Fig 2: CD163+M2 macrophages polarized by EBV suppresses T-cell function. (A) Volcano plot of differentially expressed genes between mononuclear macrophages induced with EBV-uninfected tumor supernatant and those induced with EBV-infected tumor supernatant for 3 days. (B) GO pathway enrichment analysis of differentially expressed genes between mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatant. (C, D) GSEA of differentially expressed genes between mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatant. (E) Flow cytometry analysis of T-cell functional molecules (Granzyme B, IFN γ, TNF α, Perforin) after 3 days of coculture with mononuclear macrophages polarized with EBV-uninfected or EBV-infected tumor supernatants. Mean±SD, n=3, two-tailed t-test. (F) Flow cytometry analysis of T-cell functional molecules after 3 days of coculture with mononuclear macrophages untreated (control) or polarized with 50 ng/mL CSF1+80 ng/mL IL10 (M2). (G) Statistical analysis of T-cell functional molecules in (F). Mean±SD, n=3, two-tailed t-test. (H) Flow cytometry analysis of T-cell functional molecules after 3 days of coculture with mononuclear macrophages induced by EBV-infected tumor culture medium (control), EBV-infected tumor culture medium and CSF1R inhibitor (CSF1Rin), EBV-infected tumor culture medium and anti-IL10R antibody (anti-IL10) or EBV-infected tumor culture medium and CSF1R inhibitor and anti-IL10R antibody (CSF1Rin+anti-IL10). (I) Statistical analysis of T-cell functional molecules in (H). Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus; GO, Gene Ontology; GSEA, gene set enrichment analysis.

Fig 3: Structural combination of wild-type IL-2 and an IL-10 mutein(A) Monocytes treated with LPS alone (controls) or in addition to a titration (0–13 nM) of wild-type or EBV IL-10. TNF-α reduction reported after 24 h compared to controls. Data are representative of 4 independent donor experiments. Statistical analyses performed by two-way ANOVA with Tukey’s multiple comparisons test and concentrations with statistical differences are indicated (∗p ≤ 0.05; ∗∗∗∗p ≤ 0.0001).(B) CTLs (CD8+) treated with a titration (0–13 nM) of wild-type or EBV IL-10 (0–13 nM) and IFNγ measured after 24 h. Data are representative of 14 independent donor experiments. Statistical analyses performed by two-way ANOVA with Tukey’s multiple comparisons test (∗∗p ≤ 0.01).(C) Monocytes treated with LPS alone (controls) or in addition to a titration (0–13 nM) of ScFV:EBV IL-10 wild type, ScFv:EBV IL-10 muteins (M1 or M2), or recombinant human IL-10 and TNF-α measured after 24 h. Data are reported as TNF-α reduction compared to controls. Data are representative of 2–4 independent donor experiments.(D) CTLs (CD8+) treated with a titration (0–13 nM) of ScFV:EBV IL-10 wild type, ScFv:EBV IL-10 muteins (M1 or M2), or recombinant human IL-10 and IFNγ measured after 24 h. Data are representative of 6 independent donor experiments. Statistical analyses performed by one-way ANOVA at the highest concentration (∗∗p ≤ 0.01).(E) Schematic of the full-length DK210 (EGFR) construction depicting each domain.(F) A predicted computational model of DK210 (EGFR) generated using chimera, with domains indicated.(G and H) The far- (G) and near-ultraviolet (H) spectra of DK210 (EGFR) acquired via circular dichroism.(I–K) The binding kinetics of DK210 (EGFR) measured by Octet using streptavidin (SA) immobilized biotinylated EGFR (I), IL-2Rα (J), and IL-10RA (K).Results are reported as the mean ± SEM.