Abstract
Activation of the T cell receptor (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. The TCR is commonly activated using soluble anti-TCR antibodies, but this approach is not antigen-specific. Alternatively, activating the TCR using specific antigens of a range of binding affinities in the form of a peptide-major histocompatibility complex (pMHC) is presumed to be more physiological. However, due to the lack of wide-scale phosphotyrosine (pTyr) proteomic studies directly comparing anti-TCR antibodies and pMHC, a comprehensive definition of these activated states remains enigmatic. Elucidation of the tyrosine phosphoproteome using quantitative pTyr proteomics enables a better understanding of the unique features of these activating agents and the role of ligand binding affinity on signaling. Here, we apply the recently established Broad-spectrum Optimization Of Selective Triggering (BOOST) to examine perturbations in tyrosine phosphorylation of human TCR triggered by anti-TCR antibodies and pMHC. Our data reveal that high-affinity ovalbumin (OVA) pMHC activation of the human TCR triggers a largely similar, albeit potentially stronger, pTyr-mediated signaling regulatory axis compared to the anti-TCR antibody. The signaling output resulting from OVA pMHC variants correlates well with their weaker affinities, enabling affinity-tunable control of signaling strength. Collectively, we provide a framework for applying BOOST to compare pTyr-mediated signaling pathways of human T cells activated in an antigen-independent and antigen-specific manner.