The Journal of

Maintenance of Peripheral Tolerance through Controlled Tissue Homing of -Specific T Cells in K14-mOVA Mice1

Teresa Bianchi,* Laura B. Pincus,2* Marc-Andre´ Wurbel,* Benjamin E. Rich,* Thomas S. Kupper,* Robert C. Fuhlbrigge,* and Marianne Boes3*†

Immunological tolerance is crucial to avoid autoimmune and inflammatory diseases; however, the mechanisms involved are incompletely understood. To study peripheral tolerance to skin-associated Ags, we generated new transgenic mice expressing a membrane-bound form of OVA in skin under the human keratin 14 (K14) promoter (K14-mOVA mice). In contrast to other transgenic mice expressing similar self-Ags in skin, adoptive transfer of Ag-specific T cells does not induce inflammatory skin disease in our K14-mOVA mice. OVA-specific T cells transferred into K14-mOVA mice are activated in lymphoid tissues, undergo clonal expansion, and eventually acquire effector function. Importantly, these Ag-specific T cells selectively up-regulate expression of E-selectin ligand in cutaneous lymph nodes but not in mesenteric lymph nodes and spleen, demonstrating that expression of endogenous self-Ags in skin dictates imprinting of skin tissue homing in vivo. However, an additional inflammatory signal, here induced by tape stripping, is required in K14-mOVA mice to induce migration to skin and development of inflammatory skin disease. Depletion of regulatory CD4؉CD25؉ T cells did not provoke homing of transferred T cells to skin under steady-state conditions, indicating that these cells are not the key regulators for inhibiting T cell homing in K14-mOVA mice. Both skin-derived and lymph node-resident CD8␣؉ dendritic cells are responsible for Ag presentation in vivo and induce tolerance to skin Ags, as we show by selective depletion of langerin؉ and CD11c؉ dendritic cells. Taken together, controlled skin homing of T cells is critical for the maintenance of peripheral to epidermal self-Ags. The Journal of Immunology, 2009, 182: 4665–4674.

endritic cells (DCs)4 play an important role in the de- cutaneous and mucosal immune response to infection and a prom- velopment of efficient and appropriate immune re- inent feature in the development of inflammatory skin disorders D sponses. They are highly efficient APCs that acquire Ag such as psoriasis, atopic dermatitis, allergic contact hypersensitiv- by continuously sampling their immediate surrounding tissues, in- ity, and cutaneous T cell lymphomas. In addition to DC migration cluding barrier tissues such as the skin and the intestines (1, 2). to skin, it is equally important that other leukocytes, such as T and DCs internalize and process Ags for presentation as peptide-MHC B , , and , also participate in complexes to naive T cells and thereby play a critical role in shap- the local immune response and therefore acquire the ability to ing immune responses. To this end, DCs need to display a correct migrate to peripheral tissues. profile of adhesion and tissue-homing molecules allowing them to The extravasation of leukocytes into tissue through endothelia migrate from peripheral blood to tissues and subsequently from involves a multistep cascade of molecular and cellular interactions, peripheral tissues to secondary lymphoid organs to encounter na- summarized as tethering, rolling, and activation by chemoattrac- ive T cells (3). Controlled DC migration is a crucial aspect of tants, adhesion, and diapedesis (4). The initial tethering and rolling steps are principally mediated by members of a family of type I *Department of Dermatology, Brigham and Women’s Hospital and Harvard Medical membrane proteins called selectins (5). E-selectin is constitutively † School, Boston, MA 02115; and Department of Pediatric Immunology, University expressed on human endothelium (6) and is up-regulated, along Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht, The Netherlands with P-selectin, in response to inflammatory stimuli (7–9). E- and Received for publication October 30, 2008. Accepted for publication February 22, 2009. P-selectin ligands (ESL and PSL) are critical for T cell trafficking The costs of publication of this article were defrayed in part by the payment of page to inflamed skin and together are considered key skin homing re- charges. This article must therefore be hereby marked advertisement in accordance ceptors on leukocytes. In humans, skin-homing effector/memory T with 18 U.S.C. Section 1734 solely to indicate this fact. cells express cutaneous -associated Ag, a carbohydrate 1 This research was supported by The Swiss National Science Foundation (Grant that serves as a ligand for E-selectin (10, 11) and that is PA00A3-119493, to T.B.), the Howard Hughes Medical Institute Research Training Fellowship for medical students (to L.P.), Crohn’s and Colitis Foundation of America recognized by the specific Ab HECA-452 (12). In mice, detection (to M.A.W.), the Harvard Skin Disease Research Center (to T.K.), and Netherlands of ESL with a recombinant fusion protein has allowed the eluci- Organization for Scientific Research-Veni Grant and RO1-AR052810 (to M.B.). dation of the role of ESL in homing to inflamed skin (13, 14). In 2 Current address: Department of Dermatology, University of California, San Fran- addition, the generation of mice deficient in E- and P-selectin has cisco, CA. clarified their importance in mediating the control of skin inflam- 3 Address correspondence and reprint requests to Dr. Marianne Boes, Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children’s matory disorders (7, 15). Hospital, Utrecht, The Netherlands. E-mail address: [email protected] Lymphocytes acquire their requisite tissue migratory capacity 4 Abbreviations used in this paper: DC, ; ESL, E-selectin ligand; PSL, during Ag stimulation in a lymphoid tissue environment. Indeed, T P-selectin ligand; CLN, cutaneous lymph node; MLN, mesenteric lymph node; BM, cells activated in cutaneous lymph nodes (CLN) will display skin- bone marrow; Tg, transgenic; SPL, spleen; LN, lymph node; mOVA, membrane- ␤ ␤ bound OVA; DT, diphteria toxin; WT, wild type; 2m, 2-microglobulin; Treg, reg- homing receptors, such as CCR4, ESL or PSL, whereas T cells ulatory T cell; LC, Langerhans cell; dDC, dermal DC; EGFP, enhanced GFP; ALN, activated in mesenteric lymph nodes (MLN) or Peyer’s patches auricular LN. ␣ ␤ principally express high levels of CCR9 and 4 7 and home pref- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 erentially to the gut (16, 17). The imprinting of tissue-homing www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803628 4666 PERIPHERAL TOLERANCE CONTROL THROUGH REGULATED T CELL HOMING receptors is dictated by local lymphoid organ DC subsets, as well ation signal from the human growth hormone gene was excised using re- as by the immunization route used (18, 19). Intracutaneous injec- striction enzymes. After purification by agarose gel electrophoresis, the tion of Ag-pulsed bone marrow (BM) DCs can, for example, in- 7-kb fragment was used to create Tg C57BL/6 mice by pronuclear injec- tion. The Tg allele was detected by PCR on genomic DNA prepared from duce the expression of skin-homing molecules on T cells (20). In tail biopsies. Seven founder mice were assessed for protein expression and vitro cultures of these skin-imprinted T cells with MLN-isolated two founder lines were selected for breeding and further analysis. DCs down-regulate ESL and induce CCR9 and ␣ ␤ expression, 4 7 Abs and flow cytometry demonstrating possible reprogramming of tissue-polarized T cells in the presence of specific DC subsets (20). Directly conjugated mAbs TCR␤-FITC (H57), MHC class II-FITC (M5/ In addition to their role in defining the homing properties of T 114.15.2), CD25-PECy5.5 (PC61), CD62L-allophycocyanin (MEL-14), ␣ cells during the induction of immune responses, DCs are involved CD4-PECy7 (RM4-5), CD8 -PECy7 (53-6.7) and CD44-PECy7 (IM7; eBioscience), MHC class II-PE (AF6-120.1), CD90.1-PerCP (OX7), in the maintenance of peripheral immune tolerance to self-Ags (21, CD11c-allophycocyanin (HL3) and CD4-allophycocyanin-Cy7 (GK1.5) 22). Under steady-state conditions, peripheral tolerance prevails by (BD Pharmingen), TCR␤-PE (H57), and B220-allophycocyanin (RA3- inactivation or deletion of autoreactive T cells, while in the context 6B2; BioLegend) were used to perform 6-color flow cytometry using a of infection and inflammation may be initiated. New FACSCanto flow cytometer (BD Biosciences) and data were analyzed us- ing CellQuest software (BD Biosciences). All standard stainings were per- strategies now allow for the study of the mechanisms of peripheral formed in 2.4G2 supernatant to block unspecific binding to Fc␥ receptors. tolerance in vivo to exogenous innocuous Ags. Such Ags can be ESL staining was performed using recombinant mouse E-selectin-human selectively delivered to DCs in the absence of inflammatory sig- Fc IgG chimera (R&D Systems) in HBSS supplemented with Ca2ϩ,Mg2ϩ, nals by use of chemically coupled exogenous proteins with Abs and 10 mM HEPES and was detected with anti-human IgG Fc␥ (Jackson targeting endocytic receptors expressed on DCs (23). The gener- ImmunoResearch Laboratories) as previously described (35). Each sample was subjected to 2 mM EDTA treatment as control for ESL-binding spec- ation of transgenic (Tg) mouse models expressing self-Ags under ificity. The absolute percentage of ESLϩ cells was determined by subtrac- a tissue-specific promoter also lead to great advances in this re- tion of the percentage of ESLϩ cells of the EDTA-treated sample. For DC search field (24, 25). Several Tg mice expressing epidermal self- sorting experiments, cells from CLN were first MACS purified according Ags were generated and allowed the study of immune responses to to the manufacturer’s protocol using anti-CD11c magnetic microbeads (Miltenyi Biotec), then stained with MHC class II-FITC, CD11c-allophy- skin-associated Ags (26–30). Interestingly, although these trans- cocyanin, and CD8␣-PECy7 mAbs and sorted using a FACSAria flow genic lines differ in the promoter used or in the structure of the cytometer (BD Biosciences). self-Ag expressed, they all developed inflammatory skin disease upon adoptive transfer of naive Ag-specific T cells (26–28). Adoptive transfers We developed K14-mOVA mice, a new Tg mouse model ex- Single-cell suspensions from SPL and LN were prepared. OT-I and OT-II T pressing OVA as a self-Ag in epidermis, to elucidate the imprint- cells (Thy1.1) were MACS purified according to the manufacturer’s protocol ing of skin tissue-homing characteristics on self-Ag-specific T using anti-CD8 and anti-CD4 magnetic microbeads (Miltenyi Biotec) and were checked for purity (Ͼ90%) by flow cytometry before transfer. Before cells. In addition, we propose to study the outcome of such im- CFSE staining, cells were washed twice with cold PBS and incubated at 10 ϫ printing and especially its possible role in induction of tolerance or 106 cells/ml in PBS with 2 ␮M CFSE (Molecular Probes) at 37°C for 10 min. inflammatory skin disease. In these K14-mOVA mice, no skin dis- Cells were then washed twice with cold DMEM/10% FCS. Five ϫ 106 cells/ ease was induced upon adoptive transfer of Ag-specific T cells. mouse were injected i.v. For blocking experiments, FTY720 (1 mg/kg; Cay- man Chemical) was injected i.p. 4 and 24 h after adoptive transfer of CFSE- Importantly, these OVA-specific T cells selectively up-regulated labeled OT-I and OT-II T cells. For DC depletion experiments, K14-mOVA- expression of ESL in CLN but not in MLN and spleen (SPL), CD11cDTR and K14-mOVA-Lang-DTR mice were injected i.p. with 0.25 or demonstrating that expression of endogenous self-Ags in skin can 1 ␮g/mouse diphteria toxin (DT; List Biological Laboratories), respectively dictate imprinting of skin tissue homing in vivo. However, an ad- 24 h before adoptive transfer of CFSE-labeled OT-I and OT-II T cells. For BM ditional inflammatory signal is required in K14-mOVA mice to chimeras experiments, 8 wk after reconstitution of lethally irradiated K14- mOVA mice (2 ϫ 600 rad with 3-h interval) with 10 ϫ 106 wild-type (WT) induce the migration of transferred Ag-specific T cells to skin and ␤ Ϫ/Ϫ or 2-m BM cells, CFSE-labeled OT-I T cells were adoptively transferred development of inflammatory skin disease. We identified skin-de- and mice were analyzed 48 h later. For (Treg) depletion, rived DCs and lymph ode (LN)-resident CD8␣ϩ DCs as the central anti-CD25 (PC61) mAb or control (200 ␮g/mouse; BioXCell) was players in the induction of tolerance in these mice. Therefore, our injected i.p. 5 and 3 days before adoptive transfer of OT-I T cells. Treg de- pletion was checked by flow cytometry using anti-CD25 (3C7; BioLegend) at new Tg model allowed us to show that controlled skin homing of the time of adoptive transfer and resulted in Ͼ85% depletion of CD4ϩCD25ϩ Ag-specific T cells is critical for the maintenance of peripheral Tregs. This treatment did not affect survival and proliferation of the transferred immune tolerance to epidermal self-Ag. OT-I T cells. In vivo CTL assay Materials and Methods Mice WT splenocytes were either pulsed with SIINFEKL peptide (1 ␮g/ml) for 1 h at 37°C or left untreated. The peptide-pulsed splenocytes were labeled C57BL/6, C57BL/6.PL, OT-I, and OT-II Tg (The Jackson Laboratory), with 10 ␮M CFSE, while the unpulsed splenocytes were labeled with 1 ␮M K14-mOVA (see below), CD11c-DTR (31), Lang-DTR and Lang-EGFP CFSE. The two populations were mixed at a 1:1 ratio and 10 ϫ 106 cells (32) mice were bred and maintained in the animal facility of Harvard Med- were injected i.v. into syngeneic WT and K14-mOVA mice that had been ical School (Boston, MA) under specific pathogen-free conditions. All adoptively transferred with OT-I T cells 3 days earlier. K14-mOVA recip- mouse procedures were approved by the Harvard Medical School Standing ient mice that did not receive OT-I T cells were used as control. After 5 h, Committee on the Use of Animals in Research. SPL were removed and the disappearance of the peptide-pulsed population was determined by flow cytometry. The percentage of specific lysis was Ϫ Generation of K14-mOVA Tg mice calculated with the following formula: 100 (((percent pulsedsample/per- cent unpulsed )/(percent pulsed /percent unpulsed )) ϫ 100). The membrane-bound form of OVA (mOVA) consists of a fusion protein sample ctrl ctrl containing aa 1–118 of the human transferrin transmembrane receptor Isolation of epidermal and whole ear skin cells fused to the amino acid sequence 139–386 of the chicken OVA (33). A DNA fragment containing the sequence encoding mOVA was prepared The dorsal and ventral ear halves were gently separated. For epidermal cell from pBlue-RIP-tfrOVA (provided by P. A. Gleeson, Monash University isolation, ear halves were floated dermal-side down on 0.25% trypsin/2.21 Medical School, Prahran, Australia) and inserted into the BamHI site, mM EDTA (Cellgro) for 30–45 min at 37°C. The epidermis was then which is 3Ј of the human keratin 14 promoter in the plasmid K14-HGX as peeled off, cut into small pieces, further incubated in IMDM/10% FBS for previously described (34). From the resulting plasmid, a 7-kb fragment 30 min at 37°C, and then filtered through a 70-␮m nylon filter as described containing the K14 promoter mOVA and the modified HGX polyadenyl- elsewhere (36). For in vitro culture, cells recovered from epidermal sheets The Journal of Immunology 4667

from one ear were split into three wells and incubated with 200,000 T cells was identical in CLN, MLN, and SPL in K14-mOVA mice, for 3 days in RPMI 1640supplemented with 10% FBS (Sigma-Aldrich), 10 whereas OT-II T cell proliferation was induced mainly in CLN and ␮ mM HEPES, 10 mM penicillin-streptomycin, and 50 M 2-ME (Invitro- significantly less in MLN and SPL of K14-mOVA mice (Fig. 1B). gen). For isolation of whole ear skin cells, ear halves of five mice were pooled, cut into small pieces, and incubated in HBSS/10% FBS with 5 mM No proliferation was observed when cells were transferred into EDTA for4hat4°C. Cells recovered were then filtered through a 70-␮m WT mice. Thus, in K14-mOVA mice, OT-II T cells were prefer- nylon filter. entially stimulated in CLN, whereas OT-I T cells appeared to be stimulated equally in CLN and other secondary lymphoid tissues. Skin grafts OT-I and OT-II T cells displayed activation markers when trans- Recipient WT mice were anesthetized with 1.25% Avertin (250 mg/kg; ferred into K14-mOVA mice, as shown by down-regulation of 2 Sigma-Aldrich) and shaved on the flank, after which a 1-cm piece of skin CD62L and up-regulation of CD44 and CD25 (Fig. 1C and data was removed. Back skin from shaved WT and K14-mOVA donor mice (sex matched) was removed, cut into 1-cm2 pieces, and clipped to recipient not shown). In addition, they eventually became functional effector skin using disposable skin staplers (3M). A solution of 0.9% sodium chlo- cells. Indeed, 3 days after transfer into K14-mOVA mice, but not ride (200 ␮l/mouse; Hospira) containing Banamine (1/250; Shering-Plough into WT mice, OT-I T cells acquired the ability to specifically kill Animal Health) was administered i.p. twice after surgery and mice were cells displaying their cognate antigenic peptide, as shown with an monitored daily for 5 days. Ten days after engraftment, CFSE-labeled OT-I T cells were adoptively transferred into grafted mice and analyzed 48 h in vivo CTL assay (Fig. 1E). Peptide-pulsed target cells transferred later. into K14-mOVA that did not receive OT-I T cells were unaffected. We observed that OT-I T cells in K14-mOVA mice had prolif- Skin inflammation by tape stripping erated to the same extent in CLN, MLN, and SPL, even though Mice were anesthetized with 1.25% Avertin. Each side of the ears was tape only the skin should express the transgene (Fig. 1B). This result stripped 12 times with regular adhesive tape (Staples) and painted with 25 prompted us to investigate whether OT-I T cells are preferentially ␮ l of acetone. stimulated in CLN, after which they rapidly recirculate to other Histology lymphoid organs, or whether OT-I T cell priming occurs in other secondary lymphoid tissues beside CLN. To address these ques- Skin samples were collected and fixed in 10% formalin. Embedding, sec- tions, and H&E staining were performed at the Specialized Histopathology tions, we treated K14-mOVA mice with the sphingosine 1 phos- Service Longwood Core (Boston, MA). Representative images are shown phate receptor agonist FTY720 to block the egress of activated T for each experimental condition. cells from lymphoid organs (40). We predicted that if OVA-spe- cific T cells were preferentially primed in the CLN, FTY720 treat- Statistics ment should trap stimulated T cells in CLN and prevent their mi- All graph data represent mean Ϯ SD. Statistical analysis was performed gration to MLN and SPL. FTY720 treatment did not alter the using the two-tailed unpaired Student’s t test for comparison of two groups. profiles of proliferation of OT-I T cells in K14-mOVA CLN, MLN, and SPL (Fig. 1D), indicating that OT-I T cell priming is not Results restricted to CLN, but can occur in other secondary lymphoid or- Generation of K14-mOVA mice gans as well. OT-II T cell priming occurred mainly in CLN in To study the mechanisms of peripheral tolerance to skin-associated K14-mOVA mice and treatment with FTY720 induced stronger self-Ags, we generated new Tg mice expressing a membrane- proliferation in CLN but not in MLN and SPL (Fig. 1D), suggest-

bound form of OVA, OVA139–386 (mOVA), under transcriptional ing that priming of OT-II T cells only occurs in CLN. control of the human keratin 14 (K14) promoter. This promoter Taken together, these data indicate that epidermal self-Ags can drives expression in epithelial cells of the skin, as well as thymus, reach CLN and be presented to T cells, but that the requirements tongue, and esophagus (37). In these K14-mOVA mice, keratino- for Ag presentation differ between CD8 and CD4 T cells. On the cytes in epidermis express mOVA, which contains for one hand, CD8 T cells are primed, proliferate, and become func- both MHC class I-restricted OVA-specific TCR Tg CD8 T cells tional in various lymphoid organs of K14-mOVA mice. In con- (OT-I) and MHC class II-restricted OVA-specific TCR Tg CD4 T trast, CD4 T cells are primed only in CLN where the skin-migrat- cells (OT-II) (38, 39), allowing the study of both CD8 and CD4 T ing DCs arrive and present processed epidermal Ags. Since several cell responses in the same environment. However, it should be subsets of DCs are present in the various lymphoid tissues, these noted that OT-I and OT-II Tg lines are not similar in their reac- data suggest that complementary DC subsets are involved in Ag tivity to peptide Ag and response to Ag molarity expressed in vivo. presentation in K14-mOVA mice. In addition, it is possible that priming of OT-I and OT-II T cells differs in the various lymphoid OVA-specific T cells are primed, activated, and functional in tissues tested because they respond differently to the same Ag K14-mOVA mice in vivo dose. Indeed, it is known that fewer peptide-MHC complexes are To study immune responses induced to skin-associated self-Ags, required to stimulate CD8 than CD4 T cells. we performed adoptive transfers of purified OT-I and OT-II Tg T ␣ϩ cells (Thy1.1) into WT and K14-mOVA (Thy1.2) mice. Because Skin-derived DCs and LN-resident CD8 DCs present OVA is expressed in K14-mOVA skin and skin-derived Ags epidermal Ags in vitro should be transported by local tissue-specific DCs to CLN (32), we Elucidation of the APCs involved in tissue-specific T cell priming anticipated that T cells would encounter the highest levels of pre- should yield knowledge about peripheral tolerance mechanisms. sentation of OVA within the CLN. We first analyzed the accumu- To assess Ag presentation in our K14-mOVA model, we compared lation of donor OT-I and OT-II T cells in CLN, MLN, and SPL 48 the abilities of various subsets of DCs to present self-Ags in vitro. and 72 h, respectively, after transfer. Interestingly, the accumula- First, we prepared epithelial cell suspensions from ear epidermis or tion of OT-I T cells in K14-mOVA mice was comparable in each intestine from WT and K14-mOVA mice and cultured them with of these lymphoid tissues, while OT-II T cells preferentially ac- OT-I T cells. As shown in Fig. 2A, only skin epithelial cells from cumulated in CLN rather than in MLN and SPL (Fig. 1A). We next K14-mOVA mice, but not WT mice, induced OT-I T cell prolif- monitored proliferation of donor T cells in K14-mOVA mice. To eration, as measured by [3H]thymidine incorporation and CFSE this end, we measured dilution of a cell-permeable fluorescent dye, dilution. No proliferation was observed with epithelial cells from CFSE, by flow cytometry. We found that OT-I T cell proliferation the intestine. These skin epidermal cell suspensions contain both 4668 PERIPHERAL TOLERANCE CONTROL THROUGH REGULATED T CELL HOMING

FIGURE 1. OVA-specific T cells are primed, activated, and functional in K14-mOVA mice in vivo. A, Per- centages of OT-I and OT-II T cells transferred into WT and K14-mOVA mice were determined in lymphoid tissues after 48 and 72 h, respectively, p Ͻ 0.01. B ,ءء .by flow cytometry and C, Histograms gated on CD8ϩ Thy1.1ϩ (top) and CD4ϩThy1.1ϩ (bottom) T cells represent CFSE pro- files (B) and expression of activation markers (C) after transfer into WT and K14-mOVA mice. A–C, Data are representative of three mice per group in three independent experiments. D, Histograms and bar graph gated on CD8ϩThy1.1ϩ (top) and CD4ϩ Thy1.1ϩ (bottom) T cells show CFSE profiles and percentage of divided cells after transfer into untreated or FTY720-treated K14-mOVA mice. Data are representative of three mice per group. E, In vivo CTL assay. Five hours after transfer of target cells into WT and K14-mOVA mice previously transferred with OT-I T cells, the dis- appearance of peptide-pulsed target cells was determined by flow cy- tometry in SPL. The percentage of specific lysis was calculated as de- scribed in Materials and Methods. .p Ͻ 0.01 ,ء

keratinocytes, which express OVA and Langerhans cells (LCs), LN-resident CD8␣ϩ DCs, demonstrating that in the steady-state which presumably do not. As the preponderant population of epi- epidermal self-Ags are transported and presented by skin-de- dermal APCs, it is probable that LCs retrieve Ags from keratino- rived DCs and that transfer can occur between tissue-derived cytes and upon maturation in culture present them to OT-I T cells. and LN-resident DCs in K14-mOVA mice. Second, we analyzed the ability of APCs extracted from various lymphoid organs to present epidermal self-Ags. Peripheral Ags from skin can be transported to CLN by migratory skin-resident Role for skin-derived DCs in transporting epidermal Ags to DCs, such as LCs or dermal DCs (dDCs), that can directly present CLN in vivo epidermal Ag-derived peptides to naive T cells in CLN (29, 32). To assess the role of skin-derived DCs in transporting epidermal Alternatively, skin-derived DCs can transfer Ag to LN-resident Ags to CLN in vivo, we transplanted skin from WT and K14- DCs, which are particularly efficiently at presenting Ags (41). In mOVA mice onto the flanks of WT mice. After 10 days of en- addition, it is known that small amounts of Ag can traffic to CLN graftment, CFSE-labeled OT-I T cells (Thy1.1) were transferred through lymphatics in a non-cell-associated form and be acquired into grafted WT (Thy1.2) mice and their accumulation and prolif- and presented by CLN-resident DCs. To characterize the APCs eration were monitored in CLN draining the graft and in CLN from that present epidermal Ags in lymphoid organs of K14-mOVA the contralateral side of the graft, as well as in MLN. OT-I T cells mice, we cultured purified CD11cϩ DCs from CLN, MLN, and preferentially accumulated in CLN draining the K14-mOVA skin SPL from WT and K14-mOVA mice with OT-I T cells. Only DCs graft and not in CLN from the contralateral side (Fig. 3A). Mice from CLN of K14-mOVA, and not DCs from MLN or SPL, in- grafted with WT skin served as control to show that increased duced OT-I T cell proliferation in vitro (Fig. 2B); as expected, no accumulation of OT-I T cells to the CLN draining the graft was not proliferation was induced by WT DCs (data not shown). We fur- caused by surgical trauma. In addition, proliferation of OT-I T ther investigated the CLN DC subset responsible for Ag presen- cells occurred in CLN draining the graft but not in CLN from the tation by culturing OT-I T cells with DC subsets sorted from CLN contralateral side, as measured by CFSE dilution (Fig. 3A). The of K14-mOVA mice. Both skin-derived DCs (MHC class observation that OT-I T cells failed to accumulate or proliferate in IIhighCD11clowCD8␣Ϫ) and LN-resident CD8␣ϩ DCs (MHC class MLN shows that epidermal self-Ags were not transported from IIlowCD11chighCD8␣ϩ) from K14-mOVA mice stimulated OT-I T skin to MLN, but only to CLN. Taken together, these data suggest cell proliferation, while LN-resident CD8␣Ϫ DCs (MHC class that epidermal Ags are transported by skin-resident DCs, either IIlowCD11chighCD8␣Ϫ) did not (Fig. 2C). Taken together, these epidermal LCs or dDCs, to CLN and presented to T cells to induce data show that epidermal Ags from K14-mOVA mice can be pre- immune responses in vivo. However, these experiments do not sented by both skin-derived DCs, including LCs and dDCs, and exclude the possibility that a small amount of Ag reaches CLN by The Journal of Immunology 4669

␤ Ϫ/Ϫ constituted with 2m or WT BM, demonstrating that the APCs that present epidermal Ags to OT-I T cells in K14-mOVA mice are radioresistant. The major class of professional APCs that are ra- dioresistant are skin-derived DCs, such as LCs and dermal DCs; however, certain radioresistant stromal cells can also present MHC class I-restricted Ags (45). Langerinϩ DCs include skin-resident epidermal LCs and dDCs in skin, as well as skin-derived migratory LCs and dDCs and blood-derived DCs in CLN. They can be depleted by injection of DT in Lang-DTR/EGFP knock-in mice (Lang-DTR) (32, 46). To further elucidate the role of skin-derived DCs in presenting epi- dermal self-Ags in vivo, we performed adoptive transfers of OT-I T cells into K14-mOVA-Lang-DTR previously treated with DT or left untreated. DT injection resulted in 70% depletion of langerinϩ DCs in CLN and Ͼ90% in the epidermis at 24 h after injection (Ref. 32 and data not shown). Proliferation of OT-I T cells was comparable in langerinϩ-depleted and nondepleted K14-mOVA mice (Fig. 3C). These data indicate that langerinϩ cells are not required to present epidermal self-Ags to CD8 T cells, suggesting that another APC population in the CLN beside LCs also mediates CD8 T cell stimulation in response to skin-derived self-Ags in vivo. Therefore, we next investigated whether LN-resident DCs could present epidermal Ags in K14-mOVA mice in vivo. To this end, we took advantage of CD11c-DTR Tg mice in which CD11cϩ DCs can be depleted by DT injection (31). We performed adoptive transfer experiments of OT-I and OT-II T cells into K14-mOVA- CD11cDTR mice previously treated with DT or left untreated. Injection of DT into these mice resulted in 70% depletion of CD11cϩ DCs (Ref. 31 and data not shown); in addition, since LCs express low levels of CD11c, they were largely unaffected by treat- FIGURE 2. Skin-derived and LN-resident CD8␣ϩ DCs from K14- ment with DT (supplemental Fig. 15). As shown in Fig. 3D, ab- mOVA mice display self-derived peptide-MHC complexes in vitro. A, Epi- ϩ dermal and intestinal epithelial cells from WT and K14-mOVA mice were sence of CD11c DCs had no significant effect on OT-I T cell cultured with WT and OT-I T cells for 4 days. Proliferation was measured proliferation; however, it significantly impaired proliferation of by [3H]thymidine incorporation. Data are representative of triplicates in OT-II T cells, indicating that LN-resident DCs play an important three independent experiments. Histograms show CFSE profiles of OT-I T role in vivo in CD4 T cell priming in K14-mOVA mice. cells in the presence of WT (left) and K14-mOVA (right) skin epithelial Taken together, these data revealed that in vivo langerinϩ cells cells. B, CD11cϩ DCs from CLN (E), MLN (Ⅺ), and SPL (‚) from K14- are not the only APC population capable of presenting epidermal mOVA mice were cultured with OT-I T cells (50,000 cells/well) for 3 days. Ags to CD8 T cells, suggesting that LN-resident DCs might also 3 Proliferation was measured by [ H]thymidine incorporation. Data are rep- play a role in that process; the absence of either langerinϩ DCs or resentative of triplicates in two independent experiments. C, DC subsets CD11cϩ DCs did not impair CD8 T cell stimulation. In contrast, (20,000 cells/well) from WT and K14-mOVA CLN were sorted and cul- presentation of epidermal Ags to CD4 T cells significantly in- tured with OT-I T cells (50,000 cells/well) for 3 days. Proliferation was measured by [3H]thymidine incorporation. Vertical line represents back- volves LN-resident DCs, suggesting that the role of LCs in this ground levels of [3H]thymidine incorporation in T cells in the absence of case is minor. .p Ͻ 0.01 ,ء .APCs. Data are representative of triplicates ESL up-regulation on T cells upon priming in K14-mOVA mice trafficking without DC involvement through the lymphatic net- Several Tg mouse models expressing skin-associated Ags develop work. Most importantly, skin-derived Ags are not transported skin diseases, graft-versus-host disease and , which to MLN. involve the homing of activated T cells to the skin (26–28, 30). We therefore investigated whether OVA-specific T cells transferred Role of skin-derived and LN-resident DCs in presenting into K14-mOVA mice were able to home to skin after Ag encoun- epidermal Ags in vivo ter and activation in CLN. To migrate to skin, T cells are required To define the DC subsets that present epidermal Ags in vivo in this to display the proper skin-homing receptors, such as ESL and PSL. system, we reconstituted lethally irradiated K14-mOVA mice with We therefore analyzed expression of ESL on OT-I and OT-II T ␤ ␤ Ϫ/Ϫ cells in various lymphoid organs upon transfer into WT and K14- BM from 2-microglobulin ( 2m )-deficient mice (42). In these mice, only radioresistant cells, such as LCs and langerinϩ dermal mOVA mice. Up-regulation of ESL on OT-I and OT-II T cells was DCs, are capable of presenting epidermal Ags (43, 44). K14- observed only when transferred into K14-mOVA mice, and not mOVA mice reconstituted with WT BM were generated as con- into WT mice, and specifically on T cells primed in CLN and not trol; in these mice, self-Ags can be presented by both radioresistant in MLN or in SPL (Fig. 4A, B, and D). EDTA treatment was used and radiosensitive APCs. Eight weeks after reconstitution, CFSE- in all samples to determine the specificity of ESL staining, since labeled OT-I T cells were transferred into BM chimeras and their proliferation was monitored by flow cytometry after 48 h. As shown in Fig. 3B, proliferation was comparable in chimeras re- 5 The online version of this article contains supplemental material. 4670 PERIPHERAL TOLERANCE CONTROL THROUGH REGULATED T CELL HOMING

FIGURE 3. Differential role for DC subsets in presenting epidermal Ags to CD8 and CD4 T cells. A, Skin grafts using WT or K14-mOVA skin were performed as described in Mate- rials and Methods. Forty-eight hours after transfer into grafted mice, percent- ages and numbers of OT-I T cells were determined in CLN draining the graft (G) and in CLN draining the contralat- eral side of the graft (C) as well as in MLN. Histograms show proliferation of CFSE-labeled OT-I T cells in CLN and MLN of grafted mice. Data are rep- resentative of three mice per group in p Ͻ ,ء .two independent experiments 0.01. B, Proliferation of CFSE-labeled OT-I T cells in BM chimeras Forty- eight hours after transfer. Data are rep- resentative of five mice per group. C, Proliferation of CFSE-labeled OT-I T cells in untreated or DT-treated K14- mOVA-Lang-DTR mice Forty-eight hours after transfer. Data show three mice per group. D, Proliferation of CFSE-labeled OT-I (top) and OT-II (bottom) T cells in untreated or DT- treated K14-mOVA-CD11cDTR mice Forty-eight and 72 h after transfer. Data are representative of three mice per group in two independent experiments. .p Ͻ 0.01 ,ء binding of the ESL chimeric molecule is Ca2ϩ dependent. In ad- ory T cell homing to skin under steady-state conditions. After two dition, OT-I T cells in CLN draining K14-mOVA skin transplants, injections of anti-CD25 mAb, we found that OT-I T cells did not but not in CLN draining the contralateral side, also up-regulated migrate to skin (Fig. 5D). In addition, histology examination did ESL; in contrast, OT-I T cells that were found in MLN of such not reveal any mononuclear cell infiltration or inflammation in transplant-recipient WT mice expressed the gut-associated homing these conditions (Fig. 5C). ␣ ␤ molecule 4 7 (data not shown). This is in accordance with pre- All together, these data indicate that despite significant up-reg- vious reports showing that tissue-specific homing properties on T ulation of ESL on T cells, OVA-specific T cells do not migrate to cells are dictated by the localization of priming and the origin of skin under steady-state conditions, resulting in the maintenance of the APCs (16, 17). Interestingly, CD8 T cells started the up-reg- immune tolerance to peripheral self-Ags. However, peripheral tol- ulation of ESL 48 h after transfer, while CD4 T cells required 5 erance in these mice does not involve Tregs. days (Fig. 4, A and D). In addition, the percentage of ESLϩ CD8 T cells was greater than the percentage of ESLϩ CD4 T cells in Skin inflammation by tape stripping breaks tolerance K14-mOVA mice (Figs. 5A and 6A). These results are not surpris- in K14-mOVA mice ing since CD8 and CD4 T cells present intrinsic differences in To further investigate whether tolerance can be broken to induce T terms of proliferation, differentiation, and survival. All together, cell homing to skin in K14-mOVA mice, we used tape stripping as these data show that imprinting of skin tissue homing is dictated in a method to induce skin inflammation. Tape stripping induces skin vivo in the presence of endogenously expressed epidermal self- damage by disrupting the cutaneous epithelial barrier, avoiding the Ags. In addition, our data indicate that CD8 T cells up-regulate administration of exogenous agents and resulting in the production ESL much faster and more efficiently than CD4 T cells and suggest of a specific profile of inflammatory and adhesion mol- that homing to skin might be more efficient for CD8 than CD4 T ecules, such as TNF-␣, IL-8, IFN-␥, TGF␤, IL-1␣, IL-1␤, as well cells. as E-selectin and VCAM-1 (47, 48), and suffices to induce migra- Since expression of skin homing receptors is required to allow tion of LCs from epidermis to CLN (49). Indeed, we hypothesize homing to skin, we addressed whether up-regulation of ESL on that in K14-mOVA mice tape stripping would induce activation OVA-specific T cells in CLN of K14-mOVA mice is sufficient to and migration of skin-derived DCs to CLN under inflammatory mediate T cell homing to skin under steady-state conditions. Five conditions. Accordingly, skin-derived DCs would prime T cells in days after transfer, no OT-I T cells, or at least no detectable num- an immunogenic manner, eliciting a skin-homing phenotype that bers of OT-I T cells, could be detected in K14-mOVA skin (Fig. would induce T cell homing to skin. To test this hypothesis, we 4C). As expected, OT-I T cells transferred into WT mice that did adoptively transferred OT-I and OT-II T cells into WT and K14- not up-regulate ESL did not home to the skin. Similar results were mOVA mice and either tape stripped the ears or left them un- found with OT-II T cell transfers (data not shown). treated. We then analyzed characteristics of skin homing and in- Due to the presence of a non-negligible proportion of OVA- flammation. Upon tape stripping, we observed an increase in the specific CD4ϩCD25ϩ Tregs in K14-mOVA mice, we have exam- total cellularity of auricular LN (ALN); in addition, we found in- ined the possible role for Tregs in the inhibition of effector/mem- creased numbers of OT-I T cells in ALN in K14-mOVA mice The Journal of Immunology 4671

FIGURE 4. ESL up-regulation is not sufficient for CD8 T cell homing to skin in K14-mOVA mice under steady-state conditions. A and B, ESL up-regulation on OT-I T cells in CLN (A) and SPL (B) transferred into WT and K14-mOVA mice. Histograms are representative of three mice per group in three independent experiments. Numbers represent the percent- ages of ESLϩ cells among CD8 T cells. C, Five days after OT-I T cell transfer, cells were isolated from WT and K14-mOVA ears and stained for flow cytometry. Numbers in quadrants represent the percentages of Thy1.1ϩ cells among CD8 T cells. Five pairs of ears were pooled and data FIGURE 5. Skin tape stripping induces increased ESL up-regulation on are representative of three independent experiments. D, ESL up-regulation OVA-specific CD8 T cells and homing to skin in K14-mOVA mice. A and on OT-II T cells in lymphoid organs 6 days after transfer into WT and B, After OT-I T cell transfer into WT and K14-mOVA, mice were left .p Ͻ 0.05 ,ء .K14-mOVA mice untreated or ear tape stripped. Five days later, LN cellularity, number of OT-I T cells, and ESL up-regulation (A) were determined in ALN. The presence of T cells in ear skin was analyzed by flow cytometry (B). Num- compared with nontreated K14-mOVA mice (Fig. 5A). The effects bers in quadrants represent the percentages of Thy1.1ϩ cells among CD8 of tape stripping on ALN cellularity and OT-I T cell accumulation T cells. Data are representative of five mice per group in two independent p Ͻ 0.01. C and D, Five days after OT-I ,ءء p Ͻ 0.05 and ,ء .in WT skin were not significant. experiments We further investigated the effect of tape stripping on ESL ex- T cell transfer into untreated, tape-stripped, or anti-CD25-treated WT and K14-mOVA mice, histology of ear skin sections was analyzed by H&E pression and homing to skin. Tape stripping induced significant staining (original magnification, ϫ20; C) and the percentage of OT-I T up-regulation of ESL expression on OT-I T cells transferred into cells in ear skin was determined (D). K14-mOVA, but not on OT-I T cells transferred into WT mice (Fig. 5A). In addition, tape stripping induced the migration of OT-I T cells to skin in K14-mOVA mice, as measured by flow cytom- etry of cells extracted from ear skin (1.4% vs 3.9% of CD8 T cells gest that tape stripping induced inflammation mediates Ag-specific were Thy1.1ϩ; Fig. 5B), indicating that tolerance can be broken T cell homing to skin in K14-mOVA mice. under these conditions. Histology examination revealed abundant Adoptive transfer of OT-II T cells into K14-mOVA mice pro- mononuclear cell infiltration in tape-stripped K14-mOVA skin, but voked modest, but significant increases of ESL expression on these not untreated K14-mOVA skin (Fig. 5C), indicating inflammation cells. In contrast to OT-I cells, tape stripping did not yield a further and T cell recruitment upon treatment of K14-mOVA mice. Tape increase in ESL expression on OT-II cells (Fig. 6A). Tape stripping stripping of WT skin did not induce inflammation. These data sug- of K14-mOVA skin however resulted in changes in ear thickness, 4672 PERIPHERAL TOLERANCE CONTROL THROUGH REGULATED T CELL HOMING

mOVA mice under steady-state conditions. Since dissemination of transferred T cells to nonlymphoid tissues is universal and irre- spective of a specific Ag tissue distribution (53), we believe that low levels of OVA-specific T cells do enter the skin of K14- mOVA mice in the absence of inflammation but that the numbers are below the limits of detection with the methods we have used in this study. The lack of extensive Ag-driven T cell homing to skin in K14-mOVA mice contradicts other studies that used compara- ble mouse models expressing self-Ags in skin, K5-mOVA, K14- mOVA, or K14-OVAp mice, in which transfer of OT-I T cells induces inflammatory skin disease due to effector T cell infiltration of the skin (26–28). In our new line of K14-mOVA mice, toler- ance predominates: transferred OT-I T cells do not induce skin disease unless tape stripping is applied. There are several features that might account for the discrepancy between our model and those used in the previous studies. The constructs used to generate mOVA fusion proteins for Tg mice differ in composition, resulting in structurally different expressed Ags; indeed, the existing K14- mOVA mice express OVA fused to platelet-derived growth factor receptor (27), while the K14-mOVA mice presented here express OVA fused to TfR. The level of transgene expression probably also differs between strains, since the different Tg lines employ FIGURE 6. OVA-specific CD4 T cells home to skin upon tape stripping distinct promoters (K5 vs K14) and are likely affected by their loci of K14-mOVA skin. A–C, Six days after transfer of OT-II T cells into of integration. In addition, K14-OVAp mice express only the pep- untreated or tape-stripped WT and K14-mOVA mice, ESL up-regulation in tide recognized by OT-I T cells and not the cognate peptide for ALN (A) and ear thickness (B) were determined. The presence of OT-II T OT-II T cells. Therefore, a plausible explanation for why K14- cells in ear skin was analyzed by flow cytometry (C). Numbers in quadrants ϩ OVAp mice develop severe inflammatory skin disease upon OT-I p Ͻ 0.01 ,ء .represent the percentages of Thy1.1 cells among CD4 T cells p Ͻ 0.005. T cell transfer could be that immune suppression mediated by ,ءء and OVA-specific CD4ϩCD25ϩ Tregs is lacking. In our K14-mOVA mice however, Tregs seem unlikely to play a critical role in sup- and flow cytometry of cells extracted from ear skin showed in- pression of OVA-specific T cell homing to skin and in induction of flammation and infiltration into skin (0.6% vs 1.6% of CD4 T cells skin inflammation. ϩ were Thy1.1 ; Fig. 6, B and C). This result suggests that CD4 T Our study however revealed a significant Ag-dependent T cell cell tolerance to skin-derived Ags could also be reversed by skin migration to skin when we induced mild inflammation of the skin tape stripping. by tape stripping. We conclude that tape stripping of K14-mOVA Taken together, these data indicate that tolerance is established skin induces Ag-experienced skin-resident DCs to undergo matu- in K14-mOVA mice, thereby inhibiting T cell homing to skin and ration, migrate, and present self-Ags in an immunogenic manner in autoimmunity. When tolerance is broken by tape stripping, migra- CLN. In contrast to other models, this additional signal of inflam- tion to skin occurs and results in an inflamed skin phenotype. mation is absolutely required in K14-mOVA mice to break the established peripheral tolerance and allow homing of T cells to Discussion skin and induction of tissue damage. Leukocyte migration to in- We here report for the first time that imprinting of a skin-homing flamed tissue has been widely described and is clearly dependent phenotype on T cells is dictated in vivo in mice in which the model on ESL expression (3, 13). We here demonstrate that a low level Ag OVA is endogenously expressed in skin. Indeed, the skin-hom- of inflammation in K14-mOVA mice induces a significant increase ing molecule ESL is induced on T cells upon transfer into K14- in ESL expression on adoptively transferred OT-I T cells, but no mOVA mice. Most importantly, we show that ESL up-regulation such increase is seen on OT-II T cells. The level of endogenous occurs specifically and solely in lymphoid tissues draining the skin OVA expressed in K14-mOVA skin may be a limitation to induce and not in other secondary lymphoid organs in K14-mOVA mice, extensive expression of ESL on stimulated CD4 T cells, explaining suggesting that skin tissue imprinting is critically dependent on also their lower efficiency to migrate to skin compared with CD8 skin-derived DCs that carry the Ag from skin to CLN. This is in T cells. We believe that restrained expression of homing receptors accordance with previous studies in which exogenous Ag was ad- accounts for the maintenance of peripheral tolerance in ministered via the skin in vivo or that used DCs from skin- and K14-mOVA mice. gut-associated tissues ex vivo (16, 18, 19). However, the molecular A remaining issue regarding the K14-mOVA mice presented in mechanisms that mediate imprinting of leukocyte tissue-specific this study is to identify the cells involved in Ag presentation. The homing are not fully understood but may involve vitamin metab- skin graft experiments allowed us to show that skin-expressed Ags olites through their ability to induce or suppress expression of are transported from epidermis to CLN by skin-derived DCs in tissue-associated homing molecules, such as retinoic acid or 1,25- K14-mOVA mice. Although we cannot rule out that the Ag traffics dihydroxyvitamin D, the active form of vitamin D3 (50–52). to some extent to CLN by the lymphatics and conduits network in While under steady-state conditions, the CLN microenviron- a non-cell-associated form, this scenario is considered less likely ment in K14-mOVA mice induces up-regulation of ESL expres- since tissue-derived DCs are critically important in sustaining sion on T cells; this is not sufficient to induce trafficking of T cells IL-2R expression on T cells (54). Using mice that allow specific to the skin. Although we have identified the presence of transferred depletion of DC subsets in vivo, we also showed that both skin- OVA-specific T cells in all lymphoid tissues of K14-mOVA mice, derived DCs and LN-resident CD8␣ϩ DCs present epidermal Ags we could hardly detect any of these T cells in the skin of K14- to T cells; this was also supported by our in vitro experiments; The Journal of Immunology 4673 however, depletion does not alter T cell activation, suggesting that 5. Luster, A. D., R. Alon, and U. H. von Andrian. 2005. Immune cell migration in neither of these populations is absolutely required in this model. inflammation: present and future therapeutic targets. Nat. Immunol. 6: 1182–1190. Our BM chimera experiments furthermore confirm that both ra- 6. Chong, B. F., J. E. Murphy, T. S. Kupper, and R. C. Fuhlbrigge. 2004. 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