mouse anti human cd73 percp cy5 5  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK

doi: 10.3389/fimmu.2023.1201415

Figure Lengend Snippet: Ibrutinib rejuvenates in vitro exhausted CD8+ T cell functions. (A) Scheme of testing ibrutinib effects on in vitro CTL exhaustion. By repeatedly stimulating the purified OT-І T cells with OVA(257-264) peptide for 5 days, in vitro exhausted CTLs were induced. From day 5, the cells were treated with DMSO or 1μM ibrutinib. On day 8, function and phenotype of the cells were determined. (B) Representative histograms depicting the expression of inhibitory receptors and isotype controls on DMSO or ibrutinib treated exhausted cells on day 8 of the in vitro exhaustion culture. (C) Pooled data showing the median fluorescence intensity (MFI) of the inhibitory receptors expressed on DMSO or ibrutinib treated cells. (D) Bar chart depicting frequency of cells expressing either one, two, three or four of the inhibitory receptors (IRs) PD-1, Lag3, Tim-3 and Tigit. (E) Representative FACS plots illustrating percentage of cytokine-producing CD8+ T cells after OVA(257-264) peptide re-stimulation and intracellular cytokine staining. Exhausted OT-I CD8+ T cells were re-stimulated on day 8 for 6 hours with OVA(257-264) peptide and intracellular cytokines were measured by flow cytometry. (F) Pooled data showing the frequency of cytokine producing CD8+ T cells. (G) Bar graph depicting the frequency of cells producing either one, two or three of the cytokines (IL-2, TNF-α and IFN-γ) simultaneously. Each symbol represents one animal (n=6), 4 independent experiments performed. Lines depict mean ± SE. Between the groups, paired-t test was performed, except for MFI of PD-1, CD160 and Tim3, where the Wilcoxon matched-pairs signed rank test was used “ns” indicates not significant (p>0.05) *P<0.05, **P<0.01.

Article Snippet: For surface staining, cells were first washed with FACS wash (HBSS containing 3% FBS and 0.02% sodium azide) and then incubated at 4°C in the dark for 20 minutes with appropriate concentrations of fluorochrome-conjugated monoclonal antibodies and appropriate isotype controls: anti-CD8a-eFluor 450 (clone 53-6.7, eBioscience), anti-CD160-PE-CF594 (clone CNX46-3, BD Biosciences), anti-Lag3-APC (clone C9B7W, BD Biosciences), anti-CD244-PE (clone 2B4, BD Biosciences; eBio244F4, eBioscience), anti-PD-1-APC-Cy7 (clone 19F.1A12, Biolegend), anti-Tim3-PE-Cy7 (clone RMT3-23, Invitrogen), anti-TIGIT-FITC (clone GIGD7, eBioscience); anti-CD44-BV786 (clone IM7, BD Biosciences).

Techniques: In Vitro, Purification, Expressing, Fluorescence, Staining, Flow Cytometry

Journal: Cancer Communications

Article Title: Enhancement of CAR‐T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins

doi: 10.1002/cac2.12452

Figure Lengend Snippet: Screening of immune checkpoints on CD8 + TILs in cholangiocarcinoma microenvironment. (A) Representative flow plots of immune checkpoints on CD8 + TILs. The proportion of Lag3 + cells in CD8 + TILs of tumor tissue, paratumor tissue, and normal tissue from one patient. (B‐I) Quantitative analysis of immune checkpoints on CD8 + TILs. PD‐1 (B), Tim‐3 (C), Lag3 (D), Tigit (E), BTLA (F), CTLA4 (G), CD244 (H), and CD160 (I) were detected by flow cytometric analysis respectively. The connecting lines indicated that the tissue came from one patient ( n = 15). (J) Statistical analysis of immune checkpoints on CD8 + TILs in cholangiocarcinoma tissue. Experiments were performed independently at least 3 times. Paired two‐tailed student's t‐test was used to compare two non‐independent groups. Experimental data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Abbreviations: TIL, tumor‐infiltrating lymphocyte; PD‐1, programmed death‐1; Tim‐3, T‐cell immunoglobulin and mucin domain‐3; Tigit, T cell immunoglobulin and ITIM domain; Lag3, lymphocyte activation gene 3; BTLA, B and T lymphocyte attenuator; CTLA4, cytotoxic T lymphocyte‐associated protein‐4; SEM, standard error of the mean.

Article Snippet: For T‐cell phenotypes in tumor tissues, the following antibodies were used: mouse anti‐human APC‐CD8a (Cat No. 17‐0086‐42, eBioscience, San Diego, CA, USA), PE/CY7‐PD‐1 (Cat No. 561272, BD Biosciences), Brilliant Violet 421‐Tim‐3 (Cat No. 565536, BD Biosciences), PE‐Lag3 (Cat No. 12‐2239‐41, eBioscience), PerCP‐eFluor 710‐Tigit (Cat No. 46‐9500‐42, eBioscience), PE/CY7‐BTLA (Cat No. 344516, Biolegend), PerCP‐eFluor 710‐CTLA4 (Cat No. 46‐1529‐42, eBioscience), Pacific Blue‐CD244 (Cat No. 329524, Biolegend) and PE‐CD160 (Cat No. 12‐1609‐42, eBioscience).

Techniques: Two Tailed Test, Activation Assay

Journal: Scientific Reports

Article Title: A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

doi: 10.1038/s41598-020-76069-3

Figure Lengend Snippet: Biopanning of the scFv phage display library on Streptactin magnetic beads. ( a ) Schematic representation of the biopanning process employing the StreptagII-Streptactin system. CD160 fused with Twin streptag was capture directly from cell supernatant on the surface of Streptactin magnetic beads. Phages were incubated with the antigen’ coated beads and bound phages were subsequently recovered by eluting with a 30 mM biotin solution and used to amplify the library for the next biopanning round. ( b ) The process of coating and elution of the beads throughout the biopanning process was assessed by flow cytometry analysis. These were stained during the several steps of the procedure (coating, blocking with 3%BSA and elution) using commercial anti CD160-PE. ( c ) Enrichment of the phage library for CD160 was determined after three rounds of biopanning on beads. The bacterial library from the different rounds of selection was induced to express the soluble scFv fragment in the supernatant. This was used to stain SupT1 cells overexpressing human CD160, due to the presence of a myc-tag at the C-terminus of productive scFvs we were able to detect binding using anti myc antibody. 2xTY media + anti myc-tag-DL549 and the commercial anti-CD160-PE Ab were used as controls. ( d ) The 15 individual bacterial colonies identified in the third round of biopanning carrying unique combination of CDR1,2 and 3 were screened by against SupT1 CD160 positive and negative cells (NT).

Article Snippet: Anti CD160 antibody (BY55-PE, BD Pharmigen) was used as positive control and either induced bacteria supernatant from previous round of biopanning or 2xTY medium as negative control for the staining.

Techniques: Magnetic Beads, Incubation, Flow Cytometry, Staining, Blocking Assay, Selection, Binding Assay

Journal: Scientific Reports

Article Title: A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

doi: 10.1038/s41598-020-76069-3

Figure Lengend Snippet: Characterization of the monoclonal scFv identified in the biopanning. ( a ) Alignment to the IMGT database of Rattus norvegicus germline variable sequence showed rearrange sequences originated from family 2 and 5 of the VH genes. We identified five different HCDR3 in which three were represented in multiple clones and three occurring only once. ( b ) Flow cytometry analysis of the five selected HCDR3 expressed secreted as chimeric scFv fused with mIgG2aFc. Supernatant from transfected HEK293T cells was used to stain SupT1 negative and SupT1 CD160 positive cells; the binding was detected with a secondary anti-mouseIgGFc-PE and anti CD160-PE (BY55) was used as positive control. ( c ) The affinity of these five scFv was determined using Biacore T200 technology. Individual scFvs were captured on a CM5 chip and CD160 was injected as analyte at 5 different concentration (3.70 nM, 11.11 nM, 33.33, nM, 100 nM and 300 nM) in a single-cycle kinetic study. The double-reference subtracted sensorgrams fitted with the 1:1 Langmuir binding model; kinetic and model fitting data are reported in the table.

Article Snippet: Anti CD160 antibody (BY55-PE, BD Pharmigen) was used as positive control and either induced bacteria supernatant from previous round of biopanning or 2xTY medium as negative control for the staining.

Techniques: Sequencing, Clone Assay, Flow Cytometry, Transfection, Staining, Binding Assay, Positive Control, Injection, Concentration Assay

Journal: Scientific Reports

Article Title: A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

doi: 10.1038/s41598-020-76069-3

Figure Lengend Snippet: Functional assays as chimeric antigen receptor. Four different healthy donors PBMCs were transduced with lentivirus to express the five different scFvs fused in a third generation (CD28-OX40ζ) CAR structure and tested in a cytotoxic assay. T cells were incubated with SupT1 CD160 positive and negative cells (NT) at different effector to target ratio (1:2, 1:4 and 1:8). The residual number of target cells for each donor was normalized against non-engineered PBMCs and all the five CARs demonstrated excellent killing at both 24 h and 72 h ( a ). Total cytokine production was assessed at 72 h and showed high levels of interferon-gamma (IFN-γ) and IL-2 ( b ) with no background on target-negative cells. The mean from each donor was plotted individually and analysed using two-way ANOVA with Dunnett’s post-test for comparison between CARs within the same target group. ***p < 0.0001.

Article Snippet: Anti CD160 antibody (BY55-PE, BD Pharmigen) was used as positive control and either induced bacteria supernatant from previous round of biopanning or 2xTY medium as negative control for the staining.

Techniques: Functional Assay, Transduction, Incubation

Journal: eLife

Article Title: Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells

doi: 10.7554/eLife.57869

Figure Lengend Snippet: Mucosal immune cells isolated from the colorectal tissue biopsies by enzymatic digestion were exposed to HIV-1 BaL (250 ng/mL), either free (F-HIV), complement-opsonized (C-HIV), or virions opsonized by a cocktail of complement and antibodies (CI-HIV) or mock-treated, by spinning the cultures. The effect HIV exposure had on mucosal T cells phenotype was assessed after 4 days. ( A–C ) The immune cells were stained with CD3, CD4, CD8, CD38, PD-1, LAG3, TIM3, and CD160 mAbs. ( A ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160+ CD4+ T cells, ( B ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160, and ( C ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed CD38+CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry (N = 12–15). Statistical significance was tested using repeated measures of ANOVA followed by Tukey's posttest. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM.

Article Snippet: , anti-human CD160-PE(Mouse monoclonal) , BD Biosciences , Cat#: 562118 , FACS (1 ul per test).

Techniques: Isolation, Staining, Expressing, Flow Cytometry

Journal: eLife

Article Title: Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells

doi: 10.7554/eLife.57869

Figure Lengend Snippet: Mucosal immune cells isolated from the colorectal tissue biopsies by enzymatic digestion were exposed to HIV-1 BaL (250 ng/mL), either free (F-HIV), complement-opsonized (C-HIV), or virions opsonized by a cocktail of complement and antibodies (CI-HIV) or mock-treated, by spinning the cultures. The effect HIV exposure had on mucosal T cells phenotype was assessed after 4 days. ( A–D ) The immune cells were stained with CD3, CD4, CD8, Tbet, EOMES, PD-1, LAG3, TIM3, and CD160 mAbs. ( A ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160. viSNE plot of PD-1+Tbet+CD4+ was constructed on CD4 T cells, data presented as dot plots with colored channels. Data from a donor shows CD4 T cells, subjected to tSNE algorithm, which provides cells with a unique coordinate according to its expression of PD-1+T-bet+ parameters, displayed on a two-dimensional plot (tSNE1 versus tSNE2). The heat gradient (blue to red) indicates expression level of Tbet+PD-1+ on CD4 following exposure to different conditions of viruses. ( B ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+EOMES+ CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry. Flow cytometry Zebra plots for Tbet+EOMES+ CD4+ T cells expressing PD-1. ( C ) Percentage of Tbet+CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160. viSNE plot of PD-1+Tbet+CD8+ was constructed on CD8 T cells, and data are presented as dot plots with colored channels. Data from a donor shows CD8 T cells, subjected to tSNE algorithm, which provides cells with a unique coordinate according to its expression of PD-1+T-bet+ parameters, displayed on a two-dimensional plot (tSNE1 versus tSNE2). The heat gradient (blue to red) indicates expression level of Tbet+PD-1+ on CD8 following exposure to different conditions of viruses. ( D ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+EOMES+ CD4+ T cells and percentage of Tbet+EOMES+ CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry. Flow cytometry Zebra plots for Tbet+EOMES+ CD8+ T cells expressing PD-1 (N = 12–15). Statistical significance was tested using repeated measures of ANOVA followed by Tukey's posttest. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM.

Article Snippet: , anti-human CD160-PE(Mouse monoclonal) , BD Biosciences , Cat#: 562118 , FACS (1 ul per test).

Techniques: Isolation, Staining, Expressing, Construct, Flow Cytometry

Journal: eLife

Article Title: Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells

doi: 10.7554/eLife.57869

Figure Lengend Snippet:

Article Snippet: , anti-human CD160-PE(Mouse monoclonal) , BD Biosciences , Cat#: 562118 , FACS (1 ul per test).

Techniques: Sequencing, Software, FCAP Assay