Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses.
The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.
Methi
Trond
T
The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
Ngai
Jacob
J
Mahic
Milada
M
Amarzguioui
Mohammed
M
Vang
Torkel
T
Tasken
Kjetil
K
D015152:Blotting, Northern; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000276:immunology; D006801:Humans; D047468:Immunoprecipitation; D007376:Interleukin-2 / Q000276:immunology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology / Q000378:metabolism; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000172:deficiency* / Q000276:immunology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000276:immunology / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000276:immunology / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000276:immunology / Q000378:metabolism; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000276:immunology / Q000378:metabolism; D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D018106:CD28 Antigens; D007376:Interleukin-2; D047908:Intracellular Signaling Peptides and Proteins; D050778:NFATC Transcription Factors; D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D002118:Calcium