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Short-term and long-term cultures of mouse Thy-1+ epidermal cells (EC) were established in order to characterize their phenotypic and functional properties. Concanavalin A (Con A) and Interleukin 2 (IL-2) stimulated Thy-1+ EC mediated non-MHC directed cytotoxicity preferentially against the NK-sensitive target, YAC-1 vs the NK-resistant target, P815; these cells also mediated antibody-dependent cell-mediated cytotoxicity (ADCC), indicating the presence of IgG-FcR on at least some of them. Freshly isolated Thy-1+ EC failed to lyse YAC-1 targets; however, this activity was observed after 9 d of culture with Con A and IL-2. While dendritic Thy-1+ EC, in vivo, do not express the T-cell markers, L3T4 and Lyt-2, short-term cultured cells displayed phenotypic heterogeneity with small but significant percentages of Lyt-2+ and L3T4+ cells appearing transiently. The phenotype of the effector cell(s), which mediates cytotoxic activity, was determined by utilizing flow cytometry to sort short-term cultured EC into positively and negatively stained populations. Cells which express L3T4, or which lack asialo GM1, did not lyse YAC-1 targets; maximum cytotoxic activity was found within populations of cells which are asialo GM1+, Lyt-2-, and asialo GM1+, Lyt-2+. These studies indicate that Thy-1+ cells derived from mouse epidermis when cultured in the presence of Con A and IL-2 have the capacity to generate a phenotypically heterogeneous population, some cells of which are capable of mediating cytotoxic activities.

Bulk cultures of mouse Thy-1+ dendritic epidermal cells (Thy-1+ DEC) have been shown to proliferate in response to concanavalin A (Con A) and IL-2, to secrete IL-2-like growth factors, and to lyse target cells such as YAC- 1. Limiting dilution microculture was utilized in order to determine the precursor frequency of Con A-responsive Thy-1+ DEC in suspensions of AKR/J epidermal cells as well as whether these several functional activities all reside within a single Thy-1+ DEC precursor. Precursor frequency analysis of cultures established with limiting numbers of FACS-purified Thy-1+ DEC, irradiated syngeneic splenic filler cells and exogenous IL-2 indicated that approximately 20% of Thy-1+ DEC proliferated in response to Con A. Parallel microcultures in which purified Thy-1+ DEC were plated at a density of 0.5 cells/well were used to establish clones. Twenty clones were characterized phenotypically, and ten of these were also tested for their capacities to proliferate in response to Con A or IL-2, to secrete IL-2-like growth factors, and to exhibit cytotoxicity. All clones were Thy-1+ and L3T4-, but while most were also Lyt-2-, several contained 3%-18% dull Lyt-2+ cells. Functional studies revealed that each clone displayed all of the above functional activities, albeit with substantial quantitative variation. Clones with the highest cytotoxic activity had relatively low responsiveness to Con A or IL-2 and included all clones containing dull Lyt-2+ cells; conversely, clones with the highest proliferative responses had relatively low cytotoxic activity and were all Lyt-2-. This degree of functional and phenotypic heterogeneity among cloned Thy-1+ DEC may reflect their particular states of activation or differentiation; whether it reflects the biologically relevant in vivo activities of these cells must still be determined.

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.

We have evaluated the morphology, function, and TCR mRNA and DNA profiles of Thy-1+ epidermal cells (EC) derived from athymic (nude) mice. Based on these criteria we demonstrate that Thy-1+ EC in nude mice are predominantly round and angular and located in follicular epithelium, as compared to predominantly dendritic, interfollicular Thy-1+ EC found in normal CBA or BALB/c mice. Functionally, Thy-1+ EC from normal mice proliferate and elaborate IL-2 in response to stimulation by Con A; by contrast, nude Thy-1+ EC fail to proliferate or secrete IL-2 in response to Con A. Nude Thy-1+ EC proliferate markedly in response to low-dose IL-2 alone; Thy-1+ EC from normal mice give only modest proliferative responses to IL-2. Both populations of cells proliferate and elaborate IL-2 in response to PMA and calcium ionophore. Short-term cultured Thy-1+ EC from nude mice resemble Thy-1+ EC from normal animals in that they express no detectable functional TCR-alpha and beta-RNA. Normal Thy-1+ EC express abundant levels of functional RNA for TCR-gamma and delta-chains and for the CD3 delta-chain, whereas nude Thy-1+ EC produce no detectable TCR-delta and no CD3 delta-RNA and only truncated, presumably germ-line TCR-gamma transcripts, as suggested by lack of hybridization with gamma-V region probes and by lack of detectable rearrangements in the gamma-genes. These phenotypic, functional, and genotypic characteristics of Thy-1+ EC from nude mice suggest that these cells are at a very early stage of T cell differentiation.