Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy

Abstract

Effective stimulation of immune cells is crucial for the success of cancer immunotherapies. Current approaches to evaluate the efficiency of stimuli are mainly defined by known flow cytometry-based cell activation or cell maturation markers. This method however does not give a complete overview of the achieved activation state and may leave important side effects unnoticed. Here, we used an unbiased RNA sequencing (RNA-seq)-based approach to compare the capacity of four clinical-grade dendritic cell (DC) activation stimuli used to prepare DC-vaccines composed of various types of DC subsets; the already clinically applied GM-CSF and Frühsommer meningoencephalitis (FSME) prophylactic vaccine and the novel clinical grade adjuvants protamine-RNA complexes (pRNA) and CpG-P. We found that GM-CSF and pRNA had similar effects on their target cells, whereas pRNA and CpG-P induced stronger type I interferon (IFN) expression than FSME. In general, the pathways most affected by all stimuli were related to immune activity and cell migration. GM-CSF stimulation, however, also induced a significant increase of genes related to nonsense-mediated decay, indicating a possible deleterious effect of this stimulus. Taken together, the two novel stimuli appear to be promising alternatives. Our study demonstrates how RNA-seq based investigation of changes in a large number of genes and gene groups can be exploited for fast and unbiased, global evaluation of clinical-grade stimuli, as opposed to the general limited evaluation of a pre-specified set of genes, by which one might miss important biological effects that are detrimental for vaccine efficacy.

Human Dendritic Cell Subsets Undergo Distinct Metabolic Reprogramming for Immune Response

“…Our data demonstrated that pRNA upregulated cytokines and migration-related genes in CD1c + mDCs as well as type I and III interferons (IFN-α and IFN-λ) related genes in pDC. Moreover, we demonstrated that pRNA stimulation increased expression of maturation markers (i.e., CD80, PD-L1 & CD40) in both CD1c + mDC and pDC, in addition to increase in immunostimulatory cytokines i.e., TNFα and INFα for CD1c + mDC and pDC, respectively ( 27 ). To investigate whether changes in metabolism are required for human DC-subsets immune response, we analyzed expression of OXPHOS related genes in human CD1c + mDC and pDC.…”

“…pRNA has been shown to activate CD1c + mDCs and pDCs and induces them to release IL-12 and IFN-α, respectively ( 28 ). Previously, we analyzed the whole-transcriptome of human CD1c + mDC and pDC upon TLR7/8 stimulation ( 27 ). Our data demonstrated that pRNA upregulated cytokines and migration-related genes in CD1c + mDCs as well as type I and III interferons (IFN-α and IFN-λ) related genes in pDC.…”

“…Data was analyzed using the R platform package “edgeR,” version 3.12, to analyze whole transcriptome principal coordinates analysis (using the “plotMDS” command), differential expression analysis, and GO term analysis. Differential expression was determined by fitting a generalized linear model using the “glmFit” command, and significance was determined using the likelihood ratio test provided by the “glmLRT” command ( 27 ).…”

The Application of Single-Cell RNA Sequencing in Vaccinology

“…In addition to effects modulated by vaccine regimen, platform, and immunisation route, there is ample evidence to suggest that gene expression following immunisation can be affected by adjuvant selection. Transcriptomic evaluation of nonhuman primates (NHP) and human responses to vaccine adjuvants, with and without vaccination, has largely been restricted to bulk and/or microarray analyses [66][67][68][69]. Using an NHP model to compare responses against the HIV Env antigen with eight different adjuvants, Francica et al [70] demonstrated using microarrays with whole blood RNA that TLR4 or 7 agonists had differential effects on the upregulation of inflammatory and IFN genes.…”

Naturally produced type I IFNs enhance human myeloid dendritic cell maturation and IL-12p70 production and mediate elevated effector functions in innate and adaptive immune cells

“…Additionally, we investigate how these cultures affect both innate and adaptive immune cells. As stimulus, we use protamine–RNA complexes (pR), a previously described TLR7/8 agonist that induces high levels of both IL-12p70 and IFN-α in mDCs and pDCs, respectively [ 25 – 28 ]. This will prevent possible artifact effects previously detected upon combination of different TLR ligands [ 29 ] and better resemble the clinical studies on mDCs and pDCs that are being performed with this stimulus (NCT02574377, NCT02993315, and NCT02692976).…”