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The Journal of Immunology, 2000, 165: 645-653.
Copyright © 2000 by The American Association of Immunologists

Langerhans Cells That Have Matured In Vivo in the Absence of T Cells Are Fully Capable of Inducing a Helper CD4 as Well as a Cytotoxic CD8 Response1

An De Creus, Katrien Van Beneden, Tom Taghon, Frank Stolz, Veronique Debacker, Jean Plum and Georges Leclercq2

Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University Hospital, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Langerhans cells (LCs) are immature dendritic cells (DCs) present in the skin epithelium. Upon Ag exposure, they migrate to the draining lymph nodes where they mature into potent stimulators of naive T cells. The aim of this study was to investigate the influence of T cells on LC migration and maturation. Therefore, the in vivo migration and maturation of LCs after sensitization with the hapten FITC was compared between C57BL/6 or BALB/c mice used as positive controls, and recombination activating gene (RAG) 1 knockout (-/-) mice or SCID mice used as T cell-deficient mice. Phenotypically, there was no difference between migrated LCs from RAG1-/- or SCID mice vs normal C57BL/6 or BALB/c mice: both populations of FITC+ cells had a dendritic morphology and a mature phenotype as they expressed high levels of MHC class II molecules and costimulatory molecules CD80, CD86, and CD54. Sorted migrated LCs of RAG1-/- or SCID mice were efficient stimulators of allogeneic T cells and Ag-specific CD4+ T cells. The same results were found if migrated LCs were fixed instead of irradiated, excluding the possibility that LCs derived from RAG1-/- or SCID mice would mature in the presence of T cells during the stimulation tests. Importantly, fixed migrated LCs of RAG1-/- mice were also efficient stimulators of cytotoxic CD8+ T cells. These data suggest that T cells are not required for full maturation of LCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs)3 are professional APCs capable of initiating primary T cell-mediated immune responses to a variety of Ags, including bacterial, viral and tumor-associated Ags. DCs originate from a hemopoietic progenitor and probably migrate as a precursor through the blood stream to most nonlymphoid tissues in the periphery where they reside as so-called immature DCs. Immature DCs are specialized in capturing and processing Ag, but they need to mature and migrate to the draining lymph nodes before acquiring the capacity to stimulate naive T cells (1). Upon Ag exposure, immature DCs migrate to the T cell areas of draining lymph nodes, where they may complete their maturation (2, 3, 4, 5). Mature DCs have lost their ability to pick-up and process Ags (6), express high levels of MHC class I and class II, CD80, CD86, and CD40 (7), and are capable to stimulate T cells (8, 9). DCs deliver through CD80 and CD86, and the secretion of T cell immunostimulating cytokines, activating signals to T cells (10, 11).

DCs present in nonlymphoid tissues can be induced to migrate in vivo upon systemic administration of bacterial components like LPS and inflammatory mediators like IL-1 and TNF-{alpha} (12). Different types of stimuli can lead to DC maturation, like danger signals, LPS (13), and inflammatory cytokines such as IL-1 and TNF-{alpha} (14). Indirect evidence derived from in vitro studies suggests that DCs are also susceptible to signals derived from T cells. These signals induce the up-regulation of costimulatory molecules and increase survival and cytokine secretion. CD40 triggering on DCs through CD40 ligand (CD40L), present on activated CD4+ T cells, is superior at inducing IL-12 secretion and at enhancing Ag-presenting capacities of DCs compared with inflammatory mediators such as LPS or TNF-{alpha}. It has been suggested that the interaction between CD40 and CD40L is necessary to induce final maturation of DCs (8, 15). The importance of CD40 triggering on DCs by CD40L expressed on T cells has been shown in several in vivo models (16, 17, 18). Also, TNF-related activation-induced cytokine (TRANCE), expressed on activated T cells, shows a similar function as CD40L in enhancing the survival and secretion of IL-12 by DCs (19, 20).

Langerhans cells (LCs) are DCs located in the skin epithelium. Due to their presence in epithelia interfacing the environment, LCs are challenged with invading pathogens. It has been proposed that LCs represent a lineage different from myeloid DCs present in the dermis, lymph nodes, and spleen, and lymphoid DCs present in the spleen and thymus (21). Evidence for this difference between LC and other DC subsets in mice relies on the study of different knockout mice. RelB knockout mice develop the lymphoid-related subset of DCs and normal LCs in the skin but not the myeloid-related subset of DCs (22). In Ikaros dominant-negative knockout mice, no DC subset could be found in lymphoid organs, but LCs were still present in the skin (23). TGF-ß is required for the development of LCs but not for the other DC populations. This has been clearly demonstrated in TGF-ß knockout mice, which are devoid of LCs in the skin (24).

Keratinocytes, which are the major cell population of the epidermis, probably have an important role in the migration and/or maturation of LCs through the secretion of cytokines. Stressed keratinocytes or hapten-treated keratinocytes secrete large amounts of TNF-{alpha} and GM-CSF, cytokines known to influence LC function (25). In vitro studies have also demonstrated a possible role for T cells in inducing the final maturation of LCs. Murine epidermis-derived DC lines have been shown to undergo final maturation upon interacting with T cells, which the authors referred to as T cell-mediated terminal maturation of DCs (26, 27). Geismann et al. have shown that TGF-ß1 prevents the final maturation of in vitro-derived LCs in response to nonspecific inflammatory signals such as LPS, TNF-{alpha}, and IL-1, while the CD40L signal or the presence of T cells could induce their full maturation (28).

The aim of this work was to study if T cells are necessary for the in vivo migration and in particular the maturation of LCs. Therefore, we studied LC migration and maturation in T cell-deficient mice. We compared migration and maturation of LCs in vivo between wild-type (WT) C57BL/6 or BALB/c mice used as controls and recombination activating gene (RAG) 1 knockout or SCID mice used as T cell-deficient mice after cutaneous application of the hapten FITC. It has been clearly demonstrated that after cutaneous application of a hapten, hapten-modified proteins are loaded onto LCs that migrate from the epidermis to the draining lymph nodes (29), where priming of hapten-specific T cells occurs (30). RAG1 plays an essential role in TCR as well as in Ig gene rearrangements (31). Also, SCID mice have a defect in TCR and Ig gene rearrangements due to a mutation in a component of the recombinase complex (32). Therefore, RAG1-/- and SCID mice have a defect in functional T and B cell development. It is shown here that LCs of T cell-deficient mice migrated normally to the draining lymph nodes, displayed a mature phenotype, and were potent stimulators of allogeneic and syngeneic T cells, Ag-specific CD4+ T cells, and, importantly, also of cytotoxic CD8+ T cells. Our results suggest that LCs do not need to receive a signal from T cells to become fully mature DCs capable of stimulating CD4+ T cells and cytotoxic CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

BALB/c mice, SCID mice (BALB/c background) and C57BL/6J (B6) mice were provided by Proefdierencentrum (Catholic University Leuven, Leuven, Belgium) and Harlan Netherlands (Zeist, The Netherlands), respectively. Mice were bred in our breeding facility.

RAG1 knockout (-/-) mice (C57BL/6 background) were purchased from Nederlands Kankerinstituut (Amsterdam, The Netherlands) and The Jackson Laboratory (C57BL/6J-Rag1tm1 Mom) (Bar Harbor, ME). Mice were housed in a specific pathogen-free environment. D011.10 x BALB/c mice were provided by Dr. M. Moser (Université dibre de Bruxelles, Rhode-Saint-Genèse, Belgium).

Mice were treated and used in agreement with the institutional guidelines.

Antibodies

Monoclonal Abs used for staining were anti-Fc{gamma}RII/III (unconjugated, clone 2.4G2, rat IgG2b) (kindly provided by Dr. J. Unkeless, Mount Sinai School of Medicine, New York, NY), anti I-Ab/d (biotin-conjugated, clone 25-917; mouse IgG2a; PharMingen, San Diego, CA), anti-DEC-205 (unconjugated, rat Ig, Harlan), anti-CD80 (unconjugated, hamster Ig), anti-CD86 (unconjugated, rat Ig), anti-CD54 (unconjugated, rat Ig), anti-CD45 (PE-conjugated; clone 30-F11; rat IgG2b,{kappa}; PharMingen), anti-CD11c (unconjugated, clone N418, hamster Ig) (kindly provided by Dr. M. Moser), anti-heat stable Ag (biotin-conjugated, rat IgG2b, clone M1/69; PharMingen), pan mouse NK mAb (clone DX5; PharMingen), hamster anti-mouse TCR {alpha}ß mAb (biotin-conjugated, clone H57–597), goat F(ab')2 anti-rat IgG (PE-conjugated; Tago, Camarillo, CA), goat F(ab')2 anti-hamster IgG (PE-conjugated; Caltag Laboratories, Burlingame, CA), and goat anti-mouse IgG (FITC-conjugated; Tago).

In vivo sensitization

Mice were sensitized by painting their shaved dorsal abdomens with 150 µl of 0.5% FITC (isomer I; Molecular Probes, Leiden, The Netherlands) dissolved in DMSO:acetone:dibutylphtalate (1:4.5:4.5). Twenty-four hours later, draining lymph nodes (inguinal, axillary, brachial) were removed.

Preparation of epidermal cell suspension

Skin samples were freed of fatty tissue and were floated dermal side down in a petri dish containing 0.3% trypsin-PBS solution (Difco, Detroit, MI) for 18 h at 4°C. Epidermal sheets were peeled from the underlying dermis. Epidermal skin samples were then pooled in DMEM (Life Technologogies, Paisly, U.K.) containing 0.25% DNase (Boehringer Mannheim, Mannheim, Germany). Single-cell suspensions were obtained by repeated pipetting of the skin samples. Resulting cell suspensions were filtered through a cell strainer (70 µm; Falcon, Lincoln Park, NJ) to remove hair and debris, washed three times, and resuspended in DMEM containing 0.01% DNase (Boehringer Mannheim). Cell suspensions were then applied onto a density gradient (lymphoprep; Nycomed, Oslo, Norway) and centrifuged for 15 min at 2310 rpm. This procedure removed dead cells and part of the keratinocytes and resulted in a viable LC-enriched cell suspension. Cells were resuspended in complete RPMI 1640 medium. The complete medium used was RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.03% glutamin, and 5 x 10-5 2-ME (all from Life Technologogies).

Migratory cells from skin explants

Migratory cells were obtained from ear halves of WT C57BL/6 and RAG1-/- mice that were floated dermal side down on 2 ml of complete RPMI 1640 medium in 24-well plates at 37°C. Three days after culture, migratory cells were gently resuspended, pelleted, and counted in trypan blue before use. For each experiment, migratory cells derived from ear halves of four mice were pooled.

Determination of LC densities in epidermal sheets

Skin samples were freed of fatty tissue and were floated dermal side down in a petri dish containing 3.8% ammonium thiocyanate for 20 min at 37°C. Epidermal sheets were peeled from the underlying dermis and fixed in acetone for 15 min at 4°C. Subsequently, epidermal sheets were washed in PBS and labeled with anti-DEC-205 mAb revealed with anti-rat-FITC or with biotin-conjugated anti-Ia mAb revealed with streptavidin-FITC. LCs were counted with a fluorescence microscope in a field that equaled 0.25 mm2. For each group of RAG1-/- and WT C57BL/6 mice, 30 random fields were counted. Results are expressed as mean ± SD.

Preparation of cell suspensions

Draining lymph nodes were pooled from two to six FITC-sensitized mice. A cell suspension was prepared as described (21). Briefly, tissue was cut into small fragments, suspended in 5 ml of RPMI 1640 medium (Life Technologies) supplemented with 2% FCS (Life Technologies), collagenase (1 mg/ml; type II; Worthington Biochemical, Freehold, NJ), and DNase (0.02 mg/ml; Boehringer Mannheim) and digested with intermittent agitation for 25 min at 37°C. Then, 1 ml 0.1 M EDTA, pH 7.2, was added to the digest, and incubation with agitation was continued for 5 min.

T cell enrichment using nylon wool columns

Lymph node cell suspensions of untreated mice were depleted of RBCs by NH4Cl treatment and diluted in warm (37°C) complete RPMI 1640 medium to a density of <=7.5 x 106/ml. The cell suspension was added to a nylon wool column. After complete draining, the column was incubated in an upright position in a 37°C, 5% CO2 humidified incubator for 45 min. Subsequently, the column was filled with 37°C complete RPMI 1640 medium, and nonadherent cells were collected (33). A total of 85% of nylon wool nonadherent cells were T cells as confirmed by flow cytometric (FCM) analysis.

FCM analysis and sorting

Draining lymph node cells from FITC-sensitized mice were incubated with anti-CD80, anti-CD86, anti-CD11c, anti-CD45, or anti-CD54 (ICAM-1) mAbs at 4°C for 45 min. After washing, cells were incubated with PE-conjugated anti-hamster Ig or PE-conjugated anti-rat Ig at 4°C for 45 min. Subsequently, 10% normal hamster or rat serum was added. After 15 min, cells were labeled with biotin-conjugated I-Ab/d mAb. After 45 min, cells were washed, followed by labeling with streptavidin-APC (Becton Dickinson). Cells were analyzed for fluorescence using a FACScalibur (Becton Dickinson) equipped with an argon laser (488 nm) and a helium neon laser (540 nm) with the CellQuest software program (Becton Dickinson) for data acquisition and analysis. Propidium iodide was added to the cells (2 µg/ml) just before FCM analysis. Gating was done on propidium iodide-negative cells to exclude dead cells.

Migrated FITC+ DCs were sorted from the draining lymph nodes of FITC-sensitized mice to a purity of >99% using a FACS Vantage flow cytometer (Becton Dickinson) equipped with an argon laser.

{alpha}ß T cells were sorted from the lymph nodes of untreated BALB/c mice. Lymph node cells were first labeled at 4°C with FITC-conjugated anti-mouse Ig, then 45 min later cells were washed and labeled with biotin-conjugated I-Ab/d mAb, biotin-conjugated anti-heat stable Ag mAb, and PE-labeled pan mouse NK mAb at 4°C for 45 min. After washing, cells were incubated with streptavidin-PE (Becton Dickinson). FITC- and PE-negative cells were sorted using a FACS Vantage (Becton Dickinson) to a purity of >99.5%. More than 99% of these sorted cells were TCR {alpha}ß+ as confirmed by FCM analysis using biotin-conjugated anti-TCR {alpha}ß mAb followed by labeling with streptavidin-PE.

In vitro assays

Allogeneic stimulation. A total of 250,000 sorted {alpha}ß T cells from untreated BALB/c mice were stimulated with graded numbers of mitomycin-treated migratory cells derived from explants from WT C57BL/6 and RAG1-/- mice or sorted gamma-irradiated (3000 rad) or fixed (in PBS with 1% paraformaldehyde, 20 min at room temperature) FITC+ DCs from the draining lymph nodes of FITC-sensitized WT C57BL/6 or RAG1-/- mice or with total lymph node cells from unsensitized C57BL/6 mice in round-bottom 96-well plates (Falcon; Becton Dickinson). Cultures were maintained at 37°C in a humidified incubator (5% CO2). After 3 days of culture, 1 µCi [3H]thymidine was added. [3H]Thymidine incorporation was determined after 16 h using a 96-well scintillation counter (Microbeta; Wallac, Turku, Finland).

Staphylococcal enterotoxin B (SEB) stimulation. A total of 200,000 nylon wool-purified syngeneic T cells were stimulated with graded doses of SEB in the presence of 8500 sorted gamma-irradiated (3000 rad) or fixed (1% paraformaldehyde) FITC+ DCs from the draining lymph nodes of FITC-sensitized WT C57BL/6 mice used as control mice or RAG1-/- mice. Cultures were maintained at 37°C in a humidified incubator with 5% CO2. After 24 h, supernatant was removed and added to 20,000 CTLL-2 cells. Twenty-four hours later, 1 µCi [3H]thymidine was added to the culture. [3H]Thymidine incorporation was counted after 6 h.

OVA-specific CD4+ T cell stimulation. OVA-specific TCR transgenic CD4+ T cells were purified from the lymph nodes of D011.10 x BALB/c transgenic mice. Therefore, lymph node cells were labeled with biotin-conjugated I-Ab/d mAb and biotin-conjugated heat stable Ag mAb for 45 min at 4°C. After washing, M-450 Dynabeads conjugated with streptavidin and sheep anti-mouse-IgG beads (Dynal, Olso, Norway) were added. Cells were incubated at 4°C with occasional shaking for 30 min. Non-T cells were depleted using a magnetic particle concentrater (Dynal). FITC+ DCs sorted from the draining lymph nodes of FITC-sensitized WT BALB/c mice, used as control, and SCID mice were pulsed in RPMI 1640 medium without FCS with 30 µg/ml OVA peptide specific for MHC class II I-Ad (KISQAVHAAHAEINEAG, synthesized by Prof. J. Vandekerckhove, Rÿhr Universiteit, Ghent, Belgium) or with 1 mg/ml OVA protein (Sigma, St. Louis, MO) for 2 h at 37°C. Thereafter, DCs were fixed with 1% paraformaldehyde. Then, 100,000 sorted OVA-specific CD4+ transgenic T cells were cultured with graded numbers of sorted fixed FITC+ DCs pulsed with peptide or protein in round-bottom 96-well plates (Falcon; Becton Dickinson). Cultures were maintained at 37°C in a humidified incubator (5% CO2) for 3 days. During the last 16 h of culture, [3H] thymidine was added. [3H]Thymidine incorporation was counted.

Cytotoxic assay

The tumor target used was the EL-4 cell line (H-2Kb, Db). Lymphoblast targets used were Con A-activated splenocytes from BALB/c mice or C57BL/6 mice. To generate lymphoblasts, splenocytes were cultured for 48 h in complete RPMI 1640 medium supplemented with 3 µg/ml Con A. Target cells were labeled with 100 µCi 51Cr (Amersham International, Buckinghamshire, U.K.) for 60 min at 37°C. Cells were washed three times. Effector cells used were sorted {alpha}ß T cells from lymph nodes of untreated BALB/c mice stimulated for 5 days with sorted FITC+ DCs from the draining lymph nodes of FITC-sensitized WT C57BL/6 used as control mice or RAG1-/- C57BL/6 mice. Graded effector cell numbers were cocultured in triplicate with 1000 51Cr-labeled EL4 cells or 2000 51Cr-labeled lymphoblasts in a volume of 100 µl in 96-well V-bottom plates (Nunc, Roskilde, Denmark). Alternatively, to determine the spontaneous and maximal 51Cr release, medium and 2% Triton X-100 solution, respectively, was added to the target cells instead of effector cells. After incubation for 4 h at 37°C, 70 µl supernatant was removed from each well. Then, 225 µl Optiphase Supermix (Wallac) was added to the supernatants, and radioactivity was measured using a 96-well scintillation counter (Microbeta; Wallac).

Data are expressed as the mean percent specific 51Cr release. Percent specific release was calculated as follows: 100 x [(experimental - spontaneous release)/(maximal - spontaneous release)].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic analysis of LCs in the skin of normal and RAG1-/- mice

The aim of this study was to examine whether T cells are required for the migration and maturation of skin-located LCs. As an experimental in vivo model, we chose to sensitize mice by application of the hapten FITC on the skin, which induces migration of LCs. RAG1-/- mice were used as T cell-deficient mice; WT C57BL/6 mice were used as normal controls. We first examined whether LCs are present at normal numbers in the epidermis of RAG1-/- mice and if they have the same immature phenotype as in normal C57BL/6 mice. The presence of LCs in epidermal sheets was visualized by immunofluorescent staining using a donor-specific anti-Ia mAb and anti-DEC-205 mAb specific for DCs (Fig. 1Go). Normal numbers of epidermal LCs were found in the skin of RAG1-/- mice (452 ± 12 LCs/mm2) compared with normal C57BL/6 mice (448 ± 19 LCs/mm2).



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  		FIGURE 1. RAG-/- mice have normal numbers of epidermal LCs. Epidermal LCs of WT and RAG-/- mice were visualized by immunolabeling using anti-I-Ab/d mAb (biotin-conjugated revealed with streptavidin-FITC) or anti-DEC-205 mAb (unconjugated revealed with anti-rat-FITC). Results are representative for three experiments.

 
Immature LCs in the skin of normal mice express low levels of costimulatory molecules. FCM analysis of freshly isolated epidermal cells showed that LCs from RAG1-/- mice expressed similar low levels of costimulatory molecules CD80 (B7.1), CD86 (B7.2), and CD54 (ICAM-1) as compared with LCs of normal mice (Fig. 2Go). As expected, both LC populations were positive for CD45. Histograms show a higher percentage MHC class II-positive cells in the skin of RAG1-/- mice. However, the total cell number of freshly isolated epidermal cells was higher for WT mice as compared with RAG1-/- mice, probably due to the absence of DETC in the skin of RAG1-/- mice. The final result was that the calculated absolute cell numbers of class II-positive cells were comparable in the epidermal cell suspensions of WT and RAG1-/- mice.



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  		FIGURE 2. Phenotypical analysis of LCs present in the skin of WT C57BL/6 and RAG1-/- mice. Epidermal cell suspensions were prepared from untreated WT C57BL/6 mice (left) or RAG1-/- mice (right). The mAbs used for labeling were anti-I-Ab/d (biotin-conjugated revealed with streptavidin-APC), anti-CD80 (unlabeled, anti-hamster-PE), anti-CD86 (unlabeled, anti-rat-PE), anti-CD54 (unlabeled, anti-rat-PE), or anti-CD45 (PE-conjugated). Propidium iodide was added just before analysis. Gating was done on propidium iodide-negative (upper two histograms) or on propidium iodide-negative and MHC class II-positive cells (other histograms). The white histogram represents the background staining; the black histogram represents staining by the indicated mAb. Results are representative for more than three experiments.

 
Normal migration and functional maturation of epidermal LCs from skin explants of RAG1-/- mice

To determine whether LCs from RAG1-/- mice could migrate out of the skin, skin explants of RAG1-/- mice were used as an in vitro model. Ear skin of these mice was floated directly on medium in 24-well plates. Three days later, culture medium was assessed for the presence of migrated LCs. As a positive control, we used ear skin from WT C57BL/6 mice. The presence of migratory LCs was shown by FCM analysis. A total of 36% of the migrated cells from C57BL/6 mice expressed high levels of MHC class II compared with 51% of the migrated cells from RAG1-/- mice (Fig. 3Go). This corresponded to 17,930 ± 4,070 LCs migrated from one ear of a WT C57BL/6 mice and 24,000 ± 1,500 LCs migrated from one ear of a RAG1-/- mice.



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  		FIGURE 3. Phenotypical analysis of migratory cells from explants of WT and RAG1-/- mice. Ear halves of WT and RAG-/- mice were floated on complete medium for 3 days. Migratory cells were analyzed for the expression of MHC class II. The mAb used for labeling was anti-I-Ab/d (biotin-conjugated revealed with streptavidin-APC). Propidium iodide was added just before analysis. Gating was done on propidium iodide-negative cells. The white histogram represents the background staining; the black histogram represents the MHC class II staining. Results are representative for three experiments.

 
To determine whether functional maturation of epidermal LCs derived from RAG1-/- skin explants occurred during culture, migratory cells from 3-day skin explants of WT C57BL/6 and RAG1-/- mice were assessed for the ability to stimulate allogeneic T cells. Migratory cells from RAG1-/- mice were slightly more efficient as similar cells from WT C57BL/6 mice in stimulating allogeneic T cells (Fig. 4Go). This difference in stimulation capacity between migratory cells from RAG1-/- mice and WT C57BL/6 mice is probably due to the difference in percentage of LCs present (see Fig. 3Go).



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  		FIGURE 4. Functional maturation of migratory LCs from skin explants of WT and RAG1-/- mice. Migratory cells from skin explants of WT C57BL/6 mice and RAG-/- mice were tested for their ability to stimulate allogeneic T cells. Graded numbers of irradiated migratory cells of WT or RAG1-/- mice were cocultured with 200,000 allogeneic T cells. After 3 days of culture, [3H]thymidine was added. [3H]Thymidine incorporation was measured after another 16 h. Results are representative for three experiments and expressed as the mean ± SD.

 
Normal migration and phenotypic maturation of LCs in vivo after hapten sensitization of RAG1-/- mice.

Previous results demonstrated that after application of the hapten FITC on the abdomen of mice, LCs acquire FITC and migrate to the draining lymph nodes (2, 29). We examined if DCs also appeared in the draining lymph nodes of RAG1-/- mice after FITC sensitization. As a positive control, we used WT C57BL/6 mice. Twenty-four hours after FITC sensitization, draining lymph nodes were pooled and cell suspensions were prepared. The presence of bright FITC+ cells was shown by FCM analysis for both normal C57BL/6 mice and RAG1-/- mice. By multiplying the percentage of FITC+ cells by the total lymph node cell number, we found that 128,000 and 78,000 cells were bright FITC+ in the lymph nodes of sensitized normal and RAG1-/- mice, respectively. To confirm that the cells found in the draining lymph nodes indeed belong to the LC-DC lineage, FITC+ cells were sorted. Cytospins showed that bright FITC+ cells of WT C57BL/6 mice as well as of RAG1-/- mice indeed had a dendritic morphology (data not shown).

To study if bright FITC+ cells in the draining lymph nodes were indeed migratory LCs and were not due to leakage of the hapten, we examined the draining lymph nodes of both WT C57BL/6 and RAG1-/- mice shortly after cutaneous application of the hapten FITC. Thirty minutes after FITC application, we could not find any bright FITC+ cells in the draining lymph nodes of both mice (data not shown). Also, the data obtained on skin explant-derived migratory cells of WT C57BL/6 and RAG1-/- mice (see previous results, Figs. 3Go and 4Go), support the assumption that bright FITC+ cells are likely to be LC derived.

The phenotype of the migrated bright FITC+ LCs was determined by FCM analysis. Mature DCs are known to express elevated levels of MHC class II molecules and costimulatory molecules as compared with immature DCs. It is shown in Fig. 5Go that migrated FITC+ LCs from both WT C57BL/6 as well as RAG1-/- mice had up-regulated MHC class II expression. Expression of costimulatory molecules on migrated LCs was analyzed by gating on FITC+ MHC class II + cells. Similarly elevated levels of costimulatory molecules CD80, CD86, and CD54 were observed on migrated LCs of WT mice vs RAG1-/- mice (Fig. 5Go). Migrated LCs were CD11c positive. These results clearly showed that there was no difference in the up-regulation of costimulatory molecules and MHC class II molecules by migrated LCs of WT vs RAG1-/- mice (Fig. 5Go).



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  		FIGURE 5. Phenotypical analysis of migrated LCs of WT and RAG1-/- mice. Twenty-four hours after application of FITC onto the shaved abdomen, lymph node cell suspensions were prepared from WT C57BL/6 mice (left) or RAG1-/- mice (right). The mAbs used for labeling were anti-I-Ad/b (biotin-conjugated revealed with streptavidin-APC), CD80 (unlabeled, anti-hamster-PE), CD86 (unlabeled, anti-rat-PE), CD54 (unlabeled, anti-rat-PE), or anti-CD11c (unlabeled, anti-hamster-PE). Propidium iodide was added just before analysis. Gating was done on propidium iodide-negative and FITC-positive cells (upper two histograms) or on propidium iodide-negative, FITC-positive cells and MHC class II-positive cells (other histograms). The white histogram represents the background staining; the black histogram represents staining by the indicated mAb. Results are representative for more than three experiments.

 
In vivo migrated LCs of RAG1-/- mice are functionally mature

To test whether the up-regulation of MHC class II molecules and costimulatory molecules on migrated LCs of RAG1-/- mice was indeed associated with functional maturation in vivo, bright FITC+ DCs were sorted from the draining lymph nodes 24 h after application of the hapten FITC. Sorted bright FITC+ DCs were irradiated and tested for their capability to stimulate allogeneic T cells. Migrated LCs from RAG1-/- mice were as efficient as similar cells from WT C57BL/6 mice in stimulating allogeneic T cells. As expected, total lymph node cells from normal untreated mice were much less efficient than sorted migrated LCs in allogeneic stimulation (Fig. 6GoA). Sorted bright FITC+ DCs were also tested as accessory cells for the presentation of SEB to syngeneic T cells. This superantigen does not require processing and is efficiently presented by mature DCs (7, 34). Migrated LCs of RAG1-/- mice were as potent as LCs of WT mice in SEB activation of T cells (Fig. 6GoB).



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  		FIGURE 6. Allogeneic stimulation and SEB stimulation by migrated LCs of WT and RAG1-/- mice. Twenty-four hours after application of FITC onto the shaved abdomen of normal and RAG1-/- mice, FITC+ cells were sorted from the draining lymph nodes and were irradiated with 3000 rad. A, Graded numbers of sorted irradiated LCs of WT C57BL/6 mice or RAG1-/- mice, or irradiated total lymph node cells of untreated C57BL/6 mice, were cocultured with 250,000 allogeneic T cells. After 3 days of culture, [3H]thymidine was added. [3H]Thymidine incorporation was measured after another 16 h. B, A total of 8500 sorted irradiated FITC+ LCs of WT C57BL/6 mice and RAG1-/- mice were cocultured with 200,000 nylon wool-purified syngeneic T cells in the presence of graded concentrations of SEB. Supernatant was removed after 24 h and added to 20,000 CTLL-2 cells. [3H]Thymidine was added after 24 h, and thymidine incorporation was measured after 6 h. Experiments were performed twice in triplicate. Results are expressed as the mean ± SD.

 
To exclude the possibility that migrated LCs derived from RAG1-/- mice would receive maturation signals, like CD40L, from the responder T cells that were present in the in vitro assays, we fixed the sorted FITC+ DCs with paraformaldehyde, instead of irradiating them, before coculture with responder T cells. Fixed LCs of RAG1-/- mice were still as potent as fixed LCs of WT C57BL/6 mice in stimulating allogeneic T cells (Fig. 7Go) and in presenting SEB to syngeneic T cells (data not shown).



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  		FIGURE 7. Fixed migrated LCs of WT and RAG1-/- mice are equally potent in stimulating allogeneic T cells. Twenty-four hours after application of FITC onto the shaved abdomen of normal and RAG1-/- mice, FITC+ cells were sorted from the draining lymph nodes and were fixed with 1% paraformaldehyde. Graded numbers of fixed FITC+ LCs of WT C57BL/6 mice and RAG1-/- mice were cocultured with 200,000 sorted allogeneic {alpha}ß T cells. After 3 days of culture, [3H]thymidine was added. [3H]Thymidine incorporation was measured after another 16 h. Experiments were performed twice in triplicate. Results are expressed as the mean ± SD.

 
Mature DCs have lost their capacity to process protein Ags, but they can very efficiently present peptides that do not need to be processed. Fig. 8Go shows that sorted bright FITC+ DCs from BALB/c mice, used as normal controls, and SCID mice had a decreased ability to process and to present OVA protein to OVA-specific TCR transgenic CD4+ T cells, whereas OVA peptide was well presented by migrated LCs from both mice.



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  		FIGURE 8. Efficient peptide presentation but decreased ability of protein processing by migrated LCs of WT and SCID mice. Twenty-four hours after application of FITC onto the shaved abdomen of WT BALB/c mice and SCID mice, FITC+ cells were sorted from the draining lymph nodes and pulsed for 2 h at 37°C with 1 mg/ml OVA protein or 30 µg/ml OVA peptide. Subsequently, pulsed LCs were fixed with 1% paraformaldehyde. Graded numbers of pulsed LCs were cocultured with 100,000 CD4+ OVA-specific TCR transgenic T cells. After 3 days of culture, [3H]thymidine was added. [3H]Thymidine incorporation was measured after another 16 h. Experiments were performed twice in triplicate. Results are expressed as the mean ± SD.

 
Migrated LCs of RAG1-/- mice can stimulate cytotoxic CD8+ T cells

Whereas in the previous experiments ( Figs. 5–7GoGoGo) mainly the CD4 T cell stimulatory capacity of migrated LCs was analyzed, we also tested the stimulation of CD8 cytotoxic T cells. In the case of splenic DCs, it was shown by others that these cells need to be conditioned by a Th cell through interaction between CD40 and its ligand before they are capable of activating directly cytotoxic CD8+ T cells (16, 17, 18).

Migrated LCs from FITC-sensitized RAG1-/- and WT C57BL/6 mice were sorted from the draining lymph nodes and were fixed with paraformaldehyde to exclude further maturation. Subsequently, fixed LCs were cocultured with sorted allogeneic lymph node {alpha}ß T cells. Thereafter, responder T cells were harvested and tested for their capability to lyse 51Cr-labeled allogeneic EL-4 targets and allogeneic Con A-treated spleen cells. T cells, stimulated by DCs of RAG1-/- mice, were as efficient in lysing the allogeneic targets as T cells stimulated by DCs of WT C57BL/6 mice (Fig. 9Go).



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  		FIGURE 9. Activation of cytotoxic T cells by fixed migrated LCs of WT and RAG1-/- mice. Twenty-four hours after application of FITC onto the shaved abdomen of WT C57BL/6 mice and RAG1-/- mice, FITC+ cells were sorted from the draining lymph nodes and were fixed with 1% paraformaldehyde. A total of 20,000 LCs of WT C57BL/6 mice and RAG1-/- mice were cocultured with 250,000 allogeneic sorted {alpha}ß T cells of BALB/c mice. After 5 days, responder T cells were assayed for their ability to lyse 51Cr-labeled allogeneic EL-4 cells and allogeneic Con A-cultured spleen cells. Results are presented as specific 51Cr release. Experiments were performed three times in triplicate. Results are expressed as the mean ± SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding in the present study is that in the absence of T and B cells, LCs can migrate from the skin to the draining lymph nodes where they become phenotypically and functionally mature DCs. Migrated LCs of FITC-sensitized T cell-deficient mice (RAG1-/- and SCID mice) up-regulated the expression of MHC class II molecules on their cell surface and the expression of costimulatory molecules CD80, CD86, and CD54. They were able to stimulate allogeneic T cells, Ag-specific CD4+ T cells, and cytotoxic CD8+ T cells in the absence of cognate T cell help.

It could be argued that the bright FITC+ cells found in the draining lymph nodes 24 h after cutaneous application of the hapten FITC are not LC derived but are due to leakage of the hapten through the skin. However, this is probably not the case as we did not detect bright FITC+ cells in the draining lymph nodes of WT C57BL/6 mice and RAG-/- mice 30 min or even 1 h after cutaneous FITC application. Others have also suggested that bright FITC+ DCs found in the draining lymph nodes after FITC painting are indeed derived from epidermal DCs (2, 35). Additional evidence comes from our data obtained with migratory cells from skin explants of WT C57BL/6 and RAG-/- mice. These results clearly showed that LCs from RAG-/- mice can migrate from the epidermis.

There are several indications that both skin-located as well as lymph node T cells might be able to regulate LC function. Dendritic epidermal T cells (DETCs) bearing the invariant V{gamma}3V{delta}1 TCR are the main T cell population present in the epidermis of mice. They are activated by irradiated, hapten-treated, and transformed keratinocytes, and by bacteria (36, 37). DETCs are in close contact with the LC population present in the epidermis. Due to their location, it is tempting to speculate that they can influence LC function during cutaneous immune responses. In favor for this is the fact that DETCs secrete GM-CSF (38) and TNF-{alpha} (38, 39), cytokines known to influence DC function. In vitro studies also have shown a role for DETCs in maintaining the proliferation of LCs by the production of GM-CSF and CSF-1 (40). Previous results have shown that {gamma}{delta} T cells involved in immune responses due to hapten sensitization preferentially rearrange the V{gamma}3 gene segment and can be found in the draining lymph nodes of sensitized mice (41, 42). Due to these observations, we hypothesized that DETCs might regulate LC function. T cell-deficient mice completely lack DETCs in their epidermis. We confirmed this by staining epidermal sheets with FITC-conjugated anti-CD3 mAb (data not shown). As LCs of T cell-deficient mice migrate and mature normally after application of the hapten FITC, we can conclude that DETCs do not play an essential role in this process.

After migration to the draining lymph nodes, LCs establish interactions with T cells. It might be possible that CD4+ and/or CD8+ T cells are responsible for the final maturation of migrated LCs once they have reached the draining lymph nodes. Activated CD4+ T cells express CD40L. It is known that CD40-CD40L interaction is a powerful stimulus to induce full maturation of in vitro-differentiated or spleen-derived DCs (8, 15, 16, 17). By comparing irradiated bright FITC+ LCs from T cell-deficient vs WT mice, we found no difference in their capacity to stimulate allogeneic or Ag-specific syngeneic T cells. To confirm that migrated LCs cells of T cell-deficient mice were already mature before they were cocultured with T cells during the different in vitro stimulation tests, we fixed the migrated FITC+ LCs instead of irradiating them. By fixing LCs, they are no longer able to up-regulate the expression of costimulatory molecules or other "parameters" of DC maturation. However, still no difference in stimulation of allogeneic T cells or Ag-specific syngeneic CD4+ T cells was seen using migrated bright FITC+ LCs from T cell-deficient mice compared with WT mice.

The interaction between APCs and cytotoxic precursor cells is often not enough to stimulate cytotoxic T cells, and, in these cases, a Th cell that recognizes an Ag on the same APC is also required (16, 17, 18). The activating signal, representing the interaction between CD40 on DCs and CD40L on Th cells, enables DCs to directly stimulate cytotoxic T cells. We found that in the absence of T cells, LCs could still migrate to the draining lymph nodes of FITC-sensitized mice and became very potent stimulators of cytotoxic CD8+ T cells. As fixation of the migrated LCs did not abolish their stimulation of cytotoxic T cells, it can be excluded that LCs would require additional Th signals during the in vitro test. This is in agreement with recent results showing that depending on the route of immunization, Ag-specific cytotoxic CD8+ T cell responses develop with or without CD4 help: the intradermal route does not need CD4 help, whereas help is required for the intraperitoneal route (43). This strongly indicates that it depends on the subset of DCs involved whether cognate CD4 help is required to induce stimulation of CD8 cytotoxic T cells. Also, the difference observed between our results showing that LCs do not require CD4 help to stimulate cytotoxic T cells and results from others can be due to the difference in DC type used (17, 18, 44).

Athymic nude mice, which lack mature T cells, have been reported to have defective LC functions. These LCs are much less efficient in Ag presentation in vitro compared with LCs from euthymic mice. This defect in LC function is corrected either by cytokine treatment in vitro or by adoptive transfer of thymic tissue (45). This seems to be in conflict with the results we found. However, it is important to keep in mind that most observations on maturation have been made with LC cultured in the presence of GM-CSF, secreted endogenously by contaminating keratinocytes or added exogenously. Keratinocytes from athymic mice produce less GM-CSF, and when GM-CSF is added to the culture LC function is restored (45). So, the defect in LC function in nude mice may be due to the defect found in keratinocyte function. T cell-deficient mice as RAG1-/- mice and SCID mice have normal keratinocytes, so it is possible that they have normal LC function in the absence of T cells in the skin as our results suggested. It also seems that the presence of normal thymic stroma is important for adequate LC function (45). T cell-deficient mice as RAG1-/- mice and SCID mice have normal thymic stroma. These differences between athymic and euthymic T cell-deficient mice may be responsible for the differences in LC function.

After submission of the present paper, Shreedhar et al. (46) have published that DCs do require T cells for functional maturation in vivo. These authors found, in sharp contrast to our results, that DCs from the draining lymph nodes of FITC-sensitized RAG2-/- mice are deficient in their capacity to stimulate naive T cells in vitro and in vivo. In addition, they have shown that this deficiency is corrected by reconstituting RAG2-/- mice with normal T lymphocytes. Shreedhar et al. have found that T cell-deficient mice exhibit a striking deficiency in DEC-205+ LC numbers in the epidermis, which can be corrected by adoptive transfer of normal T cells. In contrast to the results found by Shreedhar et al., we found normal numbers of LCs in T cell-deficient mice, expressing normal levels of MHC class II and DEC-205. The differences in outcome between these two studies are not easily reconciled. We do not think that the different T cell-deficient mice that were used, RAG1-/- vs RAG2-/-, are the reason. T cells are blocked at the same stage of differentiation in RAG1-/- vs RAG2-/- mice (31, 32), and neither the RAG1 nor the RAG2 gene has been described to be required for the development of cells other than lymphocytes. We carefully analyzed the thymus and the spleen of RAG1-/- mice used in our study and found them to be completely negative for T cells. A potentially important difference in this study and the one performed by Shreedhar et al. is the method used for the isolation of migrated DCs from the draining lymph nodes of FITC-sensitized mice. We sorted bright FITC+ cells from the draining lymph nodes to a purity of >99%, which were all MHC class IIbright and were probably derived from the skin. On the contrary, Shreedhar et al. enriched DCs from the draining lymph nodes of FITC-sensitized mice by density gradient centrifugation onto metrizamide. They obtained a purity of 75–90% DCs, and these DCs were not only FITC+ DCs migrated from the skin but also resident DCs. To study if the different isolation methods used might explain the contradictory results, we also enriched DCs from the draining lymph nodes of FITC-sensitized mice by using a metrizamide gradient. We obtained a purity of 43% FITCbright MHC class IIbright DCs for WT mice and 58% for RAG1-/- mice. Using these enriched DCs as stimulators for allogeneic T cells, we still could not find a difference in DC function between WT mice and RAG1-/- mice (data not shown).

We can conclude from our results that LC migration and maturation can occur in vitro and in vivo in the absence of T cells. LCs do not need help from cognate T cells to be able to directly stimulate a cytotoxic T cell response in vitro. T cells present in the draining lymph nodes may still be important in sustaining or abrogating an immune response by influencing the function of DCs through the interaction between CD40 and CD40L at the final stages in the life span of DCs. Signaling through CD40 ligation can rescue DC from Fas-induced apoptosis, which can determine this balance between sustaining or declining an immune response (47). Our results also indicate that cytokines produced by keratinocytes after Ag encounter may be sufficient to induce migration and maturation of LCs to the draining lymph nodes.

It is important to find the mechanisms responsible for inducing or inhibiting LC migration and maturation because of the clinical implications in immunological and inflammatory diseases of the skin.


   Acknowledgments
 
We thank Dr. M. Moser and Dr. H. Spits for providing us with the anti-CD11c mAb and RAG1-/- mice, respectively. We also thank M. De Smedt for purification of Abs, A. Moerman for animal care, and C. De Boever for artwork.


   Footnotes
 
1 This work was supported by grants from the research fund of the University of Ghent and the Fund for Scientific Research of Flanders. G.L. is a Senior Research Associate of the Fund for Scientific Research of Flanders. Back

2 Address correspondence and reprint requests to Dr. Georges Leclercq, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Blok A, De Pintelaan 185, B-9000 Ghent, Belgium. Back

3 Abbreviations used in this paper: DC, dendritic cell; LCs, Langerhans cells; CD40L, CD40 ligand; RAG, recombination activating gene; FCM, flow cytometric; SEB, staphylococcal enterotoxin B; WT, wild type; DETC, dendritic epidermal T cell. Back

Received for publication October 5, 1999. Accepted for publication April 24, 2000.


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