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SIFT-seq: VDJ and scRNA-seq of T cells targeting public neoantigens

ABSTRACT: We developed a modular, high-throughput discovery platform to simultaneously test whether Mut PIK3Ca is immunogenic and to retrieve paired a/b TCR gene sequences that confer specificity to this NeoAg. This method, termed Stimulation Induced Functional TCR sequencing (SIFT-seq), combines single-cell (sc) TCR V(D)J and transcriptome sequencing. Here, microwell cultures of in vitro stimulated T cells with confirmed neoantigen-specific recognition are selected for SIFT-seq. Matched aliquots of selected wells are acutely stimulated with autologous antigen-presenting cells presenting WT or Mut PI3Ka and the transcriptomic profile of individual clonotypes is assessed to identify neoantigen-specific T cells and retrieve their TCR gene sequences.

ORGANISM(S): Homo sapiens

PROVIDER: GSE172403 | GEO | 2022/03/03

REPOSITORIES: GEO

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Neoantigen-specific stem cell memory-like CD4+ T cells mediate immunotherapy of solid tumors

Project description:The expansion of neoantigen (NeoAg)-specific T cells often accompanies clinical responses to immunotherapies, highlighting the importance of these cells for antitumor immunity. While NeoAg-specific CD8+ T cells have been broadly studied, the role of NeoAg-specific CD4+ T cells is less well understood. To study the antitumor mechanisms of NeoAg-specific CD4+ T cells, we sorted single CD4+ T cells specific for an epitope derived from a mutated clathrin heavy chain gene (CLTCH129>Q) expressed by the murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer preferential recognition of CLTCH129>Q as compared to the corresponding wildtype CLTC epitope. Despite differences in TCR avidity, both moderate and high avidity CD4+ T cells were capable of proliferating to similar levels in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. Finally, we demonstrate that adoptive cellular therapy (ACT) with T stem cell memory (TSCM)-like CLTCH129>Q-specific CD4+ T cells differentiated ex vivo in culture with IL-7 and IL-15 promotes therapeutic tumor immunity associated with enhanced T cell persistence, maintenance of adoptively transferred TSCM-like cells in the tumor-draining lymph node (tdLN), and activation of CD8+ T cells in the tdLN. Overall, these findings illuminate the mechanistic role of NeoAg-specific CD4+ T cells in tumor immunity, provide insights into the impact of TCR avidity on the helper functions of CD4+ T cells, and highlight the therapeutic potential of ACT with TCR-engineered CD4+ T cells.

2023-05-24 | GSE229221 | GEO

Intravenous Nanoparticle Vaccination Generates Stem-Like TCF1+ Neoantigen-Specific CD8 T Cells

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2020-11-02 | GSE158240 | GEO

Personalized neoantigen-based T cell therapy triggers cytotoxic lymphocytes expressing polyclonal TCR against metastatic ovarian cancer

Project description:Neoantigen-reactive cytotoxic T lymphocytes play a vital role in precise cancer cell elimination. In this study, we demonstrate the effectiveness of personalized neoantigen-based T cell therapy in inducing tumor regression in two patients suffering from heavily-burdened metastatic ovarian cancer. Our approach involved the development of a robust pipeline for ex vivo expansion of neoantigen-reactive T lymphocytes. Neoantigen peptides were designed and synthesized based on the somatic mutations of the tumors and their predicted HLA binding affinities. These peptides were then presented to T lymphocytes through co-culture with neoantigen-loaded dendritic cells for ex vivo expansion. Subsequent to cell therapy, both patients exhibited significant reductions in tumor marker levels and experienced substantial tumor regression. One patient achieved repeated cancer regression through infusions of T cell products generated from newly identified neoantigens. Transcriptomic analyses revealed a remarkable increase in neoantigen-reactive cytotoxic lymphocytes in the peripheral blood of the patients following cell therapy. These cytotoxic T lymphocytes expressed polyclonal T cell receptors (TCR) against neoantigens, along with abundant cytotoxic proteins and pro-inflammatory cytokines. The efficacy of neoantigen targeting was significantly associated with the immunogenicity and TCR polyclonality. Notably, the neoantigen-specific TCR clonotypes persisted in the peripheral blood after cell therapy. Our findings indicate that personalized neoantigen-based T cell therapy triggers cytotoxic lymphocytes expressing polyclonal TCR against ovarian cancer, suggesting its promising potential in cancer immunotherapy.

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scRNAseq of Antigen Presenting Cells in severe COVID-19 patients

Project description:We isolated PBMC from healthy, moderate ( Oxygen supply < 10L/min), and severe (Oxygen supply >= 10L/min) COVID-19 patients after their admission to Intensive Care Units (ICU), at two timepoints (Day-1 and Day-4); and performed both CD14+ Monocyte enrichment followed by a Pan-DC kit to retrieve all Antigen Presenting Cell (APC) subsets from these age-matched patients. We performed single cell RNA sequencing using 10X technology on the single cell suspensions and constracted a high-resolution map of 81,643 Antigen Presenting Cells (APC) from the three COVID-19 severity groups. We were able to retrieve all the known six APC subsets and deciphered the altered pathways and ati-viral mechanisms, correlated with the disease severity.

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Gene expression and T cell repertoire profile at single cell level of peripheral blood T cells after treatment with a personalized neoantigen vaccine (GNOS-PV02) and Pembrolizumab for advanced hepatocellular carcinoma.

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Multimodal probing of T cell sensitivity with hexapod heterostructures

Project description:Studies employing antigen-presenting systems at the ensemble or single cell level can provide complementary insights into T-cell activation and signaling. However, synthetic material toolkits that probe T cells at both levels are lacking, although they would be essential in advancing basic immunology and immunotherapy. Here, we develop a biomimetic antigen-presenting system (bAPS) using hexapod heterostructures (i.e., a sub-micrometer hematite core, and nanostructured silica branches with diverse surface modifications) for single-cell, single-molecule stimulation and ensemble modulation of T-cell signaling. We report with single molecule resolution of: T-cell sensing/activation by a single agonist peptide-major histocompatibility complex; distinct T-cell receptor (TCR) responses to discriminate structurally similar peptides that differ by just one amino acid; and the superior sensitivity of TCR antigen recognition compared to chimeric antigen receptors (CARs). We also show that magnetic field-induced piconewton×micrometer-level torques on the hexapods amplify immune responses in suspension T and CAR-T cells. Furthermore, by using our bAPS, we have developed a reliable and high-throughput new method for identifying neoantigen-specific TCRs at the single-cell level. The multimodal hexapod bAPS presents an unexplored, nanotechnology-based biointerface tool for investigating recognition, signaling, and the biochemical/mechanical dual sensitivity of T cells and beyond.

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Expression data from antigen-presenting cells (APCs)

Project description:P25 is the major T cell epitope for Ag85B and enables them to induce P25-specific CD4+ Th1 cells. We used microarrays to examine the gene expression of antigen-presenting cells (APCs) stimulated with P25 and P25-specific CD4+ T cells. Splenic APCs from WT mice were cultured with P25 or medium in the presence of P25 TCR CD4+ T cells. After overnight culture, these APCs were enriched and total RNA was prepared using RNeasy (QIAGEN) according to the manufacturer's instructions. Mixed total RNA from three independent experiments was used for microarray analysis.

2012-03-01 | E-GEOD-36018 | biostudies-arrayexpress

SIFT-seq: VDJ and scRNA-seq of T cells targeting public neoantigens

Project description:SIFT-seq: VDJ and scRNA-seq of T cells targeting public neoantigens

| PRJNA723226 | ENA

Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells

Project description:Planar supported lipid bilayers (PSLB) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signaling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.

2019-10-02 | PXD007988 | Pride

Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma [Single cell]

Project description:The comparative resistance of some cancers including head and neck squamous cell carcinoma to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens. SCCVII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity within syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-γ responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-γ when stimulated by a single Cltc mutation during in vitro recall assays. Prophylactic immunization with a mixture of all stimulatory peptides or the Cltc NeoAg alone protected hosts from subsequent tumor challenge. Further, the Cltc NeoAg, eliciting both CD4+ and CD8+ T cell responses, was also therapeutically beneficial in vivo. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection via boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors.

2021-01-15 | GSE125077 | GEO

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