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Labeling and tracking of immune cells in ex vivo human skin

Nature Protocols volume 16, pages 791–811 (2021)Cite this article

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Abstract

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

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Fig. 1: Setup of the ex vivo imaging system for skin biopsies.
Fig. 2: Removal of sodium azide from conventional antibody reagent allows detection of migrating epidermal CD8+ CD103+ T cells.
Fig. 3: SHG and AF594+ signal in ex vivo human biopsies stained with control and anti-human CD8 nanobodies.
Fig. 4: Labeling and tracking of CD8+ T cells in ex vivo human skin.
Fig. 5: Visualization of immune cells and structural components in ex vivo human skin using conventional staining reagents.

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Data availability

Examples of the primary datasets underlying all data figures and Supplementary Videos presented in this article can be found at https://doi.org/10.5281/zenodo.3843094.

References

  1. Kabashima, K., Honda, T., Ginhoux, F. & Egawa, G. The immunological anatomy of the skin. Nat. Rev. Immunol. 19, 19–30 (2019).

    CAS  PubMed  Google Scholar 

  2. Pasparakis, M., Haase, I. & Nestle, F. O. Mechanisms regulating skin immunity and inflammation. Nat. Rev. Immunol. 14, 289–301 (2014).

    CAS  PubMed  Google Scholar 

  3. Clark, R. A. Resident memory T cells in human health and disease. Sci. Transl. Med. 7, 269rv261 (2015).

    Google Scholar 

  4. Gebhardt, T., Palendira, U., Tscharke, D. C. & Bedoui, S. Tissue-resident memory T cells in tissue homeostasis, persistent infection, and cancer surveillance. Immunol. Rev. 283, 54–76 (2018).

    CAS  PubMed  Google Scholar 

  5. Kumar, B. V. et al. Human tissue-resident memory t cells are defined by core transcriptional and functional signatures in lymphoid and mucosal sites. Cell Rep. 20, 2921–2934 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  8. Skon, C. N. et al. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells. Nat. Immunol. 14, 1285–1293 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 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).

    CAS  PubMed  Google Scholar 

  10. 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).

    CAS  PubMed  Google Scholar 

  11. Clark, R. A. et al. Skin effector memory T cells do not recirculate and provide immune protection in alemtuzumab-treated CTCL patients. Sci. Transl. Med. 4, 117ra117 (2012).

    Google Scholar 

  12. Muruganandah, V., Sathkumara, H. D., Navarro, S. & Kupz, A. A systematic review: the role of resident memory T Cells in infectious diseases and their relevance for vaccine development. Front. Immunol. 9, 1574 (2018).

    PubMed  PubMed Central  Google Scholar 

  13. Edwards, J. et al. CD103(+) Tumor-resident CD8(+) T cells are associated with improved survival in immunotherapy-naive melanoma patients and expand significantly during anti-PD-1 treatment. Clin. Cancer Res. 24, 3036–3045 (2018).

    CAS  PubMed  Google Scholar 

  14. Park, S. L., Gebhardt, T. & Mackay, L. K. Tissue-resident memory T cells in cancer immunosurveillance. Trends Immunol. 40, 735–747 (2019).

    CAS  PubMed  Google Scholar 

  15. Cheuk, S. et al. CD49a expression defines tissue-resident CD8(+) T cells poised for cytotoxic function in human skin. Immunity 46, 287–300 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Solimani, F., Meier, K. & Ghoreschi, K. Emerging topical and systemic JAK inhibitors in dermatology. Front. Immunol. 10, 2847 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Ariotti, S. et al. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proc. Natl Acad. Sci. USA 109, 19739–19744 (2012).

    CAS  PubMed  Google Scholar 

  18. Gebhardt, T. et al. Different patterns of peripheral migration by memory CD4+ and CD8+ T cells. Nature 477, 216–219 (2011).

    CAS  PubMed  Google Scholar 

  19. Li, J. L. et al. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat. Protoc. 7, 221–234 (2012).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  21. Beura, L. K. et al. Normalizing the environment recapitulates adult human immune traits in laboratory mice. Nature 532, 512–516 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Dijkgraaf, F. E. et al. Tissue patrol by resident memory CD8(+) T cells in human skin. Nat. Immunol. 20, 756–764 (2019).

    CAS  PubMed  Google Scholar 

  23. Wu, C. H. et al. Imaging cytometry of human leukocytes with third harmonic generation microscopy. Sci. Rep. 6, 37210 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Nishida-Aoki, N. & Gujral, T. S. Emerging approaches to study cell–cell interactions in tumor microenvironment. Oncotarget 10, 785–797 (2019).

    PubMed  PubMed Central  Google Scholar 

  25. Grivel, J. C. & Margolis, L. Use of human tissue explants to study human infectious agents. Nat. Protoc. 4, 256–269 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kennedy, L. H. & Rinholm, J. E. Visualization and live imaging of oligodendrocyte organelles in organotypic brain slices using adeno-associated virus and confocal microscopy. J. Vis. Exp. 128, 56237 (2017).

    Google Scholar 

  27. Groff, B. D., Kinman, A. W. L., Woodroof, J. F. & Pompano, R. R. Immunofluorescence staining of live lymph node tissue slices. J. Immunol. Methods 464, 119–125 (2019).

    CAS  PubMed  Google Scholar 

  28. Peranzoni, E. et al. Ex vivo imaging of resident CD8 T lymphocytes in human lung tumor slices using confocal microscopy. J. Vis. Exp., 2017, 55709 (2017).

  29. Stucker, M. et al. The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis. J. Physiol. 538, 985–994 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mort, R. L., Hay, L. & Jackson, I. J. Ex vivo live imaging of melanoblast migration in embryonic mouse skin. Pigment Cell Melanoma Res. 23, 299–301 (2010).

    PubMed  PubMed Central  Google Scholar 

  31. Weigelin, B., Bakker, G. J. & Friedl, P. Third harmonic generation microscopy of cells and tissue organization. J. Cell Sci. 129, 245–255 (2016).

    CAS  PubMed  Google Scholar 

  32. Bins, A. D. et al. A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression. Nat. Med. 11, 899–904 (2005).

    CAS  PubMed  Google Scholar 

  33. Hsiao, C. Y. et al. Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin. Microsc. Res. Tech. 69, 992–997 (2006).

    PubMed  Google Scholar 

  34. Wei, S. H. et al. Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node. J. Immunol. 179, 1586–1594 (2007).

    CAS  PubMed  Google Scholar 

  35. Platonova, E. et al. Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders. Methods 88, 89–97 (2015).

    CAS  PubMed  Google Scholar 

  36. Gurskaya, N. G. et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24, 461–465 (2006).

    CAS  PubMed  Google Scholar 

  37. Adachi, T. et al. Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma. Nat. Med. 21, 1272–1279 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Yew, E., Rowlands, C. & So, P. T. Application of multiphoton microscopy in dermatological studies: a mini-review. J. Innov. Opt. Health Sci. 7, 1330010 (2014).

    PubMed  PubMed Central  Google Scholar 

  39. Clark, R. A. et al. The vast majority of CLA+ T cells are resident in normal skin. J. Immunol. 176, 4431–4439 (2006).

    CAS  PubMed  Google Scholar 

  40. Firooz, A. et al. The influence of gender and age on the thickness and echo-density of skin. Skin Res. Technol. 23, 13–20 (2017).

    CAS  PubMed  Google Scholar 

  41. Boehncke, W. H. & Schon, M. P. Psoriasis. Lancet 386, 983–994 (2015).

    CAS  PubMed  Google Scholar 

  42. Watanabe, R. et al. Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells. Sci. Transl. Med. 7, 279ra239 (2015).

    Google Scholar 

  43. Arbabi-Ghahroudi, M. Camelid single-domain antibodies: historical perspective and future outlook. Front. Immunol. 8, 1589 (2017).

    PubMed  PubMed Central  Google Scholar 

  44. Goedhart, M. et al. CXCR4, but not CXCR3, drives CD8+ T-cell entry into and migration through the murine bone marrow. Euro. J. Immunol. 49, 576–589 (2019).

    CAS  Google Scholar 

  45. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  PubMed  Google Scholar 

  46. Khan, T. N., Mooster, J. L., Kilgore, A. M., Osborn, J. F. & Nolz, J. C. Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection. J. Exp. Med. 213, 951–966 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Guimaraes, C. P. et al. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1787–1799 (2013).

    PubMed  PubMed Central  Google Scholar 

  48. Durand, R. E. & Olive, P. L. Cytotoxicity, mutagenicity and DNA damage by Hoechst 33342. J. Histochem. Cytochem. 30, 111–116 (1982).

    CAS  PubMed  Google Scholar 

  49. Purschke, M., Rubio, N., Held, K. D. & Redmond, R. W. Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochem. Photobiol. Sci. 9, 1634–1639 (2010).

    CAS  PubMed  Google Scholar 

  50. Cahalan, M. D. & Parker, I. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. Immunol. 26, 585–626 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Beltman, J. B., Maree, A. F. & de Boer, R. J. Analysing immune cell migration. Nat. Rev. Immunol. 9, 789–798 (2009).

    CAS  PubMed  Google Scholar 

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Acknowledgements

Plasmid sequences for anti-mouse and anti-human CD8 nanobodies were kindly provided by 121Bio with support of M. Gostissa and G. Grotenbreg (Agenus subsequently acquired substantially all the assets of 121Bio). We acknowledge H. Ploegh (Harvard University) for the sortase expression vector and H. Ovaa and D. El Atmioui (Leiden University) for providing the GGGC peptide. We thank T. Venema (Slotervaart Ziekenhuis), P.G.L. Koolen (Rode Kruis Ziekenhuis), and W.G. van Selms (Onze Lieve Vrouwe Gasthuis West) and staff of the plastic surgery departments for the human skin tissue. We thank J. Beltman (LUMC) and B. van den Broek (Netherlands Cancer Institute, NKI) for analysis of migration parameters, J. Song (NKI) for histopathological analysis, M. Hoekstra (NKI) for illustration of the ex vivo imaging setup, T. Rademakers (Maastricht University), M. Rashidian (Dana-Farber Cancer Institute), L. Oomen, and L. Brocks (NKI) for technical support, and L. Perie (Curie Institute) and members of the Schumacher and Haanen laboratories for discussions. This work was supported by ERC AdG Life-His-T (to T.N.S.) and an EADV Research Fellowship (to T.R.M.).

Author information

Authors and Affiliations

  1. Division of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands

    Feline E. Dijkgraaf, Mireille Toebes, David W. Vredevoogd & Ton N. Schumacher

  2. Research Facility, Sanquin Amsterdam, Amsterdam, the Netherlands

    Mark Hoogenboezem

  3. BioImaging Facility, The Netherlands Cancer Institute, Amsterdam, the Netherlands

    Marjolijn Mertz

  4. Department of Dermatology and Netherlands Institute for Pigment Disorders, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands

    Tiago R. Matos, Marcel B. M. Teunissen & Rosalie M. Luiten

Authors
  1. Feline E. Dijkgraaf
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Contributions

F.E.D. and D.W.V. conceived the idea of the murine ex vivo imaging setup. F.E.D. designed and performed experiments with murine and human skin, and analyzed imaging data. M.T. generated nanobodies and nanobody conjugates. M.H. performed multiphoton imaging experiments. F.E.D. and M.M. designed imaging analysis. T.R.M. and M.B.M.T. organized human skin material. F.E.D., M.T., M.H., T.R.M., M.B.M.T., D.W.V., R.M.L., and T.N.S. contributed to protocol design. F.E.D. and T.N.S. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Ton N. Schumacher.

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Key reference using this protocol

Dijkgraaf, F. et al. Nat. Immunol. 20, 756–764 (2019): https://doi.org/10.1038/s41590-019-0404-3

Supplementary information

Reporting Summary

Supplementary Video 1

CD8+ T cells in murine ex vivo skin regain motility and dendricity after overnight culture. Maximum confocal microscopy projection of OT-I GFP+ CD8+ T cells (green) in mouse skin imaged directly after mounting in ex vivo imaging setup. Cells start off round and immobile and regain mobility and dendricity over time (time in minutes). N=2 recordings and 2 independent observations. Scale bar, 50 μm.

Supplementary Video 2

Removal of sodium azide from conventional antibody reagent allows detection of migrating epidermal CD8+ CD103+ T cells in human skin. First segment: Section view of MP recording showing ex vivo human skin biopsy stained with anti-hCD103-AF488 conventional antibody (green) without removal of sodium azide, also showing anti-hCD8-AF594 nanobody staining and second harmonics generation signal (SHG, blue). Note that CD8+ T cells display a round morphology and are immobile. Representative of n=2 individuals. Scale bar, 20 μm. Second segment: Section view of MP recording showing ex vivo human skin biopsy stained with anti-hCD103-AF488 conventional antibody (green) after removal of sodium azide, also showing anti-hCD8-AF594 nanobody staining and second harmonics generation signal (SHG, blue). Note that CD8+ CD103+ T cells display a variable morphology and migrate. Data in second segment adapted from Supplementary Video 11 in 22. Representative of n=3 individuals. Scale bar, 15 μm.

Supplementary Video 3

Labeling and tracking of CD8+ T cells in ex vivo human skin. First segment: Top view of MP-recordings showing examples of tracked anti-hCD8-AF594 nanobody labeled T cells (red) that migrate in ex vivo human skin. Second segment: Examples of autofluorescent and fragmented AF594+ objects in ex vivo human skin biopsies stained with anti-hCD8-AF594 nanobody (red), also showing second harmonics generation signal (SHG, collagen type I, blue). Both segments: lines indicate tracked cells. Datasets are described in Fig. 4a in 22. Data representative of n=4 individuals. Scale bars, 10 μm.

Supplementary Video 4

Visualization of immune cells and structural components in ex vivo human skin using conventional staining reagents. First segment: Top view of MP-recording showing anti-hCD8-AF594 nanobody-labeled CD8+ T cell (red) migrating in between Hoechst+ nuclei (gray) in the epidermis. Dataset described in Supplementary Video 9-II in 22. Representative of n=4 individuals. Scale bar, 10 μm. Second segment: Section view showing anti-hCD8-AF594 nanobody-labeled CD8+ T cell (red) migrating below anti-hCD1a-AF488 antibody labeled Langerhans cells (green), also showing second harmonics generation signal (collagen type I, blue). Dataset described in Supplementary Video 12-I in 22. Representative of n=4 individuals. Scale bar, 50 μm. Third segment: Section view of MP-recording showing anti-hCD8-AF594 nanobody labeled CD8+ T cells (red) migrating alongside anti-human collagen type IV positive structures (i.e., basement membrane and dermal vessels, green), also showing second harmonics generation signal (collagen type I, blue). Dataset is described in Supplementary Video 14-II in 22. Representative of n=3 individuals. Scale bar, 20 μm.

Supplementary Data 1

DNA sequence of the empty pHEN6c expression vector containing the LPETGG (i.e., Sortag) and 6xHis (i.e., Histag) sequence and the NcoI and BstEII cloning sites, which can be used to insert the desired nanobody sequence.

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Dijkgraaf, F.E., Toebes, M., Hoogenboezem, M. et al. Labeling and tracking of immune cells in ex vivo human skin. Nat Protoc 16, 791–811 (2021). https://doi.org/10.1038/s41596-020-00435-8

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