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

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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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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