RNA sequencing of fresh tumor tissues from B16F10 tumor bearing mice treated with saline or M44 vaccine

To further reveal the immune regulatory effects of MHC II-restricted vaccines on TME, bulk RNA sequencing (bulk RNA-seq) was performed to analyze the overall immune signal transformation. Differential gene expression analysis recognized a total of 1695 upregulated and 343 downregulated genes on the M44 vaccination. Specifically, a higher expression of CD74, H2-Ab1, H2-Eb1, H2-Aa was observed in the vaccinated group, indicating the active MHC class II antigen processing and presentation in tumors. The ssGSEA method was used to quantify RNA-seq data of each sample and the GO_BP (Biological Process of Gene Ontology) term scores were calculated. The results indicated that the majority of the terms within high ssGSEA scores were related to both adaptive and innate immune-related processes.We then sorted the top 300 differently upregulated genes to generated pathway enrichment analysis and clustered the pathways into 20 categories based on functional relevance. Interestingly, the majority of categories were related to the activation of antitumor immune response, such as “inflammatory response” “leukocyte activation” “antigen processing and presentation” “leukocyte migration” and “phagocytosis”. Moreover, immune infiltration estimations showed increased infiltration levels of T cells, CD8+ T cells, NK cells, memory B cells and monocytes/macrophages.