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The developmental pathway for CD103+CD8+ tissue-resident memory T cells of skin

Abstract

Tissue-resident memory T cells (TRM cells) provide superior protection against infection in extralymphoid tissues. Here we found that CD103+CD8+ TRM cells developed in the skin from epithelium-infiltrating precursor cells that lacked expression of the effector-cell marker KLRG1. A combination of entry into the epithelium plus local signaling by interleukin 15 (IL-15) and transforming growth factor-β (TGF-β) was required for the formation of these long-lived memory cells. Notably, differentiation into TRM cells resulted in the progressive acquisition of a unique transcriptional profile that differed from that of circulating memory cells and other types of T cells that permanently reside in skin epithelium. We provide a comprehensive molecular framework for the local differentiation of a distinct peripheral population of memory cells that forms a first-line immunological defense system in barrier tissues.

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Figure 1: CD103 and CD69 regulate the lodgement and persistence of TRM cells.
Figure 2: TRM cells develop from KLRG1 precursor cells that selectively infiltrate the epidermis during acute infection.
Figure 3: Opposing chemokines regulate the entry into the epidermis and differentiation of TRM cells versus egress from tissues.
Figure 4: Signaling via the TGF-β receptor and IL-15 is required for the development of CD103+ TRM cells.
Figure 5: Definition of a core transcriptional profile of TRM cells that is progressively engaged during differentiation.

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Acknowledgements

We thank W. Weninger (University of Sydney) for Ccr7−/− mice; N. Hunt (University of Sydney) for Cxcr3−/− mice; M. Bevan (University of Washington) for Tgfbr2f/fdLck-Cre OT-I mice; I. Frazer (University of Queensland) for Tcrd−/− mice; members of the Carbone and Heath laboratories for discussions; and N. McBain and J. Smith for technical assistance. Supported by National Health and Medical Research Council of Australia and Australian Research Council.

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L.K.M., F.R.C. and T.G. designed the experiments; L.K.M., A.R., J.Z.M., N.C., A.T.S., M.-L.H. and T.G. did the experiments; L.K.M., A.R., J.Z.M., A.T.S., W.R.H., M.I., F.R.C. and T.G. analyzed the data; J.V.-R., P.L., S.N.M., T.S. and D.C.T. contributed reagents; F.R.C. led the research program and, with the help of L.K.M. and T.G. wrote the manuscript.

Corresponding authors

Correspondence to Francis R Carbone or Thomas Gebhardt.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Phenotype and anatomical localization of TRM cells.

(a,b) Analysis of CD103, Bcl-2 and CD69 expression by endogenous Vα2+ T cells in skin at the indicated times p.i. Plots are gated on Vα2+ CD45.2+ cells; numbers indicate the percentage of events in the respective gates. Data representative of 2–3 experiments. (c) Mice received naïve gBT-I.GFP cells prior to HSV infection. Arrows indicate examples of gBT-I.GFP cells in the epidermis and hair follicle epithelium 14 d p.i. Photo representative of >5 experiments. (d) Wild-type (WT) and Cd69−/− gBT-I cells were transferred into WT mice prior to HSV infection. Enumeration of gBT-I cells in dorsal root ganglia (DRG) 30 d p.i. Data from one experiment (n = 5 mice/group). (e) WT (GFP-expressing, green) and Itgae−/− (DsRed-expressing, red) gBT-I cells were co-transferred into WT mice prior to HSV infection. Microscopy of skin 8 d p.i. Staining with anti-keratin antibody to delineate the epidermis and hair follicle epithelium. Arrows in insert indicate gBT-I cells in the epidermis. Photo representative of n = 3 mice analyzed.

Supplementary Figure 2 Development of virus-specific CD8+ memory T cells after infection of skin with HSV-1.

(a) Mice received naïve gBT-I cells prior to infection. Analysis of CD62L, CD127 and KLRG1 expression by gBT-I cells in the spleen at different time points after infection. Plots are gated on Vα2+CD45.2+CD45.1+ cells and are representative of n = 4–8 mice/time point. Numbers indicate the percentage of events in the respective gates (as in b). (b) Analysis of CD103 and KLRG1 expression by endogenous Vα2+ T cells in skin at the indicated times p.i. Plots are gated on Vα2+CD45.2+ cells and are representative of 3 experiments (n = 12 mice/time point). (c) Effector gBT-I cells were sorted into KLRG1+ and KLRG1 subsets from spleens of infected mice (6 d p.i.) and transferred into infected recipients (4 d p.i.). Enumeration of CD103+ gBT-I cells in dorsal root ganglia (DRG) 3 weeks p.i. Data from 3 experiments (n = 12 mice/group); *, P< 0.05 by two-tailed Mann-Whitney test.

Supplementary Figure 3 Epidermal localization of CD8+ T cells after intradermal transfer.

(a) CD103 and CD69 expression by gBT-I cells in skin at the indicated times after in vitro activation by gB peptide-coated splenocytes and intradermal transfer. Plots are gated on Vα2+CD45.2+CD45.1+ cells and are representative of 2–3 experiments (n = 6–12 mice/time point). Numbers indicate the percentage of events in the respective gates. (b) In vitro activated gBT-I.GFP cells were transferred into the skin by intradermal injection. Microscopy of skin 30 d after transfer. Staining with anti-keratin antibody to denote epithelium in the epidermis and hair follicles. Arrows indicate examples of gBT-I.GFP cells (green). Photos (2 examples shown) are representative of 2 experiments. (c) In vitro activated gBT-I cells were left untreated (Ctrl, DsRed-expressing, red) or treated with PTx (+PTx, GFP-expressing, green) and co-transferred into mice by intradermal injection. Microscopy of skin 4 weeks after transfer; anti-keratin staining denotes the epidermal layer and hair follicle epithelium. Red arrows indicate control cells in the epithelium, green arrows indicate PTx-treated cells in the dermis. Photos representative of 2 experiments. (d) In vitro activated gBT-I cells were untreated (Ctrl) or treated with PTx (+PTx). Analysis of CD103 expression following subsequent in vitro incubation of the cells in the presence (solid or dashed black lines, as indicated) or absence (orange area) of TGF-b (5 ng/ml; 24 hours). Plots are gated on Vα2+CD45.2+CD45.1+ cells and are representative of 2 experiments.

Supplementary Figure 4 Chemokine expression in skin and ex vivo migration of KLRG1+ and KLRG1 effector cells.

(a) mRNA expression for genes encoding CXCL9 and CXCL10 in CD45.2EpCAM+ keratinocytes sorted from naïve (Ctrl) or HSV-infected skin (d 6 p.i.). Data pooled from 3 independent experiments (represented by individual symbols). *, P< 0.05 by two-tailed paired t-test. (b) Ex vivo migration towards CXCL9 gradients by KLRG1+ (closed symbols) and KLRG1 (open symbols) gBT-I cells enriched from spleens 7 d after HSV skin infection (cells pooled from n = 5 donor mice).

Supplementary Figure 5 Requirement for the TGF-β receptor and IL-15 signaling in the development of CD103+ TRM cells.

(a) Wild-type (WT) and TGF-R-deficient (Tgfbr2f/f.dLck-Cre) OT-I cells were activated by culture with ovalbumin peptide-coated splenocytes and transferred into the skin by intradermal injection. Depicted are the ratios of WT relative to Tgfbr2f/f.dLck-Cre cells in the skin and spleen at the indicated times after transfer. Data representative of 2 experiments (n = 3–4 mice/group). (b,c) Effector gBT-I cells were enriched from spleens of WT mice (6 d p.i.) and transferred into infected (4 d p.i.) WT or Il15−/− recipient mice. (b) Enumeration of CD103+ gBT-I cells in dorsal root ganglia (DRG) 4 wks p.i. Data from one experiment with n = 5 mice/group. (c) CD103 and Bcl-2 expression by gBT-I cells in WT and Il15−/− mice (11 d p.i.). Plots are gated on Vα2+CD45.2+CD45.1+ cells; numbers depict percentages of events in the respective gates. Data are representative of 2 experiments (n = 8 mice/group).

Supplementary Figure 6 The developmental pathway for the formation of TRM cells.

KLRG1 TRM precursors enter the dermis where they may (i) die in situ, (ii) exit the skin and return to the blood in a CCR7-dependent manner or (iii) migrate to the epidermis, partly under the influence of CXCR3 ligands. Both tissue exit and epidermal entry are sensitive to treatment with pertussis toxin (PTx) indicating the involvement of G protein-coupled molecules such as chemokine receptors. Following epidermal entry, TRM precursors undergo maturation into long-lived CD103+ TRM cells in a TGF-β- and IL-15-dependent manner. Epithelial TRM cells and their counterparts in the circulation are depicted with different symbols to highlight their distinct transcriptional profiles.

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Mackay, L., Rahimpour, A., Ma, J. et al. The developmental pathway for CD103+CD8+ tissue-resident memory T cells of skin. Nat Immunol 14, 1294–1301 (2013). https://doi.org/10.1038/ni.2744

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