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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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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Integrated supplementary information
(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.
(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.
(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).
(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.
(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.
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 Figures 1–6 and Supplementary Tables 1–2 (PDF 2752 kb)
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)
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)
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)
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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