TCR ligand avidity determines the modeof B‐Raf/Raf‐1/ERK activation leading to the activationof human CD4⁺ T cell clone

Abstract

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR-mediated signaling, we established L cells expressing HLA-DR4 molecules covalently linked with agonistic peptide (highaffinity ligand) or altered peptide ligand (APL; low-affinity ligand) at various densities as APC for a cognate human CD4 + T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA-DR4 complexes presented at an excessive density provoked the up-regulation of CD69, IL-2 production and proliferation, but no detectable phosphorylation of ZAP-70/LAT/SLP-76. Furthermore, in contrast to the high-affinity stimulation, the low-affinity stimulation evoked delayed and sustained activation of the B-Raf/extracellular signal-regulated kinase (ERK) pathway without Raf-1 activation. The strength and duration of B-Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B-Raf activation was indispensable for the APL-induced T cell responses. These observations suggest that the differences in TCR-peptide/MHC interactions reflect the strength and duration of B-Raf/Raf-1/ERK activation in the human CD4 + T cells.

B‐Raf‐mediated signaling pathway regulates T cell development

“…Previously we reported that B-Raf activation is regulated by Ras and mediates sustained ERK activation and subsequent IL-2 production upon TCR stimulation in human T cells [22,24]. In addition, enforced expression of B-Raf in mouse CD4 + T cells resulted in augmentation of their proliferation and prevention of anergy induction [25].…”

“…1D). These results could be attributed to the lower activation threshold of B-Raf than Raf-1 in response to TCR stimulation [22]. The findings of TCR stimulationinducible B-Raf activation prompted us to hypothesize that B-Raf could play a role in TCR-mediated T cell responses in thymocytes.…”

“…Stimulation was performed by incubation at 37 C for the indicated time. Western blottings, immunoprecipitations, in vitro kinase assays for Raf-1 and B-Raf, and intracellular stainings were carried out as previously described [22,24].…”

“…Although the individual Raf genes have some apparent functional redundancies with each other in ERK regulation, the different phenotypes of each Raf kinase-deficient mouse suggest that they have specific and non-redundant functions [12,[16][17][18][19], which seem to be due to their distinct expression patterns [20] and differences in the thresholds for activation [15,21,22].…”

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

“…As in other cell types, the duration of Erk signaling plays a decisive role in multiple T cell outcomes: positive versus negative selection of thymocytes (1,2), CD4 versus CD8 lineage commitment (3), response to low-affinity versus high-affinity Ags in mature T cells (4), or Th1 versus Th2 differentiation of effector T cells (5). These developmental or differentiation processes are initiated by signaling through the TCR (6,7).…”