Fig 1: PD-L1 regulation by UL23 leads to decreased IFN-γ secretion from T cells. (A) HFFs were infected with Towne or Towne-ΔUL23 at an MOI of 3. Venn diagram and heatmap illustrating representative genes involved in the regulation of T cell activity in the Towne group compared to the ΔUL23 group. (B) HFFs were infected with Towne or Towne-ΔUL23 and treated with different indicated neutralizing antibodies or a control antibody, followed by incubation with activated Jurkat cells. Prior to co-culture, purified anti-human CD66 (CEACAM) neutralizing antibody (CEACAM1 NAb), as well as anti-human IL-10R beta neutralizing antibody (IL-10RB NAb), were individually added to the medium at a final concentration of 10 µg/mL for 30 min. qPCR analysis was performed to measure IFN-γ mRNA expression. (C and D) HFFs were transfected with NC or siR-PD-L1 for 24 h and subsequently infected with HCMV (Towne or ΔUL23). PD-L1 mRNA levels (C) and protein levels (D) were analyzed using qPCR and Western blot. Representative Western blot analysis of PD-L1 protein levels (down) and the densitometry values normalized to GAPDH (up) were presented. (E and F) siR-PD-L1-transfected HFFs were infected with HCMV (E), while HCMV-infected HFFs were treated with PD-L1 neutralizing antibodies (F). The resulting cells were then incubated with activated Jurkat cells, followed by ELISA analysis to measure IFN-γ secretion. (G) HFF-UL23 cells or HFF-C cells were infected with Towne (labeled as “T”) or Towne-ΔUL23 (labeled as “Δ”) and subsequently co-cultured with Jurkat cells in the presence of either Fc control (Fc) or PD-L1 Fc. The secretion of IFN-γ was quantified. (H–M) In the co-culture system, IL-2 secretion (H and I), T cell proliferation (J and K), and T cell apoptosis (L and M) were assessed by transfecting siR-PD-L1 or adding PD-L1 antibodies along with their respective controls. Each assay was performed three times, yielding similar results. Data are presented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01, and ***P < 0.001). Three independent experiments were conducted.