Fig 2: BRAF knockdown induced CD274 expression. (A) The RNA extracted from four PTC cell lines was used to determine the expression of BRAF, CD274, CD73, CD200, CD276, ENTPD1, and CD200. The expression in the mutant was compared with the WT cell type. (B) The immunoblot represents the expression of proteins in the BRAF-mutated and WT cell lines. (C) The bar graph represents the changes in the mRNA expression of CD274 and other immunosuppressive markers on knocking down BRAF. (D) Immunoblot represents the knockdown of BRAF and upregulation of CD274 in the BRAF-mutated cell lines T32 and T85 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA test). ANOVA, analysis of variance; PTC, papillary thyroid cancer; WT, wild-type.

Fig 3: Expression of immunosuppressive markers correlates to BRAF mutation. (A) The tumor tissues of 19 PTC cases were stained with CD274 antibody, and the expression intensity was scored by a COH pathologist. Grade 1 represents the lowest expression, and grade 3+ represents the highest. (B) The tumor tissue was processed to detect CD73, and the expression intensity was scored where grade 1 represents the lowest expression and grade 3+ represents the highest. (C) The images represent the cells positive for cancer cells (TTF-brown), T cells CD8 (blue) and CD4 (pink) in the two representative tumor tissue of BRAF-mutated and WT samples. (D) The plot illustrates the percentage of lymphocyte infiltration within the tumor area, as assessed by the pathologist. The lymphocytic infiltration ratio is notably higher for mutant patients (red dots) compared with wild-type patients (blue). Additionally, the percentages of CD4 and CD8 lymphocytes were analyzed. The findings suggest a higher contribution of CD4 lymphocytes compared with CD8, reinforcing the cell type infiltration data. PTC, papillary thyroid cancer; WT, wild-type. * indicates a p value of <0.05

Fig 4: BRAF mutation correlates with immune hot signature. (A) The heatmap represents the expression of all the T cell markers detected in the NanoString data and clustering pattern using BRAF-mutated and WT cases. (B) The graph represents differences in the expression of immunosuppressive markers between the BRAF-mutated and WT cases. (C) The heatmap represents the expression of all the T cell markers detected in the NanoString data across the TCGA thyroid cohort. (D) The box plot represents the sum of scores for the cold (C1), intermediate (C2), and hot clusters (C3). (E) The stacked bar graph represents the distribution of BRAF-mutated cases across all the three clusters. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA test). ANOVA, analysis of variance; WT, wild-type.

Fig 5: BRAF inhibitor-induced transcriptional changes upstream of CD274. (A) Cartoon showing the design of CD274-promoter-luciferase constructs used for determining the changes in transcriptional activity postdrug treatment. (B) The box plot representing the luciferase activity determined in the cells post 48 hours of transfection, where 2 Kb promoter sequence correlated with maximum activity. (C) Luciferase activity measured in the cells post 48 hours of BRAF or KRAS knockdown. (D) The bar graph represents the difference in the promoter activity between the T85 and T32 cell lines. (E, F) Luciferase (Promoter) activity correlated with increased BRAF inhibitor concentration in the T68 and T85 cells. (****p<0.0001, ANOVA test). ANOVA, analysis of variance.