(19b) Mechanistic Model of CD3ζ Immunoreceptor Tyrosine-Based Activation Motif (ITAM) Phosphorylation Sequence

Gaining a detailed understanding of T cell activation has become increasingly important as T cells are used in immunotherapeutic applications. For example, T cells engineered with modified T cell receptors (TCRs) or CD3ζ-bearing chimeric antigen receptors (CARs) have emerged as promising cancer therapies. The CD3ζ chain, a member of the TCR complex, contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which are thought to have different signaling functions: the first N-terminal ITAM is largely stimulatory, while the second and third ITAMs each exhibit some inhibitory signaling characteristics [1]. Lymphocyte-specific protein tyrosine kinase (LCK) is able to phosphorylate two tyrosine residues on each ITAM, resulting in T cell activation. The â??kinetic proofreadingâ? hypothesis is the prevailing theory regarding regulation of ITAM phosphorylation. It presumes that the presence of multiple ITAMs on the CD3ζ chain allows T cells to differentiate between foreign and self-peptides; however, the specific mechanisms that control LCK catalytic activity and its phosphorylation of the CD3ζ chain are still not well defined. Therefore, we have developed a mechanistic computational model of CD3ζ phosphorylation by LCK. The model is paired with site-specific mass spectrometry phosphorylation data from both CD3ζ and LCK in vitro to better understand the key steps that initiate T cell activation.

We constructed the model using BioNetGen, a rule-based formalism that allows us to account for the many species that arise from the multiple phosphorylation sites on CD3ζ. The model is implemented as a set of ordinary differential equations in MATLAB and fit to experimental data generated using an in vitro reconstituted membrane system that mimics the two-dimensional interactions that occur in T cells [2]. We use mass spectrometry to measure the level of phosphorylation at each individual tyrosine residue in the system over time. To determine the order in which each of the six tyrosine residues on CD3ζ are phosphorylated, we first stimulate CD3ζ with a constitutively active form of LCK and fit our mechanistic model to this data. We then combine this model with a previously developed model of LCK autoregulation [3]. The combined model will be validated using experimental data of wild-type LCK autophosphorylation and phosphorylation of CD3ζ. As a result, we generate a model that matches experimental data and can predict data not used in the fitting.

The model predicts the order and rate at which different ITAMs on the CD3ζ chain are phosphorylated, helping to decifer the kinetic proofreading mechanism that is thought to precisely regulate T cell activation. Additionally, we have applied the model to investigate how changes to the CD3ζ chain, such as removing the third ITAM, can affect overall activation. The results predicted by the model can be implemented in CD3ζ-bearing CAR-engineered T cells. Thus, the model generates new hypotheses that can be tested experimentally, allowing us to quantitatively explore T cell signaling and guide the development of immunotherapies. The model is a quantitative framework that can be used to examine the dynamics of CD3ζ chain activation by LCK and its effects on T cell activation.

Citations

[1] Chae, W., et al. Int. Immunol., 2004, 16(9), 1225-36.

[2] Hui, E. and R. Vale. Nat. Struct. Mol. Biol., 2014, 21(2), 133-142.

[3] Rohrs, J. A., Wang, P., and S. D. Finley. Cell Mol. Bioeng., 2016, In press.