Human thymic epithelial cells directly induce activation of autologous immature thymocytes.

Abstract

To study the role that epithelial cells of the thymic microenvironment play in promoting activation of immature CD7+, CD2+, CD4-, CD8-(double-negative) human thymocytes, we have isolated thymocyte subsets from normal postnatal thymus and have cocultured autologous double-negative thymocytes with pure populations of thymic epithelial (TE) cells. We report that TE cells directly activate double-negative thymocytes to proliferate and that TE cells enhance the ability of double-negative thymocytes to proliferate in response to stimulation with exogenous interleukin 2. Activated double-negative thymocytes that proliferated in vitro in the presence of TE cells and interleukin 2 remained doublenegative after 23 days in culture. Moreover, TE-cell culture supernatants in the absence of intact TE cells contain interleukin 1, interleukin 3, and granulocyte/macrophage-colonystimulating factor activity for human bone marrow cells and can activate double-negative thymocytes to proliferate. Antibodies against interleukin 1 and against granulocyte/macrophage-colony-stimulating factor inhibited TE-cell-induced thymocyte activation. These data indicate that one role of TE cells in vivo may be to activate double-negative thymocytes to proliferate.The thymus is known to be the primary site of T-cell maturation during fetal and neonatal development (1)(2)(3)(4)(5). However, specific cellular interactions and activation signals necessary for growth and differentiation of T-cell precursors within the human thymus are largely unknown (4-6). Studies of T-cell ontogeny in mice have suggested that the earliest intrathymic T cells have the surface phenotype dLytl + ("d" means that the immunofluorescence is faint, or "dull"), Lyt2-, L3T4-and that a subpopulation of dLytl +, Lyt2 -, L3T4-thymocytes are able to reconstitute the T-cell repertoire (7-13).Lobach and Haynes (14) showed that, during human fetal development, the earliest T-cell precursors are CD7+ prior to entry into the thymus, and these cells acquire the CD2, CD4, CD8, CD3, and CD1 molecules in the thymus between 82 and 14 weeks of fetal gestation. At 8½ weeks of fetal development, intrathymic T cells are CD7+, CD2+, CD4-, CD8-(double-negative) (14)(15)(16).To study the role that thymic epithelial (TE) cells might play in activating subsets of human double-negative thymocytes to proliferate and differentiate into mature T cells, we developed an in vitro coculture system of autologous TE cells and double-negative thymocytes (17). Here we show that TE cells up-regulate interleukin 2 (IL-2) receptor expression and IL-2 responsiveness of double-negative thymocytes and directly activate immature thymocytes to proliferate.Moreover, TE cells produce cytokines capable of providing activation signals to double-negative thymocytes in the absence of direct TE-cell-thymocyte contact.

Interleukin 1 binds to specific receptors on human keratinocytes and induces granulocyte macrophage colony-stimulating factor mRNA and protein. A potential autocrine role for interleukin 1 in epidermis.

“…Since it has been demonstrated that bone marrow-derived hematopoietic precursors circulate in peripheral blood, it is conceivable that human epidermis that produces (at least) IL-1, GM-CSF, and IL-6/BSF-2 may serve to induce local differentiation of these cells. Some evidence suggests that certain T cell lymphomas, leukemias, and cell lines, as well as a subpopulation of double negative thymocytes, can respond to GM-CSF (22,30,31). That epithelial tissues, including skin and thymus, produce GM-CSF, IL-1, and thymic hormones (32,33) may relate to the differentiation of T cells in these tissues from bone marrow-derived precursors.…”

Gene transcription in differentiating immature T cell receptor(neg) thymocytes resembles antigen-activated mature T cells.

“…However, the molecular signals between fetal liver (bone marrow) immigrants and thymic stromal cells that govern the activation process are as yet obscure. Recent evidence indicated that thymocyte differentiation requires interactions with more than one kind of stromal cell (52,53).…”

Synovial microenvironment‐t cell interactions

“…Purified thymocytes were teased from thymus tissue obtained from 5 control subjects (all younger than 7 years of age) during cardiovascular surgery and were purified as described previously (21). Fresh normal and activated peripheral blood mononuclear cells (PBMC) were prepared as previously described (22).…”

“…Activated T lymphocytes were prepared by activation of PBMC with the mitogen concanavalin A (Con A; 50 pg/ml for 3 days), washed in 0.3M a-methylmannoside (Sigma) to remove residual Con A, and then kept at 4°C prior to use in synovial cell-T cell binding assays (22). In some experiments, synovial lymphocytes were isolated from synovial tissue or fluid by mechanical disruption of tissue and Ficoll-Hypaque density centrifugation (21).…”

“…Suspensions containing synovial cell T cell rosettes were centrifuged onto glass slides using a cytocentrifuge (Shandon Southern Instruments, Sewickley, PA) and processed for Wright's staining or acetone-fixed for an indirect immunofluorescence assay using anti-T cell and other monoclonal antibodies followed by fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin as described (24). The following monoclonal reagents were used: NA1/34 (anti-CD1, T6) (25), OKTl1 In other experiments, purified populations of thymocytes, synovial lymphocytes, or fibroblast-like synovial cells were phenotypically characterized using the above monoclonal antibodies either on acetone-fixed cytocentrifuge preparations and indirect immunofluorescence analysis or by flow cytometry using an Ortho-SOH cytofluorograph (21). Antibodies TE-7 (35) and lBlO (Singer KH, Scearce RM, Haynes BF: unpublished observations), which specifically bind to fibroblasts, antibody M-38, which binds to the C-terminal region of type 1 procollagen (36), and Leu-M3, which specifically binds to monocyte/macrophages (37) were also used to characterize synovial cells in vitro using indirect immunofluorescence assays (24).…”