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Phosphoprotein dynamics of interacting tumor and T cells by HySic

ABSTRACT: Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. Some of the proteins involved, particularly immune checkpoints expressed by T cells, serve as promising clinical targets in immunotherapy. However, our understanding of the complexity and dynamics of the interactions between tumor cells and T cells is only rudimentary. Here we present HySic (for Hybrid quantification of SILAC (Stable Isotope Labelling by Amino acids in Cell culture)-labeled interacting cells) as an innovative method to quantify protein and phosphorylation dynamics between and within physically interacting (heterotypic) cells. We show that co-cultured HLA/antigen/TCR-matched tumor and T cells engage in stable interactions, allowing for in-depth HySic analysis. This method does not require physical separation of the two cell types for subsequent MS proteome and phosphoproteome measurements using label-free quantification (LFQ). We demonstrate that HySic can be used to identify proteins whose abundance or activation status changes upon interactions between T cells and tumor cells. We validated HySic with established signal transduction pathways, including IFN. Using HySic we also identified the RHO/RAC/PAK1 signaling pathway to be activated upon T cell:tumor cell interaction. Pharmacologic inhibition of PAK1 sensitized tumor cells to T cell killing. Thus, HySic is a simple method to study short-term protein signaling dynamics in physically interacting cells, which can be easily extended to other biological systems.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, T Cell, Lung, Epithelial Cell

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Kelly Stecker

LAB HEAD: Kelly E.

PROVIDER: PXD046734 | Pride | 2023-12-20

REPOSITORIES: Pride

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Project description:Background. Dendritic cell (DC)-based neoantigen vaccination holds potential as a safe and effective adjuvant therapy for patients with early-stage, resectable NSCLC, a tumor type typically characterized by high mutational loads. DCs have the unique ability to elicit robust antitumoral T-cell responses, while neoantigens are ideal targets to elicit high-affinity T cell responses with exquisite tumor specificity. Here, we present the results of a phase I clinical trial in which a novel DC vaccine targeting neoantigens was evaluated in six patients with early stage, resected NSCLC. Methods. Tumor samples were subjected to a comprehensive neoantigen identification approach encompassing genomics, transcriptomics and immunopeptidomics. Using genomics and transcriptopmics data, tumor-specific antigens were identified by a bioinformatics approach. Additionally, immunopeptidomics was performed by immunoprecipitation of human MHC class I molecule followed by immunopeptides enrichment and LC-MS/MS analysis. Two immunopeptidomics screens were performed. In the first immunopeptidomics screen, patient-derived tumors were analyzed to uncover neoepitopes specific for the patient. In the second, screen, patient-derived EBV-immortilized B cell lines overexpressing the selected neoepitopes were analyzed to verify that the predicted neoepitopes can bind to the HLA haplotypes of the patients. For anti-tumor vaccination, patients underwent leukapheresis for the manufacturing of monocyte-derived DCs loaded with neoantigens (Neo-mDCs) according to a four-day protocol. Neo-mDCs were injected intravenously following an intrapatient dose escalation scheme. Primary endpoint of the trial was safety. Secondary endpoints were feasibility, immunogenicity, and relapse-free survival. Results. In the first immunopeptidomics screen, one neoepitopes derived was identified from the tumor of one patient. In the second immunopeptidomics screen, several predicted neoepitopes were confirmed to be presented on the HLA haplotypes of the patients by analyzing the patient-derived EBV-immortilized B cell lines. Additionally, the vaccine was demonstrated to be feasible and safe. T cell responses were observed in 5 of 6 vaccinated patients and were dominated by CD8+ T cells, which could be detected ex vivo at high frequencies >1.5 years after the last dose. Furthermore, single cell analysis indicated that the CD8+ T cell responsive population was polyclonal and exhibited the near entire spectrum of T cell differentiation states, including a naïve-like state associated with long lasting memory but excluding exhausted cell states. Three of six vaccinated patients experienced disease relapse. Conclusion. Neo-mDC vaccination is safe and feasible. Vaccination induces large populations of neoantigen-specific T-cell responses containing long lasting memory and effector cells in early-stage NSCLC patients, suggesting clinical potential.

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Phosphoprotein dynamics of interacting tumor and T cells by HySic

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