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Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery
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SILAC Phosphoproteomics Reveals Unique Signaling Circuits in CAR-T Cells and the Inhibition of B Cell-Activating

Alijah A Griffith1, Kenneth P Callahan1, Nathan Gordo King1

  • 1Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, Rhode Island 02912, United States.

Journal of Proteome Research
|January 11, 2022
PubMed

Insights

Chimeric antigen receptor (CAR) T cells activate T cell receptor (TCR) signaling pathways against cancers. However, cancer cells show reduced signaling after CAR T cell interaction.

Area of Science:

  • Immunology
  • Oncology
  • Molecular Biology

Background:

  • Chimeric antigen receptor (CAR) T cells are a promising cancer therapy, particularly for B cell malignancies.
  • The precise intracellular signaling mechanisms driving CAR T cell efficacy and the impact on target cells are not fully understood.

Purpose of the Study:

  • To investigate the intracellular signaling events in CD19-CAR T cells and target B cells during co-culture.
  • To elucidate the signaling pathways activated by CAR T cells and their effects on cancer cell signaling.

Main Methods:

  • Utilized third-generation CD19-CAR T cells co-cultured with SILAC-labeled Raji B cells.
  • Employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) with phosphoenrichment strategies to analyze phosphopeptides.

Main Results:

  • CD19-CAR T cells showed upregulated phosphorylation events similar to canonical T cell receptor (TCR) signaling.
  • Raji B cells displayed significantly decreased B cell receptor-signaling related phosphorylation upon co-culture.
  • CD19-CAR stimulation activated a combination of unique CAR-specific and canonical TCR signaling pathways.

Conclusions:

  • CD19-CAR T cell activation involves both unique CAR-mediated and shared TCR signaling pathways.
  • CAR T cell interaction leads to a global reduction in phosphorylation signaling within target B cells.
Keywords:
SILAC cell cocultureSrc SH2 superbinderT cell signalingchimeric antigen receptorimmunotherapytumor associated antigen

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