Myra Small

Thymus cell maturation. II. Differentiation of three "mature" subclasses in vivo.

Abstract Several thymus cell subclasses may be defined on the basis of their sedimentation velocity, their light-scattering properties (a measure of cell volume), or binding of a fluoresceinated anti-Thy 1.2 antiserum. Using a multiparameter fluorescence-activated cell sorter (FACS), cells with distinguishable light-scattering or fluorescence intensity (after staining with fluorescein anti-Thy 1.2) were separable for analysis of intrathymic maturation pathways. Outer thymic cortical large and medium lymphocytes were the only cells labeled within 1 hr after transcapsular diffusion of administered [ 3 H]thymidine. These labeled cells were also entirely contained in the brightest fluorescence intensity (with fluorescein anti-Thy 1.2) subclass. Under conditions of [ 1 H] thymidine “chase” in vivo , label shifted proportionately and apparently in parallel to three “mature” subclasses: (1) small thymocytes with high surface concentrations of Thy 1.2, representing ~ 80% of all thymus cells; (2) slightly larger cells, with very low surface Thy 1.2, which are indistinguishable from cortisone-resistant thymocytes, and which make up less than 10% of all thymus cells; (3) dead or fragile cells.

Differentiation of thymus cells

The process of cellular differentiation in the thymus presumably leads to the development of immunocompetent peripheral “T” lymphocytes (1, 6). Although there are considerable data showing the existence of a small pool of immunocompetent cortisone-resistant, medullary thymocytes which, as a population, bear low concentration of Thy 1.2, lack TL antigens, and exhibit high concentrations of H-2, the identification of their immediate precursors is not yet known (1). Under conditions of parenteral hydrocortisone administration it has been demonstrated that at least some of the cortisone-resistant medullary thymocytes are derived from an intrathymic pool of cortisone-sensitive cortical precursors (7). In this paper we demonstrate the in vivo maturation of at least three lines of “mature” thymus cells from a precursor subclass using selective labeling of these precursors in situ as a marker.

Thymic aging in ICR female mice is suspended by prolonged hydrocortisone exposure

Abstract. Age-related decline of the thymus in ICR female mice was studied following long-term (three month) weekly exposure to hydrocortisone acetate. When examined one week after cortisone injections, the well-known thymic atrophy was observed. Five weeks after 12 hydrocortisone injections, the cortical volume fraction (Vc), cortical/medullary ratio (C/M), the number of thymocytes and CD4/CD8 profiles were in the range that characterizes younger mice, compared with PBS-injected mice, uninjected controls, or mice given a single hydrocortisone injection 5 weeks earlier. It seems as if thymic involution with age was suspended during the period of glucocorticoid exposure.

CD3−4−8− Thymocyte Precursors with Interleukin‐2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3−4−8− interleukin‐2 receptor positive (IL‐2R+) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL‐2R+CD3−4−8− thymocytes were obtained by positive panning of IL‐2R+ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4+8+ and CD3+4+8− cell populations developed, the latter expressing the αβ T‐cell receptor (αβ‐TCR). Thus heterogeneous stromal cell cultures support the development of IL‐2R+ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal‐dependent step.

Isolation of CD3−, CD4−, CD8−, IL-2R+ thymocyte precursors by panning

We present a panning method for isolation of thymocytes that are CD3-, CD4-, CD8- and IL-2R+. These cells have been isolated by positive selection on dishes coated with 7D4 antibody followed by treatment with biotinylated 145-2C11, GK1.5, and 53-6.7 antibodies and negative selection on avidin coated dishes.

Opposite Effects of T-Cells on Syngeneic Tumor Growth In Vivo: Tumor Inhibition by Mature Lymphocytes and Enhancement of the Same Tumors by Immature Cells

Our studies during the past few years on the interactions between host lymphocytes and several syngeneic tumors in mice have revealed that T-cells with opposing reactivities toward the same tumor can be activated by exposure to the neoplastic cells. While one population of sensitized T-cells is capable of inhibiting tumor growth in vivo, a second T-cell population can abolish this effect and bring about tumor enhancement. We have been investigating the T-cells involved in each of these responses in order to understand the cell characteristics required for each reactivity and to selectively inactivate the tumor-enhancing T-lymphocytes in situ. Our experiments indicate that these two reactivities of T-cells do not involve parallel classes of mature T-cells but rather that they represent responses characteristic of different stages in T-cell maturation. Our hypothesis is that interaction between mature T-cells and a tumor can lead to development of anti-tumor reactivity while exposure of immature T-cells to the same tumor can trigger the tumor enhancement process. Secondly we envisage that as tumor growth progresses early thymocytes with tumor enhancing activity are released from the thymus to the periphery where they counteract the activity of the tumor-inhibiting T-cells. Evidence in support of these two notions is presented below.

Isolation of adult murine thymic stromal cells that naturally express Notch ligands

This communication summarizes the procedures that enabled isolation of adult murine thymic stromal cell lines that naturally express Notch ligands Jagged-1 or Delta-1. Histochemical characterization of these cell lines, in terms of ligand and cell type, revealed epithelial cells that bear an antigen characteristic of the thymic medulla and express either Jagged-1 or Delta-1. FACS sorting of stromal cells that naturally express these and other ligands is thus feasible, and such cells can be used to investigate the activity of each ligand in Notch-mediated commitment to the T-lymphocyte pathway.

Dna repair in thymocytes of mice undergoing thymic involution.

DNA repair in mouse thymocytes before the onset of thymic involution was compared to that at successive stages during involution of the thymus. Repair of DNA in these cells after in vitro X-irradiation was evaluated by sedimentation of nucleoids in a sucrose gradient, as a measure of DNA supercoiling. DNA repair continued to function in the thymocytes even at ages when there was a dramatic reduction in the number of cells in the thymus.

Age-related changes in excision repair of cultured epithelial cells from mouse thymus

These experiments were performed to test the possibility of a link between involution of the thymus and decreased ability to repair damaged DNA. In a previous investigation this was not found to be the case with thymic lymphocytes, and in the present work the question was addressed to epithelial cells of the thymic stroma isolated by growth in tissue culture. DNA repair in the epithelial cells was measured as unscheduled DNA synthesis (UDS) and detected autoradiographically after UV irradiation of the cultures. In cultures derived from older mice, DNA repair was evident in the majority of the cells and continued after extended culture. When cultures were derived from preinvolution mice DNA repair activity, which was detected after short periods of culture, was lost from most of the cells after several days of growth. These unexpected findings raise the possibility that the ability of the stromal cells to repair DNA damage has enabled them to survive a selective pressure that is involved in thymic involution.

Culture of Mouse Thymic Epithelial Cells and Studies of Age-Related Changes

Evidence accumulating over the last few years strongly suggests a role for epithelial cells of the thymus in the development of the T cell repertoire (1,2), but relatively little is known of the mechanism of this activity. In addition, we know almost nothing on the role and fate of these epithelial cells in thymic involution, a process which results in decreased T cell activity in advanced age. Both lines of research could be advanced by tissue culture of epithelial cells. In mice this objective has been difficult to achieve as attested by the large number of unsuccessful, or complicated and only partially successful reports (3–9), which appeared before this investigation was undertaken. In recent months other procedures have also been published (10–13), and it is now possible to compare the advantages and shortcomings of each. Difficulties have been described especially with advanced donor age, and we sought a novel approach to facilitate cultures from mice of all ages. Thus, we modified a medium known to support growth of hepatic epithelial cells (14), to bring about simultaneous inhibition of fibroblasts (15) which have plagued other culture systems.