Dendritic Cell Vaccination in Conjunction with a TLR Agonist Polarizes Interferon Immune Responses in Malignant Glioma Patients

Abstract

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. 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 Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes 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. 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-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population.

Trial registration: ClinicalTrials.gov Identifier: NCT01204684.

Keywords: TLR agonist; brain cancer; glioma; immunotherapy.

Figure 1.. Combination of ATL-DC vaccine and TLR agonists results in a robust interferon pathway activation in the patient PBMCs.

A, Timeline of PBMC acquisition and analysis using CyTOF and/or RNAseq. V = vaccine, D = Day. B, Schematic of differential gene expression analysis performed on pre-treatment and post-treatment PBMCs of indicated treatment groups. Differentially expressed genes (DEGs) in TLR agonist-treated groups are compared against their changes in the placebo group to identify DEGs specific to the TLR-agonist groups. C, D, Enriched gene set terms in Gene Ontology Biological Process (C) or ARCHS4 TF Coexp datasets that significantly overlap with the union of DEGs from ATL-DC + poly-ICLC and ATL-DC + resiquimod groups (P values, FDR-adjusted, two-sided fisher exact test). E, Differential gene expression (pre vs. post-treatment fold change, in log2) of representative antigen presentation and IFN related genes across treatment groups (P values, two-sided two-sided Welch t test). F, Gene set enrichment score differences (pre vs. post-treatment, delta GSVA score) of representative IFN related genesets across treatment groups (P values, two-sided Welch t test). G, Heatmap of single-sample, gene set enrichment scores (GSVA) of type I and type II interferon genesets in pre-treatment, ATL-DC + placebo, ATL-DC+poly-ICLC and ATL-DC+resiquimod samples.

Figure 2.. Single cell analysis reveals activation of systemic T cells and monocytes as a part of interferon pathway activation in all myeloid and lymphoid populations.

A, A UMAP projection of the pre- and post-treatment PBMC sample pairs from twenty patients (placebo, n=4 pairs; poly-ICLC, n=9 pairs; resiquimod, n=7 pairs). Clustering was performed with a random sampling of 5,000 cells from each patient. B, Heatmap of normalized expression of all 27 cell markers within cell populations identified in the patient PBMCs. C, D, Normalized expression of indicated markers in monocyte (C), or T cell populations (D) within the PBMC samples of patients from indicated treatment groups. P values, two-sided Wilcoxon rank sum test. E, UMAP projection of the PBMC-derived single cells (n=99,590). Immune subset associated with each cluster is inferred based on the cluster’s differentially expressed transcripts. Canonical markers of known immune subsets are shown. F, G, Heatmaps showing the union of recurrent DEGs computed between ATL-DC treated samples (combined with placebo, resiquimod or poly-ICLC) and pre-treatment samples in the myeloid populations (F) or lymphocyte populations (G). Shown in the heatmaps are the log fold change values of the DEGs in each cell population grouped by their treatment groups.

Figure 3.. Combined ATL-DC vaccine and TLR agonist treatment show trends of improved tumor control and patient survival.

A, B, C, Progression-free survival (PFS, top) and overall survival (OS, bottom) of all patients (A), patient subset with GBM (B), or grade III glioma (C) in indicated treatment groups. P values, log-rank test. D, E, Multivariate Cox proportional hazards analysis assessing the hazard ratios of tumor progression in TLR agonist treatment groups against placebo in all patients (D) or GBM subset after adjusting for other clinical covariates (Tx_Group=treatment group, RecurNum=number of recurrences prior to ATL-DC treatment). F, MR-computed volumes of post-treatment, recurrent tumors in indicated treatment groups. P values, unpaired, two-sided Wilcoxon rank sum test.

Figure 4.. IFN pathway activation is a positive predictor of survival after ATL-DC vaccine and TLR agonist therapy.

A, Kaplan-Meier progression-free survival curves of all patients (left), GBM (center), and Grade III glioma subsets (right) stratified by their HALLMARK_INTERFERON_GAMMA_RESPONSE GSVA scores in their post-treatment PBMCs. P values, log-rank test. B, C, Multivariate Cox proportional hazards analysis assessing hazard ratios of tumor progression in patients with high HALLMARK_INTERFERON_GAMMA_RESPONSE GSVA score of in all patients (B) or GBM subset (C) after adjusting for other clinical covariates.