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U. Ritter et al.
Eur. J. Immunol. 2004. 34: 1542–1550
CD8 > - and Langerin-negative dendritic cells, but
not Langerhans cells, act as principal antigenpresenting cells in leishmaniasis
Uwe Ritter1, Anja Meißner1, Christina Scheidig1 and Heinrich Körner1,2
1
Nachwuchsgruppe 1, Interdisziplinäres Zentrum für Klinische Forschung der Universität
Erlangen-Nürnberg, Erlangen, Germany
2
Comparative Genomics Centre, James Cook University, Townsville, Australia
In the early phase of leishmaniasis three types of potential antigen-presenting cells, including epidermal Langerhans cells (LC), dermal dendritic cells (DC) and inflammatory DC, are
localized at the site of infection. Therefore, it has been a central question which cell type is
responsible for the initiation of a protective immune response. In the early stage of an antiLeishmania immune response, detectable Leishmania major antigen was localized in the
paracortex of the draining lymph nodes (LN). Characterization of antigen-positive cells
showed that L. major co-localized with DC of a CD11c+ CD8 § – Langerin– phenotype. To
determine the area of antigen uptake, dermis or epidermis, and to further define the type of
antigen-transporting cells, L. major was inoculated subcutaneously and concurrently LC
were mobilized with fluorescein isothiocyanate (FITC). After 3 days, DC carrying L. major
antigen were always FITC–, indicating a dermal and not an epidermal origin. Moreover, addition of L. major antigen to ex vivo isolated CD8 § – and CD8 § + DC from the draining LN of
L. major-infected C57BL/6 mice demonstrated that both DC subpopulations were able to
stimulate antigen-specific T cell proliferation in vitro. Without addition of exogenous antigen
only the CD8 § – Langerin– DC were capable of stimulating antigen-specific T cell proliferation. Thus, we demonstrate that CD8 § – Langerin– DC and not LC are the basis of the protective immune response to intracellular L. major parasites in vivo.
Key words: Leishmania / Langerhans cells / Parasitic protozoan / Antigen presentation
1 Introduction
The protozoan parasites Leishmania spp. are known to
infect a variety of mammalian hosts and causes diseases
ranging from localized cutaneous to systemic visceral
forms [1]. Experimental cutaneous mouse leishmaniasis
is widely used as a model for this parasitic disease. After
the subcutaneous (s.c.) infection of mice with L. major,
the obligatory intracellular parasites reside within phagocytic cells, mostly monocytes/macrophages and DC [1,
2]. It has been demonstrated that the course of mouse
leishmaniasis strongly depends on factors determined
by the genetic background [1–3]. The resistant mouse
strain C57BL/6 shows an early induction of IL-12 and an
expansion of parasite-specific IFN- + - and IL-2-pro-
[DOI 10.1002/eji.200324586]
Abbreviations: CFSE: 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester LC: Langerhans cells RT: Reverse transcription
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received
Revised
Accepted
23/9/03
17/3/04
29/3/04
ducing Th1 cells [4]. These mice are able to control the
pathogen predominantly at the site of infection and to
resolve the lesion. In contrast, susceptible BALB/c mice
show a progressive and fatal disease [1, 2]. This course
of disease is due to an expansion of IL-4-, IL-5-, and IL10-producing Th2 cells [3] propagated by the early presence of large amounts of IL-4 [5].
DC are central for antigen presentation and the priming
of naive T cells. They capture antigen and migrate to
lymphoid organs, where they induce a specific cellular
immune response [6, 7]. In experimental leishmaniasis,
L. major amastigotes interact with [8] or reside within
epidermal Langerhans cells (LC) [9]. Fetal skin-derived
DC, a cell line with the characteristics of immature DC,
take up amastigotes and respond to this challenge with
the production of IL-12, the cytokine that ultimately
determines the type of the anti-leishmanial immune
response [10]. Moreover, it has been demonstrated that
isolated epidermal LC pulsed with L. major antigen in
vitro and adoptively transferred to an infected host, have
the capacity to transport and to present L. major antigen
to T cells and to establish immunity [11].
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Eur. J. Immunol. 2004. 34: 1542–1550
It has not yet been attempted to isolate and characterize
L. major-presenting DC directly isolated ex vivo because,
after migration to the draining lymph node (LN), it was
not possible to distinguish between LC and dermal DC.
However, a set of recently defined marker molecules
enables this differentiation. Recent studies have indicated that LC up-regulate CD8 § upon migration to the
draining LN, whereas dermal DC remain CD8 § – [12–14].
Additionally, LC strongly express the C-type lectin Langerin [15] and are still positive for this molecule after they
have reached the LN [16]. Therefore, to solve the central
question, which DC subtype is transporting L. major
antigen and inducing protective immunity in vivo, we
analyzed the phenotype of L. major-harboring DC using
these novel markers. Furthermore, we separated DC
subpopulations from the draining LN of L. major-infected
mice and compared the potential of CD8 § – DC and
CD8 § + LC to induce a Leishmania-specific T cell
proliferation.
Leishmania antigen transport from skin to lymph node
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2 Results
2.1 L. major parasites are located in the
paracortex of the draining LN
We analyzed sections of the central part of draining popliteal LN of infected mice at days 3 and 6 after infection
for the presence of L. major antigen. At both of time
points after infection, L. major-specific signals could be
detected in the paracortical area of the LN (Fig. 1A). At
day 6 after infection, the LN was substantially enlarged
and a highly significant increase of the detectable
L. major antigen in the paracortical area per LN section
was visible (Fig. 1B). To quantify the number of L. majorpositive signals per LN section the signals were counted
in five 10- ? m median sections of four to six different LN
from infected mice at both time points and found to
increase from 40 signals at day 3 to 180 signals at day 6
(p X 0.001, Fig. 1C). The number of signals detected by
Fig. 1. Detectable L. major antigen is localized in the paracortex of the draining LN. (A, B) The association of L. major antigen with
the paracortical area in the draining LN of C57BL/6 mice was analyzed by two-color immunofluorescence and is shown 3 days
(A) and 6 days after infection (B). The B cell follicle is indicated with a B (red = L. major antigen, highlighted by arrows; green =
B cells; bar = 100 ? m). (C) In a quantitative immunohistological analysis the increase of L. major antigen was analyzed at days 3
and 6 after infection. The number of L. major-positive signals per LN section was calculated by counting the parasites in five 10? m median sections of four to six different infected LN. The means ± SEM are presented (*p X 0.001).
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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immunofluorescence microscopy was consistent with
results of limiting dilution assays published earlier that
showed a similar order of magnitude of viable parasites
in the draining LN in the early phase of infection [17, 18].
2.2 Characterization of the cell type harboring
L. major antigen
Popliteal LN sections of infected mice were stained with
a serum specific for L. major antigen and mAb against
either CD11c, F4/80, CD8 § , or CD11b. Analysis of sections with anti-L. major serum in combination with antiCD11c mAb indicated a co-localization of parasite antigen with CD11c+ DC (Fig. 2A). In contrast, a combination
of anti-L. major serum with anti-F4/80 (macrophages,
Fig. 2B) or anti-CD8 § mAb (T cells and LC, Fig. 2C)
revealed only distinct L. major-negative cells. Finally, the
application of anti-L. major serum in combination with an
anti-CD11b mAb (monocytes and some subpopulations
of DC) showed in some cells a co-localization (Fig. 2D,
E). To exclude an extracellular localization of L. major
parasites, a triple staining with phalloidin (f-actin and cactin [19]), anti-L. major serum, and 4’,6-diamidino-2phenylindole (cell nucleus) was performed. This staining
confirmed that the parasites resided in very close proximity to or within cells (data not shown).
To further analyze the phenotype of L. major-positive DC
populations, CD8 § + and CD8 § – DC subpopulations were
isolated from the draining LN of infected animals (sorting
purity G 95%) and analyzed using reverse transcription
(RT)-PCR. CD8 § + and CD8 § – DC were mature and
expressed both CC chemokine receptor 7 and IL-12
(p40) mRNA (Fig. 2F). The CD8 § + DC population was
L. major mRNA-negative and expressed the LC-specific
molecule Langerin (Fig. 2F). In contrast, CD8 § – DC carried L. major mRNA and where Langerin– (Fig. 2F).
To exclude that the route of infection (i.d. versus s.c.)
influenced which DC subpopulation transports L. major
to the LN, we inoculated C57BL/6 mice i.d. in the ear and
analyzed the phenotype of L. major-positive DC reaching
the draining LN 6 days after infection. After i.d. injection
L. major antigen co-localized exclusively with DC of a
CD11c+ CD8 § – Langerin– phenotype in the draining LN
(data not shown).
Fig. 2. L. major antigen and L. major-specific actin mRNA
are associated with CD8 § – DC. The phenotype of L. majorpositive cells in popliteal LN sections was determined
3 days after infection using two-color immunofluorescence
(A–E) and RT-PCR (F). L. major antigen co-localizes with
CD11c+ cells (A) but not with F4/80+ (B) or CD8 § + cells (C).
CD11b+ cells are both, L. major antigen-negative (D) and
-positive (E). CD11c, F4/80, CD8 § , and CD11b staining is
shown in green. L. major parasites are visualized in red. The
arrows indicate co-localized (A) or not-co-localized (D)
L. major parasites. Bar = 10 ? m. Representative stainings
are shown. (F) The expression of isolated CD8 § , CCR7, IL12 (p40), L. major-actin, Langerin, and g –actin of CD8 § + and
CD8 § – DC populations isolated ex vivo was analyzed by RTPCR.
2.3 L. major antigen is transported to the
draining LN by DC, but not by LC
To further analyze the DC population that transports
L. major antigen, promastigote parasites were inoculated s.c. Simultaneously, the site of infection was
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
painted with FITC, to mobilize and monitor the migration
of epidermal LC to the draining LN [20, 21]. This combined insult resulted in migration of dermal and epidermal DC to the paracortical area of the popliteal LN. Three
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Leishmania antigen transport from skin to lymph node
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days after infection the LN was analyzed by means of
immunohistology. All L. major-positive DC within the
paracortex were negative for FITC (Fig. 3A). The phenotype of FITC+ cells was analyzed with CD8 § as a marker
for epidermal LC. Staining with mAb specific for CD11c
and CD8 § disclosed that FITC+ cells were CD11c+ and
CD8 § + (Fig. 3B), demonstrating that these cells were of
epidermal origin and belonged to the LC population [14].
This confirms that not CD8 § + FITC+ LC but CD8 § – FITC–
DC transport L. major to the draining LN.
2.4 Competence of ex vivo isolated CD8 > +
and CD8 > – DC to present L. major antigen
to T cells
In the draining LN of infected mice the number of CD8 § –
DC had increased 20-fold from 2,500±600 cells/LN to
50,000±12,500 cells/LN (mean ± SEM), and the number
of CD8 § + DC sevenfold from 1,700±400 cells/LN to
12,500±3,100 cells/LN (mean ± SEM) at day 6 after
infection. To test the antigen-presenting capacity of
CD8 § + and CD8 § – DC populations, both subpopulations
of DC were isolated ex vivo from LN of L. major-infected
C57BL/6 mice at day 6 after infection (sorting purity
G 95%). In parallel, CD4+ T cells were isolated from the
same LN, labeled with 5- (and 6-) carboxyfluorescein
diacetate succinimidyl ester (CFSE) and mixed with
either of the isolated DC subpopulations with or without
adding further antigen (Fig. 4A). The addition of exogenous L. major antigen or Con A to all combinations of
cells always led to strong proliferation (Fig. 4A). This indicates that the cells were healthy and reactive (Con A)
and that the antigen-specific CD4+ T cells were able to
proliferate in the presence of abundant L. major antigen
presented by CD8 § + (34% proliferating CD4+ T cells) or
CD8 § – (37% proliferating CD4+ T cells) DC populations
equally well.
Fig. 3. FITC+ LC are negative for L. major antigen in draining
LN. Popliteal LN sections were analyzed by immunofluorescence microscopy 2–3 days after FITC painting and concurrent s.c. infection with L. major parasites. (A) L. majorpositive signals located in the paracortical area (see insert)
of the popliteal LN do not co-localize with FITC+ cells (green
= FITC+ cells, indicated by broken arrows; red = L. major
antigen, indicated by lined arrows; bar = 50 ? m). (B) FITC
particles co-localize with CD11c+ and CD8 § + cells (green =
FITC; red = CD11c and CD8 § ; bar = 20 ? m). Representative
stainings are shown.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
However, in the absence of added antigen only CD8 § –
DC were able to induce an antigen-specific T cell proliferation (21% proliferating CD4+ T cells, Fig. 4A) whereas
T cell proliferation in the presence of CD8 § + DC was not
substantially above background (9% proliferating CD4+
T cells, Fig. 4A). This is also shown by the proliferation
index, which summarizes the proliferation in the absence
of exogenous antigen of three independent experiments
(Fig. 4B) and demonstrates that the CD8 § – DC subpopulation carries L. major antigen and can induce an
immune response. Furthermore, this result is consistent
with our histological data (Fig. 1, 2) and PCR results
(Fig. 2F).
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Eur. J. Immunol. 2004. 34: 1542–1550
mal compartment and not LC that reside in the epidermis
are responsible for the transport of L. major antigen to
the draining LN. Recent observations have made this differentiation possible. It has been demonstrated that LC
up-regulate the CD8 § chain after migration to the draining LN [12–14, 16, 22, 23]. This has been used, together
with the differential expression of the surface antigens
DEC 205, CD11b, and CD4, to define at least five different DC subpopulations in the skin draining LN [16]. Furthermore, only LC of the epidermal compartment are
strongly positive for the C-type lectin Langerin [15] and
are still positive for this molecule after they have reached
the LN. This marker molecule has made it possible to
clearly identify the LC population [16].
Fig. 4. Only CD8 § – DC are able to present endogenous
L. major antigen to primed T cells and to induce proliferation. (A) The ability of CD8 § – and CD8 § + DC subpopulations
to present antigen and to induce proliferation was analyzed
by incubating CFSE-positive CD4+ T cells with CD8 § – and
CD8 § + DC subpopulations. The cells were isolated ex vivo
from draining LN of L. major-infected C57BL/6 mice (n=6–8
per experimental group) and incubated with medium (upper
panel), with L. major antigen (middle panel), and with Con A
(lower panel). Background proliferation was determined
using L. major-specific CD4+ T cells alone. (B) CD8 § – DC
significantly induce T cell proliferation (*p X 0.05). The proliferation index combines the results of three independent
experiments. The antigen-specific proliferation without adding exogenous antigen is shown. The dotted line represents
the proliferation of antigen-specific CD4+ T cells incubated
with CD8 § – DC isolated from non-infected C57BL/6 mice.
Black bars, CD8 § –; white bars, CD8 § +.
Our results show that after i.d. or s.c. injection the
L. major antigen that is detectable in the paracortical
area of the LN is associated with mature CD11c+, CD8 § –,
F4/80–, and Langerin– DC and not with CD8 § + Langerin+
LC. Furthermore, only the CD8 § – DC population can present endogenous antigen and stimulate T cell proliferation in this model of leishmaniasis. The L. major infection
with a concurrent skin painting that labels epidermal LC
also demonstrates that only DC of the dermal compartment, but not LC, take up L. major antigen. This interaction between antigen-presenting DC of one skin compartment and a pathogen occurs with a specificity that
was not anticipated. The finding that CD8 § – DC present
antigen argues against cross-presentation as the underlying mechanism of antigen presentation. The uptake of
dying DC in the LN by LN-resident DC and the subsequent presentation of their antigenic contents to T cells
has only been described for CD8 § + DC [24, 25].
It has been reported that after an inflammatory stimulus
the presentation of antigen from the skin in the draining
LN is carried out sequentially by different subpopulations
of DC [26]. In the very early stage of the inflammatory
response (3 h after transfer of antigen), both DC of dermal origin and LC that had migrated to the LN shortly
before the immunization, presented antigen that had
been swept to the LN passively. This first wave of antigen
presentation was based on a small amount of antigen,
but induced T cell priming and a T cell response in a
transfer system of transgenic T cells [26]. Later, inflammatory DC transported antigen and presented it. The
efficiency of this antigen presentation was significantly
higher [26].
3 Discussion
We investigated the phenotype of DC presenting
L. major antigen in the draining LN in vivo and studied
the potential of DC subpopulations isolated ex vivo to
induce T cell proliferation. Our results show that at
days 3 and 6 after infection with L. major, DC of the der© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In our infection model of leishmaniasis, we were neither
able to detect antigen in the LN nor to show the induction of a T cell response within the first 24 h after infection (U.R. and H.K., unpublished results). Obviously, the
threshold of detection in our experiments was much
higher since we used for detection CD4+ LN T cells
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which were not enriched for L. major-specific T cells. The
CD11c+, CD8 § –, F4/80–, and Langerin– DC that are colocalized with L. major antigen in our experiments are
consistent with the antigen-transporting DC that collect
antigen at the site of immunization, transport it to the
draining LN, and present this antigen highly efficiently
[26]. Although the antigen that leaks to the draining LN
induces a T cell response within hours after immunization, and although we cannot formally rule out a contribution of passively transported antigen that is taken up
in the LN by DC to the anti-L. major immune response,
the results by Itano and colleagues [26] regarding the
time frame of the sequential presentation of antigens
suggest that passively transported antigens do not significantly contribute to the data presented.
The origin and phenotype of the DC population in an
inflammatory situation in the skin are not yet well
defined. Inflammatory dermal DC probably descend
from monocytic precursor cells that are attracted to the
site of infection by inflammatory mediators and differentiate into dermal monocytic DC while transmigrating
from the blood to the inflamed tissue [27–29]. The use of
fluorochrome-labeled microspheres injected intracutaneously together with FITC-painting pointed to a highly
specific use of a CD8 § – DC subpopulation. DC that had
phagocytosed microspheres were positive for the fluorochrome and expressed CD11c and MHC class II, but
lacked CD8 § [29]. In contrast, a recent study investigating epidermal herpes simplex virus infection demonstrated that CD8 § + DC, but not LC, transport and present antigen [30]. The usage of CD8 § + DC in this model
could be due to the epidermal localization of the challenge. Further studies that deepen our understanding of
the early events in inflammation will be needed to resolve
these discrepancies between different models.
In conclusion, our study indicates that dermal DC or
inflammatory DC are the natural source of L. major antigen in the experimental model of leishmaniasis. This
challenges the view that LC are central in the control of
L. major infection [11]. However, this notion has been
based to a large extent on indirect studies that demonstrated the potential of LC to migrate and to present antigen in vitro [8, 9, 11, 31]. Meanwhile, different studies
demonstrated that an immune response within the skin
is based on a range of DC subpopulations residing
within, or migrating to the skin, after an inflammatory
stimulus but does not depend on skin-resident LC [26,
29]. These findings together with our study do not rule
out a role for LC in the immune response to skin infections like L. major infection. LC could, for example, be
involved in the conditioning of the tissue for an inflammatory response [32], but the role of LC has certainly to be
redefined.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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4 Materials and methods
4.1 Mice
Inbred C57BL/6 mice were purchased from Charles River
(Sulzfeld, Germany). Mice at an age of 8–16 weeks were
used.
4.2 Parasites, preparation of antigen and infection of
mice
The cloned virulent L. major isolate (MHOM/IL/81/FE/BNI)
[33] was used for infection experiments. The course of disease was monitored daily as described [34]. Alternatively,
the parasites were subjected to four cycles of rapid freezing
and thawing to prepare L. major antigen [34]. Mice were
infected s.c. in one hind footpad or i.d. in the ear with
3×106 stationary-phase promastigotes/foot pad or ear of the
third to seventh in vitro passage in a final volume of 50 ? l
(ear: 10 ? l) [34].
4.3 Antibodies
The following antibodies were used for immunofluorescence
microscopy and flow cytometry: hamster anti-mouse CD11c
(clone HL3; biotinylated or PE-conjugated), rat anti-mouse
CD11b (clone M1/70; biotinylated or FITC-conjugated), rat
anti-mouse CD8 § (clone 53–6.7; unlabeled, PE-, or FITCconjugated), rat anti-mouse pan-CD45 (clone 30-F11; unlabeled), rat anti-mouse CD45R/B220 (clone RA3–682; biotinylated). These mAb were purchased from Becton Dickinson/PharMingen (Heidelberg, Germany). Rat anti-mouse F4/
80 (clone CI.A3–1; biotinylated) was obtained from Dianova
(Hamburg, Germany). The production of a L. major-specific
polyclonal serum has been described [35]. The following
secondary reagents were used for detection of purified or
biotinylated primary mAb: Alexa Fluor 488- and 546conjugated goat anti-rat Ig and streptavidin-Alexa Fluor 488
(Mobitec, Göttingen, Germany), rhodamine-conjugated
donkey anti-rabbit IgG, Cy5-conjugated goat anti-rat IgG,
Cy5-conjugated streptavidin (Dianova), PerCP (Becton Dickinson/PharMingen).
4.4 Immunofluorescence microscopy
Tissue blocks from LN were embedded in optimal cutting
temperature compound (OCT; Diatec, Hallstadt/Bamberg,
Germany) and stored at –70°C. Tissue sections (10 ? m) were
thawed onto gelatin-coated glass slides, air-dried, fixed in
acetone (for 10–15 min, at –20°C) and rehydrated with PBS
and 0.01% Tween-20 (Sigma). Nonspecific binding sites
were blocked for 30 min at room temperature with PBS containing 1% BSA and 10% mouse serum. The sections were
first stained with rabbit anti-L. major serum in PBS/BSA and
0.1% saponin (Roth, Karlsruhe, Germany). Rabbit antiwww.eji.de
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U. Ritter et al.
L. major serum was revealed with a rhodamine-conjugated
donkey anti-rabbit IgG. A biotinylated mAb was used for a
second layer (either anti-CD11c, anti-CD11b, anti-CD45R/
B220, or anti-CD8 § ) followed by Alexa 488- or Cy5conjugated streptavidin. Alternatively, unlabeled F4/80 was
used, followed by incubation with FITC anti-rat IgG. Each
incubation step lasted 30 min at room temperature alternating with five washing steps in PBS/Tween (0.01%, 10 min,
room temperature). Sections were analyzed using an immunofluorescence microscope (Zeiss, Jena, Germany)
equipped with high-sensitivity gray scale digital camera
(Openlab System; Improvision, Heidelberg, Germany). Separate color images were collected for each section, analyzed
and merged afterwards. Final image processing was performed using Corel Photo-Paint software (Corel Corporation, Ottawa, Canada).
4.5 Isolation of cells from LN and skin
Draining popliteal LN and foot pads were harvested and
digested with collagenase D (1 mg/ml; Boehringer Mannheim, Mannheim, Germany) in Hanks’ balanced salt solution (plus Ca2+, Mg2+) for 30 min at 37°C. The reaction was
stopped with 5 mM EDTA for 10 min at 37°C. Digested tissues were passed through a stainless steel sieve and subsequently filtered through a 70- ? m cell strainer (Becton Dickinson, Heidelberg, Germany) and washed twice with FACS
buffer containing PBS, 0.1% BSA, and 0.02% sodium azide.
The number of viable cells was determined by Trypan blue
exclusion.
4.6 Flow cytometry and flow cytometric cell sorting
Multi-color flow cytometry was performed as described [36].
Cells isolated from the skin and LN (see above) were washed
and resuspended in PBS containing 0.1% BSA, sodium
azide and stained directly or indirectly with fluorochromeconjugated mAb. Cells were analyzed by flow cytometry. To
exclude cell debris, cells were electronically gated according
to light scatter properties and the hemopoietic marker
CD45. Data were collected using a FACSCalibur flow cytometer (Becton Dickinson). Analysis was performed using
CellQuest software (Becton Dickinson). Alternatively, cells
isolated from skin and draining LN were labeled and subjected to flow cytometric cell sorting using a MoFlo highspeed cell sorter (Cytomation Bioinstruments, Freiburg, Germany). After sorting, purity of cells was controlled with the
FACSCalibur.
Eur. J. Immunol. 2004. 34: 1542–1550
cells according to the following phenotypes: CD11c– and
CD4+ (CD4+ T cells), CD11c+ and CD8 § – (dermal DC),
CD11c+ and CD8 § + (LC). To monitor cell proliferation, CD4+
T cells were incubated with CFSE (Mobitec) at a final concentration of 1 ? M in PBS for 10 min at 37°C [37]. CFSElabeled T cells (1.5×105) were incubated in microtiter plates
in RPMI 1640 supplemented with 10% FCS (Sigma), glutamine, Hepes, and antibiotics (Seromed-Biochrom, Berlin,
Germany). DC (either CD8 § + or CD8 § –) were added in a ratio
of 1:10 and cultures were either stimulated with lysed
L. major promastigote total antigen (at a ratio of ten parasites per total number of cells) or with Con A (final concentration 1 ? g/ml). Background proliferation was estimated
using isolated CD11c+ CD8 § – or CD11c+ CD8 § + DC from
non-infected C57BL/6 mice together with L. major-specific
CD4+ T cells.
After 72 h of incubation, supernatants were removed and
stored at –70°C. Cells were harvested and beads (Calibrate
Beads; Becton Dickinson; 1.0×104 beads/sample) were
added to the cells to quantify the proliferating cells and to
determine the proliferation index according to the formula:
(Proliferating cells/bead in cultures plus DC) / (Proliferating
cells/bead in cultures without DC). Proliferating cells and
beads were quantified with a FACSCalibur.
4.8 RNA extraction and RT-PCR
RNA was extracted from sorted cells or tissue using the perfect RNA mini kit (Eppendorf, Hamburg, Germany) according
to the manufacturer’s instructions. All samples were treated
with RNase-free DNase (Promega, Mannheim, Germany) for
15 min followed by chloroform/phenol extraction to remove
the enzyme. First-strand cDNA was synthesized from 1–2 ? g
of total RNA using murine Moloney leukemia virus reverse
transcriptase (Promega) and oligo(dT) primer (Gibco Life
Technologies, Karlsruhe, Germany). The primers, which
were specific for CCR7 (sense: 5’-ATTTCTACAGCCCCCAGAGC-3’, antisense: 5’-TGAGCCTCTTGAAATAGATGTACG-3’), CD8 § (sense: 5’-CACGAATAATAAGTACGTTCTCACC-3’, antisense: 5’-ATGTAAATATCACAGGCGAAGTCCA3’), g -actin (sense: 5’-AATCCTGTGGCATCCATGAAAC-3’,
antisense: 5’-CGCAGCTCAGTAACAGTCCG-3’), L. major
actin (sense: 5’-TGACAACGAGCAGAGCTCCA-3’, antisense: 5’-CCCACGATCGAAGGGAAAA-3’), and mouseLangerin (sense: 5’-ACGCACCCCAAAGACCTGGTACAG3’, antisense 5’-AGACACCCTGATATTGGCACAGTG-3’)
[38], were used at a concentration of 200 nM. Amplification
(35 cycles; 20 s 94°C, 20 s 58°C, and 50 s 72°C) was performed with 1 U Taq polymerase/reaction (PAN, Aidenbach,
Germany).
4.7 Proliferation assay
4.9 Skin painting
Six days after infection mice were sacrificed and draining
popliteal LN (n=6–8 per group) were removed. Pooled popliteal LN cells were stained for CD11c, CD8 § and CD4. CD4+
T cells and antigen-presenting DC were isolated from LN
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Mice were infected with 3×106 L. major promastigotes as
described above. Immediately after infection the footpads
were painted with 400 ? l FITC (5 mg/ml dissolved in equal
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Leishmania antigen transport from skin to lymph node
1549
volumes of dibutylphtalate and acetone). After 3 and 6 days,
popliteal LN were stained with biotinylated mAb layer (either
anti-CD11c or anti-CD8 § ), or L. major-specific serum (as
described above). Sections were analyzed by immune fluorescence microscopy to determine the phenotype of
migrated FITC+ cells.
10 von Stebut, E., Belkaid, Y., Nguyen, B. V., Cushing, M., Sacks,
D. L. and Udey, M. C., Leishmania major-infected murine Langerhans cell-like dendritic cells from susceptible mice release IL-12
after infection and vaccinate against experimental cutaneous
leishmaniasis. Eur. J. Immunol. 2000. 30: 3498–3506.
4.10 Presentation of results and statistical analysis
12 Anjuere, F., Martin, P., Ferrero, I., Fraga, M. L., Martinez del
Hoyo, G., Wright, N. and Ardavin, C., Definition of dendritic cell
subpopulations present in the spleen, Peyer’s patches, lymph
nodes, and skin of the mouse. Blood 1999. 93: 590–598.
The results in the figures are expressed as means ± SEM.
Differences between experimental groups were tested for
statistical significance by Student’s t-test for unpaired samples (two-tailed).
Acknowledgements: The authors thank Prof. Bogdan and
Dr. Lutz for critical reading of the manuscript. This work was
supported by the Deutsche Forschungsgesellschaft (H.K.;
Ko 1315/3–3, Ko 1315/4–1) and by the Federal Ministry of
Education and Research (BMBF) and the Interdisciplinary
Center for Clinical Research (IZKF) at the University Hospital
of the University of Erlangen-Nürnberg, Germany (H.K., IZKF
Nachwuchsgruppe 1).
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Correspondence: Heinrich Körner, Comparative Genomics
Centre, Molecular Sciences Bld. 21, James Cook University,
Townsville, Qld 4811, Australia
Fax: +61-7-4781-6078
e-mail: heinrich.korner — jcu.edu.au
www.eji.de