Consequences of stable transduction and antigen-inducible expression of the human interleukin-7 gene on tetanus-toxoid-specific T cells.

Interleukin-7 (IL-7) has previously been shown to increase antigen-specific immune responses; the effect of IL-7 on human antigen-specific T cell lines has not directly been addressed. A tetanus-toxoid (TT)-specific T cell line exhibited increased proliferation in the presence of exogenous IL-7, suggesting that IL-7 may be useful in the potentiation of immune responses to defined microbial antigens. Murine retroviral vectors encoding the human IL-7 gene and the neomycin phosphotransferase gene (neoR) were packaged into murine retroviral particles, and supernatants containing these retroviral vectors were used to infect a CD4+ lymphoblastoid cell line. Stable integration of the retroviral vector and constitutive expression of the IL-7 gene were observed. Successful IL-7 gene transduction into TT-specific T cells was also accomplished. Detection of neoR DNA sequences and expression of IL-7-specific mRNA increased with selection in geneticin. Production of IL-7 in these cells was induced by exposure to TT. Production of IL-4, IL-6, and interferon-gamma (IFN-gamma) was detected after antigenic stimulation; there was, however, no effect of IL-7 on the pattern or kinetics of cytokine production by these cells. Human IL-7 transduced cells showed greater proliferation to TT than control T cells, particularly at subthreshold TT concentrations. These dta imply that genetic modification of antigen-specific T cells may be a plausible strategy for the study and manipulation of the immune responses to microbial pathogens.

Organoid structure of normal rat bladder in unilaminar and bilaminar histophysiologic gradient culture : methods and observations.

In lining epithelium of mammals certain recurrent architectural patterns are recognized that may be critical for epithelial organization in culture. Among these structural imperatives are three dimensional growth, restricted migration of cells, histophysiologic gradients, and continuity of epithelial membranes. Histophysiologic gradient culture procedures have been developed to comply with requirements suggested by normal tissue architecture. In a small chamber, 5 mm diam, epithelium grows attached to a thin permeable transparent collagen membrane or sandwiched between two apposed collagen membranes. The chamber is held in a plastic capsule so that metabolic exchange is limited to substances that diffuse across the collagen membranes to the adherent basal layer of epithelium. On a single membrane after 2 wk of growth, normal urothelium appears as a diffusely hypercellular urothelium, 6 to 10 cells thick. As the culture period is extended by 2 or more wk, multiple nodules of urothelium extend from the basal surface into the subepithelial space between the epithelium and the collagen membrane. Normal bladder, cultured between two apposed collagen membranes, gives rise in a few days to confluent epithelium that contains many extracellular cysts. Through an apparent merging of cysts, after 2 wk the urothelium appears as a highly organoid structure, a flattened cyst lined by completely stratified polarized urothelium. Such microbladders consist of a stratified epithelium without interruption of continuity. With histophysiologic gradient culture, processes in carcinoma and precursor lesions are accessible to study at the level of tissue organization.

Separation on Percoll density gradients of cells derived from malignant ascites of mice.

In an attempt to separate malignant from normal and reactive stromal cells, we fractionated ascites cells from BALB/c mice bearing a transplantable myeloma (MPC-11) by isopyknic centrifugation in continuous density gradients of povidone-coated silica gels (Percoll). Cells from different fractions were then analyzed by morphologic and immunologic criteria. The ability of cells from the different fractions to form colonies in soft agar and to produce tumors in BALB/c mice was also examined. Although most fractions contained morphologically identifiable plasma cells, colony-forming cells (CFC), derived from multiply passaged tumors, separated in a sharp peak at 1.072 g/ml. CFC peaked at 1.078-1.082 g/ml for tumors passed less than three times and were invariably markedly depleted from the low-density portions of the gradients. Cells recovered from different fractions of the gradients were cultured in soft agar and inoculated sc into syngeneic mice. In these experiments, a highly significant correlation was observed between the ability of cells to form colonies in soft agar and to form tumors in vivo. This correlation suggests that CFC and tumorigenic cells have similar distributions.