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Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2)

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Abstract

Human CD4+CD25highFOXP3+ T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4+CD25neg RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4+CD25highFOXP3+ Treg ± 150 or ±1,000 IU/mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p < 0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4+CD25high Treg and CD4+CD25neg RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme–perforin pathway depending on IL-2 concentrations.

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References

  1. Strauss L, Bergmann C, Szczepanski M, Gooding W, Johnson JT, Whiteside TL (2007) A unique subset of CD4+CD25highFoxp3+ T cells secreting IL-10 and TGF-beta1 mediates suppression in the tumor microenvironment. Clin Cancer Res 13:4345–4354

    Article  PubMed  CAS  Google Scholar 

  2. Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, Orban PC, Roncarolo MG (2002) Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J Exp Med 196:1335–1346

    Article  PubMed  CAS  Google Scholar 

  3. Miyara M, Sakaguchi S (2007) Natural regulatory T cells: mechanisms of suppression. Trends Mol Med 13:108–116

    Article  PubMed  CAS  Google Scholar 

  4. Strauss L, Bergmann C, Whiteside TL (2009) Human circulating CD4+CD25highFoxp3+ Treg kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis. J Immunol 182:1469–1480

    PubMed  CAS  Google Scholar 

  5. Levings MK, Sangregorio R, Roncarolo MG (2001) Human CD25(+)CD4(+) T regulatory cells suppress naïve and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med 193:1295–1302

    Article  PubMed  CAS  Google Scholar 

  6. Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A (2007) Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin. J Immunol 178:320–329

    PubMed  CAS  Google Scholar 

  7. Krammer PH, Arnold R, Laurik IN (2007) Life and death in peripheral T cells. Nat Rev Immunol 7:532–542

    Article  PubMed  CAS  Google Scholar 

  8. Gondek DC, Lu LF, Quezada SA, Sakaguchi S, Noelle RJ (2005) Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J Immunol 174:1783–1786

    PubMed  CAS  Google Scholar 

  9. Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ (2004) Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity 21:589–601

    Article  PubMed  CAS  Google Scholar 

  10. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M et al (2007) Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 204:1257–1265

    Article  PubMed  CAS  Google Scholar 

  11. Borsellino G, Kleinewietfeld M, DiMitri D, Sternjak A, Diamantini A, Giometto R, Hopner S, Centonze D, Bernardi G, Dell’Acqua ML et al (2007) Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood 110:1225–1232

    Article  PubMed  CAS  Google Scholar 

  12. Mandapathil M, Szczepanski MJ, Szajnik M, Ren J, Lenzner DE, Jackson EK, Gorelik E, Lang S, Johnson JT, Whiteside TL (2009) Increased ectonucleotidase expression and activity in Treg of patients with head and neck cancer. Clin Cancer Res 15(20):6348–6357

    Article  PubMed  CAS  Google Scholar 

  13. Davis JE, Sutton VR, Browne KA, Trapani JZ (2003) Purification of natural killer cell cytotoxic granules for assaying target cell apoptosis. J Imunol Meth 276:59–68

    Article  CAS  Google Scholar 

  14. Givan AL, Fisher JL, Waugh M, Ernstoff MS, Wallace PK (1999) A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens. J Immunol Meth 230:99–112

    Article  CAS  Google Scholar 

  15. Strauss L, Bergmann C, Gooding W, Johnson JT, Whiteside TL (2007) The frequency and suppressor function of CD4+CD25highFoxp3+ T cells in the circulation of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 13:6301–6311

    Article  PubMed  CAS  Google Scholar 

  16. Wang RF (2006) Immune suppression by tumor specific CD4(+) regulatory T cells in cancer. Semin Cancer Biol 16:73–79

    Article  PubMed  CAS  Google Scholar 

  17. Zou W (2005) Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer 5:263–274

    Article  PubMed  CAS  Google Scholar 

  18. Wei WZ, Morris GP, Kong YC (2004) Anti-tumor immunity and autoimmunity: a balancing act of regulatory T cells. Cancer Immunol Immunother 53:73–78

    Article  PubMed  CAS  Google Scholar 

  19. Shevach EM (2002) CD4+CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol 2:389–400

    PubMed  CAS  Google Scholar 

  20. Rafaeli Y, Van Parijs L, London CA, Tschopp J, Abbas AK (1998) Biochemical mechanisms of IL-2-regulated Fas-mediated apoptosis. Immunity 8:615–623

    Article  Google Scholar 

  21. Trapani JA, Sutton VR (2003) Granzyme B: pro-apoptotic, antiviral and antitumor functions. Curr Opin Immunol 15:533–543

    Article  PubMed  CAS  Google Scholar 

  22. Lenardo M, Chan KM, Hornung F, McFarland H, Siegel R, Wang J, Zheng L (1999) Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev Immuno 17:221–253

    Article  CAS  Google Scholar 

  23. Cao X, Cai SF, Fehniger TA, Song J, Collins LI, Piwnica-Worms DR, Ley TJ (2007) Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity 27:635–646

    Article  PubMed  CAS  Google Scholar 

  24. Gondek DC, Devries V, Nowak EC, Lu LF, Bennett KA, Scott ZA, Noelle RJ (2008) Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells. J Immunol 181:4752–4760

    PubMed  CAS  Google Scholar 

  25. Kurschus FC, Kleinschmidt M, Fellows E, Dornmair K, Rudolph R, Lilie H, Jenne DE (2004) Killing of target cells by redirected granzyme B in the absence of perforin. FEBS let 562:87–92

    Article  CAS  Google Scholar 

  26. Smyth MJ, Kelly JM, Sutton VR, Davis JE, Browne KA, Sayers TJ, Trapani JA (2001) Unlocking the secrets of cytotoxic granule proteins. J Leukoc Biol l70:18–21

    Google Scholar 

  27. Hellerstein MK, Hoh RA, Hanley MB, Cesar D, Lee D, Neese RA, McCune JM (2003) Subpopulations of long-lived and short-lived T cells in advanced HIV infection. J Clin Invest 112:821–824

    Google Scholar 

  28. Kuss I, Schaefer C, Godfrey TE, Ferris RL, Harris JM, Gooding W, Whiteside TL (2005) Recent thymic emigrants and subsets of naïve and memory T cells in the circulation of patients with head and neck cancer. Clin Immunol 116:27–36

    Article  PubMed  Google Scholar 

  29. Hoyer KK, Dooms H, Barron L, Abbas AK (2008) Interleukin-2 in the development and control of inflammatory disease. Immunol Rev 226:19–28

    Article  PubMed  CAS  Google Scholar 

  30. Josefowicz SZ, Rudensky A (2009) Control of regulatory T cell lineage commitment and maintenance. Immunity 30:616–625

    Article  PubMed  CAS  Google Scholar 

  31. Zhang H, Chua KS, Guimond M, Kapoor V, Brown MV, Fleisher TA, Long LM, Bernstein D, Hill BJ, Douek DC et al (2005) Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells. Nat Med 11:1238–1243

    Article  PubMed  CAS  Google Scholar 

  32. Cesana GC, DeRaffele G, Cohen S, Moroziewicz D, Mitcham J (2006) Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol 24:1169–1177

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

Supported in part by National Institutes of Health (NIH) grants PO1 CA109688, PO1 DE12321 and RO1 DE13918 to TLW. Dr. Bergmann was supported in part by Philip Morris USA, Inc. and Philip Morris International.

Disclosure statement

All authors have read the manuscript and concur with its content. The authors do not have any financial/commercial conflict of interest that could be considered to have influenced the content of this paper.

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Authors and Affiliations

  1. Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, 15213, USA

    Malgorzata Czystowska, Laura Strauss, Christoph Bergmann, Marta Szajnik & Hannah Rabinowich

  2. Departments of Pathology, Immunology and Otolaryngology, Suite L1.27 at the Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Suite 1.27, Pittsburgh, PA, 15213-1863, USA

    Theresa L. Whiteside

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  1. Malgorzata Czystowska
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  2. Laura Strauss
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  3. Christoph Bergmann
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  6. Theresa L. Whiteside
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Correspondence to Theresa L. Whiteside.

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Czystowska, M., Strauss, L., Bergmann, C. et al. Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2). J Mol Med 88, 577–588 (2010). https://doi.org/10.1007/s00109-010-0602-9

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  • DOI: https://doi.org/10.1007/s00109-010-0602-9

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