Yeast surface display libraries containing the randomly mutated IgV domain of human CD80 (residues 35-141) were generated by error-prone PCR as described21 (link). Multiple selection strategies were employed to generate a diverse set of candidates. All intermediate selection outputs were assayed for CD28, CTLA-4, and PD-L1 binding by flow cytometry. The pool of mutants from which the lead candidate domain (H2393) was isolated and evolved as follows. A randomly mutated library (2.7 × 108 clones, avg 4.6 mutations/clone) was screened with 2 parallel flow sorting progressions: (A) 3 nM CTLA-4-Fc+ (produced at Alpine Immune Sciences) followed by two successive 300 nM CD28-Fc− (R&D Systems) sorts and (B) two successive 300 nM CD28-Fc− sorts followed by a 3 nM CTLA-4-Fc+ sort. Yeast plasmid DNA isolated from the A and B terminal sorts was pooled and used as template to build a subsequent error-prone PCR library. This second-round library was screened via the following sort progression: 50 nM PD-L1-Fc+ (R&D Systems) followed by 20 mM CTLA-4-Fc+, followed by 10 nM PD-L1-Fc+ followed by 25 nM CD28-Fc. Each sort step was followed by outgrowth in YPD media (Himedia) at 30 °C and expression induction in media derived from Benatuil et al. (2010), with the substitution of dextrose/galactose with dextrose only at 22 °C before progressing to the next sort.
Pd l1 fc
Manufactured by R&D Systems
Sourced in United States
PD-L1-Fc is a recombinant protein consisting of the extracellular domain of human Programmed Death-Ligand 1 (PD-L1) fused to the Fc region of human IgG1. It is a laboratory tool used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pdl2 fc, by R&D Systems (2 mentions)
Ig control, by R&D Systems (2 mentions)
Epoch 2 microplate reader, by Agilent Technologies (2 mentions)
Hrp conjugated streptavidin, by Vector Laboratories (2 mentions)
Ctla 4 fc, by R&D Systems (1 mentions)
Cd28 fc, by R&D Systems (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Tcrγδ isolation kit, by Miltenyi Biotec (1 mentions)
Cxcl13, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
Anti icos, by BioLegend (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Negative selection kit, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
11 protocols using pd l1 fc
1
Generating Diverse CD80 Mutant Library
2
Smad7 Antisense Oligonucleotide Inhibition in DSS Colitis
3
CD4+ and CD8+ T Cell Proliferation
4
Engineering and Characterization of PD-1 Blocking scFv
5
Engineering and Characterization of PD-1 Blocking scFv
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The RMP1–14 scFv was engineered from the RMP1–14 hybridoma. The PD-1-blocking scFv, termed E27, was identified from a human antibody scFv phage library (Eureka Therapeutics Inc., Emeryville CA). The scFv library was screened for binding to human PD-1. Briefly, biotinylated PD-1-Fc fusion protein was mixed with the human scFv phage library. The antigen-scFv complexes were pulled down by streptavidin-conjugated beads and bound clones were eluted and transformed into bacteria. The binding and affinity of scFv to PD-1 was determined using ForteBio Octet QK (Pall Life Sciences, Menlo Park, CA, USA). The ability of the scFv to block PD-1 from interacting with PD-L1 was determined using an ELISA assay. Briefly, PDL1-Fc (R&D Systems, Minneapolis, MN, USA), was loaded onto a plate. PD-1-Fc was mixed with dilutions of antibody derived from the E27 scFv clones or human IgG1 isotype control mAb (Eureka Therapeutics Inc.) and then added to the PDL1-Fc-coated plate. Binding of the clones was determined by a standard ELISA method against biotinylated PD-1. Streptavidin-conjugated HRP (Vector Laboratories, Burlingame, CA, USA) was used to detect binding on an Epoch-2 microplate reader (BioTek Instruments, Winooski, VT, USA).
6
Regulation of T-cell Activation by PD-1
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The effects of PD-1 crosslinking during T-cell activation were determined according to the method described by Bertsias et al. [20 (link)]. CD4+ T cells (1 × 105/well) stimulated in 96-well plates with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (250 ng/ml) were incubated with a pool of L929-PD-L1 cells (PD-L1 transgenic cell line) or with various concentrations of plate-bound PD-L1-Fc (0.5 μg/ml; R&D Systems), a chimeric protein containing the extracellular part of human PD-L1 linked to the Fc fragment of human immunoglobulin G1 (IgG1). Then the above CD4+ T cells were incubated in the presence or absence of the recombinant fusion protein PD-1-Fc for 4 days. After 3 days of coculture supernatants were collected for cytokine measurements, and after 4 days cells were pulsed with the reagent of Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) for another 4 h to measure proliferation.
7
Modulation of γδ T Cell IL-17 by PD-1/PD-L1
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Standard laboratory protocols were used to prepare the splenocytes and isolate the γδ T cells by MACS using a TCRγδ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instruction. We diluted the anti-PD-1 antibody (eBioscience, San Diego, CA, USA) into 10 and 20 µg/mL, the PD-L1-Fc fusion protein (PD-L1-Fc, R&D Systems, Minneapolis, MN, USA) into 5 and 10 µg/mL with PBS respectively. According to the report (21 (link)), The anti-PD-1 antibody and PD-L1-Fc were coated in 96-well plates overnight at 4 °C. In each well, 2×105 MACS-purified γδ T cells (purity was greater than 90.0%) were added, and cultured in RPMI 1640 medium containing 10% FBS with 5 µg/mL anti-CD3 antibody for 48 hours. In control group, the cells were cultured in the plate precoated with rat IgG. IL-17A-producing γδ T cells were examined by flow cytometry and the level of IL-17A in culture supernatant was determined by ELISA.
8
Modulation of CD8+ PBTs by PD-1/PD-L1 Blockade
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CD8+ PBTs from HNSCC patients were activated for 72–96 h using PMA and ionomycin followed by treatment with the αPD-1 antibody pembrolizumab (10 μg/ml) and/or the αPD-L1 antibody atezolizumab (1 and 10 μg/ml) for 6 h prior to performing functional studies. CD8+ PBTs from HDs were plated on cell culture plates coated with PD-L1 (PD-L1-Fc, R&D Systems) (10 μg/ml) and activated with either PMA and ionomycin or (with anti-CD3/CD28 antibodies for 72–96 h followed by treatment with the αPD-1 antibody pembrolizumab (10 μg/ml) for 6 h before performing functional studies. For prolonged treatment with PD-L1, cells were activated for 120 h with plate-bound anti-CD3/CD8 antibodies along with PD-L1-Fc in T cell medium supplemented with 20 IU/ml of IL-2.
9
Smad7 Antisense Oligonucleotide Inhibition in DSS Colitis
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We used a 21-base Smad7 antisense oligonucleotide with the sequence 5′-GTC GCC CCT TCT CCC CGC AGC-3′ complementary to the mRNA of Smad7 (purchased from Integrated DNA Technology). The same sequence Smad7-as inhibitor has previously been validated by others in vitro and in vivo in models of Crohn’s disease (CD) (Boirivant et al., 2006 (link)) and neuroinflammation (Kleiter et al., 2007 (link)), and in CD patients (Monteleone et al., 2001 (link)). Mice were treated with Smad7-as or oligonucleotide control inhibitor i.p. (250 μg/mouse/day) in PBS every other day for four days at the beginning of a 7 day 3% DSS colitis induction, as indicated in Figure 5A . For PDL1/2-Fc treatment, mice were treated with PDL1-Fc, PDL2-Fc, or Ig control (R&D systems) i.p. (20 μg/mouse/day) every other day for four days at the beginning of a 7 day 3% DSS colitis induction, as indicated in Figure 5A .
10
ICOS and CXCL13 Signaling in T Cells
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Previously activated or retrovirally transduced T cells were deprived of serum for 3 h. Approximately 2 × 106 T cells were suspended in serum-free RPMI-1640 medium, incubated with purified anti-ICOS (Biolegend) or CXCL13 (PeproTech) with or without PD-L1-Fc (R&D) of indicated concentration for 30 min in 37°C. Stimulated cells were quickly spun down and lysed by 2% SDS and loading buffer and then boiled at 100 °C for 15 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with TBS containing 5% milk and 0.1% Tween-20. Antibodies for immunoblotting included rabbit anti–pAKT (S473), anti-AKT, anti-actin, anti-pSHP-2 (Y542), anti-SHP2 (Cell Signaling Technology). Appropriate HRP-conjugated secondary reagents were from Jackson Immunoresearch Laboratories.
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