SILAC Phosphoproteomics Reveals Unique Signaling Circuits in CAR-T Cells and the Inhibition of B Cell-Activating Phosphorylation in Target Cells

Abstract

Chimeric antigen receptor (CAR) is a single-pass transmembrane receptor designed to specifically target and eliminate cancers. While CARs prove highly efficacious against B cell malignancies, the intracellular signaling events which promote CAR T cell activity remain elusive. To gain further insight into both CAR T cell signaling and the potential signaling response of cells targeted by CAR, we analyzed phosphopeptides captured by two separate phosphoenrichment strategies from third generation CD19-CAR T cells cocultured with SILAC labeled Raji B cells by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Here, we report that CD19-CAR T cells upregulated several key phosphorylation events also observed in canonical T cell receptor (TCR) signaling, while Raji B cells exhibited a significant decrease in B cell receptor-signaling related phosphorylation events in response to coculture. Our data suggest that CD19-CAR stimulation activates a mixture of unique CD19-CAR-specific signaling pathways and canonical TCR signaling, while global phosphorylation in Raji B cells is reduced after association with the CD19-CAR T cells.

Phospho-proteomic analysis of CAR-T cell signaling following activation by antigen-presenting cancer cells

“…Nevertheless, among the CAR phosphorylation sites that we did quantify, it is interesting that both were slightly downregulated (CD3ζ pTyr142 and CD28 pTyr209). This contrasts with previous phospho-proteomic studies where these phosphorylation sites were significantly upregulated by stimulation of second-generation CARs with antibody-coated beads (9, 11) or activation of a third-generation CAR with cell displayed-antigen (7). Potentially, this discrepancy could reflect differences in experimental design or that our CD19-expressing cancer cells have not maximally activated the CAR.…”

“…5,6). Many of the observed signaling pathways are consistent with other analyses of CAR signaling, supporting the validity of our approach (7,8,11,15). One of the strongest signals we observed was the activation of the ERK/MAPK signaling pathway, both at the level of individual ERK1/2 activation sites and PTM-SEA pathway analysis.…”

“…Notably, many of these phosphorylation events occur on tyrosine residues (e.g., ITAM domains, ZAP70 pTyr492, LCK pTyr192, etc. ), suggesting that inclusion of a parallel phospho-tyrosine enrichment (e.g., phospho-tyrosine antibody Phospho-proteomic analysis of CAR-T cell signaling (15), Src SH2 superbinder (7)) would generate a more comprehensive view of the CAR phospho-proteome. Nevertheless, among the CAR phosphorylation sites that we did quantify, it is interesting that both were slightly downregulated (CD3ζ pTyr142 and CD28 pTyr209).…”

“…Because CARs are built from endogenous proteins involved in T cell receptor (TCR) signaling, it has been thought that their activation pathways share many similarities. However, emerging data suggests that CARs activate a mixture of canonical TCR signaling and unique CAR-specific pathways (7–10). In addition, the choice of CAR stimulatory domains (e.g., CD28, 4-1BB, or both) can influence the strength of CAR signaling, thereby affecting CAR-T cell function (11–13).…”

“…For comprehensive, unbiased, and quantitative characterization of protein phosphorylation, liquid chromatography-mass spectrometry (LC-MS)-based phospho- proteomics is the technology of choice (14), and these methods have been applied to CAR signaling (7, 9, 11, 15). However, phospho-proteomic studies of CAR signaling have been limited by the challenge of stimulating CAR-expressing cells with their in vivo stimulus, antigen-presenting cancer cells.…”