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Glucocorticoid-induced Tumor Necrosis Factor Receptor Negatively Regulates Activation of Human Primary Natural Killer (NK) Cells by Blocking Proliferative Signals and Increasing NK Cell Apoptosis

2008; Elsevier BV; Volume: 283; Issue: 13 Linguagem: Inglês

10.1074/jbc.m708944200

ISSN

1083-351X

Autores

Baoying Liu, Zhuqing Li, Sankaranarayana P. Mahesh, Seth Pantanelli, Frank S. Hwang, Willie O. Siu, Robert B. Nussenblatt,

Tópico(s)

T-cell and B-cell Immunology

Resumo

Glucocorticoid-induced tumor necrosis factor receptor (GITR), found constitutively expressed on human primary natural killer (NK) cells at low levels was up-regulated upon stimulation by either Toll-like receptor ligand or NK cell growth factor, interleukin (IL)-15. cDNA microarray analysis showed that engagement of GITR primarily suppressed the activation of NF-KB pathway of NK cells and up-regulated anti-inflammatory genes heme oxygenase-1 and IL-10. Further analysis revealed that GITR activation suppressed NK cell proliferation in response to IL-15. GITR activation also suppressed proinflammatory cytokine secretion and increased NK cell apoptosis. GITR activation resulted in blocked phosphorylation of Stat5 and Akt, which may have contributed to the observed antiproliferative effect of GITR on NK cells. Increased apoptosis was independent of the Fas-FasL pathway, but Bcl-XL and phospho-Bad protein expressions were diminished, suggesting involvement of the mitochondrial apoptosis pathway. The results suggest that although GITR is an activation marker for NK cells similar to that for T cells, GITR serves as a negative regulator for NK cell activation. Our studies demonstrate a novel physiological role of GITR on NK cells. Glucocorticoid-induced tumor necrosis factor receptor (GITR), found constitutively expressed on human primary natural killer (NK) cells at low levels was up-regulated upon stimulation by either Toll-like receptor ligand or NK cell growth factor, interleukin (IL)-15. cDNA microarray analysis showed that engagement of GITR primarily suppressed the activation of NF-KB pathway of NK cells and up-regulated anti-inflammatory genes heme oxygenase-1 and IL-10. Further analysis revealed that GITR activation suppressed NK cell proliferation in response to IL-15. GITR activation also suppressed proinflammatory cytokine secretion and increased NK cell apoptosis. GITR activation resulted in blocked phosphorylation of Stat5 and Akt, which may have contributed to the observed antiproliferative effect of GITR on NK cells. Increased apoptosis was independent of the Fas-FasL pathway, but Bcl-XL and phospho-Bad protein expressions were diminished, suggesting involvement of the mitochondrial apoptosis pathway. The results suggest that although GITR is an activation marker for NK cells similar to that for T cells, GITR serves as a negative regulator for NK cell activation. Our studies demonstrate a novel physiological role of GITR on NK cells. Natural killer (NK) 3The abbreviations used are:NKnatural killerGITRglucocorticoid-induced tumor necrosis factor receptorGITRLGITR ligandIFNinterferonPBMCperipheral blood mononuclear cellILinterleukinFITCfluorescein isothiocyanate7-AAD7-amino-actinomycin DTLRToll-like receptor. 3The abbreviations used are:NKnatural killerGITRglucocorticoid-induced tumor necrosis factor receptorGITRLGITR ligandIFNinterferonPBMCperipheral blood mononuclear cellILinterleukinFITCfluorescein isothiocyanate7-AAD7-amino-actinomycin DTLRToll-like receptor. cells are an important component of the innate immune system. They use cytolytic granules and cytokine production to mediate their effector functions (1Chan S.H. Perussia B. Gupta J.W. Kobayashi M. Pospisil M. Young H.A. Wolf S.F. Young D. Clark S.C. Trinchieri G. J. Exp. Med. 1991; 173: 869-879Crossref PubMed Scopus (957) Google Scholar). By dynamic interactions with other immune cells, such as dendritic cells, macrophages, and T cells, NK cells can also mediate the transition from innate to adaptive immune response (2Bancroft G.J. Kelly J.P. Immunobiology. 1994; 191: 424-431Crossref PubMed Scopus (44) Google Scholar, 3Marcenaro E. Ferranti B. Moretta A. Autoimmun. Rev. 2005; 4: 520-525Crossref PubMed Scopus (48) Google Scholar, 4Shi F.D. Van Kaer L. Nat. Rev. Immunol. 2006; 6: 751-760Crossref PubMed Scopus (119) Google Scholar). NK cell activity is regulated delicately by a large number of putative activating and inhibitory receptors (5Karre K. Ljunggren H.G. Piontek G. Kiessling R. Nature. 1986; 319: 675-678Crossref PubMed Scopus (1684) Google Scholar, 6Lanier L.L. Curr. Opin. Immunol. 2003; 15: 308-314Crossref PubMed Scopus (296) Google Scholar). Under normal physiological conditions, mature NK cells are relatively quiescent. Following infection, NK cells undergo proliferative expansion that is either nonspecific (e.g. mediated by cytokines) or specific (e.g. induced by engagement of activating receptors), after which these cells return to relative quiescence. natural killer glucocorticoid-induced tumor necrosis factor receptor GITR ligand interferon peripheral blood mononuclear cell interleukin fluorescein isothiocyanate 7-amino-actinomycin D Toll-like receptor. natural killer glucocorticoid-induced tumor necrosis factor receptor GITR ligand interferon peripheral blood mononuclear cell interleukin fluorescein isothiocyanate 7-amino-actinomycin D Toll-like receptor. Glucocorticoid-induced tumor necrosis factor receptor (GITR, or TNFRSF18) is a recently identified member of the tumor necrosis factor receptor superfamily. It was initially identified as a glucocorticoid-responsive gene in a murine hybridoma T cell line (7Nocentini G. Giunchi L. Ronchetti S. Krausz L.T. Bartoli A. Moraca R. Migliorati G. Riccardi C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6216-6221Crossref PubMed Scopus (366) Google Scholar). Initial studies suggested that GITR was selectively expressed on CD4+CD25+ regulatory T cells, and receptor engagement abrogated the suppressive function of T regulatory cells (8Ji H.B. Liao G. Faubion W.A. Abadia-Molina A.C. Cozzo C. Laroux F.S. Caton A. Terhorst C. J. Immunol. 2004; 172: 5823-5827Crossref PubMed Scopus (179) Google Scholar). Subsequent studies showed GITR expression on CD4+CD25– and CD8+ T cells (9Nocentini G. Riccardi C. Eur. J. Immunol. 2005; 35: 1016-1022Crossref PubMed Scopus (163) Google Scholar, 10Watts T.H. Annu. Rev. Immunol. 2005; 23: 23-68Crossref PubMed Scopus (1109) Google Scholar). It is markedly induced in T cells after TCR activation in mice, and it is a general consensus that GITR provides a strong co-stimulatory signal for T cells when stimulated by its natural ligand or its agonistic antibody (8Ji H.B. Liao G. Faubion W.A. Abadia-Molina A.C. Cozzo C. Laroux F.S. Caton A. Terhorst C. J. Immunol. 2004; 172: 5823-5827Crossref PubMed Scopus (179) Google Scholar, 11Gurney A.L. Marsters S.A. Huang R.M. Pitti R.M. Mark D.T. Baldwin D.T. Gray A.M. Dowd A.D. Brush A.D. Heldens A.D. Schow A.D. Goddard A.D. Wood W.I. Baker K.P. Godowski P.J. Ashkenazi A. Curr. Biol. 1999; 9: 215-218Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 12Kwon B. Yu K.Y. Ni J. Yu G.L. Jang I.K. Kim Y.J. Xing L. Liu D. Wang S.X. Kwon B.S. J. Biol. Chem. 1999; 274: 6056-6061Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 13Li Z. Mahesh S.P. Kim B.J. Buggage R.R. Nussenblatt R.B. J. Autoimmun. 2003; 21: 83-92Crossref PubMed Scopus (51) Google Scholar, 14Ronchetti S. Nocentini G. Riccardi C. Pandolfi P.P. Blood. 2002; 100: 350-352Crossref PubMed Scopus (142) Google Scholar, 15Ronchetti S. Zollo O. Bruscoli S. Agostini M. Bianchini R. Nocentini G. Ayroldi E. Riccardi C. Eur. J. Immunol. 2004; 34: 613-622Crossref PubMed Scopus (308) Google Scholar, 16Shimizu J. Yamazaki S. Takahashi T. Ishida Y. Sakaguchi S. Nat. Immunol. 2002; 3: 135-142Crossref PubMed Scopus (1466) Google Scholar). GITR is not confined to T cells. It is expressed on macrophages, B cells (8Ji H.B. Liao G. Faubion W.A. Abadia-Molina A.C. Cozzo C. Laroux F.S. Caton A. Terhorst C. J. Immunol. 2004; 172: 5823-5827Crossref PubMed Scopus (179) Google Scholar, 13Li Z. Mahesh S.P. Kim B.J. Buggage R.R. Nussenblatt R.B. J. Autoimmun. 2003; 21: 83-92Crossref PubMed Scopus (51) Google Scholar, 15Ronchetti S. Zollo O. Bruscoli S. Agostini M. Bianchini R. Nocentini G. Ayroldi E. Riccardi C. Eur. J. Immunol. 2004; 34: 613-622Crossref PubMed Scopus (308) Google Scholar, 16Shimizu J. Yamazaki S. Takahashi T. Ishida Y. Sakaguchi S. Nat. Immunol. 2002; 3: 135-142Crossref PubMed Scopus (1466) Google Scholar, 17Shin H.H. Lee M.H. Kim S.G. Lee Y.H. Kwon B.S. Choi H.S. FEBS Lett. 2002; 514: 275-280Crossref PubMed Scopus (49) Google Scholar), and NK cells (18Baltz K.M. Krusch M. Bringmann A. Brossart P. Mayer F. Kloss M. Baessler T. Kumbier I. Peterfi A. Kupka S. Kroeber S. Menzel D. Radsak M.P. Rammensee H.G. Salih H.R. FASEB J. 2007; 21: 2442-2454Crossref PubMed Scopus (79) Google Scholar, 19Hanabuchi S. Watanabe N. Wang Y.H. Wang Y.H. Ito T. Shaw J. Cao W. Qin F.X. Liu Y.J. Blood. 2006; 107: 3617-3623Crossref PubMed Scopus (131) Google Scholar), whereas its cognate ligand (GITRL) is constitutively expressed on antigen-presenting cells, such as dendritic cells and B cells (20Mackay F. Kalled S.L. Curr. Opin. Immunol. 2002; 14: 783-790Crossref PubMed Scopus (90) Google Scholar, 21Tuyaerts S. Van Meirvenne S. Bonehill A. Heirman C. Corthals J. Waldmann H. Breckpot K. Thielemans K. Aerts J.L. J. Leukocyte Biol. 2007; 82: 93-105Crossref PubMed Scopus (56) Google Scholar). Manipulation of the GITR-GITRL interaction is suggested as a target for tumor immunotherapy, treatment of viral infection, and treatment of autoimmune diseases (22Bushell A. Wood K. Am. J. Transplant. 2007; 7: 759-768Crossref PubMed Scopus (55) Google Scholar, 23Cohen A.D. Diab A. Perales M.A. Wolchok J.D. Rizzuto G. Merghoub T. Huggins D. Liu C. Turk M.J. Restifo N.P. Sakaguchi S. Houghton A.N. Cancer Res. 2006; 66: 4904-4912Crossref PubMed Scopus (175) Google Scholar, 24Kim J. Choi W.S. Kim H.J. Kwon B. Exp. Mol. Med. 2006; 38: 94-99Crossref PubMed Scopus (11) Google Scholar, 25Ko H.J. Kim Y.J. Kim Y.S. Chang W.S. Ko S.Y. Chang S.Y. Sakaguchi S. Kang C.Y. Cancer Res. 2007; 67: 7477-7486Crossref PubMed Scopus (173) Google Scholar, 26Kohm A.P. Williams J.S. Miller S.D. J. Immunol. 2004; 172: 4686-4690Crossref PubMed Scopus (132) Google Scholar, 27La S. Kim E. Kwon B. Exp. Mol. Med. 2005; 37: 193-198Crossref PubMed Scopus (33) Google Scholar, 28Patel M. Xu D. Kewin P. Choo-Kang B. McSharry C. Thomson N.C. Liew F.Y. Eur. J. Immunol. 2005; 35: 3581-3590Crossref PubMed Scopus (53) Google Scholar, 29Ramirez-Montagut T. Chow A. Hirschhorn-Cymerman D. Terwey T.H. Kochman A.A. Lu S. Miles R.C. Sakaguchi S. Houghton A.N. van den Brink M.R. J. Immunol. 2006; 176: 6434-6442Crossref PubMed Scopus (152) Google Scholar, 30Suvas S. Kim B. Sarangi P.P. Tone M. Waldmann H. Rouse B.T. J. Virol. 2005; 79: 11935-11942Crossref PubMed Scopus (57) Google Scholar). This possibility has intensified the significance of studying GITR-GITRL interactions. Although there is a consensus for the role of GITR in T cells, its role in NK cells remains controversial. Hanabuchi et al. (19Hanabuchi S. Watanabe N. Wang Y.H. Wang Y.H. Ito T. Shaw J. Cao W. Qin F.X. Liu Y.J. Blood. 2006; 107: 3617-3623Crossref PubMed Scopus (131) Google Scholar) showed that GITRL expression on activated plasmacytoid dendritic cell precursors enhanced both NK cell cytotoxic activity and IFN-γ production by NK cells via GITR-GITRL interactions. A recent report by Baltz et al. (18Baltz K.M. Krusch M. Bringmann A. Brossart P. Mayer F. Kloss M. Baessler T. Kumbier I. Peterfi A. Kupka S. Kroeber S. Menzel D. Radsak M.P. Rammensee H.G. Salih H.R. FASEB J. 2007; 21: 2442-2454Crossref PubMed Scopus (79) Google Scholar) suggested that GITRL-expressing tumor cells diminish NK cell cytotoxicity and inhibit IFN-γ release. In light of the role of GITR in the regulation of T cells and its constitutive and increased expression upon activation on human NK cells, we investigated GITR signaling mechanisms and the potential role of GITR in regulating NK cell activation. Normal Donors—A total of 20 healthy human donors were studied. Samples from normal donors were obtained from the National Institutes of Health blood bank after informed consent (protocol 99-C-0168). Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) and NK Cells and Cell Culture—Human PBMCs from normal donors were isolated from their buffy coat using Ficoll gradient centrifugation as previously described (31Li Z. Lim W.K. Mahesh S.P. Liu B. Nussenblatt R.B. J. Immunol. 2005; 174: 5187-5191Crossref PubMed Scopus (89) Google Scholar). NK cells were obtained from isolated PBMCs by a magnetic sorting technique using a negative NK isolation kit II (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. For cell stimulation, PBMCs (2 × 106/ml) or purified NK cells (2 × 106/ml) in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA) supplemented with 2 mm glutamine and 1× antibiotics were plated in 6-well plates (Nalge Nunc International, Rochester, NY) precoated with anti-GITR at 10 μg/ml in the presence or absence of either 10 ng/ml IL-15 (Peprotech Inc., Rocky Hill, NJ) or poly(I-C) (Sigma) at 25 μg/ml for stimulation. Cells were cultured at 37 °C in 5% CO2. Flow Cytometry Analysis—To study GITR expression in resting and activated NK cells, PBMCs were cultured for 48 h with or without stimulation. Whole blood or PBMCs were analyzed using a three-color flow cytometry staining protocol. Specifically, 2 × 106/ml cells were stained with 5 μg/ml biotinylated anti-human GITR antibody (R & D Systems, Minneapolis, MN) in staining buffer (1× phosphate-buffered saline, 0.5% bovine serum albumin (Sigma)) at 4 °C for 30 min. After two washes with phosphate-buffered saline, cells were stained with 4 μg/ml allophycocyanin-labeled streptavidin (BD Biosciences) and counterstained with anti-human PE-CD56 and FITC-CD3 antibodies (BD Biosciences) at room temperature for 15 min. After two washes with phosphate-buffered saline and fixation with 300 μl of 1% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA), cells were acquired by a FACSCalibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (TreeStar, San Jose, CA). Briefly, lymphocytes were gated based on cell optic characteristics (forward scatter versus side scatter). CD3+, CD56+, CD3–, and CD56– populations were gated based on antibody staining. A subpopulation was derived from its parent population. The gate for the positive staining of GITR was set based on the nonspecific staining of an isotype-matched biotinylated control antibody. The percentage of GITR-positive cells in a defined subpopulation was calculated as the number of GITR-positive cells in the subpopulation divided by the total number of cells in the subpopulation. For example, the percentage of GITR in the CD3–CD56+ population was calculated as follows: (number of GITR+CD3–CD56+ cells/number of CD3–CD56+ cells) × 100%. Microarray Analysis—Human NK cells were stimulated with poly(I-C) (25 μg/ml) and cultured in plates with or without precoated anti-GITR antibody (10 μg/ml). After 6 h of stimulation, NK cells were collected and lysed, and total RNA was purified using an RNAeasy isolation kit according to the manufacturer's instructions (Qiagen). For cDNA microarray analysis, an array of genes selectively involved in the NF-κB pathway (SuperArray Inc., Gaithersburg, MD) was used for analysis. The cDNA microarray analysis was performed following the manufacturer's instructions. Briefly, an equal amount of total RNA was biotinylated, and probes were then hybridized onto the membranes containing NF-κB pathway-specific genes. After extensive washing, specific hybridization onto the membrane was detected by chemiluminescence and recorded by a digital CCD camera (UVP, Upland, CA). Differential gene expression was then analyzed using GEArray Expression Analysis Suite software (SuperArray Inc.) designed by the manufacturer. NK Cell Proliferation Assay—Purified NK cells were cultured in 96-well flat bottom plates (Nalge Nunc International) at 2 × 106/ml in 0.2 ml/well and stimulated with or without IL-15 in the presence of either 0.5 or 5 μg/ml anti-GITR agonist antibody. All experiments were done in triplicates. Cells were cultured at 37 °C in 5% CO2 for 3 or 5 days. Eight to ten hours before harvest, cells were pulsed with [3H]thymidine (2.5 μCi/ml) (Amersham Biosciences). Cells were then harvested, and [3H]thymidine uptake was measured with a β counter (PerkinElmer Life Sciences). The extent of proliferation was expressed as stimulation index (SI), which is represented by the ratio of radioactive [3H]thymidine (cpm) incorporated by cells treated with the agonist antibody to that by cells without treatment. SDS-PAGE and Western Blotting—A total of 5 million NK cells were lysed in 100 μl of lysis buffer (50 mm Tris-Cl, 1% Triton X-100, 100 mm NaCl, 2 mm EDTA, 50 mm NaF, 50 mm glycerol-phosphate, 1 mm NaVO4, and 1× protease inhibitor mixture) (Roche Applied Science). Complete cell lysis was achieved by immediately vortexing the cells and then boiling in an equal amount of 2× SDS protein loading buffer at 95 °C for 5 min. Cell debris was removed by centrifugation at 12,000 rpm for 3 min. Twenty microliters of each sample was loaded into a 12% SDS-polyacrylamide gel containing a 4% stacking gel. Immunoblotting was carried out. Primary antibodies of anti-Bcl-XL, anti-cleaved caspase 3, anti-cleaved PARP, anti-phospho-Bad, anti-phospho-Akt, anti-Akt, and anti-phospho-PDK1 were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Apoptosis Assay—Apoptotic cells were detected by staining cells with the combination of annexin-V-FITC and Via-probe™ (7-amino-actinomycin D; 7-AAD) according to the manufacturer's instructions (BD Biosciences). Early apoptotic cells (FITC+7-AAD–) and late apoptotic cells (FITC+7-AAD+) were counted for total apoptosis (32Lecoeur H. Ledru E. Prevost M.C. Gougeon M.L. J. Immunol. Methods. 1997; 209: 111-123Crossref PubMed Scopus (158) Google Scholar). Briefly, 2.5 × 105 cells were incubated with saturating concentrations of annexin V-FITC and 7-AAD for 15 min at room temperature and immediately analyzed by flow cytometry as described above. Fas Neutralization Assay—Purified NK cells were preincubated with a Fas neutralization antibody, clone ZB4 (10 μg/ml; Upstate Biotechnology, Inc., Lake Placid, NY) for 1 h at 37 °Cin 5% CO2 to allow antibody/cell interaction. Cells were then plated in 6-well plates, precoated with anti-GITR at 10 μg/ml, in the presence or absence of IL-15. After 48 h of incubation, cells were collected for apoptosis assays. Statistical Analysis—Analysis of NK proliferation was done using independent Student's t test. Analysis of cytokine secretion and apoptosis were performed using Student's t test, paired two samples for means. Gene Expression Profiling Revealed Predominantly Negative Effects of GITR on Activated Human NK Cells—To assess the impact of GITR on NK cell activation, we used an NF-κB-selective microarray assay to study overall effects of GITR on human NK cells. Purified human NK cells were activated by TLR3 ligand poly(I-C) in the presence or absence of GITR stimulation. Surprisingly, the overall effect of GITR engagement was to inhibit human NK cell activation. Of 113 genes analyzed, 21 genes qualified as differentially regulated by GITR when a typical 2-fold difference standard was applied as cut-off. Among those 21 genes differentially regulated by GITR, only 4 of 21 (19%) genes were up-regulated by GITR, whereas 17 of 21 (81%) genes were suppressed by GITR stimulation (Table 1). It was noteworthy that among the four genes that were up-regulated by GITR, heme oxygenase-1 and IL-10 are both well known protective genes against oxidative stress and inflammatory response (33Moore K.W. de Waal Malefyt R. Coffman R.L. O'Garra A. Annu. Rev. Immunol. 2001; 19: 683-765Crossref PubMed Scopus (5267) Google Scholar, 34Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1011) Google Scholar). Table 1 lists all 21 genes that were differentially regulated by GITR and their -fold changes.TABLE 1Genes differentially regulated by GITR in human NK cellsGene symbolDescriptionRefSeq numberPoly(I-C) aloneGITR + poly(I-C)Up-regulated by GITR HMOX1Heme oxygenase (decycling) 1NM_0021331.111.6 IL-10Interleukin 10NM_0005720.46.4 PPP5CProtein phosphatase 5, catalytic subunitNM_0062470.52.0 IL-8Interleukin 8NM_0005843.12.0Down-regulated by GITR GPR89G protein-coupled receptor 89NM_0163340.50.4 RHOCRas homolog gene family, member CNM_1757441.60.4 RHOARas homolog gene family, member ANM_0016643.50.4 MYD88Myeloid differentiation primary response gene 88NM_0024681.50.4 MAP3K14Mitogen-activated protein kinase kinase kinase 14NM_0039541.30.3 STAT1Signal transducer and activator of transcription 1, 91 kDaNM_0073151.60.3 BIRC2Baculoviral IAP repeat-containing 2NM_0011661.50.3 TLR8Toll-like receptor 8NM_0166102.10.3 TNFAIP3Tumor necrosis factor, α-induced protein 3NM_0062902.20.3 RELAV-rel reticuloendotheliosis viral oncogene homolog ANM_0219751.60.3 TLR2Toll-like receptor 2NM_0032642.00.2 TRADDTNFRSF1A-associated via death domainNM_0037891.60.2 TNFRSF10BTumor necrosis factor receptor superfamily, member 10bNM_0038421.90.2 TNFRSF1ATumor necrosis factor receptor superfamily, member 1ANM_0010651.80.2 IL-1BInterleukin 1, βNM_0005764.40.2 C3Complement component 3NM_0000642.70.1 CCL2Chemokine (C-C motif) ligand 2NM_0029823.50.1 Open table in a new tab GITR Expression Is Up-regulated by either TLR Ligand Poly(I-C) or NK Cell Growth Factor IL-15—We stimulated NK cells with either TLR3 ligand poly(I-C) or IL-15 to test their ability and effectiveness at regulating GITR expression in activated NK cells. Human PBMCs were treated with IL-15 or poly(I-C) as described, and GITR expression in CD3–CD56+ NK cells was monitored by flow cytometry. As shown in Fig. 1, there was constitutively low GITR expression (23%) in resting human primary NK cells (Fig. 1, control) compared with nonspecific staining of an isotype-matched biotinylated control antibody (Fig. 1, control IgG isotype), which is consistent with a recent report (19Hanabuchi S. Watanabe N. Wang Y.H. Wang Y.H. Ito T. Shaw J. Cao W. Qin F.X. Liu Y.J. Blood. 2006; 107: 3617-3623Crossref PubMed Scopus (131) Google Scholar). In addition, both poly(I-C) and IL-15 up-regulated GITR expression in NK cells by more than 2-fold over control (medium versus stimuli). Interestingly, the up-regulation of GITR by poly(I-C) seemed more rapid than that seen with IL-15, since GITR expression plateaued after 24 h of stimulation by poly(I-C), whereas it took 48 h to reach a plateau following IL-15 stimulation. Same staining has been repeated in three donors, and similar results have been obtained. GITR Inhibited NK Cell Proliferation and Production of Proinflammatory Cytokines upon Activation—We next examined NK cell proliferation in response to IL-15, with or without GITR engagement (Fig. 2). An agonist monoclonal antibody (clone 110416) against GITR was used to stimulate NK cells. IL-15, along with 0.5 μg/ml or 5 μg/ml anti-GITR antibody, was included in the culture medium for 3 or 5 days. As shown in Fig. 2A, after 3 days, the mean stimulation index was significantly less in the anti-GITR-treated groups (24.2 ± 1.9 for 0.5 μg/ml anti-GITR (p < 0.01) and 18.7 ± 4.0 for 5 μg/ml anti-GITR (p < 0.05)) when compared with cultures receiving IL-15 and an irrelevant mouse IgG negative control (34.6 ± 3.3), suggesting that GITR activation inhibited IL-15-induced NK proliferation. Similarly, in the day 5 treatment group (Fig. 2B), the stimulation index for the irrelevant IgG control (41.1 ± 0.8) and anti-GITR antibody treatment of 0.5 μg/ml (32.6 ± 0.9) and 5 μg/ml (26.2 ± 2.4) showed similar patterns. Secretion of proinflammatory cytokines is one of the hallmarks for NK cell activation and its functions. To further understand the functional impact of GITR on NK cell activation, culture supernatants of NK cells, stimulated in the presence or absence of GITR, were analyzed. Fig. 3 showed two sets of data side by side; GITR engagement significantly reduced poly(I-C)-induced production of proinflammatory cytokines (e.g. IFN-γ and IL-6 (p = 0.03 in both groups)). Productions of IL-12 and IL-1α were also slightly reduced (p = 0.06 and p = 0.05 separately). There was no significant difference of cytokine production between GITR engagement compared with control group (IFN-γ, p = 0.5; IL-1a, p = 0.49; IL-6, p = 0.43; IL-12p40, p = 0.28). GITR Engagement Blocked Phosphorylation of Stat5 and Akt but Did Not Affect the Mitogen-activated Protein Kinase Pathway—To investigate possible molecular mechanisms for the antiproliferative effect of GITR on activated NK cells, we next examined the signaling pathways of IL-15 on NK cells that may have been affected by the engagement of GITR, such as the Jak3/Stat5 and the phosphatidylinositol 3-kinase/Akt pathways. As shown in Fig. 4A, GITR activation abrogated IL-15-induced Stat5 phosphorylation at time points of 20 and 30 min following treatments. As shown in Fig. 4B, IL-15 induced Akt phosphorylation at 1 and 2 days after the initiation of treatments. GITR activation abrogated IL-15-induced phosphorylation. Consistent with the Akt results, another molecule in the phosphatidylinositol 3-kinase pathway, the phosphorylation of PDK1 induced by IL-15, was also abrogated by GITR activation. However, there was little change in phosphorylated p38 (Fig. 4A) or ERK1/2 (data not shown). GITR Activation Induced NK Cell Apoptosis—Earlier studies suggested that GITR prevents T cells from undergoing T cell receptor-induced apoptosis (14Ronchetti S. Nocentini G. Riccardi C. Pandolfi P.P. Blood. 2002; 100: 350-352Crossref PubMed Scopus (142) Google Scholar). Therefore, we wanted to determine if GITR engagement affects apoptosis of activated NK cells. NK cells stimulated with either poly(I-C) or IL-15 were stained with FITC-annexin V in conjunction with 7-AAD and subsequently analyzed by flow cytometry. This method allows discrimination between living (FITC–7-AAD–), early apoptotic (FITC+7-AAD–), and late apoptotic cells (FITC+7-AAD+) (32Lecoeur H. Ledru E. Prevost M.C. Gougeon M.L. J. Immunol. Methods. 1997; 209: 111-123Crossref PubMed Scopus (158) Google Scholar). Fourteen normal donors were used for IL-15 treatment, and six normal donors were used for poly(I-C) treatment. As shown in Tables 2 and 3, we summarized the apoptosis information of 20 donors that have been analyzed. Briefly, after 48 h, it showed a significant increase of apoptosis compared with IL-15/poly(I-C) plus GITR engagement with IL-15/poly(I-C) treatment only (p = 0.0016 < 0.01 in the IL-15 group and p = 0.04 < 0.05 in the poly(I-C) group). GITR activation in 10 of 14 donors (71.5%) in the IL-15 treatment group and four of six (66.7%) in the poly(I-C) treatment group showed more apoptosis if we used 5% more apoptosis as a cut-off line. Fig. 5 shows a typical flow cytometry analysis for NK cell apoptosis. Early apoptotic cells (FITC+7-AAD–) and late apoptotic cells (FITC+7-AAD+) were counted for total apoptosis (32Lecoeur H. Ledru E. Prevost M.C. Gougeon M.L. J. Immunol. Methods. 1997; 209: 111-123Crossref PubMed Scopus (158) Google Scholar).TABLE 2Apoptosis occurred in IL-15 and IL-15 plus GITR treatment groups of human NK cellsDonor numberControlGITRIL-15IL-15 + GITRaSignificant increase occurred in IL-15 plus anti-GITR treatment group compared with IL-15 treatment only (p < 0.01).128.955.045763.45216.0214.3328.1246.3327.8532.4733.648.6442.54650.163.1518.878.569.5313.85665.767.171.978.4754.4977.0954.481.69834.6172.1944.288.6495052.5866.375.631036.735.339.241.51125.6623.7233.740.6124145.543.751.7136255.2869.974.71456.349.237.340.9a Significant increase occurred in IL-15 plus anti-GITR treatment group compared with IL-15 treatment only (p < 0.01). Open table in a new tab TABLE 3Apoptosis occurred in poly(I-C) and poly(I-C) plus GITR treatment groups of human NK cellsDonor numberControlGITRPoly(I-C)Poly(I-C) + GITRaSignificant increase occurred in poly(I-C) plus anti-GITR treatment group compared with poly(I-C) treatment only (p < 0.05).142.244.439.948.7257.959.943.963.47384.4582.6972.6579.63447.363.0842.2161.93546.695.984891.34659.3363.1659.3961.36a Significant increase occurred in poly(I-C) plus anti-GITR treatment group compared with poly(I-C) treatment only (p < 0.05). Open table in a new tab GITR-enhanced Apoptosis Is Independent of the Fas-FasL Pathway but Mediated by a Mitochondria-dependent Pathway—Fas-FasL interaction is responsible for cytokine-induced T cell death (35Green D.R. Droin N. Pinkoski M. Immunol. Rev. 2003; 193: 70-81Crossref PubMed Scopus (479) Google Scholar, 36Lenardo M.J. J. Exp. Med. 1996; 183: 721-724Crossref PubMed Scopus (165) Google Scholar). To test whether the Fas-FasL pathway causes GITR-induced NK cell apoptosis, purified NK cells were preincubated with a Fas-neutralizing antibody before IL-15 treatment and GITR engagement. As shown in Fig. 6A, consistent with our finding that GITR enhances cytokine-induced apoptosis, the level of activated/cleaved caspase 3 for IL-15 plus GITR-treated NK cells was greatly increased when compared with the IL-15 treatment group (Fig. 6A, lane 5 versus lane 3). After Fas was blocked, the level of cleaved caspase 3 was dramatically decreased in the IL-15 treatment group (Fig. 6A, lane 4 versus lane 3), indicating that Fas-FasL interaction contributed to IL-15-induced cell death of NK cells. However, Fas blockade did not further block caspase 3 cleavage induced by GITR engagement (Fig. 6A, lane 5 versus lane 6), indicating that Fas-FasL interaction is not involved in the enhanced apoptosis seen with GITR engagement. FITC-annexin V, in conjunction with 7-AAD staining, showed similar results (data not shown). We next exa

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