Strategies for modulation of dendritic cell responses

With increased knowledge in dendritic cell (DC) biology, innate immune receptors and their ligands, and the shaping of adaptive responses, refined approaches to modulate our immune system are today emerging as treatment strategies for chronic infections and severe cancers. At the center of attention stand DCs – the innate immune cells that orchestrate the adaptive immune responses. In this thesis, strategies to activate and to inhibit DC activation are described, and the effect of different types of activation of DCs on HIV-1 infection is also investigated. In paper I, we have characterized a novel strategy of TLR3 inhibition in DCs and in other TLR3 expressing cells. The TLR3 ligand poly I:C normally activates DCs to upregulate maturation markers CD80 and CD86 and to secreted pro-inflammatory cytokines. We found that simultaneous addition of oligodeoxynucleotides (ODNs) based on a phosphorothioate (PS) backbone together with poly I:C inhibited the TLR3-mediated DC activation. This inhibition was dependent on the structure of the ODN backbone, since ODNs built on a phosphodiester backbone did not have inhibitory effects, but independent of the sequence, since both CpG and non-CpG containing PS-ODNs had the ability to inhibit the effect of poly I:C. We could repeat the PS-ODN-mediated inhibition on poly I:C activation in three additional non-hematopoietic cell types. Upon investigation of the mechanism behind this observation, we determined that PS-ODNs are preferably taken up into DCs over poly I:C, and are thereby inhibiting the ligand interaction with TLR3. To confirm this finding in vivo, we treated cynomolgus macaques intranasally with the ligands, either alone or in combination, and measured the secreted cytokine levels. Significantly reduced levels of IL-12p40 were detected in animals receiving PS-ODNs compared to animals treated with poly I:C alone, and a similar trend was observed also for additional pro-inflammatory cytokines and chemokines measured. Hence, these findings encourage the development of PS-ODNs as a treatment strategy during TLR3- mediated pathology. Our group has previously reported that irradiated activated PBMCs have the ability to induce DC maturation. In paper II, we set out to determine the underlying mechanism for this finding. First, we investigated whether the activated apoptotic cells (ACs) had to be phagocytosed for mediating their effect, but cell-cell contact was shown to be enough for DC maturation when co-cultured with ACs. We then tested if both cellular and supernatant fractions of activated ACs had the ability to mature DCs. Activated ACs were previously shown to release low levels of TNF-α, and we could confirm that the cytokine was a maturing agent in the supernatant fraction. The cellular fraction also matured DCs, and to investigate what molecules could be involved, we neutralized receptors previously shown to be stimulated by endogenous substances. We found that DC-SIGN, TLR4, and β2-integrins all were involved in AC-induced DC maturation, and a plausible ligand for TLR4 was shown to be heat shock protein 60. When investigating the intracellular signaling pathways mediating this effect, we determined that activated ACs induced signaling via Src family of tyrosine kinases, PI3K/Akt, JNK, and p38, and activated the NF-κB and AP-1 transcription factors. We further investigated the effect of activated apoptotic T cells on DC and HIV-1 infection in paper III. These activated ACs, either HIV-1 infected or uninfected, had the ability to mature DCs, and also to reduce HIV-1 infection in DCs. This reduction was partly due to TNF-α produced by stimulated DCs, but mainly due to the increased expression of the HIV-1 host restriction factor APOBEC3G in DCs. In paper IV, we continued to investigate the expression of APOBEC3 family members in DCs upon treatment with TNF-α or IFN-α. We could confirm previous reports on expression of APOBEC3A, F, and G in DCs, and we also concluded that TNF-α, despite induction of DC activation, did not induce expression of APOBEC3 molecules, but more probably stimulated additional host restriction factors in DCs

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

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. GMCSF 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 clinicalgrade 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

A Comparative Study of the T Cell Stimulatory and Polarizing Capacity of Human Primary Blood Dendritic Cell Subsets

Dendritic cells (DCs) are central players of immune responses; they become activated upon infection or inflammation and migrate to lymph nodes, where they can initiate an antigen-specific immune response by activating naive T cells. Two major types of naturally occurring DCs circulate in peripheral blood, namely, myeloid and plasmacytoid DCs (pDCs). Myeloid DCs (mDCs) can be subdivided based on the expression of either CD1c or CD141. These human DC subsets differ in surface marker expression, Toll-like receptor (TLR) repertoire, and transcriptional profile, suggesting functional differences between them. Here, we directly compared the capacity of human blood mDCs and pDCs to activate and polarize CD4 + T cells. CD141 + mDCs show an overall more mature phenotype over CD1c + mDC and pDCs; they produce less IL-10 and more IL-12 than CD1c + mDCs. Despite these differences, all subsets can induce the production of IFN-in naive CD4 + T cells. CD1c + and CD141 + mDCs especially induce a strong T helper 1 profile. Importantly, naive CD4 + T cells are not polarized towards regulatory T cells by any subset. These findings further establish all three human blood DCs-despite their differences-as promising candidates for immunostimulatory effectors in cancer immunotherapy

Exposure to Apoptotic Activated CD4+ T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4+ T cells (ApoInf) or apoptotic uninfected activated CD4+ T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4+ T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection

Paradigm Shift in Dendritic Cell-Based Immunotherapy: From in vitro Generated Monocyte-Derived DCs to Naturally Circulating DC Subsets

Contains fulltext : 136469.pdf (publisher's version ) (Open Access)Dendritic cell (DC)-based immunotherapy employs the patients' immune system to fight neoplastic lesions spread over the entire body. This makes it an important therapy option for patients suffering from metastatic melanoma, which is often resistant to chemotherapy. However, conventional cellular vaccination approaches, based on monocyte-derived DCs (moDCs), only achieved modest response rates despite continued optimization of various vaccination parameters. In addition, the generation of moDCs requires extensive ex vivo culturing conceivably hampering the immunogenicity of the vaccine. Recent studies, thus, focused on vaccines that make use of primary DCs. Though rare in the blood, these naturally circulating DCs can be readily isolated and activated thereby circumventing lengthy ex vivo culture periods. The first clinical trials not only showed increased survival rates but also the induction of diversified anti-cancer immune responses. Upcoming treatment paradigms aim to include several primary DC subsets in a single vaccine as pre-clinical studies identified synergistic effects between various antigen-presenting cells

A Comparative Study of the T Cell Stimulatory and Polarizing Capacity of Human Primary Blood Dendritic Cell Subsets

Dendritic cells (DCs) are central players of immune responses; they become activated upon infection or inflammation and migrate to lymph nodes, where they can initiate an antigen-specific immune response by activating naive T cells. Two major types of naturally occurring DCs circulate in peripheral blood, namely, myeloid and plasmacytoid DCs (pDCs). Myeloid DCs (mDCs) can be subdivided based on the expression of either CD1c or CD141. These human DC subsets differ in surface marker expression, Toll-like receptor (TLR) repertoire, and transcriptional profile, suggesting functional differences between them. Here, we directly compared the capacity of human blood mDCs and pDCs to activate and polarize CD4+ T cells. CD141+ mDCs show an overall more mature phenotype over CD1c+ mDC and pDCs; they produce less IL-10 and more IL-12 than CD1c+ mDCs. Despite these differences, all subsets can induce the production of IFN-γ in naive CD4+ T cells. CD1c+ and CD141+ mDCs especially induce a strong T helper 1 profile. Importantly, naive CD4+ T cells are not polarized towards regulatory T cells by any subset. These findings further establish all three human blood DCs—despite their differences—as promising candidates for immunostimulatory effectors in cancer immunotherapy

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis

Abstract Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses

Single-stranded DNA oligonucleotides inhibit TLR3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques

Item does not contain fulltextTLR3 is a key receptor for recognition of double-stranded RNA and initiation of immune responses against viral infections. However, hyperactive responses can have adverse effects, such as virus-induced asthma. Strategies to prevent TLR3-mediated pathology are therefore desired. We investigated the effect of single-stranded DNA oligonucleotides (ssDNA-ODNs) on TLR3 activation. Human monocyte-derived dendritic cells up-regulate maturation markers and secrete proinflammatory cytokines on treatment with the synthetic TLR3 ligand polyinosine-polycytidylic acid (poly I:C). These events were inhibited in cultures with ssDNA-ODNs. Poly I:C activation of nonhematopoietic cells was also inhibited by ssDNA-ODNs. The uptake of poly I:C into cells was reduced in the presence of ssDNA-ODNs, preventing TLR3 engagement from occurring. To confirm this inhibition in vivo, we administered ssDNA-ODNs and poly I:C, alone or in combination, via the intranasal route in cynomolgus macaques. Proinflammatory cytokines were detected in nasal secretions in the poly I:C group, while the levels were reduced in the groups receiving ssDNA-ODNs or both substances. Our results demonstrate that TLR3-triggered immune activation can be modulated by ssDNA-ODNs and provide evidence of dampening proinflammatory cytokine release in the airways of cynomolgus macaques. These findings may open novel perspectives for clinical strategies to prevent or treat inflammatory conditions exacerbated by TLR3 signaling