Москва, г. Москва и Московская область, Россия
Россия
Россия
ФГБУ «Федеральный научно- клинический центр специализированных видов медицинской помощи и медицинских технологий Федерального медико-биологического агентства» (Кафедра дерматовенерологии и косметологии, Профессор)
Россия
Способность γδТ-лимфоцитов расСВИТИЧ О.А., академик РАН, д.м.н., профессор РАН, директор ФГБНУ «Научно-исследовательский институт вакцин и сывороток им. И.И. Мечникова», пропознавать антигены независимо от главного комплекса гистосовместимости, а также продуцировать цитокины и цитотоксические молекулы делает их перспективными объектами для иммунотерапии. Тканерезидентные γδТ-клетки играют важную роль в иммунном надзоре, поддержании гомеостаза и патогенезе различных заболеваний, включая инфекционные, онкологические и аутоиммунные процессы. Однако методы выделения и культивирования γδТ-лимфоцитов остаются сложными и недостаточно стандартизированными, особенно для тканевых источников. В данном обзоре рассматриваются современные подходы к получению этих клеток из различных тканей человека (биоптатов, операционного материала) и животных (преимущественно мышей).
γδΤ-лимфоциты, тканерезидентные лимфоциты, выделение клеток, культура лимфоцитов
1. Hu Y., Hu Q., Li Y., Lu L., Xiang Z., Yin Z., Kabelitz D., Wu Y. γδT cells: origin and fate, subsets, diseases and immunotherapy // Signal Transduct Target Ther. – 2023 Nov 22; 8(1):434. Doi:https://doi.org/10.1038/s41392-023-01653-8
2. Ribot J.C., Lopes N., Silva-Santos B. γδT cells in tissue physiology and surveillance // Nat Rev Immunol. – 2021 Apr; 21(4):221–232. Doi:https://doi.org/10.1038/s41577-020-00452-4
3. Qu G., Wang S., Zhou Z., Jiang D., Liao A., Luo J. Comparing Mouse and Human Tissue-Resident γδT cells // Front Immunol. – 2022 Jun 8; 13:891687. Doi:https://doi.org/10.3389/fimmu.2022.891687
4. Nielsen M.M., Witherden D.A., Havran W.L. γδT cells in homeostasis and host defence of epithelial barrier tissues // Nat Rev Immunol. – 2017 Dec; 17(12):733–745. Doi:https://doi.org/10.1038/nri.2017.101
5. Cheng M., Hu S. Lung-resident γδ T cells and their roles in lung diseases // Immunology. – 2017 Aug; 151(4):375–384. Doi:https://doi.org/10.1111/imm.12764
6. Kang I., Kim Y., Lee H.K. Double-edged sword: γδ T cells in mucosal homeostasis and disease // Exp Mol Med. – 2023 Sep; 55(9):1895–1904. Doi:https://doi.org/10.1038/s12276-023-00985-3
7. Gray J.I., Caron D.P., Wells S.B., Guyer R., Szabo P., Rainbow D., Ergen C., Rybkina K., Bradley M.C., Matsumoto R., Pethe K., Kubota M., Teichmann S., Jones J., Yosef N., Atkinson M., Brusko M., Brusko T.M., Connors T.J., Sims P.A., Farber D.L. Human γδT cells in diverse tissues exhibit site- specific maturation dynamics across the life span // Sci Immunol. – 2024 Jun 7; 9(96):eadn3954. Doi:https://doi.org/10.1126/sciimmunol.adn3954
8. Ribeiro M., Brigas H.C., Temido-Ferreira M., Pousinha P.A., Regen T., Santa C., Coelho J.E., Marques-Morgado I., Valente C.A., Omenetti S., Stockinger B., Waisman A., Manadas B., Lopes L.V., Silva-Santos B., Ribot J.C. Meningeal γδ T cell-derived IL-17 controls synaptic plasticity and short-term memory // Sci Immunol. – 2019 Oct 11; 4(40):eaay5199. Doi:https://doi.org/10.1126/sciimmunol.aay5199
9. Jensen I.J., Wells S.B., Gras J., Farber D.L. Isolation and Characterization of Human Tissue Resident Memory T cells // Curr Protoc. – 2025 Mar; 5(3):e70120. Doi:https://doi.org/10.1002/cpz1.70120
10. Upton N.E.G., Hayday T.S., James L.K. Isolation and Characterization of Lymphocytes from Human Mucosal Biopsies // Methods Mol Biol. – 2019; 2020:165–174. Doi:https://doi.org/10.1007/978-1-4939-9591-2_12
11. Iwamuro M., Takahashi T., Watanabe N., Okada H. Isolation of lymphocytes from the human gastric mucosa // World J Methodol. – 2021 Jul 20; 11(4):199–207. Doi:https://doi.org/10.5662/wjm.v11.i4.199
12. Shacklett B.L., Critchfield J.W., Lemongello D. Isolating mucosal lymphocytes from biopsy tissue for cellular immunology assays // Methods Mol Biol. – 2009; 485:347–56. Doi:https://doi.org/10.1007/978-1-59745-170- 3_23
13. Flynn L., Carton J., Byrne B., Kelehan P., O'Herlihy C., O'Farrelly C. Optimisation of a technique for isolating lymphocyte subsets from human endometrium // Immunol Invest. – 1999 Jul; 28(4):235–46. Doi:https://doi.org/10.3109/08820139909060858
14. Depierreux D.M., Seshadri E., Shmeleva E.V., Kieckbusch J., Hawkes D.A., Colucci F. Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry // J Vis Exp. – 2021 Oct 14; (176). Doi:https://doi.org/10.3791/62670
15. Parasar P., Bernard M., Ahn S.H., Kshirsagar S.K., Nguyen S.L., Grzesiak G.R., Vettathu M., Martin D., Petroff M.G. Isolation and characterization of uterine leukocytes collected using a uterine swab technique // Am J Reprod Immunol. – 2022 Nov; 88(5):e13614. Doi:https://doi.org/10.1111/aji.13614
16. Duong A., Wong A., Ramendra R., Sebben D., Moshkelgosha S., MacParland S., Liu M., Juvet S., Martinu T. A Rapid Human Lung Tissue Dissociation Protocol Maximizing Cell Yield and MinimizingCellular Stress // Am J Respir Cell Mol Biol. – 2024 Nov; 71(5):509–518. Doi:https://doi.org/10.1165/rcmb.2023- 0343MA
17. Zakeri N., Hall A., Swadling L., Pallett L.J., Schmidt N.M., Diniz M.O., Kucykowicz S., Amin O.E., Gander A., Pinzani M., Davidson B.R., Quaglia A., Maini M.K. Characterisation and induction of tissue- resident gamma delta T-cells to target hepatocellular carcinoma // Nat Commun. – 2022 Mar 16; 13(1):1372. Doi:https://doi.org/10.1038/s41467-022-29012-1
18. Hunter S., Willcox C.R., Davey M.S., Kasatskaya S.A., Jeffery H.C., Chudakov D.M., Oo Y.H., Willcox B.E. Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations // J Hepatol. – 2018 Sep; 69(3):654–665. Doi:https://doi.org/10.1016/j.jhep.2018.05.007
19. He X., de Oliveira V.L., Keijsers R., Joosten I., Koenen H.J. Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture // J Vis Exp. – 2016 Apr 8; (110):e52564. Doi:https://doi.org/10.3791/52564
20. Polakova A., Hudemann C., Wiemers F., Kadys A., Gremke N., Lang M., Zwiorek L., Pfützner W., Hertl M., Möbs C., Zimmer C.L. Isolation of Lymphocytes from Human Skin and Murine Tissues: A Rapid and Epitope-Preserving Approach // JID Innov. – 2022 Sep 7; 3(1):100155. Doi:https://doi.org/10.1016/j.xjidi.2022.100155
21. Du W., Lenz D., Köhler R., Zhang E., Cendon C., Li J., Massoud M., Wachtlin J., Bodo J., Hauser A.E., Radbruch A., Dong J. Rapid Isolation of Functional ex vivo Human Skin Tissue-Resident Memory T Lymphocytes // Front Immunol. – 2021 Mar 22; 12:624013. Doi:https://doi.org/10.3389/fimmu.2021.624013
22. Salimi M., Subramaniam S., Selvakumar T., Wang X., Zemenides S., Johnson D., Ogg G. Enhanced isolation of lymphoid cells from human skin // Clin Exp Dermatol. – 2016 Jul; 41(5):552-6. Doi:https://doi.org/10.1111/ced.12802
23. Gargas S., Bshara-Corson S., Cruz M., Jameson J. Isolation and Analysis of Mouse and Human Skin γδT Cells // Curr Protoc Immunol. – 2019 Dec; 127(1):e92. Doi:https://doi.org/10.1002/cpim.92
24. Bettin L., Darbellay J., van Kessel J., Dhar N., Gerdts V. Porcine γδT cells express cytotoxic cell- associated markers and display killing activity but are not selectively cytotoxic against PRRSV- or swIAV-infected macrophages // Front Immunol. – 2024 Jul 31; 15:1434011. Doi:https://doi.org/10.3389/fimmu.2024.1434011
25. Stepanova K., Sinkora M. Porcine γδT lymphocytes can be categorized into two functionally and developmentally distinct subsets according to expression of CD2 and level of TCR // J Immunol. – 2013 Mar 1; 190(5):2111-20. Doi:https://doi.org/10.4049/jimmunol.1202890
26. Le Page L., Gillespie A., Schwartz J.C., Prawits L.M., Schlerka A., Farrell C.P., Hammond J.A., Baldwin C.L., Telfer J.C., Hammer S.E. Subpopulations of swine γδ T cells defined by TCRγ and WC1 gene expression // Dev Comp Immunol. – 2021 Dec; 125:104214. Doi:https://doi.org/10.1016/j.dci.2021.104214
27. Hoek A., Rutten V.P., Kool J., Arkesteijn G.J., Bouwstra R.J., Van Rhijn I., Koets A.P. Subpopulations of bovine WC1(+) gammadelta T cells rather than CD4(+)CD25(high) Foxp3(+) T cells act as immune regulatory cells ex vivo // Vet Res. – 2009 Jan–Feb; 40(1):6. Doi:https://doi.org/10.1051/vetres:2008044
28. McGill J.L., Sacco R.E. γδT cells and the immune response to respiratory syncytial virus infection // Vet Immunol Immunopathol. – 2016 Nov 15; 181:24–29. Doi:https://doi.org/10.1016/j.vetimm.2016.02.012
29. Baldwin C.L., Damani-Yokota P., Yirsaw A., Loonie K., Teixeira A.F., Gillespie A. Special features of γδ T cells in ruminants // Mol Immunol. – 2021 Jun; 134:161–169. Doi:https://doi.org/10.1016/j.molimm.2021.02.028
30. Wu H., Knight J.F., Alexander S.I. Regulatory gamma delta T cells in Heymann nephritis express an invariant Vgamma6/Vdelta1 with a canonical CDR3 sequence // Eur J Immunol. – 2004 Aug; 34(8):2322–30. Doi:https://doi.org/10.1002/eji.200324780
31. Williams L., Dery K.J., Lee W.H., Li H., Shively J.E., Kujawski M. Isolation and expansion of murine γδT cells from mouse splenocytes // J Immunol Methods. – 2022 Sep; 508:113322. Doi:https://doi.org/10.1016/j.jim.2022.113322
32. https: //www.miltenyibiotec.com/UN-en/products/tcrg-d-t-cell-isolation-kit-mouse.html
33. Wang Y., Yan J. Intracellular ATP delivery to in vitro expanded mouse CD27- γδT cells // STAR Protoc. – 2023 Sep 15; 4(3):102532. Doi:https://doi.org/10.1016/j.xpro.2023.102532 34. Ribot J.C., deBarros A., Pang D.J., Neves J.F., Peperzak V., Roberts S.J., Girardi M., Borst J., Hayday A.C., Pennington D.J., Silva-Santos B. CD27 is a thymic determinant of the balance between interferon-gamma- and interleukin 17-producing gammadelta T cell subsets // Nat Immunol. – 2009 Apr; 10(4):427–36. Doi:https://doi.org/10.1038/ni.1717
34. Sheridan B.S., Lefrançois L. Isolation of mouse lymphocytes from small intestine tissues // Curr Protoc Immunol. – 2012 Nov; Chapter 3:3.19.1-3.19.11. Doi:https://doi.org/10.1002/0471142735.im0319s99
35. Qiu Z., Sheridan B.S. Isolating Lymphocytes from the Mouse Small Intestinal Immune System // J Vis Exp. – 2018 Feb 28; (132):57281. Doi:https://doi.org/10.3791/57281
36. James O.J., Vandereyken M., Swamy M. Isolation, Characterization, and Culture of Intestinal Intraepithelial Lymphocytes // Methods Mol Biol. – 2020; 2121:141–152. Doi:https://doi.org/10.1007/978-1-0716- 0338-3_13
37. Xu H., Agalioti T., Zhao J., Steglich B., Wahib R., Vesely M.C.A., Bielecki P., Bailis W., Jackson R., Perez D., Izbicki J., Licona-Limón P., Kaartinen V., Geginat J., Esplugues E., Tolosa E., Huber S., Flavell R.A., Gagliani N. The induction and function of the anti-inflammatory fate of TH17 cells // Nat Commun. – 2020 Jul 3; 11(1):3334. Doi:https://doi.org/10.1038/s41467-020-17097-5
38. Kim E., Tran M., Sun Y., Huh J.R. Isolation and analyses of lamina propria lymphocytes from mouse intestines // STAR Protoc. – 2022 May 6; 3(2):101366. Doi:https://doi.org/10.1016/j.xpro.2022.101366
39. Scheuermann S., Schäfer A., Langejürgen J., Reis C. A step towards enzyme-free tissue dissociation // Current Directions in Biomedical Engineering. – 2019 5(1), 545–548. Doi:https://doi.org/10.1515/cdbme-2019-0137
40. Zhang J., Dong Z., Zhou R., Luo D., Wei H., Tian Z. Isolation of lymphocytes and their innate immune characterizations from liver, intestine, lung and uterus // Cell Mol Immunol. – 2005 Aug; 2(4):271–80.
41. Ye Y., Yue M., Jin X., Chen S., Li Y. Isolation of murine small intestinal intraepithelial γδT cells // Immunol Invest. – 2010; 39(7):661–73. Doi:https://doi.org/10.3109/08820131003753026
42. Снегирева Н., Гаврилова М., Сидорова Е. Выделение γδT-клеток из тонкой кишки мыши //Открытый журнал иммунологии. – 2013; 3, 221–223. Doi:https://doi.org/10.4236/oji.2013.34028
43. Rana I., Badarinath K., Zirmire R.K., Jamora C. Isolation and Quantification of Mouse γδT-cells in vitro and in vivo // Bio Protoc. – 2021 Sep 5; 11(17):e4148. Doi:https://doi.org/10.21769/BioProtoc.4148
