Keratinocytes (KCs) may play important roles for maintenance of peripheral tolerance in the upper layers of the skin. Coinhibitory signals mediated via the programmed death (PD)-1 and its ligand B7-H1 (PD-L1/CD274) are crucial for the downregulation of T cell immune responses and for the maintenance of peripheral tolerance. In this study, to investigate the role of KC-expressed B7-H1 in the regulation of T cell immune responses, we generated transgenic (tg) mice overexpressing B7-H1 under the control of keratin 14 (K14) promoter (K14-B7-H1 tg). K14-B7-H1 tg mice displayed impaired contact hypersensitivity (CH) responses to primary and secondary hapten challenges. The K14-B7-H1 tg mice did not exhibit substantial impairment of cutaneous dendritic cell migration after sensitization and of hapten-specific proliferation and IFN-γ production of CD4+ and CD8+ T cells in the draining lymph nodes, suggesting that overexpression of B7-H1 on KCs did not affect the induction phase of the CH response. The systemic or s.c. injection of hapten-sensitized T cells into the K14-B7-H1 tg mice did not efficiently induce the CH response. IFN-γ expression and apoptosis of KCs in the challenged ears were impaired in K14-B7-H1 tg mice. IFN-γ production by presensitized CD8+ T cells stimulated with hapten-pulsed KCs was markedly impaired for the KCs obtained from the K14-B7-H1 tg mice but was restored by the addition of an anti–B7-H1 mAb. These results suggest that KC-associated B7-H1 directly downregulates the effector function of CD8+ T cells by associating with PD-1 at local inflammatory sites and that it plays a role in peripheral T cell tolerance against exogenous Ags.

Antigen-specific T cell responses are controlled by various cosignaling molecules that are critical for T cell activation and regulation (14). The best-characterized cosignaling molecules are the costimulatory receptors CD28 and ICOS and the coinhibitory receptors CTLA-4 and programmed death (PD)-1. CTLA-4 and PD-1 play crucial roles in the induction and maintenance of peripheral tolerance (3, 4). The CTLA-4 ligands CD80 and CD86 are expressed only on APCs, whereas the PD-1 ligand B7-H1 (PD-L1/CD274) is broadly induced on various nonlymphoid tissue cells, including pancreatic islet cells, smooth muscle cells, corneal epithelial cells, hepatocytes, heart endothelial cells, and liver sinusoidal endothelial cells, at the inflammatory disease sites (511).

The results of studies using B7-H1–deficient mice or treatment with anti–B7-H1 or anti–PD-1 mAb have suggested that tissue-associated B7-H1 is involved in the PD-1–mediated regulatory pathway in autoimmunity and allotransplantation (79, 1214). However, the separate contributions of tissue and lymphoid B7-H1 were not clearly discriminated in these studies. Furthermore, studies using B7-H1–transgenic (tg) mice have produced contradictory results (15, 16). B7-H1–overexpressing islet β cells in C57BL/6 mice promoted organ-specific autoimmunity and transplant rejection, whereas similar B7-H1 overexpression in NOD mice played a protective role against autoimmune diabetes. Transfer of prediabetic T cells into NOD/SCID mice deficient in both PD-1 ligands or into bone marrow (BM) chimeras expressing PD-1 ligands solely on nonlymphoid cells revealed that B7-H1 expression on parenchymal cells, rather than on hematopoietic immune cells, mediates tissue tolerance and protects autoimmunity (4). All of these studies focused on peripheral T cell tolerance against tissue or self-Ags. The role of tissue-associated B7-H1 in local T cell tolerance against foreign Ags has not been clarified.

The skin and type II mucosal surfaces, including oral mucosae, cornea, and vagina, are covered by stratified squamous epithelial cells known as keratinocytes (KCs). KCs play important roles in protection against foreign invaders and in the maintenance of homeostasis (17). Immune responses to foreign Ags are evoked in the skin and mucosae. These Ag-specific responses are primed and amplified in secondary lymphoid organs, and the final effector functions of T cells are executed at the local sites (1820). KCs are able to induce MHC class II molecules in some conditions but lack expression of potent costimulators, such as CD86, and produce immunoregulatory cytokines, such as IL-10 and TGF-β. Thus, KCs generally induce T cell anergy or tolerance rather than T cell activation (2022). However, the regulatory mechanisms in the interactions with T cells and KCs are poorly understood. Although KCs cannot prime naive T cells, they can potentially induce a recall immune response in Ag-experienced T cells and seem to act as nonprofessional APCs (23).

We previously reported that B7-H1 is expressed on KCs of the oral mucosa and skin from patients with lichen planus, a chronic inflammatory mucocutaneous disease characterized by massive T cell infiltration under the epithelium (24). We found that the addition of IFN-γ to human KCs in culture upregulated the level of B7-H1 on cultured human KCs and that B7-H1–expressing KCs downregulated the proliferative response and the production of IFN-γ of activated T cells in vitro (24). When we examined the regulatory role of the PD-1:B7-H1 pathway in hapten-induced contact hypersensitivity (CH), a T cell-mediated inflammatory response, we were able to confirm that B7-H1 expression is induced on KCs of hapten-painted skin but were unable to elucidate the contribution of the KC-expressed B7-H1 to the CH response (25).

In the current study, to evaluate the contribution of KC-associated B7-H1 to effector T cell regulation in inflammatory skin lesions, we newly generated tg mice for B7-H1 under the control of human keratin 14 (K14) promoter such that B7-H1 was overexpressed only on KCs (K14-B7-H1 tg) and investigated the role of KC-associated B7-H1 in the CH response.

BALB/c and C57BL/6 (B6) mice were purchased from Japan SLC (Shizuoka, Japan), maintained under specific pathogen-free conditions and used at 8–16 wk of age. All procedures were reviewed and approved by the Animal Care and Use Committee of Tokyo Medical and Dental University.

K14 promoter (2.4 kb) was amplified from Jurkat cell genomic DNA with addition of 5′ KpnI and 3′ XhoI sites. Murine B7-H1 cDNA (0.9 kb) was amplified from splenic cDNA by RT-PCR with addition of 5′ XhoI and 3′ NotI sites. The KpnI–XhoI fragment of K14 promoter, the XhoI–NotI fragment of B7-H1, and the NotI–SacI fragment of β-globin poly A were assembled into pBluescript KS+ plasmid. The KpnI–SacI fragment of the resulting construct was purified and injected into B6 eggs to establish K14-B7-H1 tg mice. The founder was backcrossed to B6 mice, and transgene-positive and -negative mice were used as K14-B7-H1 tg and littermate (Lm) control mice, respectively.

For genomic DNA analyses, mouse-tail DNA was digested with EcoRV, and Southern blotting was performed with a 0.3-kb B7-H1 cDNA probe using a standard method. Alternatively, genotyping of extracted mouse tail genomic DNA was performed by PCR amplification of the regions between the K14 promoter and B7-H1 exon 2 (K14-B7-H1) and between B7-H1 exons 3 and 4 (B7-H1 exon 3–4) using the primer pairs 5′-gaa agc cca aaa cac tcc aa-3′/5′-ttg act ttc agc gtg att cg-3′ and 5′-cag aag ctg agg taa tct gga-3′/5′-tct caa gaa gag gag gac cg-3′, respectively.

Mouse KCs were isolated from ear skin by trypsinization as described previously (26). Briefly, dorsal halves of ear skin were incubated with 0.5% trypsin-PBS at 37°C for 1 h. Epidermal sheets were separated from the dorsal ear halves and incubated in DNase I (10 U/ml; Sigma-Aldrich, St. Louis, MO) at 37°C for 10 min to obtain a single-cell suspension of KCs. Contaminating CD11c+ or MHC class II+ cells made up <2% of the cells in the preparation.

Epidermal KCs were cultured in KC serum-free medium (Invitrogen, Carlsbad, CA) in collagen type I-coated dishes (Iwaki, Tokyo, Japan). In some experiments, cultured KCs were stimulated with or without IFN-γ (10 ng/ml; eBioscience, San Diego, CA) for 2 d.

Draining lymph node (dLN) cells were collected from cervical, axillary, and inguinal LNs of sensitized mice 3 d after the final sensitization. The cells were isolated using the collagenase method, and the dLN T cell population was enriched by negative selection using a mixture of biotinylated mAbs against CD49b integrin α2 (HMα2), CD45R (RA3-6B2), and I-A/I-E (M5/114.15.2) with magnetic beads (IMag; BD Biosciences, Franklin Lakes, NJ), according to the manufacturer’s protocol. To isolate CD4+ and CD8+ LN T cells, biotinylated anti-CD8 (53-6.7) and anti-CD4 (GK1.5) mAbs were added to the mixture, respectively. The purity of the CD3+, CD4+, and CD8+ T cell preparations was confirmed to be >95% by flow cytometry.

BM-derived dendritic cells (BMDCs) were generated as previously described (27) with some modifications. Briefly, isolated BM cells were cultured with GM-CSF and IL-4 (10 ng/ml each; eBioscience) for 6 d.

PE-conjugated anti–H-2Kd (SF1-1.1), anti-ICAM (YN1/1.7.4), anti-CD40 (1C10), anti-CD80 (1G10), anti-CD86 (GL1), anti–B7-H1 (MIH5), anti–B7-DC (TY25), anti-CD11c (HL3), anti-CD4 (RM4-5), and anti-MHC class II (M5/114.15.2), as well as FITC-conjugated anti-CD8 (53-6.7) and PerCP-Cy5.5–conjugated CD3 (145-2C11) mAbs and control Abs were obtained from eBioscience or BD Biosciences. Stained cells were analyzed on a FACSCalibur flow cytometer using the CellQuest (BD Biosciences) or the FlowJo (Tree Star, Ashland, OR) software.

CH to 2,4-dinitro-1-fluorobenzene (DNFB; Sigma-Aldrich) was induced as described previously (28, 29). For sensitization, 20 μl 0.5% DNFB dissolved in acetone:olive oil (4:1) was painted onto shaved abdominal skin on days 0 and 1. For B6 mice, the concentration of DNFB was doubled to 1% to induce a similar level of CH responses. For experimental challenge, 20 μl 0.2% DNFB was applied to either ear on day 5. For secondary challenge experiments, ear painting was performed on day 35. Ear thickness was measured before and 24, 48, and 72 h after the challenge. Differences between the Lm control and K14-B7-H1 tg mouse groups were analyzed for statistical significance using the Mann-Whitney U test.

CD4+ and CD8+ T cell responses against haptens were measured as described previously (28). Briefly, BMDCs or KC stimulator cells were pulsed with 20 mM dinitrobenzene sulfonate (DNBS, a water-soluble analog of DNFB; Sigma-Aldrich) in serum-free medium at 37°C for 30 min. Purified T cell fractions (3 × 105 cells/well) were cocultured with various ratios of hapten-pulsed stimulator cells in complete RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% FBS and 50 μM 2-ME and 10 μg/ml gentamicin (Sigma-Aldrich) in 96-well flat-bottomed plates. In some experiments, either anti–mB7-H1 mAb (MIH6) or control rat IgG (20 μg/ml) was added at the start of the assay.

The proliferative response was determined by measuring [3H]thymidine incorporation (1 μCi/well) during the final 18 h of the indicated culture periods. The incorporated radioactivity was measured using a Microplate beta counter. IFN-γ production was measured using ELISA as previously described to quantify the IFN-γ in supernatants collected after 3 d of culture (30).

dLN T cells were isolated from DNFB-sensitized wild-type (Wt) B6, Lm control and K14-B7-H1 tg mice at day 5 and transferred systemically (2.5 × 107 cells) or s.c. into the ear skin (1 × 106 cells) of intact mice. Ears were challenged with DNFB 1 d after systemic injection or immediately after s.c. injection, and ear thickness was measured 24 and 48 h after challenge.

Ear tissues from Lm control and K14-B7-H1 tg mice and abdominal skin from intact and DNFB-sensitized mice were surgically removed, embedded in Tissue-Tek (Sakura, Tokyo, Japan), frozen, and stored at –80°C until use. Cryostat sections (4–5 μm thick) were stained with anti–B7-H1 mAb (MIH5) as described previously (31). For TUNEL staining, cryostat sections were stained with Apoptosis in situ Detection Kit Wako (Wako). Digital images were obtained using an inverted microscope and camera system (IX71 and Pro600ES-D; Olympus, Tokyo, Japan).

mRNA was prepared from whole ear skin using Micro-FastTrack 2.0 (Invitrogen). First-strand cDNA was synthesized using oligo(dT) primers and Superscript III reverse transcriptase (Invitrogen). Quantitative PCR was performed as described previously (28). The primers used were β-actin (Actb), 5′-agg gaa atc gtg cgt gac at-3′ and 5′-aac cgc tcg ttg cca ata gt-3′, and IFN-γ (Ifng), 5′-cct tct tca gca aca gca agg cg-3′ and 5′-ccc acc ccg aat cag cag cg-3′.

To confirm induction of B7-H1 on epidermal KCs, we examined B7-H1 expression after exposure to several stimuli in vitro and in vivo. In immunohistochemical analyses, KCs in intact abdominal skin did not express substantial levels of B7-H1, but painting of the skin with DNFB hapten dramatically induced B7-H1 expression, with KC expansion and enlargement (Fig. 1A). We also observed upregulation of B7-H1 expression on isolated KCs after skin painting with DNFB or PMA in flow cytometric analyses (data now shown). B7-H1 was expressed at a low level in cultured KCs isolated from ear skin but was markedly upregulated by the addition of IFN-γ (Fig. 1B). A minor population of cells that expressed much higher levels of B7-H1 appeared to be contaminating cutaneous DCs. Thus, the KC-associated B7-H1 was easily induced by various stimuli.

FIGURE 1.

Induction of B7-H1 expression on KCs. A, Cryostat sections were prepared from abdominal skin of intact mice and DNFB-painted mice 24 h after application of DNFB and immunostained with anti–B7-H1 mAb (original magnification ×400). Scale bars, 20 μm. Representative sections from each group of two mice are shown. B, Cultured KCs isolated from ear skin were treated with or without IFN-γ (10 ng/ml) for 48 h, stained with PE-anti–B7-H11 mAb, and analyzed by flow cytometry. The data are shown as histograms with the control staining (shaded histograms). Values are the mean fluorescence intensity (MFI) ± SD from three independent experiments. Representative data are shown.

FIGURE 1.

Induction of B7-H1 expression on KCs. A, Cryostat sections were prepared from abdominal skin of intact mice and DNFB-painted mice 24 h after application of DNFB and immunostained with anti–B7-H1 mAb (original magnification ×400). Scale bars, 20 μm. Representative sections from each group of two mice are shown. B, Cultured KCs isolated from ear skin were treated with or without IFN-γ (10 ng/ml) for 48 h, stained with PE-anti–B7-H11 mAb, and analyzed by flow cytometry. The data are shown as histograms with the control staining (shaded histograms). Values are the mean fluorescence intensity (MFI) ± SD from three independent experiments. Representative data are shown.

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To investigate the functional role of B7-H1 in epidermal KCs, we established B7-H1 tg mice expressing a high level of B7-H1 on their KCs. Because K14 is widely used to investigate the function of specific KC molecules, we selected K14 as the promoter to drive the expression of B7-H1. A K14 promoter-B7-H1 cDNA construct with β-globin polyA (Fig. 2A) was injected into B6 eggs, and three founders with the K14-B7-H1 transgene (numbers 4, 15, and 30) were obtained. Southern blotting showed that founder number 4 has 8–10 copies of the transgene, as estimated by intensity of the bands (Fig. 2B). Therefore, offsprings of the founder number 4 were used.

FIGURE 2.

Generation of K14-B7-H1 tg mice. A, Schematic diagram of the K14-B7-H1 expression construct composed of the human K14 promoter (□), B7-H1 cDNA (▪), and β-globin polyadenylation (poly A) site (hatched). B, Southern blot analysis of the B7-H1 gene in Wt B6 (Wt), transgene-negative Lm control (Lm), and transgene-positive tg mice. C, Ear tissue sections from Lm control and K14-B7-H1 tg mice were stained with anti–B7-H1 mAb (original magnification ×400). Scale bars, 25 μm. D, Epidermal KCs freshly isolated from the ear skin of Lm control or K14-B7-H1 tg mice were stained with PE-anti–B7-H1 mAb or control rat IgG and analyzed by flow cytometry. The data are displayed as histograms with the control histograms nearest the ordinate (shaded). Data shown are representative of five independent experiments.

FIGURE 2.

Generation of K14-B7-H1 tg mice. A, Schematic diagram of the K14-B7-H1 expression construct composed of the human K14 promoter (□), B7-H1 cDNA (▪), and β-globin polyadenylation (poly A) site (hatched). B, Southern blot analysis of the B7-H1 gene in Wt B6 (Wt), transgene-negative Lm control (Lm), and transgene-positive tg mice. C, Ear tissue sections from Lm control and K14-B7-H1 tg mice were stained with anti–B7-H1 mAb (original magnification ×400). Scale bars, 25 μm. D, Epidermal KCs freshly isolated from the ear skin of Lm control or K14-B7-H1 tg mice were stained with PE-anti–B7-H1 mAb or control rat IgG and analyzed by flow cytometry. The data are displayed as histograms with the control histograms nearest the ordinate (shaded). Data shown are representative of five independent experiments.

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K14-B7-H1 tg mice exhibited normal development and hemato/lymphopoiesis with typical ratios and numbers of lymphocyte subpopulations including B, T, NK cells, and APCs (data not shown). Immunohistochemical analysis of ear tissues (Fig. 2C) and flow cytometric analysis of isolated KCs (Fig. 2D) revealed a considerably high level of B7-H1 expression on the tg mouse KCs. No obvious abnormalities were seen in the skin and hair, even in those aged mice. Overexpression of B7-H1 was not found in other lymphoid or nonlymphoid cells (data not shown). In addition, splenocytes and LN cells from K14-B7-H1 tg mice exhibited normal levels of MHC class II, CD80, CD86, B7-H1, and B7-DC (data not shown).

We previously demonstrated the regulatory role of the PD-1:B7-H1 pathway in the CH response (25). In this study, to examine the role of KC-associated B7-H1 in this regulation, we first compared the hapten-induced CH responses of K14-B7-H1 tg and Lm control mice. Ear swelling at 24, 48, and 72 h after DNFB challenge was markedly impaired in K14-B7-H1 tg mice (Fig. 3A). An impaired CH response was consistently observed upon rechallenge performed 1 mo after the primary challenge (Fig. 3B).

FIGURE 3.

Impairment of the CH response in K14-B7-H1 tg mice. CH against DNFB was induced as described in 1Materials and Methods, and ear swelling was measured 24, 48, and 72 h after primary challenge (A) and after rechallenge performed 30 d after the primary challenge (B). Data shown represent the means ± SD for five or six Lm control (◯) or K14-B7-H1 tg (●) mice and are representative of three independent experiments. *p < 0.05, statistically different from the Lm control group.

FIGURE 3.

Impairment of the CH response in K14-B7-H1 tg mice. CH against DNFB was induced as described in 1Materials and Methods, and ear swelling was measured 24, 48, and 72 h after primary challenge (A) and after rechallenge performed 30 d after the primary challenge (B). Data shown represent the means ± SD for five or six Lm control (◯) or K14-B7-H1 tg (●) mice and are representative of three independent experiments. *p < 0.05, statistically different from the Lm control group.

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CH is invoked by the induction phase of hapten sensitization and by the effector phase of hapten challenge. To clarify the mechanisms of impaired CH response in the K14-B7-H1 tg mice, we first focused on the activation status of dLN T cells 3 d after the final sensitization. The total cell numbers (10.1 ± 1.8 and 9.5 ± 2.3 × 107; mean ± SD) and percentages of CD4+ (36.5 ± 5.1 and 40.5 ± 2.7) and CD8+ (17.4 ± 2.1 and 19.0 ± 1.5) T cells are comparable between K14-B7-H1 tg and Lm control mice. CD4+ and CD8+ dLN T cells from the sensitized Lm control or K14-B7-H1 tg mice were cocultured with DNBS-pulsed BMDCs and examined for their proliferative response and IFN-γ production. The proliferative responses of the CD4+ and CD8+ T cells from K14-B7-H1 tg mice were comparable to those of the cells from Lm control mice (Fig. 4A). IFN-γ production by CD4+ and CD8+ T cells from K14-B7-H1 tg mice was slightly lower than in those from the control mice (Fig. 4B).

FIGURE 4.

Activation status of dLN-T cells after sensitization. A and B, dLN T cells from Lm control (◯) or K14-B7-H1 tg (●) mice were cocultured with hapten-pulsed BM-DCs at the indicated responder/stimulator (R/S) ratios and assessed for their proliferative responses during the final 18 h of the 4-d culture (A) and for production of IFN-γ (B). Cultures containing T cell only, pulsed BMDCs only, or T cells plus unpulsed BMDCs consistently exhibited a low proliferative response (<500 cpm). The data shown represent means ± SD of triplicate wells and are representative of two independent experiments. C, dLN T cells from Lm control or K14-B7-H1 tg mice were systemically transferred into intact B6 mice. The mice were hapten challenged 24 h after the transfer, and ear swelling was measured 24 h after the challenge. The data shown represent means ± SD for each group of three mice and are representative of two independent experiments. *p < 0.05, statistically different from the Lm control group.

FIGURE 4.

Activation status of dLN-T cells after sensitization. A and B, dLN T cells from Lm control (◯) or K14-B7-H1 tg (●) mice were cocultured with hapten-pulsed BM-DCs at the indicated responder/stimulator (R/S) ratios and assessed for their proliferative responses during the final 18 h of the 4-d culture (A) and for production of IFN-γ (B). Cultures containing T cell only, pulsed BMDCs only, or T cells plus unpulsed BMDCs consistently exhibited a low proliferative response (<500 cpm). The data shown represent means ± SD of triplicate wells and are representative of two independent experiments. C, dLN T cells from Lm control or K14-B7-H1 tg mice were systemically transferred into intact B6 mice. The mice were hapten challenged 24 h after the transfer, and ear swelling was measured 24 h after the challenge. The data shown represent means ± SD for each group of three mice and are representative of two independent experiments. *p < 0.05, statistically different from the Lm control group.

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To further analyze the status of sensitized dLN T cells, dLN T cells from Lm control or K14-B7-H1 tg mice were isolated 3 d after the final sensitization and systemically transferred into intact B6 mice, which were then hapten challenged 24 h after the transfer. The two groups of mice exhibited comparable levels of ear swelling (Fig. 4C), suggesting that overexpression of B7-H1 on the KCs at the sensitized sites did not substantially affect the activation or priming status of dLN T cells.

We also examined the migratory capacity of cutaneous DCs after hapten sensitization using an FITC-induced CH model. The numbers of FITC-carrying CD11c+ DCs in the dLNs from Lm control and K14-B7-H1 tg mice were comparable (data not shown), suggesting that overexpression of B7-H1 does not alter the migratory capacity of hapten-carrying DCs. Taken together, our results suggest that overexpression of B7-H1 on KCs does not affect the induction phase of CH responses mediated by hapten-carrying migrating DCs and dLN T cells.

The above results indicate that impairment of the CH response in B7-H1 tg mice occurs at the effector phase of the response. Hapten-primed effector T cells are recruited to hapten-challenged local sites and execute their effector functions in situ. The already primed T cells might be modified by B7-H1–expressing KCs at such local sites. To investigate this possibility, sensitized dLN T cells were systemically transferred into either intact Lm control or K14-B7-H1 tg mice, and the ear skin of the mice was hapten challenged 24 h after the transfer. Lm control mice who received primed dLN T cells showed obvious ear swelling 24 h after the challenge, whereas the tg mice showed a significantly lower ear swelling (Fig. 5A).

FIGURE 5.

KC overexpression of B7-H1 regulates the CH effector phase. A, Lm control or K14-B7-H1 tg mice received systemic transfer of dLN T cells from sensitized Wt B6 mice and were then challenged with DNFB 24 h after the transfer. B, dLN cells or dLN T cells from the sensitized Wt B6 mice were s.c. transferred into Lm control or K14-B7-H1 tg mice, and DNFB was challenged immediately. Ear swelling was measured 24 and 48 h after challenge. The data shown represent means ± SD for each group of five mice and are representative from two independent experiments. *p < 0.05, statistically different from the Lm control group.

FIGURE 5.

KC overexpression of B7-H1 regulates the CH effector phase. A, Lm control or K14-B7-H1 tg mice received systemic transfer of dLN T cells from sensitized Wt B6 mice and were then challenged with DNFB 24 h after the transfer. B, dLN cells or dLN T cells from the sensitized Wt B6 mice were s.c. transferred into Lm control or K14-B7-H1 tg mice, and DNFB was challenged immediately. Ear swelling was measured 24 and 48 h after challenge. The data shown represent means ± SD for each group of five mice and are representative from two independent experiments. *p < 0.05, statistically different from the Lm control group.

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To examine more localized activity, sensitized dLN T cells were injected s.c. into the ear skin of intact Lm control and K14-B7-H1 tg mice, and the ears were immediately painted with hapten. Local injection of primed dLN T cells into the tg mice, but not the Lm control mice, consistently and markedly impaired ear swelling (Fig. 5B), suggesting that the primed T cells were considerably affected by local regulation at the ear skin effector sites of the K14-B7-H1 tg mice. To further confirm regulation of effector CD8+ T cells at the local ears, we first examined the number of infiltrating CD8+ T cells in the ears 24 h after the challenge by immunohistochemistry and flow cytometric analyses using single-cell suspensions of epidermal sheets. However, no clear difference was observed between Lm control and K14-B7-H1 tg mice (data not shown). We next examined functional differences of CD8+ T cells. Because CD8+ T cells are a major source of IFN-γ in the early time point after DNFB challenge (32), we investigated IFN-γ mRNA expression in the challenged ears by quantitative RT-PCR. Although the sensitized and vehicle-treated ears showed very low levels of Ifng in both groups, this was markedly upregulated after DNFB challenge (Fig. 6A). However, the enhanced level in K14-B7-H1 tg mice was lower (approximately half) compared with that of Lm control mice. It has been shown that apoptosis of KCs in DNFB-challenged ears is induced by infiltrating CD8+ T cells (33). We further analyzed apoptosis of KCs in ear skin by TUNEL. In vehicle-treated ear skin, few and weak TUNEL-positive cells were found in epidermis of both Lm control and K14-B7-H1 tg mice (Fig. 6B). DNFB challenge in Lm control mice markedly induced TUNEL-positive cells at the basal cell layer of epidermis, but positive cells in the ears of K14-B7-H1 tg mice were clearly less, suggesting decreased apoptosis of KCs in K14-B7-H1 tg mice. These results suggest that effector functions, presumably mediated by infiltrating CD8+ T cells are functionally impaired in the K14-B7-H1 tg mice.

FIGURE 6.

Effector function of CD8+ T cells is impaired in K14-B7-H1 tg mice. A, mRNA from the ear skin challenged with DNFB or vehicle alone at 24 h was extracted, and quantitative RT-PCR was performed. Relative gene expression was calculated by normalization with β-actin (Actb). B, Cryostat sections from the ear skin challenged with DNFB or vehicle alone were stained for TUNEL (original magnification ×400). Representative images are shown. Arrowheads indicate TUNEL-positive (apoptotic) cells. Scale bars, 100 μm.

FIGURE 6.

Effector function of CD8+ T cells is impaired in K14-B7-H1 tg mice. A, mRNA from the ear skin challenged with DNFB or vehicle alone at 24 h was extracted, and quantitative RT-PCR was performed. Relative gene expression was calculated by normalization with β-actin (Actb). B, Cryostat sections from the ear skin challenged with DNFB or vehicle alone were stained for TUNEL (original magnification ×400). Representative images are shown. Arrowheads indicate TUNEL-positive (apoptotic) cells. Scale bars, 100 μm.

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In inflamed ear skin, interactions between KCs, DCs, and T cells may modulate the respective functions of the cells. To investigate whether KCs modulate the responses of CD8+ T cells mediated by Ag-pulsed DCs, CD8+ T cells from sensitized B6 mice were stimulated by DNBS-pulsed BMDCs in the presence of various numbers of KCs from Lm control or K14-B7-H1 tg mice. The addition of KCs suppressed the proliferative response and IFN-γ production in a KC cell number-dependent manner, but the suppressed levels were comparable for the Lm and the K14-B7-H1 tg KCs (data not shown).

We next examined the possibility that the B7-H1–overexpressing KCs directly inhibit the effector function of T cells. KCs isolated from intact ear skin of Lm control or K14-B7-H1 tg mice did not express substantial levels of costimulatory molecules CD40, CD80, CD86, or B7-DC but did express comparable levels of MHC class I (H-2Kd) and ICAM-1, suggesting that overexpression of B7-H1 in the tg mice did not intrinsically affect the expression of other cell surface molecules on the KCs (Fig. 7A). Then, hapten-primed CD4+ or CD8+ dLN T cells were isolated from sensitized B6 mice and cocultured with DNBS-pulsed KCs obtained from either Lm control or K14-B7-H1 tg KCs for 92 h, and proliferative responses and IFN-γ production were measured. Although the primed CD4+ T cells did not exhibit obvious proliferation and IFN-γ production after stimulation by either Lm control or the tg mice KCs (data not shown), the primed CD8+ T cells exhibited a weak proliferative response after stimulation with KCs from either group (Fig. 7B). In contrast, IFN-γ production from CD8+ T cells was markedly enhanced by Lm control KC in a KC cell number-dependent manner (Fig. 7C, left panel). However, coculture with hapten-pulsed B7-H1–overexpressing KCs failed to induce IFN-γ production. The addition of anti–B7-H1 mAb into the cultures had no obvious effect on the response induced by Lm control KCs, but it efficiently restored IFN-γ production to a level similar to that observed in cultures with Lm control KCs (Fig. 7C, right panel).

FIGURE 7.

KC-associated B7-H1 directly inhibits the effector function of CD8+ T cells. A, Expression of functional molecules on the cell surface of KCs. KCs freshly isolated from ears of Lm control or K14-B7-H1 tg mice were stained with control Ig, anti–H-2Kd, anti–ICAM-1 (CD54), anti-CD40, anti-CD80, anti-CD86, anti–B7-H1, or anti–B7-DC mAb, and the stained cells were analyzed by flow cytometry. The data are shown as histograms. B and C, CD8+ dLN T cells from sensitized B6 Wt mice were cocultured with DNBS-pulsed KCs from Lm control (◯) or K14-B7-H1 tg (●) mice at the indicated KC/T ratios. Proliferative responses in the final 18 h of 4-d culture (B) and IFN-γ production after the 3 d culture (C) were shown. For blocking experiments, either control rat IgG or anti–B7-H1 (MIH6) mAb was added at the start of the assay. Values shown are means ± SD from triplicate cultures in which the KC/T ratio = 2. The proliferative responses of CD8+ T cells without KCs were <40 cpm. IFN-γ production in the cultures with T cells alone, KCs alone, and T cells plus unpulsed KCs was undetectable (<0.01 ng/ml). The data shown are representative of two independent experiments. *p < 0.05, statistically different from the Lm control group.

FIGURE 7.

KC-associated B7-H1 directly inhibits the effector function of CD8+ T cells. A, Expression of functional molecules on the cell surface of KCs. KCs freshly isolated from ears of Lm control or K14-B7-H1 tg mice were stained with control Ig, anti–H-2Kd, anti–ICAM-1 (CD54), anti-CD40, anti-CD80, anti-CD86, anti–B7-H1, or anti–B7-DC mAb, and the stained cells were analyzed by flow cytometry. The data are shown as histograms. B and C, CD8+ dLN T cells from sensitized B6 Wt mice were cocultured with DNBS-pulsed KCs from Lm control (◯) or K14-B7-H1 tg (●) mice at the indicated KC/T ratios. Proliferative responses in the final 18 h of 4-d culture (B) and IFN-γ production after the 3 d culture (C) were shown. For blocking experiments, either control rat IgG or anti–B7-H1 (MIH6) mAb was added at the start of the assay. Values shown are means ± SD from triplicate cultures in which the KC/T ratio = 2. The proliferative responses of CD8+ T cells without KCs were <40 cpm. IFN-γ production in the cultures with T cells alone, KCs alone, and T cells plus unpulsed KCs was undetectable (<0.01 ng/ml). The data shown are representative of two independent experiments. *p < 0.05, statistically different from the Lm control group.

Close modal

To verify that the inhibition mediated by B7-H1–overexpressing KCs was dependent on PD-1 signaling, we performed similar experiments using CD8+T cells from PD-1–deficient mice and the B7-H1 tg KCs. IFN-γ production by PD-1−/−CD8+ T cells was not impaired by stimulation with B7-H1 tg KCs (data not shown). Collectively, these results indicate that KC-associated B7-H1 directly interacts with PD-1 expressed on effector CD8+ T cells and inhibits their effector function.

We have established B7-H1 tg mice that constitutively and specifically overexpress B7-H1 on their KCs and have an impaired CH response to haptens. Overexpression of B7-H1 on the KCs in primary sensitized skin affected neither the migratory capacity of cutaneous DC nor priming ability of T cells in the dLNs. Transfer of sensitized T cells to the B7-H1 tg mice significantly reduced ear swelling. The effector function of infiltrating CD8+ T cells assessed by IFN-γ expression and apoptosis of KCs was impaired in K14-B7-H1 tg mice. The downregulation of effector CD8+ T cell function by B7-H1 was confirmed by the observation that IFN-γ production by CD8+ T cells was inhibited by coculture with hapten-pulsed KCs from B7-H1 tg mice. These results suggest that KC-associated B7-H1 at the challenge site directly inhibited the effector function of CD8+ T cells at the local site, resulting in downregulation of the CH response.

In a general model for hapten-induced CH, the CH response is evoked by an afferent phase of the primary sensitization and an efferent phase of the subsequent challenge. Topical application of the hapten initially triggers production of various epidermal-derived cytokines, including IL-1α, IL-1β, TNF-α, GM-CSF, MIP-1α, IFN-γ, and IFN-γ–inducible 10 kDa protein, and induces both phases of the CH response (34). KCs, which make up the bulk of epidermal cells, are a rich source of cytokines, and epidermal Langerhans cells (LCs) also secrete certain cytokines. Expression of B7-H1 is induced by various stimuli, including certain cytokines and chemicals (4). We found that stimulation with IFN-γ induced the expression of B7-H1 on cultured KCs and that DNFB application rapidly induced B7-H1 expression and KC activation in situ (Fig. 1). Although IFN-γ is known to be a potent inducer of B7-H1, other epidermal-derived cytokines may augment the expression of KC-associated B7-H1 in hapten-painted skin. B7-H1 tg mice constitutively express B7-H1 on their KCs, and freshly isolated KCs from the tg mouse ear skin expressed approximately two to four times as much B7-H1 as did IFN-γ–treated cultured KCs, as assessed by mean fluorescence intensity. Although the tg mice express more B7-H1 than is normally induced physiologically, their level of B7-H1 is within a range that should be relevant to the examination of the exact role of KC-associated B7-H1 in cutaneous immune responses.

In the afferent phase of CH, KC-associated B7-H1 may initially interact with epidermal LCs in the epidermis. We recently reported that one of the TNF family costimulatory pathways, the glucocorticoid-induced TNF receptor-related protein (GITR):GITR ligand pathway, interacts with KCs and LCs to mediate cutaneous DC migration (28). PD-1 might be inducible on a subset of cutaneous DCs, where its interactions modify their function, as PD-1 expression on myeloid cells has been reported (35). A recent report has shown that PD-1 is induced on splenic DCs, where it negatively regulates DC-derived cytokine production after Listeria monocytegenes infection or TLR ligand stimulation (36). We observed PD-1 expression on migratory DCs in LNs from skin (data not shown). However, the overexpression of B7-H1 in sensitized skin did not obviously affect the migratory capacity of cutaneous DCs or the priming state of dLN T cells assessed by transfer experiments (Fig. 4 and data not shown). Thus, we conclude that KC-associated B7-H1 is unlikely to be a contributing factor in the impaired CH responses of sensitized skin in our tg mice.

In this study, we demonstrated that the ear swelling induced by the transfer of hapten-sensitized T cells into ear skin was significantly reduced in B7-H1 tg mice. Impairment of the response was also observed upon systemic transfer, suggesting that KC-associated B7-H1 in challenged skin modulates the activation state of infiltrating effector T cells that may react with either cutaneous DCs or KCs. However, the addition of B7-H1–expressing KCs failed to affect either the proliferative response or the production of IFN-γ by primed CD8+ T cells when they were stimulated with hapten-pulsed DCs (Fig. 4). In contrast, hapten-pulsed KCs substantially induced the proliferative response and the production of IFN-γ by hapten-primed CD8+ T cells, and KC-associated B7-H1 clearly suppressed IFN-γ production in a B7-H1:PD-1 pathway-dependent manner (Fig. 7). In addition, hapten-pulsed KCs could not directly stimulate unprimed CD8+ T cells (data not shown) but did stimulate already primed CD8+ T cells. These results suggest that KCs are not able to exert Ag-presenting ability in the presence of professional APCs like DCs; however, KCs can present the hapten Ags to already primed CD8+ T cells and thus stimulate their function. The B7-H1 expressed on KCs directly interacts with PD-1 on primed CD8+ T cells to suppress their activation.

Induction of PD-1 on T cells occurs after activation (37). In particular, PD-1 induced on CD8+ T cells contributes to T cell exhaustion in chronic viral infection, to the induction of tolerance against self-Ags or alloantigens, and to the attenuation of antitumor immunity (12, 15, 3844). Hapten challenge of LC-depleted skin has been shown to elicit an enhanced CH response through Ag presentation by the KCs expressing CD86, a costimulatory molecule (45). KCs activated by IFN-γ may provide costimulatory signals required for T cell activation and act as APCs (46, 47). Akiba et al. (33) reported that the early recruitment of CD8+ T cells to the site of hapten skin challenge and the initial production of IFN-γ are critical events in the early CH response; the recruited CD8+ T cells induce KC apoptosis in the CH response. A more recent report demonstrated an initial small number of effector CD8+ T cell recruitment into the skin requires prior neutrophil infiltration and this was critical for triggering sequential infiltration of neutrophils and effector T cells after hapten challenge (32). Our results demonstrated the effector function of recruiting CD8+ T cells was impaired in K14-B7-H1 tg mice, although the recruitment of CD8+ T cells was comparable. KC-associated B7-H1 may modulate such CD8+ T cell function prior to neutrophil infiltration. LCs are not the relevant APCs during the elicitation phase of CH; rather, by directly interacting with skin infiltrating CD8+ T cells, KCs play a crucial role in CH response elicitation (45). Consistent with the above reports, we conclude that KC-associated B7-H1 plays an inhibitory role in cutaneous immune responses and is involved in peripheral tolerance mediated by skin-infiltrating CD8+ T cells.

We previously used anti-B7-H1 and anti–PD-1 mAbs to demonstrate PD-1:B7-H1–mediated regulation of the CH responses (25). In that study, we were unable to demonstrate the involvement of KC-associated B7-H1 in PD-1:B7-H1–mediated regulation, although this regulation was definitely involved in the interactions with hapten-pulsed DCs and T cells in the responses in the dLNs. The present study clearly demonstrates that B7-H1 expressed on KCs contributes to peripheral tolerance induction in CD8+ T cell-mediated inflammatory skin responses. B7-H1 is easily inducible, even in human KCs, by various stimuli (24); therefore, B7-H1:PD-1–mediated regulation may be involved in the pathogenesis of various T cell-mediated skin diseases. The targeting of B7-H1 in the skin to manipulate cutaneous immune responses is a promising strategy for the treatment of refractory inflammatory skin diseases, such as psoriasis and lichen planus.

We thank the staffs of Laboratory of Recombinant Animals, Medical Research Institute, Tokyo Medical and Dental University for the establishment of the transgenic animal.

Disclosures The authors have no financial conflict of interest.

This work was supported by a grant from the Japan Society for the Promotion of Science (to M.H. and M.A.) and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M.A).

Abbreviations used in this paper:

BM

bone marrow

BMDC

bone marrow-derived dendritic cell

CH

contact hypersensitivity

DC

dendritic cell

DNBS

dinitrobenzene sulfonate

DNFB

2,4-dinitro-1-fluorobenzene

K14

human keratin 14

KC

keratinocyte

LC

Langerhans cell

Lm

littermate

MFI

mean fluorescence intensity

PD

programmed death

R/S

responder/stimulator

tg

transgenic

Wt

wild-type.

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