title: Evidence implicating the Ras pathway in multiple CD28 costimulatory functions in CD4+ T cells.
abstract: CD28 costimulation is a critical event in the full activation of CD4(+) T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L) to transduce normal, Coxsackie-Adenovirus Receptor (CAR) transgenic CD4(+) T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.
authors.affiliation: Department of Pathology, The University of Chicago, Chicago, Illinois, United States of America.
authors.forename: Sujit V
authors.initials: SV
authors.lastname: Janardhan
authors.forename: Kesavannair
authors.initials: K
authors.lastname: Praveen
authors.forename: Reinhard
authors.initials: R
authors.lastname: Marks
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Gajewski
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D018106:CD28 Antigens / Q000276:immunology* / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D049109:Cell Proliferation; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008213:Lymphocyte Activation; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D008822:Mice, Transgenic; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011991:Receptors, Virus / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D018106:CD28 Antigens; C568771:CLMP protein, human; C568772:CLMP protein, mouse; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D007376:Interleukin-2; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D011991:Receptors, Virus; D008156:Luciferases; D051057:Proto-Oncogene Proteins c-akt; D048670:MAP Kinase Kinase 4; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)