Journal: Frontiers in Immunology

Article Title: Exploration of T cell immune responses by expression of a dominant-negative SHP1 and SHP2

doi: 10.3389/fimmu.2023.1119350

Figure Lengend Snippet: dSHP2 but not dSHP1 alleviates PD1-mediated suppression of CAR T activity. (A) Schematic of CAR constructs. Retroviral constructs were designed consisting of an anti-GD2 CAR with a CD28-CD3ζ endodomain linked to RQR8 as a marker of transduction and including PD1 and/or dSHP1 or dSHP2 separated by viral 2A sequences (shown in red). (B) Effect of dSHP1 (left panel) and dSHP2 (right panel) on PD1/PDL1-mediated suppression of CAR cytotoxicity. CAR T cells were co-cultured with 2x10 5 SupT1 cells lacking antigen expression (SupT1-NT) or engineered to express GD2 alone (SupT1-GD2) or in the presence of PDL1 (SupT1-GD2-PDL1) at an effector:target (E:T) ratio of 1:4 for 72 hours. Surviving target cells were enumerated and normalized to the respective co-cultures with non-transduced T cells (100%). (C, D) Effect of dSHP1 (left panel) and dSHP2 (right panel) on PD1/PDL1-mediated suppression of IFNγ and IL2 secretion. Supernatants from (B) were harvested and cytokine secretion measured by ELISA. (E) Effect of dSHP1 (left panel) and dSHP2 (right panel) on PD1/PDL1-mediated suppression of proliferation. CAR T cells were labelled with Cell Trace Violet and incubated with 2x10 5 of the indicated SupT1 target cells at an E:T ratio of 1:2 for 4 days and CAR T cells enumerated. Bars represent the mean of 5-6 biologically independent replicates. Statistical significance was measured by two-way ANOVA. Significance is defined as: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Antibodies used were: CCR7-PE (Miltenyi Biotec; 130-119-583), CD19–APC Cy7 (BioLegend; 302218), CD25-BV421 (Biolegend; 356114), CD27-VioBright515 (Miltenyi Biotec; 130-120-028), CD3–PE Cy7 (BioLegend; 344816), CD3-VioGreen (Miltenyi Biotec; 130-113-142), CD45RA-APCVio770 (Miltenyi Biotec; 130-117-747), CD69-FITC (Biolegend; 310904), CD8-APC-Cy7 (Biolegend; 301016), CD8-Vioblue (Miltenyi Biotec; 130-110-683), CD95-PEVio770 (Miltenyi Biotec 130-113-006), HA–AF488 (Biolegend; 901509), KLRG1-APC-Vio77 (Miltenyi Biotec; 130-120-423), LAG3-VioBright515 (Miltenyi Biotec; 130-120-012), PD1-PE (Miltenyi Biotec; 130-120-382), CD34 (RQR8)-PE (R&D Systems; FAB7227P), CD34 (RQR8)-APC (R&D Systems; FAB7227A), SYTOXTM AADvancedTM Dead Cell Stain (ThermoFisher; S10274), TIM3-PEVio770 (Miltenyi Biotec; 130-121-334), GD2-APC (Biolegend; 357306), HVEM-PE (Biolegend; 318806), PDL1-PE (Biolegend; 329706), CD48-PE (Biolegend; 336708), CD80-FITC (Biolegend; 305206), CD155-PE (Biolegend; 337610).

Techniques: Activity Assay, Construct, Retroviral, Marker, Transduction, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cells

Article Title: IMG-A1: A Novel Immortalized Granulosa Cell Line for Investigating FSH-Dependent Folliculogenesis and Ovarian Pathophysiology

doi: 10.3390/cells14241940

Figure Lengend Snippet: Basic Characterization of the IMG-A1 Granulosa Cell Line. ( a , b ) Phase-contrast micrographs of the IMG-A1 cell culture. ( c ) Staining for senescence-associated β-galactosidase activity in passage 18 cells. ( d – f ) Immunocytochemical staining for the proliferation marker Ki67, showing a phase-contrast image ( d ), Ki67 immunofluorescence ( e ), and a merged image with DAPI nuclear counterstain ( f ). ( g , h ) Representative metaphase spreads showing a normal diploid karyotype (40 chromosomes) ( g ) and a near-tetraploid karyotype (78 chromosomes) from the IMG-A1 line ( h ). ( i – k ) Ploidy analysis by flow cytometry. Control blood cells ( i ) and primary granulosa cells ( j ) show characteristic diploid (2n) and tetraploid (4n, G2/M phase) peaks. The IMG-A1 culture ( k ) displays a predominantly near-tetraploid and near-octaploid cell population. ( l ) Flow cytometry analysis confirming the homogeneity of the IMG-A1 culture (passages 10 and 40) based on staining for the stromal marker CD29 and the absence of hematopoietic markers CD45, CD34, and the fibroblast marker CD90. Quadrant gates were set based on unstained controls. Scale bars = 100 µm.

Article Snippet: PE CD34 Rat mAb [RAM34] , Elabscience Biotechnology Co., Ltd., Wuhan, China , E-AB-F1284D.

Techniques: Cell Culture, Staining, Activity Assay, Marker, Immunofluorescence, Flow Cytometry, Control

Journal: Stroke

Article Title: Trametinib decreased intracerebral hemorrhages and endothelial-to-mesenchymal transition in KRAS G12V -induced brain arteriovenous malformations in mice

doi: 10.1161/STROKEAHA.125.052418

Figure Lengend Snippet: (A) Immunostaining images of mesenchymal markers (N-cad, Slug, and CD44), endothelial marker (VE-Cad), (B) proliferation (Ki-67), and angiogenesis markers (CD34 and pVEGFR2), and the respective intensity in CD31+ intact and bAVM vessels. n=ROIs (dots) in 2–3 (Veh) or 3–6 (TM) mice (1mg/kg and 2mg/kg combined), Mice were sacrificed at 10 weeks after treatments, Data expressed as mean value of dots ± SEM, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 vs KRAS G12V mice treated with vehicle. Student’s t -test. Scale bar = 100μm. Veh, Vehicle; TM, Trametinib.

Article Snippet: The fixed samples were incubated with primary antibodies: CD31 (AF3628, R&D Systems, Minneapolis, MN), Ter119 (MAB1125, R&D systems), VE-Cad (361900, Thermo Fisher Scientific, Waltham, MA), p-ERK1/2 (9101S), N-Cad (13116S), Slug (9585S), CD44 (37259S), and Ki-67 (9129S) from Cell Signaling Technology, CD34 (NB6001071, Novus Biologicals, Centennial, CO), or pVEGFR2 (SAB4504567, Sigma-Aldrich, St. Louis, MO), followed by secondary antibodies: 488-conjugated donkey anti-goat (705-545-147) or anti-rabbit IgG (711-545-152), Rhodamine Red-X-AffiniPure donkey anti-mouse (715-295-151), anti-rabbit (711-295-152), or anti-goat IgG (705-295-147), 647-conjugated donkey anti-rabbit (711-605-152), anti-mouse (715-605-150), or anti-rat IgG (712-605-153).

Techniques: Immunostaining, Marker