LTA drives NF-κB and JAK/STAT activation and constitutes a mixed lymphoid/myeloid gene-expression program in cHL cells. (A) Whole-cell extracts of L1236 wild-type (WT), control (v2), and 2 LTA-KO clones for each gRNA (LTAg2 and LTAg3) were analyzed in western blots for expression and phosphorylation of indicated proteins. Nuclear extracts of control and KO cells were assessed for NF-κB DNA binding activity by EMSA (bottom panel). (B) L1236 control and LTA clonal KO cells (g2_1) were cultivated at a cell density of 5 × 10⁵ cells per milliliter in RPMI 1640 without fetal calf serum. The medium was collected after 24 hours, and cytokine secretion was analyzed using the Cytokine Array C3. (C) L1236 control cells or the indicated LTA-KO clones were cultured for 72 hours. Expression levels of indicated target genes were verified by real-time quantitative polymerase chain reaction. Error bars represent the standard error of the mean. (D) PD-L1 and PD-L2 cell surface expression levels were analyzed by flow cytometry. Filled curve: isotype control. (E) Coexpression analysis of LTA target genes in healthy PBMCs.²¹  Heat map shows the similarity between expression profiles of each pair of LTA target genes. Positive correlation between 2 genes is designated by the intensity of the red color, whereas the blue gradient designates negative correlation (genes with more similar expression profiles are positioned closer on the heat map). Red blocks show the existence of gene-regulatory modules. Based on their level of expression, genes were annotated as expressed primarily in the lymphoid lineage or the myeloid lineage (orange/green annotation). (F) Expression of LTA and LTA receptors in PBMCs. The x-axis shows the percentage of cells in a cell population that express the corresponding gene. The y-axis shows the average gene expression in expressing cells. Cell populations expressing the gene in <1% of the cells were removed from the visualization. The color depicts the cell type.