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Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin

Nature Immunology volume 15, pages 1064–1069 (2014)Cite this article

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Abstract

It remains largely unclear how antigen-presenting cells (APCs) encounter effector or memory T cells efficiently in the periphery. Here we used a mouse contact hypersensitivity (CHS) model to show that upon epicutaneous antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of skin T cells in a manner dependent on antigen and the integrin LFA-1. We found that DCs accumulated in perivascular areas and that DC clustering was abrogated by depletion of macrophages. Treatment with interleukin 1α (IL-1α) induced production of the chemokine CXCL2 by dermal macrophages, and DC clustering was suppressed by blockade of either the receptor for IL-1 (IL-1R) or the receptor for CXCL2 (CXCR2). Our findings suggest that the dermal leukocyte cluster is an essential structure for elicitating acquired cutaneous immunity.

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Figure 1: The formation of DC–T cell clusters is responsible for epidermal eczematous conditions.
Figure 2: Antigen-dependent T cell proliferation in DC–T cell clusters.
Figure 3: LFA-1 is essential for the persistence of DC–T cell clustering and for T cell activation in the skin.
Figure 4: Macrophages are essential for DC cluster formation.
Figure 5: Macrophages mediate the perivascular formation of DC clusters.
Figure 6: IL-1α upregulates the expression of CXCR2 ligands in M2 macrophages to induce the formation of DC clusters.

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Acknowledgements

We thank H. Yagita (Juntendo University) for the KBA neutralizing antibody to LFA-1; P. Bergstresser and J. Cyster for critical reading of our manuscript. Supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Author notes

  1. Yohei Natsuaki and Gyohei Egawa: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan

    Yohei Natsuaki, Gyohei Egawa, Satoshi Nakamizo, Sachiko Ono, Sho Hanakawa, Nobuhiro Kusuba, Atsushi Otsuka, Akihiko Kitoh, Tetsuya Honda, Saeko Nakajima, Yoshiki Miyachi & Kenji Kabashima

  2. Department of Dermatology, Kurume University School of Medicine, Fukuoka, Japan

    Yohei Natsuaki & Takashi Hashimoto

  3. Research Unit for Immunodynamics, RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan

    Takaharu Okada

  4. Department of Pharmaceutical Biochemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

    Soken Tsuchiya & Yukihiko Sugimoto

  5. Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Osaka, Japan

    Ken J Ishii

  6. Laboratory of Vaccine Science, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Ken J Ishii

  7. Department of Microbiology, Hyogo College of Medicine, Hyogo, Japan

    Hiroko Tsutsui

  8. Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

    Hideo Yagita

  9. Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan

    Yoichiro Iwakura

  10. Medical Mycology Research Center, Chiba University, Chiba, Japan

    Yoichiro Iwakura

  11. Laboratory for Cytokine Regulation, RIKEN center for Integrative Medical Science, Kanagawa, Japan

    Masato Kubo

  12. Division of Molecular Pathology, Research Institute for Biomedical Science, Tokyo University of Science, Chiba, Japan

    Masato Kubo

  13. Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore

    Lai guan Ng

  14. Department of Dermatology, Icahn School of Medicine at Mount Sinai School Medical Center, New York, New York

    Judilyn Fuentes & Emma Guttman-Yassky

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  1. Yohei Natsuaki
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Contributions

Y.N., G.E. and K.K. designed this study and wrote the manuscript; Y.N., G.E., S. Nakamizo, S.O., S.H., N.K., A.O., A.K., T. Honda and S. Nakajima performed the experiments and analyzed data; S.T. and Y.S. did experiments related to microarray analysis; K.J.I., H.T., H.Y., Y.I., M.K. and L.g.N. developed experimental reagents and gene-targeted mice; J.F. and E.G.-Y. did experiments related to immunohistochemistry of human samples; T.O., T. Hashimoto, Y.M. and K.K. directed the project and edited the manuscript; and all authors reviewed and discussed the manuscript.

Corresponding author

Correspondence to Kenji Kabashima.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 DC motility in the elicitation phase of CHS.

(a) Superimposed 30-min tracks of 30 randomly selected dermal DCs in the x–y plane, setting the starting coordinates to the origin. Tracks of a steady state, 6, 12, and 24 h after the elicitation with DNFB are shown. (b and c) Velocity (b) and displacement (c) of dDCs at each time point (n=30). Each bar represents the mean + SD. *, P < 0.05.

Supplementary Figure 2 Subset-specific depletion of cutaneous DCs.

(a) A schematic representation of our strategy to generate subset-specific cutaneous DC depletion models. To deplete all cutaneous DC subsets, Langerin-DTR mice were transferred with BM cells from CD11c-DTR mice, and DT was injected. To selectively deplete LCs, Langerin-DTR mice were transferred with BM cells from C57BL/6 mice, and DT was injected. To selectively deplete dDCs, C57BL/6 mice were transferred with BM cells from CD11c-DTR mice, and DT was injected. BMT; BM transplantation. (b) FACS plots of each group of mice after DT treatment. In dermis, the percentages in CD45+ cells were indicated. (c) Histological findings of the ear skin after CHS. HE staining of the ears of mice 24 h after challenge with DNFB. Mice were pretreated with or without sensitization, depleted of LCs and/or dDCs, and challenged with DFNB. Scale bar = 100 μm. (d) CHS response was induced on the ear skin, and skin-infiltrating cells were stained and analyzed with TCR beta, CD44, and CD62L antibodies by flow cytometry.

Supplementary Figure 3 dDC clusters are formed in response to various stimuli.

(a) The scoring criteria for DC clusters by numbers and diameters of clusters. (b) DC (green) cluster formation 24 h after topical application without (NT) or with acetone, olive oil, 3% TNCB, 2% DNTB, 0.3% DNFB, or Mycobacterium bovis BCG-inoculation (n=4, each). (c) Scores of DC cluster numbers of each group 24 h after each stimuli. (d) DC (green) cluster formation 24 h after topical application without (NT) or with 0.5% DNFB on the back skin and footpad. Scale bar = 100 μm. (e) Mobility of DCs and T cells of the cluster by treatment with anti-LFA-1 treatment. Anti-LFA-1 neutralizing antibody, KBA, was injected intravenously 14 h after elicitation. T cell (red) clustering was dissolved but DC (green) clustering persisted 10 h after KBA-treatment. Scale bar = 100 μm. (f) Score of DC cluster number 24 h after DNFB application with KBA (red) or control IgG (black) treatment (n=5, each).

Supplementary Figure 4 Analysis of M1 and M2 macrophage markers.

(a) TRITC-conjugated dextran was injected and dermal suspension was prepared 24 h later. CD45+ and TRITC+ cells were further analyzed with CD11b and F4/80 antibodies by flow cytometry. (b) M1 macrophage markers, such as TNF-a, Nos2, and IL-12a, and M2 macrophage markers, such as arginase (Arg)-1, Retnla, and Chi313, were examined in BM-derived M1 and M2 macrophages. Each bar represents the mean + SD (n=3). A.U., arbitrary units. *, P < 0.05. ***P < 0.0001.

Supplementary Figure 5 Neutrophils are not essential for CXCL2 expression in DNFB-painted skin.

(a) Relative amount of Il1r1 24 h after with or without DNFB-sensitization (n=5). (b) Relative amount of Cxcl2 in DNFB-painted skin in 1A8- or control IgG-treated mice (n=5, each). (c) FACS plot of DNFB-painted skin prepared from 1A8- or control IgG-treated mice. CD11b+ Gr-1+ neutrophils were significantly depleted with 1A8-treatment. (d) Relative amount of Il1r1 from dermal macrophages cultured with or without IL-1α (n=4, each). (e) RT-PCR analysis of chemokine receptor mRNA expression in BM-derived DCs.

Supplementary Figure 6 A schema of immunological events in CHS response.

Sensitization phase. Epidermal contact with antigens triggers release of IL-1 in the skin, which activates macrophages that subsequently attract dDCs to perivascular area via CXCL2 to form clusters. In the absence of antigen-specific effector/memory T cells, DC clustering is a transient event, and hapten-carrying DCs migrate into draining LNs to establish sensitization. (b) Elicitation phase. In the presence of antigen-specific effector/memory T cells, the antigen is recognized efficiently in the DC clusters by antigen-specific effector T cells to form clusters, and inflammation is induced promptly via activation and proliferation of antigen-specific effector T cells.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–2 (PDF 2752 kb)

Leukocyte cluster formation in elicitation phase of DNFB-induced CHS response

CMTMR-labeled DNFB-sensitized T cells were transferred into CD11c-YFP mice and then challenged with DNFB to the ear. CD11c+ dermal DCs (green) and T cells (red) formed clusters approximately 6 h after hapten application. The images were taken every 7 min for 24 h. (WMV 41200 kb)

High magnification view of leukocyte cluster in the elicitation phase of CHS

CMTMR-labeled DNFB-sensitized T cells were transferred into CD11c-YFP mice and then challenged with DNFB to the ear. Sixteen hours later, the established DC–T cell cluster was observed in high magnification view for 2 h every 1 min. In this leukocyte cluster, some of T cells (red) interacted with dermal DCs (green) for more than 2 h. The pale yellow debris are melanin granules. Fragmented red and green debris seems to be indicative of dead T cells and DCs engulfed by macrophages, respectively. (WMV 28045 kb)

T cell division in the skin

CMTMR-labeled DNFB-sensitized T cells divided in DNFB-challenged site. The mean frequency of T cell division was 1.67±1.81 /h/mm2 (calculated from 5 movies which recorded more than an hour). (WMV 3003 kb)

Macrophages attracted dermal DCs

TRITC-conjugated dextran was intravenously injected to DNFB-sensitized CD11c-YFP mice to label skin macrophages. The next day, ear skin was challenged with DNFB and examined using two-photon microscopy. In this representative movie, a dermal DC (green) migrated toward TRITC-positive macrophages (red). (WMV 9892 kb)

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Natsuaki, Y., Egawa, G., Nakamizo, S. et al. Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin. Nat Immunol 15, 1064–1069 (2014). https://doi.org/10.1038/ni.2992

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