Cutting edge: TCR ligation triggers digital activation of NF-kappaB

Abstract

TCR-mediated activation of the transcription factor NF-κB is required for T cell proliferation, survival, and effector differentiation. Although this pathway is the subject of intense study, it is not known whether TCR signaling to NF-κB is digital (switch-like) or analog in nature. Through analysis of the phosphorylation and degradation of IκBα and the nuclear translocation and phosphorylation of the NF-κB subunit RelA, we show that TCR-directed NF-κB activation is digital. Furthermore, digitization occurs well upstream of the IκB kinase complex, as protein kinase C translocation to the immunologic synapse and activation-associated aggregation of Bcl10 and Malt1 also demonstrate both digital behavior and high correlation with RelA nuclear translocation. Thus, similar to the TCR-to-MAPK signaling cascade, analog Ag inputs are converted to digital activation outputs to NF-κB at an early step downstream of TCR ligation.

Figure 1. TCR stimulation triggers digital IκBα degradation

A, Jurkat T cells and the indicated mutants of Jurkat were stimulated for 20 min with 5 μg/mL anti-CD3 + 1μg/mL anti-CD28. Flow cytometry was used to detect levels of intracellular IκBα. Histograms show unstimulated cells (solid line) and stimulated cells (gray fill). B, Jurkat stimulation was as in A, but with the indicated concentrations of anti-CD3. Dotted line marks peak intensity of IκBα staining in unstimulated cells, and dashed line marks the peak intensity of IκBα staining in stimulated cells (Left). Graph shows the percentage of cells in the stimulated cell peak at each anti-CD3 concentration (Right). C, The experiment of B was repeated using the indicated concentrations of PMA plus 1 μm ionomycin as the stimulus.

Figure 2. POLKADOTS formation and PKCθ translocation are highly correlated with RelA nuclear translocation

D10 T cells were stimulated with the indicated concentrations of anti-CD3. A, Percentage of cells with RelA enrichment in the nucleus was evaluated at the indicated time points for three different anti-CD3 concentrations. Data are representative of two experiments. B, In cells that formed POLKADOTS, the ratio of nuclear to cytoplasmic RelA was calculated at 0, 0.5, and 4 h for the indicated anti-CD3 concentrations (Left), and at 0 and 0.5 h using the indicated concentrations of PMA plus 1 μM ionomycin (Right). Unstimulated cells formed no POLKADOTS and were randomly selected for analysis. Asterisk indicates p<0.001 vs. 10 ng/mL PMA (one-way ANOVA, Tukey’s multiple comparison test). C, The 0 and 0.5 h anti-CD3 and PMA data from B are shown as scatter plots, with 0.7, the empirically determined maximum for nuclear/cytoplasmic RelA in unstimulated (or unactivated) cells, indicated by a dashed line. D, The percentage of T cells in T cell-APC conjugates having either RelA nuclear translocation (white bars), PKCθ translocation to the IS (gray bars), or both nuclear RelA and PKCθ translocation (black bars), 25 min after conjugate formation. The D10 T cell lines used in these experiments expressed either Bcl10-CFP and Malt1-YFP (A-C) or PKCθ-GFP (D). Error bars are SEM.

Figure 3. Antigen stimulation triggers digital phosphorylation of IκBα and RelA

A, C57BL/6 lymphocytes were stimulated for 48 h with the indicated concentrations of anti-TCRβ. Phospho-IκBα was measured in CD4⁺ and CD8⁺ T cells by flow cytometry (histograms). Dashed line indicates peak intensity of phospho-IκBα after stimulation. Graphs show mean fluorescence intensity (MFI) of responding CD4⁺ (black bars) and CD8⁺ (shaded bars) T cells, and the percentage of responding CD4⁺ (black bars) and CD8⁺ (shaded bars) T cells. B, Same experiment as in A, except phospho-RelA was quantified. C, OTII CD4⁺ T cells were stimulated with the indicated concentrations of OVA_325–339, and phospho-IκBα was quantified as in A. Adjusted MFI = (MFI of the pIκBα peak) – (MFI of the 2°-only control). Each panel is representative of 4 similar experiments.