Fig 1: Evolutionary conservation of CD274-L2A genomic features in hominids.(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 the leporine Cd274 gene were removed to aid the visual representation of alignment (both these species are highlighted in A. Base substitutions are indicated by highlighting and absence of highlighting denotes base conservation. (C) Comparison of the human and porcine genes, illustrating the SINE/tRNA insertion in the latter. (D) Comparison of the human and leporine genes, illustrating an insertion in the polyadenylation site of the latter. (E) Comparison of the human and murine genes, illustrating a 24-nucleotide deletion in the latter and mutations at the splice and polyadenylation sites. (F) Alignment tree depicting the distance of the consensus L2A element sequence from the respective human, chimpanzee, murine and rat elements. (G) Sequence divergence of coding and non-coding exons, introns and of the 100 nucleotides covering the exonised part of intron 4 and embedded L2A element in CD274 genomic sequences from 10 primate species. The individual segments of the CD274 gene compared were scaled to the same width.
Fig 2: CD274-L2A-derived soluble PD-L1 acts as a receptor antagonist in the presence of transmembrane PD-L1.(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 transfected cells in the presence of conditioned media from HEK293T.CD274-L2A transduced cells or a PD-L1-blocking antibody. Mean (± SEM) proportion of CTVlo and GzmB+ cells of three healthy donors is shown. (B) Percentage of activated (CD25+CD69+) Jurkat cells in the presence of conditioned media from parental HBL-1 cells, IFN-?-stimulated HBL-1 cells, and transduced B-3T3.CD274-L2A cells. Cells were stimulated with CD3- and CD28-coated beads for 24 hr, with 10 µg/mL of a PD-L1-blocking antibody added where indicated. Mean (± SEM) proportion from three independent experiments are shown. (C) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells in the presence of conditioned media from parental HBL-1 cells, IFN-?-stimulated HBL-1 cells, and transduced HEK293T.CD274-L2A cells. Cells were stimulated with CD3- and CD28-coated beads for 24 hr, with 10 µg/mL of a PD-L1-blocking antibody added where indicated. Mean (± SEM) proportion from three independent experiments are shown. (D) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells following co-culture with parental HEK293T or HEK293T.CD274v1 transfected cells in the presence of conditioned media from HEK293T.CD274-L2A or a PD-L1-blocking antibody. Cells were stimulated with CD3- and CD28-coated beads for 24 hr. Mean (± SEM) proportion from three independent experiments are shown. (E) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells following co-culture with HEK293T cells transfected with varying ratios of CD274v1 and CD274-L2A as shown. The concentration of the CD274v1 plasmid is kept constant across all conditions at 1000 ng. Cells were stimulated with CD3- and CD28-coated beads for 24 hr. Mean (± SEM) proportion from three independent experiments are shown.
Fig 3: CD274 protein domains and splice variants.(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. Variant numbers correspond to the following NCBI Reference Sequence accessions: variant 1 (NM_014143), variant 2 (NM_001267706), variant 3 (NR_052005) and variant 4 (NM_001314029). (C) Recently-described novel variants encoding sPD-L1 (Zhou et al., 2017). (D) CD274 splice variants de novo assembled in this study and overlapping one or more EREs. (E) Inclusion of an L2A element as a terminal exon and polyadenylation site in splice variant CD274-L2A. The relative position of the intronic L2A element, as well as the novel C-terminal amino acid created from its exonisation are also indicated. (F) RNA-seq traces representative of each of the ERE-overlapping CD274 variants, CD274-MIRB, CD274-FLAM_A and CD274-L2A. For the low recurrence CD274-MIRB and CD274-FLAM_A variants, the samples with the highest expression are shown, whereas for the high recurrence CD274-L2A, a representative ESCA sample is shown. (G) Box plot of CD274 variant 1 and CD274-L2A expression (in TPMs) in the indicated cancer patient (n = 24 for each indication) and healthy control samples (n between 2 and 156).Figure 1—source data 1.Expression of CD274 variant 1 and CD274-L2A in TCGA and GTEx samples.
Fig 4: CD274-L2A and CD274v1 expression are decoupled under certain stimuli.(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 samples. (B) CD274 variant 1 and CD274-L2A expression (in TPMs) across the CCLE dataset. Dashed lines denote a 10:1 and 1:1 ratio of CD274v1:CD274-L2A, respectively. (C) Expression of CD274 variant 1 and CD274-L2A, measured by qRT-PCR using variant-specific primers in five leukocyte cell lines. Cells were stimulated with IFN-a or IFN-? for 48 hr or were left untreated. Mean (± SEM) expression normalized to HPRT from three independent experiments is shown. (D) Expression of CD274 variant 1 and CD274-L2A (in TPMs), calculated using RNA-seq data (SRP045500) from B cells, monocytes and neutrophils isolated from peripheral blood of healthy individuals of patients with Sepsis, ALS or T1D or from MS patients before and 24 hr after the first treatment with IFN-ß.Figure 3—source data 1.Expression of CD274 variants in the CCLE collection.
Fig 5: CD274-L2A-derived soluble PD-L1 is not immunosuppressive.(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 shown. (B) Coomassie Brilliant Blue stain, under native or reducing (ß-ME/SDS) PAGE conditions, of serum-free supernatants from HEK293T, CHO and B3 cells transfected or not with CD274-L2A. (C–D) Primary CD8+ T cells were labelled with CTV and stimulated with CD3- and CD28-coated beads for 72 hr, alone or co-cultured with HEK293T, HEK293T.CD274v1 or HEK293T.CD274-L2A cells transfected with the indicated amount of plasmid DNA. T cells were stained for intracellular GzmB at the end of the culture period. Representative histograms and scatter plots are shown in C; quantification of CTVlo and GzmB+ cells of three healthy donors according to the amount of transfected plasmid DNA is shown in D. (E) Percentage of activated (CD25+CD69+) Jurkat cells in the presence of conditioned media from parental HBL-1 cells, IFN-?-stimulated HBL-1 cells, parental B-3T3 cells or B-3T3.CD274-L2A transduced cells. Cells were stimulated with CD3- and CD28-coated beads for 24 hr, with 10 µg/mL of a PD-L1-blocking antibody added where indicated. Mean (± SEM) proportion from three independent experiments are shown. (F) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells after co-cultured with parental HEK293T, HEK293T.CD274v1, or HEK293T.CD274-L2A cells. Cells were stimulated with CD3- and CD28-coated beads for 24 hr, with 10 µg/mL of a PD-L1-blocking antibody added where indicated. Mean (± SEM) proportion from three independent experiments are shown.
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