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. 2018 Sep 27;36:367–375. doi: 10.1016/j.ebiom.2018.09.040

Fig. 4.

Fig. 4

Combined STAT1 plus 2 or IRF1 silencing decrease interferon-α-induced PDL1 expression.

(A to G) EndoC-βH1 cells were transfected with control siRNA (siCTL) or siRNAs targeting STAT1 (siSTAT1) and/or STAT2 (siSTAT2). After 48 h of recovery, the cells were exposed or not to interferon-α (IFNα, 2000 U/ml) for 24 h. The knockdown efficiency (A, C, E, F) and the PDL1 expression (B, D, G) were determined by real time RT-PCR. The values were normalized by the housekeeping gene beta-actin and are represented as fold induction compared to non-treated cells (NT) (n = 3–6, * p < .05, ** p < .01, *** p < .001, ANOVA with Bonferroni correction).

(H) EndoC-βH1 cells were transfected with a luciferase reporter construct containing the wild type human PDL1 promoter or the same construct presenting a deletion in the IRF1 binding site plus a pRL-CMV plasmid (used as internal control); cells were then exposed to IFNα for 24 h and luciferase activity was assayed. The values were corrected for the activity of the internal control (pRL-CMV) and are presented as normalized relative luciferase units (RLUs) in relation to non-treated cells (NT) considered as 1 (n = 4, ** p < .01, *** p < .001, ANOVA with Bonferroni correction).

(I and J) EndoC-βH1 cells were transfected with control siRNA (siCTL) or two independent siRNAs targeting IRF1 (siIRF1 #1 and #2) plus the wild type PDL1 promoter reporter and the pRL-CMV plasmid. The cells were then exposed to IFNα for 24 h and luciferase activity was assayed (J). The knockdown efficiency (I) was determined by real time RT-PCR. (n = 4, *** p < .001, ANOVA with Bonferroni correction).

(K) Immunostaining of PDL1 (green), IRF1 (red), insulin (light blue) and nuclei (dapi; dark blue) in the islets of an individual with T1D. The insets show the regions highlighted in each islet with a white box at higher magnification.

(L) 15 islets were selected at random from each of 6 individuals with type 1 diabetes (from the DiViD) cohort) and analysed for the proportion that were immunonegative for both PDL1 and IRF1, or were immunopositive for PDL1 alone, IRF1 alone or both PDL1 and IRF1.

(M) The proportion of islets that were immunonegative for PDL1 and IRF1 was scored in relation to islet inflammation measured as the mean number of CD8+ T-cells present in each individual patient studied.

The mean values ± SEM are shown.