Fig. 5

The expression of CD244 is increased in response to endoplasmic reticulum (ER) stress in immature monocytes. (A-F) The single cell RNA sequencing (scRNA-seq) data (GSE121861) was downloaded and re-analyzed. GSE121861 contained scRNA-seq data of 6 syngeneic mice tumor model (CT-26, EMT-6 : BALB/C; MC-38, LL2, B16F10 : C57B6/J; Sa1N : A/J). Each mouse tumor was harvested when it reached 100–200 mm. (A) Uniform manifold approximation and projection (UMAP) plot showing 4 clusters of myeloid cells among total 16 clusters containing 3 lymphoid [11, 13, 15] and 4 myeloid [2, 3, 5, 7] clusters, cancer associated fibroblasts (CAF), and tumor cells (CT26, LL2, MC-38, Sa1N, B16F10 and EMT-6). (B) The dotplot illustrated markers for classical monocytes (CM), immunosuppressive monocytes (IM), M1-like macrophages (M1), and M2-like macrophages (M2). (C) Gene set enrichment analysis (GSEA) result predicted from differentially expressed genes (DEGs) of IM compared to CM. (D) The UMAP plot depicted CD244 expression in 4 monocyte-lineage cell clusters. (E) A graph presented CD244 expression levels on CM, IM, M1, and M2. (F) GSEA result was predicted from DEGs of CD244-high IMs compared to CD244-low IMs. (G-H) Thapsigargin (THG) and Taurosodeoxycholic acid (TUDCA), an inducer and an inhibitor of ER stress, were administered to BMDMs along with M-CSF, and the cells were cultured for 3 days. (G) Representative flow cytometry plots displayed the monocyte and macrophage populations. (H) MFI of CD244 in monocytes (left) and the proportion of macrophages (right) were evaluated after treatment with THG alone or co-treatment with THG and TUDCA. (I) WT and CD244^−/− BMDMs were stained with Hoechst 33,342 and rabbit anti-mouse LC3B antibody, followed by a secondary anti-rabbit IgG-AlexaFlour555 antibody. LC3B expression on BMDMs was assessed using Immunofluorescence (left) and flow cytometry (right; top). Autophagosome formation in WT and CD244^−/− BMDMs was measured by Cyto-ID staining (right; bottom). (J) BMDMs were treated with chloroquine, an inhibitor of autophagolysosome formation, and M-CSF and cultured for 3 days. Representative flow cytometry plots demonstrated changes in the monocyte/macrophage population (left) and the number of macrophages (right). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; one-way ANOVA (E, H) or unpaired Student’s t-test (I) or two-way ANOVA (J). The data represent two (G-J) independent experiments