TCR activation kinetics and feedback regulation in primary human T cells

Abstract

BackgroundSignaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses.ResultsHere, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.ConclusionsIn summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

ERK Positive Feedback Regulates a Widespread Network of Tyrosine Phosphorylation Sites across Canonical T Cell Signaling and Actin Cytoskeletal Proteins in Jurkat T Cells

“…Profound changes in the pattern of TCR signaling was observed in U0126-treated Jurkat T cells relative to control cells, as demonstrated in SILAC heatmaps, with overall decreases in phosphorylation of TCR proximal signaling proteins, including CD3 ε, δ, γ, ζ, Lck, and ZAP-70, as well as the adaptor and downstream signaling proteins VAV1, IL-2-inducible T cell kinase (Itk), phospholipase C γ1 (PLCγ1), and NCK1 (Figure 6, Figure 7). Despite deficiencies in the lipid phosphatases PTEN and SHIP1 in the Jurkat cell line [37], [38], the quantitative perturbations of phosphopeptide abundance observed with inhibition of ERK feedback in this study are consistent with observations made in primary T cells by Stefanova et al [4] and Poltorak et al [12] and support the hypothesis that ERK is a positive feedback regulator in TCR proximal signaling.…”

“…Positive feedback mechanisms that promote T cell activation have also been observed in T cells, but are less defined [4], [5], [10], [11]. In particular, it has been reported that in response to TCR interaction with high affinity agonists, ERK is activated to positively regulate TCR signaling through Lck (Figure 1) [4], [12]. Upon TCR agonist engagement, Lck becomes phosphorylated at Ser 59 by ERK [13], [14] leading to the modification of Lck’s Src homology 2 (SH2) domain, and consequently, a reduction in the accessibility or affinity for phosphoproteins to bind [15].…”

A Synthetic CD8α:MyD88 Coreceptor Enhances CD8+ T-cell Responses to Weakly Immunogenic and Lowly Expressed Tumor Antigens

“…CD8α:MyD88 led to the enhanced and more rapid activation (phosphorylation) of downstream signaling proteins ERK1/2, JNK, and p38 (Figure 1F). Increased ERK1/2 activation is advantageous as ERK2 drives CD8 + T cell proliferation and survival(13). CD8α:MyD88 also enhanced the activation of the proximal TCR protein ZAP-70, a CD3 receptor–associated protein tyrosine kinase (Figure 1F).…”

Sos1 regulates sustained TCR‐mediated Erk activation

“…To address this issue, we took advantage of anti‐CD3‐coated microbeads. This stimulation condition induces sustained TCR‐mediated signaling, T‐cell activation, and proliferation, thus mimicking physiological stimulation by APCs . Using this method and RNAi‐mediated suppression of Sos1 expression, we have revealed that Sos1 significantly contributes to Erk activation during sustained TCR signaling and T‐cell activation.…”