(A) Depiction of reference genome EREs and other repeats in the genomic locus spanning the CD274 gene, relative to CD274 exons. (B) GENCODE or RefSeq annotated splice variants of the CD274 gene. …
Expression of CD274 variant 1 and CD274-L2A in TCGA and GTEx samples.
(A) CD274 exons encoding receptor binding and transmembrane domains. (B) Transcript structure of the full-length CD274 variant 1 and variants 3/12, nine and CD274-L2A lacking the transmembrane …
(A) Minigenes comprising the full-length CD274 cDNA with an intact intron 4 (CD274 IS4), or an intron 4 lacking the L2A element (CD274 IS4ΔL2A) or CD274 variant 1 cDNA (CD274 v1), were expressed in …
Box plots of CD274-MIRB (top) and CD274-FLAM_A (bottom) expression in cancer patient samples (listed by TCGA abbreviations) and in healthy control samples from the indicated tissues.
Expression of CD274-MIRB and CD274-FLAM_A in TCGA and GTEx samples.
Annotated splice junctions in the CD274 gene (top) were numbered according to the linked exons (e.g. 1–2); 4int refers to splicing internal to exon 4, specific to variants 3 and 12; mid5-6 refers to …
(A–B) Genomic alignment of the indicated portion of the CD274 gene using nucleotide sequences from 65 eutherian mammals. A large SINE insertion in the porcine Cd274 gene and a smaller insertion in …
(A) Flow cytometric detection of cell surface PD-L1 in murine EL4 and MCA-38 cells. (B) Quantification of soluble murine PD-L1 by ELISA in the supernatants murine EL4 and MCA-38 cells. The dashed …
(A) CD274 variant 1 and CD274-L2A expression across TCGA tumour and GTEx healthy samples. Average TPM is shown per tissue type, with linear regression performed separately for tumour and healthy …
Expression of CD274 variants in the CCLE collection.
CD274 variant 1 and CD274-L2A expression across TCGA tumour and GTEx healthy samples. Each symbol represents an individual sample. Dashed blue lines represent linear regressions for each cancer type …
(A) Quantification of soluble PD-L1 by ELISA in the supernatant of HBL-1 cells treated with exosome inhibitor GW4869 or DMSO as denoted. Mean (± SEM) concentration from three independent experiments …
(A) Quantification of soluble PD-L1 by ELISA in supernatants of B-3T3 and HEK293T cells retrovirally transduced with CD274-L2A. Mean (± SEM) concentration from three independent experiments are …
(A) Surface expression of PD-1 on sorted PDCD1-transduced Jurkat cells (red) and parental Jurkat cells (blue). Representative histograms are shown. (B) Immunostaining of Jurkat.PDCD1 and parental …
(A) Primary CD8+ T cells were labelled with Cell Trace Violet (CTV) and stimulated with CD3- and CD28-coated beads for 72 hr. T cells were co-cultured with parental HEK293T or HEK293T.CD274v1 …
Quantification of surface and soluble PD-L1 on HEK293T cells transfected with CD274v1 or CD274-L2A. Surface expression was quantified by flow cytometry on transfected cells 24 hr post-transfection. …
Surface expression of PD-L1 on HEK293T cells transfected with CD274v1 and CD274-L2A. The concentration of the CD274v1 plasmid is kept constant across all conditions at 1000 ng. Transfected HEK293T …
(A–B) Tumour growth following subcutaneous inoculation of 1 × 106 MCA-38 cells expressing human sPD-L1 (MCA-38.hCD274-L2A), a constructed murine sPD-L1 variant (MCA-38.mCd274-L2A) or GFP. One group …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (Mus musculus) | C57BL/6J | The Jackson Laboratory | RRID: IMSR_JAX:000664 | |
Cell line (Homo sapiens) | HEK293T | Cell Services Facility, Francis Crick Institute | RRID: CVCL_0063 | |
Cell line (Homo sapiens) | Jurkat | Cell Services Facility, Francis Crick Institute | RRID: CVCL_0065 | |
Cell line (Chlorocrbus sabaeus) | Vero | Cell Services Facility, Francis Crick Institute | RRID: CVCL_0059 | |
Cell line (Chlorocebus aethiops) | CV-1 | Cell Services Facility, Francis Crick Institute | RRID: CVCL_0229 | |
Cell line (Oryctolagus cuniculus) | R9ab | Cell Services Facility, Francis Crick Institute | RRID: CVCL_3782 | |
Cell line (Cricetulus griseus) | CHO | Cell Services Facility, Francis Crick Institute | RRID: CVCL_0213 | |
Cell line (Mus musculus) | B-3T3 | Cell Services Facility, Francis Crick Institute | RRID: CCL-163 | |
Cell line (Mus musculus) | EL4 | Cell Services Facility, Francis Crick Institute | RRID: CVCL_0255 | |
Cell line (Mus musculus) | MCA-38 | Cell Services Facility, Francis Crick Institute | RRID: CVCL_B288 | |
Cell line (Mus musculus) | B3 | Cell Services Facility, Francis Crick Institute | RRID: CVCL_RP56 | |
Antibody | Rat monoclonal anti-mouse PD-L1 (clone 10F.9G2) | Biolegend (FACS), BioXCell (in vivo) | Cat: 124315 (Biolegend) Cat: BE0101 (BioXCell) | FACS (1:200) In vivo injection (200 ug i.p.) |
Antibody | Mouse monoclonal anti-human PD-L1 (clone 29E.2A3) | Biolegend (FACS) | Cat: 329706 | FACS (1:200) |
Antibody | Mouse monoclonal anti-human PD-1 (clone EH12.2H7) | Biolegend (FACS) | Cat: 329908 | FACS (1:200) |
Antibody | Mouse monoclonal anti-human CD25 (clone BC96) | Biolegend (FACS) | Cat: 302642 | FACS (1:200) |
Antibody | Mouse monoclonal anti-human CD69 (clone FN50) | Biolegend (FACS) | Cat: 310906 | FACS (1:200) |
Antibody | Mouse monoclonal anti-human CD8 (clone SK1) | Biolegend (FACS) | Cat: 344710 | FACS (1:200) |
Antibody | Mouse monoclonal anti-human Granzyme B (clone QA16A02) | Biolegend (FACS) | Cat: 372204 | FACS (1:200) |
Antibody | Mouse monoclonal anti-human HGS (clone C-7) | Santa Cruz (WB) | Cat: sc-271455 | WB (1:1000) |
Antibody | HRP-conjugated mouse monoclonal anti-human Actin (clone AC-15) | Abcam (WB) | Cat: ab49900 | WB (1:25000) |
Chemical compound, drug | GW4869 | Sigma Aldrich | Cat: D1692 | |
Transfected construct (Homo sapiens, Mus musculus) | pRV-IRES-GFP (lentiviral vector) | This paper | Lentiviral construct expressing GFP; used as empty vector control | |
Transfected construct (Homo sapiens, Mus musculus) | pRV-CD274-L2A-IRES-GFP (lentiviral vector) | This paper | Lentiviral construct expressing human CD274-L2A | |
Transfected construct (Mus musculus) | pRV-mCd274-L2A-IRES-GFP (lentiviral vector) | This paper | Lentiviral construct expressing murine-human chimeric Cd274-L2A | |
Transfected construct (Homo sapiens) | pRV-PDCD1-IRES-GFP (lentiviral vector) | This paper | Lentiviral construct expressing human PDCD1 (PD-1) | |
Recombinant DNA reagent | pcDNA3.1-CD274 (plasmid) | This paper | Mammalian expression plasmid encoding human CD274 | |
Recombinant DNA reagent | pcDNA3.1-CD274-L2A (plasmid) | This paper | Mammalian expression plasmid encoding human CD274-L2A | |
Recombinant DNA reagent | pcDNA3.1-CD274 IS4 (plasmid) | This paper | Mammalian expression plasmid encoding human CD274 with intron 4 | |
Recombinant DNA reagent | pcDNA3.1-CD274 IS4ΔL2A (plasmid) | This paper | Mammalian expression plasmid encoding human CD274 with intron four with L2A sequence deleted | |
Commercial assay or kit | Human PD-L1 ELISA kit | Abcam | Cat: ab214565 | |
Commercial assay or kit | Mouse PD-L1 ELISA kit | Biomatik | Cat: EKU06803 | |
Commercial assay or kit | RNeasy Mini RNA extraction kit | Qiagen | Cat: 74104 | |
Commercial assay or kit | High Capacity cDNA Reverse Transcription kit | Applied Biosystems | Cat: 4368814 |