Dendritic Cell Vaccination in Conjunction with TLR Agonist Polarizes Interferon Immune Responses Associated with Long-term Survival in Malignant Glioma Patients [scRNA-seq]
Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. Pre-clinical studies demonstrate that toll-like receptor (TLR) agonists can enhance the anti-tumor immune response from cancer vaccines.  To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a randomized, open-label multi-arm Phase 2 clinical trial to evaluate immune responses and survival in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. Patients were randomized to receive ATL-DC vaccination with either a placebo, a TLR-7/8 agonist (resiquimod, topical 0.2%), or a TLR-3 agonist (poly-ICLC, 20 mcg/kg intramuscular).  Peripheral blood was obtained at baseline and following the vaccination cycle for immune monitoring. Mass cytometry, bulk and single-cell RNA sequencing analyses were performed. Analysis of the bulk RNAseq data (pre-treatment vs. post-treatment) demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant TLR agonist (either poly-ICLC or resiquimod) together with ATL-DC.  CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased levels of memory PD-1+ T-cell and monocyte populations after ATL-DC + TLR agonist administration.  In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes. Median progression-free survival (PFS) was 8.1 months (5.5 placebo vs. 8.1 resiquimod vs. 31.4 poly-ICLC), and median overall survival (OS) was 26.6 months (7.7 placebo vs. 16.7 resiquimod vs. 52.5 poly-ICLC).   Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression.  The results suggest that ATL-DC in conjunction with adjuvant poly-ICLCTL induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population. This combination was associated with extended survival in this patient population. This interferon response signature may represent an important blood biomarker for immunotherapy in patients with malignant glioma. Overall design: The cells for scRNAseq analysis were resuspended in PBS at a concentration of 1,000 cells/µl. We only selected representative patients from each treatment group whose PBMC quality were sufficient for single cell RNAseq processing (Placebo: 2 pairs, Poly-ICLC: 3 pairs (1 sample failed and is not included), Resiquimod (2 pairs); PBMC of two normal donors are added to provide a normal reference). Cell preparation, library preparation, and sequencing were carried out according to Chromium product-based manufacturer protocols (10X Genomics), targeting for a total of 10,000 cells sequenced. Cells from post-treatment samples (Placebo, Poly-ICLC, or Resiquimod), were compared to the pre-treatment samples to identify differentially expressed genes associated with each treatment.