Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: Ascites blocks NK cell degranulation but not TRAIL-dependent HPMC apoptosis (A and B) TAL were pre-cultured in RPMI/5% AB media or 100% ascites pool (+/− α-CD3 Ab stimulation) prior to co-culture with HPMC. (A) Apoptosis of HPMC is shown as percentage of annexin V+ cells after gating on CD45 − cells (n = 7 patients). (B) Degranulating NK cells in response to HPMC were measured via flow cytometry and depicted as percentage of CD335+/CD107a+ cells (n = 6 patients). (C and D) TAL were pretreated with α-TRAIL blocking Ab prior to HPMC co-culture. An irrelevant mouse IgG was included as control. Experiments were conducted with TAL cultured in RPMI/5% AB media (n = 11 patients) (C) and 100% ascites pool (n = 5 patients) (D). The amount of Annexin V+ HPMC was determined as described previously. (E) Schematic representation of co-culture experiments applying T cell CM for NK cell activation. CM were collected from CD3 + , CD3+/CD4+, and CD3+/CD8+ T cell subsets cultured in media +/− α-CD3 Ab stimulation. Purified NK cells were then stimulated with T cell CM prior to HPMC co-cultures. (F) The amount of apoptotic HPMC induced by NK cells activated with CM of CD3 + T cells was compared with CM of CD3+/CD4+ and CD3+/CD8+ T cells (n = 4 matched pairs of different patients). (G) Analysis of TRAIL signaling was performed by applying an α-TRAIL blocking Ab to NK cells stimulated with CD3 + T cell CM (α-CD3 Ab activated) prior to the co-culture with HPMC (n = 5 patients). An irrelevant mouse IgG was included as control. The mean is shown by horizontal bars or boxes; vertical error bars represent the standard deviation in A–D, F, and G. ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; determined by paired t test and Benjamini-Hochberg adjustment (ns, not significant).

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Blocking Assay, Control, Activation Assay, Purification, Standard Deviation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet:

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Control, Recombinant, Polymer, Software

Journal: PLoS ONE

Article Title: CXCL10/IP-10 Neutralization Can Ameliorate Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Rats

doi: 10.1371/journal.pone.0169100

Figure Lengend Snippet: Monoclonal antibodies used for the analysis of inflammatory cells.

Article Snippet: CD8 , PE , Miltenyi Biotec , REA222 , 130-103-322 , 1 in 100.

Techniques: Bioprocessing, Control

Journal: Translational Lung Cancer Research

Article Title: Targeting complement C5a to improve radiotherapy sensitivity in non-small cell lung cancer

doi: 10.21037/tlcr-23-258

Figure Lengend Snippet: Infiltration of recruited CD8 + T cells increased after treatment of RT. (A) Mice were inoculated subcutaneously with LLC tumor cells and treated with 8 Gy of local RT daily for 3 times. Tumor tissues were collected at the indicated time points for subsequent analysis. (B) Tumor tissues in RT and control groups. (C) Immunofluorescence of tumors in RT and control groups. Sections were stained with an antibody recognizing CD8 (green) and DAPI (blue). Scale bars represent 50 µm. Representative images are shown. (D) Flow cytometry assays displayed that infiltration of CD8 + T cells were upregulated after RT. RT, radiotherapy; DAPI, 4',6-diamidino-2-phenylindole; FSC-A, forward scatter-A; FITC-A, fluorescein isothiocyanate-A; LLC, Lewis lung carcinoma; con, control.

Article Snippet: After processes of dewaxing, rehydration, and antigen retrieval with ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer (pH 8.0) (Servicebio, Wuhan, China; G1206), the slides were then blocked with blocking solution [PBS with 10% fetal calf serum (FCS) and 0.1% TritonX] at room temperature for 1 hour, then washed in PBS and incubated overnight at 4 oC in the appropriate primary anti-C5aR antibody (Abcam, 1:500) and anti-CD8a antibody (Boster, 1:50) diluted in blocking solution.

Techniques: Control, Immunofluorescence, Staining, Flow Cytometry

Journal: Translational Lung Cancer Research

Article Title: Targeting complement C5a to improve radiotherapy sensitivity in non-small cell lung cancer

doi: 10.21037/tlcr-23-258

Figure Lengend Snippet: RT upregulated C5aR1 expression in RT-recruited CD8 + T cells. (A) CD8 + T cells were sorted by magnetic beads for RNA-seq. (B) RNA-seq analysis was displayed and the differential genes are shown in the heat map. (C) DEG counts in RT and control groups. (D) Top 20 of GO enrichment of the up-regulated genes. The red arrows indicate that these functional pathways exist C5aR1 gene enrichment. (E) Volcano map of RNA-seq showing the DEGs. The red dots indicate significantly upregulated genes. RNA-seq, RNA sequencing; RT, radiotherapy; DEGs, differentially expressed genes; GO, Gene Ontology.

Article Snippet: After processes of dewaxing, rehydration, and antigen retrieval with ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer (pH 8.0) (Servicebio, Wuhan, China; G1206), the slides were then blocked with blocking solution [PBS with 10% fetal calf serum (FCS) and 0.1% TritonX] at room temperature for 1 hour, then washed in PBS and incubated overnight at 4 oC in the appropriate primary anti-C5aR antibody (Abcam, 1:500) and anti-CD8a antibody (Boster, 1:50) diluted in blocking solution.

Techniques: Expressing, Magnetic Beads, RNA Sequencing, Control, Functional Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Anti-CD8 antibodies can trigger CD8 + T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining

doi: 10.4049/jimmunol.1003941

Figure Lengend Snippet: (A) 5 × 104 naïve mouse transgenic F5 T-cells were either mock treated or incubated with 100 μg/ml CT-CD8a, 100 μg/ml 53.6.7, 100 μg/ml CT-CD8b or 100 μg/ml KT112 at 4°C for 25 minutes, then stained with cognate PE-conjugated H-2Db tetramer (25μg/ml) as described in the Materials & Methods. No staining was observed under any of the conditions shown with a control H-2Db tetramer folded around the lymphocytic choriomeningitis virus GP1-derived epitope KAVYNFATC (residues 33-41). Data were acquired using a modified FACSAriaII flow cytometer and analyzed with FlowJo software. Results were obtained by gating on the CD3+CD4− population. (B) 3×104 naïve mouse transgenic F5 T-cells were incubated at 37°C for 18 hours with either 100 μg/ml CT-CD8a, 100 μg/ml 53.6.7, 100 μg/ml CT-CD8b, 100 μg/ml KT112 or PMA (50 ng/ml) and ionomycin (1 μg/ml). Supernatants were harvested and assayed for MIP1β by ELISA. Error bars represent standard deviations.

Article Snippet: For experiments with mouse cells, the following unconjugated anti-mouse CD8 antibodies were used: (i) anti-CD8α clone CT-CD8a (Caltag-Medsystems, Buckingham, UK); (ii) anti-CD8α clone 53.6.7 (Biolegend, Cambridge, UK); (iii) anti-CD8β clone KT112 (hybridoma kindly provided by Prof. Rose Zamoyska); and, (iv) anti-CD8β clone CT-CD8b (Caltag-Medsystems).

Techniques: Transgenic Assay, Incubation, Staining, Derivative Assay, Modification, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Anti-CD8 antibodies can trigger CD8 + T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining

doi: 10.4049/jimmunol.1003941

Figure Lengend Snippet: The heterogeneity of anti-mouse CD8 antibodies Summary of the effects exerted by anti-mouse CD8 antibodies on pMHCI tetramer binding and CD8 + T-cell activation in the absence of TCR engagement.

Article Snippet: For experiments with mouse cells, the following unconjugated anti-mouse CD8 antibodies were used: (i) anti-CD8α clone CT-CD8a (Caltag-Medsystems, Buckingham, UK); (ii) anti-CD8α clone 53.6.7 (Biolegend, Cambridge, UK); (iii) anti-CD8β clone KT112 (hybridoma kindly provided by Prof. Rose Zamoyska); and, (iv) anti-CD8β clone CT-CD8b (Caltag-Medsystems).

Techniques: Binding Assay, Activation Assay