Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4⁺ T cells, but also on MHC-class I molecules to CD8⁺ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4⁺ and CD8⁺ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin⁺ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin⁺ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4⁺ and CD8⁺ T cell responses emphasizes their potential for epicutaneous immunization strategies. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications,” Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Cytology of primary cutaneous Langerhans cell histiocytosis with a malignant phenotype. A case report

BACKGROUND: Langerhans cell histiocytosis (LCH) is a proliferative disorder of Langerhans cells, but the nature of LCH, whether reactive, benign, or malignant and neoplastic, is controversial. We encountered a case of LCH showing a malignant phenotype initially localized in the skin of an elderly woman. Since there is no other report on the cytologic appearance of primary cutaneous LCH or on LCH with a malignant phenotype, we compared the cytologic features of this case with those of benign cases at other sites reported in the literature. CASE: A 74-year-old woman presented with a gradually enlarging and partially ulcerated skin lesion expanding both sides of her right hand. On histologic and ultrastructural analyses of surgically resected tissue, we diagnosed the lesion as Langerhans cell histiocytosis originating in the skin. Although the patient had no recurrence or metastases for six months after surgical resection of the primary skin lesion and radiation therapy, the tumor extended multisystemically, and the patient died of multiple organ failure 14 months after the initial diagnosis. CONCLUSION: Imprint and scrape cytology of multiple skin lesions six months after surgery was useful in immediately diagnosing the recurrent LCH. The tumor cells had indented, twisted or grooved nuclei, and some had intranuclear inclusions. Immunocytochemically the cells were positive for CD1a and S-100 protein. Numerous eosinophils were seen in the background.     

Anti-Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage

Anti-Leu-3a, anti-Leu-3b, OKT4, and anti-T4 murine monoclonal antibodies react with a membrane component expressed by mature peripheral blood helper T cells and certain thymocyte subsets. Using a variety of immunologic staining techniques, we have demonstrated the reactivity of these antibodies with other cell types. Normal and neoplastic cells of monocyte/macrophage lineage bear the Ia+/Leu-6-/Leu-3+ phenotype, whereas histiocytosis X cells bear the Ia+/Leu-6+/Leu-3+ phenotype. The Ia+/Leu-6- cells of malignant histiocytosis and the Ia+/Leu-6+ epidermal Langerhans cells were variably Leu-3+. Normal monocyte/macrophage reactivity with anti-Leu-3/T4 appears to be primarily intracytoplasmic, whereas on U937 monocyte tumor cells, marked membrane reactivity is also observed. These results strongly suggest that certain cells other than helper T cells and thymocytes can express and, at least in some cases, synthesize a component previously regarded as T-lineage specific.     

The Langerhans cell: from in vitro production to use in cellular immunotherapy

Dendritic cells constitute a family of antigen presenting cells defined by their morphology and their capacity to initiate primary immune response. Langerhans cells are paradigmatic dendritic cells, described in 1868 by a young medical student, Paul Langerhans in Berlin. Langerhans cells are present with epithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge, Langerhans cells migrate into the T cell areas of proximal lymph nodes where they act as professional antigen-presenting cells. Langerhans cells originate in the bone marrow and CD34+ hematopo?etic progenitors are present in cord blood or circulating blood. They are actively involved in skin lesions of allergic contact dermatitis or atopic dermatitis, in cancer immunosurveillance and are infected by HIV in AIDS. Since 1992, Langerhans cells may be generated in vitro from CD34+ cord blood or circulating blood progenitors by culture with GM-CSF and TNF alpha, as well as from peripheral blood monocytes by culture with GM-CSF, IL4 and TGF beta 1. The possibility to obtain from the blood, the circulating progenitors of dendritic cells and the subsequent possibility to harvest a large number of these cells through in vitro culture using growth factors, have given rise to several very interesting therapeutic perspectives, especially in the field of anti-cancer immunotherapy. In dermatology advanced studies have concerned malignant melanomas. Anti-melanoma immunization trials were performed in patients, through dendritic cells charged with melanoma antigens. Side effects appear to be limited. Injections of antigenically charged dendritic cells were performed subcutaneously, intravenously or in the lymph nodes. Positive clinical responses were obtained with, in some cases, complete remission of the metastasis. These results open a particularly interesting perspective in the field of cancer treatment.     

Evidence that Langerhans cells in adult pulmonary Langerhans cell histiocytosis are mature dendritic cells: importance of the cytokine microenvironment.

Because Langerhans cells (LC) in peripheral tissues are generally "immature" cells with poor lymphostimulatory activity, the contribution of immune responses initiated by LC to the pathogenesis of pulmonary LC histiocytosis (LCH) has been uncertain. In this study we demonstrate that LC accumulating in LCH granulomas are phenotypically similar to mature lymphostimulatory dendritic cells present in lymphoid organs. LC in LCH granulomas intensely expressed B7-1 and B7-2 molecules, whereas normal pulmonary LC and LC accumulating in other pathologic lung disorders did not express these costimulatory molecules. The presence of B7+ LC in LCH granulomas was associated with the expression in these lesions, but not at other sites in the lung, of a unique profile of cytokines (presence of GM-CSF, TNF-alpha, and IL-1beta and the absence of IL-10) that is known to promote the in vitro differentiation of LC into cells expressing a lymphostimulatory phenotype. Finally, LCH granulomas were the only site where CD154-positive T cells could be identified in close contact with LC intensely expressing CD40 Ags. Taken together, these results strongly support the idea that an abnormal immune response initiated by LC may participate in the pathogenesis of pulmonary LCH, and suggest that therapeutic strategies aimed at modifying the lymphostimulatory phenotype of LC may be useful in the treatment of this disorder.     

Skin-derived dendritic cells can mediate deletional tolerance of class I-restricted self-reactive T cells

Skin-draining lymph nodes contain a number of dendritic cell (DC) subsets of different origins. Some of these are migratory, such as the skin-derived epidermal Langerhans cells and a separate dermal DC subset, whereas others are lymphoid resident in nature, such as the CD8+ DCs found throughout the lymphoid tissues. In this study, we examine the DC subset presentation of skin-derived self-Ag by migratory and lymphoid-resident DCs, both in the steady state and under conditions of local skin infection. We show that presentation of self-Ag is confined to skin-derived migrating DCs in both settings. Steady state presentation resulted in deletional T cell tolerance despite these DCs expressing a relatively mature phenotype as measured by traditional markers such as the level of MHC class II and CD86 expression. Thus, self-Ag can be carried to the draining lymph nodes by skin-derived DCs and there presented by these same cells for tolerization of the circulating T cell pool.     

Histiocytose langerhansienne osseuse multifocale : intérêt de la scintigraphie osseuse planaire

Langerhans cell histiocytosis is a disease of the reticuloendothelial system characterized by a clonal proliferation of dendritic cells of Langerhans. This is a rare disease that primarily affects children and young adults. It can take many forms, from a single eosinophilic granuloma to widespread lesions involving multisystem life-threatening. Bone involvement is the most common, it can be uni- or multifocal. We report a case of multifocal bone Langerhans cell histiocytosis, revealed in a child aged 3 years and touched his left temporal bone and right femur. The bone scintigraphy has found a third location in right scapular. The outcome was favorable after chemotherapy. The bone lesions are characterized by lytic lesions and are responsible for pain, swelling and fractures. All the imaging techniques (radiographs, CT, and MRI) contribute to the formation of the diagnosis. Bone scintigraphy is a very sensitive examination that allows the initial staging and follow-up on treatment of skeletal manifestations.     

The vesicular monoamine transporter 2 (VMAT2) is expressed by normal and tumor cutaneous mast cells and Langerhans cells of the skin but is absent from Langerhans cell histiocytosis.

Monoamine storage in secretory granules is mediated by the vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2). The aim of our study was to identify monoamine-handling normal and neoplastic inflammatory cells in the skin by their expression of VMAT1 and VMAT2. Normal skin from various parts of the body, as well as 21 cases of cutaneous mastocytosis and 10 cases of cutaneous Langerhans cell histiocytosis were analyzed by immunohistochemistry, radioactive in situ hybridization, and double-fluorescence confocal microscopy. VMAT2-positive cells in the subepidermal layer were identified as mast cells by their expression of tryptase. Neoplastic mast cells in all cases of cutaneous mastocytosis retained their VMAT2 positivity. The intraepidermal VMAT2-expressing cells were identified as Langerhans cells by their CD1a positivity. VMAT2 was absent from Langerhans cell histiocytosis. VMAT2 is an excellent marker for normal and neoplastic mast cells. The expression of VMAT2 demonstrates the capacity of mast cells for monoamine storage and handling. The presence of VMAT2 in epidermal Langerhans cells revealed a previously unrecognized monoamine-handling phenotype of these cells and indicates possible involvement of amine storage and release associated with antigen presentation. Absence of VMAT2 in neoplastic Langerhans cells indicates a loss of monoamine handling capacity of these cells during tumorigenesis.     

Mononuclear cell subpopulations and infiltrating lymphocytes in erythema dyschromicum perstans and vitiligo

Erythema dyschromicum perstans (EDP) and vitiligo are two cutaneous pigmentary dermatoses of unknown etiology. In the present study, the leukocyte infiltrates in the affected skin of EDP and vitiligo patients were studied using the avidin-biotin (ABC) immunoperoxidase technique and monoclonal antibodies which recognise the following mononuclear cell subgroups: T-suppressor/cytotoxic (CD8-Leu-2), T-helper (CD4 = OKT4), T-suppressor + macrophages (Leu-15), Pan T (CD3 = Leu-4), macrophages (Leu-M3) and Langerhans cells (CD1 = Leu-6), and other cellular markers such as Ia antigens and the Interleukin-2 receptor (CD25 = TAC). The immunocytochemical analysis showed a selective accumulation of CD3+, CD8+, Leu-15-, T-cytotoxic cells in the epidermis of both EDP and early lesions of vitiligo. In addition, an increase in the number of epidermal Langerhans cells (CD1+) was observed in some cases of EDP and vitiligo. The CD4/CD8 ratios in affected and uninvolved skin for both disorders were not significantly different, although values lower than unity were only observed in the infiltrates of affected skin. Ia antigen positivity was observed in the dendritic cells of the dermis and epidermis, as well as in most of the lymphoid cells within the infiltrates for both diseases. Macrophages (Leu-M3) in EDP dermal infiltrates were generally found adjacent to extracellular melanin pigment. Lymphocytes expressing TAC (CD25) surface antigens were also present in the dermal infiltrates. These morphological observations suggest a possible immune cell participation in the dyschromia of such cutaneous disorders.     

朗格罕细胞组织细胞增生症颅内垂体影像表现及相关临床表现

目的:探讨朗格罕细胞组织细胞增生症累及垂体的MR表现及相关临床表现。方法:搜集了6例确诊为朗格罕细胞组织细胞增生症并垂体表现异常的患儿,男5例,女1例,年龄2～11岁,平均(6±3)岁,对其影像及临床表现进行回顾性分析。结果:临床患儿主要以头面部包块,多饮、多尿等就诊。头颅MR平扫(T1WI)表现6例患儿神经垂体高信号全部消失,垂体柄增粗5例,垂体柄著征1例,垂体饱满1例,其中3例治疗后复查垂体及垂体柄均有变化。结论:神经垂体高信号消失,垂体柄增粗或著征为朗格罕细胞组织细胞增生症累及垂体的头颅MR表现。累及垂体者临床几乎都有多饮、多尿表现。目前,MRI是诊断朗格罕细胞组织细胞增生症累及垂体的唯一可靠的影像学检查方法,并对治疗后病情随访有重要作用。     

Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.     

Inhibition of functional T cell priming and contact hypersensitivity responses by treatment with anti-secondary lymphoid chemokine antibody during hapten sensitization

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.     

Langerhans cell histiocytosis: report of a single organ involvement in a child

Langerhans cell histiocytosis is a rare disorder characterized by abnormal proliferation of Langerhans cells that can affect various organ systems. The disease usually presents as a unifocal lytic bone lesion and can affect any age group. Less frequently it presents as a disseminated disease with multisystem involvement. Hepatic manifestation in Langerhans cell histiocytosis is relatively rare and usually presents as a part of a disseminated process. We report a case of Langerhans cell histiocytosis involving only the liver in a 9-years-old child.     

Limited diversity of gamma delta antigen receptor genes of Thy-1+ dendritic epidermal cells

T cells bearing gamma delta antigen receptors constitute minor populations in most peripheral lymphoid tissues but represent the major populations of T cells in certain epithelia, including the epidermis. We show that murine dendritic epidermal cell (dEC) clones express V gamma and V delta gene segments, which are rare in adult T cells but predominate in fetal thymocytes. Analysis of the junctions of the rearranged gamma and delta genes shows a striking homogeneity among the receptors of five dEC clones. Our data support a model in which dECs represent one of perhaps several waves of emigrants from the early fetal thymus, and imply a role for dECs in immune surveillance that is distinct from that of alpha beta- and other gamma delta-bearing T cells.     

Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.     

Human thymomas: evidence of immunohistologically defined normal and abnormal microenvironmental differentiation

Fifteen human thymomas were analyzed by immunoperoxidase studies on frozen and paraffin-embedded tissue sections in an attempt to identify the existence of immunologically defined microenvironments. All nine lymphocyte predominant thymomas contained a predominance of lymphocytes bearing the phenotype of cortical thymocytes and dendritic Class II major histocompatibility complex antigen-positive epithelial cells, thus defining cortical-like microenvironments. Medullary-like foci were also seen in all of these cases. Minor phenotypic abnormalities in Leu-2 and -3 antigen expression were seen in three cases. In contrast, the two epithelial predominant thymomas and four mixed thymomas all exhibited features of aberrant microenvironmental differentiation, with only two cases showing demarcation into cortical and medullary foci. A lack of Class II major histocompatibility complex antigens was associated with a decrease in the lymphoid populations and an increase in Leu-1 antigen expression by T cells of otherwise normal cortical phenotype when lymphocytes were present. In contrast, lack of Class I antigen on epithelial cells was not associated with any abnormality in lymphocyte phenotype or microenvironmental organization. We document for the first time abnormal microenvironments in thymomas that may offer insights into understanding normal thymic differentiation.     

Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology

The objectives of the present study were to determine the cytological features of isolated follicular dendritic cells (FDC), which distinguish them from other leukocytes or dendritic cell types. Consequently, we have developed methods for the fixation, peroxidase cytochemistry, and visualization of FDC, which are applicable to cytological evaluations by Nomarski optics, scanning, and transmission electron microscopy. A functionally supported identification of FDC in vitro was made possible by utilizing, in conjunction with the dendritic morphology, the cytochemically identifiable antigen, horseradish peroxidase (HRP), and the known capacity of FDC to sequester immune complexes (i.e. HRP-anti-HRP) on their plasma membranes. The observations showed that FDC constitute a relatively pleomorphic, nonphagocytic group, distinct from other dendritic type cells such as lymphoid dendritic cells, Langerhans cells, and interdigitating cells (LDC, LC, and IDC), as well as typical leukocytes. Morphologically distinct FDC were identified as cells either with filiform dendrites or with "beaded" dendrites. FDC possessed a single or sometimes a double, lymphocyte-size cell body, which contained an irregular, lobated nucleus, Golgi apparatus, numerous small vesicles, and some mitochondria. Mitochondria were not abundant in the dendritic processes. Filiform dendrites tended to branch and anastomose near the cell body and form a radiating "sunburst"-like pattern. On the average, dendrites measured 15-20 microns in length and 0.1-0.3 micron in diameter. Occasional dendrites were extremely elongated, reached several hundred microns in length, and terminated in an enlargement measuring nearly a micron in diameter. Other filiform dendrites usually had a club-shaped terminal enlargement. The microspheres of "beaded" dendrites ranged between 0.3 and 0.6 micron in diameter. The dendritic processes were also shown to have a highly ordered pattern of immune complex attachment on their surface, suggestive of a periodic arrangement of receptor sites.     

The Cytology of Langerhans Cell Histiocytosis (Histiocytosis X)

The cytomorphology of 13 cases of Langerhans cell histiocytosis is described. The most striking features were the presence of intranuclear clefts, pale nuclei and inconspicuous nucleoli, together with ample pale cytoplasm, only slight cellular pleomorphism, and an admixture of varying numbers of eosinophils, macrophages and degenerated cells. In 13 of 16 cases investigated ultrastructurally, characteristic Birbeck granules were detected. Out of six cases tested, four exhibited positivity for S-100, and of three cases tested, all were positive for CD1a (leu 6) and HLA-DR. In one case malignant transformation occurred, terminating in monocytic leukaemia.     

The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus

The surface of dendritic cells from mouse spleen, thymus, and epidermis has been compared with a panel of monoclonal antibodies and the FACS. A method was first developed to isolate populations of large, adherent, thymic dendritic cells that were greater than 90% pure. These were released by collagenase digestion and separated from adherent macrophages after overnight culture. Enrichment was based on the facts that most macrophages remained plastic adherent and rosetted strongly with antibody-coated erythrocytes. As in spleen, thymic dendritic cells were stellate in shape, had abundant class I and II MHC products, lacked many standard macrophage and lymphocyte markers, and actively stimulated the mixed leukocyte reaction. Most spleen and thymic dendritic cells could be lysed by the 7D4 mAb, to the low-affinity IL-2 receptor, and complement but the levels of 7D4 by FACS were low and sometimes not above background. Differences among dendritic cells from different tissues were noted with other mAb. Adherent dendritic cells from thymus all expressed the J11d "B cell" antigen and the NL145 interdigitating cell marker, but lacked the 33D1 spleen dendritic cell antigen. Eighty to ninety percent of spleen dendritic cells were J11d-, NL145-, 33D1+ but the remainder expressed the J11d+, NL145+, 33D1- thymic phenotype. The latter phenotype also was identical to that of epidermal Langerhans' cells. We postulate that the major 33D1+ cell in spleen represents a migratory stage in which dendritic cells are moving from tissues to lymphoid organs.     

Increased blood myeloid dendritic cells and dendritic cell-poietins in Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH), previously known as histiocytosis X, is a reactive proliferative disease of unknown pathogenesis. Current therapies are based on nonspecific immunosuppression. Because multiple APCs, including Langerhans cells and macrophages, are involved in the lesion formation, we surmised that LCH is a disease of myeloid blood precursors. We found that lin(-) HLA-DR(+)CD11c-+ precursors of dendritic cells, able to give rise to either Langerhans cells or macrophages, are significantly (p = 0.004) increased in the blood of LCH patients. The analysis of serum cytokines in 24 patients demonstrated significantly elevated levels of hemopoietic cytokines such as fms-like tyrosine kinase ligand (FLT3-L, a dendritic cell-mobilizing factor, approximately 2-fold) and M-CSF ( approximately 4-fold). Higher levels of these cytokines correlated with patients having more extensive disease. Serum levels of FLT3-L and M-CSF were highest in high risk patients with extensive skin and/or multisystem involvement. Finally, patients with bone lesions had relatively higher levels of M-CSF and of stem cell factor. Thus, early hemopoietic cytokines such as FLT3-L, stem cell factor, and M-CSF maybe relevant in LCH pathogenesis and might be considered as novel therapeutic targets.