Journal: Cells

Article Title: SHIP1 Is Present but Strongly Downregulated in T-ALL, and after Restoration Suppresses Leukemia Growth in a T-ALL Xenotransplantation Mouse Model

doi: 10.3390/cells12131798

Figure Lengend Snippet: Reduced expression of SHIP1 in primary T-ALL samples. ( A ) SHIP1 mRNA expression was analyzed in peripheral blood mononuclear cells (PBMC) from healthy donors, healthy CD3-enriched (CD3+) cells and primary T-ALL patient samples. ( B ) SHIP1 protein expression was analyzed in Jurkat cells containing a Tet-regulated wild-type SHIP1 cDNA, in healthy CD3-positve cells and in primary T-ALL samples. Ponceau S staining of the membrane was performed as a loading and blotting control.

Article Snippet: CD3-positive T cells were isolated from fresh peripheral blood mononuclear cells of healthy donors using EasySep Human CD3 positive selection kit (STEMCELL Technologies) with the associated EasySep magnet (STEMCELL Technologies) for the selection of magnetic particles.

Techniques: Expressing, Staining

Journal: iScience

Article Title: Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure

doi: 10.1016/j.isci.2024.109443

Figure Lengend Snippet: in vivo efficacy and toxicity of RKI-1447 (A) CD3-depleted frozen PBMC from three SRSF2 -mutated acute myeloid leukemia (AML) samples were injected into SGM3 (#800667) or NSG mice (#209945 and #830163) intrafemorally (i.f.); after 5 weeks transplantation mice were treated with RKI-1447 (50 mg/kg) or DMSO control for 21 days. (B) NSG mice (n = 5–10/sample) were injected with 80,000 to 150,000 CD34 + cells from three mobilized peripheral blood samples (i.f.). On day 35 the animals were randomized to RKI-1447 or a carrier control. RKI-1447 was administered i.p. at a dose of 50 mg/kg daily for 21 days. On day 56 mice were sacrificed and analyzed for human CD45 + (hCD45) cells engraftment by flow cytometry. Mann-Whitney U test with FDR correction for multiple hypothesis testing, ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005. (C) 5 ∗ 10ˆ6 MOLM14 mutated cells were injected into 225 rad irradiated NSG mice. The mice were treated with RKI-1447 (50 mg/kg/day), starting from day 3 following cell transplantation. The mice were treated every day via intraperitoneal (i.p.) injection for 21 days. The Kaplan-Meier test p = 0.01.

Article Snippet: Frozen primary SRSF2 Mut/WT AML samples were thawed and CD3 + cells were depleted with CD3 positive selection kit (EasySep Human CD3 Positive Selection Kit II, STEMCELL Cat.17851).

Techniques: In Vivo, Injection, Transplantation Assay, Control, Flow Cytometry, MANN-WHITNEY, Irradiation

Journal: iScience

Article Title: Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure

doi: 10.1016/j.isci.2024.109443

Figure Lengend Snippet:

Article Snippet: Frozen primary SRSF2 Mut/WT AML samples were thawed and CD3 + cells were depleted with CD3 positive selection kit (EasySep Human CD3 Positive Selection Kit II, STEMCELL Cat.17851).

Techniques: Selection, Sequencing

Journal: Science Advances

Article Title: Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells

doi: 10.1126/sciadv.adg1610

Figure Lengend Snippet: ( A ) Schematic representation describes the experimental procedure. Blood was taken from eight healthy volunteers at five time points: 7 days before DI exposure (day 0, orange); 7 (blue), 14 (teal), and 21 (green) days during DI exposure; and 7 days after DI exposure (day 28, pink). CD3 + T cells were isolated from the blood, and bulk RNA-seq was performed. ( B ) Horizontal bars show the fraction of granulocytes, lymphocytes, and monocytes in blood isolates before CD3 + T cell isolation as determined by flow cytometry. Values are indicated as means + 1.96 × SEM. ( C ) Horizontal bars display the estimated fraction of T cell populations detectable in the bulk RNA-seq data as determined by deconvolution analysis with the quanTIseq algorithm. Values are indicated as means + 1.96 × SEM. ( D ) Factorial map of the PC analysis separates bulk RNA-seq gene expression data of each individual volunteers (small dots) and mean values across all volunteers (large dots). The proportion of variance explained by each PC is indicated in parentheses. Color-coding is according to the different time points. ( E ) The dot plot shows PC1 separation of bulk RNA-seq samples by time point. Individual values (small dots) and mean values (large dots) are shown per time point (color-coded). Error bars indicate means ± 1.96 × SEM. ( F ) Illustration displays differentially expressed genes (DEGs) identified by pairwise time point comparisons using DESeq2. The number of significantly (adjusted P < 0.01) up- [positive log 2 FC (fold change), red] and down-regulated (negative log 2 FC, blue) genes is indicated. ( G ) Line charts show the two k -means clusters and number of differentially expressed genes per cluster detected in at least one time point comparison. Standardized gene expression profiles for individual genes (gray lines) and the cluster center (connected dots) are shown.

Article Snippet: Human T cells were enriched by the positive CD3 cell selection Kit (EasySep Human CD3 Positive Selection Kit II, STEMCELL Technology) with a purity of 90 to 95%.

Techniques: Isolation, RNA Sequencing, Cell Isolation, Flow Cytometry, Gene Expression, Comparison

Journal: Science Advances

Article Title: Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells

doi: 10.1126/sciadv.adg1610

Figure Lengend Snippet: ( A ) Heatmap demonstrates expression of overall down-regulated genes in CD3 + T cells identified upon DI (cluster 3 in fig. S9, n = 148) (left ) that overlap with genes in CD4 + (middle) and CD8 + (right) T cells obtained in the NASA twin study. Gene expression changes are shown as log 2 FC to day 0 for DI and log 2 FC for relevant comparisons in the NASA study (gray if missing). Relevant genes are highlighted. ( B ) Vertical bars display changes in expression for selected genes [ x axis annotated according to (A)]. Log 2 FC values show up- (red) and down-regulation (blue). ( C ) The dot plot denotes mean fluorescence intensity (MFI) ratio of cell surface marker CD25 compared to control as determined by flow cytometry. Values for individual volunteers (small dots) and mean values across all volunteers (large dots) are shown for each time point (color-coded). Error bars indicate means ± 1.96 × SEM.

Article Snippet: Human T cells were enriched by the positive CD3 cell selection Kit (EasySep Human CD3 Positive Selection Kit II, STEMCELL Technology) with a purity of 90 to 95%.

Techniques: Expressing, Gene Expression, Fluorescence, Marker, Control, Flow Cytometry

Journal: bioRxiv

Article Title: speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

doi: 10.1101/2021.08.23.457342

Figure Lengend Snippet: Left panel: First, an initial CAR architecture is chosen based on functionality and encoded in a DNA plasmid. Next, one or more modular domains of the CAR are swapped for alternative natural immune intracellular signaling domain in a semi-random combinatorial fashion, resulting in a plasmid library of CAR variants with diverse combinations. Middle panel: The plasmid library of CAR variants is genomically integrated at the TRAC locus of primary human T cells via CRISPR/Cas9 genome editing. This ensures that each cell expresses a single CAR variant, and simultaneously deletes the endogenous TCR. A pooled library of CAR T cells is co-cultured in the presence of tumor cells expressing cognate antigen. Right panel: The functional screening of the pooled CAR T cell library is performed by scRNA-seq and scCAR-seq, revealing the transcriptional phenotype. This dataset is used to select promising variants for in-depth characterization with functional assays.

Article Snippet: The T cells were isolated using the EasySep human T cell isolation kit (Stemcell) and activated with human T-activator CD3/CD28 Dynabeads (Gibco) at a bead:cell ratio of 1:1.

Techniques: Plasmid Preparation, CRISPR, Variant Assay, Cell Culture, Expressing, Functional Assay

Journal: bioRxiv

Article Title: speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

doi: 10.1101/2021.08.23.457342

Figure Lengend Snippet: A) Schematic representation of CAR architecture for the shuffling of signaling domains. The CAR variant library is derived from an initial second-generation CAR featuring the intracellular signaling domains of CD28 and CD3ζ; the scFv (4D5) is based on the variable domains of the clinical antibody trastuzumab (specificity to the antigen HER2). The entire CD28 signaling domain and a segment of the CD3ζ domain are exchanged with signaling domains from two pools: Domain A and B, which possess either zero or one ITAM, respectively. A truncated CD3ζ (tCD3Z) possessing two ITAMs is retained. B) Combinatorial shuffling of Domain A and Domain B intracellular signaling domains yields a library of 180 possible CAR variants. C) Schematic representation of the cloning strategy for domain shuffling. A CAR chassis vector was designed encoding a conserved 4D5 scFv sequence, a cloning cassette and a part of CD3ζ. The cloning cassette replaced the co-signaling domain and the remainder of CD3ζ with outward-facing recognition sequences of the Type IIS restriction enzyme AarI . A restriction digest yields unique overhangs that are compatible with the ligation of domain from pool A and one domain from pool B, in that order (5’ to 3’). D) Long-read deep sequencing was performed following cloning and assembly of the signaling domain shuffling library. Circos plot shows that 179/180 possible combinations are present and in generally balanced proportions. E) Schematic of the strategy for targeted genomic integration of CAR library into the TRAC locus of primary human T cells by CRISPR-Cas9 HDR. F) Flow cytometry of human primary T cells before transfection, after transfection and after selection for CAR expression. G) Similar to panel C, circos plots display deep sequencing of the sorted library of CAR T cells, revealing a reduced diversity with unbalanced combinations of signaling domains.

Article Snippet: The T cells were isolated using the EasySep human T cell isolation kit (Stemcell) and activated with human T-activator CD3/CD28 Dynabeads (Gibco) at a bead:cell ratio of 1:1.

Techniques: Variant Assay, Derivative Assay, Clone Assay, Plasmid Preparation, Sequencing, Ligation, CRISPR, Flow Cytometry, Transfection, Selection, Expressing

Journal: Cell reports

Article Title: DNA Damage Baseline Predicts Resilience to Space Radiation and Radiotherapy

doi: 10.1016/j.celrep.2020.108434

Figure Lengend Snippet: (A) Baseline number of foci per nucleus as a function of age in CD3 + lymphocytes extracted from finger prick collection in 339 healthy donors (***p < 0.0001, deviation from zero of the slope of linear regression). Four donors in this cohort were considered healthy but were in remission from cancer at the time of the finger prick (shown as red dots). See also . (B) Distribution of spontaneous foci per nucleus after bead-based extraction of CD3 + lymphocytes from finger prick samples (finger prick), Ficoll-based extraction of PBMCs from buffy coat samples (PBMC baseline), and a further freeze-thaw cycle and plating in the cell culture media (PBMC 0 Gy). ***p < 0.0001, one-way ANOVA, Tukey’s post hoc test for multiple comparisons among all three groups: 1–2, 2–3, and 1–3. Boxplots show median ± quartiles ± min/max values.

Article Snippet: EasySep CD3+ Whole Blood Selection Kit , StemCell Technologies , Cat# 18081.

Techniques: Extraction, Cell Culture

Journal: Cell reports

Article Title: DNA Damage Baseline Predicts Resilience to Space Radiation and Radiotherapy

doi: 10.1016/j.celrep.2020.108434

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EasySep CD3+ Whole Blood Selection Kit , StemCell Technologies , Cat# 18081.

Techniques: Recombinant, Red Blood Cell Lysis, Selection, Flow Cytometry, Multiplex Assay, Software