Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) Expression by qRT-PCR analysis of NPM-ALK mRNA in the transformed CD4+ T cells (CD4-NPM/ALK+ lane shows the mean from 9 independent cell lines) and 3 positive control NPM-ALK+ ALCL cell lines: KARPAS-299, SU-DHL-1, and COST.CD4+ T cells preactivated with CD3/CD28 antibody-coated beads were used as negative controls (preactivated CD4). MLNS1 was used as an internal control. Relative NPM-ALK expression was expressed as the 2–ΔCt relative to MLN51. Data represent mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.001; unpaired 2-tailed Student’s t test with Welch’s correction. (B) Suppressive effect of the ALK inhibitor crizotinib (500 nmol/L) on ALK and STAT3 phosphorylation in transformed CD4+ T cells and control NPM-ALK+ KARPAS-299 cells. The GAPDH protein served as an internal control to ensure equal loading. Blots from 1 representative experiment are shown.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Expressing, Quantitative RT-PCR, Transformation Assay, Positive Control, Control, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) We used publicly available methylation data sets (30) generated from different developmental T cell stages (multipotent ETPs [CD34+/CD1a–; n = 2]; T cell–committed progenitors [CD34+/CD1a+; n = 1]; pre-TCR T cells [n = 2]; TCR-expressing CD4+/CD8+ double-positive T cells [DP-TCR+, n = 2]; and single positive [SP] CD8+ or CD4+ cells [SP-CD4+; n = 2 or SP-CD8+; n = 2]) to identify a cluster of 510 DMRs available to discriminate each different stage of T cell differentiation in the thymus. (B) Hierarchical clustering dendrogram using a cluster of 510 DMRs revealed that NPM-ALK–transformed CD4+ T cells were distant to the healthy CD4+ lymphocyte profile and clustered with primary NPM-ALK+ ALCL biopsies. Heatmaps also showed a similarity of NPM-ALK+ cells (NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL) with CD34+/CD1a– cells corresponding to the ETP stage.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Methylation, Generated, Expressing, Cell Differentiation, Transformation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: Two hundred and forty-three among the 510 DMRs within NPM-ALK–transformed CD4+ T cells (CD4+/NPM-ALK+), primary patient–derived NPM-ALK+ ALCL cells, and CD34+/CD1a– cells corresponding to the ETP stage. Venn diagram reveals that the ETP and both the NPM-ALK+ tumor cell entities (CD4+/NPM-ALK+ lymphoma cells and primary NPM-ALK+ ALCLs) share 38 DMRs with similar expression patterns.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Transformation Assay, Derivative Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: mRNA expression profiles from several cell populations isolated ex vivo from the neonatal human thymus defining in vivo maturation stages, multipotent ETPs (CD34+/CD1a–/CD7–; n = 3), late thymic precursor (CD34+/CD1a–/CD7+; n = 3), T cell–committed progenitors (CD34+/CD1a–/CD7+; n = 3), CD3–/CD4+ immature single-positive (ISP) (ISP-CD4+, n = 4), CD4+/CD8+ double-positive TCR– cells (DP-TCR–; n = 3), and TCR-expressing CD4+/CD8+ double-positive T cells (DP-TCR+, n = 3) were integrated with our previous findings from the gene expression array data of 55 primary NPM-ALK+ ALCL samples (NPM-ALK+ ALCL) (35) and RNA-Seq data from the NPM-ALK–transformed CD4+ T cells (CD4+/NPM-ALK+; n = 9). NPM-ALK CD4+/NPM-ALK+ cells were distant to the healthy CD4+ lymphocyte but close to NPM-ALK+ ALCL. Moreover, NPM-ALK+ cells (NPM-ALK–transformed CD4+ T cells and primary patient–derived NPM-ALK+ ALCL) showed a similarity with the ETP stage.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Expressing, Isolation, Ex Vivo, In Vivo, Gene Expression, RNA Sequencing, Transformation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: ALK-transformed mature T lymphocytes restore early thymus progenitor features

doi: 10.1172/JCI134990

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of ALK and EPAS1 mRNA was performed in primary patient–derived NPM-ALK+ ALCL cells (n = 29). Relative mRNA expression was expressed as the 2–ΔΔCt relative to MLN51, S5, ABL, GAPDH, S14, or RPL0 genes for normalization and compared with preactivated healthy CD4+ lymphocytes (n = 5). Data represent mean ± SEM. ***P < 0.001; unpaired 2-tailed Student’s t test. (B) Quantitative RT-PCR analysis of EPAS1 mRNA expression in NPM-ALK+ lymphoma cell lines, COST, KARPAS-299 (KARPAS), and SU-DHL1, treated for 72 hours or not (PBS) with crizotinib or transfected with either an irrelevant siRNA as the negative control (si-CTL) or a siRNA targeting ALK mRNA (si-ALK) or STAT3 (si-STAT3). Relative EPAS1 mRNA expression was expressed as the 2–ΔCt relative to MLN51. Data represent mean ± SEM from 3 independent experiments. *P < 0.05, ***P < 0.001; unpaired 2-tailed Student’s t test with Welch’s correction. (C) Western blotting analysis of HIF2A expression (top) in NPM-ALK+ COST, KARPAS-299, and SU-DHL1 cells treated with crizotinib (crizo) or not (PBS), transfected by si-CTL, si-ALK, or si-STAT3. The GAPDH protein (bottom) served as an internal control to ensure equal loading. Results from 1 representative experiment are shown.

Article Snippet: The human NPM-ALK + ALCL cell line COST was established in the laboratory ( 40 ) and KARPAS-299 and SU-DHL-1 NPM-ALK + ALCL cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Quantitative RT-PCR, Derivative Assay, Expressing, Transfection, Negative Control, Western Blot, Control

Journal: Nature medicine

Article Title: Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma.

doi: 10.1038/s41591-018-0262-9

Figure Lengend Snippet: Fig. 1 | WASP and WIP are selectively down-regulated in ALCL. a, Gene-expression profiling analysis of ALK, WASP, WIP, N-WASP, CDC42 and TNFRS8 (CD30) on different cases of human T cell lymphomas: AITL, n = 40; PTCL-NOS, n = 74; ALK– ALCL, n = 24, ALK+ ALCL, n = 30. The boxes represent the first and third quartiles and the line represents the median. The whiskers represent the upper and lower limits of the range (ALK+ ALCL versus AITL, ****P = 5.85 × 10−6; ALK+ ALCL versus PTCL-NOS, ****P = 6.08 × 10−12; ALK+ ALCL versus ALK– ALCL, **P = 0.0075; significance was determined by unpaired, two-tailed Student’s t-test). TNFRS8 (CD30) is strongly expressed in ALK– and ALK+ ALCL but not in other TCL subtypes. b, Representative H&E staining and immunohistochemistry stainings performed with the indicated antibody on human T cell lymphoma subtypes. The number of human T cell lymphoma samples analyzed is reported in Fig. 1c. WASP antibody was validated in formalin-fixed samples on samples with inducible WASP expression (Supplementary Fig. 7f). WIP antibody cross reacts with mouse WIP and was validated on WIP knockout cells (Supplementary Fig. 1b). Scale bar, 100 μm. Insets: high-magnification images. c, WASP and WIP expression in human T cell lymphoma subtypes. AITL, n = 20; PTCL-NOS, n = 20; ALK– ALCL, n = 29; ALK+ ALCL, n = 43 for WASP and n = 31 for WIP; other TCL are natural killer (NK)/T cell lymphoma, nasal-type, n = 3 and hepatosplenic γδ T cell lymphoma, n = 3. The number of patient samples is indicated for each lymphoma subtype. WASP and WIP expressions were quantified by immunostaining. Normal expression, expression equal to surrounding reactive T cells; low expression, decreased expression compared to surrounding reactive T cells; and no expression, absence of expression in lymphoma cells.

Article Snippet: Human ALK+ ALCL cell lines (TS, SU-DHL1, JB6, Karpas-299, DEL, SUP-M2 and L82) and ALK− cell lines (MAC-1, FePD and Jurkat) ALCL cell lines were obtained from DSMZ (German collection of Microorganisms and Cell Cultures).

Techniques: Gene Expression, Two Tailed Test, Staining, Immunohistochemistry, Expressing, Knock-Out, Immunostaining

Journal: Haematologica

Article Title: Histone acetylation and DNA demethylation of T cells result in an anaplastic large cell lymphoma-like phenotype

doi: 10.3324/haematol.2011.054619

Figure Lengend Snippet: Expression of RYBP, TCF1, LEF1 and GATA3 in 15 cases of primary anaplastic large cell lymphoma (ALCL).

Article Snippet: Differentially expressed genes in T-cell and anaplastic large cell lymphoma cell lines To establish a gene expression signature which characteristically describes the cell type-specific differences between the T-cell lines (CCRF-CEM, Jurkat, MOLT-3 and MOLT-4) and the ALCL cell lines (FE-PD, JB6, Karpas 299 and SU-DHL-1) we compared their gene expression profiles (untreated cells).

Techniques: Expressing

Journal: Haematologica

Article Title: Histone acetylation and DNA demethylation of T cells result in an anaplastic large cell lymphoma-like phenotype

doi: 10.3324/haematol.2011.054619

Figure Lengend Snippet: The ID2 promoter region is demethylated in 5-aza-dC/TSA-treated T cells and in tumor cells of primary ALCL. The methylation status of the ID2 promoter was analyzed by bisulfite sequencing. The sequenced region in the ID2 promoter contained six CpG dinucleotides. Clones were sequenced from 5-aza-dC/TSA-treated and untreated Jurkat and MOLT-3 cells and four primary ALCL cases. Every row represents a single PCR clone.

Article Snippet: Differentially expressed genes in T-cell and anaplastic large cell lymphoma cell lines To establish a gene expression signature which characteristically describes the cell type-specific differences between the T-cell lines (CCRF-CEM, Jurkat, MOLT-3 and MOLT-4) and the ALCL cell lines (FE-PD, JB6, Karpas 299 and SU-DHL-1) we compared their gene expression profiles (untreated cells).

Techniques: Methylation, Methylation Sequencing, Clone Assay

Journal: Haematologica

Article Title: Histone acetylation and DNA demethylation of T cells result in an anaplastic large cell lymphoma-like phenotype

doi: 10.3324/haematol.2011.054619

Figure Lengend Snippet: (A) Promoter H3K27 trimethylation status of T-cell signaling pathway genes and T-cell transcription factor genes after ChIP and quantification by real-time DNA-PCR in T- and ALCL cell lines. β-ACTIN was chosen as the endogenous reference and the input was calculated relative to the ChIP input control. (B) Immunohistochemistry for GATA3 (original magnification, 400×), LEF1 and TCF1 (original magnification 200×) in primary ALCL cases. All three T-cell transcription factors were strongly expressed in the nuclei of the reactive T cells whereas the tumor cells displayed no or only faint expression.

Article Snippet: Differentially expressed genes in T-cell and anaplastic large cell lymphoma cell lines To establish a gene expression signature which characteristically describes the cell type-specific differences between the T-cell lines (CCRF-CEM, Jurkat, MOLT-3 and MOLT-4) and the ALCL cell lines (FE-PD, JB6, Karpas 299 and SU-DHL-1) we compared their gene expression profiles (untreated cells).

Techniques: Control, Immunohistochemistry, Expressing