A logical model provides insights into T cell receptor signaling

Abstract

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.

Figure 1. Logical Model of T Cell Activation (Screenshot of CellNetAnalyzer)

Each arrow pointing at a species box is a so-called hyperarc representing one possibility to activate that species (see Methods). All the hyperarcs pointing at a particular species box are OR connected. Yellow species boxes denote output elements, while green ones represent (co)receptors. In the shown “early-event” scenario, the feedback loops were switched off, and only the input for the costimulatory molecule CD28 is active (scenario in column 2 of Table 1). The resulting logical steady state was then computed. Small text boxes display the signal flows along the hyperarcs (blue boxes: fixed values prior to computation; green boxes: hyperarcs activating a species (signal flow is 1); red boxes: hyperarcs which are not active (signal flow is 0)).

Figure 2. Dependency Matrix of the Logical T Cell Signaling Model (Figure 1) for the Early Events Scenario (τ = 1)

The color of a matrix element M_xy has the following meaning [13]: (i) dark green: x is a total activator of y; (ii) dark red: x is a total inhibitor of y; (iii) white: no (direct or indirect) influence from x on y.

Figure 3. In Vitro Analysis of Model Predictions

(A) Activation of ERK and JNK upon CD28, TCR (CD3), or TCR + CD28 stimulation in mouse splenic T cells. (B) Activation of PKB upon TCR, TCR+ CD4, and TCR + CD28 stimulation in Fyn-deficient and heterozygous splenic mouse T cells. (C) Inhibition of PI3K with both Ly294002 and Wortmannin blocks the phosphorylation of PKB, ERK, and JNK, but not ZAP-70 in human T cells. (D) Inhibition of PI3K with both Ly294002 and Wortmannin blocks the phosphorylation of PKB, but not of JNK in human T cells upon CD28 stimulation. As a control, the total amount of ZAP70 (A) or β-actin (B–D) was determined. One representative experiment (of three) is shown.

Figure 4. In Vitro Analysis of Src-Kinase Inhibition

Inhibition of Src-Kinases (Lck and Fyn) with PP2 blocks TCR-induced but affects only moderately CD28-induced PKB and JNK activation in human T cells; therefore, we concluded that CD28 signaling is not strictly Src-kinase– dependent. The effect was compared with PI3K inhibition via Wortmannin (ccf. Figure 3C and 3D), which blocks the phosphorylation of PKB but not of JNK. β-actin was included as the loading control. One representative experiment (of three) is shown.

Figure 5. Considering Different Time Scales, a Rough Description of the Dynamics Can Be Obtained

The activation of key elements upon activation of the TCR, the coreceptor CD4, and the costimulatory molecule CD28 is represented at the resting state, τ = 0 (no inputs); early events τ = 1 (input(s), no feedback loops); and later-time events, τ = 2 (input(s), feedback loops). The black lines correspond to a wild type while the green ones to a PAG KO. Note that the absence of PAG has no effect on key downstream elements of the cascade, due to the redundant role of other negative regulatory mechanisms (degradation via c-Cbl and Cbl-b, Gab-2 mediated inhibition of PLCγ1). Multiple knock-out of these regulatory molecules leads to sustained activation of key elements (red lines).