Transcriptome analysis of ITGA3+ and ITGA3- from expanded CB cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA542151 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9036401_1.fastq.gz | Fastqsanger.gz | |||
| SRR9036401_2.fastq.gz | Fastqsanger.gz | |||
| SRR9036402_1.fastq.gz | Fastqsanger.gz | |||
| SRR9036402_2.fastq.gz | Fastqsanger.gz | |||
| SRR9036403_1.fastq.gz | Fastqsanger.gz |
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Project description:Cell purification technology combined with whole transcriptome sequencing and small molecule agonist of hematopoietic stem cell self-renewal has allowed us to identify ITGA3 as a surface maker that defines a rare subpopulation of human cells which is highly enriched for stem cell activity in vivo. ITGA3-positive cells (within the EPCR+CD90+CD133+CD34+CD45RA- fraction) exhibit a robust multi-lineage differentiation potential and serial reconstitution in immunocompromised mice. In culture, most if not all of the HSC activity is detected in the ITGA3+ subset, arguing for the stability of this marker on the surface of cultured cells, a feature not found with more recently described markers such as CD49f. Functionally ITGA3 is essential for human HSC activity in vivo. Its exploitation should open new possibilities in our effort to understand the molecular bases behind HSC self-renewal.
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Project description:A study of immunotherapy with expanded CB-NK cells in combination with cetuximab, to evaluate activity against minimal residual disease in patients with colon cancer that have completed adjuvant treatment but are positive for ctDNA
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Project description:Our team had previously explored the potential of expanding cord blood (CB) derived γδ T cells (CB-gdT) as well as their corresponding ability to target primary acute myeloid leukemia (AML) cells. Using a feeder cell line-based in vitro expansion protocol, we achieved a clinically relevant scale expansion of γδ T cells over a period of 14 days. These cells exhibit variable degree of potency against a range of human AML cell lines and primary patient samples. In order to dissect the cellular and molecular programs governing the activation, differentiation and functional states of our in vitro expanded CB-gdT, we performed multiplex single cell sequencing analysis using the 10X Genomics Chromium System. After initial quality check and filtering, data from a total of 4,276 cells were retrieved. Among which, we identified 742 unique TCRγδ clonotypes, representing 18.6% of the starting 4,000 FACS purified γδ T cells seeded for expansion. Consistent to our FACS analysis, Vδ1 is the predominant TRD chain in the expanded cultures, accounting for 61.2% of all clones. Based on uniform manifold approximation and projection for dimension reduction (UMAP), all cells were clustered into 11 subsets. Key cytotoxic genes including GZMB, GZMA and NKG7 were all highly expressed across all clusters, indicating that the expanded cells were indeed functionally cytotoxic. Comparing against multiple curated gene sets, we have identified 3 main subsets of γδ T cells: the Proliferative, Cytotoxic γδ T cells (P-CT), Differentiated Cytotoxic γδ T cells (D-CT) and Late Activated Cytotoxic γδ T cells (LA-CT). P-CT (~46% of all cells) shows an expression profile positively associated with cell proliferation as well as increased cell surface expression of memory T cell markers CD27, CCR7 and CD62L. Similar to cytotoxic genes, genes associated with TCR signaling and interferon response were found to be expressed across all cell clusters, yet with elevated levels in D-CT and LA-CT. Furthermore, cell surface expression of different NK receptors including NKG2D, DNAM1 and NKp30 are more enriched in LA-CT compared to the other 2 subsets, suggesting the acquisition of additional NK receptor related functions in this group of cells. Consistent with the concept of progressive γδ T cell differentiation and activation in culture, we found that in 85 (11.5%) of the γδ T cell clones bearing more than 10 cells each, all clones contain cells distributed across the 3 different γδ T cell subsets. This is the first report on single cell immune profiling of in vitro expanded gdT cells derived from human CB.
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Project description:Single Cell Immune Profiling of in vitro expanded cord blood derived gamma-delta T cells (CB-gdT)
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Project description:Since the role of cord blood (CB) regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is under investigation, we analyzed and compared the functional properties and gene expression profile of Tregs expanded from CB units or from the peripheral blood (PB) of helathy donors. Tregs were purified from 23 CB units and from the PB of 13 donors and expanded for 6 days with anti-CD3, anti-CD28 and IL-2. Immunophenotypic analyses were performed, and suppressor activity of expanded Tregs was measured in mixed lymphocyte reaction (MLR) cultures. The IL-10 production capacity was tested and gene expression profile experiments were performed on 6 Tregs from PB and 4 from CB. CB and PB Tregs had similar immunophenotypic features. Tregs from CB presented a higher expansion capacity and genomic characterization showed in CB-derived Tregs a significant enrichments of genes involved in cell proliferation, chromatin modification and regulation of gene expression in CB-derived Tregs. All samples were positive for the Foxp3 gene and protein after expansion. CB and PB expanded Tregs exerted a comparable and potent suppressive function of MLR and presented a high in vitro IL-10 production capacity. Gene profile analysis also revealed for PB Tregs a significant enrichments of genes involved in the adaptive immune response. Comparison of the transcriptional profile of T-REGS derived either from cord blood or peripheral blood. 6 highly purified T-regs samples derived from peripheral blood of healthy donors were compared to highly purified T-regs from cord blood.
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