Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells
Main Article Content
Keywords
Tregs, flow cytometry, antibody panel, peripheral blood mononuclear cells
Abstract
T regulatory cells (Tregs) are a cell subset that can suppress immune responses to maintain homeostasis and self-tolerance. In some scenarios, the immunosuppressive nature could be associated to other pathological developments such as autoimmune diseases and cancers. Due to the importance of Tregs in disease pathogenesis, we developed and validated an 11-color flow cytometry panel for phenotypic and functional detection of Treg markers using healthy human donor peripheral blood mononuclear cells (PBMCs). Our panel contains 4 Treg surface proteins and 2 functional cytokines as well as T-lymphocyte lineage markers CD3, CD4, and CD8. Our data shows an increase in expression of markers CD25, FoxP3, CTLA4, GITR and intracellular cytokines IL4 and TGFβ when comparing unstimulated samples to CD3/CD28 bead stimulated samples. This 11-color panel can be used to functionally evaluate immunosuppressive Tregs in human PBMC samples.
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References
1. Chatenoud L, Bach JF. Adaptive human regulatory T cells: myth or reality? J Clin Invest. 2006;116(9):2325-7.
2. Liu Y, Amarnath S, Chen W. Requirement of CD28 signaling in homeostasis/survival of TGF-beta converted CD4+CD25+ Tregs from thymic CD4+CD25- single positive T cells. Transplantation. 2006;82(7):953-64.
3. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat Rev Immunol. 2003;3(3):253-7.
4. Bin Dhuban K, d'Hennezel E, Nashi E, Bar-Or A, Rieder S, Shevach EM, et al. Coexpression of TIGIT and FCRL3 identifies Helios+ human memory regulatory T cells. J Immunol. 2015;194(8):3687-96.
5. Beyer M, Classen S, Endl E, Kochanek M, Weihrauch MR, Debey-Pascher S, et al. Comparative approach to define increased regulatory T cells in different cancer subtypes by combined assessment of CD127 and FOXP3. Clin Dev Immunol. 2011;2011:734036.
6. Davids MS, Kim HT, Bachireddy P, Costello C, Liguori R, Savell A, et al. Ipilimumab for Patients with Relapse after Allogeneic Transplantation. N Engl J Med. 2016;375(2):143-53.
7. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med. 2004;199(7):971-9.
8. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood. 2007;110(8):2983-90.
9. d'Hennezel E, Piccirillo CA. Analysis of human FOXP3+ Treg cells phenotype and function. Methods Mol Biol. 2011;707:199-218.
10. Ronchetti S, Ricci E, Petrillo MG, Cari L, Migliorati G, Nocentini G, et al. Glucocorticoid-induced tumour necrosis factor receptor-related protein: a key marker of functional regulatory T cells. J Immunol Res. 2015;2015:171520.
11. Nettenstrom L, Alderson K, Raschke EE, Evans MD, Sondel PM, Olek S, et al. An optimized multi-parameter flow cytometry protocol for human T regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood. J Immunol Methods. 2013;387(1-2):81-8.
12. Baron U, Floess S, Wieczorek G, Baumann K, Grutzkau A, Dong J, et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol. 2007;37(9):2378-89.
13. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007;5(2):e38.
14. Polansky JK, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, et al. DNA methylation controls Foxp3 gene expression. Eur J Immunol. 2008;38(6):1654-63.
15. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009;30(6):899-911.
16. McClymont SA, Putnam AL, Lee MR, Esensten JH, Liu W, Hulme MA, et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol. 2011;186(7):3918-26.
17. Su H, Longhi MS, Wang P, Vergani D, Ma Y. Human CD4+CD25(high)CD127 (low/neg) regulatory T cells. Methods Mol Biol. 2012;806:287-99.
18. Walker LS. Treg and CTLA-4: two intertwining pathways to immune tolerance. J Autoimmun. 2013;45:49-57.
19. Ephrem A, Epstein AL, Stephens GL, Thornton AM, Glass D, Shevach EM. Modulation of Treg cells/T effector function by GITR signaling is context-dependent. Eur J Immunol. 2013;43(9):2421-9.
20. Dahmani A, Delisle JS. TGF-beta in T Cell Biology: Implications for Cancer Immunotherapy. Cancers (Basel). 2018;10(6).
21. Yang WC, Hwang YS, Chen YY, Liu CL, Shen CN, Hong WH, et al. Interleukin-4 Supports the Suppressive Immune Responses Elicited by Regulatory T Cells. Front Immunol. 2017;8:1508.
22. Patel T, Cunningham A, Holland M, Daley J, Lazo S, Hodi FS, et al. Development of an 8-color antibody panel for functional phenotyping of human CD8+ cytotoxic T cells from peripheral blood mononuclear cells. Cytotechnology. 2018;70(1):1-11.
23. Canavan JB, Afzali B, Scotta C, Fazekasova H, Edozie FC, Macdonald TT, et al. A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy. Blood. 2012;119(8):e57-66.
24. Cunningham R HM, McWilliams E, Hodi FS, Severgnini M. Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry. Journal of Biological Methods [Internet]. 2019; 6(2).
25. Heylmann D, Badura J, Becker H, Fahrer J, Kaina B. Sensitivity of CD3/CD28-stimulated versus non-stimulated lymphocytes to ionizing radiation and genotoxic anticancer drugs: key role of ATM in the differential radiation response. Cell Death Dis. 2018;9(11):1053.
26. Li Y, Kurlander RJ. Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation. J Transl Med. 2010;8:104.
2. Liu Y, Amarnath S, Chen W. Requirement of CD28 signaling in homeostasis/survival of TGF-beta converted CD4+CD25+ Tregs from thymic CD4+CD25- single positive T cells. Transplantation. 2006;82(7):953-64.
3. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat Rev Immunol. 2003;3(3):253-7.
4. Bin Dhuban K, d'Hennezel E, Nashi E, Bar-Or A, Rieder S, Shevach EM, et al. Coexpression of TIGIT and FCRL3 identifies Helios+ human memory regulatory T cells. J Immunol. 2015;194(8):3687-96.
5. Beyer M, Classen S, Endl E, Kochanek M, Weihrauch MR, Debey-Pascher S, et al. Comparative approach to define increased regulatory T cells in different cancer subtypes by combined assessment of CD127 and FOXP3. Clin Dev Immunol. 2011;2011:734036.
6. Davids MS, Kim HT, Bachireddy P, Costello C, Liguori R, Savell A, et al. Ipilimumab for Patients with Relapse after Allogeneic Transplantation. N Engl J Med. 2016;375(2):143-53.
7. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med. 2004;199(7):971-9.
8. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood. 2007;110(8):2983-90.
9. d'Hennezel E, Piccirillo CA. Analysis of human FOXP3+ Treg cells phenotype and function. Methods Mol Biol. 2011;707:199-218.
10. Ronchetti S, Ricci E, Petrillo MG, Cari L, Migliorati G, Nocentini G, et al. Glucocorticoid-induced tumour necrosis factor receptor-related protein: a key marker of functional regulatory T cells. J Immunol Res. 2015;2015:171520.
11. Nettenstrom L, Alderson K, Raschke EE, Evans MD, Sondel PM, Olek S, et al. An optimized multi-parameter flow cytometry protocol for human T regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood. J Immunol Methods. 2013;387(1-2):81-8.
12. Baron U, Floess S, Wieczorek G, Baumann K, Grutzkau A, Dong J, et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol. 2007;37(9):2378-89.
13. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007;5(2):e38.
14. Polansky JK, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, et al. DNA methylation controls Foxp3 gene expression. Eur J Immunol. 2008;38(6):1654-63.
15. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009;30(6):899-911.
16. McClymont SA, Putnam AL, Lee MR, Esensten JH, Liu W, Hulme MA, et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol. 2011;186(7):3918-26.
17. Su H, Longhi MS, Wang P, Vergani D, Ma Y. Human CD4+CD25(high)CD127 (low/neg) regulatory T cells. Methods Mol Biol. 2012;806:287-99.
18. Walker LS. Treg and CTLA-4: two intertwining pathways to immune tolerance. J Autoimmun. 2013;45:49-57.
19. Ephrem A, Epstein AL, Stephens GL, Thornton AM, Glass D, Shevach EM. Modulation of Treg cells/T effector function by GITR signaling is context-dependent. Eur J Immunol. 2013;43(9):2421-9.
20. Dahmani A, Delisle JS. TGF-beta in T Cell Biology: Implications for Cancer Immunotherapy. Cancers (Basel). 2018;10(6).
21. Yang WC, Hwang YS, Chen YY, Liu CL, Shen CN, Hong WH, et al. Interleukin-4 Supports the Suppressive Immune Responses Elicited by Regulatory T Cells. Front Immunol. 2017;8:1508.
22. Patel T, Cunningham A, Holland M, Daley J, Lazo S, Hodi FS, et al. Development of an 8-color antibody panel for functional phenotyping of human CD8+ cytotoxic T cells from peripheral blood mononuclear cells. Cytotechnology. 2018;70(1):1-11.
23. Canavan JB, Afzali B, Scotta C, Fazekasova H, Edozie FC, Macdonald TT, et al. A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy. Blood. 2012;119(8):e57-66.
24. Cunningham R HM, McWilliams E, Hodi FS, Severgnini M. Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry. Journal of Biological Methods [Internet]. 2019; 6(2).
25. Heylmann D, Badura J, Becker H, Fahrer J, Kaina B. Sensitivity of CD3/CD28-stimulated versus non-stimulated lymphocytes to ionizing radiation and genotoxic anticancer drugs: key role of ATM in the differential radiation response. Cell Death Dis. 2018;9(11):1053.
26. Li Y, Kurlander RJ. Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation. J Transl Med. 2010;8:104.
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Published
Apr 13, 2020
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1.
Manuszak C, Brainard M, Thrash E, Hodi FS, Severgnini M. Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells. J Biol Methods [Internet]. 2020 Apr. 13 [cited 2024 Apr. 19];7(2):e131. Available from: https://jbmethods.org/index.php/jbm/article/view/325
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Author Biography
Mariano Severgnini, Dana-Farber Cancer Institute Center for Immuno-Oncology
I am the Lead Scientist and Director of the Center for Immuno-Oncology Immune Assessment Laboratory
