TLR and nucleotide-binding oligomerization domain-like receptor signals differentially regulate exogenous antigen presentation

The effect of dendritic cell (DC) maturation on MHC class II-restricted Ag presentation is well studied, but less is known about the effects of DC maturation on MHC class I-restricted cross-presentation. We investigated the ability of mature DCs to present Ags from cells infected with HSV-1. Pretreatment with pure LPS increased cross-presentation in a manner dependent on both MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-β, whereas a similar dose of a less pure LPS preparation inhibited cross-presentation. The difference could not be attributed to differences in uptake or phenotypic maturation. The likely contaminant responsible for shutting down cross-presentation is peptidoglycan (PGN). Addition of PGN to pure LPS abrogated its ability to enhance cross-presentation. Direct activation of DCs with PGN inhibited cross-presentation through nucleotide-binding oligomerization domain-like receptor signaling. These results demonstrate that different maturation stimuli can have opposite impacts on the ability of DCs to cross-present viral Ags.     

The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells

The FcγRs found on macrophages (Ms) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Ms and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8(+) T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4(+) T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Ms but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4(+) T cells in the endosomal versus phagosomal compartments of Ms versus DCs.     

The acquisition of antigen cross-presentation function by newly formed dendritic cells

The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8(+) DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8(+) DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.     

Regulated expression of FcgammaR in human dendritic cells controls cross-presentation of antigen-antibody complexes

Receptors for IgG (FcgammaR) expressed in dendritic cells (DCs) influence the initiation of Ab-mediated immunity. Dynamic variations in FcgammaR expression allow DCs to adjust their capacity to capture Ab-opsonized Ag. The current paradigm predicts a progressive decline in FcgammaR-mediated phagocytic function upon DC maturation. Surprisingly, we find that expression of the phagocytic receptor FcgammaRIIa is preserved in immature and mature DCs at comparable levels with macrophages. Moreover, phagocytosis of antigenic peptides directed to FcgammaRIIa on DCs leads to dramatic increases in Ag cross-presentation and T cell activation. In immature DCs, high expression of inhibitory FcgammaRIIb correlates with decreased uptake and cross-presentation of Ab-Ag complexes. In contrast, engagement of FcgammaRIIb is not associated with changes in cross-presentation in mature DCs. We provide evidence that FcgammaRIIb expression is patently reduced in mature DCs, an effect that is modulated by treatment with cytokines. The regulated expression of activating and inhibitory FcgammaRs in DCs emerges as a critical checkpoint in the process of Ag uptake and cross-presentation.     

Dendritic cells matured in the presence of the lycopodium alkaloid annotine direct T cell responses toward a Th2/Treg phenotype

Annotine is a lycopodane-type alkaloid isolated from the Icelandic club moss Lycopodium annotinum ssp. alpestre. Annotine does not inhibit acetylcholinesterase, as some other lycopodium alkaloids do, and other bioactivities have not been reported. The aim of this study was to determine the effects of annotine on maturation of dendritic cells (DCs) and their ability to activate allogeneic CD4⁺ T cells. Human monocyte-derived DCs were matured in the absence or presence of annotine at a concentration of 1, 10 or 100 μg/ml. The effect of the annotine on maturation of the DCs was determined by measuring concentration of cytokines in culture supernatant by ELISA and expression of surface molecules by flow cytometry. DCs matured in the absence or presence of annotine at 100 µg/ml were also co-cultured with allogeneic CD4⁺ T cells and concentration of cytokines in supernatants determined by ELISA and expression of surface molecules by flow cytometry. When cultured alone, DCs matured in the presence of annotine secreted less of the pro-inflammatory cytokines IL-6 and IL-23 and had a tendency toward less secretion of IL-12p40 than DCs matured in the absence of annotine. However, when DCs were matured in the presence of annotine and then co-cultured with allogeneic CD4⁺ T cells they secreted more IL-12p40 and had a tendency toward secreting more IL-6 than DCs matured in the absence of annotine and then co-cultured with T cells. Allogeneic CD4⁺ T cells co-cultured with DCs matured in the presence of annotine secreted more IL-13 than T cells co-cultured with DCs matured in the absence of annotine, but stimulating the DCs in the presence of annotine did not affect T cell secretion of IFN-γ and IL-17. There was also more IL-10 in co-cultures of T cells and DCs matured in the presence of annotine than in co-cultures of T cells and DCs matured in the absence of annotine. These results show that annotine increases the ability of DCs to direct the differentiation of allogeneic CD4⁺ T cells toward a Th2/Treg phenotype, which may be of interest in the development of new treatments for Th1- and/or Th17-mediated inflammatory diseases.     

Cutting edge: Toll-like receptor 9 expression is not required for CpG DNA-aided cross-presentation of DNA-conjugated antigens but essential for cross-priming of CD8 T cells

Covalent linkage of immunostimulatory CpG DNA to OVA results in CpG DNA-aided cross-presentation of OVA by dendritic cells (DCs). In vivo, cross-presentation is conditional for cross-priming of OVA-specific CD8 T cells. In this study, we investigated the involvement of the CpG DNA receptor Toll-like receptor (TLR)9 in CpG DNA-aided cross-presentation and cross-priming. Although CpG DNA-aided cross-presentation is not altered in TLR9-deficient cells, TLR9 is required for maturation of APC allowing cross-priming, as resulting in CTL function. These findings imply that TLR9 does not trigger endocytosis of CpG-OVA conjugates, but activates DCs downstream of endocytosis.     

NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells

To initiate adaptative cytotoxic immune responses, proteolytic peptides derived from phagocytosed antigens are presented by dendritic cells (DCs) to CD8+ T lymphocytes through a process called antigen "crosspresentation." The partial degradation of antigens mediated by lysosomal proteases in an acidic environment must be tightly controlled to prevent destruction of potential peptides for T cell recognition. We now describe a specialization of the phagocytic pathway of DCs that allows a fine control of antigen processing. The NADPH oxidase NOX2 is recruited to the DC's early phagosomes and mediates the sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosomal acidification and increased antigen degradation, resulting in impaired crosspresentation. Therefore, NOX2 plays a critical role in conferring DCs the ability to function as specialized phagocytes adapted to process antigens rather than kill pathogens.     

Variable Processing and Cross-presentation of HIV by Dendritic Cells and Macrophages Shapes CTL Immunodominance and Immune Escape

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

Histamine improves antigen uptake and cross-presentation by dendritic cells

Previous studies have shown that histamine is able to modulate the function of dendritic cells (DCs). Histamine seems to be required for the normal differentiation of DCs. Moreover, it is capable of stimulating the chemotaxis of immature DCs and of promoting the differentiation of T CD4+ cells into a Th2 profile. In this study, we analyzed whether histamine was able to modulate endocytosis and cross-presentation mediated by immature DCs. Our results show that both functions are stimulated by histamine. Endocytosis of soluble HRP and FITC-OVA and cross-presentation of soluble OVA were markedly increased by histamine. Interestingly, stimulation of endocytosis and cross-presentation appeared to be mediated through different histamine receptors. In fact, the enhancement of endocytosis was prevented by the histamine2 receptor (H2R) antagonist cimetidine, whereas the stimulation of cross-presentation was prevented by the H3R/H4R antagonist thioperamide. Of note, contrasting with the observations made with soluble Ags, we found that histamine did not increase either the uptake of OVA-attached to latex beads, or the cross-presentation of OVA immobilized on latex beads. This suggests that the ability of histamine to increase endocytosis and cross-presentation is dependent on the Ag form and/or the mechanisms through which the Ag is internalized by DCs. Our results support that histamine may favor cross-presentation of soluble allergens by DCs enabling the activation of allergen-specific T CD8+ cells, which appears to play an important role in the development of allergic responses in the airway.     

Extracellular ATP induces a distorted maturation of dendritic cells and inhibits their capacity to initiate Th1 responses

Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1alpha, IL-1beta, TNF-alpha, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA(+) T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-gamma and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-alpha and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses.     

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.     

Mixture of fibroblast, epithelial and endothelial cells conditioned media induce monocyte-derived dendritic cell maturation

Fully matured DCs with large amount cytoplasm and copious dendritic projections were visible at the end of culturing period in the presence of MCM, TNF-α and poly (I:C), with or without FEECM. Thus, DCs generated with these maturation factors are nonadherent and have typical satellite morphology. Flow cytometric analysis using anti-CD14, -CD80, -CD86, -HLA-DR and -CD83 revealed that expression of CD14 is decreased in particular in FEECM treated DCs, on day 5 and expression of CD80, CD86 and HLA-DR was the higher when FEECM are added to maturation factor. Functionally, when DCs matured in the presence of FEECM elicited stronger MLR, reduced phagocytic activity. These results support the use of the FEECM with MCM, TNF-α and poly (I–C) as maturation factor in DC generation that could result in functionally mature monocyte-derived DCs in comparison to either alone.     

Cellubrevin alterations and Mycobacterium tuberculosis phagosome maturation arrest

The intracellular trafficking processes controlling phagosomal maturation remain to be fully delineated. Mycobacterium tuberculosis var. bovis BCG, an organism that causes phagosomal maturation arrest, has emerged as a tool for dissection of critical phagosome biogenesis events. In this work, we report that cellubrevin, a v-SNARE functioning in endosomal recycling and implicated in endosomal interactions with post-Golgi compartments, plays a role in phagosomal maturation and that it is altered on mycobacterial phagosomes. Both mycobacterial phagosomes, which undergo maturation arrest, and model phagosomes containing latex beads, which follow the normal pathway of maturation into phagolysosomes, acquired cellubrevin. However, the mycobacterial and model phagosomes differed, as a discrete proteolytic degradation of this SNARE was detected on mycobacterial phagosomes. The observed cellubrevin alteration on mycobacterial phagosomes was not a passive event secondary to a maturation arrest at another checkpoint of the phagosome maturation pathway, since pharmacological inhibitors of phagosomal/endosomal pathways blocking phagosomal maturation did not cause cellubrevin degradation on model phagosomes. Cellubrevin status on phagosomes had consequences on phagosomal membrane and lumenal content trafficking, involving plasma membrane marker recycling and delivery of lysosomal enzymes. These results suggest that cellubrevin plays a role in phagosomal maturation and that it is a target for modification by mycobacteria or by infection-induced processes in the host cell.     

Influenza A Virus Infection of Human Primary Dendritic Cells Impairs Their Ability to Cross-Present Antigen to CD8 T Cells

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.     

Ascaris lumbricoides pseudocoelomic body fluid induces a partially activated dendritic cell phenotype with Th2 promoting ability in vivo

Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do not follow conventional definitions of DC maturation processes. While a number of models of DC maturation by Th2 stimuli are postulated, further studies are required if we are to clearly define DC maturation processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides pseudocoelomic body fluid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage inflammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hallmarks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1 and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken together, we believe this is the first paper to demonstrate A. lumbricoides murine DC-Th cell-driven responses shedding further light on DC maturation processes by helminth antigens.     

Vascular endothelial growth factor inhibits maturation of dendritic cells induced by lipopolysaccharide,but not by proinflammatory cytokines

Purpose: Dendritic cells (DCs) play an important role in the hosts immunosurveillance against cancer. It has been shown that the function of DCs is impaired and their population decreased in a cancer-bearing host. In the present study, we investigated the mechanism of down-regulation of DCs in a cancer-bearing host. Methods: We evaluated the relationship between DC infiltration and production of vascular endothelial growth factor (VEGF) in carcinoma tissue by immunohistochemistry. Furthermore, functional and phenotypical alterations of DCs were evaluated when monocyte-derived, mature DCs were treated with VEGF in vitro. Monocyte-derived DCs were generated in a culture of monocyte with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and the maturation of DCs was induced by either lipopolysaccharide (LPS) or a proinflammatory cytokine cocktail: tumor-necrosis factor , prostaglandin E₂, IL-6, and IL-1. Results: A significant inverse correlation was found between the density of DCs and the quantity of VEGF production in gastric carcinoma tissue (r=–0.39, p<0.05). In LPS-induced maturation, the ability of mature DCs to stimulate allogenic T cells and produce IL-12 (p70 heterodimer) was suppressed by the addition of VEGF in a dose-dependent manner. A lesser expression of costimulatory molecules (CD80 and CD86) was seen in DCs treated with exogenous VEGF than in DCs not treated with VEGF. The population of dead DCs (early and late apoptosis) treated with VEGF increased more than that without VEGF treatment, using the annexin V and propidium iodide evaluation in DCs matured by LPS. In contrast, in DCs matured by the proinflammatory cytokine cocktail, the down-regulation of costimulatory molecules and induction of DC apoptosis was not seen. Conclusions: These findings show that the inhibition of DC maturation by VEGF differs depending on the maturation status of the DCs.Abbreviations APC antigen-presenting cells - DC dendritic cells - ELISA enzyme-linked immunosorbent assay - FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - HLA human leukocyte antigen - IL interleukin - LPS lipopolysaccharide - mAb monoclonal antibody - MHC major histocompatibility complex - PBS phosphate-buffered saline - PCNA proliferative cell nuclear antigen - PE phycoerythrin - PG prostaglandin - PI propidium iodide - TNF tumor-necrosis factor - VEGF vascular endothelial growth factor This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan.     

Extracellular acidosis triggers the maturation of human dendritic cells and the production of IL-12

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-gamma, but not IL-4, by Ag-specific CD4(+) T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.     

Increased expression of HSP70 by colon cancer cells is not always associated with access to the dendritic cell cross-presentation pathway

Dendritic cells (DCs) are highly specialized antigen-presenting cells endowed with the unique ability to not only present exogenous antigens upon exposure to MHC II, but also to cross-present these upon exposure to MHC I. This property was exploited to generate the tumor-specific CD8 cytotoxic lymphocyte (CTL) response in DCs-based cancer vaccine protocols. In this context, the source of tumor antigens remains a critical challenge. A crude tumor in the context of danger signals is believed to represent an efficient source of tumor antigens (TAs) for DCs loading. In our previous work, increased DCs cross-presentation of antigens from necrotic gastric carcinoma cells paralleled up-regulation of the heat shock protein hsp70. We studied the expression of hsp70 on primary colon carcinoma cells and its relevance in the cross-priming of anti-tumor CTL by tumor-loaded DCs. Hsp70 was expressed on all three of the tumors studied, but was never detected in the peritumoral normal mucosa (NM). The uptake of the tumor induced a trend towards down-modulation of the monocyte-specific marker CD14, but had no effect on the chemokine receptors CCR4 and CCR7. The IFN-γ enzyme-linked immunospot assay (ELIspot) showed cross-priming of CTL by tumor-loaded but not NM-loaded DCs in four of the six cases studied. The CTL response generated in DC+tumor cultures was directed towards the tumor, but not towards NM, and it was characterized by refractoriness to polyclonal (Ca ionophores, PKC activators) stimuli. Of the three CTL-generating tumors, only one expressed hsp70. This data indicates a tumor-specific expression of hsp70, but does not support its relevance in the DC cross-presentation of TAs.