Circulating CD3+/T cell receptor V gamma 3+ fetal murine thymocytes home to the skin and give rise to proliferating dendritic epidermal T cells.

The presence of CD3/TCR V gamma 3 moieties on both dendritic epidermal T cells (DETC) and fetal murine thymocytes has led to the concept that fetal thymocytes expressing this particular TCR phenotype are the actual DETC precursors. To test this assumption, we injected i.v. thymocyte suspensions prepared from day 16 and day 19 fetal mice as well as from adult animals, into syngeneic and Thy-1-disparate nude mice, the epidermis of which contains only Thy-1+/CD3- lymphocytes. Phenotypic analysis of the recipient epidermis by in situ immunolabeling revealed that injection of day 16 and day 19 fetal, but not of adult, thymocytes resulted in the appearance of distinct clusters of DETC as judged by their dendritic morphology and uniform expression of CD3/TCR V gamma 3 receptors. The presence of CD3+/TCR V gamma 3+ cells in the fetal, but not in the adult, thymocyte population(s) together with the failure to detect DETC after transfer of Thy-1+/CD3- fetal thymocytes strongly suggest that CD3+/TCR V gamma 3+ thymocytes are the DETC precursors. Kinetic studies of the DETC population from 2 to 12 wk after cell transfer revealed a substantial increase in the cell density within the DETC clusters that was not accompanied by an increase in the number of clusters. Thus, it appears that newly arriving DETC undergo proliferative activity in situ. Collectively, our results show that, under the experimental conditions chosen, CD3+/TCR V gamma 3+ fetal thymocytes are actual DETC precursors. Although it is not clear whether these experimental conditions are representative of the in vivo situation, they may serve as a useful model for studying the mechanisms underlying the homing properties of different lymphocyte subsets.     

Effects of physicochemical agents on murine epidermal Langerhans cells and Thy-1-positive dendritic epidermal cells

The possibility that Thy-1-positive dendritic epidermal cells (Thy-1+DEC) may contribute to the immunologic functions of murine epidermal cells (EC) prompted us to simultaneously assess the effects of certain immunomodulating physicochemical agents on both Thy-1+DEC and Ia-bearing Langerhans cells (LC). C3H/He mice received one of the following treatment modalities: UV-B irradiation (four consecutive days); psoralen plus UV-A (PUVA; three times a week for three consecutive weeks); topically and systemically applied glucocorticosteroids (GCS). Beginning 2 days after the last treatment, animals were sacrificed and the structure and surface marker expression of Ia+EC and Thy-1+DEC were assessed by immunohistologic means on epidermal sheet preparations from ear skin by using appropriate monoclonal antibodies. Whereas low-dose UV-B irradiation (4 X 100 or 200 J/m2) had little, if any, effect on either Ia+EC or Thy-1+DEC, high-dose UV-B (4 X 700 or 1000 J/m2) or PUVA treatment led to an almost complete disappearance of both surface characteristics. Immunoelectron microscopic studies revealed that in the case of LC, high-dose UV-B or PUVA treatment results in the disappearance of their anti-Ia reactivity but leaves their ultrastructural morphology intact. In sharp contrast, Thy-1+DEC escape ultrastructural detection after PUVA treatment and are greatly reduced in number after high-dose UV-B. Ia+EC continuously reappeared with both treatment modalities over a course of 4 to 6 wk, whereas even after 14 to 22 wk Thy-1+DEC were present only in negligible numbers. Similar to high-dose UV-B or PUVA therapy, administration of GCS resulted in the disappearance of both anti-Thy-1- and anti-Ia-reactive cells. Ultrastructural studies disclosed, however, that these steroid-induced alterations in the surface characteristics were accompanied by a dramatic reduction of the LC population but were not paralleled by morphologic changes of Thy-1+DEC. In the course of 7 wk after cessation of steroid treatment, the number of both Ia+EC and Thy-1+DEC had returned to normal values. The selective removal of either of these two dendritic epidermal cell populations by physicochemical agents may provide an excellent strategy to further clarify the functional properties of both LC and Thy-1+DEC.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

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)     

Induction and regulation of contact hypersensitivity by resident, bone marrow-derived, dendritic epidermal cells: Langerhans cells and Thy-1+ epidermal cells

Circumstantial evidence suggests strongly that epidermal Langerhans cells (LC) alone among epidermal cells (EC) are responsible for generating an immunogenic signal for contact hypersensitivity (CH) after epicutaneous application of hapten. However, data obtained from previous studies performed with intact skin or isolated EC do not address the immunogenic capacity of a second dendritic, bone marrow-derived population of cells that resides within the epidermis, Thy-1+ epidermal cells. To identify the cellular source(s) of the antigenic signals emerging from the epidermis, purified preparations of LC, Thy-1+ cells, and keratinocytes were prepared from CBA/J mouse skin. Each cell type was derivatized in vitro with TNBS and inoculated via various routes into syngeneic mice that were assayed for the induction of CH and specific unresponsiveness. IA+ LC, when derivatized with hapten and inoculated into mice, induced CH without evidence of down-regulation regardless of the route of immunization. Derivatized Thy-1+ EC did not deliver a positive signal for CH. Rather, Thy-1+ EC possessed the capacity to initiate down-regulation of the CH response when they were delivered i.v. We conclude that all cellular elements necessary for the induction and regulation of CH after epicutaneous application of hapten to skin reside within the epidermis. The resident, dendritic, bone marrow-derived populations within the epidermis have the capacity to determine the outcome of an epicutaneous antigenic encounter.     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.     

Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in V delta 1-deficient mice

One of the most intriguing features of gammadelta T cells that reside in murine epithelia is the association of a specific Vgamma/Vdelta usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" Vgamma5+ fetal thymocytes and overwhelmingly express the canonical Vgamma5/Vdelta1-TCRs lacking junctional diversity. Targeted disruption of the Vdelta1 gene resulted in a markedly impaired development of Vgamma5+ fetal thymocytes as precursors of DETCs; however, gammadeltaTCR+ DETCs with a typical dendritic morphology were observed in Vdelta1-/- mice and their cell densities in the epidermis were slightly lower than those in Vdelta1+/- epidermis. Moreover, the Vdelta1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vgamma5+ DETCs were predominant in the Vdelta1-/- epidermis, though Vgamma5- gammadeltaTCR+ DETCs were also detected. The Vgamma5+ DETCs showed a typical dendritic shape, gammadeltaTCR(high), and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vgamma5- gammadeltaTCR+ DETCs showed a less dendritic shape, gammadeltaTCR(low), and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vgamma/Vdelta-chain usage but requires expression of a limited diversity of gammadeltaTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.     

T cell receptor gamma delta expression of Thy-1+ dendritic epidermal cells--an update

Thy-1+ dendritic epidermal cells (Thy-1+DEC) are present in the murine epidermis. They are morphologically dendritic and express Thy-1, CD3 and asialoGM1, but not CD4 or CD8. T cell receptor (TCR) of Thy-1+DEC is TCR gamma delta. Allison et al and Tonegawa et al recently found that TCR of Thy-1+DEC is V gamma 5 J gamma C gamma -V delta 1D2J2C delta and has no junctional diversity. This TCR gamma delta of Thy-1+DEC is identical to TCR expressed on the earliest fetal thymocytes. It is distinct from that of other epithelial associated lymphocytes or other thymocytes. The ligand of Thy-1+DEC is not known, although TCR gamma delta of adult type could recognize allogenic major histocompatibility complex(MHC) class I or class II and mycobacterium antigen, especially heat shock protein. The TCR of Thy-1+DEC may not be the homing receptor to epidermis. The further studies are needed to elucidate the ligands or functions of Thy-1+DEC.     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

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.     

Demonstration of a CD3+ lymphocyte subset in the epidermis of athymic nude mice. Evidence for T cell receptor diversity.

The existence of CD3/TCR-bearing lymphocytes in athymic and thymectomized chimeric mice implies that T cell maturation can occur in the absence of a thymus. Considering the possibility that the epidermis may be one of the organs providing T cell educating stimuli, we attempted to characterize the Thy-1+ epidermal lymphocyte population of athymic mice. Immunohistologic studies of epidermal sheets revealed (1) that Thy-1+ epidermal cells of C57BL/6 nu/nu mice are CD5-, CD4-, and predominantly CD8-, and (2) that a minor subset of these cells displays anti-CD3 epsilon reactivity. Although these CD3+ epidermal cells could hardly be detected at 6 wk of age, they comprised approximately 2% of all Thy-1+ epidermal cells in 12-mo-old athymic mice. Most of these CD3+ cells expressed TCR-gamma/delta, but TCR-alpha/beta+ cells were also present. TCR-gamma/delta+ epidermal T cells of athymic mice preferentially expressed TCR V gamma 2, V gamma 4, and V gamma 5 specificities rather than TCR V gamma 3 as found on DETC of euthymic mice. Using mitogenic stimuli, we have succeeded in establishing cell lines and clones from BALB/c nu/nu and C57BL/6 nu/nu epidermis. Their marker profile corresponds to that seen on resident CD3+ epidermal cells, as well as on a very small subset of CD3+ splenic and lymph node lymphocytes of athymic mice. The ontogenetic relationship, if any, between the epidermal and lymphoid CD3+, CD5-, CD4-, CD8- cells, has yet to be clarified. Cell lines/clones representative of resident CD3+ epidermal cells of nu/nu mice should provide a useful tool in the elucidation of homing patterns and functional properties of extrathymically matured T cells.     

Detection of in situ mitotic activity of dendritic epidermal T-cells by BrdU labeling.

We analyzed mitotic dendritic epidermal T-cells (DETC) in the epidermis of C3H/He (Thy-1.2+) mice, using double immunoenzymatic labeling. Ear skin was incubated with 100 microM bromodeoxyuridine (BrdU) for 5 hr and then either directly studied or cultured for an additional 12 hr in BrdU-free medium. After BrdU labeling, with or without additional culture, epidermal sheets were obtained by ethylenediaminetetraacetic acid separation. The epidermal specimens were immunostained by the peroxidase method to visualize nuclear BrdU and then by the biotin-streptavidin-alkaline phosphatase method for surface Thy-1.2 antigen. In specimens processed immediately after BrdU labeling, a mean 3.0% of all basal cells were labeled with BrdU and a mean 1.1% of the BrdU-labeled cells were also positive for Thy-1.2. Moreover, a mean 2.1% of the DETC had incorporated BrdU. BrdU-labeled DETC had a variety of appearances; they were dendritic and round in the BrdU-treated specimens, while oval and paired cells were also found in the specimens after additional culture. These morphological changes of BrdU-labeled DETC demonstrate that resident DETC can become mother cells undergoing mitosis through the retraction of their dendrites, and it appears that DETC divide at a relatively high rate, i.e., up to 10% of the DETC may enter the S-phase of the cell cycle every 24 hr.     

Purification and identification of murine epidermal dendritic cells: flow cytometry and immunogold labeling

We report on application of flow cytometric and immunogold labeling techniques to purify and identify two types of murine epidermal dendritic cells: Langerhans cells (LC) and Thy-1-positive dendritic epidermal cells (Thy 1+-dEC). After density centrifugation of epidermal cell (EC) suspensions through Ficoll gradients. IA-positive LC and Thy 1+-dEC are labeled with monoclonal antibodies (fluorescein-conjugated anti-IAd for LC and anti-Thy 1.2-biotin, followed by avidin-phycoerythrin, for Thy 1+-dEC). The fluorescence-activated cell sorter (FACS) is then used to obtain 95-98% pure populations of these dendritic cells with a yield of 2-4 X 10(6) cells and a viability of 80-90%. A post-fixation, pre-embedding immunogold labeling technique using 15 nm and 40 nm colloidal gold particles is employed to identify LC and Thy 1+-dEC, respectively, to confirm the purity of the sorting and to estimate the number of IA antigenic sites per LC. With transmission electron microscopy, ultrastructural morphology of sorted LC is preserved; however, Birbeck granules are markedly diminished compared to the pre-sorted population of LC. In contrast, characteristic dense-core granules are readily visualized in sorted Thy 1+-dEC. Purification of epidermal dendritic cells by flow cytometry may be a useful technique to employ in functional studies of epidermal dendritic cells.     

Rejection of skin allografts by CD4+ T cells is antigen-specific and requires expression of target alloantigen on Ia- epidermal cells

The effector mechanism of skin allograft rejection has been characterized as Ag specific, rejecting cells that express the target alloantigen but sparing those that do not. However, the rejection of MHC class II disparate skin grafts, in which very few cells (Langerhans cells) actually express the target Ia Ag could conceivably proceed by either one of two distinct rejection mechanisms. One possibility is that Ia- cells are destroyed by a sequence of events in which CD4+ T cells, activated by Ia+ LC, elaborate soluble factors that are either directly cytolytic or that recruit and activate non-specific effector cells. The alternative possibility is that activated CD4+ T cells elaborate soluble factors which induce Ia expression on Ia- cell populations, and that these Ia+ cells are subsequently destroyed by effector cells specific for the induced Ia alloantigens. We found that rejection of Ia+ LC was not of itself sufficient to cause rejection of skin grafts, indicating that skin allograft rejection is contingent on the destruction not only of LC but of other graft cell populations as well. We then investigated whether CD4+ T cells rejected allogeneic skin grafts in an antigen specific fashion. To do so, we engrafted immunoincompetent H-2b nude mice with trunk skin grafts from B6----A/J allophenic mice because such skin is composed of mutually exclusive cell populations expressing either H-2a or H-2b histocompatibility Ag, but not both. The engrafted mice were subsequently reconstituted with H-2b CD4+ T cells. The CD4+ T cells destroyed keratinocytes of A/J origin but spared keratinocytes of B6 origin, even though neither cell population constitutively expresses target IAk alloantigen. The targeted rejection of A/J keratinocytes but not of B6 keratinocytes indicates that the target Ia alloantigen must have been induced on Ia- A/J keratinocytes, rendering them susceptible to destruction by anti-Iak-specific CD4+ effector cells. These data demonstrate that CD4+ T cell rejection of skin allografts is mediated by Ag-specific CD4+ cytolytic T cells and hence, requires the induction of target Ia alloantigens on epidermal cells within the graft.     

Enhancement by various cytokines or 2-beta-mercaptoethanol of Ia antigen expression on Langerhans cells in skin from normal aged and young mice. Effect of cyclosporine A

The number of Ia+ Langerhans cells (LC) in skin from aged (16- to 18-mo old) BALB/c mice is approximately 40% lower than in skin from young (2- to 3-mo old) mice. Overnight incubation at 37 degrees C with a variety of cytokines, including IL-1, IL-2, IL-3, IL-4, IL-6, TNF-alpha, IFN-gamma, and granulocyte/macrophage-CSF, causes a significant increase in the Ia expression on LC and raises the number of Ia+ cells by at least 300/mm2 in skin from both young and old mice. At 1 to 5 x 10(-5) M, 2-ME has a similar effect. The percentage increment in Ia+ cells is much higher for skin from aged mice than from young mice, particularly with IL-3, which appears to reconstitute the number of Ia+ LC in skin from aged mice to that of IL-3 exposed skin from young mice. Under the incubation conditions of our experiments, neither keratinocytes nor Thy-1+ dendritic cells acquire Ia Ag. Addition of 10 micrograms/ml cyclosporine A to the medium abolishes the effect of 2-ME and of all of the cytokines except IL-2 and IL-6. These results demonstrate the presence of a significant population of Ia- LC in the epidermis of both young and aged mice. It is suggested that epidermal production of cytokines may be of importance in the maintenance of constitutive Ia expression on LC. The possible interaction between keratinocytes and LC and the effect of aging on this process are discussed.     

A keratinocyte-responsive gamma delta TCR is necessary for dendritic epidermal T cell activation by damaged keratinocytes and maintenance in the epidermis

A unique population of T lymphocytes, designated dendritic epidermal T cells (DETC), homes to the murine epidermis during fetal development. DETC express a canonical gammadelta TCR, Vgamma3/Vdelta1, which recognizes Ag expressed on damaged, stressed, or transformed keratinocytes. Recently, DETC were shown to play a key role in the complex process of wound repair. To examine the role of the DETC TCR in DETC localization to the epidermis, maintenance in the skin, and activation in vivo, we analyzed DETC in the TCRdelta(-/-) mouse. Unlike previous reports in which the TCRdelta(-/-) skin was found to be devoid of any DETC, we discovered that TCRdelta(-/-) mice have alphabeta TCR-expressing DETC with a polyclonal Vbeta chain repertoire. The alphabeta DETC are not retained over the life of the animal, suggesting that the gammadelta TCR is critical for the maintenance of DETC in the skin. Although the alphabeta DETC can be activated in response to direct stimulation, they do not respond to keratinocyte damage. Our results suggest that a keratinocyte-responsive TCR is necessary for DETC activation in response to keratinocyte damage and for DETC maintenance in the epidermis.     

Aryl hydrocarbon receptor is critical for homeostasis of invariant gammadelta T cells in the murine epidermis

An immunoregulatory role of aryl hydrocarbon receptor (AhR) has been shown in conventional αβ and γδ T cells, but its function in skin γδ T cells (dendritic epidermal T cells [DETC]) is unknown. In this study, we demonstrate that DETC express AhR in wild-type mice, and are specifically absent in the epidermis of AhR-deficient mice (AhR-KO). We show that DETC precursors are generated in the thymus and home to the skin. Proliferation of DETC in the skin was impaired in AhR-KO mice, resulting in a >90% loss compared with wild type. Surprisingly, DETC were not replaced by αβ T cells or conventional γδ T cells, suggesting a limited time frame for seeding this niche. We found that DETC from AhR-KO mice failed to express the receptor tyrosine kinase c-Kit, a known growth factor for γδ T cells in the gut. Moreover, we found that c-kit is a direct target of AhR, and propose that AhR-dependent c-Kit expression is potentially involved in DETC homeostasis. DETC are a major source of GM-CSF in the skin. Recently, we had shown that impaired Langerhans cell maturation in AhR-KO is related to low GM-CSF levels. Our findings suggest that the DETCs are necessary for LC maturation, and provide insights into a novel role for AhR in the maintenance of skin-specific γδ T cells, and its consequences for the skin immune network.     

Loss of epidermal integrity by T cell-mediated attack induces long term local resistance to subsequent attack. II. Thymus dependency in the induction of the resistance

Our previous study showed that in cutaneous graft-vs-host disease (GVHD) induced by intradermal injection of autoreactive T cells the epidermal structures spontaneously recovered from the destruction became resistant to the subsequent attempts to induce the cutaneous GVHD and that the resistance correlated well with a 30-fold increase in the number of Thy-1+ epidermal cells (Thy-1+EC). We show that the resistance to the cutaneous GVHD was never induced in athymic nude mice and adult thymectomy lethal radiation and bone marrow reconstitution (ATXBM) mice under the same conditions, indicating that the induction of the resistance is dependent on the presence of thymus. A great increase in the number of Thy-1+EC was similarly observed in the epidermis of the athymic nude and ATXBM mice that spontaneously recovered from the cutaneous GVHD and that remained susceptible to the induction of the cutaneous GVHD. However, the results with B10Thy-1.1----B6 radiation chimeras clearly demonstrate that the vast majority of the increased Thy-1+EC found in the "susceptible" epidermis of the ATXBM mice were of donor bone marrow origin and there was no increase in the number of host-derived Thy-1+EC, whereas in the "resistant" epidermis of the XBM mice both Thy-1+EC populations were equally increased. The overall results indicate that the expansion of Thy-1+EC that mature in the thymus is crucial to the induction of the resistance, although the migration of bone marrow-derived Thy-1+EC precursors to the epidermis occurs quite independently of the presence of thymus.     

Enrichment of epidermal Langerhans cells by immunoadsorption to Staphylococcus aureus cells

In addition to keratinocytes and melanocytes, the mammalian epidermis harbors the so-called Langerhans cells (LC)2 as a third cell population, which is thought to participate in immune reactions involving the epidermis (1, 2). LC are dendritic cells located above the basal cell layer, have a characteristic ultrastructural appearance (3), and originate from a bone marrow precursor (4, 5). They lack membrane-incorporated surface immunoglobulin and sheep red blood cell receptors, but are the only epidermal cells (EC) that bear receptors for the Fc portion of IgG (Fc-IgG) and for C3 and express Ia antigens (1, 2). Because LC constitute only 3 to 5% of all EC, enrichment procedures are important for functional studies. Moderate enrichment of LC to 18 to 35% by separation of Fc-IgG rosetting EC on density gradients was sufficient to show the critical role of LC in EC-induced T cell proliferation (6). More powerful isolation procedures are needed, however, for more exacting analysis of LC functions, such as their role in immune induction, their secretory capacities including production of EC-derived thymocyte-activating factor (7, 8) and prostaglandins, immune endocytosis, the role of LC granules, etc. Methods hitherto available for enriching LC beyond 60% (9, 10) are time consuming and of low yield and viability, and thus are of limited practical value. In this report we describe a simple and efficient procedure to obtain viable LC suspensions of high purity based on the use of monolayers of protein A-bearing Staphylococcus aureus cells as a solid-phase immunoadsorbent (11).