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: Frontiers in Immunology

Article Title: Molecular determinants of STEC-HUS: from complement activation to microvascular thrombosis

doi: 10.3389/fimmu.2026.1749811

Figure Lengend Snippet: Prothrombotic effects of STEC-HUS serum are dependent on WPB exocytosis from endothelial cells. P-selectin (A) and vWF (B) expression on unstimulated HMEC-1 exposed to serum from patients with acute STEC-HUS (n = 5 for P-selectin experiments; n = 6 for vWF experiments), in the presence or in the absence of different complement inhibitors (sCR1, 150 µg/mL; factor B inhibitor, iptacopan,10 µM; eculizumab 100 µg/mL) or to a pool of control sera (normal human serum, NHS), run in parallel. Results are shown as pixel 2 /high-power field (HPF) of stained surface area. Data are mean ± SD. Circles represent single patients’ data. The addition of either sCR1, or iptacopan or eculizumab to patient’s serum significantly decrease both P-selectin and vWF expression induced by patient’s serum alone. *P < 0.05 vs STEC-HUS serum alone (ANOVA, followed by Tukey’s multiple comparisons test for data of P-selectin expression and by Holm-Šídák’s multiple comparisons test for data of vWF expression). (C) Endothelial surface area covered by thrombi on ADP-activated HMEC-1 exposed to serum from STEC-HUS patients collected during the acute phase of the disease and then perfused with whole blood. Before the experiments, HMEC-1 were left for 16 hours with medium added or not with the RalA inhibitor BQU57 (10 µM). Data are expressed as mean ± SD of percentages of serum-induced thrombus formation in respect to a pool of control sera (normal human serum, NHS), run in parallel in each experiment and set as 100% (n = 3 independent experiments). Circles indicate single patients’ data. Horizontal dashed lines indicate upper and lower limits of the normal range . *P < 0.05 (paired Student’s t test). (D) Representative confocal microscopy images (original magnification X200) of experiments of thrombus formation (green staining) relative to Figure 6C . Scale bar: 100 µm.

Article Snippet: Cells were washed again and treated with the following specific antibodies: FITC-conjugated rabbit anti-human C3c-complement (Dako, that recognizes C3c, part of C3 and C3b, 1:300 final dilution in Dapi 1 μg/mL); or rabbit anti-human complement C5b-9 complex (Calbiochem, 1:200 final dilution in PBS1X) followed by FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:50 final dilution in 1 μg/mL Dapi); or goat anti-human C4 (Abcam, 1:100 final dilution in PBS1X) followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:200 final dilution in Dapi 1 μg/mL); or FITC-conjugated anti-human IgG (Sigma Aldrich, 1:32 final dilution in 1 μg/mL Dapi); or mouse anti-human P-selectin (R&D System, 20 μg/mL final concentration in PBS1X), followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:60 final dilution in 1 μg/mL Dapi); or rabbit anti-human vWF (Dako, 10 μg/mL final concentration in PBS1X), followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:50 final dilution in 1 μg/mL Dapi).

Techniques: Expressing, Control, Staining, Confocal Microscopy

Journal: Cancer Research Communications

Article Title: Phase I First-in-Human Study of TRK-950, an IgG1 Antibody Specific to CAPRIN-1, in Patients with Advanced Solid Tumors

doi: 10.1158/2767-9764.CRC-25-0123

Figure Lengend Snippet: Graphs depicting the density of macrophage subsets pre- and posttreatment with TRK-950. Tumor specimens obtained with core-needle biopsies at the screening and C1D22 time points were subjected to multi-immunofluorescence to detect macrophage infiltrates. Quantitative analyses were performed on digital images of CD68 and CD163 staining. A, total CD68 + cells; ( B ) CD68 + /CD163 − subsets; ( C ) CD68 + /CD163 + subsets. Error bars indicate SD. C1D22, cycle 1 day 22; SCR, screening.

Article Snippet: After antigen retrieval (36 minutes at 95°C, pH 8.4), 4-μm-thick sections of tumor specimens obtained from core-needle biopsies at the screening and C1D22 time points were stained with mouse monoclonal anti-human CD68 (IgG2b, clone 298807, R&D Systems) and mouse monoclonal anti-human CD163 (IgG1, clone 10D6, Leica Biosystems) and then incubated with AF647 goat anti-mouse IgG2b and AF488 goat anti-mouse IgG1 (both from Invitrogen).

Techniques: Immunofluorescence, Staining

Journal: iScience

Article Title: βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

doi: 10.1016/j.isci.2022.103758

Figure Lengend Snippet: rβig-h3-structured collagen I fibers modulate the phenotype and function of macrophages (A) Representative photographs of immunofluorescence staining of pancreata from three-months-old KC mice stained for βig-h3 (green) and CD206 (red). Nuclear counterstaining in DAPI (blue). (B) Representative photographs of βig-h3, F4/80, CD206, and Arg1 staining on serial section of pancreata from three-months-old KC mice. (C) FACS analysis of BMMCs cultured alone or on collagen I structured in the absence (BMMCs + Col I) or presence of rβig-h3 (BMMCs + Col I rβig-h3 ). Representative data of three independent experiments with three in vitro replicates per group are shown. ∗p<0.05, ∗∗p<0.01. Error bars 50 μm, 25 μm.

Article Snippet: The following monoclonal Abs were used in flow cytometry: anti-F4/80 Pe-Cy7 (clone BM8-Biolegend, 123114) or PerCP Cy5.5 (clone BM8-Biolegend, 123128), anti-MHCII PE (clone M5/114.15.2- eBioscience, 12-5321-81), anti-CD206 BV650 (clone C068C2-Biolegend, 141723), anti-CD86 BV605 (clone GL-1- Biolegend, 105029), anti-CD80 APC-Cy7 (clone 16-10A1-Biolegend, 104729), anti-CSFRI AF488 (clone AFS98- Biolegend, 135511), anti-INOS PE (clone CXNFT, Life Techno, 12-5920-82), anti-Arginase1 APC (RD, IC5868A), anti-TGF-β B421 (clone TW7-16B4-Biolegend, 141408) and anti-TNFα BV605 (clone MP6-XT22-Biolegend, 506329).

Techniques: Immunofluorescence, Staining, Cell Culture, In Vitro

Journal: iScience

Article Title: βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

doi: 10.1016/j.isci.2022.103758

Figure Lengend Snippet: βig-h3 Ab depletion in vivo reprograms macrophage phenotype in the tumor microenvironment (A) Experimental setting. (B) Tumor weight of mice treated in A. (C-F) FACS analysis of the percentages of CD45 + (C), F4/80 + CD45 + (D), CD206 + F4/80 + (E), and Arg1 + F4/80 + (F) cells collected in A. ∗p<0.05, ∗∗p<0.01. Representative of two independent experiments with fivemice per group.

Article Snippet: The following monoclonal Abs were used in flow cytometry: anti-F4/80 Pe-Cy7 (clone BM8-Biolegend, 123114) or PerCP Cy5.5 (clone BM8-Biolegend, 123128), anti-MHCII PE (clone M5/114.15.2- eBioscience, 12-5321-81), anti-CD206 BV650 (clone C068C2-Biolegend, 141723), anti-CD86 BV605 (clone GL-1- Biolegend, 105029), anti-CD80 APC-Cy7 (clone 16-10A1-Biolegend, 104729), anti-CSFRI AF488 (clone AFS98- Biolegend, 135511), anti-INOS PE (clone CXNFT, Life Techno, 12-5920-82), anti-Arginase1 APC (RD, IC5868A), anti-TGF-β B421 (clone TW7-16B4-Biolegend, 141408) and anti-TNFα BV605 (clone MP6-XT22-Biolegend, 506329).

Techniques: In Vivo

Journal: iScience

Article Title: βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

doi: 10.1016/j.isci.2022.103758

Figure Lengend Snippet:

Article Snippet: The following monoclonal Abs were used in flow cytometry: anti-F4/80 Pe-Cy7 (clone BM8-Biolegend, 123114) or PerCP Cy5.5 (clone BM8-Biolegend, 123128), anti-MHCII PE (clone M5/114.15.2- eBioscience, 12-5321-81), anti-CD206 BV650 (clone C068C2-Biolegend, 141723), anti-CD86 BV605 (clone GL-1- Biolegend, 105029), anti-CD80 APC-Cy7 (clone 16-10A1-Biolegend, 104729), anti-CSFRI AF488 (clone AFS98- Biolegend, 135511), anti-INOS PE (clone CXNFT, Life Techno, 12-5920-82), anti-Arginase1 APC (RD, IC5868A), anti-TGF-β B421 (clone TW7-16B4-Biolegend, 141408) and anti-TNFα BV605 (clone MP6-XT22-Biolegend, 506329).

Techniques: Recombinant, Modification, Plasmid Preparation, Activation Assay, Concentration Assay, Software