Journal: Nature Biomedical Engineering

Article Title: Generation of antigen-specific mature T cells from RAG1 −/− RAG2 −/− B2M −/− stem cells by engineering their microenvironment

doi: 10.1038/s41551-023-01146-7

Figure Lengend Snippet: a , Schematic showing ESI017 TKO + TCR PSC generation. ESI017 DKO + TCR PSC clones were gene edited to KO B2M to generate the polyclonal ESI017 TKO + TCR line. Following haematopoietic induction, EMOs were collected and then aggregated with either hDLL4 or hDLL4-A02BI stroma for T cell differentiation in ATOs. b , Differentiation kinetics of WT + TCR and TKO + TCR T cells with hDLL4 or hDLL4-A02BI at the indicated time points of ATO differentiation. The gating strategy is indicated above the plots, and the numbers within the plots indicate the percentage of cells within each gate. c , The percentage of T cell populations from the DAPI − mCD29 − CD56 − CD45 + V β 13.1 + CD3 + gate in b at the indicated time points (data are shown as mean ± s.e.m.; WT + TCR with hDLL4 stroma, n = 4 independent experiments; WT + TCR with hDLL4-A02BI stroma, n = 3 independent experiments; TKO + TCR with hDLL4 stroma, n = 4 independent experiments; TKO + TCR with hDLL4-A02BI stroma, n = 5 independent experiments). The populations are defined as DN (CD8α − CD4 − ), DP (CD8α + CD4 + ) and SP8 (CD8αβ + CD4 − ).

Article Snippet: Collected cells were counted, and then 2 × 10 4 cells were combined with 2.5 × 10 5 engineered MS5-hDLL4 stromal cell lines per ATO in 1.5 ml Eppendorf tubes.

Techniques: Clone Assay, Cell Differentiation

Journal: Nature Biomedical Engineering

Article Title: Generation of antigen-specific mature T cells from RAG1 −/− RAG2 −/− B2M −/− stem cells by engineering their microenvironment

doi: 10.1038/s41551-023-01146-7

Figure Lengend Snippet: a , Output of SP8 T cells per WT + TCR or TKO + TCR PSC ATO aggregated with hDLL4 or hDLL4-A02BI stroma over the 7 week course of T cell differentiation (mean ± s.e.m.; P values are shown compared with TKO + TCR with hDLL4-A02BI stroma; * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed unpaired t -test; test is not significant if no P value is indicated; WT + TCR with hDLL4 stroma, n = 4 independent experiments; WT + TCR with hDLL4-A02BI stroma, n = 3 independent experiments; TKO + TCR with hDLL4 stroma, n = 4 independent experiments; TKO + TCR with hDLL4-A02BI stroma, n = 5 independent experiments). b , SP8 max T cell output per ATO reached over the 7 week course of T cell differentiation. The mean ± s.e.m. (* P < 0.05, two-tailed Mann–Whitney U -test) is shown for each group (WT + TCR with hDLL4 stroma, n = 4 independent experiments; WT + TCR with hDLL4-A02BI stroma, n = 3 independent experiments; TKO + TCR with hDLL4 stroma, n = 4 independent experiments; TKO + TCR with hDLL4-A02BI stroma, n = 5 independent experiments). c , At 7 weeks of T cell differentiation, TKO + TCR PSC-derived SP8 T cells were analysed for maturation markers of conventional T cells. The gating strategy is indicated above the plots, and the numbers within the plots indicate the percentage of cells within each gate.

Article Snippet: Collected cells were counted, and then 2 × 10 4 cells were combined with 2.5 × 10 5 engineered MS5-hDLL4 stromal cell lines per ATO in 1.5 ml Eppendorf tubes.

Techniques: Cell Differentiation, Two Tailed Test, MANN-WHITNEY, Derivative Assay

Journal: Nature Biomedical Engineering

Article Title: Generation of antigen-specific mature T cells from RAG1 −/− RAG2 −/− B2M −/− stem cells by engineering their microenvironment

doi: 10.1038/s41551-023-01146-7

Figure Lengend Snippet: a , UMAP visualization of ATO-derived SP8 T cells from WT PSCs with hDLL4 stroma ( n = 1 independent experiment, 4,710 cells total), WT + TCR PSCs with hDLL4 ( n = 2 independent experiments, 8,618 cells total) and hDLL4-A02BI ( n = 3 independent experiments, 12,797 cells total) stroma, and TKO + TCR PSCs with hDLL4-A02BI stroma ( n = 3 independent experiments, 7,930 cells total) compared with thymic SP8 ( n = 2 independent experiments, 4,793 cells total), PB SP8 ( n = 1 independent experiments, 2,083 cells total), PB NK ( n = 2 independent experiments, 684 cells total) and PB monocytes ( n = 2 independent experiments, 2,630 cells total). b , Average expression of specific genes from each scRNA-seq sample group. c , Dendrogram of hierarchical clustering analysis and heatmap showing Pearson’s correlation of global gene expression for all pairwise combinations between each sample, listed individually to the right of the heatmap.

Article Snippet: Collected cells were counted, and then 2 × 10 4 cells were combined with 2.5 × 10 5 engineered MS5-hDLL4 stromal cell lines per ATO in 1.5 ml Eppendorf tubes.

Techniques: Derivative Assay, Expressing

Journal: Nature Biomedical Engineering

Article Title: Generation of antigen-specific mature T cells from RAG1 −/− RAG2 −/− B2M −/− stem cells by engineering their microenvironment

doi: 10.1038/s41551-023-01146-7

Figure Lengend Snippet: a , After 6 weeks of T cell differentiation, mature, conventional SP8 T cells were isolated for TCR repertoire analysis at a single-cell resolution from the following ATO conditions: WT PSCs with hDLL4 stroma ( n = 1 independent experiment), WT + TCR PSCs with hDLL4 stroma ( n = 2 independent experiments), WT + TCR PSCs with hDLL4-A02BI stroma ( n = 3 independent experiments) and TKO + TCR PSCs with hDLL4-A02BI stroma ( n = 3 independent experiments). Pie charts showing the frequency of barcoded cells that expressed full contigs of only endogenous TCR chains (grey), only 1G4 TCR chains (black), 1G4 TCR and endogenous TRAV chains (pink), 1G4 TCR and endogenous TRBV chains (green), and 1G4 TCR and both endogenous TRAV and TRBV chains (purple). b , c , TCR V α ( b ) and TCR V β ( c ) diversity from mature, conventional SP8 T cells at single-cell resolution. WT PSCs with hDLL4 stroma (grey; n = 1 independent experiment, 4,710 cells in total), WT + TCR PSCs with hDLL4 stroma (orange; n = 2 independent experiments, 9,619 cells in total), WT + TCR PSCs with hDLL4-A02BI stroma (green; n = 3 independent experiments, 12,797 cells in total) and TKO + TCR PSCs with hDLL4-A02BI stroma (red; n = 3 independent experiments, 7,930 cells in total).

Article Snippet: Collected cells were counted, and then 2 × 10 4 cells were combined with 2.5 × 10 5 engineered MS5-hDLL4 stromal cell lines per ATO in 1.5 ml Eppendorf tubes.

Techniques: Cell Differentiation, Isolation

Journal: Nature Biomedical Engineering

Article Title: Generation of antigen-specific mature T cells from RAG1 −/− RAG2 −/− B2M −/− stem cells by engineering their microenvironment

doi: 10.1038/s41551-023-01146-7

Figure Lengend Snippet: a – f , WT + TCR and TKO + TCR (1G4 TCR) SP8 T cells were isolated from week 6 ATOs, both generated with hDLL4-A02BI stroma and expanded with K562 aAPCs expressing the cognate antigen (NYESO), IL-2 and IL-7 before in vitro functional assays. The numbers indicate the percentage of cells within each gate. a , Expanded T cells were rested and then stimulated for 6 hours with K562 aAPCs presenting non-specific (MART1) or cognate (NYESO) antigen as an SCT to assay cytokine production and CD107α upregulation (gated on Zombie NIR − mStrawberry − TCRαβ + CD3 + CD8α + cells). Data are representative of three independent experiments. b , Upregulation of activation markers CD25 and 4-1BB on WT + TCR and TKO + TCR SP8 T cells in response to stimulation with MART1 or NYESO aAPCs for 24 hours (gated on DAPI − V β 13.1 + CD3 + CD8α + cells). c , Proliferation of SP8 T cells, measured by carboxyfluorescein succinimidyl ester (CFSE), in response to stimulation with MART1 or NYESO aAPCs for 5 days (gated on DAPI − mStrawberry − CD8α + cells). d , Staining with 1G4 tetramer and V β 13.1 from 1G4 TCR on SP8 T cells, after 6 days of expansion with NYESO aAPCs (gated on DAPI − CD8α + cells). e , Cell-surface expression of transgenic 1G4 TCR (measured by V β 13.1) and CD3 on SP8 T cells in response to co-culture with MART1 or NYESO aAPCs for 24 hours (gated on DAPI − CD8α + cells). f , In vitro cytotoxicity of WT + TCR and TKO + TCR SP8 T cells against MART1 or NYESO aAPCs based on Apotracker Green assay at 6 hours of co-culture (data are representative of n = 3 independent experiments). g , Fold expansion (from initial input) of WT + TCR and TKO + TCR SP8 T cells immediately after isolation from ATOs in response to NYESO aAPCs, IL-2 and IL-7 (data are shown as mean ± s.e.m.; n = 2 independent experiments).

Article Snippet: Collected cells were counted, and then 2 × 10 4 cells were combined with 2.5 × 10 5 engineered MS5-hDLL4 stromal cell lines per ATO in 1.5 ml Eppendorf tubes.

Techniques: Isolation, Generated, Expressing, In Vitro, Functional Assay, Activation Assay, Staining, Transgenic Assay, Co-Culture Assay

Journal: Cell Death & Disease

Article Title: Mtu1 defects are correlated with reduced osteogenic differentiation

doi: 10.1038/s41419-020-03345-5

Figure Lengend Snippet: A Mtu1 was silenced in MS5 stromal cells using distinct Mtu1 shRNA-encoding lentivirus or scramble lentivirus. Western blot analyses show lower Mtu1 expression levels in shMtu1_2 cells. B Flow cytometric analysis for cell-surface markers. Green peaks, isotype control; red peaks, specific antibodies. C 2-Thiouridylation level analysis of mt-tRNAs by APM gel. D Proportion of tRNA 2-thiouridine modification levels. The proportion values are expressed as ratios (%) of the average of 2-thiouridine modified tRNA levels to total levels. E , F Alizarin Red S staining and quantification of mineralized calcium deposits in cells after 14 days of osteogenic differentiation. The figures show one representative result of at least three experiments. G RT-qPCR at 0, 7, 14, and 21 days for mRNA levels of osteoblast differentiation markers, including Runx2 , Ocn , and Alp . * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: MS5 stromal cell line, a type of mouse adherent fibroblastic cells growing in monolayers, was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: shRNA, Western Blot, Expressing, Control, Modification, Staining, Quantitative RT-PCR