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Proteomics analysis reveal early event molecular effectors of anti-CD19 CAR-T cell therapy in hematological cancer

ABSTRACT: Chimeric antigen receptor T-cell (CAR-T) therapy is at the forefront of cell immunotherapy. In this study, we generated an anti-CD19 CAR-Jurkat T cell line using a locally produced second-generation CD19 CAR construct, enabling us to analyze early proteomic changes crucial for understanding the signaling pathways and mechanisms of action of this CAR-T cell. SILAC-heavy tagged RAJI B-cells and anti-CD19 CAR-Jurkat T-cells were co-cultured for ten minutes. The proteomic profiles were obtained via DIA methodology on the Orbitrap Astral LC-MS/MS platform. The proteome was extensively covered, resulting in approximately 8800 protein identifications at 1% FDR. The effector CAR-Jurkat cells showed proteomic changes involving antigen presentation by CD74. The target RAJI B-cells exhibited more significant alterations, such as CD28, essential for T-cell survival and activation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, T Cell, Cell Culture

DISEASE(S): B-lymphoblastic Leukemia/lymphoma,B-cell Acute Lymphoblastic Leukemia

SUBMITTER: John Oluwafemi Teibo

LAB HEAD: Vitor Marcel

PROVIDER: PXD062748 | Pride | 2025-07-30

REPOSITORIES: Pride

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			Action	DRS
	240428_RN-JT_SILAC-DIA_TP_01.raw	Raw
	240428_RN-JT_SILAC-DIA_TP_02_20240429151755.raw	Raw
	240428_RN-JT_SILAC-DIA_TP_03_20240429160550.raw	Raw
	240428_RN-JT_SILAC-DIA_TP_04_20240429165337.raw	Raw
	240428_RN-JT_SILAC-DIA_TP_05_20240429174125.raw	Raw

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Project description:Adoptive cell therapy, a subset of cancer immunotherapy, is collection of therapeutic approaches which aim to redirect the immune system by reprogramming patient T-cells to target antigenic molecules differentially and specifically expressed in certain cancers. One promising immunotherapy technique is CAR T-cell therapy, where cancer cells are targeted through the expression a chimeric antigen receptor (CAR), a synthetic trans- membrane receptor that functionally compensates for the T-cell receptor (TCR) but targets a tumor associated antigen on the cancer cell surface. While CAR T-cell therapy is promising with two clinically approved second-generation CARs (Kymriah and Yescarta), few studies have investigated the mechanism of signal propagation in T-cells and no studies have investigated the potential signaling response in the target cells. To gain further insight to CAR-based signaling, we stimulated third generation CD19 CAR-expressing Jurkat T-cells by co-culture with SILAC labeled CD19HI Raji B-cells and used two phosphoenrichment strategies coupled with liquid chromatography-tandem mass spec- trometry (LC-MS/MS) to detect and analyze global phosphorylation changes in both cell populations. Analysis of the phosphopeptides originating from the CD19-CAR T cells revealed an increase in many phosphorylation events necessary for canonical TCR signaling. We also observed for the first time a significant decrease in B-cell receptor- related phosphopeptide abundance in CD19HI Raji B-cells after co-culture with CD19-targetted CAR T-cells.

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