BackgroundVaccines are a promising therapeutic for patients with advanced cancer, but achieving robust T-cell responses remains a challenge. Melanoma-associated antigen-A3 (MAGE-A3) in combination with adjuvant AS15 (a formulation of Toll-Like-Receptor (TLR)-4 and 9 agonists and a saponin), induced systemic CD4+ T-cell responses in 50% of patients when given subcutaneously/intradermally. Little is known about the transcriptional landscape of the vaccine-site microenvironment (VSME) of patients with systemic T-cell responses versus those without. We hypothesized that patients with systemic T-cell responses to vaccination would exhibit increased immune activation in the VSME, higher dendritic cell (DC) activation/maturation, TLR-pathway activation, and enhanced Th1 signatures.MethodsBiopsies of the VSME were obtained from participants on the Mel55 clinical trial (NCT01425749) who were immunized with MAGE-A3/AS15. Biopsies were taken 8 days after immunization. T-cell response to MAGE-A3 was assessed in PBMC after in-vitro stimulation with recMAGE-A3, by IFNγ ELISPOT assay. Gene expression was assessed by RNAseq using DESeq2. Comparisons were made between immune-responders (IR), non-responders (NR), and normal skin controls. FDR p<0.01 was considered significant.ResultsFour IR, four NR, and three controls were evaluated. The 500 most variable genes were used for principal component analysis (PCA). Two IR samples were identified as outliers on PCA and excluded from further analysis. There were 882 differentially expressed genes (DEGs) in the IR group vs the NR group (figure 1A). Unsupervised clustering of the top 500 DEGs revealed clustering according to the experimental groups (figure 1B). Of the 10 most highly upregulated DEGs, 9 were immune-related (figure 1C). Gene-set enrichment analysis revealed that immune-related pathways were highly enriched in IRs vs NRs (figure 1D). CD4 and CD8 expression did not differ between IR and NR (figure 2A), though both were higher in IR compared to control. Markers of DC activation/maturation were higher in IR vs NR (figure 2B), as were several Th1 associated genes (figure 2C). Interestingly, markers of exhaustion were higher in IR v NR (figure 2D). Expression of numerous TLR-pathway genes was higher in IR vs NR, including MYD88, but not TICAM1 (figure 2E).Abstract 611 Figure 1Gene expression profiling of vaccine site samples from patients immunized with MAGE-A3/AS15. (A) Volcano plots showing the distribution of differentially expressed genes (DEGs) between immune responders (IR) and non-responders (NR), IR and control, and NR and control. (B) Heatmap of the top 500 most differentially expressed genes demonstrating hierarchical clustering of sequenced samples according to IR, NR, and control. (C) Table showing the 10 most highly up and down-regulated genes in IR compared to NR. 9 of the top 10 most highly up-regulated genes are related to the immune response. (D) Enrichment plots from a gene set enrichment analysis highlighting the upregulation of immune related pathways in IR compared to NR. Gene set enrichment data was generated from the Reactome gene set database and included all expressed genes. Significance was set at FDR p <0.01Abstract 611 Figure 2Expression of T-cell markers in IR vs NR vs Control samples in the vaccine site microenvironment (VSME). (A) T-cell markers showing similar expression in IR vs NR but higher expression in IR vs control. (B) Markers of dendritic cell activation and maturation in the VSME which are higher in IR vs control but not IR vs NR. (B) Transcription factors and genes associated with Th1/Th2 responses within the VSME. (D) Genes associated with T-cell exhaustion at the VSME. (E) Expression of TLR pathway genes in the VSME. Expression data is provided in terms of normalized counts. Bars demonstrate median and interquartile range. N=9. IR = immune responder, NR = non-responder, TLR = Toll-like Receptor. * = <0.01, ** < 0.001, *** <0.0001, **** < 0.00001ConclusionsThese findings suggest a unique immune-transcriptional landscape in the VSME is associated with circulating T-cell responses to immunization, with differences in DC activation/maturation, Th1 response, and TLR signaling. Thus, immunologic changes in the VSME are useful predictors of systemic immune response, and host factors that modulate immune-related signaling at the vaccine site may have concordant systemic effects on promoting or limiting immune responses to vaccines.Trial RegistrationSamples for this work were collected from patients enrolled on the Mel55 clinical trial NCT01425749.Ethics ApprovalThis work was completed after approval from the UVA institutional review board IRB-HSR# 15398.
Testing Proximity of Genomic Regions to Transcription Start Sites and Enhancers Complements Gene Set Enrichment Testing