Isolation of NK Cells and NK-Like Cells from the Intestinal Lamina Propria

  • Stephanie L. Sanos
  • Andreas Diefenbach
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 612)

Abstract

Being exposed to food products, pathogens and harmless commensal bacteria, the mucosal immune system faces a constant challenge. Therefore, maintenance of a homeostatic balance is required to achieve tolerance to harmless bacteria and their products and to induce potent immunity to infection with pathogenic bacteria. Until recently, the literature on mucosal natural killer (NK) cells residing in the intestinal lamina propria was scarce and phenotype and function of gut mucosal NK cells did not receive much attention. Recently, data have become available identifying two distinct subsets of mucosal NKp46+ lymphocytes based on the expression of the orphan transcription factor RORγt. In many ways, the RORγt− subset resembled “classical” NK cells in that it was developmentally dependent on IL-15 but not on RORγt and displayed NK cell function (e.g., cell-mediated cytotoxicity, IFN-γ production). In contrast, the RORγt+ subset developed independent of IL-15 but required RORγt, suggesting that this subset may be related to lymphoid tissue inducer (LTi) cells. Interestingly, these RORγt+ NKp46+ NK-LTi cells constitutively produced large amounts of IL-22, a cytokine regulating antimicrobial protection and regeneration of epithelial cells. In this chapter, we provide experimental procedures to isolate “classical” NK cells from the intestinal lamina propria as well as the newly described lymphoid tissue inducer-like (LTi-like) cells producing IL-22 and co-expressing NK cell receptors.

Key words

Lymphoid tissue inducer cells RORγt IL-22, IL-15 mucosal immunity cryptopatches 
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References

  1. 1.
    Herberman, R. B., Nunn, M. E., Holden, H. T., and Lavrin, D. H. (1975) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer 16, 230–239.CrossRefPubMedGoogle Scholar
  2. 2.
    Kiessling, R., Klein, E., Pross, H., and Wigzell, H. (1975) “Natural” killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristic of the killer cell. Eur J Immunol 5, 117–121.CrossRefPubMedGoogle Scholar
  3. 3.
    Trinchieri, G. (1989) Biology of natural killer cells. Adv Immunol 47, 187–376.CrossRefPubMedGoogle Scholar
  4. 4.
    Mowat, A. M., and Viney, J. L. (1997) The anatomical basis of intestinal immunity. Immunol Rev 156, 145–166.CrossRefPubMedGoogle Scholar
  5. 5.
    Newberry, R. D., and Lorenz, R. G. (2005) Organizing a mucosal defense. Immunol Rev 206, 6–21.CrossRefPubMedGoogle Scholar
  6. 6.
    Artis, D. (2008) Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat Rev Immunol 8, 411–420.CrossRefPubMedGoogle Scholar
  7. 7.
    Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., and Medzhitov, R. (2004) Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241.CrossRefPubMedGoogle Scholar
  8. 8.
    Cash, H. L., Whitham, C. V., Behrendt, C. L., and Hooper, L. V. (2006) Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313, 1126–1130.CrossRefPubMedGoogle Scholar
  9. 9.
    Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J. P., and Ricciardi-Castagnoli, P. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2, 361–367.CrossRefPubMedGoogle Scholar
  10. 10.
    Nenci, A., Becker, C., Wullaert, A., Gareus, R., van Loo, G., Danese, S., Huth, M., Nikolaev, A., Neufert, C., Madison, B., Gumucio, D., Neurath, M. F., and Pasparakis, M. (2007) Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 446, 557–561.CrossRefPubMedGoogle Scholar
  11. 11.
    Dobson, G. E. (1884) On the presence of Peyer’s patches (Glandulae Agminatae) in the caecum and colon of certain mammals. J Anat Physiol 18, 388–392.PubMedGoogle Scholar
  12. 12.
    Kanamori, Y., Ishimaru, K., Nanno, M., Maki, K., Ikuta, K., Nariuchi, H., and Ishikawa, H. (1996) Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J Exp Med 184, 1449–1459.CrossRefPubMedGoogle Scholar
  13. 13.
    Hamada, H., Hiroi, T., Nishiyama, Y., Takahashi, H., Masunaga, Y., Hachimura, S., Kaminogawa, S., Takahashi-Iwanaga, H., Iwanaga, T., Kiyono, H., Yamamoto, H., and Ishikawa, H. (2002) Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 168, 57– 64.PubMedGoogle Scholar
  14. 14.
    Ivanov, II, McKenzie, B. S., Zhou, L., Tadokoro, C. E., Lepelley, A., Lafaille, J. J., Cua, D. J., and Littman, D. R. (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133.CrossRefPubMedGoogle Scholar
  15. 15.
    Sanos, S. L., Bui, V. L., Mortha, A., Oberle, K., Heners, C., Johner, C., and Diefenbach, A. (2009) RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells. Nat Immunol 10, 83–91.CrossRefPubMedGoogle Scholar
  16. 16.
    Luci, C., Reynders, A., Ivanov, II, Cognet, C., Chiche, L., Chasson, L., Hardwigsen, J., Anguiano, E., Banchereau, J., Chaussabel, D., Dalod, M., Littman, D. R., Vivier, E., and Tomasello, E. (2009) Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin. Nat Immunol 10, 75–82.CrossRefPubMedGoogle Scholar
  17. 17.
    Satoh-Takayama, N., Vosshenrich, C. A., Lesjean-Pottier, S., Sawa, S., Lochner, M., Rattis, F., Mention, J. J., Thiam, K., Cerf-Bensussan, N., Mandelboim, O., Eberl, G., and Di Santo, J. P. (2008) Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity 29, 958–970.CrossRefPubMedGoogle Scholar
  18. 18.
    Zhou, L., Lopes, J. E., Chong, M. M., Ivanov, II, Min, R., Victora, G. D., Shen, Y., Du, J., Rubtsov, Y. P., Rudensky, A. Y., Ziegler, S. F., and Littman, D. R. (2008) TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 453, 236–240.CrossRefPubMedGoogle Scholar
  19. 19.
    Iwasaki, A. (2007) Mucosal dendritic cells. Annu Rev Immunol 25, 381–418.CrossRefPubMedGoogle Scholar
  20. 20.
    Tagliabue, A., Befus, A. D., Clark, D. A., and Bienenstock, J. (1982) Characteristics of natural killer cells in the murine intestinal epithelium and lamina propria. J Exp Med 155, 1785–1796.CrossRefPubMedGoogle Scholar
  21. 21.
    Hogan, P. G., Hapel, A. J., and Doe, W. F. (1985) Lymphokine-activated and natural killer cell activity in human intestinal mucosa. J Immunol 135, 1731–1738.PubMedGoogle Scholar
  22. 22.
    Gibson, P. R., and Jewell, D. P. (1985) The nature of the natural killer (NK) cell of human intestinal mucosa and mesenteric lymph node. Clin Exp Immunol 61, 160–168.PubMedGoogle Scholar
  23. 23.
    Walzer, T., Blery, M., Chaix, J., Fuseri, N., Chasson, L., Robbins, S. H., Jaeger, S., Andre, P., Gauthier, L., Daniel, L., Chemin, K., Morel, Y., Dalod, M., Imbert, J., Pierres, M., Moretta, A., Romagne, F., and Vivier, E. (2007) Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46. Proc Natl Acad Sci U S A 104, 3384–3389.CrossRefPubMedGoogle Scholar
  24. 24.
    Sivori, S., Vitale, M., Morelli, L., Sanseverino, L., Augugliaro, R., Bottino, C., Moretta, L., and Moretta, A. (1997) p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med 186, 1129–1136.CrossRefPubMedGoogle Scholar
  25. 25.
    Sun, Z., Unutmaz, D., Zou, Y. R., Sunshine, M. J., Pierani, A., Brenner-Morton, S., Mebius, R. E., and Littman, D. R. (2000) Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288, 2369–2373.CrossRefPubMedGoogle Scholar
  26. 26.
    Kurebayashi, S., Ueda, E., Sakaue, M., Patel, D. D., Medvedev, A., Zhang, F., and Jetten, A. M. (2000) Retinoid-related orphan receptor gamma (RORgamma) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc Natl Acad Sci U S A 97, 10132–10137.CrossRefPubMedGoogle Scholar
  27. 27.
    Eberl, G., and Littman, D. R. (2004) Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells. Science 305, 248–251.CrossRefPubMedGoogle Scholar
  28. 28.
    Zheng, Y., Valdez, P. A., Danilenko, D. M., Hu, Y., Sa, S. M., Gong, Q., Abbas, A. R., Modrusan, Z., Ghilardi, N., de Sauvage, F. J., and Ouyang, W. (2008) Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 14, 282–289.CrossRefPubMedGoogle Scholar
  29. 29.
    Cella, M., Fuchs, A., Vermi, W., Facchetti, F., Otero, K., Lennerz, J. K., Doherty, J. M., Mills, J. C., and Colonna, M. (2008) A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature. [epub ahead of print]Google Scholar
  30. 30.
    Zenewicz, L. A., Yancopoulos, G. D., Valenzuela, D. M., Murphy, A. J., Stevens, S., and Flavell, R. A. (2008) Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease. Immunity 29, 947–957.CrossRefPubMedGoogle Scholar
  31. 31.
    Eberl, G., Marmon, S., Sunshine, M. J., Rennert, P. D., Choi, Y., and Littman, D. R. (2004) An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 5, 64–73.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Stephanie L. Sanos
    • 1
  • Andreas Diefenbach
    • 1
  1. 1.Institute of Medical Microbiology & HygieneUniversity of Freiburg Medical CenterFreiburgGermany

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