CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells.
ABSTRACT Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.
- [show abstract] [hide abstract]
ABSTRACT: Design and evaluate a new micro-machining based approach for fabricating dissolvable microneedle arrays (MNAs) with diverse geometries and from different materials for dry delivery to skin microenvironments. The aims are to describe the new fabrication method, to evaluate geometric and material capability as well as reproducibility of the method, and to demonstrate the effectiveness of fabricated MNAs in delivering bioactive molecules. Precise master molds were created using micromilling. Micromolding was used to create elastomer production molds from master molds. The dissolvable MNAs were then fabricated using the spin-casting method. Fabricated MNAs with different geometries were evaluated for reproducibility. MNAs from different materials were fabricated to show material capability. MNAs with embedded bioactive components were tested for functionality on human and mice skin. MNAs with different geometries and from carboxymethyl cellulose, polyvinyl pyrrolidone and maltodextrin were created reproducibly using our method. MNAs successfully pierce the skin, precisely deliver their bioactive cargo to skin and induce specific immunity in mice. We demonstrated that the new fabrication approach enables creating dissolvable MNAs with diverse geometries and from different materials reproducibly. We also demonstrated the application of MNAs for precise and specific delivery of biomolecules to skin microenvironments in vitro and in vivo.Pharmaceutical Research 08/2013; · 4.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Transplantation of allogeneic or xenogeneic skin grafts can evoke strong immune responses that lead to acute rejection of the graft tissues. In this process, donor-derived dendritic cells play crucial roles in the triggering of such immune responses. Both the innate and acquired host immune systems participate in graft rejection. At present, the rejection of skin grafts cannot be well-controlled by ordinary systemic immunosuppression therapy. Although several strategies for the long-term survival of allogeneic or xenogeneic skin grafts have been demonstrated in animal models, the induction of long-term tolerance to skin grafts is still a great challenge in clinical settings. In this article, we review the progress in the understanding of immune responses to skin grafts and discuss the possible methods that can decrease the immunogenicity of graft tissues and improve the survival of skin grafts, especially those included in preoperative pre-treatments.Archivum Immunologiae et Therapiae Experimentalis 05/2013; · 2.38 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 μm beneath the dermoepidermal junction), Mφ were located deeper (40-60 μm), and CD3(+) lymphocytes were observed throughout (0-60 μm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 μm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.Journal of Investigative Dermatology advance online publication, 19 December 2013; doi:10.1038/jid.2013.481.Journal of Investigative Dermatology 11/2013; · 6.19 Impact Factor
CD4?T Cell Responses Elicited by Different Subsets of
Human Skin Migratory Dendritic Cells1
Adrian E. Morelli,‡§J. Peter Rubin,‡Geza Erdos,* Olga A. Tkacheva,*†Alicia R. Mathers,*†
Alan F. Zahorchak,‡§Angus W. Thomson,†‡§Louis D. Falo, Jr.,* and Adriana T. Larregina2*†
Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we
have analyzed the CD4?T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin
explants, including CD1a?CD14?Langerhans’ cells (LC), CD1a?CD14?dermal DC (DDC), and CD1a?CD14?LC precursors.
Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-?1, IL-23p19, and
IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated
APC able to stimulate allogeneic naive CD4?T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4?
T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and
costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast,
CD1a?CD14?LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4?T
cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function
of CD14?LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with re-
sponding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the
function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations,
pathogen infections, or allograft transplantation. The Journal of Immunology, 2005, 175: 7905–7915.
surface of APC. Dendritic cells (DC)3are professional APC with
the unique ability to prime naive T cells (1). Immunogenic DC
stimulate and bias naive CD4?Th lymphocytes into Th1/Th2 cells
and naive CD8?CTL into T cytotoxic type 1 or type 2 cells,
respectively (2–6). By contrast, regulatory DC trigger apoptosis/
anergy of Ag-specific T cells and induce/amplify T regulatory
(Treg) cells (7–10). During the DC:T cell interaction, several fac-
tors affect the ability of DC to activate/bias Th cells, including 1)
the density/affinity of the MHC-peptide complex for the TCR, 2)
the level of costimulatory molecules expressed by DC, 3) the
length of DC:T cell contact, 4) the DC:T cell ratio, and 5) the
cytokines secreted by DC and neighboring cells (11–19). The se-
cretion of IL-12p70 or the presence of IL-4 induces differentiation
of Th1 and Th2 cells, respectively, whereas the release of IL-10
and TGF-?1 is associated with generation of Tregcells (19–21). In
o maintain self-tolerance or to initiate an immune re-
sponse against foreign Ag, T cells must recognize MHC
molecules loaded with self or non-self peptides on the
the steady state, immature/semimature DC trafficking constitu-
tively from peripheral tissues to secondary lymphoid organs are
responsible for maintenance of peripheral T cell tolerance (7–10).
By contrast, DC that have matured in response to proinflammatory
mediators and/or signaling through pathogen recognition receptors
are responsible for activation/polarization of T cells (19,
Considered one of the most immunogenic organs, the skin reacts
to antigenic stimuli by triggering inflammation and potent T cell
responses (26). These properties make the skin an ideal site for
vaccination, but cutaneous DC represent a major drawback for
acceptance of skin allografts (26–30). The immunogenic function
of the skin correlates with the high number of epidermal- and
dermal-resident DC (26–28). We and others have shown that the
DC population that migrates spontaneously from human skin ex-
plants (skin migratory DC (smiDC)) is heterogeneous (31, 32).
Based on their expression of CD1a and CD14, smiDC are classi-
fied as 1) CD1a?CD14?DC or Langerhans’ cells (LC), 2)
CD1a?CD14?DDC, and 3) CD1a?CD14?LC precursors (31,
32). Although LC (the prototype of peripheral tissue-resident DC)
have been the subject of numerous studies, the capacity of different
populations of human skin DC to stimulate, bias, or inhibit T cell
responses remains controversial. In humans, it is still unclear
whether 1) epidermal LC display different T cell stimulatory func-
tion than DDC; 2) skin DC produce bioactive IL-12p70; 3) skin
DC-derived IL-12p70 is critical for Th1-biased responses as de-
scribed for monocyte-derived DC (moDC) (20, 31–36); and 4) skin
harbors tolerogenic DC (31).
In the present study, we have demonstrated that smiDC include
DC at distinct stages of maturation and with different stimulatory
functions for allogeneic (allo) naive CD4?T cells. Despite these
differences, all smiDC expressed the secondary lymphoid organ-
homing receptor CCR7 and produced IL-10, TGF-?1, and IL-
23p19/IL-12/23p40, but did not secrete IL-12p70. Treatment of
*Department of Dermatology,†Department of Immunology, and‡Department of Sur-
gery, and§Thomas E. Starzl Transplantation Institute, University of Pittsburgh School
of Medicine, Pittsburgh, PA 15213
Received for publication April 28, 2005. Accepted for publication October 3, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the National Institutes of Health:
R01CA100893 and R21AI57958 (to A.T.L.); R01HL075512, R01HL077545,
R21HL69725, and R21AI55027 (to A.E.M.); U01AI056488 and R01AI06008 (to
L.D.F.); and R01AI41011 and AI51698 (to A.W.T.).
2Address correspondence and reprint requests to Dr. Adriana T. Larregina, Suite 145
Lothrop Hall, 190 Lothrop Street, Pittsburgh, PA 15213-2193. E-mail address:
3Abbreviations used in this paper: DC, dendritic cell; allo, allogeneic; DDC, dermal
DC; hu, human; LC, Langerhans cell; moDC, monocyte-derived DC; poly(I:C),
polynosinic:polycytidylic acid; rhu, recombinant human; RPA, RNase protection as-
say; Treg, T regulatory cell; smiDC, skin migratory DC.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
smiDC with DC1-driving signals (known to increase the produc-
tion of IL-12p70 by moDC) (19) failed to augment IL-12p70 se-
cretion. Regardless of their lack of IL-12p70, LC and DDC were
CD45ROhigh, CD62Llow, CCR7low) Th1 cells. The Th1-biasing
function of smiDC depended on their ability to produce IL-23,
because blockade of IL-12/23p40 or specific inhibition of (human)
IL-23 heterodimer by mAb abrogated IFN-? secretion by allo
CD4?T cells. The ability of LC and DDC to stimulate the pro-
liferation and cytokine secretion of allo CD4?T cells correlated
with the expression of MHC-II, CD11a, CD54, CD80, and CD86.
By contrast, CD14?smiDC secreted the highest amounts of IL-10
and TGF-?1 and were weakly stimulatory for allo CD4?T cells,
both characteristics previously reported in regulatory DC. How-
ever, CD14?smiDC became potent stimulators of allo CD4?T
cells and Th1 inducers when incubated at high DC:T cell ratios, for
longer periods of time with responder T cells, or in the presence of
recombinant human (rhu) IL-23 during MLC.
Materials and Methods
Isolation of smiDC and naive CD4?T cells
Samples of normal skin were obtained from healthy donors undergoing
abdominal plastic surgery. Human peripheral blood samples (leukopacks)
from healthy volunteers were obtained from the blood bank. Both skin and
blood samples were obtained under institutional review board approval and
were used according to the University of Pittsburgh Medical Center
SmiDC were obtained from human skin samples as previously described
(31). Skin explants composed of epidermis and a thin layer of dermis were
cultured, epidermal side up, on top of 1-mm pore size steel meshes and
placed in 100-mm tissue culture petri dishes (Falcon) with RPMI 1640
(Irvine Scientific) supplemented with 10% heat-inactivated normal human
AB serum (Nabi), 20 mM HEPES, 2 mM L-glutamine, 200 U/ml penicillin/
streptomycin (Invitrogen Life Technologies), and 20 ?g/ml gentamicin
(Sigma-Aldrich; complete medium) at 37°C in 5% CO2. Depending on the
experiment performed, total skin migratory cells were collected at 24, 48,
or 72 h after culture of skin explants. Cell viability was ?90% according
CD1a?CD14?, and CD1a?CD14?smiDC, total skin migratory cells were
first depleted of CD3?T cells by incubation with bead anti-CD3 mAb,
followed by negative selection by MACS (Miltenyi Biotec). Next, CD3-
depleted cells were incubated with bead anti-CD1a mAb to isolate CD1a?
smiDC by positive selection, and the negative fraction was incubated with
bead anti-CD14 mAb to purify CD14?smiDC by positive selection, in
both cases by passage through paramagnetic columns (Miltenyi Biotec).
The resulting negative fraction was composed of CD1a?CD14?smiDC.
Human CD4?CD45RA?T cells were purified from PBMC by negative
selection using human naive CD4 T cell enrichment columns (R&D Sys-
tems; purity, ?90% by flow cytometric analysis).
Generation of moDC
MoDC were generated from peripheral blood monocytes purified from
human PBMC by positive selection using immunomagnetic bead sort-
ing after incubation with bead-conjugated anti-CD14 mAb (Miltenyi
Biotec) according to the manufacturer’s protocol (?90% purity by flow
cytometry). Peripheral blood monocytes were cultured in T75 tissue
culture flasks (Falcon) in serum-free AIM-V medium (Invitrogen Life
Technologies) supplemented with rhuGM-CSF and rhuIL-4 (both 1000
kine secretion profile of smiDC. A,
SmiDC were individualized by their
high size and granularity (high for-
ward scatter (FSC) and side scatter
(SSC), respectively). Numbers in his-
tograms denote the percentage of
positive cells. One representative
study of five is shown. B, Pattern of
cytokines secreted by smiDC. C,
High amounts of IL-10, but no IL-
12p70, were detected in 10-fold con-
centrated supernatants of smiDC cul-
tured for 24 h. The means ? 1 SD of
five different experiments are dis-
played. D, After exposure to different
DC1-driving signaling smiDC signif-
icantly increased the secretion of IL-
12p40 (p ? 0.001), but they did not
secret significant levels of IL-12p70.
E, Under the same experimental con-
ditions, moDC secreted significant
high amounts of IL-12p70. NT, non-
treated DC. The means ? 1 SD of
three independent experiments are
Phenotype and cyto-
7906T CELL STIMULATORY FUNCTION OF HUMAN SKIN DC
IU/ml; R&D Systems) for 5 days. On day 3, 50% of the culture medium
was replaced by fresh medium supplemented with cytokines. On day 5,
the nonadherent cell fraction was harvested, and their phenotype was
analyzed by flow cytometry.
Analysis by flow cytometry
Skin migratory cells were blocked with normal human AB serum (1/10; 20
min) and incubated with PE-conjugated anti-HLA-DR, -CD1a, -CD40,
-CD80, -CD83, -CD86, or -CCR7 mAb in combination with FITC-conju-
gated anti-CD14 mAb (BD Pharmingen). T cells were labeled simulta-
neously with FITC-anti-CD4 mAb and PE-anti-CD3, -CD25, -CD69,
-CD45RO, -CD62L, or -CCR7 mAb (BD Pharmingen). Cells were fixed in
2% paraformaldehyde and analyzed by flow cytometry. Appropriate spe-
cies and Ig isotype controls were included.
The generation of moDC was assessed on day 5 DC by flow cytometric
analysis. MoDC were blocked with normal human AB serum (1/10; 20
min) and incubated with PE-conjugated anti-CD11c, HLA-DR, -CD40,
-CD80, -CD83, and -CD86 mAb in combination with FITC-anti-CD14
mAb (BD Pharmingen). A total of 80 ? 5% of cells showed a phenotype
of immature DC determined by their expression of CD11c; intermediate
levels of HLA-DR, CD80, and CD86; low CD40; and absence of CD83
and CD14 (data not shown).
MACS-purified CD1a?CD14?, CD1a?CD14?, or CD1a?CD14?smiDC
were spun onto slides using a Shandon cytocentrifuge (at 230 ? g), air
dried, and stained with May-Gru ¨nwald-Giemsa as previously described (31).
Proliferation of allogeneic naive T cells
CD1a?CD14?, or CD1a?CD14?smiDC were gamma irradiated (20 Gy)
and used as stimulators of naive CD4?CD45RA?T cells at different stimu-
lator:responder cell ratios in MLC. Cultures were maintained in 96-well,
round-bottomed plates for 5 or 7 days. For the final 18 h, individual wells
were pulsed with 1 ?Ci of [3H]thymidine. The amount of radioisotope
incorporated was determined using a beta scintillation counter. Assays
were performed in triplicate, and the results are expressed as the mean
cpm ? 1 SD.
(total) smiDCor MACS-purifiedCD1a?CD14?,
IL-5, IL-10, IL-12p40, IL-12p70, IFN-?, and TGF-?1 secretion was quan-
tified by ELISA (OptEIA (BD Pharmingen) and EmaxImmunoassay Sys-
tem (Promega)). Plates were analyzed using a Spectramax 340 PC plate
reader (Molecular Devices), and results were expressed as the mean ? 1
SD of duplicate wells. For smiDC, cytokine secretion was assessed in 24-h
culture supernatants of total smiDC or MACS-purified CD1a?CD14?,
CD1a?CD14?, or CD1a?CD14?smiDC. For some experiments, 24-h cul-
ture supernatants were concentrated using Centricom Plus-80 PL-10 cen-
trifugal filters (Millipore). For studies analyzing DC1 polarization, total
smiDC or moDC were cultured for 24 h with one or a combination of the
following DC1-driving stimuli: 1) LPS (500 ng/ml; Escherichia coli 011:
B4; Sigma-Aldrich), 2) polyinosinic:polycytidylic acid (poly(I:C); 20 ?g/
ml; Sigma-Aldrich), 3) rhuTNF-? (50 ng/ml), 4) rhuIL-1? (25 ng/ml; R&D
Systems), 5) agonist anti-huCD40 mAb (14G7; 10 ?g/ml; Caltag Labora-
tories), and 6) rhuIFN-? (1000 U/ml; R&D Systems). For T cell cytokine
secretion assays, smiDC were cocultured with responder naive CD4?T
cells at a DC:T cell ratio of 1:10. After 5 days of culture, the secretion of
cytokines was analyzed in culture supernatants by ELISA. Controls in-
cluded cytokine analysis of culture supernatants of T cells, smiDC, or
RNase protection assays (RPAs) for smiDC-derived cytokines
Total RNA was isolated from imunobead-sorted CD1a?CD14?or
CD1a?CD14?smiDC using a total RNA Isolation Kit (BD Pharmingen) as
described previously (25). cDNA encoding huIL-10, huIL-12p35, huIL-
12p40, huIL-23p19, and the housekeeping genes L32 and GAPDH were
used as templates for the T7 polymerase-directed synthesis of [?-32P]UTP-
labeled antisense RNA probes. Hybridization (16 h at 56°C) of each
mRNA with the antisense RNA probe sets was followed by RNase and
proteinase K treatment, phenol-chloroform extraction, and ammonium ac-
etate precipitation of protected RNA duplexes. In each RPA, the corre-
sponding antisense RNA probe set was included as the m.w. standard.
Yeast tRNA served as a negative control. Samples were electrophoresed on
acrylamide-urea sequencing gels. Quantification of bands was performed
by densitometry (Molecular Dynamics).
cell stimulatory and Th-biasing func-
tion of smiDC. A, Proliferation of
allo naive CD4?T cells stimulated
with smiDC (a representative exper-
iment of 42 is illustrated). B, The pat-
tern of cytokines secreted by re-
sponder CD4?T cells was mostly
Th1 biased (80.9% of samples stud-
ied). C, The comparative phenotype
between naive CD4?T cells (filled
histograms) and CD4?T cells stim-
ulated with total smiDC (thick lines)
is displayed. Negative controls in-
cluded species- and IgG isotype-
matched, PE-conjugated irrelevant
Ab. A representative result of four in-
dependent experiments is shown.
7907 The Journal of Immunology
of smiDC were distinguished: CD1a?CD14?(R1), CD1a?CD14?(R2), and CD1a?CD14?(R3). Cytological examination of these three populations
showed that CD1a?CD14?and CD1a?CD14?smiDC exhibit dendritic processes on their cell surface and bean-shaped nuclei (typical of mature
DC), whereas CD1a?CD14?smiDC showed fewer surface membrane processes, round nuclei, and cytoplasmic granules (May Gru ¨nwald Giemsa
stain; magnification, ?1000). B, CD1a?CD14?and CD1a?CD14?smiDC displayed a phenotype of mature DC, whereas CD1a?CD14?cells
showed a phenotype of immature/semimature DC. Numbers in histograms represent percentages of positive cells and mean fluorescence intensity
(in parentheses). A and B, One representative study of five is illustrated. C, Pattern of cytokines secreted by CD1a?CD14?, CD1a?CD14?, and
CD1a?CD14?smiDC. The mean ? 1 SD of triplicate results from one representative experiment of five are displayed.
Characterization of different populations of smiDC. A, According to their surface expression of CD1a and CD14, three populations
7908T CELL STIMULATORY FUNCTION OF HUMAN SKIN DC
Blockade of DC surface molecules and DC-derived cytokines
Inhibition of DC surface molecules was performed in dose-dependent fash-
ion by adding 25, 12.5, 6.25, or 3.12 ?g/ml purified nonazide/low endo-
toxin blocking anti-CD11a/LFA-1 (HI111), anti-CD54 (HA58), anti-CD80
(BB1), anti-CD86 (2331(FUN-1)), or anti-HLA-DR/DP/DQ (TU¨39) mAb
(BD Pharmingen). Inhibition of DC-secreted cytokines was performed by
adding an optimal concentration of anti-IL-12p40/p70 (10 ?g/ml; C11.5),
anti-IL-10 (25 ?g/ml; JES3-19F1; BD Pharmingen), anti-TGF-?1 mAb (25
?g/ml; 90.16.2; R&D Systems), or irrelevant Ig (as a control). Blocking
mAb or irrelevant Ig were added to MLC on days 1 and 3. Specific block-
ade of huIL-23p19 was performed by adding 0.1, 1, or 10 ?g/ml anti-
huIL-23 blocking mAb (clone MAB1290; R&D Systems), which binds
specifically the huIL-23 heterodimer without binding huIL-12p70 or huIL-
12p35. IL-23-blocking agents were added on days 1 and 3 during MLC.
Addition of rhuIL-23 to MLC using CD14?smiDC as
The effect of IL-23 on the T cell stimulatory and biasing function of
CD14?smiDC was determined by adding rhuIL-23 (100 ng/ml; R&D Sys-
tems) on days 1 and 3 of MLC.
Means ? 1 SD were compared by ANOVA, followed by the Student-
Newman-Keuls test. Comparisons between two different means ? 1 SD
from migration inhibitory assays were performed by Student’s t test. A
value of p ? 0.05 was considered significant.
Characterization of subsets of smiDC
We analyzed the phenotype and function of smiDC mobilized
from human epidermal/dermal explants. We and other groups have
demonstrated that this experimental model allows harvesting of
skin-resident DC and T lymphocytes that migrate spontaneously
from human skin through dermal lymphatic vessels (31, 32, 37,
38). SmiDC collected from skin explants (cultured for 48–72 h)
expressed high levels of surface MHC class-II (HLA-DR), CD86,
CD40, CD83, and the lymph node-homing receptor CCR7, as as-
sessed by flow cytometry (Fig. 1A). The bimodal distribution of
the fluorescence intensity of expression of these molecules indi-
cates that smiDC were at different states of maturation/activation.
Purified smiDC (by negative selection using MACS) secreted
IL-10 (350 ? 45 pg/106DC), TGF-?1 (453 ? 57 pg/106DC), very
low amounts of IL-12p40 (32 ? 3.5 pg/106DC), and no detectable
IL-12p70 (Fig. 1B), as determined in smiDC culture supernatants
by ELISA. To determine whether the absence of IL-12p70 un-
der our experimental conditions was due to protein degradation
or exhaustion of DC by the time point that smiDC were har-
vested from the explants (48–72 h after culture) (18), we as-
sessed the level of IL-12p70 in 10-fold concentrated culture
supernatants obtained 24 h after skin explant cultures. Although
the amount of IL-10 (measured as a positive control) increased
in concentrated culture supernatants, the levels of IL-12p70 re-
mained below the limit of detection, indicating that DC exhaus-
tion or cytokine degradation were not responsible for the lack of
IL-12p70 detection (Fig. 1C).
Next, we tested whether the absence of IL-12p70 secretion by
smiDC was due to the lack of exposure of smiDC to DC1-driving
signals during their mobilization from skin explants. To address
this question, we compared the abilities of smiDC and peripheral
blood moDC to secrete IL-12 p70 after DC1 signaling. SmiDC or
moDC from three different donors were exposed for 24 h to 1)
LPS; 2) poly(I:C); 3) IL-1? and TNF-?; 4) LPS, IL-1?, and
TNF-?; 5) IL-1?, TNF-?, and poly(I:C); or 6) agonist anti-CD40
mAb and IFN-?. Pretreatment of smiDC with DC1-driving stimuli
up-regulated the surface expression of HLA-DR, CD86, CD40,
and CD83 (data not shown) and significantly increased the secre-
tion of IL-12p40 (p ? 0.001), but did not induce the production of
IL-12p70 (Fig. 1D). However, exposure of moDC to the same
DC1-driving stimuli induced the secretion of high levels of IL-
12p70, ranging from 298 ? 10 pg/ml (agonist anti-CD40 mAb and
IFN-?) to 5395 ? 128 pg/ml (IL-1?, TNF-?, and poly(I:C); Fig.
1E), demonstrating that the lack of secretion of IL-12p70 by
smiDC was not caused by an inability of our experimental ap-
proach to detect IL-12 p70 secretion.
SmiDC stimulated consistently high proliferation of allo naive
(CD62Lhigh, CD45RAhigh) CD4?T cells in 5-day MLC (Fig. 2A).
Thirty-four (80.9%) of 42 samples of smiDC harvested from dif-
ferent skin donors induced a Th1 bias (IFN-?/IL-5 ratio, ?2.5), six
(14.2%) induced a Th2 response (IL-5/IFN-? ratio, ?2), and two
(4.7%) induced a mixed Th1/Th2 profile (IFN-?/IL-5 ratio, ?2;
Fig. 2B). These results demonstrate that smiDC unable to secrete
detectable levels of IL-12p70 are strong stimulators of allo CD4?
T cells that exhibit a predominant Th1-biased response (Fig. 2B).
In all cases, the subpopulation of responder T cells up-regulated
the activation markers CD25 and CD69, acquired the T cell mem-
ory marker CD45RO, and decreased the lymph node-homing re-
ceptor CCR7 and CD62L (Fig. 2C).
smiDC. A, CD1a?CD14?smiDC and CD1a?CD14?smiDC stimulated
stronger proliferation of CD4?T cells than that induced by CD1a?CD14?
smiDC in 5-day MLC. B, The pattern of cytokine secretion by responder
T cells stimulated by different smiDC revealed that
CD1a?CD14?smiDC and CD1a?CD14?smiDC stimulated greater se-
cretion of IFN-? by responder T cells compared with that induced by
CD1a?CD14?smiDC. The mean ? 1 SD of triplicate results from one
representative experiment of three are displayed.
CD4?T cell stimulatory function of different subsets of
7909The Journal of Immunology
T cell allostimulatory function of subsets of smiDC
Because smiDC include APC at distinct stages of maturation/ac-
tivation, we analyzed whether these subsets of smiDC exhibit dif-
ferent T cell allostimulatory function. Based on the surface ex-
pression of CD1a and CD14, smiDC are composed of the
following cellular subpopulations: 1) CD1a?CD14?epidermal
LC; 2) CD1a?CD14?DDC, and 3) CD1a?CD14?LC precursors
(Fig. 3A). Analysis by flow cytometry showed that LC and DDC
were mature/activated APC with similar high levels of HLA-DR,
CD80, CD86, CD40, and CD83. CD1a?CD14?LC precursors
expressed similar amounts of CD80 to LC and DDC and lower
amounts of HLA-DR, CD86, and CD40; most were CD83?(Fig.
3B). SmiCD14?DC did not adhere to plastic surfaces and differed
from peripheral monocytes as described previously (31, 32). These
phenotypic differences between CD14?and CD14?smiDC indi-
cate that unlike CD14?cells, CD14?smiDC were immature/semi-
mature APC. All smiDC expressed CCR7, an indication that these
cells were capable of homing to skin draining lymph nodes re-
gardless of their maturation state (Fig. 3B).
We then compared the ability of each smiDC subpopulation to
1) secrete cytokines that polarize (IL-12p70) or suppress the T cell
response (IL-10 and TGF-?1), and 2) induce proliferation and po-
larization of allo naive CD4?T cells.
MACS-purified CD1a?CD14?(LC), CD1a?CD14?(DDC),
and CD1a?CD14?(LC precursors) cells (purity, ?90%) were cul-
tured for 24 h, and the level of cytokines secreted in culture su-
pernatants was assessed by ELISA. The three subsets of smiDC
secreted IL-10 and TGF-?1, very low amounts of IL-12p40, and
no IL-12p70 (Fig. 3C). CD14?smiDC produced the highest
amounts of IL-10 and TGF-?1 (p ? 0.001 and p ? 0.05, respec-
tively; Fig. 3C).
Both LC and DDC triggered high proliferation of allo naive
CD4?T cells in 5-day MLC, whereas CD14?smiDC induced
only a weak CD4?T cell alloresponse (Fig. 4A). In all cases,
responder T cells up-regulated CD69, CD70, and CD154
(CD40L); acquired the T cell memory marker CD45RO; and de-
creased CCR7 and CD62L (data not shown). LC and DDC induced
a potent Th1 alloresponse, whereas CD14?smiDC stimulated a
lower secretion of IFN-?, resulting in a mixed Th1/Th2 allore-
sponse (Fig. 4B). Thus, smiDC are composed of subsets of pro-
fessional APC at distinct stages of maturation/activation and with
different abilities to release key immunoregulatory cytokines and
bias the Th alloresponse.
CD14?smiDC acquire a similar T cell stimulatory ability as
We have demonstrated previously that the subpopulation of
CD1a?CD14?smiDC represents immature precursors with the
ability to differentiate into LC in response to TGF-?1 (31). In this
study we compared the ability to secrete cytokines and stimulate T
cells of CD14?smiDC with that of CD14?smiDC (LC plus
DDC). For this analysis, we did not discriminate between LC and
DDC because both smiDC induced similar proliferation and Th
polarization of allo naive CD4?T cells. After purification and
functions of CD14?and CD14?smiDC.
A, RPA showing that CD14?and CD14?
smiDC transcribed IL-10 and IL-23p19
and IL-12/23 p40 mRNA. Neither CD14?
nor CD14?smiDC synthesized IL-12p35
mRNA. B, Densitometric analysis showed
that CD14?smiDC synthesized higher
amounts of IL-10 and lower amounts of
IL-23p40 and IL-23p19 than CD14?
smiDC. A and B show representative ex-
periments of three performed. C, Secretion
of IL-10, TGF-?1, IL-12p70, and IL-
12p40 was confirmed by ELISA. CD14?
amounts of TGF-?1 and IL-10 than
CD14?smiDC. D, CD14?smiDC are high
stimulators, whereas CD14?smiDC were
low stimulators of allo naive CD4?T cells.
The mean ? 1 SD of triplicate results from
one representative experiment is shown.
Comparative analysis of the
7910 T CELL STIMULATORY FUNCTION OF HUMAN SKIN DC
24-h culture, CD14?smiDC produced higher levels of IL-10
mRNA/protein and TGF-?1 protein and lower amounts of mRNA
transcripts for IL-23p19 and IL-12/23p40 than CD14?cells (Fig.
5, A–C). We did not detect IL-12p35 mRNA/protein in any subset
of smiDC (Fig. 5, A–C). CD14?smiDC induced greater prolifer-
ation of allo naive CD4?T cells than CD14?smiDC in 5-d MLC
(Fig. 5D). Together, these results suggest that CD14?smiDC
might exert a regulatory effect on T cell responses. If so, increasing
the number of CD14?smiDC in MLC would correlate with stron-
ger inhibition of the Th cell response in vitro. To address this
question, we performed 5-day MLC using smiDC at higher APC:T
cell ratios than usually examined. Under these conditions, allo na-
ive CD4?T cell proliferation and IFN-? secretion increased sig-
nificantly when CD14?smiDC were used as stimulators at higher
APC:T cell ratios (p ? 0.001; Fig. 6, A and B).
Next, we investigated whether CD14?smiDC require a longer
period of incubation with T cells to develop into potent APC. As
shown in Fig. 6, C and D, CD14?smiDC increased the prolifer-
ation and IFN-? secretion of allo naive CD4?T cells when used
as stimulators in 7-day MLC compared with 5-day MLC (p ?
0.0001). Together, the previous results demonstrate that CD14?
smiDC represent immature/semimature APC that are able to ac-
quire the function of fully mature, highly stimulatory DC.
Variables that influence the T cell stimulatory function of
Our results demonstrated that CD14?smiDC (LC and DDC) that
were not exposed to DC1-driving signals and did produce IL-23,
but did not secrete IL-12p70, were able to induce a Th1-biased
response. In addition, greater secretion of IFN-? by responding T
cells correlated with the higher numbers of stimulatory CD14?
smiDC during 5-day MLC. Thus, we next investigated what fac-
tors may control the capacity of CD14?smiDC to stimulate/bias
allo naive CD4?T cells. We focused on the role of MHC class II,
adhesion, and costimulatory molecules and cytokines produced by
Inhibition of MHC-II (HLA-DR/DP/DQ), CD54 (ICAM-1),
or the CD54 ligand CD11a (the latter not shown) with blocking
mAb decreased the proliferation of and secretion of IFN-? and
IL-5 (p ? 0.001) by allo naive CD4?T cells in a dose-
dependent manner (Fig. 7, A–C). Simultaneous blockade of
CD80 and CD86 completely abrogated T cell proliferation (p ?
0.001) and secretion of IFN-? and IL-5 (p ? 0.001; Fig. 7, A
and D). By contrast, inhibition of IL-12p40, TGF-?1, or IL-10
by neutralizing mAb did not affect the proliferation of allo naive
CD4?T cells (p ? 0.259; Fig. 8A). Blockade IL-12/23p40
proliferation induced by CD14?smiDC (p ? 0.0001). However, CD4?T cell stimulation induced by CD14?smiDC increased with higher DC:T ratios
(p ? 0.001). B, Increase in the allo CD4?T stimulatory function of CD14?smiDC correlated with greater secretion of IFN-? by responder T cells. C and
D, The allo CD4?T cell stimulatory function of CD14?smiDC was increased by prolonging the duration of DC:T cell contact. C, Proliferation of allo
CD4?T cells increased 8-fold in 7-day MLC compared with 5-day MLC (p ? 0.0001); D, secretion of IFN-? increased 2-fold in 7-day MLC compared
with 5-day MLC. A–D, The mean ? 1 SD of triplicate results from representative experiments are illustrated. Three independent experiments were
Allostimulatory function of CD14?smiDC. A, The proliferation of allo naive CD4?T cells induced by CD14?smiDC was higher than the
7911 The Journal of Immunology
significantly diminished the secretion of IFN-? (p ? 0.0001)
without affecting IL-5 production. Inhibition of TGF-?1 or
IL-10 did not influence IFN-? or IL-5 secretion compared with
controls (p ? 0.05; Fig. 8B). Taken together, these results
suggest that the ability of CD14?smiDC to induce the prolif-
eration of CD4?T cells depends on the levels of MHC class II,
CD54, and CD80/86 on the DC surface, whereas their ability to
induce the secretion of IFN-? by responder T cells relies mainly
on the production of IL-23.
Role of IL-23 in the T cell stimulatory capacity of smiDC
The role played by IL-23 in the T cell activation induced by dif-
ferent smiDC populations was analyzed by adding rhuIL-23 to
MLC stimulated with CD14?smiDC or by specifically blocking
IL-23 in those MLC stimulated with CD14?smiDC. Addition of
rhuIL-23 significantly enhanced the proliferation and IFN-? secre-
tion of responder allo naive CD4?T cells stimulated by CD14?
smiDC in 5-day MLC (Fig. 9, A and B).
Specific blockade of IL-23 was performed by using an anti-
huIL-23-neutralizing mAb (clone MAB1290; R&D Systems),
which binds specifically to the soluble huIL-23 heterodimer with-
out interacting with huIL-12p70 or huIL-12p35. As shown in Fig.
9C, IL-23 mAb significantly inhibited, in a dose-dependent man-
ner, the ability of CD14?smiDC to induce secretion of IFN-? by
responder allo naive CD4?T cells. Together, these results dem-
onstrate that IL-23 plays a critical role in the T cell stimulatory and
Th1-biasing capabilities of smiDC.
Peripheral tissue-resident DC have been implicated in both the
initiation of T cell immunity and peripheral T cell tolerance (1).
Based on the plasticity of these professional APC, it has been
proposed that mature DC that express high levels of MHC and
costimulatory molecules and are able to secrete proinflammatory
cytokines are immunogenic DC (22). Conversely, immature/semi-
mature DC with low amounts of surface MHC and costimulatory
molecules, bearing lymph node-homing receptors, and secreting
IL-10 and/or TGF-?1 have been described as tolerogenic/regula-
tory DC. In fact, it has been shown in vitro that immature human
moDC have the ability to generate allo Tregcells (33, 39).
Immunogenic DC that have undergone type 1 polarization
(known as DC1) are responsible for driving IFN-?-secreting Th1
cells (19, 40). IL-12p70, a cytokine produced in high amounts by
in vitro-generated human moDC, is considered the critical factor
that directs Th1 polarization (40). Accordingly, studies to promote
Th1 responses for the purpose of vaccine development are cur-
rently focused on the generation of IL-12p70-producing DC1 (40).
Conversely, in vitro generation of regulatory DC secreting IL-10
and TGF-?1 is being pursed to prevent/ameliorate allograft rejec-
tion and treat autoimmune disorders (21, 22, 33, 39).
Despite the fact that LC and DDC are prototypic peripheral
tissue-resident DC with extraordinary capacity to stimulate allo
naive CD4?T cells, several aspects of the mechanisms that stim-
ulate/bias the Th response by smiDC remain controversial, in par-
ticular those regarding the secretion of IL-12p70 (20, 34–36).
Herein, we analyzed the allo CD4?T cell stimulatory/biasing
function of smiDC mobilized spontaneously from normal human
skin explants. Surprisingly, smiDC secreted high amounts of
TGF-?1 and IL-10, but not IL-12p70. In the present study the
absence of IL-12p70 was not due to DC “exhaustion” (18), cyto-
kine degradation, the lack of exposure to DC1-driving signals, or
the inability to detect IL-12p70 under our experimental conditions,
as demonstrated by the high amounts of IL-12p70 secreted by
moDC exposed to the same DC1-driving signals as smiDC. Al-
though the lack of IL-12p70 by smiDC might be ascribed to the
fact that in our model DC were terminally differentiated and there-
fore resistant to DC1-polarizing signals, a recent publication has
shown that freshly isolated human LC (which did not undergo
terminal differentiation) did not produce IL-12p70 in response to
signaling through the CD40 molecule (34). Accordingly, our data
indicate that smiDC were responsive to DC1-polarizing signals,
but instead of secreting IL-12p70, they increased the secretion of
IL-12p40, TGF-?1, and IL-10. Interestingly, in our model smiDC
unable to secrete IL-12p70 induced potent Th1-polarized re-
sponses when cultured with allo naive CD4?T cells.
volved in the immunological synapse
between smiDC and T cells in the
CD4?T cell alloresponse. A–C, Spe-
cific inhibition of CD54 or HLA-DR/
DP/DQ by blocking mAb diminished
the proliferation (A) and secretion of
IFN-? and IL-5 (B and C) by allo na-
ive CD4?T cells in a dose-dependent
manner. Simultaneous blockade of
CD80 and CD86 abrogated both allo
CD4?T cell proliferation (A) and
IFN-? and IL-5 secretion (D) even at
very low concentrations of blocking
Role of molecules in-
7912T CELL STIMULATORY FUNCTION OF HUMAN SKIN DC
Several groups have documented that mature human moDC are
immunogenic APC that elicit Th1-biased allo responses, whereas
their immature counterparts induce T cell tolerance (22, 33, 39).
Because smiDC include DC at different stages of maturation, it is
tempting to hypothesize that mature LC and DDC are the immu-
nogenic migratory DC of the skin, whereas the more immature
CD14?DC population (secreting high amounts of IL-10 and TGF-
?1) might play a role during the induction/maintenance of periph-
eral T cell tolerance. Although we confirmed that LC and DDC
trigger potent allo CD4?T cell proliferation and Th1 differentia-
tion, we were unable to generate CD4?Tregcells using CD14?
DC as APC. Moreover, CD14?DC induced strong allo CD4?T
cell proliferation and Th1 biasing at high APC:T cell ratios or after
prolonged interaction with T cells. These results indicate that
CD14?DC are immature APC with the ability to develop a T cell
allostimulatory function similar to that induced by LC and DDC.
Moreover, we demonstrated that the potent T cell stimulatory func-
tion of LC and DDC correlates with their APC maturation stage,
because blockade of MHC class II, adhesion, or costimulatory
molecules resulted in abrogation of allo CD4?T cell proliferation
and secretion of IFN-? and IL-5.
In our system the secretion of IL-23 and not IL-12p70 was re-
sponsible for the secretion of IFN-? by responder naive CD4?T
cells. Our comparative studies demonstrated that CD14?and, to a
lesser extent, CD14?smiDC transcribed IL-23p19 mRNA and se-
creted IL-12/23p40 protein. Importantly, blockade of IL-12/23p40
or specific inhibition of IL-23 resulted in significant decrease in
IFN-? secretion by responder allo CD4?T cells. In addition, IL-23
was responsible for increasing the T cell stimulatory and biasing
function of CD14?smiDC.
In mice, IL-12p70 and IL-23 exhibit complementary functions,
whereas IL-12p70 exerts its effects mainly on naive T cells, IL-23
plays a key role during the generation of T cell memory (41–44).
However in humans, in vitro studies with moDC have suggested
that IL-23 may affect the function of naive and memory T cells
(42). Our results demonstrate that in humans and in the absence of
rived cytokines on proliferation and
Th bias of responding allo CD4?T
cells. A, Blockade of IL-12/23p40,
TGF-?1, or IL-10 did not modify the
proliferation of T cells (p ? 0.259).
B, The secretion of IFN-? by re-
sponder T cells was significantly in-
hibited by blockade of IL-12/23p40
(p ? 0.0001) and was not influenced
by TGF-?1 or IL-10 blockade com-
pared with control cultures in the
matched Ig (p ? 0.05). The secretion
of IL-5 by responder T cells was not
affected by cytokine blockade. The
mean ? 1 SD of triplicate results
from one representative experiment
of three are illustrated.
Effects of smiDC-de-
7913 The Journal of Immunology
IL-12p70, smiDC producing IL-23 induce differentiation of naive
CD4?T cells into effector/memory IFN-?-secreting cells. Inter-
estingly, and in agreement with our results, recent reports have
suggested the importance of IL-23 in the generation of Th1-me-
diated cutaneous immunity as well as IFN-? secretion by cord
blood-derived CD4?T cells (45–47). Besides the role of IL-23, it
is possible that other DC-derived factors that influence the signal-
ing of responding T cells at the immunological synapse may be
coresponsible for the Th1-driving capacity that smiDC exhibited in
80.9% of our samples. Variations in the affinity/density of MHC
class II-peptide complexes for the TCR and/or expression of dif-
ferent ligands by the APC (e.g., Notch ligands) may explain why
in a small percentage of skin samples, smiDC induced Th2-polar-
ized or mixed Th1/Th2 responses (11–18, 48).
Our data demonstrate that both immature and mature DC that
spontaneously migrate from human skin explants become potent
stimulators of allo naive CD4?T cells in vitro. Our results seem
to contradict previous observations regarding the potential ability
of immature DC to induce T cell tolerance. However, in our ex-
perimental model CD14?smiDC represent nonterminally differ-
entiated APC able to undergo further maturation and become po-
tent APC. In addition, the presence of an immature APC
phenotype and secretion of high levels IL-10 and TGF-?1 by
CD14?smiDC did not suffice for induction of anergy, immuno-
deviation, or Tregcells against alloantigen. To become tolerogenic
APC before leaving the skin, immature CD14?smiDC may need
to receive tolerogenic signals (e.g., exposure to regulatory cyto-
kines/neuropeptides/complement factors, UV-B irradiation, or li-
gands present on the surface of apoptotic cells) (49–52).
In summary, our findings support the hypothesis that the human
skin is populated with heterogeneous populations of DC that mi-
grate via lymphatic vessels at different stages of maturation. All
smiDC have the potential to stimulate IFN-? secretion by CD4?
allo T cells. This potent T cell stimulatory function of smiDC may
be explained by their plasticity, which makes them able to function
as biosensors of the cutaneous microenvironment able to recognize
self from non-self Ag. The immunological mechanisms by which
smiDC stimulate or suppress allo T cell responses may differ from
those used by in vitro-generated human moDC, as demonstrated by
the lack of IL-12p70 secretion. Taken together, our results shed
light on the complex network of skin-resident DC that is set in
motion after transplantation of skin allografts, cutaneous immuni-
zations, and pathogen infections.
The authors have no financial conflict of interest.
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