Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8+ tissue-resident memory T cells (TRM cells) require active transforming growth factor-β1 (TGF-β) for epidermal residence. Here we found that integrins αvβ6 and αvβ8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-β. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvβ6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-β, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-β by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.
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We thank D. Sheppard (University of California, San Francisco) for Itgb6−/− and Itgb8loxP mice and the isotype-matched control antibody ADWA-21; A. Glick, N. Blazanin and A. Ravindran for technical assistance with mouse KC culture; S. Violette (Biogen Idec) for antibody clones 6.3g9 and ch2A1 (both specific for αvβ6); J. Mitchell for technical assistance with confocal and epifluorescence microscopy; T. Martin, J. Motl and P. Champoux for technical assistance with flow cytometry and cell sorting; D. Rifkin and M. Vassallo for providing detailed protocols of an in vitro latent TGF-β-activation assay; the Research Animal Resources staff at the University of Minnesota for animal care; the Mayo Clinic division of Biostatistics and Bioinformatics for assistance in searching control and losartan-treated skin samples; and M. Jenkins for critical reading of the manuscript. Supported by the US National Institutes of Health (AR060744 to D.H.K., AI084913 to D.M.), the Dermatology Foundation (J.M.) and the American Skin Association (J.M.).
The authors declare no competing financial interests.
Integrated supplementary information
(a-c) Archived pathology skin samples from controls and patients treated with losartan for at least 6 months were immunostained for CD1a to identify the presence of Langerhans cells. Representative examples of LC (brown) in skin of controls (a) and losartan treated patients with near-complete (b) and modest (c) depletion are shown.
Supplementary Figure 2 αvβ6 alters TGF-β signaling and affects epidermal LC residence but not dermal DC residence.
(a) WT and TGF-βRI-CALC mice were treated with TAM i.p. at 0.05 mg/g of mouse weight for 2 consecutive days. Epidermal cells were analyzed the following day to detect expression of hemagglutinin (HA) epitope tagged to constitutively active TGF-βRI. Representative histograms from 3 mice show expression of HA on LC but not KC (WT, grey filled; TGF-βRI-CALC, red). (b) To eliminate the possibility that constitutive TGF-β signaling could induce LC apoptosis, WT and TGF-βRI-CALC mice were treated with TAM as above for 5 consecutive days and epidermal LC analyzed 2 days later for annexin V and viability dye (see methods) binding (n=5). (c) Dermal CD103+ DC and CD103- DC were quantified by flow cytometry after treating WT mice with 10 mg/kg of isotype control or anti-αvβ6 once a week for 4 weeks. (d) WT, Itgb6−/− and Itgb6−/− Χ TGF-βRI-CALC were treated with TAM i.p. at 0.05 mg/g of mouse weight for 5 consecutive days. Two weeks later, the number of LC in the epidermis was determined by immunofluorescence imaging of MHC-II (green) in epidermal whole mounts. Representative images are shown. Scale bars, 100 μm. (e) The number of LC per high power field (HPF) was quantified. Each symbol represents the number of LC averaged from at least 5 fields from an individual animal. * p<0.001 (Tukey’s multiple comparisons test). (Error bars, mean ± s.e.m.).
(a) Transverse section of back skin from adult WT mouse stained for langerin (red) and DAPI (blue). LC distribution within regions of epidermis containing different KC subsets are highlighted. Scale bar, 25 μm. (b) Gating strategy to identify different populations within epidermal cells prepared from the skin of adult WT mice. Single cell suspensions were stained as described in Methods section and sorted on FACSAria cell sorter. Populations of dendritc epidermal T cells (DETC), LC, IFE, IM and bulge were selected (as described in Methods) for sorting from viable single cells.
(a-c) Six-weeks old Itgb6−/− and wild-type C57BL/6 CD45.2 mice were lethally irradiated using X-ray irradiator receiving two split doses at 500 cGy each. The following day, 5X106 BM cells from congenically marked C57BL/6 CD45.1 mice were injected intravenously into irradiated mice. Mice were rested for at least 6 weeks and percent chimerism was tested by flow cytometry based on expression of CD45.1 (donor) and CD45.2 (host) in blood (a), LN (b) and epidermis (c). Each symbol represents data from an individual animal.
(a) Representative micrographs of back epidermis whole mounts of adult WT and Itgb8ΔLC mice stained with MHC-II (green) to identify LC (n=7-10). Scale bars, 100 μm. (b-c) LC in epidermis (b) and LN (c) were quantified by flow cytometry in WT and Itgb8ΔLC mice. Each symbol represents data from an individual animal.
Supplementary Figure 6 αvβ6 alters the residence of CD103+ DCs in the epithelium but not the lamina propria of the small intestine in mice.
(a) Expression of the indicated mRNA transcripts normalized to hprt as assessed by RT-qPCR of intestinal epithelial cells from WT mice is shown. (b) Identification of CD103+ DC in a section of SI stained for MHC-II, CD103, Collagen IV (ColIV) and DAPI showing intraepithelial CD103+ DC located above the ColIV and lamina propria CD103+ DC located below the ColIV is shown. (c-d) WT mice were treated i.p. with 10 mg/kg of isotype control or anti-αvβ6 once a week for 4 weeks and SI sections stained for MHC-II, CD103, ColIV and DAPI. The number of CD103+ DC per 106 nucleated cells in SI epithelium (c) and (lamina propria) (d) were manually counted. Each symbol represents data from an individual animal and is representative of 2 independent experiments. * p<0.01 (two-tailed unpaired Students’ t test). (Error bars, mean ± s.e.m.).
Supplementary Figure 7 Loss of TGF-β signaling is required for efficient hapten-induced LC migration.
Cohorts (n=3) of TGF-βRI-CALC mice were treated with tamoxifen or vehicle for 5 days. Mice were then painted with 20ul TRITC (1ug/ul in 1:1 acetone/dibutylphthalate) or vehicle alone (control) on shaved back skin. Four days later skin draining LN were harvested and analyzed by flow cytometry. Migratory LC were identified based on expression of CD11c, MHC-II, Langerin, CD11b and TRITC. Each symbol represents data from an individual animal. Data are representative of 3 independent experiments. * p<0.05 (two-tailed unpaired Students’ t test). (Error bars, mean ± s.e.m.).
Supplementary Figure 8 Systemic proliferation of P14 Thy1.1+ cells is not altered in Itgb6−/− or Itgb6−/−Itgb8ΔKC mice or following blockade of αvβ6.
(a) Blood and skin-draining LN from WT, Itgb6−/− and Itgb6−/−Itgb8ΔKC mice used in Fig. 5a,b and Fig. 6a-c were analyzed for Thy1.1+ cells at the time of harvest. (b) As in a, Thy1.1+ cells were analyzed in blood and skin-draining LN of mice used in Fig. 5c-e and Fig. 6d-f at the time of harvest. (c) As in a, Thy1.1+ cells were analyzed in blood and skin-draining LN of WT mice treated with isotype or anti-αvβ6 neutralizing antibody shown in Fig. 5f-g and Fig. 6g-i. (Error bars, mean ± s.e.m.).
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Mohammed, J., Beura, L., Bobr, A. et al. Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-β. Nat Immunol 17, 414–421 (2016). https://doi.org/10.1038/ni.3396
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