Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-β

Abstract

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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Figure 1: Activation of latent TGF-β by αvβ6 inhibits homeostatic LC migration.
Figure 2: Epidermal residence of LCs requires expression of αvβ6 by IFE epidermal KCs.
Figure 3: LC residence is controlled by spatially distinct expression of αvβ6 and αvβ8 on KC subsets through activation of latent-TGF-β.
Figure 4: UV irradiation promotes LC migration through diminished integrin expression and TGF-β activation.
Figure 5: αvβ6 and αvβ8 are required for the residence of CD8+ TRM cells in epidermis.
Figure 6: αvβ6 is required for residence of TRM cells in intestinal epithelium.

References

  1. 1

    Merad, M., Ginhoux, F. & Collin, M. Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells. Nat. Rev. Immunol. 8, 935–947 (2008).

  2. 2

    Merad, M. et al. Langerhans cells renew in the skin throughout life under steady-state conditions. Nat. Immunol. 3, 1135–1141 (2002).

  3. 3

    Haley, K. et al. Langerhans cells require MyD88-dependent signals for Candida albicans response but not for contact hypersensitivity or migration. J. Immunol. 188, 4334–4339 (2012).

  4. 4

    Igyártó, B.Z. et al. Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific T helper cell responses. Immunity 35, 260–272 (2011).

  5. 5

    Kobayashi, T. et al. Dysbiosis and staphylococcus aureus colonization drives inflammation in atopic dermatitis. Immunity 42, 756–766 (2015).

  6. 6

    King, J.K. et al. Langerhans cells maintain local tissue tolerance in a model of systemic autoimmune disease. J. Immunol. 195, 464–476 (2015).

  7. 7

    Kautz-Neu, K. et al. Langerhans cells are negative regulators of the anti-Leishmania response. J. Exp. Med. 208, 885–891 (2011).

  8. 8

    Kaplan, D.H., Jenison, M.C., Saeland, S., Shlomchik, W.D. & Shlomchik, M.J. Epidermal langerhans cell-deficient mice develop enhanced contact hypersensitivity. Immunity 23, 611–620 (2005).

  9. 9

    Obhrai, J.S. et al. Langerhans cells are not required for efficient skin graft rejection. J. Invest. Dermatol. 128, 1950–1955 (2008).

  10. 10

    Schenkel, J.M. & Masopust, D. Tissue-resident memory T cells. Immunity 41, 886–897 (2014).

  11. 11

    Gebhardt, T. et al. Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus. Nat. Immunol. 10, 524–530 (2009).

  12. 12

    Jiang, X. et al. Skin infection generates non-migratory memory CD8+ TRM cells providing global skin immunity. Nature 483, 227–231 (2012).

  13. 13

    Ariotti, S. et al. T cell memory. Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert. Science 346, 101–105 (2014).

  14. 14

    Rashighi, M. et al. CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo. Sci. Transl. Med. 6, 223ra23 (2014).

  15. 15

    Bertolini, M., Uchida, Y. & Paus, R. Toward the clonotype analysis of alopecia areata-specific, intralesional human CD8+ T lymphocytes. J. Investig. Dermatol. Symp. Proc. 17, 9–12 (2015).

  16. 16

    Li, M.O. & Flavell, R.A. TGF-β: a master of all T cell trades. Cell 134, 392–404 (2008).

  17. 17

    Bobr, A. et al. Autocrine/paracrine TGF-β1 inhibits Langerhans cell migration. Proc. Natl. Acad. Sci. USA 109, 10492–10497 (2012).

  18. 18

    Kel, J.M., Girard-Madoux, M.J.H., Reizis, B. & Clausen, B.E. TGF-β is required to maintain the pool of immature Langerhans cells in the epidermis. J. Immunol. 185, 3248–3255 (2010).

  19. 19

    Yasmin, N. et al. Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation. J. Exp. Med. 210, 2597–2610 (2013).

  20. 20

    Mackay, L.K. et al. The developmental pathway for CD103+CD8+ tissue-resident memory T cells of skin. Nat. Immunol. 14, 1294–1301 (2013).

  21. 21

    Travis, M.A. & Sheppard, D. TGF-β activation and function in immunity. Annu. Rev. Immunol. 32, 51–82 (2014).

  22. 22

    Yang, Z. et al. Absence of integrin-mediated TGFβ1 activation in vivo recapitulates the phenotype of TGFβ1-null mice. J. Cell Biol. 176, 787–793 (2007).

  23. 23

    Aluwihare, P. et al. Mice that lack activity of αvβ6- and αvβ8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice. J. Cell Sci. 122, 227–232 (2009).

  24. 24

    Cohn, R.D. et al. Angiotensin II type 1 receptor blockade attenuates TGF-β-induced failure of muscle regeneration in multiple myopathic states. Nat. Med. 13, 204–210 (2007).

  25. 25

    Lanz, T.V. et al. Angiotensin II sustains brain inflammation in mice via TGF-β. J. Clin. Invest. 120, 2782–2794 (2010).

  26. 26

    Bartholin, L. et al. Generation of mice with conditionally activated transforming growth factor β signaling through the TGFβ1/ALK5 receptor. Genesis 46, 724–731 (2008).

  27. 27

    Nagao, K. et al. Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin. Nat. Immunol. 13, 744–752 (2012).

  28. 28

    Melton, A.C. et al. Expression of αvβ8 integrin on dendritic cells regulates Th17 cell development and experimental autoimmune encephalomyelitis in mice. J. Clin. Invest. 120, 4436–4444 (2010).

  29. 29

    Travis, M.A. et al. Loss of integrin αvβ8 on dendritic cells causes autoimmunity and colitis in mice. Nature 449, 361–365 (2007).

  30. 30

    Abe, M. et al. An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal. Biochem. 216, 276–284 (1994).

  31. 31

    Farache, J. et al. Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity 38, 581–595 (2013).

  32. 32

    Seré, K. et al. Two distinct types of Langerhans cells populate the skin during steady state and inflammation. Immunity 37, 905–916 (2012).

  33. 33

    El-Asady, R. et al. TGF-β-dependent CD103 expression by CD8+ T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease. J. Exp. Med. 201, 1647–1657 (2005).

  34. 34

    Casey, K.A. et al. Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues. J. Immunol. 188, 4866–4875 (2012).

  35. 35

    Masopust, D. et al. Dynamic T cell migration program provides resident memory within intestinal epithelium. J. Exp. Med. 207, 553–564 (2010).

  36. 36

    Zaid, A. et al. Persistence of skin-resident memory T cells within an epidermal niche. Proc. Natl. Acad. Sci. USA 111, 5307–5312 (2014).

  37. 37

    Park, C.O. & Kupper, T.S. The emerging role of resident memory T cells in protective immunity and inflammatory disease. Nat. Med. 21, 688–697 (2015).

  38. 38

    Merad, M., Sathe, P., Helft, J., Miller, J. & Mortha, A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu. Rev. Immunol. 31, 563–604 (2013).

  39. 39

    Harberts, E., Zhou, H., Fishelevich, R., Liu, J. & Gaspari, A.A. Ultraviolet radiation signaling through TLR4/MyD88 constrains DNA repair and plays a role in cutaneous immunosuppression. J. Immunol. 194, 3127–3135 (2015).

  40. 40

    Wilson, N.S. et al. Normal proportion and expression of maturation markers in migratory dendritic cells in the absence of germs or Toll-like receptor signaling. Immunol. Cell Biol. 86, 200–205 (2008).

  41. 41

    Zhang, N. & Bevan, M.J. Transforming growth factor-β signaling controls the formation and maintenance of gut-resident memory T cells by regulating migration and retention. Immunity 39, 687–696 (2013).

  42. 42

    Knight, P.A. et al. Enteric expression of the integrin αvβ6 is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1. Am. J. Pathol. 161, 771–779 (2002).

  43. 43

    Akhurst, R.J. & Hata, A. Targeting the TGFβ signalling pathway in disease. Nat. Rev. Drug Discov. 11, 790–811 (2012).

  44. 44

    Kaplan, D.H. et al. Autocrine/paracrine TGFβ1 is required for the development of epidermal Langerhans cells. J. Exp. Med. 204, 2545–2552 (2007).

  45. 45

    Weinreb, P.H. et al. Function-blocking integrin αvβ6 monoclonal antibodies: distinct ligand-mimetic and nonligand-mimetic classes. J. Biol. Chem. 279, 17875–17887 (2004).

  46. 46

    Beura, L.K. et al. Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution. J. Leukoc. Biol. 97, 217–225 (2015).

  47. 47

    Steinert, E.M. et al. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance. Cell 161, 737–749 (2015).

  48. 48

    Dlugosz, A.A., Glick, A.B., Tennenbaum, T., Weinberg, W.C. & Yuspa, S.H. Isolation and utilization of epidermal keratinocytes for oncogene research. Methods Enzymol. 254, 3–20 (1995).

  49. 49

    Annes, J.P., Chen, Y., Munger, J.S. & Rifkin, D.B. Integrin αvβ6-mediated activation of latent TGF-β requires the latent TGF-β binding protein-1. J. Cell Biol. 165, 723–734 (2004).

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Acknowledgements

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.).

Author information

J.M., D.M. and D.H.K. designed and interpreted experiments; J.M. performed most experiments; L.K.B., B.A., B.C., S.W.K., N.E.W., B.Z.I., S.W. and E.A.T. performed experiments and provided technical assistance; C.M. and W.D.S. provided technical and conceptual assistance; L.B. and D.S. provided reagents and technical assistance; A.B., A.K. and A.G.B. collected and analyzed data from control and losartan treated patients; and J.M. and D.H.K. wrote the manuscript and all authors edited it.

Correspondence to Daniel H Kaplan.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fewer epidermal LCs in losartan-treated patients.

(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.).

Supplementary Figure 3 Epidermal LC distribution and KC subset gating strategy.

(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.

Supplementary Figure 4 Establishment of chimerism in wild-type and Itgb6−/− mice.

(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.

Supplementary Figure 5 Ablation of Itgb8 in LCs does not affect epidermal residence.

(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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