title: Nonapoptotic and extracellular activity of granzyme B mediates resistance to regulatory T cell (Treg) suppression by HLA-DR-CD25hiCD127lo Tregs in multiple sclerosis and in response to IL-6.
abstract: In autoimmune patients, regulatory T cells (Tregs) are increasingly found to be unable to suppress patient-derived T cells, an outcome referred to as Treg resistance. In this study, we show that CD4 T cells from patients with multiple sclerosis resist suppression by patient-derived or healthy donor-derived ex vivo Tregs. Importantly, we report that granzyme B (GzmB) contributes to this Treg resistance via a novel, apoptosis-independent mechanism. We show that memory CD4(+)CD127(lo)FOXP3(+) Treg subsets do not express GzmB, whereas activated, nonregulatory CD4 T cells isolated from patients with multiple sclerosis express higher levels of GzmB than do cells from healthy donors. In contrast to the intracellular GzmB that mediates apoptosis, GzmB can be found in extracellular fluids where it is hypothesized to regulate other cellular processes. In this study, we show that providing extracellular GzmB strongly inhibits Treg suppression, without altering Treg viability. However, when GzmB and GzmB-specific inhibitor are both provided to the cocultures, Treg suppression occurs. Thus, these data suggest that a novel activity of extracellular GzmB is to regulate Treg suppression. Additionally, we find that the suppression-abrogating cytokine IL-6 augments GzmB expression by human CD4 T cells, and it inhibits Treg suppression via this nonapoptotic GzmB-mediated mechanism. Lastly, in examining the mechanism whereby GzmB inhibits Treg function, we show that extracellular GzmB reduces Treg expression of CD39 and programmed death ligand 1. Collectively, these data indicate that extracellular GzmB plays an unexpected, nonapoptotic role in regulating Treg suppression and suggest that inactivation of specifically the extracellular activity of GzmB may be an efficacious therapeutic in autoimmunity.
authors: Center for Neurologic Disease, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; and.
authors: Siddheshvar
authors: S
authors: Bhela
authors: Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors: Christine
authors: C
authors: Kempsell
authors: Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors: Monali
authors: M
authors: Manohar
authors: Center for Neurologic Disease, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; and.
authors: Margarita
authors: M
authors: Dominguez-Villar
authors: Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors: Russell
authors: R
authors: Griffin
authors: Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors: Pooja
authors: P
authors: Bhatt
authors: Center for Neurologic Disease, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; and.
authors: Pia
authors: P
authors: Kivisakk-Webb
authors: Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors: Robert
authors: R
authors: Fuhlbrigge
authors: Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors: Thomas
authors: T
authors: Kupper
authors: Center for Neurologic Disease, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; and.
authors: Howard
authors: H
authors: Weiner
authors: Center for Neurologic Disease, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; and Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115 callan@rics.bwh.harvard.edu.
authors: Clare
authors: C
authors: Baecher-Allan
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D005260:Female; D005786:Gene Expression Regulation; D053804:Granzymes / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology*; D015850:Interleukin-6 / Q000494:pharmacology*; D053699:Interleukin-7 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D020529:Multiple Sclerosis, Relapsing-Remitting / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D061251:Primary Cell Culture; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology* / Q000473:pathology
chemical_list: D006684:HLA-DR Antigens; C508017:IL2RA protein, human; C508600:IL6 protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D015850:Interleukin-6; D053699:Interleukin-7 Receptor alpha Subunit; C509213:GZMB protein, human; D053804:Granzymes
references: J Leukoc Biol. 2007 Aug;82(2):354-60
references: 17505023
references: J Immunol Methods. 2007 Jul 31;324(1-2):92-104
references: 17582431
references: Science. 2003 Feb 14;299(5609):1033-6
references: 12532024
references: Biochem J. 2011 Jul 15;437(2):313-22
references: 21548883
references: J Immunol. 2009 Dec 1;183(11):7602-10
references: 19917691
references: Clin Exp Dermatol. 2000 Nov;25(8):643-7
references: 11167981
references: J Med Genet. 2002 Aug;39(8):537-45
references: 12161590
references: J Biol Chem. 2008 Aug 15;283(33):22498-504
references: 18577516
references: J Biol Chem. 2005 Jun 24;280(25):23549-58
references: 15843372
references: J Immunol. 2007 Jan 15;178(2):683-92
references: 17202328
references: Clin Exp Immunol. 1999 May;116(2):366-70
references: 10337032
references: Clin Exp Immunol. 2004 Jun;136(3):463-71
references: 15147348
references: Clin Cancer Res. 2009 Oct 15;15(20):6348-57
references: 19825957
references: Blood. 2007 Aug 15;110(4):1225-32
references: 17449799
references: J Immunol. 2005 Aug 1;175(3):1686-93
references: 16034109
references: Purinergic Signal. 2014 Dec;10(4):529-64
references: 25352330
references: J Invest Dermatol. 2013 Jun;133(6):1677-9
references: 23321921
references: PLoS One. 2013 Dec 23;8(12):e83139
references: 24376655
references: Biochem J. 2006 Mar 15;394(Pt 3):563-73
references: 16336214
references: J Biol Chem. 2004 Jun 18;279(25):26581-7
references: 15069086
references: J Immunol. 2009 Sep 1;183(5):3170-6
references: 19648274
references: J Immunol. 2008 Nov 15;181(10):7350-5
references: 18981158
references: Proc Natl Acad Sci U S A. 2011 May 10;108(19):7914-9
references: 21518876
references: Immunity. 2005 Sep;23(3):249-62
references: 16169498
references: J Immunol. 2007 Jan 1;178(1):271-9
references: 17182564
references: Science. 2009 Nov 13;326(5955):986-91
references: 19797626
references: J Biol Chem. 2006 Sep 22;281(38):28326-35
references: 16798735
references: Nat Med. 2007 Oct;13(10):1173-5
references: 17828272
references: Blood. 2011 Sep 29;118(13):3538-48
references: 21828127
references: Nat Med. 2007 Apr;13(4):423-31
references: 17384649
references: J Immunol. 2002 Dec 1;169(11):6210-7
references: 12444126
references: J Immunol. 2000 Jan 1;164(1):183-90
references: 10605010
references: Blood. 2004 Nov 1;104(9):2840-8
references: 15238416
references: J Immunol. 2011 Mar 15;186(6):3317-26
references: 21300823
references: Nat Med. 2011 Jul 17;17(8):989-95
references: 21765403
references: J Immunol. 2017 Feb 1;198(3):981-985
references: 28115586
references: J Immunol. 2006 Apr 15;176(8):4622-31
references: 16585553
references: Immunol Rev. 2009 May;229(1):67-87
references: 19426215
references: Biol Chem. 2006 Jul;387(7):827-37
references: 16913832
references: Med Sci Monit. 2000 Nov-Dec;6(6):1104-8
references: 11208463
references: Diabetes. 2005 Jan;54(1):92-9
references: 15616015
references: J Neurosci Res. 2006 Jun;83(8):1432-46
references: 16583400
references: Sci Transl Med. 2013 Jan 30;5(170):170ra15
references: 23363979
references: Science. 2010 Apr 16;328(5976):372-6
references: 20339032
references: J Exp Med. 2004 Apr 5;199(7):971-9
references: 15067033
references: Immunol Rev. 2010 Jul;236:219-42
references: 20636820
references: J Exp Med. 2005 Mar 7;201(5):737-46
references: 15753207
references: Nat Rev Immunol. 2010 Dec;10(12):849-59
references: 21107346
references: J Chromatogr. 1985 Jun 19;326:357-61
references: 3875623
references: J Immunol. 2009 Oct 15;183(8):4843-7
references: 19801510
references: Cancer Res. 2002 Sep 1;62(17):4884-9
references: 12208735
references: Mol Cell Biol. 1999 Dec;19(12):8604-15
references: 10567584
references: J Immunol. 2005 Feb 15;174(4):1783-6
references: 15699103
references: J Immunol. 2001 Aug 1;167(3):1245-53
references: 11466340
references: Eur J Immunol. 2005 Nov;35(11):3343-52
references: 16206232
references: Eur J Immunol. 2003 Jan;33(1):215-23
references: 12594850
references: Arthritis Rheum. 2007 Mar;56(3):732-42
references: 17328044