The IgV domain of the poliovirus receptor alone is immunosuppressive and binds to its receptors with comparable affinity

Figures

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  Figure 1. Design and purification of murine vdPVR-Fc and mutant vdPVR-Fc. (A) Diagram depicting the structure of IgV-domain PVR-Fc (vdPVR-Fc) and mutant vdPVR-Fc. The vdPVR-Fc recombinant construct consists of the N-terminal murine IgV portion of the PVR extracellular domain (residues 29–147; Accession # NP_081790.1) connected by a short linker (IEGRMDP) to the human IgG1 Fc domain (residues 100–330; Accession # P01857) located at its C-terminus. Mutant vdPVR-Fc contains two point mutations in the PVR IgV domain (Q62R and F128R). (B) Ribbon structure representation of the mutant vdPVR-Fc construct generated using ColabFold. Color pattern: vdPVR (in green), linker (in purple) and Fc domain (in blue). Q62R and F128R mutations in vdPVR are highlighted in red. (C) The purity and molecular mass of both proteins were confirmed by SDS-PAGE (left panel) in the presence or absence of a reducing agent (DTT). Western blot (right panel) of both constructs in the absence of DTT (blot detected using an anti-human Fc fragment antibody conjugated to HRP). Both expressed proteins migrated as a 94 kDa band in their dimeric form.

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  Figure 2. Surface plasmon resonance (SPR) titration profiles of vdPVR-Fc, mutant vdPVR-Fc and PVR-Fc binding to mouse TIGIT, mouse CD226 and mouse CD96. (A-C) SPR single-cycle kinetics sensorgrams (in red) and fitted curves (in black) with equilibrium association (KD) constant showing the binding of vdPVR-Fc, mutant vdPVR-Fc and full length PVR-Fc to immobilized recombinant (A) mouse TIGIT-Fc-his, (B) mouse CD226-his and (C) CD96-his. Five concentrations (indicated on the sensorgrams) of vdPVR-Fc, mutant vdPVR-Fc and PVR-Fc were flown over the immobilized recombinant proteins. Kinetic parameters were calculated from 1:1 Langmuir binding model using Biacore evaluation software.

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  Figure 3. vdPVR-Fc binds to primary mouse T-cells. Stimulated T-cells in mixed splenocyte cultures were incubated with 2.5 µg of either vdPVR-Fc and mutant vdPVR-Fc. Histograms depicting vdPVR-Fc (blue) and mutant vdPVR-Fc (blue) binding to CD4+ and CD8+ T-cells compared with a human IgG1isotype control (red). Binding of vdPVR-Fc and mutant vdPVR-Fc to T-cells was detected with an anti-human IgG1 Fc antibody conjugated to PE. Data is representative of 2 independent experiments.

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  Figure 4. vdPVR-Fc as a ligand suppresses T-cell activity in mouse splenocyte cultures in-vitro. (A) Representative CFSE profiles showing the proliferation percentages of murine T-cells in splenocyte cultures stimulated with anti-CD3/CD28 beads in the presence or absence of vdPVR-Fc (10 µg/mL) for 3 days. (B) Graph showing the mean proliferation percentages of CD4+ and CD8+ T-cells unstimulated, stimulated with anti-CD3/CD28 coated beads, or with anti-CD3/CD28 beads in the presence of either vdPVR-Fc (10 µg/mL) or mutant vdPVR-Fc (10 µg/mL) after 3 days of culture. Proliferation was measured by flow cytometry as a measure of the percentage of CFSE signal associated with cell divisions over 3 days. The culture supernatants were harvested from the murine splenocyte culture after 3 days and the level of (C) IL-2 and (D) IFNγ were quantified by ELISA. Red line represents maximum stimulation using anti-CD3/CD28 coated beads. Each point on the graph represents a biological replicate. Error bars represent mean ± SEM, *p < 0.05, **p < 0.01 and ****p < 0.0001 evaluated by one-way ANOVA relative to anti-CD3/CD28 coated beads alone.

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  Figure 5. Expression of PVR cognate receptors on T-cells and cell binding competition. (A) TIGIT, CD226 and CD96 expression on CD3+ T-cells was assessed after 1 day of stimulation with anti-CD3/CD28 beads. (B) Flow cytometry histograms showing vdPVR-Fc binding to stimulated CD4+ and CD8+ T-cell subsets (blue) and vdPVR-Fc binding after the addition of 2-molar excess anti-TIGIT antibody (red). Data are representative of 2 independent experiments.

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  Figure 6. vdPVR-Fc delays the onset of psoriatic symptoms in a murine model of imiquimod-induced psoriasis. Female C57BL/6 mice received a daily topical application of either Vaseline or 5% imiquimod (IMQ) cream on their shaved backs for 5 days. IMQ-treated mice received intraperitoneal doses of either vdPVR-Fc or mutant vdPVR-Fc every other day starting from Day 0. (A) Representative images of mouse back skin on Day 2 after treatment with either Vaseline, imiquimod alone, imiquimod and vdPVR-Fc or imiquimod and mutant vdPVR-Fc. (B) Individual (erythema, scaling, and thickness) and cumulative scores of the treatment groups (n = 5). Graphs show an average score of 3 independent scorers comparing each group to IMQ alone (adjusted p value, evaluated by two-way ANOVA). (C) Hematoxylin and eosin staining of back skin sections of Vaseline, imiquimod alone, vdPVR-Fc and mutant vdPVR-Fc treated mice. Back skin samples were collected on Day 3. Epidermal thickness was quantified from histology sections (n = 9). Error bars represent mean ± SD, *p < 0.05, **p < 0.01 and ****p < 0.0001 relative to IMQ alone group evaluated by ANOVA.