Soluble PD-L1 generated by endogenous retroelement exaptation is a receptor antagonist

  1. Kevin W Ng
  2. Jan Attig
  3. George R Young
  4. Eleonora Ottina
  5. Spyros I Papamichos
  6. Ioannis Kotsianidis
  7. George Kassiotis  Is a corresponding author
  1. Retroviral Immunology, The Francis Crick Institute, United Kingdom
  2. Retrovirus-Host Interactions, The Francis Crick Institute, United Kingdom
  3. Democritus University of Thrace Medical School, Greece
  4. Faculty of Medicine, Imperial College London, United Kingdom
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 4 supplements
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. …

Figure 1—source data 1

Expression of CD274 variant 1 and CD274-L2A in TCGA and GTEx samples.

https://cdn.elifesciences.org/articles/50256/elife-50256-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
The CD274-L2A protein product retains receptor binding domains but not transmembrane domain.

(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 …

Figure 1—figure supplement 2
The L2A element in intron 4 of the CD274 gene is essential for sPD-L1 production.

(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 …

Figure 1—figure supplement 3
Expression of CD274-MIRB and CD274-FLAM_A variants.

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.

Figure 1—figure supplement 3—source data 1

Expression of CD274-MIRB and CD274-FLAM_A in TCGA and GTEx samples.

https://cdn.elifesciences.org/articles/50256/elife-50256-fig1-figsupp3-data1-v2.xlsx
Figure 1—figure supplement 4
Splice junction analysis of CD274 variant expression.

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 …

Figure 2 with 1 supplement
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 …

Figure 2—figure supplement 1
Expression of CD274-L2A and production of sPD-L1 in mammalian species.

(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 …

Figure 3 with 2 supplements
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 …

Figure 3—source data 1

Expression of CD274 variants in the CCLE collection.

https://cdn.elifesciences.org/articles/50256/elife-50256-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
CD274-L2A and CD274v1 expression in individual cancer and healthy samples.

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 …

Figure 3—figure supplement 2
CD274-L2A-derived soluble PD-L1 is exosome independent.

(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 …

Figure 4 with 1 supplement
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 …

Figure 4—figure supplement 1
Generation of Jurkat.PDCD1 cells.

(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 …

Figure 5 with 2 supplements
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

Figure 5—figure supplement 1
CD274v1 and CD274-L2A predominantly produce transmembrane and soluble PD-L1 respectively.

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. …

Figure 5—figure supplement 2
Surface PD-L1 is not lost upon co-transfection with CD274-L2A.

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 …

Figure 6 with 1 supplement
CD274-L2a-derived soluble PD-L1 delays in vivo tumour growth.

(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 …

Figure 6—figure supplement 1
Characterisation of sPD-L1-producing MCA-38 cells.

(A) MCA-38 cells were transduced with retroviral vectors encoding human sPD-L1 (MCA-38.hCD274-L2A) or a chimeric murine sPD-L1 (exons 1–4) with the 18 C-terminal amino acids of human sPD-L1 …

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (Mus musculus)C57BL/6JThe Jackson LaboratoryRRID: IMSR_JAX:000664
Cell line (Homo sapiens)HEK293TCell Services Facility, Francis Crick InstituteRRID: CVCL_0063
Cell line (Homo sapiens)JurkatCell Services Facility, Francis Crick InstituteRRID: CVCL_0065
Cell line (Chlorocrbus sabaeus)VeroCell Services Facility, Francis Crick InstituteRRID: CVCL_0059
Cell line (Chlorocebus aethiops)CV-1Cell Services Facility, Francis Crick InstituteRRID: CVCL_0229
Cell line (Oryctolagus cuniculus)R9abCell Services Facility, Francis Crick InstituteRRID: CVCL_3782
Cell line (Cricetulus griseus)CHOCell Services Facility, Francis Crick InstituteRRID: CVCL_0213
Cell line (Mus musculus)B-3T3Cell Services Facility, Francis Crick InstituteRRID: CCL-163
Cell line (Mus musculus)EL4Cell Services Facility, Francis Crick InstituteRRID: CVCL_0255
Cell line (Mus musculus)MCA-38Cell Services Facility, Francis Crick InstituteRRID: CVCL_B288
Cell line (Mus musculus)B3Cell Services Facility, Francis Crick InstituteRRID: CVCL_RP56
AntibodyRat 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.)
AntibodyMouse monoclonal anti-human PD-L1 (clone 29E.2A3)Biolegend (FACS)Cat: 329706FACS (1:200)
AntibodyMouse monoclonal anti-human PD-1 (clone EH12.2H7)Biolegend (FACS)Cat: 329908FACS (1:200)
AntibodyMouse monoclonal anti-human CD25 (clone BC96)Biolegend (FACS)Cat: 302642FACS (1:200)
AntibodyMouse monoclonal anti-human CD69 (clone FN50)Biolegend (FACS)Cat: 310906FACS (1:200)
AntibodyMouse monoclonal anti-human CD8 (clone SK1)Biolegend (FACS)Cat: 344710FACS (1:200)
AntibodyMouse monoclonal anti-human Granzyme B (clone QA16A02)Biolegend (FACS)Cat: 372204FACS (1:200)
AntibodyMouse monoclonal anti-human HGS (clone C-7)Santa Cruz (WB)Cat: sc-271455WB (1:1000)
AntibodyHRP-conjugated mouse monoclonal anti-human Actin (clone AC-15)Abcam (WB)Cat: ab49900WB (1:25000)
Chemical compound, drugGW4869Sigma AldrichCat: D1692
Transfected construct (Homo sapiens, Mus musculus)pRV-IRES-GFP (lentiviral vector)This paperLentiviral construct expressing GFP; used as empty vector control
Transfected construct (Homo sapiens, Mus musculus)pRV-CD274-L2A-IRES-GFP (lentiviral vector)This paperLentiviral construct expressing human CD274-L2A
Transfected construct (Mus musculus)pRV-mCd274-L2A-IRES-GFP (lentiviral vector)This paperLentiviral construct expressing murine-human chimeric Cd274-L2A
Transfected construct (Homo sapiens)pRV-PDCD1-IRES-GFP (lentiviral vector)This paperLentiviral construct expressing human PDCD1 (PD-1)
Recombinant DNA reagentpcDNA3.1-CD274 (plasmid)This paperMammalian expression plasmid encoding human CD274
Recombinant DNA reagentpcDNA3.1-CD274-L2A (plasmid)This paperMammalian expression plasmid encoding human CD274-L2A
Recombinant DNA reagentpcDNA3.1-CD274 IS4 (plasmid)This paperMammalian expression plasmid encoding human CD274 with intron 4
Recombinant DNA reagentpcDNA3.1-CD274 IS4ΔL2A (plasmid)This paperMammalian expression plasmid encoding human CD274 with intron four with L2A sequence deleted
Commercial
assay or kit
Human PD-L1 ELISA kitAbcamCat: ab214565
Commercial assay or kitMouse PD-L1 ELISA kitBiomatikCat: EKU06803
Commercial assay or kitRNeasy Mini RNA extraction kitQiagenCat: 74104
Commercial assay or kitHigh Capacity cDNA Reverse Transcription kitApplied BiosystemsCat: 4368814

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