After completing this course, the reader should be able to:1. Describe the subtypes of Toll-like receptor 7 and 8 agonists and their effect on the different components of the antitumor immune response.2. Argue why they are used as stand-alone immunotherapeutic agents.3. Evaluate their potential to improve current approaches of active and passive immunotherapy.This article is available for continuing medical education credit at CME.TheOncologist.com. CME CME ABSTRACT

Dendritic cells (DC), professional antigen-presenting cells of the immune system, exert important functions both in induction of T cell immunity, as well as tolerance. It is well established that the main function of immature DC (iDC) in their in vivo steady-state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. Previously, it was believed that T cell unresponsiveness induced after stimulation with iDC is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. Moreover, several reports indicate that traditional DC maturation can no longer be used to distinguish tolerogenic and immunogenic properties of DC. This review will focus on the complementary role of dendritic cells in inducing both tolerance and immunity, and we will discuss the clinical implications for dendritic cell-based therapies.

The main function of our immune system is to protect us from invading pathogens and microorganisms by destroying infected cells, while minimizing collateral damage to tissues. In order to maintain this balance between immunity and tolerance, current understanding of the immune system attributes a major role to regulatory T cells (Tregs) in controlling both immunity and tolerance. Various subsets of Tregs have been identified based on their expression of cell surface markers, production of cytokines, and mechanisms of action. In brief, naturally occurring thymic-derived CD4+CD25+ Tregs are characterized by constitutive expression of the transcription factor FOXP3, while antigen-induced or adaptive Tregs are mainly identified by expression of immunosuppressive cytokines (interleukin-10 (IL-10) and/or transforming growth factor-β (TGF-β)). While Tregs in normal conditions regulate ongoing immune responses and prevent autoimmunity, imbalanced function or number of these Tregs, either enhanced or decreased, might lead, respectively, to decreased immunity (e.g., with tumor development or infections) or autoimmunity (e.g., multiple sclerosis). This review will discuss recent research towards a better understanding of the biology of Tregs, their interaction with other immune effector cells, such as dendritic cells, and possible interventions in human disease.

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally, we investigated possible mediators of this effect, namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1, n = 11; group 2, n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis, either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise, EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise, with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile.

Recently, human dendritic cells ( DCs ) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti -tumor -specific T -cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow -derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70 -80% with a mean fluorescence intensity of 100 -200. A maximal expression level was reached 3 hours after electroporation. A clear dose -response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNAelectroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose -dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

BackgroundCell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.ResultsIn this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.ConclusionWe here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.

SUMMARYDendritic cells (DC) are the most professional antigen-presenting cells of the immune system and are capable of initiating immune responses in vitro and in vivo . One of the great challenges in immunotherapy protocols is to introduce relevant antigens into DC for stimulation of major histocompatibility complex (MHC) class I-and class II-restricted anti-tumour or anti-viral immunity. This review will focus on the development of mRNA-loaded DC-based immunotherapy vaccines. First, several published results concerning mRNA transfection efficiency in DC are compared. Next, an overview is given for several published studies describing CD8+ and CD4 + T-cell clone activation using RNA-loaded DC. These data show that RNA-loaded DC efficiently process and present antigenic epitopes. Next, published data from in vitro T-cell activation studies using RNA-loaded DC are summarized and provide evidence that RNA-loaded DC can efficiently stimulate in vitro primary and secondary immune responses. Finally, the summarized data provide evidence that RNA-loaded DC are a promising strategy for the development of future cancer vaccination strategies.

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.