Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling

BMC Immunology

Wynand Alkema¹  Annemieke MH Boots²  Irma Joosten³  Hans JPM Koenen³  Anja Garritsen⁶  Sussane Bauerschmidt⁵  Henri Klop⁶  Monika Gorecka⁶  Frank Wijnands⁶  Paul M Vink⁶  Xuehui He³  Wilco WM Fleuren⁷  Ruben L Smeets⁴ 

[1]NIZO Food Research, Ede, The Netherlands

[2]Department of Rheumatology and Clinical Immunology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands

[3]Department of Laboratory Medicine, laboratory for Medical Immunology, Radboud University Medical Centre, Nijmegen, The Netherlands

[4]Department of Laboratory Medicine, laboratory for Clinical Chemistry, Radboud University Medical Centre, Nijmegen, Geert Grooteplein 10, Postbus 9101, 6500 HB Nijmegen, The Netherlands

[5]Department of Molecular Design and Informatics, Merck Research Laboratories (MRL), MSD, Oss, the Netherlands

[6]Department of Immune Therapeutics, Merck Research Laboratories (MRL), MSD, Oss, the Netherlands

[7]Netherlands Bioinformatics Centre (NBIC), Nijmegen, The Netherlands

关键词: Th1 and Th2 development;    T lymphocytes;    Gene expression profiling;    Signal transduction pathways;   

Others  :  1077934
DOI  :  10.1186/1471-2172-13-12

 received in 2011-12-14, accepted in 2012-03-14,  发布年份 2012

【 摘 要 】

Background

T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.

Results

Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells.

Conclusions

This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.

【 授权许可】

   
2012 Smeets et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 7.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Murphy KM, Reiner SL: The lineage decisions of helper T cells. Nat Rev Immunol 2002, 2:933-944.
• [2]Szabo SJ, Sullivan BM, Peng SL, Glimcher LH: Molecular mechanisms regulating Th1 immune responses. Annu Rev Immunol 2003, 21:713-758.
• [3]Dardalhon V, Korn T, Kuchroo VK, Anderson AC: Role of Th1 and Th17 cells in organ-specific autoimmunity. J Autoimmun 2008, 31:252-256.
• [4]Wan YY, Flavell RA: How diverse-CD4 effector T cells and their functions. J Mol Cell Biol 2009, 1:20-36.
• [5]Brogdon JL, Leitenberg D, Bottomly K: The potency of TCR signaling differentially regulates NFATc/p activity and early IL-4 transcription in naive CD4+ T cells. J Immunol 2002, 168:3825-3832.
• [6]Constant SL, Bottomly K: Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 1997, 15:297-322.
• [7]Sloan-Lancaster J, Steinberg TH, Allen PM: Selective loss of the calcium ion signaling pathway in T cells maturing toward a T helper 2 phenotype. J Immunol 1997, 159:1160-1168.
• [8]Tao X, Constant S, Jorritsma P, Bottomly K: Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell differentiation. J Immunol 1997, 159:5956-5963.
• [9]Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell costimulation. Annu Rev Immunol 1996, 14:233-258.
• [10]Green JM, Noel PJ, Sperling AI, Walunas TL, Gray GS, Bluestone JA, Thompson CB: Absence of B7-dependent responses in CD28-deficient mice. Immunity 1994, 1:501-508.
• [11]Lenschow DJ, Herold KC, Rhee L, Patel B, Koons A, Qin HY, Fuchs E, Singh B, Thompson CB, Bluestone JA: CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes. Immunity 1996, 5:285-293.
• [12]Gonzalo JA, Tian J, Delaney T, Corcoran J, Rottman JB, Lora J, Al-garawi A, Kroczek R, Gutierrez-Ramos JC, Coyle AJ: ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. Nat Immunol 2001, 2:597-604.
• [13]Salek-Ardakani S, Song J, Halteman BS, Jember AG, Akiba H, Yagita H, Croft M: OX40 (CD134) controls memory T helper 2 cells that drive lung inflammation. J Exp Med 2003, 198:315-324.
• [14]Palacios EH, Weiss A: Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 2004, 23:7990-8000.
• [15]Rudd CE, Taylor A, Schneider H: CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol Rev 2009, 229:12-26.
• [16]Wang D, Matsumoto R, You Y, Che T, Lin XY, Gaffen SL, Lin X: CD3/CD28 costimulation-induced NF-kappaB activation is mediated by recruitment of protein kinase C-theta, Bcl10, and IkappaB kinase beta to the immunological synapse through CARMA1. Mol Cell Biol 2004, 24:164-171.
• [17]Wang D, You Y, Case SM, McAllister-Lucas LM, Wang L, DiStefano PS, Nunez G, Bertin J, Lin X: A requirement for CARMA1 in TCR-induced NF-kappa B activation. Nat Immunol 2002, 3:830-835.
• [18]Yokosuka T, Kobayashi W, Sakata-Sogawa K, Takamatsu M, Hashimoto-Tane A, Dustin ML, Tokunaga M, Saito T: Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation. Immunity 2008, 29:589-601.
• [19]Dong C, Davis RJ, Flavell RA: MAP kinases in the immune response. Annu Rev Immunol 2002, 20:55-72.
• [20]June CH, Ledbetter JA, Gillespie MM, Lindsten T, Thompson CB: T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression. Mol Cell Biol 1987, 7:4472-4481.
• [21]Ledbetter JA, Parsons M, Martin PJ, Hansen JA, Rabinovitch PS, June CH: Antibody binding to CD5 (Tp67) and Tp44 T cell surface molecules: effects on cyclic nucleotides, cytoplasmic free calcium, and cAMP-mediated suppression. J Immunol 1986, 137:3299-3305.
• [22]Badou A, Savignac M, Moreau M, Leclerc C, Foucras G, Cassar G, Paulet P, Lagrange D, Druet P, Guery JC, Pelletier L: Weak TCR stimulation induces a calcium signal that triggers IL-4 synthesis, stronger TCR stimulation induces MAP kinases that control IFN-gamma production. Eur J Immunol 2001, 31:2487-2496.
• [23]Furue M, Ishibashi Y: Differential regulation by dexamethasone and cyclosporine of human T cells activated by various stimuli. Transplantation 1991, 52:522-526.
• [24]Mosmann TR, Sad S: The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996, 17:138-146.
• [25]Muller K, Bischof S, Sommer F, Lohoff M, Solbach W, Laskay T: Differential production of macrophage inflammatory protein 1gamma (MIP-1gamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro and in vivo. Infect Immun 2003, 71:6178-6183.
• [26]Porter CM, Clipstone NA: Sustained NFAT signaling promotes a Th1-like pattern of gene expression in primary murine CD4+ T cells. J Immunol 2002, 168:4936-4945.
• [27]Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH: A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000, 100:655-669.
• [28]Djuretic IM, Levanon D, Negreanu V, Groner Y, Rao A, Ansel KM: Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells. Nat Immunol 2007, 8:145-153.
• [29]Bishop B, Lloyd CM: CC chemokine ligand 1 promotes recruitment of eosinophils but not Th2 cells during the development of allergic airways disease. J Immunol 2003, 170:4810-4817.
• [30]Romagnani S: Cytokines and chemoattractants in allergic inflammation. Mol Immunol 2002, 38:881-885.
• [31]Kang HS, Blink SE, Chin RK, Lee Y, Kim O, Weinstock J, Waldschmidt T, Conrad D, Chen B, Solway J, et al.: Lymphotoxin is required for maintaining physiological levels of serum IgE that minimizes Th1-mediated airway inflammation. J Exp Med 2003, 198:1643-1652.
• [32]Zheng W, Flavell RA: The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 1997, 89:587-596.
• [33]Stephensen CB, Rasooly R, Jiang X, Ceddia MA, Weaver CT, Chandraratna RA, Bucy RP: Vitamin A enhances in vitro Th2 development via retinoid X receptor pathway. J Immunol 2002, 168:4495-4503.
• [34]Badou A, Bennasser Y, Moreau M, Leclerc C, Benkirane M, Bahraoui E: Tat protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway. J Virol 2000, 74:10551-10562.
• [35]Gajewski TF, Lancki DW, Stack R, Fitch FW: "Anergy" of TH0 helper T lymphocytes induces downregulation of TH1 characteristics and a transition to a TH2-like phenotype. J Exp Med 1994, 179:481-491.
• [36]Al-Ramadi BK, Nakamura T, Leitenberg D, Bothwell AL: Deficient expression of p56(lck) in Th2 cells leads to partial TCR signaling and a dysregulation in lymphokine mRNA levels. J Immunol 1996, 157:4751-4761.
• [37]Molina TJ, Bachmann MF, Kundig TM, Zinkernagel RM, Mak TW: Peripheral T cells in mice lacking p56lck do not express significant antiviral effector functions. J Immunol 1993, 151:699-706.
• [38]Baine I, Abe BT, Macian F: Regulation of T-cell tolerance by calcium/NFAT signaling. Immunol Rev 2009, 231:225-240.
• [39]Takeda K, Harada Y, Watanabe R, Inutake Y, Ogawa S, Onuki K, Kagaya S, Tanabe K, Kishimoto H, Abe R: CD28 stimulation triggers NF-kappaB activation through the CARMA1-PKCtheta-Grb2/Gads axis. Int Immunol 2008, 20:1507-1515.
• [40]Matsumoto R, Wang D, Blonska M, Li H, Kobayashi M, Pappu B, Chen Y, Lin X: Phosphorylation of CARMA1 plays a critical role in T Cell receptor-mediated NF-kappaB activation. Immunity 2005, 23:575-585.
• [41]Das J, Chen CH, Yang L, Cohn L, Ray P, Ray A: A critical role for NF-kappa B in GATA3 expression and TH2 differentiation in allergic airway inflammation. Nat Immunol 2001, 2:45-50.
• [42]Rodriguez-Palmero M, Hara T, Thumbs A, Hunig T: Triggering of T cell proliferation through CD28 induces GATA-3 and promotes T helper type 2 differentiation in vitro and in vivo. Eur J Immunol 1999, 29:3914-3924.
• [43]Salek-Ardakani S, So T, Halteman BS, Altman A, Croft M: Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase C theta. J Immunol 2004, 173:6440-6447.
• [44]Keane-Myers A, Gause WC, Linsley PS, Chen SJ, Wills-Karp M: B7-CD28/CTLA-4 costimulatory pathways are required for the development of T helper cell 2-mediated allergic airway responses to inhaled antigens. J Immunol 1997, 158:2042-2049.
• [45]Shahinian A, Pfeffer K, Lee KP, Kundig TM, Kishihara K, Wakeham A, Kawai K, Ohashi PS, Thompson CB, Mak TW: Differential T cell costimulatory requirements in CD28-deficient mice. Science 1993, 261:609-612.
• [46]Korn T, Bettelli E, Gao W, Awasthi A, Jager A, Strom TB, Oukka M, Kuchroo VK: IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature 2007, 448:484-487.
• [47]Yang L, Anderson DE, Baecher-Allan C, Hastings WD, Bettelli E, Oukka M, Kuchroo VK, Hafler DA: IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 2008, 454:350-352.
• [48]Borhani DW, Calderwood DJ, Friedman MM, Hirst GC, Li B, Leung AK, McRae B, Ratnofsky S, Ritter K, Waegell W: A-420983: a potent, orally active inhibitor of lck with efficacy in a model of transplant rejection. Bioorg Med Chem Lett 2004, 14:2613-2616.
• [49]Evenou JP, Wagner J, Zenke G, Brinkmann V, Wagner K, Kovarik J, Welzenbach KA, Weitz-Schmidt G, Guntermann C, Towbin H, et al.: The potent protein kinase C-selective inhibitor AEB071 (sotrastaurin) represents a new class of immunosuppressive agents affecting early T-cell activation. J Pharmacol Exp Ther 2009, 330:792-801.
• [50]Mihara K, Almansa C, Smeets RL, Loomans EE, Dulos J, Vink PM, Rooseboom M, Kreutzer H, Cavalcanti F, Boots AM, Nelissen RL: A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a comparison with neutralization of mouse TNFalpha. Br J Pharmacol 2008, 154:153-164.