title: Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling.
abstract: 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.
authors.affiliation: Department of Immune Therapeutics, Merck Research Laboratories-MRL, MSD, Oss, the Netherlands. RL.Smeets@labgk.umcn.nl
authors.forename: Ruben L
authors.initials: RL
authors.lastname: Smeets
authors.forename: Wilco W M
authors.initials: WW
authors.lastname: Fleuren
authors.forename: Xuehui
authors.initials: X
authors.lastname: He
authors.forename: Paul M
authors.initials: PM
authors.lastname: Vink
authors.forename: Frank
authors.initials: F
authors.lastname: Wijnands
authors.forename: Monika
authors.initials: M
authors.lastname: Gorecka
authors.forename: Henri
authors.initials: H
authors.lastname: Klop
authors.forename: Sussane
authors.initials: S
authors.lastname: Bauerschmidt
authors.forename: Anja
authors.initials: A
authors.lastname: Garritsen
authors.forename: Hans J P M
authors.initials: HJ
authors.lastname: Koenen
authors.forename: Irma
authors.initials: I
authors.lastname: Joosten
authors.forename: Annemieke M H
authors.initials: AM
authors.lastname: Boots
authors.forename: Wynand
authors.initials: W
authors.lastname: Alkema
mesh_terms: D018106:CD28 Antigens / Q000378:metabolism*; D017252:CD3 Complex / Q000378:metabolism; D054402:Chemokine CCL1 / Q000235:genetics / Q000378:metabolism; D016000:Cluster Analysis; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D018417:Th1 Cells / Q000276:immunology* / Q000378:metabolism; D018418:Th2 Cells / Q000276:immunology* / Q000378:metabolism
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; D054402:Chemokine CCL1; D016207:Cytokines; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell