Integrin-driven monocyte to dendritic cell conversion in modified extracorporeal photochemotherapy.

Due to clinical efficacy and safety profile, extracorporeal photochemotherapy (ECP) is a commonly used cell treatment for patients with cutaneous T cell lymphoma (CTCL) and graft-versus-host disease (GVHD). The capacity of ECP to induce dendritic antigen-presenting cell (DC)-mediated selective immunization or immunosuppression suggests a novel mechanism involving pivotal cell signalling processes that have yet to be clearly identified as related to this procedure. In this study we employ two model systems of ECP to dissect the role of integrin signalling and adsorbed plasma proteins in monocyte-to-DC differentiation. We demonstrate that monocytes that were passed through protein-modified ECP plates adhered transiently to plasma proteins, including fibronectin, adsorbed to the plastic ECP plate and activated signalling pathways that initiate monocyte-to-DC conversion. Plasma protein adsorption facilitated 54·2 ± 4·7% differentiation, while fibronectin supported 29·8 ± 7·2% differentiation, as detected by DC phenotypic expression of membrane CD80 and CD86, as well as CD36, human leucocyte antigen D-related (HLA-DR) and cytoplasmic CD83. Further, we demonstrate the ability of fibronectin and other plasma proteins to act through cell adhesion via the ubiquitous arginine-glycine-aspartic (RGD) motif to drive monocyte-to-DC differentiation, with high-density RGD substrates supporting 54·1 ± 5·8% differentiation via αVß3 and α5ß1integrin signalling. Our results demonstrate that plasma protein binding integrins and plasma proteins operate through specific binding domains to induce monocyte-to-DC differentiation in ECP, providing a mechanism that can be harnessed to enhance ECP efficacy.

Synergy among lymphoid cells mediating the graft-versus-host response. V. Derivation by migration in lethally irradiated recipients of two interacting subpopulations of thymus-derived cells from normal spleen.

Spleen cells from normal adult mice were injected into lethally irradiated adult syngeneic recipients. 24 h later, cell suspensions were prepared from the recipients' spleens or peripheral lymph nodes and tested either alone or combined for their capacity to elicit graft-versus-host (GVH) reactions in neonatal F(1) recipients, using the Simonsen spleen weight assay. Either the lymph node-seeking subpopulation or the spleen-seeking subpopulation alone was markedly deficient in its ability to provide a GVH reaction when compared with the normal population from which it was derived. However, an appropriate mixture of the two had a reactivity characteristic of the parent population. Both subpopulations were sensitive to treatment with anti-theta antibody and complement in vitro. These results provide a convincing demonstration of the functional heterogeneity within the pool of thymus-derived cells present in a single normal lymphoid tissue. They strongly suggest that the normal expression of GVH reactivity of such a tissue involves an interaction among distinct subpopulations of thymus-derived cells.

Synergy among lymphoid cells mediating the graft-versus-host response. IV. Synergy in the GVH reaction quantitated by a mortality assay in sublethally irradiated recipients.

A mortality assay was used to quantitate graft-versus-host (GVH) reactions in sublethally irradiated (400 R) neonatal (C57BL/6 x BALB/c)F(1) recipients of BALB/c lymphoid cells from various tissues. The probit of the 35 day cumulative per cent of mortality was a linear function of the logarithm of the cell inoculum for any tissue; reactivities of different tissues fell on a series of parallel lines. Peripheral blood leukocytes (PBL), the most active cells, were about 30 times as active as thymocytes, the least active cells studied; femoral lymph node cells and spleen cells were about 23 and 8 times as reactive as thymocytes, respectively. The average survival time of recipients of thymocytes who eventually died was nearly a week longer than that of recipients of comparably lethal numbers of PBL, lymph node, or spleen cells. Mixtures of PBL and thymocytes gave levels of 35 day mortality significantly greater than those expected if the reactivities of the mixture had been merely the sum of the reactivities of the components measured separately, thereby confirming in any assay independent of host splenomegaly the synergistic interaction of thymocytes and PBL in the GVH reaction. Both populations of cells in the mixture had to be allogeneic to the host in order to observe this synergy. The kinetics of cumulative mortality observed for mixtures of PBL and thymocytes were indistinguishable from those seen with thymocytes alone, indicating activation of the latter cell type. Finally, comparison of the relative abilities of different cell populations to cause splenomegaly on the one hand and lethal runting on the other has raised the possibility that expression of different effector functions of cell-mediated immune reactions may in fact be initiated by distinct cells.

Separable populations of activated thymus-derived lymphocytes identified in two assays for cell-mediated immunity to murine tumor allografts.

The immune response of C57BL mice to a DBA/2 tumor allograft has been assessed in two assays of cell-mediated immunity, the in vitro lysis of (51)Cr-labeled target cells and the antigen-mediated inhibition of macrophage migration. Both assays were shown to be measuring a T-cell-mediated reaction. Three types of experiments suggested that distinct subpopulations of T cells mediate these reactions. The tissue distributions of these activities was distinctive; both activities were present in spleens from i.p. immunized mice, but only macrophage migration inhibition activity was found in the peripheral lymph nodes (PLN) of such mice. Adoptive transfer of immune spleen cells into irradiated syngeneic recipients revealed that while a substantial amount of migration inhibition activity could subsequently be found in PLN, cytotoxic activity was found predominantly in the spleens of these adoptive hosts. Velocity sedimentation analysis of immune cells 14 days after i.p. immunization indicated that while the majority of cytotoxic activity was associated with small and medium lymphocytes, the majority of migration inhibition activity was associated with medium and large lymphocytes. In addition, normal spleen cells were fractionated by velocity sedimentation immediately before allosensitization in vitro. Subsequent analysis of the sensitized fractions revealed that the activity profiles for cytotoxicity and macrophage migration inhibition were not coincident. The implications of these observations are discussed.

Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation.

Acute, low dose ultraviolet B radiation of murine body wall skin followed by local application of DNFB produces a state of antigen-specific unresponsiveness. This state is maintained at least in part by an Lyt-1+ T cell that effects unresponsiveness by impairing the induction phase of contact hypersensitivity. The absence of suppressor cells capable of acting at the effector stage of immunity suggests that tolerogenic signals derived from the skin establish suppressor networks that are incomplete and perhaps different from networks that are induced by systemic administration of tolerogens. It is proposed that ultraviolet radiation produces its effects by impairing the antigen-presenting potential of resident Langerhans cells in whose absence hapten-derivatized keratinocytes (or their products) are able to deliver a tolerogenic signal.

Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches.

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.

Conservation of T cell receptor conformation in epidermal gammadelta cells with disrupted primary Vgamma gene usage.

A feature that distinguishes gammadelta T cell subsets from most alphabeta T cells and B cells is the association of expression of single T cell receptor (TCR) gamma and delta variable (V) region gene segments with specific anatomic sites. Mice lacking the TCR Vgamma5 chain normally expressed by most dendritic epidermal T cells were shown to retain a conformational determinant (idiotype) ordinarily expressed exclusively by such Vgamma5+ cells. Conservation by shuffled gammadelta TCR chains of an idiotype associated with a specific anatomic site indicates that for TCRgammadelta, as for immunoglobulin, conformation is associated to a greater extent with the function or development of lymphocyte repertoires than is the use of particular gene segments.

Regulation of cutaneous malignancy by gammadelta T cells.

The localization of gammadelta T cells within epithelia suggests that these cells may contribute to the down-regulation of epithelial malignancies. We report that mice lacking gammadelta cells are highly susceptible to multiple regimens of cutaneous carcinogenesis. After exposure to carcinogens, skin cells expressed Rae-1 and H60, major histocompatibility complex-related molecules structurally resembling human MICA. Each of these is a ligand for NKG2d, a receptor expressed by cytolytic T cells and natural killer (NK) cells. In vitro, skin-associated NKG2d+ gammadelta cells killed skin carcinoma cells by a mechanism that was sensitive to blocking NKG2d engagement. Thus, local T cells may use evolutionarily conserved proteins to negatively regulate malignancy.

Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4.

Atopic individuals are predisposed to mounting vigorous Th2-type immune responses to environmental allergens. To determine the factors responsible, animal models that closely mimic natural modes of allergen exposure should prove most informative. Therefore, we investigated the role of IL-4, a known Th2-promoting cytokine, in generation of Th2 responses after exposure of either the skin or airway to soluble protein. Compared with wild-type (WT) mice, IL-4-deficient (IL-4(-/-)) mice showed markedly impaired Th2 activation after primary exposure to inhaled ovalbumin (OVA), with decreased OVA-specific IgG1 and IgE, and significantly fewer eosinophils in bronchoalveolar lavage (BAL) fluid after airway challenge. In contrast, IL-4(-/-) mice initially exposed to epicutaneous (e.c.) OVA mounted Th2 responses equivalent to responses in WT mice, with high numbers of eosinophils in BAL fluid. Because Th2 responses were not induced by e.c. OVA exposure in Stat6(-/-) mice (mice lacking signal transducer and activator of transcription 6), the role of IL-13 was tested. In vivo depletion of IL-13 prevented Th2 responses induced by e.c. OVA exposure in IL-4(-/-) mice. These data demonstrate a marked difference in the IL-4 dependence of Th2 responses generated at two anatomic sites of natural allergen encounter and identify the skin as a particularly potent site for Th2 sensitization.

The intracutaneous growth of murine lymphomas: epidermal invasion is characteristic of multiple tumor phenotypes.

Affinity of lymphoid cells for the epidermis (epidermotropism) is characteristic of the cutaneous T-cell lymphomas, mycosis fungoides and the Sézary syndrome. Consistent with numerous studies indicating that mycosis fungoides is a neoplasm of OKT4+T8- ("helper/inducer") T lymphocytes is the possibility that epidermotropism is a phenotypic hallmark of this subset of malignant T cells. This proposal was investigated in mice using 8 phenotypically characterized lymphomas of BALB/c origin: 3 histiocytic (phagocytic, lysozyme-positive, FcR+, Ig-, Thy 1-), 1 B-cell (IgM+, FcR+, Thy 1-), and 4 T-cell (Ig-, Thy 1+) lines, including 1 with markers of mouse helper/inducer T cells (Lyt1+23-), 2 with suppressor/cytotoxic markers (Lyt1-23+), and 1 with markers of immature thymocytes (Lyt1+23+). The intracutaneous growth pattern of these lines was studied on hematoxylin and eosin-stained sections through the centers of tumors obtained at times after intradermal injection into parallel groups of syngeneic mice. All of these lymphomas manifested variable epidermotropism that followed a typical sequence. Following dermal growth and spread to the dermal-epidermal junction, tumor cells appeared within the stratum spinosum. Subsequently, collections of cells appeared in spaces within the epidermis (Pautrier-like microabscesses) in tumors greater than 2 cm in diameter, coincident with early epidermal necrosis. Thus, in this animal model it is clear that the intraepidermal invasion/growth does not correlate with the helper/inducer T-cell surface phenotype. These observations are nonetheless consistent with recent studies using monoclonal antibodies to cell surface antigens which have demonstrated a heterogeneity of lymphoid cell subsets within the epidermis in lesions of mycosis fungoides and of other malignant and benign dermatoses.

Immunobiology of mouse dendritic epidermal T cells: a decade later, some answers, but still more questions.

Over the past decade, overwhelming evidence has accumulated in many species, most notably in mice, that epithelial sites such as skin, intestine, and reproductive tract are populated with relatively discrete subsets of gamma delta cells. Such studies have identified several distinguishing and, in some cases, unique features of the dendritic epidermal T cells (DETC) populating the skin of all normal mice: homogeneous V5-J1-C gamma 1/V1-D2-J2-C delta T-cell receptors devoid of junctional diversity, apparent tissue restriction in adult mice to the skin, an important role for active hair growth in their localization and/or proliferation in the skin, and a capacity to recognize an antigen expressed on stressed epidermal cells. These properties have led to the hypothesis that DETC play distinctive roles in cutaneous immune surveillance and/or immunoregulation via recognition of a common self-antigen expressed by adjacent cells under various potentially harmful circumstances. Despite substantive advances in our knowledge about gamma delta cells in general (e.g., recent evidence that their manner of antigen recognition may be fundamentally different from that used by conventional alpha beta T cells) and about epithelial-specific subsets such as murine DETC in particular, it is clear that, compared with our understanding of alpha beta cells, major gaps still exist in our understanding of these cells. Persisting questions about DETC include: precise identification of the ligands for their homogenous T-cell receptors, the cellular and molecular requirements for their activation, their full range of functional activities, the reason(s) for the absence in normal human skin of a precise morphologic and phenotypic homologue, and, perhaps most important, their biologically relevant role(s) in cutaneous physiology, immunity, and/or pathology.

Specific suppression of lupus-like graft-versus-host disease using extracorporeal photochemical attenuation of effector lymphocytes.

(C57BL/6 x DBA/2)F1 (B6D2F1) mice inoculated with parental DBA/2 (D2) splenocytes develop chronic stimulatory graft-versus-host reaction with many of the clinical manifestations of systemic lupus erythematosus. This investigation tested the ability of 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) light-treated D2 cells, primed to contain an expanded population of T cells specific for B6D2F1 major histocompatability complex antigens, to treat and/or prevent such systemic lupus erythematosus-like disease. 8-MOP/UVA-treated cells from B6D2F1-primed D2 donors were inoculated into B6D2F1 recipients weekly six to ten times, either before or after initiating graft-versus-host disease with normal D2 cells. A third group of B6D2F1 recipients were vaccinated weekly six times before disease initiation using 8-MOP/UVA-attenuated, B6D2F1-primed D2 cells that had been secondarily stimulated and expanded in vitro in the presence of irradiated B6D2F1 targets and interleukin-2. Control B6D2F1 mice were vaccinated with 8-MOP/UVA-treated D2 cells stimulated in vitro and/or in vivo with (C3H/HeJ x DBA/2)F1 cells. Only mice vaccinated with 8-MOP/UVA-attenuated D2-anti-B6D2F1 cells that were secondarily stimulated and expanded in vitro exhibited differences from controls when measured by the clinical parameters of ascites formation, and mean survival (p < 0.025). These groups also differed significantly in mean antinuclear antibody titer measured 14 weeks after disease initiation (p < 0.05). At 28 weeks, histologic evidence of systemic lupus erythematosus-like kidney disease was found only in the control group. These results indicate that photochemically attenuated D2-anti-B6D2F1 cells primed in vivo and secondarily stimulated and expanded in vitro are capable of vaccinating recipients against progression of graft-versus-host reaction-initiated systemic lupus erythematosus-like disease.

Conjugated avidin identifies cutaneous rodent and human mast cells.

Avidin conjugated to the fluorescent dyes rhodamine or fluorescein binds to mast cell granules in rodent and human skin. Sequential staining of tissue mast cells first with conjugated avidin, and then with a metachromatic stain demonstrated that both techniques identify the same mast cell granules. Specificity for mast cells was confirmed by the absence of avidin-positive cells in the skin of mast cell-deficient (W/Wv) mice. Binding of conjugated avidin to mast cells was inhibited by pretreating tissue specimens with unconjugated avidin but not by pretreating conjugated avidin with biotin, indicating that avidin does not bind to biotin or a biotin-like molecule. Within murine dermis, unique patterns of mast cell distributions were observed, with a prominent perivascular localization in ear skin, and a complete absence of mast cells underlying the scales in tail skin. In tissue sections of guinea pig skin undergoing basophil hypersensitivity reactions and in murine and human skin specimens infiltrated with eosinophils, conjugated avidin selectively stained only dermal mast cells, demonstrating specificity for mast cells in sites of inflammation. Conjugated avidin also readily stained rat peritoneal mast cells, demonstrating its utility for identifying extracutaneous mast cells. Unlike the metachromatic stains, avidin binding to mast cells in tissues is not limited by methods of fixation or special embedding and cutting procedures. Thus, mast cell identification with conjugated avidin is a reliable, specific, and simple method with important clinical and investigative applications.

Origin and function of Thy-1+ dendritic epidermal cells in mice.

The epidermis of normal mouse skin incorporates a newly-recognized population of dendritic cells which express relatively large amounts of the cell surface glycoprotein, Thy-1 antigen. These cells, termed Thy-1+dEC, are distinct from both epidermal Langerhans cells (LC) and melanocytes, and they populate cutaneous sites in surface densities which range to as high as 580 cells/mm2, approximately two-thirds that of LC. Studies of lethally irradiated mice which were reconstituted with semiallogeneic bone marrow cells and mice which received grafts of semiallogeneic skin have demonstrated that some, if not all, Thy-1+dEC are of bone marrow origin, and that they are capable of migrating into epidermis from a vascular source. Thy-1+dEC expressed both asialo GM1 and a cell surface determinant recognized by the monoclonal antibody 20-10-5S, further suggesting their functions will be included among those normally ascribed to lymphoreticular cells. Isolation of epidermal cells with the Fluorescence Activated Cell Sorter (FACS) was successful in producing relatively pure populations of Thy-1+dEC and LC. Such technological advances as this should facilitate testing several hypotheses concerning the ultimate function of these cells, including the possibilities that they are antigen-presenting cells which selectively activate down-regulating signals, T lymphocytes, natural killer (NK) cells, or natural suppressor (NS) cells.

TCR gamma/delta+ dendritic epidermal T cells as constituents of skin-associated lymphoid tissue.

The epidermis of all strains of normal mice is populated by two distinct dendritic, bone marrow-derived cells: Langerhans cells and CD4-CD8- Thy-1+ dendritic epidermal T cells (DETC). The overwhelming majority of DETC are an unusually homogeneous population of thymic-dependent cells which express CD3-associated T-cell antigen receptors (TCRs) of the gamma/delta type, thereby distinguishing them from conventional CD4+CD8- or CD4-CD8+ T cells expressing CD3-associated alpha/beta TCR. Most DETC are ontogenetically primitive, derived from early fetal thymocytes with a preferential, but poorly understood tropism for the epidermis. Like the TCR on other populations of gamma/delta cells, which preferentially populate other epithelia such as in the gut and lung, the TCR on most DETC selectively utilize particular variable (V) gene segments (i.e., V gamma 3 and V delta 1 for DETC vs V gamma 5 and V delta 4 or V delta 6 for intestinal intraepithelial lymphocytes). However, unlike other gamma/delta populations whose TCR junctional regions exhibit marked heterogeneity, DETC junctional diversity is extremely limited. This lack of TCR heterogeneity among DETC suggests they recognize a narrow range of physiologic ligands (antigens) and that this recognition is restricted not by conventional polymorphic class-I or class-II MHC molecules, but rather by relatively nonpolymorphic self MHC-like molecules of the class Ib MHC type [e.g., Qa, TL, and CD1 (T6)]. Additional studies are required to clarify precisely what DETC recognize, their relevant biological functions, as well as their relationship(s) to the gamma/delta cells recently identified in human skin.

Thy-1 antigen-bearing dendritic cells in murine epidermis are derived from bone marrow precursors.

Thy-1 antigen is expressed on a dendritic subpopulation of cells in murine epidermis. Numbering between 200 and 500/mm2 surface area in abdominal skin, they are distinct from the dendritic Langerhans cells (LCs) and melanocytes. Since immigrant lymphoid cells as well as constitutive cells in various organs have been demonstrated to be Thy-1+, their origin and function are not certain. To assess these issues, two experimental protocols were established. First, grafts of whole skin from AKR mice were placed orthotopically on (AKD2)F1 recipients. Immigration of recipient-derived cells into graft epidermis was assessed histologically by fluorescence microscopy employing monoclonal anti-Thy-1.2 and anti-I-Ad antibodies. Second, bone marrow chimeras were established in AKR recipients after lethal irradiation and reconstitution with cells from (AKD2)F1 donors. In the first protocol, dendritic I-Ad+ LCs of donor origin infiltrated each graft to normal densities within 2 weeks. Thy-1.2+ cells also immigrated into the same grafts, but at much slower rates. In the second protocol, bone marrow-derived Thy-1.2+ cells populated normal skin epidermis slowly over several months, with densities reaching 70/mm2. We conclude that some, if not all, Thy-1+ cells in normal murine epidermis are derived from bone marrow precursors, that their infiltration rates differ substantially from those of LCs, and that those factors which govern immigration rates into adult skin derive from the skin itself rather than from the systemic availability of their precursors. We suggest that the function of Thy-1+ epidermal cells will therefore reside among those usually ascribed to recirculating hematogenous cells, including the possibility that they may down-regulate immunizing signals that emerge from skin.

FACS purification of bone marrow-derived epidermal populations in mice: Langerhans cells and Thy-1+ dendritic cells.

A method was developed which allows for the separation and purification of Langerhans cells (LC) and Thy-1+ cells (Thy-1+dEC) from mouse epidermis. Epidermal cell (EC) suspensions were subjected to Ficoll separation, and the resulting interface EC were harvested. These EC were then "tagged" with the appropriate monoclonal antibody and sorted into positive and negative populations using the Fluorescence Activated Cell Sorter (FACS). Preparations of viable LC and Thy-1+dEC were obtained with 94-98% and 94-99% purities, respectively.

Phenotypic heterogeneity and cytotoxic activity of Con A and IL-2-stimulated cultures of mouse Thy-1+ epidermal cells.

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.

Skin-selective lymphocyte homing mechanisms in the pathogenesis of leukemic cutaneous T-cell lymphoma.

The concept of skin-associated lymphoid tissue embraces those cells and functions that are integrated in the cutaneous host defense. Recently, it has been possible to identify those circulating T-cells that are skin associated. These cells display the cell-surface phenotype of memory T cells (CD45RO+) and express the cutaneous lymphocyte antigen, a tissue-selective homing receptor involved in directing T-cell traffic to inflamed skin. To investigate the participation of this skin-associated T-cell subset in the pathogenesis of cutaneous T-cell lymphoma, we studied 16 patients with erythrodermic cutaneous T-cell lymphoma for the presence of these surface proteins on circulating cells. Results were compared with eight patients in remission and eight with minimal patch-plaque cutaneous T-cell lymphoma. The mean expression of both CD45RO and cutaneous lymphocyte antigen were significantly greater in the erythrodermic patients than in the other two patient groups. Expression of these markers was shown to be on the cells of the malignant clone by two-color flow cytometry. These results demonstrate that the malignant cells of cutaneous T-cell lymphoma express the markers of skin homing lymphocytes and that their levels are increased in the erythrodermic cutaneous T-cell lymphoma patients. Moreover, the findings suggest a critical role for the skin-selective homing receptor cutaneous lymphocyte antigen in the pathogenesis of cutaneous T-cell lymphoma.

Thy-1+ dendritic epidermal cells in mice: precursor frequency analysis and cloning of concanavalin A-reactive cells.

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.