Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response

Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes na?ve CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Y. enterocolitica inhibits antigen degradation in dendritic cells

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.     

Emerging evidence indicates that physiologically relevant thermal stress regulates dendritic cell function

Summary Elevations in temperature that are associated with inflammation or fever have been linked to improved survival from infections, enhanced immunological functions, and increased control of tumor growth. Over the past few years, several groups have begun to explore the possible linkage among these observations and have tested the hypothesis that various immune cells are especially sensitive to thermal stimulation. However, relatively little is known regarding the effects of thermal stimulation on antigen presenting cells (APCs), such as dendritic cells (DCs). Very recently, several groups have begun to examine the ability of thermal stimuli to regulate the function of these cells which are known to play a pivotal role in the efficacy of vaccines and other immunotherapies. In this review, we summarize what has been discovered about the role of mild thermal stress in regulating various Dendritic cell (DC) activities. Excitingly, it appears that mild elevations of temperature have the potential to enhance antigen uptake, activation associated migration, maturation, cytokine expression and T cell stimulatory activity of DCs. While these studies reveal that the timing, temperature and duration of heating is important, they also set the stage for essential questions that now need to be investigated regarding the molecular mechanisms by which elevated temperatures regulate DC function. With this information, we may soon be able to maximize the strategic use of thermal therapy as an adjuvant, i.e., combining its use with cancer immunotherapies such as vaccines, which depend upon the function of DCs. Several possible strategies and timepoints involving the clinical application of hyperthermia in combination with immunotherapy are presented. This article forms part of the Symposium in Writing “Thermal stress-related modulation of tumor cell physiology and immune responses”, edited by Elfriede Noessner.     

Delivery of a heterologous antigen by a registered Salmonella vaccine (STM1)

STM1 is an aro A(-) attenuated mutant of Salmonella enterica serovar Typhimurium, and is a well-characterised vaccine strain available to the livestock industry for the prevention of salmonellosis in chickens. This strain has potential for heterologous antigen delivery, and here we show that the strain can be used to deliver a model antigen, ovalbumin, to immune cells in vitro and in vivo. Two plasmid constructs expressing the ovalbumin gene were utilised, one of which uses a prokaryotic promoter and the other the CMV promoter (DNA vaccine). In vitro, STM1 carrying ovalbumin-encoding plasmids was able to invade dendritic cells and stimulate a CD8(+) cell line specific for the dominant ovalbumin epitope, SIINFEKL. In vivo, spleen cells were responsive to SIINFEKL after vaccination of mice with ovalbumin-encoding plasmids in STM1, and finally, humoral responses, including IgA, were induced after vaccination.     

Myosin IIa activation is crucial in breast cancer derived galectin-1 mediated tolerogenic dendritic cell differentiation

Background

Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.

Methods

Circulating CD14⁺CD16⁺ of breast cancer patients and induced CD14⁺CD16⁺ DCs were identified as tDCs by treating CD14⁺ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.

Results

The CD14⁺CD16⁺ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c⁺ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c⁺/Ly6G⁺/IL-10⁺ in the BALB/c mice.

Conclusions

Galectin-1 can induce tDCs after internalizing into CD14⁺ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.

General significance

Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.     

Classical CD11c dendritic cells, not plasmacytoid dendritic cells, induce T cell responses to Plasmodium chabaudi malaria

Dendritic cells play an important role in the development of immune responses in malaria, but the contribution of plasmacytoid dendritic cells (pDC) to CD4 T cell activation and immunopathology is unknown. We have investigated pDC in a Plasmodium chabaudi infection in mice. During infection, pDC increased in number and transiently up-regulated expression of Major Histocompatibility Complex class II and co-stimulatory molecules. However, in contrast to classical CD11c^high DC, pDC could not phagocytose parasites or process parasite proteins, to activate CD4 T cells. Activation of naïve pDC, but not CD11c^high DC, by infected red blood cells induced IFNα in vitro, which was dependent on the Toll-like receptor, TLR9. However, inactivation of TLR9 in knock-out mice had no effect on a P. chabaudi infection suggesting that TLR9 was not crucial for parasite elimination or pathology. Neither pDC nor IFNαβ were essential for parasite clearance as mice depleted of pDC or IFNαβ Receptor-knock-out mice could control infection. However, these mice lost significantly more weight than untreated or wild-type mice. We conclude that classical DC are the major antigen-presenting cells for CD4 T cells in this infection, but that pDC and IFNαβ may play minor roles in controlling the magnitude of acute stage pathology.     

An assay for functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors of human dendritic cell adhesion

We report a new dendritic cell adhesion assay, using either immature or mature dendritic cells, for identifying functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors. Because immature dendritic cells are responsible for pathogen binding and invasion, this in vitro assay provides an important link between in vitro results and pathogen-based in vivo assays. Furthermore, this assay does not require laborious expression, refolding, and purification of DC-SIGN carbohydrate recognition domain or extracellular domain as receptor-based assays. The assay power evaluated with Z and Z′ parameters enables screening of compound libraries and determination of IC₅₀ values in the first stage of DC-SIGN inhibitor development.     

Mycobacterium bovis BCG decreases MHC-II expression in vivo on murine lung macrophages and dendritic cells during aerosol infection

Mycobacterium tuberculosis and M. bovis BCG infect APCs. In vitro, mycobacteria inhibit IFN-gamma-induced MHC-II expression by macrophages, but the effects of mycobacteria on lung APCs in vivo remain unclear. To assess MHC-II expression on APCs infected in vivo, mice were aerosol-infected with GFP-expressing BCG. At 28 d, ∼1% of lung APCs were GFP+ by flow cytometry and CFU data. Most GFP+ cells were CD11b^high/CD11c^neg-mid lung macrophages (58-68%) or CD11b^high/CD11c^high DCs (28-31%). Lung APC MHC-II expression was higher in infected mice than naïve mice. Within infected lungs, however, MHC-II expression was lower in GFP+ cells than GFP− cells for both macrophages and DCs. MHC-II expression was also inhibited on purified lung macrophages and DCs that were infected with BCG in vitro. Thus, lung APCs that harbor mycobacteria in vivo have decreased MHC-II expression relative to uninfected APCs from the same lung, possibly contributing to evasion of T cell responses.     

Scavenger receptor-A functions in phagocytosis of E. coli by bone marrow dendritic cells

Class-A scavenger receptors (SR-A) are cellular pattern recognition receptors that bind and traffic a variety of endogenous and microbial ligands. However, despite an emerging role for SR-A as a contributor to the innate immune system, little is known of the regulation or function of SR-A on dendritic cells (DCs). Here we show that SR-A expression is upregulated during murine DC differentiation and that SR-A expression levels correlate with the expression of the murine DC marker CD11c. Using bone marrow-derived DCs (BMDCs) from SR-A knockout (SR-A(-/-)) mice, we investigated the contribution of SR-A to BMDC particulate phagocytosis. Functional analyses demonstrated that SR-A is a critical phagocytic receptor for BMDC internalization of the gram-negative bacteria E. coli. SR-A(-/-) BMDCs were impaired in their ability to phagocytose bacteria, and this deficit varied with the bacteria:BMDC cell ratio. Microscopic and biochemical analyses revealed that SR-A is broadly distributed on the surface of BMDCs and is not physically associated with lipid rafts. However, cholesterol depletion demonstrated dependence of SR-A-mediated phagocytosis upon lipid rafts. These data demonstrate a functional contribution for SR-A in the BMDC phagocytic pathway.     

Accelerated antigen presentation and elicitation of humoral response in vivo by FcgammaRIIB- and FcgammaRI/III-mediated immune complex uptake

It is well established that activating-type Fc receptors for IgG (FcgammaR), such as FcgammaRI and FcgammaRIII, are essential for inducing inflammatory responses, whereas a unique inhibitory FcgammaR, FcgammaRIIB, inhibits intracellular signaling upon ligation of IgG-immune complexes, and can suppress inflammation and autoimmunity. Although antigen presentation is a crucial step for evoking inflammatory responses, the contribution of FcgammaRIIB to antigen presentation is controversial as to whether it regulates antigen-presenting cells (APC), particularly dendritic cells (DC), positively or negatively. In the present report, we show that the antigen targeting to both activating-type FcgammaRs, FcgammaRI/III, and inhibitory FcgammaRIIB on bone marrow-derived DC and macrophages and primary epidermal Langerhans' cells augmented T cell proliferation in vitro and elicited humoral responses upon adoptive transfer of the antigen-pulsed DC. The DC lacking FcgammaRIIB showed a reduction in IC-uptake ability and a decreased T-cell stimulation, and induced less efficient IgG production than those of DC from wild-type mice. On the other hand, the DC lacking FcR common gamma subunit, which only expresses FcgammaRIIB, showed significant up-regulations of IC-uptake, T-cell proliferation, and IgG production compared to those of FcgammaR null DC, demonstrating a positive regulation of FcgammaRIIB for the efficient antigen presentation of IgG-complexed antigens. These results support the therapeutic benefits of antigen-targeting to FcgammaR on APC in the various inflammatory disorders.     

Distinctive dendritic cell modulation by vitamin D(3) and glucocorticoid pathways

Dendritic cell (DC) maturation plays a central role in regulating immunity. We show that glucocorticoid and 1alpha,25(OH)(2)D(3) agonists modulate DCs via distinct and additive signaling pathways. Phenotypic and functional indices were examined in DCs treated with dexamethasone (DEX) and/or a 1alpha,25(OH)(2)D(3) analog (D(3) analog). DEX potently attenuated pro-inflammatory cytokines and chemokines but had modest, reversible effects on T-cell stimulatory capacity. D(3) analog produced significantly greater inhibition of T-cell stimulation in vitro and in vivo and, unlike DEX, increased expression of the chemokines MCP-1 and MIP-1alpha. Both DEX and D(3) analog were associated with reduced expression of the NF-kappaB proteins c-Rel and Rel B but not Rel A. Combined DEX and D(3) analog treatment of DCs resulted in significant additive inhibition of pro-inflammatory cytokines, T-cell stimulation, chemokines, chemokine receptors, and NF-kappaB components. Additive inhibition was most striking for RANTES, CCR5, CCR7, and Rel B. The combined effects of the two hormonal pathways on DCs have unique immunomodulatory potential.     

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4

All-trans-retinoic acid (atRA) appears to affect Th1-Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-α. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor.     

NK cell activation by dendritic cell vaccine: a mechanism of action for clinical activity

Recent reports revealed that dendritic cell (DC)–natural killer (NK) cell interaction plays an important role in tumor immunity, but few DC vaccine studies have attempted to evaluate the non-specific, yet potentially clinically relevant, NK response to immunization. In this study, we first analyzed in vitro activation of NK cells by DCs similar to those used in clinical trials. Subsequently, NK cell responses were analyzed in a phase I clinical trial of a vaccine consisting of autologous DCs loaded with a fowlpox vector encoding CEA. The data were compared with the clinical outcome of the patients. DC enhances NK activity in vitro, partly by sustaining NK cell survival and by enhancing the expression of NK-activating receptors, including NKp46 and NKG2D. Among nine patients in our clinical trial, NK cytolytic activity increased in four (range 2.5–5 times greater lytic activity) including three who had increased NK cell frequency, was stable in two and decreased in three. NKp46 and NKG2D expression showed a good correlation with the patients’ NK activity. When patients were grouped by clinical activity (stable disease/no evidence of disease (stable/NE, n=5) vs progressive disease (N=4) at 3 months), the majority in the stable/NE group had increases in NK activity (P=0.016). Anti-CEA T cell response was enhanced in all the nine patients analyzed, but was not significantly different between the two groups (P=0.14). Thus, NK responses following DC vaccination may correlate more closely with clinical outcome than do T cell responses. Monitoring of NK response during vaccine studies should be routinely performed.     

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4–Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4–Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.Dhanalakshmi Chinnasamy and Matt Tector contributed equally to this work     

Selected allogeneic dendritic cells markedly enhance human tumour antigen-specific T cell response in vitro

Background  Alloreaction is known to accumulate several theoretical advantages that can improve dendritic cell (DC)-based anti-infective or antitumour strategies. Allogeneic DC have already been tested in experimental and clinical studies, but their efficacy compared with their autologous counterparts was rarely investigated and conclusions diverge. Objective  This study compared antigen-specific T cell responses following priming with autologous versus allogeneic DC and examined the possibility of screening these responses in order to select allogeneic DC that lead to a great amplification. Results  Allogeneic DC obtained from donors matched with the single HLA-A2 allele were efficient in generating in vitro peptide-specific T cell responses. When randomly chosen, allogeneic DC generated a broad range of antigen-specific T cell responses in comparison with autologous DC. When screened and selected, allogeneic DC markedly enhanced peptide-specific T cell priming and allowed a more efficient boosting of resulting T cells. These selected allogeneic DC provided a favourable cytokinic and cellular environment that can help concurrent antigen-specific responses. Conclusion  Ex vivo selected allogeneic DC provide adjuvant effects that lead to amplification of concomitant antigen-specific T cell responses. A. Gervais and J.-C. Eymard contributed equally to this work.     

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.