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

// Till S.M. Mathan 1 , Johannes Textor 1 , Annette E. Skold 1, 2 , Inge Reinieren-Beeren 1 , Tom van Oorschot 1 , Mareke Bruning 3 , Carl G. Figdor 1 , Sonja I. Buschow 1, 4, * , Ghaith Bakdash 1, * , I. Jolanda M. de Vries 1, 5 1 Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands 2 Department of Oncology and Pathology, Karolinska University Hospital Solna, Karolinska Institute, Stockholm, Sweden 3 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany 4 Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 5 Department of Medical Oncology, Radboud University Medical Centre, Nijmegen, The Netherlands * Authors contributed equally Correspondence to: I. Jolanda M. de Vries, email: Jolanda.deVries@radboudumc.nl Keywords: dendritic cells, immunotherapy, RNA sequencing, adjuvants, protamine-RNA, transcriptomics Received: September 09, 2016     Accepted: December 05, 2016     Published: February 08, 2017 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 Fruhsommer 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.