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Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University Hospital, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
(12).
Different types of stimuli can lead to DC maturation, like danger
signals, LPS (13), and inflammatory cytokines such as IL-1
and TNF- (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-. 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- 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-, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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,
; 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 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.
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 ß+ as
confirmed by FCM analysis using biotin-conjugated anti-TCR ß
mAb followed by labeling with streptavidin-PE.
In vitro assays
Allogeneic stimulation.
A total of 250,000 sorted 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 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)].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). 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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). 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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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. 3). 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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).
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. 3
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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. 3 and 4), 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. 5 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. 5).
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. 5).
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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. 6A). 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. 6B).
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) and in presenting SEB to
syngeneic T cells (data not shown).
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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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Whereas in the previous experiments (
Figs. 5–7) 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 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. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 V3V
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- (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 T cells involved in immune responses due to hapten
sensitization preferentially rearrange the V3 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.
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Acknowledgments |
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Footnotes |
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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.
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.
Received for publication October 5, 1999. Accepted for publication April 24, 2000.
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