Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs
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PROVIDER: PRJNA798987 | ENA |
REPOSITORIES: ENA
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Project description:The goal of this study was to compare gene expressions on the single cell level in (i) freshly isolated PBMCs (no stimulation and no sorting), (ii) PBγδT cells; PBMCs were stimulated with HMBPP in vitro and CD3(+) γδTCR(+) cells were sorted and (iii) iγδT cells; differentiated cells from γδT-iPSC clone were stimulated with HMBPP and CD3(+) γδTCR(+) cells were sorted or not sorted.
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Project description:γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αβT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αβT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer.
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Project description:Numerous lymphocytes seed the body surfaces of metazoans promoting tissue protection and integrity. These tissue-resident populations differ substantially from circulating lymphocytes, as they adopt a unique phenotype and do not recirculate. They span the innate-adaptive continuum, ranging from innate lymphoid cells (ILCs) to unconventional T cells (NKT, MAIT, γδT cells and IELs) and tissue-resident memory (TRM) T cells, and while differ in the particulars of their biology, all contribute to barrier immunity, tissue homeostasis, and immune regulation. Although they share the same microenvironment, little is known about how these tissue-resident lymphocyte populations interact with each other and with the non-lymphoid cellular environment. As such, there is an emerging view of tissues as varied ‘‘ ecosystems ’’, whereby protection against inflammatory challenges is orchestrated through the interplay between tissue-resident and infiltrating-lymphocyte subtypes. In mice, barrier tissues are populated during early life by innate-like γδT lymphocytes. These cells provide tissue protection, including limiting conventional T cell-driven inflammation. Therefore, elucidating the contributions of tissue-resident T cells, particularly γδT cells, to the basic pathophysiology of tissue function and protection against infection, inflammation, and cancer is a priority. To this aim, we have induced allergic contact dermatitis in the back skin of different strains and we analyzed the behavior of γδT cells
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Project description:We report the application of single cell RNA sequencing for profiling the fingerprints of γδT cells of hepatocellular carcinoma compared with healthy donors. By comparing the transcriptome and TCR clonality of γδT cells from 3 HCC patients and expanded γδT cells from 3 healthy donors, we found that expanded γδ T cells derived from different donors shared great transcriptomic similarity and we found higher enrichments of major γδ TCRs in expanded γδ T cells compared to infiltrated but not peripheral ones in HCC. Analysis of differentiation trajectory demonstrated a branched structure in expanded γδ T cells, implying functional and differentiation stage diversity, and γδ T cells from HCC patients are localized to the far end of the trajectory. Furthermore, GVSA analyses made on metabolic pathways demonstrated strong enrichments in major metabolic pathways such as OXPHOS, glycolysis, and fatty acid metabolism in expanded γδ T cells versus peripheral ones of HCC. The comparison indicated higher cytotoxicity score of expanded γδ T cells compared to infiltrated or peripheral ones of HCC. The successful application of allogeneic γδ T cells immunotherapy in late-stage liver cancer patients was supported by our current scRNA transcriptomic analyses, which suggested strong anti-tumor functional complementation by the ex-vivo expanded γδ T cells.
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Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.
2018-06-06 | PXD009142 | Pride
Project description:Leptin receptors (Lepr) are expressed by various types of stem cells including mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells. Leptin/lepr signaling is also a central regulator of metabolism. However, the role of Lepr in pluripotency, metabolic disease progression and growth development is still controversial and poorly understood. In the present study, we explored the Lepr function in disease progression, pluripotency and metabolism using day 14.5 mouse embryonic fibroblasts (MEFs) and their reprogrammed induced pluripotent stem cells (iPSCs) as model system. We successfully reprogrammed mouse embryonic fibroblasts into iPSCs from control and db/db (Lepr deficient) mice. Using a global quantitative proteomic approach, we identified key pathways regulating pluripotency, metabolic homeostasis and protein synthesis during fetal growth and development. The Lepr MEFs show abnormal metabolic abnormalities and mitochondrial dysfunction as compared to control MEFs, while Lepr iPSCs show upregulated elongated factor 4 e (eIF4e) protein synthesis pathway and altered Oct4 and Stat3 pathways which are involved in normal fetal growth development. Furthermore, chip analysis revealed that higher Stat3 binding on the promoter of eIF4e in Lepr iPSCs leads to higher protein synthesis in these cell types as compared to control iPSCs. Finally, point mutation corrected Lepr iPSCs using CRISPR/Cas9 gene editing method showed recovered pluripotency, metabolic and protein synthesis pathways. In conclusion, we have shown that Lepr signaling is involved in the regulation of the metabolic properties and key developmental pathways in MEFs and stemness of pluripotent stem cells. Disruption of Lepr signaling has been shown to involve in the pathophysiology of various diseases including obesity and diabetes. The generated MEFs and iPSCs in this present study provide valuable tools to explore the role of Lepr in the progression of obesity, diabetes and metabolic abnormalities, and to find the putative targets of Lepr signaling during the development of these diseases.
2021-09-09 | PXD020959 | Pride
Project description:Whereas cytotoxic T lymphocytes (CTLs) represent the most promising therapeutic avenue in cancer immunotherapy, adaptive transfer of antigen-specific CTLs has faced difficulty in efficient expansion of CTLs from patients in ex vivo culture. To solve this issue, several groups have proposed that the induced pluripotent stem cell (iPSC) technology can be applied for the expansion of antigen-specific CTLs: when iPSCs are produced from antigen-specific CTLs and CTLs are induced from these iPSCs, all regenerated CTLs express the same TCR as original CTLs. However, in these previous studies, one critical issue remains to be solved. With current methods, the regenerated CTLs are mostly of the CD8αα+ innate type and have less antigen-specific cytotoxic activity than primary CTLs. Here we report that, by stimulating purified iPSC-derived CD4/CD8 double positive (DP) cells with anti-CD3 antibody, T cells expressing CD8αβ were generated and they had cytotoxic activity comparable to primary CTLs.
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Project description:Fresh human HCC tissue and paired pertumor liver tissue were acquired, isolation of the infiltrating lymphocytes was conducted by discontinous density gradient centrifugation in Percoll. After removal of dead cells and incubation with components of TCRγ/δ isolation kit (Mitenyi Biotec), γδ T cells were positively selected from the single-cell suspension using autoMACS Separator (Miltenyi Biotec) with recommended programs. Then we employed whole genome microarray expression profiling as a discovery platform to indentify genes which are differentially expressed between HCC-derived and paired peritumor-derived γδT cells.
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