Journal: Frontiers in Immunology

Article Title: Structural and functional characterization of IgG- and non-IgG-based T-cell-engaging bispecific antibodies

doi: 10.3389/fimmu.2024.1376096

Figure Lengend Snippet: Granzyme/perforin activity and cytokine production by anti-EGFR/CD3 DVD-Ig and BiTE (A) Representative anti-granzyme B immunoblots using the WCL from co-culture of tumor cells (CaOV3 or MDA-MB-231) and T cells exposed to DVD-Ig or BiTE for 2 days. First lane represents tumors cell only. Actin was used as loading control. (B, C) Granzyme B and perforin measurement in co-culture supernatants was performed using ELISA kit. This experiment was performed in biological triplicates, and data is presented as mean ± SD. **p < 0.01 (D) Detection of human cytokines in co-culture supernatant of CaOV3 was performed using array kit. Green boxes (and associated green cytokine coordinates) represent increased dot intensity under BiTE treatment condition; blue boxes (and associated blue cytokine coordinates) represent increased array dot intensity under both DVD-Ig and BiTE treatment conditions.

Article Snippet: Quantitative measurement of Granzyme B was performed using Human Granzyme B Quantikine ELISA Kit (cat# DGZB00, R and D Systems, Minneapolis, MN) and perforin was measured using Perforin (PRF1) Human ELISA Kit (cat# ab46068, Abcam, Waltham, MA).

Techniques: Activity Assay, Western Blot, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Translational Cancer Research

Article Title: Increase in GPIHBP1 expression in advanced stage colorectal cancer indicates poor immune surveillance

doi: 10.21037/tcr-23-1766

Figure Lengend Snippet: CD8 + or CD56 + cells infiltrated in CRC. (A) IHC staining results of CD8, CD56, and GZMB (scale bar: 50 µm). (B) Comparisons of CD8, CD56, or GZMB presence in patients with different stages of CRC. Correlational study of GPIHBP1 presence in cancer foci (C) or stroma (D) with infiltrations of CD8 + , CD56 + , and GZMB + cells. ***, P<0.001. GPIHBP1, GPI-anchored high-density lipoprotein-binding protein 1; GPI, glycosylphosphatidylinositol; CRC, colorectal cancer; IHC, immunohistochemistry; GZMB, granzyme B.

Article Snippet: The following antibodies were used for immunohistochemistry: GPIHBP1 antibody from Abcam (ab224725; Cambridge, UK); F4/80 (70076S) and CD56 (99746S) from Cell Signalling Technology (Danvers, MA, USA); CD68 (macrophage marker; 66231-2-IG), CD8 (66868-1-IG), arginase 1 (Arg1; M2 macrophage marker; 66129-1-IG) , and S100A8 (MDSCs marker, 15792-1-AP) ( ) from Proteintech (Wuhan, China); and antibody (MA5-17139) against inducible nitric oxide synthase (iNOS; M1 macrophage marker) ( ) from ThermoFisher Scientific; granzyme B (GZMB) antibody (NBP2-76414) from Novus Biologicals (Littleton, CO, USA).

Techniques: Immunohistochemistry, Binding Assay

Journal: bioRxiv

Article Title: Rapid receptor internalization potentiates CD7-targeted lipid nanoparticles for efficient mRNA delivery to T cells and in vivo CAR T-cell engineering

doi: 10.64898/2026.01.23.701374

Figure Lengend Snippet: (A) Experimental scheme for evaluating aCD7/tLNP-mediated CAR T-cell generation in vivo and their cytolytic activity against target cells. (B) CAR expression in CD4⁺ and CD8⁺ T cells in the spleen and levels of remaining CD20⁺ B cells 24-hours after treatment with aCD7/tLNP-ZsGreen (control) or aCD7/tLNP-CAR mRNA (2.5 μg per mouse), analyzed by flow cytometry. (C and D) Quantification of CAR expression in CD4⁺ and CD8⁺ T cells. (E) Quantification of CD20⁺ B cells in the spleen. (F) Representative immunohistochemistry images showing CD20⁺ B cells (upper panel) and Granzyme B (lower panel) in spleens from aCD7/tLNP-ZsGreen- or aCD7/tLNP-CAR-treated humanized mice. (G) Quantification of CD20⁺ B-cell density shown in (F). n = 6 mice for (B-E); n = 3 mice for (F and G). Data are shown as mean ± SD. Scale bar, 100 μm.

Article Snippet: Tissue sections were blocked with 10% goat serum for 15 minutes and incubated overnight at 4 °C with primary antibodies against CD20 (SP32, Abcam, Cat# ab64088; 1:100) and Granzyme B (D6E9W, Cell Signaling Technology, Cat. 46890; 1:100).

Techniques: In Vivo, Activity Assay, Expressing, Control, Flow Cytometry, Immunohistochemistry

Journal: Cancer Cell International

Article Title: Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

doi: 10.1186/s12935-024-03557-1

Figure Lengend Snippet: ADSCs-Exos IFN−γ+TNF−α promoted immune escape of TNBC cells by transporting UCHL1. ADSCs were treated with 10 ng/mL IFN-γ and TNF-α, and transfected with si-NC or si-UCHL1. The exosomes were extracted from the supernatant. TNBC cells were treated with 10 μg/mL of the ADSCs-CM and different exosomes for 24 h, and then co-cultured with THP-1 cells for 48 h or with T cells for 16 h. A UCHL1 protein levels in TNBC cells in the indicated groups. B Viability of TNBC cells in the indicated groups. C Representative images showing Ki-67 expression in TNBC cells in the indicated groups. Scale bar = 100/25 μm. D Representative images showing migration of TNBC cells in the indicated groups. Scale bar = 100 μm. * P < 0.05 vs . MDA-MB-231 or MDA-MB-468. # P < 0.05 vs . Exos + MDA-MB-231 or Exos + MDA-MB-468. & P < 0.05 vs . Exos IFN−γ+TNF−α+con + MDA-MB-231 or Exos IFN−γ+TNF−α+con + MDA-MB-468. E The proportion of CD68 + CD206 + THP-1 cells in the indicated groups. F Arg-1, CD206, TNF-α, and iNOS expression in THP-1 cells. * P < 0.05 vs . THP-1. # P < 0.05 vs . MDA-MB-231 + THP-1 or MDA-MB-468 + THP-1. & P < 0.05 vs . Exos + THP-1. @ P < 0.05 vs . Exos IFN−γ+TNF−α+con + THP-1. G The proportion of CD3 + CD8 + T cells in the indicated groups. H Perforin and GZMB levels in T cells. * P < 0.05 vs . T cells. # P < 0.05 vs . MDA-MB-231 + T cells or MDA-MB-468 + T cells. & P < 0.05 vs . Exos + T cells. @ P < 0.05 vs . Exos IFN−γ+TNF−α+con + T cells

Article Snippet: Granular enzyme B (GZMB) and perforin levels were analyzed using specific GZMB (CSB-E08718h, CUSABIO, China) and perforin (CSB-E09313h, CUSABIO, China) ELISA kits according to the instructions.

Techniques: Transfection, Cell Culture, Expressing, Migration

Journal: Cancer Cell International

Article Title: Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

doi: 10.1186/s12935-024-03557-1

Figure Lengend Snippet: ADSCs-Exos IFN−γ+TNF−α promoted tumor growth in vivo. BALB/c mice were subcutaneously injected with 4T1 cells (2 × 10 6 ) and treated with 100 μg of the different exosomes every 3 days. A Tumor weight and volume in the indicated groups. B In-situ expression of Ki-67 in the tumor tissues of indicated groups. Scale bar = 100/25 μm. C The F4/80 + /CD206 + cells in the tumor tissues of indicated groups. Scale bar = 25 μm. D The proportion of CD3 + CD8 + T cell in the tumor tissues of indicated groups. E Perforin and GZMB levels in the tumor tissues of indicated groups. F and G CD8 + /GZMB + cells and CD8 + /perforin + cells in the tumor tissues of indicated groups. (H) HDAC6, UCHL1, PD-L1, p-STAT3 and STAT3 levels in the tumors of indicated groups. * P < 0.05 vs . 4T1. # P < 0.05 vs . Exos + 4T1. & P < 0.05 vs . Exos IFN−γ+TNF−α+con + 4T1

Article Snippet: Granular enzyme B (GZMB) and perforin levels were analyzed using specific GZMB (CSB-E08718h, CUSABIO, China) and perforin (CSB-E09313h, CUSABIO, China) ELISA kits according to the instructions.

Techniques: In Vivo, Injection, In Situ, Expressing