WO2007031273A2

WO2007031273A2 - Artificial immune-reconstituted epidermis equivalents

Info

Publication number: WO2007031273A2
Application number: PCT/EP2006/008872
Authority: WO
Inventors: Bernhard Moser; Patrick Schaerli
Original assignee: Universität Bern
Priority date: 2005-09-16
Filing date: 2006-09-12
Publication date: 2007-03-22
Also published as: WO2007031273A3

Abstract

The invention relates to a method of preparation of a novel human immune-reconstituted epidermis equivalent comprising culturing epidermis precursor cells from skin biopsies or outer root sheaths of hair follicles and then co-culturing this epidermis equivalent with human peripheral blood mononuclear cells expressing CD14 on a solid support, and to such novel human immune-reconstituted epidermis equivalent prepared by this method and comprising progeny of human peripheral blood mononuclear cells originally expressing CD14. The invention further relates to the use of these immune-reconstituted epidermis equivalents in assays relating to immune reactivity and skin-damaging properties of immunogenic and potentially harmful substances as well as to the use of these immune-reconstituted epidermis equivalents in immunotherapy of skin lesions, inflammation and skin diseases caused by pathogens or tumors.

Description

Artificial Immune-reconstituted Epidermis Equivalents

Field of the invention

The invention relates to a novel human immune-reconstituted epidermis equivalent, to a method of preparation of such human epidermis equivalents by in vitro reconstitution of epidermal stem cells in the presence of human blood-derived monocytes expressing CD14, and the use of these immune-reconstituted epidermis equivalents in novel skin tests relating to the examination of immune reactivity and skin-damaging properties of immunogenic and potentially harmful substances.

Background of invention

Dendritic cells (DCs) and Langerhans cells (LCs) DCs form a distinct class of leukocytes, are derived from hematopoietic progenitor cells in the bone marrow, and primarily reside in extravascular sites that include epithelial / mucosal tissues (skin, airways and gastrointestinal/urogenital tracts, among others) and secondary lymphoid tissues (spleen, lymph nodes and Peyer's patches). In peripheral blood, DCs or DC precursors make up less than 1% of mononuclear leukocytes. Distinct DC subsets differ in their tissue localization, as exemplified by interstitial DCs that primarily reside in soft tissues bordering epithelia, Langerhans cells (LCs) present in the epidermis and plasmacytoid DCs with homing preferences for lymph nodes. These DC subsets are fully differentiated non-proliferating cells with a limited life-span of several days to several weeks, indicating that they are continuously replaced under steady-state conditions by bone marrow-derived precursors.

The principal function of tissue-resident DCs (including LCs) is the uptake and processing of local antigens, their relocation via afferent lymphatic vessels to draining lymph nodes and the initiation of antigen-specific adaptive immune responses. DCs are also referred to as "professional" antigen presenting cells (APCs) because they are capable of stimulating naive αβ T cells during primary immune responses.

The immune system of the human skin

The skin fulfils many critical functions that are required for sustaining life. First, it forms a physical barrier between the body and the environment, providing a shield from environmental substances, preventing desiccation and rapid temperature loss and absorbing radiation energy. Second, it is equipped with an expert local immune system for initiation of rapid responses against immunogenic substances that have penetrated the skin by crossing the outermost layer called "stratum corneum". Third, it features efficient molecular and cellular repair mechanisms for rapid restoration of skin integrity. Skin tissue is divided into (inner-to-outer direction) subcutis, containing larger blood and lymphatic vessels, reticular dermis, papillary dermis where postcapillary venules feed the skin with nutrients and immune cells and afferent lymphatic capillaries drain skin tissue fluids and exiting immune cells, and epidermis. The epidermis is a stratified epithelium composed of a basal layer of cuboidally shaped keratinocytes, followed by multiple layers of more flattened keratinocytes and ending in the stratum corneum that is devoid of live cells. The epidermis is a self-renewing tissue and contains epidermal stem cells (precursor cells) that constantly produce basal-layer keratinocytes, which follow an unidirectional path of differentiation, and probably other cells, such as melanocytes. Melanocytes reside in the basal part of the epidermis and produce radiation-neutralizing melanin in response to UV light exposure and, in addition, may also have some antigen-presenting function. Finally, Langerhans cells (LCs) are in suprabasal position and, thus, constitute the first line of defense against any sort of external threat. LCs are epidermal sentinel cells that take up and process antigen and quickly mature in response to inflammatory stimuli. This process enables their efficient relocation to draining lymph nodes where appropriate T and B cell responses are initiated.

LCs in the epidermis, like interstitial DCs in the dermis, are present in a fully differentiated but "immature" state. These immature LCs/DCs are poorly immunogenic, i.e. are not capable of inducing primary adaptive immune responses. Instead they are experts in antigen uptake by means of receptor-mediated endocytic or fluid phase pinocytic mechanisms. A multitude of environmental signals induce LC/DC "maturation", including virus- or bacteria-derived stimuli that trigger toll-like receptors (TLRs), host cell-derived inflammatory mediators (interferon IFN-γ, tumor necrosis factor TNF-α, interleukin IL-1 , prostaglandin PGE2, tissue growth factors, among others), and T cell co-stimulatory molecules (CD40-ligand/CD154) (Banchereau, J., et al., Annu. Rev. Immunol. 18:767- 81 1 , 2000). LC/DC stimulation triggers efficient antigen processing and peptide loading onto intracellular MHC-I/II molecules for subsequent cell surface presentation. Furthermore, LC/DC maturation is accompanied by the installment of a lymph node homing migration program characterized by the expression of the chemokine receptor CCR7. Skin tests in the development of pharmaceutical products and cosmetics Issues relating to immunological safety and skin damage are important aspects in the development of pharmaceutical and cosmetic products, which require time-consuming and costly tests. Without addressing these questions by performing relevant tests (hereafter referred to as skin tests), novel substances do not get governmental approval for marketing. Substances that show positive reactions in skin tests are removed from drug and cosmetic development programs despite the fact that they may have favorable target selectivity and pharmacokinetic properties. Consequently, for economic reasons, immunological safety and otherwise skin-damaging issues need to be addressed early in drug and cosmetic development programs. This also means that these skin tests are carried out with a large number of potential new pharmaceutical and cosmetic products worldwide. Current industrial skin tests include a large variety of animal models. The present invention provides an alternative to these animal models and, thus, may contribute to lessening the number of animals used in such studies. Furthermore, the skin test presented in this invention comprises exclusively human cells and, therefore, results obtained from skin tests using immune-reconstituted epidermis equivalents are highly relevant to immune processes within human skin. The immune systems of man and e.g. mice (which is the most common laboratory animal) differ greatly, making results obtained in animal models less relevant.

Human epidermal equivalents

Human epidermal equivalents have been the focus in numerous laboratories for many years, with the goal to understand human skin physiology as well as for their use in skin reconstitution therapies (transplantation) and for substitution of animal experimentation in toxicology tests (for a review see Ponec, M., Adv. Drug Deliv. Rev. 54 Suppl 1 :S19-S30, 2002). Human epidermal equivalents are generated ex vivo by culturing epidermal progenitor cells on various types of substrata, including dermal fibroblasts, de- epidermized dermis and artificial membrane inserts either bare or reconstituted with extracellular matrix components. The growth media for generating epidermal equivalents vary greatly but always include a combination of growth and differentiation factors. Human epidermal equivalents resemble natural epidermis in their morphology and epidermal marker expression. Of note, epidermal equivalents prepared from epidermal progenitor cells are devoid of immune cells, i.e. do not have LCs or any other type of immune cells and, therefore, can not be used in immunological skin tests for determining the immune- stimulatory potential of pharmaceutical and cosmetic products. Instead, their use is restricted to toxicology tests, examining morphological and barrier-function changes in epidermal equivalents in response to topically applied substances.

The generation of immune-reconstituted epidermis equivalents is extremely difficult, as evidenced by the scarcity of reports. The state of progress in this area of research is described in Facy, V., et al., J. Invest. Dermatol. 122:552-553, 2004, and one patent (US Pat. 5,861 ,153 and equivalent European Patent EP 789 094 B) protects the commercial use of these results. In essence, untreated or treated CD34⁺ hematopoietic progenitor cells (HPCs) are used as LC precursors for generation of immune-reconstituted epidermis equivalents. The immune system comprises a large number of distinct immune cells

(including T and B lymphocytes, natural killer cells, monocytes, phagocytes, dendritic cells and LCs, among others), and they all derive from CD34⁺ HPCs present in the bone marrow. CD34⁺ HPCs even give rise to non-immune cells, such as erythrocytes. The use of "dedicated" LC precursors that are committed to develop uniformly into functional LCs under well defined conditions would represent an important progress in the generation of immune-reconstituted epidermis equivalents.

Summary of invention

The present invention relates to a novel human immune-reconstituted epidermis equivalent comprising a support, progeny of cultured epidermis precursor cells from skin biopsies or outer root sheaths of hair follicles, and progeny of co-cultured human peripheral blood mononuclear cells originally expressing CD14.

The invention further relates to a method of preparation of such human epidermis equivalents comprising a) culturing epidermis precursor cells from skin biopsies or outer root sheaths of hair follicles and b) co-culturing progeny from step a) with human peripheral blood mononuclear cells expressing CD14 on a solid support.

In particular, the invention relates to the type of cells within said novel human immune- reconstituted epidermis equivalent arising out of CD14⁺ monocytes obtained by the method of the invention of preparing human epidermal equivalents, which include dendritic cells (DCs) with Langerhans cell (LC) characteristics as assessed by cell morphology criterion, cell surface marker expression and antigen-presentation function. The invention further relates to the particular conditions of growth (addition, reduction, omission or substitution of growth factors with regard to standard conditions used for the generation of epidermal equivalents) allowing the generation of epidermal equivalents while eliminating or reducing inhibitory factors that interfere with CD14⁺ DC precursor differentiation.

The invention further relates to the use of these immune-reconstituted epidermis equivalents in assays relating to immune reactivity and skin-damaging properties of immunogenic and potentially harmful substances, in particular to an assay for the determination of immune reactivity and skin-damaging properties of a substance comprising applying the compound to the stratum corneum or the culture medium of an immune-reconstituted epidermis equivalent according to the invention and determining the mobilization of antigen presenting cells, as well as the induction of DC/LC differentiation and activation markers as well as expression of antigen-presentation functions in these cells.

The invention further relates to a method of treating skin lesions, inflammation and skin diseases caused by pathogens or tumors with human immune-reconstituted epidermis equivalents of the invention, and to the use of such human immune-reconstituted epidermis equivalents for the preparation of a medicament for the treatment of skin lesions, inflammation and skin diseases caused by pathogens or tumors.

Brief description of the figures

Fig. 1. CXCL14 is a selective chemoattractant for CD14⁺ monocytes.

A) Results of a flow cytometric analysis depicting the three major subsets of monocytes present in human peripheral blood, characterized by the cell surface markers CD4 plus either CD14^hιghCD16- (R1), CD14^highCD16⁺ (R2) or CD14^dιmCD16⁺ (R3).

B) In vitro migration responses of the three subsets of monocytes (R1 , R2, R3) mediated by CXCL14 or other chemokines with known chemoattractant function in human myeloid cells. The in vitro migration responses were determined in a standard two-chamber chemotaxis assay. Chemotaxis activities are expressed as migration indices (Ml), which is the ratio between the number of cells migrated in response to CXCL14 and the background migration (number of cells in the absence of a chemokine). Fig. 2. CXCL14 is expressed at high levels in normal human skin tissue. This figure illustrates the distribution of CXCL14 expression in normal healthy human skin. CXCL14 protein expression was analyzed in human skin sections by immunohistochemistry, using an antibody specific for human CXCL14 (CXCL14); the lower inset shows the staining with an unrelated antibody (C, control). Highest CXCL14 expression is seen in the epidermis but blood vessels also stained positive for this chemokine. E, epidermis; D, dermis; B, blood vessels.

Fig. 3. Human epidermis equivalents produce CXCL14. As detailed in "Examples", human epidermis equivalents were generated in a co-culture system using dermal fibroblasts as feeder cells and either single cell suspension from epidermis or epidermal stem cells derived from the outer root sheaths of live hair. A) Hematoxylin-eosin (HE) staining of a cross-section of a typical human epidermis equivalent. This artificial epidermis resembles normal epidermis in human skin in morphology, and contains epidermis-typical cellular structures: 1) stratum corneum ; 2) stratum granulosum; 3) flattened keratinocytes in suprabasal location; 4) cuboidal keratinocytes in basal location; 5) membrane of tissue culture insert; 6) irradiated fibroblast monolayer. The artificial epidermis differs from normal epidermis in the complete absence of immune cells. B) Human epidermis equivalents stain strongly for CXCL14, indicating similar degree of CXCL14 production as in normal (non-inflamed) human skin (Fig. 2). CXCL14, immunostaining using an antibody for CXCL14; C, control staining with an unrelated antibody.

Fig. 4. Co-culture with epidermis equivalents induces CDI 4^* DC precursor relocation and survival.

The conditions for co-culturing CD14⁺ DC precursors with epidermis equivalents are detailed in "Examples".

A) Distribution and morphology of CD14⁺ DC precursors within epidermis equivalents. The CD14⁺ DC precursors were initially labeled with the green fluorescence dye CFSE (CFSE) in order to be identified by fluorescence microscopy in suprabasal location within epidermis equivalents. The inset shows an enlarged section to illustrate the dendritic morphology of CFSE-labeled cells.

B) Degree of survival of CD14⁺ DC precursors (S, in % of total input cells) during co- culture with epidermis equivalents (black bar) or with monolayer cultures of keratinocytes (grey bar). In the absence of epidermis equivalents or keratinocytes but in the presence of supplements used for generating epidermis equivalents the survival of CD14⁺ DC precursors was <5% (white bar on top).

Fig. 5. Distribution of LC-like cells within epidermis equivalents. The conditions for co-culturing CD14⁺ DC precursors with epidermis equivalents are detailed in "Examples". Frozen sections of immune-reconstituted epidermis equivalents were stained with antibodies for hematopoietic cells (CD45). This figure shows that CD45⁺ cells (LC-like cells) are similarly distributed within the layers of keratinocytes as LCs within normal human epidermis. LC₁ LC-like cells.

Fig. 6. Co-culture with epidermis equivalents induces changes in cell size in CD14^* DC precursors.

A) Marker expression and size characteristics in untreated CD14⁺ DC precursors, i.e. freshly isolated from peripheral blood. As assessed by flow cytometry, the cells are uniformly positive for CD14 and HLA-DR and are of small size, i.e. have low forward (FSC) and side scatter (SSC) values.

B) After 10 days of co-culture with epidermis equivalents, the CD14⁺ DC precursors have undergone dramatic changes, including upregulation of HLA-DR and dramatic increases in size, as indicated in increased FSC-and SSC-values. CD14⁺ DC precursors loose CD14 expression during co-culture within epidermis equivalents and, therefore, CD45, the global marker identifying all hematopoietic cells, was used to examine the progeny of CD14⁺ DC precursors.

Fig. 7. Co-culture with epidermis equivalents induces differentiation of CD14^* DC precursors into LC-like cells.

The conditions for co-culturing CD14⁺ DC precursors with epidermis equivalents are detailed in "Examples". After 10 days of co-culture hematopoietic cells (CD45⁺) were extracted from epidermis equivalents and analyzed by flow cytometry for expression of dendritic cell markers. Numbers within the histograms refer to the percent of cells that are positive for the respective markers. The cells lack CD14, indicating a complete transition of the CD14⁺ DC precursors into CD14^' cells. Instead the cells express high levels of HLA-DR, CD80 and CD86, which are typical markers for antigen-presenting cells. Expression of CD1a and CD1c indicates the presence of DCs. Langerin, a C-type lectin associated with Birbeck granules, is typical for LCs. LCs are specialized DCs that are uniquely present in the epidermis. DC-SIGN and CD205 (DEC-205) are important molecules for antigen-uptake on DCs. These data illustrate that during co-culture with epidermis equivalents CD14⁺ DC precursors differentiated into DCs with LC characteristics, here termed LC-like cells.

Fig. 8. LC-like cells exhibit potent antigen-presenting function. LC-like cells extracted from epidermis equivalents (circles), monocyte-derived DCs

(triangles) and freshly isolated CD14⁺ DC precursors (squares), all from the same donor, were analyzed for their ability to stimulate naive heterologous CD4⁺ T cells (primary mixed leukocyte reaction). LC-like cells isolated from co-cultures with epidermis equivalents were previously matured in situ with 500 ng/ml LPS, IL-1β and TNF-α (each 10 ng/ml) for 3 days, whereas in vitro generated DCs were matured for 2 days. Proliferation of T cells was measured by ³H-thymidine incorporation in a standard proliferation assay. The data show that LC-like cells isolated from epidermis equivalents exhibit antigen-presenting functions similar to professional APCs. In contrast, CD14⁺ DC precursors lack efficient APC function.

Detailed description of the invention

The present invention relates to a novel human immune-reconstituted epidermis equivalent and a method of preparing the same by in vitro reconstitution of epidermis stem cells in the presence of human peripheral blood mononuclear cells expressing CD14.

The present invention describes that suitable precursors of human epidermal Langerhans cells (LCs) are the subset of blood monocytes which express high levels of CD14 (referred to here as "CD14⁺ dendritic cell (DC) precursors"). CD14⁺ DC precursors are readily isolated from human peripheral blood. This invention further describes a new method for the preparation of immune-reconstituted epidermis equivalents, comprising a fully developed artificial epidermis with LC-like cells in suprabasal position. Cells used to generate these immune-reconstituted epidermis equivalents are CD14⁺ DC precursors and epidermal stem cells, preferably derived from the outer root sheaths of healthy human hair follicles. Modifications in the culture medium supplement are described herein below, allowing for efficient development of LC-like cells out of CD14⁺ DC precursors. Finally, it is documented that LC-like cells extracted from immune-reconstituted epidermis equivalents according to the invention show morphologic features of DCs, carry many cell surface markers for LCs and have potent antigen presenting functions. Furthermore, this invention describes the use of these immune-reconstituted human epidermis equivalents in in vitro skin tests for determining the immune reactivity and skin-damaging properties of potentially harmful substances, including but not limited to pharmaceutical products and cosmetics.

The invention is based on a study describing a chemokine (a cytokine with chemoattractant properties), which selectively attracts DC precursors. This chemokine is called CXCL14 and is produced under steady-state (non-inflamed) conditions in human skin, notably in the epidermis. These findings are highly relevant to the present invention since they provide a rational for formulating a method for the generation of immune- reconstituted epidermis equivalents. The following documentation entails some of the novel scientific results that are of critical importance to the present invention.

The chemokine CXCL14 is highly selective for the subset of human monocytes characterized by the cell surface phenotype of CD4 and CD14^hi9hCD16^" (the gate R1 in Fig. 1). This subset of monocytes is also called "early" monocytes, in order to indicate that these cells are in transition to become "intermediate" monocytes and "late" monocytes. It may be noted that CD14^hιghCD16^' monocytes (gate R1 cells) also respond well to the monocyte-typical chemokines CCL2 and CCL5. These chemokines were included as positive control; they regulate the traffic of monocytes under inflammatory, as opposed to homeostatic conditions. The subset of CD14^h'^9hCD16^' monocytes (gate R1 cells) is herein referred to as CD14⁺ DC precursors.

CXCL14 does not induce migration responses in any other type of human myeloid cells, including committed DC precursors present in peripheral blood or in vitro generated monocyte-derived DCs, LCs or macrophages. The responses with CD14⁺ DC precursors are observed at micromolar concentrations of CXCL14, which is unusual for chemokines. The CD14⁺ DC precursors resemble the CD14⁺ cells generated during in vitro culture of CD34⁺ hematopoietic cells in the presence of survival and differentiation factors.

It is important to know where and under what conditions CXCL14 is produced. Clearly, this information identifies the target location of CD14⁺ DC precursors and, thus, may help to understand the physiological function of CD14⁺ DC precursors. It is possible that the CXCL14-producing cells may influence the differentiation of CD14⁺ DC precursors, either through direct cell-to-cell contact and/or through secretion of survival and differentiation factors. CXCL14 is extensively expressed in human skin, notably in the epidermis (keratinocytes) and blood vessels, as detected in human skin sections by immunohistochemistry using an antibody specific for human CXCL14. CXCL14 is constitutive, i.e. CXCL14 production occurs under steady-state (non-inflamed or healthy) conditions. These facts suggest that CXCL14 controls the co-localization of CD14⁺ DC precursors with CXCL14-producing keratinocytes, resulting in the generation of CD14⁺ DC precursor-derived DCs or LCs under steady-state conditions.

Co-culture of freshly isolated CD14⁺ DC precursors with monolayers of primary human keratinocytes lead to the generation of mostly macrophages and a few interstitial DCs, which is similar to a previous report (Chomarat, P., et al., Nat. Immunol. 1 :510-514, 2000). LCs are not detected under such conditions. In addition, cultured keratinocytes cease to produce CXCL14, indicating epidermis-untypical behavior.

The situation is different if epidermis equivalents are used, e.g. when generated under conditions as detailed in "Examples". Epidermis equivalents are artificially grown outer- skin sections that resemble human epidermis. Typically, such epidermis equivalents are composed of a basal layer of cuboidal keratinocytes, followed by flattened keratinocytes in suprabasal position, a stratum granulosum and ending in a stratum corneum. The epidermis equivalents used in this invention are generated either from single cell suspensions of human epidermis or from epidermal stem cells derived from the outer root sheaths of live human hair. Fig. 4 illustrates the morphology of such epidermis equivalents. Further, such epidermis equivalents produce high levels of CXCL14, indicating that these artificial structures resemble human epidermis not only in morphology but also in cytokine production.

In order to generate immune-reconstituted artificial epidermis according to the invention, such epidermis equivalents are used in a co-culture system together with CD14⁺ DC precursors. Regular epidermis equivalents do not contain immune cells, such as LCs. According to the method of the invention, CD14⁺ human peripheral blood monocytes are added to the epidermis equivalents, and differentiation of CXCL14-responsive, CD14⁺ DC precursors into LC-like cells then occurs within these epidermis equivalents. The experimental conditions favoring the formation of such immune-reconstituted epidermis equivalents are detailed in "Examples".

More specifically, in sandwich epidermal coculture experiments precursor cells are seeded into a cell culture insert carrying a comparable number of irradiated dermal fibroblasts underneath. After 2 to 4 days, the insert is "air lifted" and further cultured to obtain fully stratified epidermis equivalents after 2 to 3 weeks. This epidermis equivalent is transferred onto a cell culture insert with irradiated dermal fibroblasts underneath and with CD14⁺ DC precursors on top. Thus, the CD14⁺ DC precursors are "sandwiched" between a fully developed epidermis equivalent and a membrane of the cell culture insert. This type of experiment is used to determine the effect of fully developed epidermis on CD14⁺ DC precursor relocation and survival.

Alternatively, and more specifically, for the generation of immune-reconstituted epidermal equivalents, precursor cells from epidermal tissue or, preferably, from outer root sheaths of live hair, are cultured until confluence on cell culture inserts containing irradiated dermal fibroblasts underneath. After washing, the cultured epidermal cell monolayer is overlaid with isolated CD14⁺ DC precursors and further cultured for 2 hours in suitable medium. Then, all medium in the upper well is carefully removed ("air lifting" of growing epidermal equivalents), and the medium in the lower well is replaced every day until full development of immune-reconstituted epidermis equivalents. The numbers of cells is adapted to the size of the cell culture inserts. Also, the ratio between epidermal precursor-derived cells and CD14⁺ DC precursors can be varied to optimize the yield in LC-like cell development. For instance, when using a 24-well cell culture insert, CD14⁺ DC precursors to be added may range between 20'0OO and 10OOOOO per 200¹OOO epidermal precursor-derived cells of one 24-well cell culture insert. Similarly, the time optimal for generating fully developed immune-reconstituted epidermis equivalents may vary between 5 and 30 days, preferably 7 and 21 days. Of note, the culture medium for generating immune-reconstituted epidermis equivalents differs from the standard culture medium used for generating plain epidermis equivalents because of the inhibitory effects of some supplements within the latter medium on CD14⁺ DC precursor differentiation. For instance, the supplements adenine, insulin, hydrocortisone and triiodothyronine inhibit the in vitro generation of monocyte-derived DCs. The quality of immune-competent epidermis equivalents can be further improved by additional modifications in the composition of the medium supplement, as described below under "Examples".

The CD14⁺ DC precursors to be added are human peripheral blood mononuclear cells expressing CD14, preferably freshly isolated monocytes characterized by the cell surface expression of CD14. Furthermore, the human peripheral blood mononuclear cells expressing CD14 may be stimulated in vitro before use, for example, with stimuli selected from bacterial products, cytokines, mitogens, and arachidonic acid metabolites, either alone or in combination. Cell culture inserts are obtained from commercial suppliers and consist each of a membrane support with pores of defined diameters (which allow the exchange of nutrients, growth factors and other soluble components but not cells), which is suspended/submersed in a plastic culture well containing culture medium. For optimal growth of outer root sheath cells of live hair, dermal fibroblasts are grown on the underside of cell culture inserts until confluence. After irradiation of the fibroblasts to prevent further growth, the cell culture inserts are inserted into the cell culture wells (fibroblasts facing down) and then outer root sheath of live hair are placed on top of cell culture inserts to allow growth of outer root sheath cells. In the absence of irradiated fibroblast monolayers, growth of outer root sheath cells as well as subsequent formation of epidermal equivalents is not optimal. An adequate substitute for fibroblasts is not known.

Fig. 5 documents that co-culture of CD14⁺ DC precursors with epidermis equivalents under the conditions specified for sandwich epidermal coculture experiments have a tremendous effect on CD14⁺ DC precursors. First, CD14⁺ DC precursors (or their progeny, respectively) move into the epidermis equivalents to locations where LCs in normal human epidermis typically reside. Second, these conditions result in survival of numerous CD14⁺ DC precursors (or their progeny, respectively), which is in contrast to the rapid death of CD14⁺ DC precursors during culture in the absence of epidermis equivalents. Monolayer cultures of primary human keratinocytes have a similar survival effect. The fluorescence dye CFSE has an inhibitory effect on CD14⁺ DC precursor differentiation; therefore, the investigations are performed with freshly isolated but unmarked CD14⁺ DC precursors. The distribution of hematopoietic (CD45⁺) cells within a cross-section of an epidermis equivalent derived from CD14⁺ DC precursors is similar to the distribution of LCs within normal human epidermis, i.e. density and distribution of CD45⁺ cells resemble LCs within normal human epidermis.

Freshly isolated CD14⁺ DC precursors express moderate levels of MHC class Il molecules (HLA-DR), are uniformly positive for CD14 and are of small size (Fig. 6). During co-culture with epidermis equivalents these cells undergo profound changes, including loss of CD14, enhanced expression of HLA-DR and large increase in cell size. Since the cells have lost CD14, the pan-hematopoietic cell marker CD45 is used to identify the progeny of CD14⁺ DC precursors. Further examination of the CD45⁺ cells recovered from epidermis equivalents after 10 days of co-culture with CD14⁺ DC precursors indicates that most cells have acquired LC-characteristics (Fig. 7). As mentioned above, the progeny of CD14⁺ DC precursors have completely lost CD14, indicating that they lack a typical marker for monocytes or macrophages. Instead, they have upregulated HLA-DR, CD80 and CD86, which are numerous on antigen-presenting cells, such as DCs and LCs. Also, these cells now express the DC/LC markers CD1a and CD1c and, of note, largely (but not uniformly) express Langerin, the C-type lectin associated with Birbeck granules within LCs.

When analyzed by phase contrast microscopy, CD45⁺ cells recovered from immune- reconstituted epidermal equivalents show typical morphologic features of DCs, including increased cell granularity and multiple cell membrane protrusions (membrane filaments, lamellapodia). When further analyzed by immunocytochemistry, the recovered CD45⁺ cells have intracellular Langerin and HLA-DR but lack DC-Lamp, resembling immature LCs. When immune-reconstituted epidermal equivalents are treated for 2-3 days with inflammatory stimuli (e.g. lipopolisaccharide, TNF-α), then the recovered CD45⁺ cells show tremendous cell surface expression of HLA-DR, upregulation of Langerin and uniform expression of intracellular DC-Lamp, resembling mature LCs. Thus, the progeny of CD14⁺ DC precursors present in immune-reconstituted epidermis equivalents as described in this invention show characteristics of LCs and are referred to here as LC-like cells. These LC-like cells are in an immature state, similar to DCs that have not encountered pathogens or inflammatory conditions. However, the LC-like cells within immune-reconstituted epidermis equivalents readily mature in response to inflammatory stimuli. These important data indicate that LC-like cells within immune-reconstituted epidermal equivalents can act as sensors in a novel skin test, by upregulating maturation markers and antigen-presentation function in response to test substances with immunostimulatory properties.

In order to demonstrate that the LC-like cells recovered from epidermis equivalents according to the invention have potent (DC-like) antigen presentation function, mixed leukocyte proliferation assays are performed using MHC mismatched (heterologuos) naϊve CD4⁺ T cells as responder cells. Because of the elevated threshold of activation, naive T cells require antigen presentation by "professional" antigen presenting cells

(APCs), such as DCs and LCs (Lanzavecchia, A. and Sallusto, F., Nat. Immunol. 2:487- 492, 2001). This is in contrast to memory T cells that respond more readily to antigen. As negative and positive control, freshly isolated CD14⁺ DC precursors and mature monocyte-derived DCs from the same donor, respectively, are used as APCs. Of note, mature (LPS/TNF-α-treated) LC-like cells are potent stimulators of naϊve CD4⁺T cells and prominent proliferation responses are already observed at an APC: responder ratio of 1 :1000 (Fig. 9). LC-like cells are less potent than control DCs, which may be explained by the harsh treatment (trypsin digestion) employed for extracting these APCs out of epidermal equivalents. In contrast to LC-like cells, freshly isolated CD14⁺ DC precursors fail to induce substantial proliferation responses in naϊve T cells. In conclusion, the epidermal equivalents according to the invention characterized by co-culture of human epidermal precursor cells with CD14+ human peripheral blood monocytes allow efficient in situ differentiation of CD14⁺ DC precursors into LC-like cells with potent APC function.

It is known that immune-reconstituted epidermis equivalents may be generated by inclusion of treated or untreated CD34⁺ hematopoietic progenitor cells (HPCs) instead of CD14⁺ DC precursors as precursors of immune cells within the epidermis equivalents (EP 0 789 074, Facy, V., et al. J. Invest. Dermatol. 122:552-553, 2004). However, CD34⁺ HPCs give rise to all mature hematopoietic cells present in blood and peripheral tissues, such as skin. The lineage of differentiation (myeloid, lymphoid, etc.) is growth factor dependent, and initial steps in CD34⁺ HPC differentiation occur in the bone marrow.

Further, CD34⁺ HPCs are extremely rare (<1% of mononuclear cells) in peripheral blood, and there is no experimental evidence that CD34⁺ HPCs can reach the epidermis. The state of function and differentiation of CD34⁺ HPC-derived cells present in these epidermis equivalents is not clear. Importantly, it is not clear how much these cells resemble DCs or LCs and whether they are able to induce primary T cell responses. In contrast, the present invention identifies CD14⁺ DC precursors as the immediate precursors of DCs and LCs. It is shown that the progeny cells carry many markers of DCs and LCs and that they have potent antigen-presentation function. CD14⁺ DC precursors are committed to become DCs/LCs or macrophages, i.e. are much further differentiated than CD34⁺ HPCs. Also, they can be recruited by a skin chemokine (CXCL14) to the epidermis in normal human skin. Importantly, CD14⁺ DC precursors are numerous (10-15% of mononuclear cells) in peripheral blood of healthy individuals and, therefore, are readily isolated in high purity for use in the development of immune-reconstituted epidermis equivalents. Collectively, the present invention entails a simple method for the successful generation of immune- reconstituted epidermis equivalents that are reconstituted with functional LC-like cells.

The present invention concerns the generation of immune-reconstituted epidermis equivalents for use in in vitro skin tests. In such a skin test according to the invention, the immune reactivity and skin-damaging properties of potentially harmful substances, for example pharmaceutical compositions, cosmetics, detergents, natural products, and the like, can be tested. Such factors are either proteins or related cleavage products, carbohydrates, lipids, synthetic compounds or a combination of such substances. Such factors also include known and novel allergens, solvents, radiation (UV-light, heat) and pressure. These factors are applied topically onto immune-reconstituted epidermis equivalents, added to the culture medium or added directly to the LC-like cells after their extraction from immune-reconstituted epidermis equivalents. Apart from using the novel immune-reconstituted epidermis equivalent as described herein, the in situ skin test makes use of standard experimental set-ups for assessing in vitro immune activation of leukocytes, notably activation of APCs, such as DCs and LCs. For example, changes in phenotype and localization of LC-like cells can be examined using antibody reagents to detect by flow cytometry or immunocytochemistry cell surface maturation, migration and antigen-presentation molecules.

Parameters, for example those related to DC or LC mobilization and activation (as described above) or those related to tissue integrity (barrier function, cell death, tissue disruption), may be determined, describing the degree of immune-reactivity and skin- damaging properties of individual substances that are being tested. Substances to be tested are added onto the stratum corneum of immune-reconstituted epidermis equivalents or are added into the culture medium. Activity (read-out) assays include but are not limited to the numerical evaluation of the mobilization of LC-like cells within immune-reconstituted epidermis equivalents, by counting the LC-like cells that have emigrated out of immune-reconstituted epidermis equivalents in response to substances to be tested. Alternatively, the degree of maturation in LC-like cells in response to substances to be tested is evaluated by flow cytometry, measuring the reduction or enhanced expression of markers for DC maturation (HLA-DR, CD80, CD86, CD83, CCR7, and the like), in cells recovered from immune-reconstituted epidermis equivalents.

Alternatively, induction of antigen-presentation functions in LC-like cells in response to substances to be tested are examined in in vitro activation, proliferation and differentiation assays, using peripheral blood lymphocytes or cultured T cell lines as responder cells. Alternatively, the effect of substances to be tested on the degree of survival of LC-like cells can be determined by staining for markers of cell death followed by analysis in situ or by flow cytometry. Alternatively, immune-reconstituted epidermis equivalents are grafted onto normal or immunosuppressed (e.g. SCID) mice and substances are tested for their effect on the human graft, e.g. degree of graft rejection, wound healing, and the like.

In particular, the determination of immune reactivity and skin-damaging properties of a substance comprises applying the substance to the stratum corneum or the culture medium of an immune-reconstituted epidermis equivalent according of the invention and determining the mobilization of antigen presenting cells. For this purpose the epidermis equivalent may be disrupted, cells expressing CD45 selected and markers for mobilized antigen presenting cells detected and quantified. Markers for mobilized antigen presenting cells are selected, for example, from CD11a, CD11 b, CD11c, CD18, CD40, CD50, CD54, CD70, CD80, CD83, CD86, DEC205, MMR, and HLA-DR.

Determining the mobilization of antigen presenting cells may be measured by a decrease in residual LC-like cells within the immune-reconstituted epidermis equivalent or by their accumulation in the matrix or medium supporting the immune-reconstituted epidermis equivalent, or, alternatively, by the secretion of cytokines and chemokines by LC-like cells, or by mixed leukocyte responses measuring activation, proliferation or differentiation of naive heterologous T cells in response to recovered LC-like cells.

More specifically, cellular extracts are generated by cutting the immune-reconstituted epidermis equivalents into small pieces, digestion in trypsin/EDTA for a few minutes at around 37⁰C, and mechanical dispersion by vigorous pipetting and/or filtering trough a suitable mesh. LC-like cells are identified in cellular extracts by flow cytometry by gating on viable (propidium iodide-negative) CD45⁺HLA-DR⁺ cells. LC-like cells can be purified by positive selection, e.g. antibody-based magnetic and/or fluorescent cell sorting, or by negative selection (elimination of contaminating cells using antibody-based magnetic and/or fluorescent cell sorting). Cellular extracts containing LC-like cells or purified LC-like cells can be examined for antigen-presentation functions in in vitro cell assays with human naϊve and/or memory T cells or B cells as responder cells. Effects in responder cells to be measured include but are not limited to responder cell proliferation, responder cell differentiation, induction of effector functions (cytokine/chemokine production, target cell lysis), and memory cell formation. Alternatively, LC-like cells within cellular extracts or purified LC-like cells are examined for phenotypic changes directly after processing of immune-reconstituted epidermis equivalents. For instance, phenotypic changes are examined by flow cytometry. APC-related cell surface markers that are modulated during activation include but are not limited to differentiation markers (e.g. CD14, CD1a, CD1c, Langerin, Fcε-Rlα), maturation markers (CD83, CCR7), co-stimulatory markers (e.g. CD40, CD80, CD86) and markers for antigen uptake and processing (e.g. HLA-DR, DC- SIGN, DC-LAMP, Langerin, DEC-205, mannose receptor). Alternatively, the in situ skin test includes immune-reconstituted epidermis equivalents prepared with CD14⁺ DC precursors that were labeled with CFSE or any other fluorescent cell tracking substance. Functional and phenotypic changes in such labeled LC-like cells can be tracked in situ by immunofluorescence or after cell extraction (see above) by flow cytometry in combination with markers for LC/DC activation and differentiation. Alternatively, labeled or unlabeled LC-like cells can also be tested in in vitro migration assays, e.g. a transwell assay. Finally, the effects of substances on LC-like cells within immune-reconstituted epidermis equivalents can be evaluated in isolated LC-like cells in specific (single gene or protein expression) or global gene expression analyses, e.g. genomic and proteomic approaches including but not limited to genechip analysis and fingerprinting of secreted proteins.

The method of the invention is superior to conventional skin tests in animal models in that it reduces the number of animals used in such tests. Furthermore, since the skin test described in the present invention is based on human epidermal cells, including human keratinocytes and human LC-like cells, the results obtained are highly relevant to the potential application of the tested substances in humans.

The invention further relates to a method of treating skin lesions, inflammation and skin diseases caused by pathogens or tumors with human immune-reconstituted epidermis equivalents of the invention. Pathogens causing skin diseases may e.g. be immunostimulatory substances damaging the skin. The method may consist of standard immunotherapy protocols using the immune-reconstituted epidermis equivalents, in particular by applying the epidermis equivalent to skin lesions and wounds under sterile conditions.

The invention furthermore relates to the use of human immune-reconstituted epidermis equivalents of the invention for the preparation of a medicament for the treatment of skin lesions, inflammation and skin diseases caused by pathogens or tumors. The preparation of such medicament follows standard protocols, and is performed under sterile conditions.

Examples

Abbreviations

DC dendritic cell

LC Langerhans cell

HPC hematopoietic progenitor cell

TCR T cell antigen receptor

MHC major histocompatibility complex

APC antigen-presenting cell

FACS fluorescence-activated cell sorter

MFI mean fluorescence intensity

SD standard deviation

CFSE carboxyfluorescein diacetate succinimidyl ester

Example 1 : Cell isolation and generation

1.1 Epidermal precursor cells

Epidermal precursor cells are either isolated from a) skin biopsies or b) hair follicles of healthy individuals. a) First, split skin is digested by dispase Il giving raise to an epidermal sheet which is further trypsinized to release a single cell extract containing epidermal precursor cells.

The extracted cells are either used directly to initiate a culture of an epidermis equivalent or can be propagated first in subcultures with post-mitotic (e.g. irradiated) fibroblasts (e.g. from dermis) as feeder cells or alone in serum-free keratinocytes growth medium (e.g. SFM-K-medium from GibcoBRL, Invitrogen). Single cell extracts containing epidermal precursor cells can also be cryo-preserved (e.g. in RPMI 1640 medium/FCS/DMSO at

5:4:1) for later use. b) Epidermal precursor cells from hair follicles are isolated from primary outer root sheaths cultures following the methods as described (Limat, A. and Hunziker, T., Cells Tissues Organs 172:79-85, 2002). Single cell extracts containing epidermal precursor cells, either from epidermis tissue or outer root sheaths of live hair, can also be cryo-preserved (e.g. in

RPMI 1640 medium/FCS/DMSO at 5:4:1) for later use.

1.2 CDU^* DC precursors Human peripheral blood mononuclear cells (PBMCs) are isolated from heparin-treated donor blood buffy coats or fresh blood by Ficoll-Paque centrifugation according to standard protocols. CD14⁺ DC precursors are isolated from PBMCs by positive selection of CD14⁺ cells by anti-CD14 antibody-based magnetic and/or fluorescent cell sorting or by negative depletion (elimination of cells not expressing CD14⁺ using antibody-based magnetic and/or fluorescent cell sorting. Large numbers (>10⁶ cells) of high purity (>98% CD14⁺ cells) monocytes are routinely obtained, and show characteristic flow cytometric features of CD14⁺ DC precursors, i.e. CD14^hi9h, HLA-DR⁺, and small size (low forward (FSC) and side scatter (SSC) values).

CD14⁺ DC precursors, generated either from CD34⁺ hematopoietic progenitor cells or isolated from human peripheral blood, are also functionally characterized by their responsiveness to the chemokine CXCL14. CXCL14-induced responses measured in in vitro assays include but are not limited to chemotactic migration and Ca²⁺ mobilization in CD14⁺ DC precursors, as recently described (Kurth, I., et al., J. Exp. Med. 194:855-861 , 2001). CXCL14 is either prepared by chemical synthesis (Kurth, I., et al., loc. cit.), bought from commercial sources or isolated by sequential chromatography from natural sources, including supernatant of keratinocyte cultures or CXCL14-DNA-transfected cell lines. Antibodies to CXCL14 are from commercial sources.

1.3 Dermal fibroblasts Primary dermal fibroblast lines are preferentially generated in "F medium" (see Example 2) from cultures of adherent dermal cells which derive from digestion of split skin by 0.2 U/ml collagenase D and 1.25 U/ml dispase II. Alternatively, immortalized fibroblast lines can be used (e.g. by spontaneous immortalization or transfection of oncogenes / tumor suppressor genes), and fibroblasts can also be derived from other human tissues.

Example 2: Generation of immune-reconstituted epidermis equivalents

2.1 Cell culture media

"RPMI medium" consists of RPMI 1640 plus 2 mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin, 5 x 10^"5 M 2-ME (all

Invitrogen, Basel, Switzerland) and 10% FCS (Biological Ind., Israel). "DMEM medium" consists of DMEM (high glucose; Invitrogen) plus 10% FCS, 2 mM L-glutamine, 1% nonessential amino acids, 50 μg/ml penicillin/streptomycin, 5 x 10^"5 M 2-ME. "F medium" consists of 3 parts DMEM and 1 part Ham's F12 (PAA Labs, Austria) plus 20 mM HEPES, 0.135 mM adenine (Sigma, MO), 5 μg/ml insulin (Invitrogen), 10 ng/ml EGF, 0.4 μg/ml hydrocortisone (Sigma), 2 nM triiodothyronine (Sigma), 100 U/ml penicillin, 100 μg /ml streptomycin (Invitrogen), 10% FCS. "K medium" is "F medium" plus 10 ng/ml cholera toxin (Sigma), "K+ medium" is "K medium" plus 50 μg/ml L-ascorbic acid (Sigma). "RPMI/F12 medium" consists of 3 parts "RPMI medium" and 1 part Ham's F12 plus 20 ng/ml EGF, 5 mM HEPES and 3.5 mg/ml D-glucose.

2.2 Epidermis equivalents

200'0OO epidermal precursor cells are seeded in 500 μl of K medium into a cell culture insert (3460; Corning Costar) carrying 200'0OO irradiated (70 Gy) dermal fibroblasts underneath. After 2 - 4 days, the insert is air lifted and "K+ medium" is used to obtain fully stratified epidermis equivalents after 2 - 3 weeks which can be used in "sandwich cultures", where an intact epidermis equivalent is lifted with forceps and transferred onto a cell culture insert with irradiated dermal fibroblasts underneath and with 200'0OO CD14⁺ DC precursors on top. Thus, the CD14⁺ DC precursors are sandwiched between a fully developed epidermis equivalent and a membrane of the cell culture insert. Alternatively, epidermal precursor cells, either from epidermal tissue or outer root sheaths of live hair, are cultured on cell culture inserts with irradiated dermal fibroblasts underneath in "K+ medium" until confluency (approximately 4 days if 200'0OO precursors/well were seeded initially). After washing of epidermal precursor cell-derived monolayers several times with RPMI 1640, the cultured epidermal cells are overlaid with 200'0OO freshly isolated CD14⁺ DC precursors and further cultured for 2 hours in "RPMI/F12" medium. Then, all medium in the upper well is carefully removed and 1 ml "RPMI/F12" in the lower well is replaced every day until full development of immune-reconstituted epidermis equivalents.

2.3 Flow cytometry

Flow cytometric analysis was done by gating on live (propidium iodide-negative) CD45⁺HLA-DR⁺ cells. CD45 is an established marker for leukocytes and is not expressed on keratinocytes and therefore allows the identification of co-cultured HLA-DR⁺ leukocytes in the heterogeneous cell suspension. To track leukocytes in epidermis equivalents, CD14⁺ DC precursors were labeled in PBS containing 0.5-5 μM CFSE (Molecular Probes, Leiden, The Netherlands) and 5% FCS for 4 min at room temperature. Example 3: Analysis of LC-like cells

Functional readouts determining the effect of substances to be tested on LC-like cells within immune-reconstituted epidermal equivalents are numerous and include (but are not limited to) assays listed below.

1) Isolation.

Cellular extracts are generated by cutting the immune-reconstituted epidermis equivalents into small pieces, digestion in PBS/trypsin/10mM EDTA for 5-30 min at 37°C, mechanical dispersion by vigorous pipetting and filtering trough a 70 μm pore mesh. LC-like cells are identified in cellular extracts by flow cytometry by gating on viable (propidium iodide- negative), CD45⁺HLA-DR⁺ cells. LC-like cells can be purified by positive or negative selection, or by collecting migratory LC-like cells. Positive selection of e.g. CD45⁺ cells can be achieved by using magnetically labeled antibodies to CD45 (e.g. MACS system from Miltenyi Biotech, Germany) and/or by cell sorting using fluorescently labeled antibodies to CD45. Negative selection is achieved by depletion of all non-leukocytes, using antibodies against keratinocytes- and other tissue cell-specific proteins.

2) Immunohistochemistry. Frozen or paraffin-embedded sections of immune-reconstituted epidermis equivalents are stained with a selection of antibodies that may include antibodies against DC/LC differentiation markers (e.g. CD14, CD1a, CD1c, Langerin, Fc-epsilon-RI-alfa, DC-SIGN, E-Cadherin, integrins) and activation/maturation markers (e.g. CD83, DC-LAMP, CCR5, CCR6, CCR7, HLA-DR, CD40, CD80, CD86, Toll-like Receptors, chemokines, cytokines such as IL-12, interferons, TGF-beta, IL-10, IL-13, IL-4).

3) Phenotypic analysis of isolated LC-like cells after application (e.g. topical or in solution) of substances to be tested.

LC-like cells can be analyzed by flow cytometry for their size and granularity (forward and side scatter profiles) and their expression of differentiation markers (e.g. CD14, CD1a,

CD1c, Langerin, Fc-epsilon-RI-alfa, DC-SIGN, E-Cadherin, integrins) and activation/maturation markers (e.g. CD83, DC-LAMP, CCR5, CCR6, CCR7, HLA-DR,

CD40, CD80, CD86, Toll-like Receptors, chemokines, cytokines).

The morphologic and ultrastructural phenotype of LC-like cells can be examined in cytospin preparations containing LC-like cells. In addition to positive or negative selection (see above), LC-like cells can be obtained by collecting cells that have migrated out of immune-reconstituted epidermis equivalents in response to activation by substances to be tested, whereby kinetics and efficiency of migration is dependent on substances to be tested. Similarly, the influence of substances on the migratory capacity of LC-like cells to chemokines, e.g. CCL19 or CCL21 , can be tested in this way.

4) Functional analysis of isolated LC-like cells after application of substances to be tested. The immuno-stimulatory and -regulatory function of LC-like cells after application of substances can be tested in co-cultures of isolated LC-like cells with naive or memory T cells, either autologous or heterologous, by measuring T cell activation by flow cytometry (e.g. CD25, CD69), proliferation (e.g. ³H-thymidine incorporation or CFSE dilution or cell count), differentiation/polarization (e.g. cytokine [IL-4 and IFN-gamma, among others] mRNA or protein production, chemokine receptor and integrin expression, regulation of gene expression by gene chip analysis), as well as survival and cytolytic activity in co- cultured T cells (e.g. chromium release assay).

Collectively, the evaluation of functions and phenotypic changes in T cells in response to stimulation by isolated LC-like cells after application of substances to be tested follow standard protocols used in current immunologic research.

Claims

1. A human immune-reconstituted epidermis equivalent comprising a support, progeny of cultured epidermis precursor cells from skin biopsies or outer root sheaths of hair follicles, and progeny of co-cultured human peripheral blood mononuclear cells originally expressing CD14.

2. The epidermis equivalent according to claim 1 wherein the epidermis precursor cells are cells from outer root sheaths of hair follicles.

3. The epidermis equivalent according to claim 1 wherein the progeny of human peripheral blood mononuclear cells originally expressing CD14 has been co-cultured for 5 to 30 days.

4. The epidermis equivalent according to claim 1 wherein the progeny of co-cultured cells originally expressing CD14 expresses HLA-DR, CD80, CD86, CD205 and DC-SIGN.

5. A method of preparation of a human immune-reconstituted epidermis equivalent comprising a) culturing epidermis precursor cells from skin biopsies or outer root sheaths of hair follicles and b) co-culturing progeny from step a) with human peripheral blood mononuclear cells expressing CD14 on a support.

6. The method of claim 5 wherein in step a) epidermis precursor cells from outer root sheaths of hair follicles are used.

7. The method of claim 5 wherein in step b) co-culturing is for 5 to 30 days.

8. The method of claim 5 comprising a) culturing epidermis precursor cells from outer root sheaths of hair follicles on a solid support with irradiated dermal fibroblasts, and b) placing the epidermal equivalent from step a) on top of human peripheral blood mononuclear cells expressing CD14 on a solid support with irradiated dermal fibroblasts and co-culturing for 5 to 30 days.

9. The method of claim 5 wherein the human peripheral blood mononuclear cells expressing CD14 are freshly isolated monocytes characterized by the cell surface expression of CD14.

10. The method of claim 5 wherein the human peripheral blood mononuclear cells expressing CD14 are in vitro stimulated monocytes characterized by the cell surface expression of CD14.

11. The method of claim 10 wherein in vitro stimulated monocytes are freshly isolated monocytes treated with stimuli selected from bacterial products, cytokines, mitogens, and arachidonic acid metabolites, either alone or in combination, before use.

12. A method for the determination of immune reactivity and skin-damaging properties of a substance comprising applying the substance to the stratum corneum or the culture medium of an immune-reconstituted epidermis equivalent according to claim 1 and determining the mobilization of antigen presenting cells.

13. The method according to claim 12 wherein for determining the mobilization of antigen presenting cells the immune-reconstituted epidermis equivalent is disrupted, cells expressing CD45 are selected and markers for mobilized antigen presenting cells are detected and quantified.

14. The method according to claim 13 wherein markers for mobilized antigen presenting cells are selected from CD11a, CD11 b, CD11c, CD18, CD40, CD50, CD54, CD70, CD80, CD83, CD86, DEC205, MMR, and HLA-DR.

15. The method according to claim 12 wherein determining the mobilization of antigen presenting cells is measured by a decrease in residual Langerhans cell-like cells within the immune-reconstituted epidermis equivalent or by their accumulation in the matrix or medium supporting the immune-reconstituted epidermis equivalent.

16. The method according to claim 12 wherein determining the mobilization of antigen presenting cells is measured by the secretion of cytokines and chemokines by

Langerhans cell-like cells.

17. The method according to claim 12 wherein determining the mobilization of antigen presenting cells is measured by mixed leukocyte responses measuring activation, proliferation or differentiation of naive heterologous T cells in response to recovered Langerhans cell-like cells.

18. A method of treating skin lesions, inflammation and skin diseases caused by pathogens or tumors in a patient, characterized in that human immune-reconstituted epidermis equivalent according to claim 1 is applied to the patient in need thereof in an effective amount.

19. The use of human immune-reconstituted epidermis equivalent according to claim 1 for the preparation of a medicament for the treatment of skin lesions, inflammation and skin diseases caused by pathogens or tumors.