Cells Subsets of Human Skin Migratory Dendritic T Cell Responses ...

The Journal of Immunology

CD4ⴙ 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 CD4ⴙ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1aⴙCD14ⴚ Langerhans’ cells (LC), CD1aⴚCD14ⴚ dermal DC (DDC), and CD1aⴚCD14ⴙ 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 CD4ⴙ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4ⴙ 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, CD1aⴚCD14ⴙ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4ⴙ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14ⴙ 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. The Journal of Immunology, 2005, 175: 7905–7915.

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o maintain self-tolerance or to initiate an immune response against foreign Ag, T cells must recognize MHC molecules loaded with self or non-self peptides on the surface of APC. Dendritic cells (DC)3 are 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 factors 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 secretion 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 Treg cells (19 –21). In *Department of Dermatology, †Department of Immunology, and ‡Department of Surgery, 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. 1 This 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.). 2 Address correspondence and reprint requests to Dr. Adriana T. Larregina, Suite 145 Lothrop Hall, 190 Lothrop Street, Pittsburgh, PA 15213-2193. E-mail address: [email protected] 3 Abbreviations 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 assay; Treg, T regulatory cell; smiDC, skin migratory DC.

Copyright © 2005 by The American Association of Immunologists, Inc.

the steady state, immature/semimature DC trafficking constitutively 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, 20, 22–25). 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 explants (skin migratory DC (smiDC)) is heterogeneous (31, 32). Based on their expression of CD1a and CD14, smiDC are classified 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 function than DDC; 2) skin DC produce bioactive IL-12p70; 3) skin DC-derived IL-12p70 is critical for Th1-biased responses as described 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 organhoming receptor CCR7 and produced IL-10, TGF-␤1, and IL23p19/IL-12/23p40, but did not secrete IL-12p70. Treatment of 0022-1767/05/$02.00

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T CELL STIMULATORY FUNCTION OF HUMAN SKIN DC

smiDC with DC1-driving signals (known to increase the production of IL-12p70 by moDC) (19) failed to augment IL-12p70 secretion. Regardless of their lack of IL-12p70, LC and DDC were able to induce activated/memory (CD25high, CD69high, 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 proliferation 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. However, 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.

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 to trypan blue exclusion. For purification of CD1a⫹CD14⫺, 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, CD3depleted 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 Systems; purity, ⬎90% by flow cytometric analysis).

Materials and Methods

Generation of moDC

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 guidelines.

MoDC were generated from peripheral blood monocytes purified from human PBMC by positive selection using immunomagnetic bead sorting 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

Isolation of smiDC and naive CD4⫹ T cells

FIGURE 1. Phenotype and cytokine secretion profile of smiDC. A, SmiDC were individualized by their high size and granularity (high forward scatter (FSC) and side scatter (SSC), respectively). Numbers in histograms 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 IL12p70, were detected in 10-fold concentrated supernatants of smiDC cultured for 24 h. The means ⫾ 1 SD of five different experiments are displayed. D, After exposure to different DC1-driving signaling smiDC significantly increased the secretion of IL12p40 (p ⬍ 0.001), but they did not secret significant levels of IL-12p70. E, Under the same experimental conditions, moDC secreted significant high amounts of IL-12p70. NT, nontreated DC. The means ⫾ 1 SD of three independent experiments are displayed.

The Journal of Immunology 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-conjugated anti-CD14 mAb (BD Pharmingen). T cells were labeled simultaneously 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 species 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).

7907 Cytokine detection IL-5, IL-10, IL-12p40, IL-12p70, IFN-␥, and TGF-␤1 secretion was quantified by ELISA (OptEIA (BD Pharmingen) and Emax Immunoassay System (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 culture supernatants were concentrated using Centricom Plus-80 PL-10 centrifugal 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 Laboratories), 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 included cytokine analysis of culture supernatants of T cells, smiDC, or medium alone.

Cytospin preparation 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 CD3-depleted (total) smiDC or MACS-purified CD1a⫹CD14⫺, CD1a⫺CD14⫺, or CD1a⫺CD14⫹ smiDC were gamma irradiated (20 Gy) and used as stimulators of naive CD4⫹CD45RA⫹ T cells at different stimulator: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.

FIGURE 2. Allogeneic CD4⫹ T cell stimulatory and Th-biasing function of smiDC. A, Proliferation of allo naive CD4⫹ T cells stimulated with smiDC (a representative experiment of 42 is illustrated). B, The pattern of cytokines secreted by responder CD4⫹ T cells was mostly Th1 biased (80.9% of samples studied). C, The comparative phenotype between naive CD4⫹ T cells (filled histograms) and CD4⫹ T cells stimulated with total smiDC (thick lines) is displayed. Negative controls included species- and IgG isotypematched, PE-conjugated irrelevant Ab. A representative result of four independent experiments is shown.

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, huIL12p40, huIL-23p19, and the housekeeping genes L32 and GAPDH were used as templates for the T7 polymerase-directed synthesis of [␥-32P]UTPlabeled 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 acetate precipitation of protected RNA duplexes. In each RPA, the corresponding 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).

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FIGURE 3. Characterization of different populations of smiDC. A, According to their surface expression of CD1a and CD14, three populations 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.


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Blockade of DC surface molecules and DC-derived cytokines Inhibition of DC surface molecules was performed in dose-dependent fashion by adding 25, 12.5, 6.25, or 3.12 ␮g/ml purified nonazide/low endotoxin blocking anti-CD11a/LFA-1 (HI111), anti-CD54 (HA58), anti-CD80 ¨ 39) mAb (BB1), anti-CD86 (2331(FUN-1)), or anti-HLA-DR/DP/DQ (TU (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 blockade of huIL-23p19 was performed by adding 0.1, 1, or 10 ␮g/ml antihuIL-23 blocking mAb (clone MAB1290; R&D Systems), which binds specifically the huIL-23 heterodimer without binding huIL-12p70 or huIL12p35. IL-23-blocking agents were added on days 1 and 3 during MLC.

Addition of rhuIL-23 to MLC using CD14⫹ smiDC as stimulators 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 Systems) on days 1 and 3 of MLC.

Statistical analysis Means ⫾ 1 SD were compared by ANOVA, followed by the StudentNewman-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.

Results 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 assessed by flow cytometry (Fig. 1A). The bimodal distribution of the fluorescence intensity of expression of these molecules indicates that smiDC were at different states of maturation/activation. Purified smiDC (by negative selection using MACS) secreted IL-10 (350 ⫾ 45 pg/106 DC), TGF-␤1 (453 ⫾ 57 pg/106 DC), very low amounts of IL-12p40 (32 ⫾ 3.5 pg/106 DC), and no detectable IL-12p70 (Fig. 1B), as determined in smiDC culture supernatants by ELISA. To determine whether the absence of IL-12p70 under our experimental conditions was due to protein degradation or exhaustion of DC by the time point that smiDC were harvested from the explants (48 –72 h after culture) (18), we assessed 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 remained below the limit of detection, indicating that DC exhaustion 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 secretion of IL-12p40 ( p ⬍ 0.001), but did not induce the production of

FIGURE 4. CD4⫹ T cell stimulatory function of different subsets of 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 allo CD4⫹ T cells stimulated by different smiDC revealed that CD1a⫹CD14⫺ smiDC and CD1a⫺CD14⫺ smiDC stimulated greater secretion 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.

IL-12p70 (Fig. 1D). However, exposure of moDC to the same DC1-driving stimuli induced the secretion of high levels of IL12p70, 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 approach 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 different 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 memory marker CD45RO, and decreased the lymph node-homing receptor CCR7 and CD62L (Fig. 2C).

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FIGURE 5. Comparative analysis of the 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 experiments of three performed. C, Secretion of IL-10, TGF-␤1, IL-12p70, and IL12p40 was confirmed by ELISA. CD14⫹ smiDC secreted significantly higher 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. Five independent experiments were performed.

T cell allostimulatory function of subsets of smiDC Because smiDC include APC at distinct stages of maturation/activation, we analyzed whether these subsets of smiDC exhibit different T cell allostimulatory function. Based on the surface expression 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 indicate that unlike CD14⫺ cells, CD14⫹ smiDC were immature/semimature APC. All smiDC expressed CCR7, an indication that these cells were capable of homing to skin draining lymph nodes regardless 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 polarization of allo naive CD4⫹ T cells. MACS-purified CD1a⫹ CD14⫺ (LC), CD1a⫺CD14⫺ (DDC), and CD1a⫺CD14⫹ (LC precursors) cells (purity, ⱖ90%) were cultured for 24 h, and the level of cytokines secreted in culture supernatants 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, respectively; 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 decreased 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 alloresponse (Fig. 4B). Thus, smiDC are composed of subsets of professional 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 CD14⫺ smiDC 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

The Journal of Immunology 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 proliferation 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 stronger 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 naive CD4⫹ T cell proliferation and IFN-␥ secretion increased significantly 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 proliferation 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 acquire the function of fully mature, highly stimulatory DC.

7911 Variables that influence the T cell stimulatory function of CD14⫺ smiDC 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 factors 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 CD14⫺ smiDC. 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 dosedependent 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

FIGURE 6. Allostimulatory function of CD14⫹ smiDC. A, The proliferation of allo naive CD4⫹ T cells induced by CD14⫺ smiDC was higher than the 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 performed.

7912


FIGURE 7. Role of molecules involved in the immunological synapse between smiDC and T cells in the CD4⫹ T cell alloresponse. A–C, Specific 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 naive CD4⫹ T cells in a dose-dependent manner. Simultaneous blockade of the DC costimulatory molecules 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 mAb.

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 proliferation 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 different 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-␥ secretion 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 antihuIL-23-neutralizing mAb (clone MAB1290; R&D Systems), which binds specifically to the soluble huIL-23 heterodimer without interacting with huIL-12p70 or huIL-12p35. As shown in Fig. 9C, IL-23 mAb significantly inhibited, in a dose-dependent manner, the ability of CD14⫺ smiDC to induce secretion of IFN-␥ by responder allo naive CD4⫹ T cells. Together, these results demonstrate that IL-23 plays a critical role in the T cell stimulatory and Th1-biasing capabilities of smiDC.

Discussion 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/semimature 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 Treg cells (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 currently 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 rejection 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 stimulate/bias the Th response by smiDC remain controversial, in particular 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), cytokine 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. Although the lack of IL-12p70 by smiDC might be ascribed to the fact that in our model DC were terminally differentiated and therefore 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 responses when cultured with allo naive CD4⫹ T cells.


7913

FIGURE 8. Effects of smiDC-derived 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 responder T cells was significantly inhibited by blockade of IL-12/23p40 (p ⬍ 0.0001) and was not influenced by TGF-␤1 or IL-10 blockade compared with control cultures in the presence of irrelevant, isotypematched 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.

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 immunogenic 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 peripheral T cell tolerance. Although we confirmed that LC and DDC trigger potent allo CD4⫹ T cell proliferation and Th1 differentiation, we were unable to generate CD4⫹ Treg cells 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 function 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 responsible 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 secreted 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

7914


FIGURE 9. Effects of IL-23 on the proliferation and IFN-␥ secretion of responding allo naive CD4⫹T cells. A and B, Addition of rhuIL-23 to MLC where CD14⫹smiDC were used as stimulators significantly increased both T cell proliferation (A) and secretion of IFN-␥ (B) of responder T cells. C, Specific inhibition of the huIL-23 heterodimer by antihuIL-23 neutralizing mAb significantly decreased IFN-␥ secretion by responder T cells in a dose-dependent manner. Isotype-matched Ig was included as a control. The secretion of IL-5 by responder T cells was not affected by IL-23 blockade. The mean ⫾ 1 SD of triplicate results from one representative experiment of four are illustrated.

IL-12p70, smiDC producing IL-23 induce differentiation of naive CD4⫹ T cells into effector/memory IFN-␥-secreting cells. Interestingly, and in agreement with our results, recent reports have suggested the importance of IL-23 in the generation of Th1-mediated 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 signaling 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 different ligands by the APC (e.g., Notch ligands) may explain why in a small percentage of skin samples, smiDC induced Th2-polarized 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 experimental model CD14⫹ smiDC represent nonterminally differentiated APC able to undergo further maturation and become potent 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, immunodeviation, or Treg cells against alloantigen. To become tolerogenic APC before leaving the skin, immature CD14⫹ smiDC may need to receive tolerogenic signals (e.g., exposure to regulatory cytokines/neuropeptides/complement factors, UV-B irradiation, or ligands 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 migrate 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 immunizations, and pathogen infections.

Disclosures The authors have no financial conflict of interest.

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