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Cited by 29T-cell and B-cell ImmunologyImmune Cell Function and InteractionCellular transport and secretion

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

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Mouchacca, Pierre, Schmitt-Verhulst, Anne-Marie, Boyer, Claude·2013·PLoS ONE
DOI 10.1371/journal.pone.0067239PMC PMC3695958
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Abstract

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells' plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.

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  • Cytolytic granule polarization and degranulation are two steps in lymphocyte-mediated cytotoxicity.

    “Cytolytic granule polarization and degranulation are two steps in lymphocyte-mediated cytotoxicity(Bryceson, March, Barber, Ljunggren, & Long, 2005;Mouchacca, Schmitt-Verhulst, & Boyer, 2013).”

    From Cuprizone‐induced demyelination triggers a <scp>CD8</scp>‐pronounced T cell recruitment (2021) · cites this paper as Mouchacca, Schmitt-Verhulst, & Boyer, 2013

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  1. [1]Fehniger TA, Cai SF, Cao X, Bredemeyer AJ, Presti RM, et al (2007) Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs. Immunity 26: 798– 811. 17540585
  2. [2]de Saint Basile G, Menasche G, Fischer A (2010) Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nat Rev Immunol 10: 568– 579. 20634814
  3. [3]Peters PJ, Borst J, Oorschot V, Fukuda M, Krahenbuhl O, et al (1991) Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med 173: 1099– 1109. 2022921 PMC2118839
  4. [4]Jenne DE, Tschopp J (1988) Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation. Immunol Rev 103: 53– 71. 3292396
  5. [5]Chowdhury D, Lieberman J (2008) Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol 26: 389– 420. 18304003 PMC2790083
  6. [6]Law RH, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, et al (2010) The structural basis for membrane binding and pore formation by lymphocyte perforin. Nature 468: 447– 451. 21037563
  7. [7]Kondos SC, Hatfaludi T, Voskoboinik I, Trapani JA, Law RH, et al (2010) The structure and function of mammalian membrane-attack complex/perforin-like proteins. Tissue Antigens 76: 341– 351. 20860583
  8. [8]Pardo J, Bosque A, Brehm R, Wallich R, Naval J, et al (2004) Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis. J Cell Biol 167: 457– 468. 15534000 PMC2172484
  9. [9]Keefe D, Shi L, Feske S, Massol R, Navarro F, et al (2005) Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 23: 249– 262. 16169498
  10. [10]Kagi D, Ledermann B, Burki K, Seiler P, Odermatt B, et al (1994) Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369: 31– 37. 8164737
  11. [11]Walsh CM, Matloubian M, Liu CC, Ueda R, Kurahara CG, et al (1994) Immune function in mice lacking the perforin gene. Proc Natl Acad Sci U S A 91: 10854– 10858. 7526382 PMC45124
  12. [12]Badovinac VP, Hamilton SE, Harty JT (2003) Viral infection results in massive CD8+ T cell expansion and mortality in vaccinated perforin-deficient mice. Immunity 18: 463– 474. 12705850
  13. [13]Jordan MB, Hildeman D, Kappler J, Marrack P (2004) An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood 104: 735– 743. 15069016
  14. [14]Feldmann J, Le Deist F, Ouachee-Chardin M, Certain S, Alexander S, et al (2002) Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis. Br J Haematol 117: 965– 972. 12060139
  15. [15]Masson D, Tschopp J (1987) A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell 49: 679– 685. 3555842
  16. [16]Waterhouse NJ, Sedelies KA, Browne KA, Wowk ME, Newbold A, et al (2005) A central role for Bid in granzyme B-induced apoptosis. The Journal of biological chemistry 280: 4476– 4482. 15574417
  17. [17]Brunet J-F, Dossetto M, Denizot F, Mattei M-G, Clark WR, et al (1986) The inducible cytotoxic-T-lymphocyte-associated gene transcript CTLA-1: sequence and gene localization to mouse chromosome 14. Nature 322: 268– 271. 3090449
  18. [18]Pham CT, MacIvor DM, Hug BA, Heusel JW, Ley TJ (1996) Long-range disruption of gene expression by a selectable marker cassette. Proc Natl Acad Sci U S A 93: 13090– 13095. 8917549 PMC24051
  19. [19]Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ (1994) Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76: 977– 987. 8137431
  20. [20]Moffat JM, Gebhardt T, Doherty PC, Turner SJ, Mintern JD (2009) Granzyme A expression reveals distinct cytolytic CTL subsets following influenza A virus infection. Eur J Immunol 39: 1203– 1210. 19404988
  21. [21]Kolset SO, Tveit H (2008) Serglycin–structure and biology. Cell Mol Life Sci 65: 1073– 1085. 18066495 PMC11131666
  22. [22]Lovo E, Zhang M, Wang L, Ashton-Rickardt PG (2012) Serine protease inhibitor 6 is required to protect dendritic cells from the kiss of death. J Immunol 188: 1057– 1063. 22227570 PMC3270301
  23. [23]Holt OJ, Gallo F, Griffiths GM (2006) Regulating secretory lysosomes. J Biochem 140: 7– 12. 16877763
  24. [24]D'Angelo ME, Bird PI, Peters C, Reinheckel T, Trapani JA, et al (2010) Cathepsin H is an additional convertase of pro-granzyme B. J Biol Chem. 285: 20514– 20519. PMC2898313 20435891
  25. [25]Pham CT, Ivanovich JL, Raptis SZ, Zehnbauer B, Ley TJ (2004) Papillon-Lefevre syndrome: correlating the molecular, cellular, and clinical consequences of cathepsin C/dipeptidyl peptidase I deficiency in humans. J Immunol 173: 7277– 7281. 15585850
  26. [26]Tran TV, Ellis KA, Kam CM, Hudig D, Powers JC (2002) Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity. Arch Biochem Biophys 403: 160– 170. 12139965
  27. [27]Bertrand F, Esquerre M, Petit AE, Rodrigues M, Duchez S, et al (2010) Activation of the ancestral polarity regulator protein kinase C zeta at the immunological synapse drives polarization of Th cell secretory machinery toward APCs. J Immunol 185: 2887– 2894. 20679531
  28. [28]Ma JS, Monu N, Shen DT, Mecklenbrauker I, Radoja N, et al (2007) Protein kinase Cdelta regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL. J Immunol 178: 7814– 7821. 17548619 PMC3712856
  29. [29]Pardo J, Buferne M, Martinez-Lorenzo MJ, Naval J, Schmitt-Verhulst AM, et al (2003) Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression. Int Immunol 15: 1441– 1450. 14645153
  30. [30]Maul-Pavicic A, Chiang SC, Rensing-Ehl A, Jessen B, Fauriat C, et al (2011) ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis. Proc Natl Acad Sci U S A 108: 3324– 3329. 21300876 PMC3044412
  31. [31]Stinchcombe JC, Griffiths GM (2007) Secretory Mechanisms in Cell-Mediated Cytotoxicity. Annu Rev Cell Dev Biol 23: 495– 517. 17506701
  32. [32]Stinchcombe JC, Majorovits E, Bossi G, Fuller S, Griffiths GM (2006) Centrosome polarization delivers secretory granules to the immunological synapse. Nature 443: 462– 465. 17006514
  33. [33]Lasserre R, Charrin S, Cuche C, Danckaert A, Thoulouze MI, et al (2010) Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse. Embo J 29: 2301– 2314. 20551903 PMC2910277
  34. [34]Kannan K, Stewart RM, Bounds W, Carlsson SR, Fukuda M, et al (1996) Lysosome-associated membrane proteins h-LAMP1 (CD107a) and h-LAMP2 (CD107b) are activation-dependent cell surface glycoproteins in human peripheral blood mononuclear cells which mediate cell adhesion to vascular endothelium. Cell Immunol 171: 10– 19. 8660832
  35. [35]Liu D, Bryceson YT, Meckel T, Vasiliver-Shamis G, Dustin ML, et al (2009) Integrin-dependent organization and bidirectional vesicular traffic at cytotoxic immune synapses. Immunity 31: 99– 109. 19592272 PMC2740634
  36. [36]Pipkin ME, Lieberman J (2007) Delivering the kiss of death: progress on understanding how perforin works. Curr Opin Immunol 19: 301– 308. 17433871 PMC11484871
  37. [37]Thiery J, Keefe D, Boulant S, Boucrot E, Walch M, et al (2011) Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Nat Immunol 12: 770– 777. 21685908 PMC3140544
  38. [38]Veugelers K, Motyka B, Goping IS, Shostak I, Sawchuk T, et al (2006) Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate. Mol Biol Cell 17: 623– 633. 16280358 PMC1356574
  39. [39]Mouchacca P, Guyot V, Gregoire C, Schmitt-Verhulst AM, Boyer C (2012) Granzyme B-tdTomato, a new probe to visualize cytolytic effector cell activation. Eur J Immunol 42: 264– 266. 22120827
  40. [40]Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22: 1567– 1572. 15558047
  41. [41]Breart B, Lemaitre F, Celli S, Bousso P (2008) Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J Clin Invest 118: 1390– 1397. 18357341 PMC2268880
  42. [42]Muyrers JP, Zhang Y, Testa G, Stewart AF (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27: 1555– 1557. 10037821 PMC148353
  43. [43]Carbone FR, Bevan MJ (1990) Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo. J Exp Med 171: 377– 387. 2137512 PMC2187713
  44. [44]Wiedemann A, Muller S, Favier B, Penna D, Guiraud M, et al (2005) T-cell activation is accompanied by an ubiquitination process occurring at the immunological synapse. Immunol Lett 98: 57– 61. 15790509
  45. [45]Bajenoff M, Narni-Mancinelli E, Brau F, Lauvau G (2010) Visualizing early splenic memory CD8+ T cells reactivation against intracellular bacteria in the mouse. PLoS ONE 5: e11524. 20634957 PMC2902518
  46. [46]Thompson CB, Lindsten T, Ledbetter JA, Kunkel SL, Young HA, et al (1989) CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines. Proc Natl Acad Sci USA 86: 1333– 1337. 2465550 PMC286684
  47. [47]Menager MM, Menasche G, Romao M, Knapnougel P, Ho CH, et al (2007) Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13–4. Nat Immunol 8: 257– 267. 17237785
  48. [48]Stinchcombe JC, Bossi G, Booth S, Griffiths GM (2001) The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15: 751– 761. 11728337
  49. [49]Veugelers K, Motyka B, Frantz C, Shostak I, Sawchuk T, et al (2004) The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis. Blood 103: 3845– 3853. 14739229
  50. [50]McGavern DB, Christen U, Oldstone MB (2002) Molecular anatomy of antigen-specific CD8(+) T cell engagement and synapse formation in vivo. Nat Immunol 3: 918– 925. 12352968 PMC2481514
  51. [51]Wiedemann A, Depoil D, Faroudi M, Valitutti S (2006) Cytotoxic T lymphocytes kill multiple targets simultaneously via spatiotemporal uncoupling of lytic and stimulatory synapses. Proc Natl Acad Sci U S A 103: 10985– 10990. 16832064 PMC1544161
  52. [52]Voskoboinik I, Thia MC, Fletcher J, Ciccone A, Browne K, et al (2005) Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain: A critical role for aspartate residues 429, 435, 483, and 485 but not 491. J Biol Chem 280: 8426– 8434. 15576364
  53. [53]Henkart PA (1985) Mechanism of lymphocyte-mediated cytotoxicity. Ann Rev Immunol 3: 31– 58. 3904772
  54. [54]Podack ER (1986) Molecular mechanisms of cytolysis by complement and by cytolytic lymphocytes. J Cell Biochem 30: 133– 170. 2422185
  55. [55]Purbhoo MA, Irvine DJ, Huppa JB, Davis MM (2004) T cell killing does not require the formation of a stable mature immunological synapse. Nat Immunol 5: 524– 530. 15048111
  56. [56]Jenkins MR, Tsun A, Stinchcombe JC, Griffiths GM (2009) The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse. Immunity 31: 621– 631. 19833087 PMC2791175
  57. [57]Lopez JA, Susanto O, Jenkins MR, Lukoyanova N, Sutton VR, et al.. (2013) Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack. Blood. In press. 23377437
  58. [58]Caramalho I, Faroudi M, Padovan E, Muller S, Valitutti S (2009) Visualizing CTL/melanoma cell interactions: multiple hits must be delivered for tumour cell annihilation. J Cell Mol Med 13: 3834– 3846. 19017355 PMC4516531
  59. [59]Poenie M, Tsien RY, Schmitt-Verhulst A-M (1987) Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets. EMBO J 6: 2223– 2232. 3499312 PMC553622
  60. [60]Faroudi M, Utzny C, Salio M, Cerundolo V, Guiraud M, et al (2003) Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold. Proc Natl Acad Sci U S A 100: 14145– 14150. 14610278 PMC283560
  61. [61]Stewart SE, D'Angelo ME, Bird PI (2012) Intercellular communication via the endo-lysosomal system: translocation of granzymes through membrane barriers. Biochim Biophys Acta 1824: 59– 67. 21683168

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Authors
Mouchacca, P., Schmitt-Verhulst, A.-M., & Boyer, C.
Affiliations
Centre National de la Recherche Scientifique (FR)
Corresponding author
Boyer, Claude
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-06-28
Volume
8
Issue
6
Pages
e67239
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0067239
PMC
PMC3695958
OpenAlex
W2046188384
Cited by
29
References
0
Citations by year
2026: 2 · 2025: 2 · 2024: 3 · 2023: 2 · 2022: 1 · 2021: 3 · 2020: 3 · 2019: 1 · 2018: 2 · 2017: 1 · 2016: 2 · 2015: 4 · 2014: 3
FWCI
1.66
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Association pour la Recherche sur le Cancer
  • Institut National de la Santé et de la Recherche Médicale
  • Centre National de la Recherche Scientifique
  • Institut National Du Cancer
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Cell Function and Interaction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
granzyme B, cytotoxic T lymphocytes, granule exocytosis, perforin, CD8+ T cell differentiation, CD4+ T cells, natural killer cells, synaptogenesis, calcium signaling, T cell activation, dendritic cells, Listeria monocytogenes infection, fluorescent protein fusion, cytotoxicity
MeSH terms
Red Fluorescent Protein, Animals, Cell Differentiation, Cells, Cultured, Killer Cells, Natural, Kinetics, Listeriosis, Luminescent Proteins, Lymph Nodes, Lymphocyte Activation, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Microscopy, Fluorescence, Recombinant Fusion Proteins, T-Lymphocytes, Cytotoxic, Cell Degranulation, Gene Expression, Microscopy, Confocal, Secretory Vesicles, Mice, Granzymes, Gene Knock-In Techniques, Single-Cell Analysis

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