Protease‐activated receptor‐2 signalling by tissue factor on dendritic cells suppresses antigen‐specific CD4+ T‐cell priming

The precise function of tissue factor (TF) expressed by dendritic cells (DC) is uncertain. As well as initiating thrombin generation it can signal through protease‐activated receptor 2 (PAR‐2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow (BM) ‐derived DC; 20% of BM‐derived DC expressed TF, which did not vary after incubation with lipopolysaccharide (LPS) or dexamethasone (DEX). However, the pro‐coagulant activity of DEX‐treated DC in recalcified plasma was 30‐fold less than LPS‐treated DC. In antigen‐specific and allogeneic T‐cell culture experiments, the TF on DEX‐treated DC provided a signal through PAR‐2, which contributed to the reduced ability of these cells to stimulate CD4⁺ T‐cell proliferation and cytokine production. In vivo, an inhibitory anti‐TF antibody and a PAR‐2 antagonist enhanced antigen‐specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR‐2‐dependent mechanism on DEX‐DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen‐specific CD4⁺ T‐cell activation.     

Immunotherapy using lipopolysaccharide‐stimulated bone marrow‐derived dendritic cells to treat experimental autoimmune encephalomyelitis

Lipopolysaccharide (LPS) produced by Gram‐negative bacteria induces tolerance and suppresses inflammatory responses in vivo; however, the mechanisms are poorly understood. In this study we show that LPS induces apoptosis of bone marrow‐derived dendritic cells (DCs) and modulates phenotypes of DCs. LPS treatment up‐regulates expression of tolerance‐associated molecules such as CD205 and galectin‐1, but down‐regulates expression of Gr‐1 and B220 on CD11c⁺ DCs. Moreover, LPS treatment regulates the numbers of CD11c⁺CD8⁺, CD11c⁺CD11b^low and CD11c⁺CD11b^hi DCs, which perform different immune functions in vivo. Our data also demonstrated that intravenous transfer of LPS‐treated DCs blocks experimental autoimmune encephalomyelitis (EAE) development and down‐regulates expression of retinoic acid‐related orphan receptor gamma t (ROR‐γt), interleukin (IL)‐17A, IL‐17F, IL‐21, IL‐22 and interferon (IFN)‐γ in myelin oligodendrocyte glycoprotein (MOG)‐primed CD4⁺ T cells in the peripheral environment. These results suggest that LPS‐induced apoptotic DCs may lead to generation of tolerogenic DCs and suppress the activity of MOG‐stimulated effector CD4⁺ T cells, thus inhibiting the development of EAE in vivo. Our results imply a potential mechanism of LPS‐induced tolerance mediated by DCs and the possible use of LPS‐induced apoptotic DCs to treat autoimmune diseases such as multiple sclerosis.     

CD4+ T‐cell activation is differentially modulated by bacteria‐primed dendritic cells,but is generally down‐regulated by n‐3 polyunsaturated fatty acids

Appropriate activation of CD4⁺ T cells is fundamental for efficient initiation and progression of acquired immune responses. Here, we showed that CD4⁺ T‐cell activation is dependent on changes in membrane n‐3 polyunsaturated fatty acids (PUFAs) and is dynamically regulated by the type of signals provided by dendritic cells (DCs). Upon interaction with DCs primed by different concentrations and species of gut bacteria, CD4⁺ T cells were activated according to the type of DC stimulus. The levels of CD80 were found to correlate to the levels of expression of CD28 and to the proliferation of CD4⁺ T cells, while the presence of CD40 and CD86 on DCs inversely affected inducible costimulator (ICOS) and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) levels in CD4⁺ T cells. For all DC stimuli, cells high in n‐3 PUFAs showed reduced ability to respond to CD28 stimulation, to proliferate, and to express ICOS and CTLA‐4. Diminished T‐cell receptor (TCR) and CD28 signalling was found to be responsible for n‐3 PUFA effects. Thus, the dietary fatty acid composition influences the overall level of CD4⁺ T‐cell activation induced by DCs, while the priming effect of the DC stimuli modulates CD80, CD86 and CD40 levels, thereby affecting and shaping activation of acquired immunity by differential regulation of proliferation and costimulatory molecule expression in CD4⁺ T cells.     

GM‐CSF therapy inhibits chronic graft‐versus‐host disease via expansion of regulatory T cells

Regulatory T cells (Tregs) attenuate excessive immune responses, making their expansion beneficial in immune‐mediated diseases, including allogeneic bone marrow transplantation associated with graft‐versus‐host disease (GVHD). In addition to interleukin‐2, Tregs require T‐cell receptor and costimulatory signals from antigen‐presenting cells, such as DCs, for their optimal proliferation. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) increases DC number and may promote DC‐dependent Treg proliferation. Here, we demonstrate that GM‐CSF treatment increases CD4⁺CD8^– DCs, which are associated with Treg expansion. In a mouse model of chronic GVHD (cGVHD), GM‐CSF therapy expanded Tregs, protected against the development of skin GVHD, and regulated both Th1 and Th17 responses in the peripheral lymph nodes, resulting in an attenuation of skin cGVHD. Notably, the expanded Tregs were instrumental to GM‐CSF‐mediated cGVHD inhibition, which was dependent upon an increased ratio of Tregs to conventional T cells rather than augmentation of suppressive function. These data suggest that GM‐CSF induces Treg proliferation by expanding CD4⁺CD8^? DCs, which in turn regulate alloimmune responses in a cGVHD mouse model. Thus, GM‐CSF could be used as a therapeutic DC modulator to induce Treg expansion and to inhibit excessive alloimmune responses in immune‐related diseases.     

Butyrate and retinoic acid imprint mucosal‐like dendritic cell development synergistically from bone marrow cells

Accumulating data show that the phenotypes and functions of distinctive mucosal dendritic cells (DCs) in the gut are regulated by retinoic acid (RA). Unfortunately, the exact role of butyrate in RA‐mediated mucosal DC differentiation has not been elucidated thoroughly to date. Mucosal‐like dendritic cell differentiation was completed in vitro by culturing bone marrow cells with growth factors [granulocyte–macrophage colony‐stimulating factor (GM‐CSF/interleukin (IL)‐4], RA and/or butyrate. The phenotypes, cytokine secretion, immune functions and levels of retinal dehydrogenase of different DCs were detected using quantitative polymerase chain reaction (qPCR), enzyme‐linked immunosorbent assay (ELISA) and flow cytometry, respectively. The results showed that RA‐induced DCs (RA‐DCs) showed mucosal DC properties, including expression of CD103 and gut homing receptor α₄β₇, low proinflammatory cytokine secretion and low priming capability to antigen‐specific CD4⁺ T cells. Butyrate‐treated RA‐DCs (Bu‐RA‐DCs) decreased CD11c, but increased CD103 and α₄β₇ expression. Moreover, the CD4⁺ T priming capability and the levels of retinal dehydrogenase of RA‐DCs were suppressed significantly by butyrate. Thus, butyrate and retinoic acid have different but synergistic regulatory functions on mucosal DC differentiation, indicating that immune homeostasis in the gut depends largely upon RA and butyrate to imprint different mucosal DC subsets, both individually and collectively.     

Carbon monoxide impairs mitochondria‐dependent endosomal maturation and antigen presentation in dendritic cells

Heme‐oxygenase 1 (HO‐1) prevents T cell‐mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m‐chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome‐to‐lysosome fusion, and antigen processing, dampening the production of peptide‐MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4⁺ T‐cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8⁺ T cell‐dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T‐cell priming by blocking an unknown mitochondria‐dependent antigen‐processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen‐containing endosomes. In conclusion, CO produced by HO‐1 impairs antigen‐dependent inflammation by regulating DC immunogenicity by a mitochondria‐dependent mechanism.     

In vivo targeting of human DC‐SIGN drastically enhances CD8+ T‐cell‐mediated protective immunity

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T‐cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC‐SIGN (DC‐specific‐ICAM3‐grabbing‐nonintegrin/CD209) is a C‐type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC‐SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC‐SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti‐DC‐SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti‐DC‐SIGN antibodies conjugated to OVA induced strong and persistent antigen‐specific CD4⁺ and CD8⁺ T‐cell responses, which efficiently protected from infection with OVA‐expressing Listeria monocytogenes. Thus, we propose DC targeting via DC‐SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.     

Triggering of NF‐κB in cytokine‐matured human DCs generates superior DCs for T‐cell priming in cancer immunotherapy

Understanding the signaling that governs the immunogenicity of human dendritic cells (DCs) is a prerequisite for improving DC‐based therapeutic vaccination strategies, in which the ability of DCs to induce robust and lasting Ag‐specific CTL responses is of critical importance. Cytokine‐matured DCs are regularly used, but to induce memory‐type CTLs, they require additional activation stimuli, such as CD4⁺ T‐cell help or TLR activation. One common denominator of these stimuli is the activation of NF‐κB. Here, we show that human monocyte‐derived, cytokine cocktail‐matured DCs transfected with constitutively active mutants of IκB kinases (caIKKs) by mRNA electroporation, further upregulated maturation markers, and secreted enhanced amounts of cytokines, including IL‐12p70, which was produced for more than 48 h after transfection. Most importantly, cytotoxic T cells induced by caIKK‐transfected DCs combined high CD27 expression, indicating a more memory‐like phenotype, and a markedly enhanced secondary expandability with a high lytic capacity. In contrast, CTLs primed and expanded with unmodified cytokine cocktail‐matured DCs did not maintain their proliferative capacity upon repetitive stimulations. We hypothesize that “designer” DCs expressing constitutively active IκB kinases will prove highly immunogenic also in vivo and possibly emerge as a new strategy to improve the clinical efficacy of therapeutic vaccinations against cancer and other chronic diseases.     

Enhanced cross‐priming of naive CD8+ T cells by dendritic cells treated by the IMiDs® immunomodulatory compounds lenalidomide and pomalidomide

The IMiDs^® immunomodulatory compounds lenalidomide and pomalidomide are agents with anti‐inflammatory, immunomodulatory and anti‐cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti‐tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow‐derived DCs treated with 5 or 10 μm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD^® immunomodulatory compounds increased expression of Class I (H2‐Kb), CD86, and pomalidomide also increased Class II (I‐Ab) expression in bone marrow‐derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μm pomalidomide or lenalidomide compared with non‐treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin‐specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8⁺ T‐cell cross‐priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4⁺ T‐cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.     

Chemokine programming dendritic cell antigen response: part II – programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype

In a companion article to this study,¹ the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre‐treatment of DCs with a specific ‘cocktail’ of select chemokines. Chemokine pre‐treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4⁺ T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre‐treated murine bone marrow‐derived DCs were examined in long‐term co‐culture with antigen‐specific CD4⁺ T cells to quantify how chemokine pre‐treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine‐treated DCs, OVA‐biased CD4⁺T‐cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon‐γ, interleukin‐1β, interleukin‐2 and interleukin‐10 in the CD4⁺ T cell : DC co‐culture (with or without chemokine pre‐treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC‐based vaccine efficiency.     

Vaccine molecules targeting Xcr1 on cross‐presenting DCs induce protective CD8+ T‐cell responses against influenza virus

Targeting antigens to cross‐presenting dendritic cells (DCs) is a promising method for enhancing CD8⁺ T‐cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross‐presenting murine CD8α⁺ DCs, and that the expression is conserved on homologous DC subsets in humans (CD141⁺ DCs), sheep (CD26⁺ DCs), and macaques (CADM1⁺ DCs). We therefore tested if targeting antigens to Xcr1 on cross‐presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α⁺ DCs and increased proliferation of antigen‐specific T cells. DNA vaccines encoding dimeric Xcl1‐hemagglutinin (HA) fusion proteins induced cytotoxic CD8⁺ T‐cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8⁺ T‐cell responses, targeting of antigen to Xcr1 induced CD4⁺ Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α⁺ DCs using Xcl1 represents a novel and promising method for induction of protective CD8⁺ T‐cell responses.     

TRAF2 regulates peripheral CD8+ T‐cell and NKT‐cell homeostasis by modulating sensitivity to IL‐15

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8⁺ T‐cell and NKT‐cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8⁺ T‐cell subsets. IL‐15‐dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8⁺CD44^hiCD122⁺ T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8⁺CD44^hi T cells exhibited impaired dose‐dependent proliferation to exogenous IL‐15. In contrast, TRAF2TKO CD8⁺ T cells proliferated normally to anti‐CD3 and TRAF2TKO CD8⁺CD44^hi T cells exhibited normal proliferation to exogenous IL‐2. TRAF2TKO CD8⁺ T cells expressed normal levels of IL‐15‐associated receptors and possessed functional IL‐15‐mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8⁺CD44^hiCD122⁺ and NKT cells was mechanistically linked to an inability to respond to IL‐15. The reduced CD8⁺CD44^hiCD122⁺ T‐cell and NKT‐cell populations in TRAF2TKO mice were rescued in the presence of high dose IL‐15 by IL‐15/IL‐15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8⁺ CD44^hiCD122⁺ T‐cell and NKT‐cell homeostasis by modulating sensitivity to T‐cell intrinsic growth factors such as IL‐15.     

Significant anti-tumour activity of adoptively transferred T cells elicited by intratumoral dendritic cell vaccine injection through enhancing the ratio of CD8+ T cell/regulatory T cells in tumour

We have shown that immunization with dendritic cells (DCs) pulsed with hepatitis B virus core antigen virus‐like particles (HBc‐VLP) packaging with cytosine–guanine dinucleotide (CpG) (HBc‐VLP/CpG) alone were able to delay melanoma growth but not able to eradicate the established tumour in mice. We tested whether, by modulating the vaccination approaches and injection times, the anti‐tumour activity could be enhanced. We used a B16‐HBc melanoma murine model not only to compare the efficacy of DC vaccine immunized via footpads, intravenously or via intratumoral injections in treating melanoma and priming tumour‐specific immune responses, but also to observe how DC vaccination could improve the efficacy of adoptively transferred T cells to induce an enhanced anti‐tumour immune response. Our results indicate that, although all vaccination approaches were able to protect mice from developing melanoma, only three intratumoral injections of DCs could induce a significant anti‐tumour response. Furthermore, the combination of intratumoral DC vaccination and adoptive T cell transfer led to a more robust anti‐tumour response than the use of each treatment individually by increasing CD8⁺ T cells or the ratio of CD8⁺ T cell/regulatory T cells in the tumour site. Moreover, the combination vaccination induced tumour‐specific immune responses that led to tumour regression and protected surviving mice from tumour rechallenge, which is attributed to an increase in CD127‐expressing and interferon‐γ‐producing CD8⁺ T cells. Taken together, these results indicate that repeated intratumoral DC vaccination not only induces expansion of antigen‐specific T cells against tumour‐associated antigens in tumour sites, but also leads to elimination of pre‐established tumours, supporting this combined approach as a potent strategy for DC‐based cancer immunotherapy.     

Dendritic cell homeostasis is maintained by nonhematopoietic and T‐cell‐produced Flt3‐ligand in steady state and during immune responses

Lymphoid‐tissue dendritic cells (DCs) are short‐lived and need to be continuously replenished from bone marrow‐derived DC progenitor cells. Fms‐related tyrosine kinase 3 is expressed during cellular development from hematopoietic progenitors to lymphoid‐tissue DCs. Fms‐related tyrosine kinase 3 ligand (Flt3L) is an essential, nonredundant cytokine for DC progenitor to lymphoid tissue DC differentiation and maintenance. However, which cells contribute to Flt3L production and how Flt3L cytokine levels are regulated in steady state and during immune reactions remains to be determined. Here we demonstrate that besides nonhematopoietic cells, WT T cells produce Flt3L and contribute to the generation of both classical DCs (cDCs) and plasmacytoid DCs in Flt3L^?/? mice. Upon stimulation in vitro, CD4⁺ T cells produce more Flt3L than CD8⁺ T cells. Moreover, in vivo stimulation of naïve OT‐II CD4⁺ T cells with OVA leads to increase of pre‐cDCs and cDCs in draining lymph nodes of Flt3L^?/? mice in a partially Flt3L‐dependent manner. Thus, Flt3L‐mediated lymphoid tissue DC homeostasis is regulated by steady‐state T cells as well as by proliferative T cells, fostering local development of lymphoid organ resident DCs.     

Combination of CTL‐associated Antigen‐4 Blockade and Depletion of CD25+ Regulatory T Cells Enhance Tumour Immunity of Dendritic Cell‐based Vaccine in a Mouse Model of Colon Cancer

Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via CTL‐associated antigen‐4 (CTLA‐4) blockade or removal of CD4⁺ CD25⁺ regulatory T (Treg) cells appears to be a promising strategy for cancer immunotherapy. In this study, we tested the hypothesis that the combination of CTLA‐4 blockade and depletion of Treg cells would improve the potency of dendritic cell (DC)‐based vaccine in a clinically relevant mouse model, which is transgenic for both carcinoembryonic antigen (CEA) and HLA‐A2 for the treatment of colon carcinoma in a therapeutic setting. We found that administration of anti‐CD25 antibody prior to vaccination or systemic administration of anti‐CTLA‐4 antibody with the vaccine improved tumour‐free survival against CEA‐expressing tumours compared with mice immunized with DC‐based vaccine alone. However, the efficacy of the vaccine proved to be most effective when anti‐CTLA‐4 antibody was combined with Treg inhibition. This vaccination strategy dramatically improved the tumour‐free survival and allowed the development of long‐lasting immune responses. The combined vaccination strategy resulted in increased secretion of IFN‐γ and enhanced HLA‐A2‐restricted CEA‐specific CTL responses. Furthermore, coadministration of anti‐CD25 and anti‐CTLA‐4 antibodies along with the vaccine was effective against more advanced tumours. These results provide evidence that simultaneous blockade of T‐cell regulatory pathways is a promising approach for the induction of therapeutic antitumour immunity against CEA⁺ colon carcinoma.     

Sustained cross‐presentation capacity of murine splenic dendritic cell subsets in vivo

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α⁺ DCs showed sustained MHC class I‐peptide specific CD8⁺ T‐cell activation for more than 4 days. CD8α^? DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α^? DCs were able to present antigen in MHC class II to specific CD4⁺ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.     

HIV gag protein is efficiently cross‐presented when targeted with an antibody towards the DEC‐205 receptor in Flt3 ligand‐mobilized murine DC

DC present exogenous proteins to MHC class I‐restricted CD8⁺ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8⁺ T‐cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8⁺ or DEC‐205⁺ DC are specialized for cross‐presentation, and this subset can be increased 10‐fold in numbers following Fms‐like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross‐presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti‐CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11c^high DC, particularly the DEC‐205⁺ subset. DC cross‐present HIV gag to primed CD8⁺ T cells, but when the antigen is delivered within an antibody to DEC‐205 receptor, cross‐presentation becomes 100‐fold more efficient than non‐targeted antigen. This finding requires gag to be engineered into anti‐DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross‐presentation, since their use overcomes the obstacle posed by the low number of cross‐presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC‐targeted vaccines.     

Topical rather than intradermal application of the TLR7 ligand imiquimod leads to human dermal dendritic cell maturation and CD8+ T‐cell cross‐priming

Toll‐like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra‐)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara‐treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro‐inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara‐stimulated DCs showed enhanced cross‐presentation of the melanoma antigen MART‐1, which resulted in increased priming and activation of MART‐1‐specific CD8⁺ T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.     

Binding immunoglobulin protein-treated peripheral blood monocyte-derived dendritic cells are refractory to maturation and induce regulatory T-cell development

Binding immunoglobulin protein (BiP) has been shown previously to have immunomodulatory functions. Herein we investigated whether BiP could affect the differentiation of monocytes into dendritic cells (DCs) and thence the development of regulatory T cells. Peripheral blood monocyte-derived DCs were matured with lipopolysaccharide in the presence or absence of BiP. DC development and T-cell changes were monitored by flow cytometry and regulatory T-cell function was measured by uptake of tritiated thymidine. More BiP-treated DCs (DC_(BiP)s) expressed amounts of intracellular indoleamine 2,3-dioxygenase (IDO) and cell surface leucocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), retained CD14 expression but down-regulated expression of human leucocyte antigen (HLA)-DR and CD86, and produced copious amounts of interleukin (IL)-10, when compared with control DCs. T cells co-cultured with DC_(BiP)s developed regulatory function with increased surface expression of CD4⁺ CD25^hi CD27^hi but with no concomitant increase in forkhead box P3 (Foxp3). These T cells also showed significantly higher levels of intracellular cytotoxic T-lymphocyte antigen (CTLA)-4. The latter could be inhibited by the presence of the IDO inhibitor 1 methyl tryptophan. The addition of neutralizing anti-IL-10 antibody or the specific mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 reversed the inhibition of DC differentiation by BiP. In conclusion, BiP is an immunomodulator able to arrest inflammation through induction of tolerogenic DCs and subsequent generation of T regulatory cells.     

Virulence‐dependent induction of interleukin‐10‐producing‐tolerogenic dendritic cells by Mycobacterium tuberculosis impedes optimal T helper type 1 proliferation

Mycobacterium tuberculosis inhibits optimal T helper type 1 (Th1) responses during infection. However, the precise mechanisms by which virulent M. tuberculosis limits Th1 responses remain unclear. Here, we infected dendritic cells (DCs) with the virulent M. tuberculosis strain H37Rv or the attenuated strain H37Ra to investigate the phenotypic and functional alterations in DCs and resultant T‐cell responses. H37Rv‐infected DCs suppressed Th1 responses more strongly than H37Ra‐infected DCs. Interestingly, H37Rv, but not H37Ra, impaired DC surface molecule expression (CD80, CD86 and MHC class II) due to prominent interleukin‐10 (IL‐10) production while augmenting the expression of tolerogenic molecules including PD‐L1, CD103, Tim‐3 and indoleamine 2,3‐dioxygenase on DCs in a multiplicity‐of‐infection (MOI) ‐dependent manner. These results indicate that virulent M. tuberculosis drives immature DCs toward a tolerogenic phenotype. Notably, the tolerogenic phenotype of H37Rv‐infected DCs was blocked in DCs generated from IL‐10^−/− mice or DCs treated with an IL‐10‐neutralizing monoclonal antibody, leading to restoration of Th1 polarization. These findings suggest that IL‐10 induces a tolerogenic DC phenotype. Interestingly, p38 mitogen‐activated protein kinase (MAPK) activation predominantly mediates IL‐10 production; hence, H37Rv tends to induce a tolerogenic DC phenotype through expression of tolerogenic molecules in the p38 MAPK–IL‐10 axis. Therefore, suppressing the tolerogenic cascade in DCs is a novel strategy for stimulating optimal protective T‐cell responses against M. tuberculosis infection.