of this study is to determine whether SCCBs arise from a pre-existent urothelial carcinoma or share a molecular pathogenesis in common with small cell lung cancer.
To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
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