cd160 pe antibody  (Thermo Fisher)

Journal: Oncoimmunology

Article Title: Toll-like receptor agonist combinations augment mouse T-cell anti-tumor immunity via IL-12- and interferon ß-mediated suppression of immune checkpoint receptor expression

doi: 10.1080/2162402X.2022.2054758

Figure Lengend Snippet: Combinations of TLR agonists at the time of T-cell activation in vitro affect expression of T-cell checkpoint receptors: Splenocytes were prepared from the spleens of OT-1 mice and stimulated in vitro with the high-affinity SIINFEKL (OVA) peptide in the presence or absence of TLR agonists [TLR 1/2 (Pam3CSK4), TLR 3 (Poly I:C), TLR 4 (MPLAs), TLR 5 (FLA-ST), TLR 2/6 (FSL-1), TLR 7 (Gardiquimod), TLR 7/8 (R848), TLR 9 (ODN1826), or TLR 13 (ORN Sa19)] or their pairwise combinations. The median fluorescence intensity (MFI) of 4–1BB and T-cell checkpoint receptor expression on CD8 + T-cells were determined by flow cytometry, collected daily for 4 days, and computed as Area Under the Curve (AUC) using the trapezoid rule. (a) Calculation of AUC ratio to compare AUC of each receptor expression for each pairwise combination with that obtained following OVA stimulation alone, without TLR activation. (b) Heat-map demonstrating AUC ratio of each pairwise combination for 4–1BB and multiple T-cell checkpoint receptors. (c) Representative MFI plots showing 4–1BB, PD-1, LAG-3, and CD160 expression with the combinations of TLR1/2, TLR3, and TLR9 agonists. Results are from one experiment, with samples assessed in triplicate, and are representative of three similar, independent experiments. Asterisks represent significant (p < .05) differences as assessed by ANOVA.

Article Snippet: At the time points indicated, cells were analyzed via flow cytometry (BD LSRFortessa) with the following antibodies: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BV786 (BD 563332), LAG-3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17–5871-82), CTLA4–PE-Cy7 (Tonbo 60–1522-U100), CD160-PE (eBioscience 12–1601-81), TIGIT-BV605 (BD 744212), CD244.2-BV510 (740115), VISTA-BV421 (BD 150212), 41BB–PerCPeF710 (eBioscience 46–1371–82), and Live/Dead Ghost dye 780 (Tonbo, San Diego, CA 13–0865–T100).

Techniques: Activation Assay, In Vitro, Expressing, Fluorescence, Flow Cytometry

Journal: Oncoimmunology

Article Title: Toll-like receptor agonist combinations augment mouse T-cell anti-tumor immunity via IL-12- and interferon ß-mediated suppression of immune checkpoint receptor expression

doi: 10.1080/2162402X.2022.2054758

Figure Lengend Snippet: Changes in CD8 ± T-cell expression of PD-1 following TLR stimulation depends on both IL-12 and type-1 interferons. (a) Purified DCs or B cells were stimulated in the presence of TLR1/2 agonist (Pam3CSK4), TLR3 agonist (HMW PolyI:C), TLR9 agonist (ODN 1826), or media only (Control) for 24 hours. Culture supernatants were then evaluated for the presence of IFNβ by ELISA. (b) OT-1 splenocytes were stimulated in vitro with the high-affinity SIINFEKL peptide in the presence of recombinant IL-12 and/or IFNβ. The median fluorescence intensity (MFI) of 4–1BB, PD-1, CD160, LAG-3 expression on CD8 + T-cells were assessed after 4 days, by flow cytometry. (c) OT-1 splenocytes were stimulated in vitro with the high-affinity SIINFEKL peptide in the presence of specific TLR agonists, anti-IFNAR1 and/or anti-IL-12Rβ2. The median fluorescence intensity (MFI) of PD-1 expression on CD8 + T-cells was assessed after 3 days, by flow cytometry. Asterisks demonstrate significant (p < .05) differences as assessed by ANOVA. Results are from one experiment and are representative of two independent experiments.

Article Snippet: At the time points indicated, cells were analyzed via flow cytometry (BD LSRFortessa) with the following antibodies: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BV786 (BD 563332), LAG-3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17–5871-82), CTLA4–PE-Cy7 (Tonbo 60–1522-U100), CD160-PE (eBioscience 12–1601-81), TIGIT-BV605 (BD 744212), CD244.2-BV510 (740115), VISTA-BV421 (BD 150212), 41BB–PerCPeF710 (eBioscience 46–1371–82), and Live/Dead Ghost dye 780 (Tonbo, San Diego, CA 13–0865–T100).

Techniques: Expressing, Purification, Control, Enzyme-linked Immunosorbent Assay, In Vitro, Recombinant, Fluorescence, Flow Cytometry

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: Blood Cancer Journal

Article Title: Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia

doi: 10.1038/bcj.2014.92

Figure Lengend Snippet: MRD detection by CD160FCA and correlation with PCR in the pilot cohort. ( a ) The theoretical MRD expression (calculated, x axis) is plotted against the observed MRD expression (actual analyzed MRD analysis, y axis) by CD160FCA. The results are the percentage expression of CD160 of the total leukocyte population. The comparative RT-PCR results are demonstrated at the top of the graph above the corresponding dilution. The R 2 linear regression value for observed expression vs theoretical expression was R 2 =0.98. ( b ) Separation of MRD-positive and MRD-negative populations based on CD160FCA assessment ( P =0.01). ( c ) MRD assessment in paired peripheral blood and bone marrow CLL cells as determined by the CD160FCA ( n =47), irrespective of the treatment regimen (Spearman Rank R =0.87, P <0.0001).

Article Snippet: The CD160FCA incorporates CD2-FITC (Clone S5.2), CD5-APC (Clone L17F12), CD19-PerCP cy5.5 (Clone SJ25C1), CD23-APC (Clone EBVCS-5) and CD45 V500 (Clone Hi30) (BD Biosciences, Oxford, UK); CD160-PE (Clone BY55; IgM isotype; Immunotech, Beckman Coulter, Marseilles, France).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Blood Cancer Journal

Article Title: Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia

doi: 10.1038/bcj.2014.92

Figure Lengend Snippet: MRD detection by CD160FCA vs International Standardized Protocols in the validation cohort. ( a ) Comparative assessment of CD160FCA MRD positivity as a percentage of total leucocytes against the ISA methodology ( n =48 samples, P <0.001, R =0.91). ( b ) MRD-positive and -negative cohorts based on the median fluorescence intensity (MFI) of the CD2 neg /CD5 + /CD19 + /CD23 + /CD160 + population.

Article Snippet: The CD160FCA incorporates CD2-FITC (Clone S5.2), CD5-APC (Clone L17F12), CD19-PerCP cy5.5 (Clone SJ25C1), CD23-APC (Clone EBVCS-5) and CD45 V500 (Clone Hi30) (BD Biosciences, Oxford, UK); CD160-PE (Clone BY55; IgM isotype; Immunotech, Beckman Coulter, Marseilles, France).

Techniques: Biomarker Discovery, Fluorescence