Resiquimod

Total RNA was isolated from frozen PBMC of the patients isolated at baseline and after three biweekly vaccines with ATL-DC plus adjuvant using the ZYMO quick RNA extraction kit. We utilized the TruSeq RNA exome kit to construct the RNA sequencing libraries in samples that passed QC (placebo: 5 pairs, resiquimod: 8 pairs, and poly-ICLC: 8 pairs; see Supplementary Data 1C). Paired-end, 2 × 100 base pair (bp) transcriptome reads were mapped to the Genome Reference Consortium Human Build 38 (GRCh38) reference genome using HISAT2 (version 2.0.6)^{54 (link)}. The gene-level counts were generated by the HTSeq-count program (version 0.5.4p5)^{55 (link)}. We utilized the DESeq2 R package’s counts function (version 1.24.0)^{56 (link)} to compute the normalized gene expression values from the raw gene expression counts. DESeq2 normalized gene expression was log2 transformed after adding a pseudo count of 1. For subsequent differentially expressed genes (DEGs) and gene set enrichment analyses, we only included the known genes (i.e., genes with RefSeq transcripts ID starting with “NM_“, that satisfy: 1) normalized expression IQR ≥ 1; and 2) normalized log2 expression ≥1 in at least one of the samples.
Based on the filtered gene list, we first obtained the patient-specific, log2 fold change of each gene before and after the ATL-DC vaccine treatment. Next, the mean of the log2 fold changes in the poly-ICLC or resiquimod group is compared to those in the placebo group. Genes showing at least 2-fold upregulation in any of the TLR agonist-treated group (resiquimod or poly-ICLC, nominal t-test p value ≤ 0.05) with respect to the placebo were tested for significant overlap with gene ontology and known gene sets using the web-based tools, ENRICHR^{57 (link)}.
To calculate single sample gene set enrichment of the interferon-related genes, we used the Gene Set Variation Analysis (GSVA) package (version 1.32.0)^{58 (link)}. To compute the GSVA scores, the filtered, log2 normalized gene expression were supplied to the GSVA program using the ‘kcdf=Gaussian’ mode. We manually selected gene sets that are related to interferon pathway activation from the c2.cgp, c6, c7, and hallmark geneset collections of the Broad Institute’s Molecular Signatures Database (version 7.0)^{59 (link)}.

This was a single-center, randomized, open-label multi-arm phase II clinical trial. The study protocol was approved by an independent ethics committee, institutional review board and internal scientific peer review committee at the University of California, Los Angeles. Patients were recruited and completed treatment between 2010 and 2014, with survival follow-up until the present date. Subjects were not compensated, and all patients gave written informed consent before enrollment.
Twenty-three patients with high-grade WHO Grade III or IV gliomas were enrolled in this protocol. To be eligible for the primary cohort, patients had to be >18 years and have newly diagnosed or recurrent WHO Grade III or IV malignant glioma, as determined through central pathology review. For all patients, a Karnofsky Performance Score (KPS) of ≥60, adequate bone marrow, liver, and renal function, life expectancy of ≥8 weeks, no other prior malignancy within the last 5 years, no active viral infections, and sufficient resected tumor material to produce the autologous vaccine were required. All newly diagnosed patients underwent surgical resection followed by radiation and chemotherapy with temozolomide for 6 weeks, per standard of care. Patients in the recurrent setting proceeded to trial treatment after recovery from surgery. All patients were scheduled to receive ATL-DC. Patients were then randomized to receive either placebo, resiquimod (topical 0.2%, 3 M), or poly-ICLC (20 μg/kg i.m., Oncovir) as an adjuvant to the DC vaccine. Patients underwent leukapheresis to obtain adequate numbers of PBMC for DC generation. For the study treatment, we processed the resected tumor tissue into a lysate, then prepared and cryopreserved the autologous DCs as we previously described^{2 (link),3 (link)}. Patients were then treated with three intradermal injections of autologous tumor lysate-pulsed DC plus adjuvant TLRs/placebo on days 0, 14, and 28. The TLR agonists were delivered as separate injections. Poly ICLC (20 ug/kg) was given as an intramuscular injection at the same site as the intradermal ATL-DC vaccine. Resiquimod (0.2%) was applied as a topical gel directly over the intradermal ATL-DC vaccine site. The placebo was a gel without any resiquimod and administered similarly over the intradermal vaccine site. All vaccines were administered on the upper arm. Follow-up for patients was conducted at the study site for vital signs, KPS, hematology and serum chemistries, as well as neurological and physical examinations.

Preparation of stimuli was described previously.¹² (link)^,15 The final concentrations of stimuli in the assay strips were: RPMI 1:2, 1:10 γ-irradiated (50 kGy) mock infected vero-cell supernatants (iVero), 1:10 γ-irradiated (50 kGy) SARS-CoV-2 infected vero-cell supernatants (iSARS) 10^6.2 TCID₅₀/mL, BCG-Denmark (Serum Statens Institut, Denmark) 75 μg/mL, heat-killed (HK) Candida albicans 1.0 × 10⁶ CFU/mL, HK Escherichia coli 1.0 × 10⁶ CFU/mL, HK Staphylococcus aureus 1.0 × 10⁷ CFU/mL and resiquimod (R848) 3.5 μg/mL.