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: Molecular Therapy. Nucleic Acids

Article Title: Efficient and in situ correction of hemoglobin Constant Spring mutation by prime editing in human hematopoietic cells

doi: 10.1016/j.omtn.2024.102371

Figure Lengend Snippet: Prime editing efficiently creates the Hb CS mutations in human HSPCs for long-term transplantation (A) To determine whether prime-edited HSPCs can repopulate BM in vivo , prime-edited HSPCs were electroporated into CD34 + HSPCs and transplanted after 24 h into NCG-Kit-V831M mice by tail-vein injection. Bone marrow (BM) was harvested for further analysis at 16 weeks of transplantation. (B) Editing efficiency evaluation at the HBA2 and HBA1 loci in HSPCs before transplantation by NGS. (C) Abnormal α CS -globin chains detection by western blot. (D) Flow cytometry analysis in mouse BM 16 weeks after transplantation for determination of human engraftment. (E) Editing efficiency analysis in CD34 + human HSPCs derived from the BM of engrafted mice 16 weeks after transplantation. The error bars indicate the SDs of two healthy donors.

Article Snippet: Cells were harvested from BM; stained with antibodies against human surface markers human CD45 (BioLegend, 304008), human CD235a (BD Pharmingen, 559943), human CD34 (Elabscience, E-AB-F1143E), human CD19 (BioLegend, 392504), human CD33 (BioLegend, 366614); and analyzed by flow cytometry using a Gallios analyzer and FlowJo software (BD Biosciences).

Techniques: Transplantation Assay, In Vivo, Injection, Western Blot, Flow Cytometry, Derivative Assay

Journal: PLoS ONE

Article Title: Murine Mesenchymal Stem Cells Exhibit a Restricted Repertoire of Functional Chemokine Receptors: Comparison with Human

doi: 10.1371/journal.pone.0002934

Figure Lengend Snippet: A. Flow cytometry analysis of all primary murine MSC cultures (upper panel) and human MSC (lower panel) cultures showed they were CD34 and CD45 negative, and CD105 positive. CD molecule antibody staining is represented by the filled histogram; isotype control staining is represented by the green line. B. Murine MSCs (CD45 − , CD34 − , CD105 + ) incubated in osteogenic medium for 21 days stained positive for alkaline phosphatase activity (top right), whereas murine MSCs incubated in culture medium alone did not stain positive (top left). Murine MSCs incubated in adipogenic medium for 21 days showed fat droplets in the cells stained with Oil Red O (bottom right), whereas murine MSCs incubated in culture medium alone showed no positive staining (bottom left). The black bar represents 200 µm in the top panels and 100 µm in the bottom two panels.

Article Snippet: Antibodies used in this study were as follows: anti-human CCR1 (used at 1 in 100 dilution), CCR2 (1 in 200), CCR3 (1 in 100), CCR5 (1 in 200), CCR6 (1 in 200), CCR7 (1 in 200), CCR8 (1 in 20), CCR9 (1 in 20), CXCR1 (1 in 100), CXCR2 (1 in 100), CXCR3 (1 in 200), CXCR4 (1 in 50), CXCR5 (1 in 50), and CXCR6 (1 in 50), anti-mouse CCR6 (1 in 50), CCR9 (1 in 50), CXCR2 (1 in 50), CXCR3 (1 in 50), and CXCR6 (1 in 50) and anti-mouse CD105 (all from R&D Systems, UK), anti-human CCR4 (1 in 100), anti-mouse CCR3 (1 in 50), CCR5 (1 in 50), CXCR4 (1 in 50), CXCR5 (1 in 50) and CD45 PE/Cy5 (all from BD Pharmingen, UK), anti-human CCR10 and anti-mouse CCR4 (1 in 50) and CCR8 (1 in 50) (Abcam, UK), anti-human CD105 FITC (1 in 50), CD34 PE (1 in 100), CD45 PE/Cy5 (1 in 100) and anti-mouse CD34 PE (1 in 100) (Immunotools, Germany), and anti-mouse CCR7 (BioLegend, UK).

Techniques: Flow Cytometry, Staining, Control, Incubation, Activity Assay