Ovalbumin antigen-specific activation of T cell receptor closely resembles soluble antibody stimulation as revealed by BOOST phosphotyrosine proteomics

Abstract

Activation of T cell receptors (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. TCR are 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 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 TCR triggered by anti-TCR antibodies and pMHC. Our data reveals that high-affinity ovalbumin (OVA) pMHC activation of the TCR triggers a largely similar, albeit potentially stronger, pTyr-mediated signaling regulatory axis compared to anti-TCR antibody. 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 T cells activated in an antigen-independent and antigen-specific manner.

SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells

“…The settings for PTM-SEA of transformed q-values were as follows: the values were not rank normalized and the weight parameter was set to 1, incorporating replicate variance into the enrichment score calculation. 28,43 Normalized Enrichment Scores (N ES)…”

“…The phosphorylation events identified by Salter et al exhibited minor exceptions from those commonly observed by antibody-stimulated TCR phosphoproteomic studies, namely a lack of CD3ϵ/δ phosphorylation post-stimulation. 17,28 While notable as the first proteomic investigation of CAR signaling, antibody stimulation disregards the potential influence of the interaction between other surface molecules expressed by both the T and target cell and, therefore, may not fully recapitulate physiological activation of the CAR.…”