Small rho GTPases regulate antigen presentation in dendritic cells

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms maintaining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was significantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo.     

Intracellular Salmonella inhibit antigen presentation by dendritic cells

Dendritic cells (DC) are important APCs linking innate and adaptive immunity. During analysis of the intracellular activities of Salmonella enterica in DC, we observed that viable bacteria suppress Ag-dependent T cell proliferation. This effect was dependent on the induction of inducible NO synthase by DC and on the function of virulence genes in Salmonella pathogenicity island 2 (SPI2). Intracellular activities of Salmonella did not affect the viability, Ag uptake, or maturation of DC, but resulted in reduced presentation of antigenic peptides by MHC class II molecules. Increased resistance to reinfection was observed after vaccination of mice with SPI2-deficient Salmonella compared with mice vaccinated with SPI2-proficient Salmonella, and this correlated with an increased amount of CD4(+) as well as CD8(+) T cells. Our study is the first example of interference of an intracellular bacterial pathogen with Ag presentation by DC. The subversion of DC functions is a novel strategy deployed by this pathogen to escape immune defense, colonize host organs, and persist in the infected host.     

Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.     

Murine gammaherpesvirus-68 inhibits antigen presentation by dendritic cells

Dendritic cells (DCs) play a central role in initiating adaptive immunity. Murine gammaherpesvirus-68 (MHV-68), like many persistent viruses, infects DCs during normal host colonization. It therefore provides a means to understanding what host and viral genes contribute to this aspect of pathogenesis. The infected DC phenotype is likely to depend on whether viral gene expression is lytic or latent and whether antigen presentation is maintained. For MHV-68, neither parameter has been well defined. Here we show that MHV-68 infects immature but not mature bone marrow-derived DCs. Infection was predominantly latent and these DCs showed no obvious defect in antigen presentation. Lytically infected DCs were very different. These down-regulated CD86 and MHC class I expression and presented a viral epitope poorly to CD8(+) T cells. Antigen presentation improved markedly when the MHV-68 K3 gene was disrupted, indicating that K3 fulfils an important function in infected DCs. MHV-68 infects only a small fraction of the DCs present in lymphoid tissue, so K3 expression is unlikely to compromise significantly global CD8(+) T cell priming. Instead it probably helps to maintain lytic gene expression in DCs once CD8(+) T cell priming has occurred.     

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

BACKGROUND: Transplantable B16 melanoma is widely used as a tumor model to investigate tumor immunity. We wished to characterize the leukocyte populations infiltrating B16 melanoma tumors, and the functional properties of tumor-infiltrating dendritic cells (TIDC). MATERIALS AND METHODS: We used the B16 melanoma cell line expressing ovalbumin protein (OVA) to investigate the phenotype and T cell stimulatory capacity of TIDC. RESULTS: The majority of leukocytes in B16 melanoma were macrophages, which colocalized with TIDCs, B and T cells to the peripheral area of the tumor. Both myeloid and plasmacytoid DC populations were present within tumors. Most of these DCs appeared immature, but about a third expressed a mature phenotype. TIDCs did not present tumor-derived antigen, as they were unable to induce the proliferation of tumor-specific CD4+ and CD8+ T cells in vitro unless in the presence of specific peptides. Some presentation of tumor-derived antigen could be demonstrated in the tumor-draining lymph node using in vivo proliferation assays. However, while proliferation of CD8+ T cells was reproducibly demonstrated, no proliferation of CD4+ T cells was observed. CONCLUSION: In summary, our data suggest that DCs in tumors have limited antigen-presenting function. Inefficient antigen presentation extends to the tumor-draining lymph node, and may affect the generation of antitumor immune responses.     

LILRA2 activation inhibits dendritic cell differentiation and antigen presentation to T cells

The differentiation of monocytes into dendritic cells (DC) is a key mechanism by which the innate immune system instructs the adaptive T cell response. In this study, we investigated whether leukocyte Ig-like receptor A2 (LILRA2) regulates DC differentiation by using leprosy as a model. LILRA2 protein expression was increased in the lesions of the progressive, lepromatous form vs the self-limited, tuberculoid form of leprosy. Double immunolabeling revealed LILRA2 expression on CD14+, CD68+ monocytes/macrophages. Activation of LILRA2 on peripheral blood monocytes impaired GM-CSF induced differentiation into immature DC, as evidenced by reduced expression of DC markers (MHC class II, CD1b, CD40, and CD206), but not macrophage markers (CD209 and CD14). Furthermore, LILRA2 activation abrogated Ag presentation to both CD1b- and MHC class II-restricted, Mycobacterium leprae-reactive T cells derived from leprosy patients, while cytokine profiles of LILRA2-activated monocytes demonstrated an increase in TNF-alpha, IL-6, IL-8, IL-12, and IL-10, but little effect on TGF-beta. Therefore, LILRA2 activation, by altering GM-CSF-induced monocyte differentiation into immature DC, provides a mechanism for down-regulating the ability of the innate immune system to activate the adaptive T cell response while promoting an inflammatory response.     

Effect of HIV on antigen presentation by dendritic cells and macrophages

The antigen-presenting function of dendritic cells (DC) and macrophages (MO) following infection with HIV in vitro was examined. Using non-infected cells, DC, but not MO, stimulated primary proliferative responses in allogeneic lymphocytes in the mixed leukocyte reaction. Both DC and MO stimulated secondary responses to influenza virus and to tetanus toxoid in autologous T lymphocytes. After exposure of DC and MO to HIV1 in vitro for 2 days, 27 % of DC but < 1 % MO became infected as assessed by in situ hybridization. DC were blocked in their capacity to stimulate responses to alloantigens or to the recall antigens. By contrast, MO retained the ability to stimulate responses to the recall antigens. Similar effects during in vivo infection would allow activated T-cell clones to respond to antigens presented by MO early in infection. However, any loss of activated T cells might prove cumulative and damaging in the absence of an effective DC recruitment mechanism for resting T cells.     

The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells

Dendritic cells (DCs) capture Ags or viruses in peripheral tissue to transport them to lymphoid organs to induce cellular T cell responses. Recently, a DC-specific C-type lectin was identified, DC-specific ICAM-grabbing non-integrin (DC-SIGN), that functions as cell adhesion receptor mediating both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R that captures HIVgp120 and facilitates DC-induced HIV transmission of T cells. Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function of DC-SIGN as endocytic receptor. In this study we demonstrate that on DCs DC-SIGN is rapidly internalized upon binding of soluble ligand. Mutating a putative internalization motif in the cytoplasmic tail reduces ligand-induced internalization. Detailed analysis using ratio fluorescence imaging and electron microscopy showed that DC-SIGN-ligand complexes are targeted to late endosomes/lysosomes. Moreover, ligands internalized by DC-SIGN are efficiently processed and presented to CD4+ T cells. The distinct pattern of expression of C-type lectins on DCs in situ and their nonoverlapping Ag recognition profile hint to selective functions of these receptors to allow a DC to recognize a wide variety of Ags and to process these to induce T cell activation. These data point to a novel function of the adhesion receptor DC-SIGN as an efficient DC-specific Ag receptor that can be used as a target to induce viral and antitumor immunity.     

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.     

Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells

Vaccination with dendritic cells (DCs) pulsed with antigenic peptides derived from various tumor antigens has great, but as yet significantly unrealized, potential in cancer treatment. Here, we describe a strategy for prolonged presentation of an MHC class I-restricted self-peptide on DCs through linkage of it to a cell penetrating peptide (CPP). DCs loaded with a peptide derived from tyrosinase-related protein 2 (TRP2) covalently linked to a CPP1 sequence retained full capacity to stimulate T cells for at least 24 h, completely protected immunized mice from subsequent tumor challenge, and significantly inhibited lung metastases in a 3-day tumor model. DCs pulsed with TRP2 alone failed to provide any of these protections. In addition, we demonstrate that both CD4+ and CD8+ T cells were required for potent antitumor immunity. This CPP-based approach may be generally applicable to enhance the efficacy of DC-based peptide vaccines against cancer and other diseases.     

B cells drive early T cell autoimmunity in vivo prior to dendritic cell-mediated autoantigen presentation

Both B cells and dendritic cells (DCs) have been implicated as autoantigen-presenting cells in the activation of self-reactive T cells. However, most self-proteins are ubiquitously and/or developmentally expressed, making it difficult to determine the source and the exposure of autoantigens to APCs in a controlled manner. In this study, we have used an Ig transgenic mouse model to examine the mechanisms by which B cells and other APCs acquire and present lupus autoantigens in vivo. Targeting a lupus autoantigen, the small nuclear ribonucleoprotein particle D protein, to the BCR activates autoreactive T cells in the periphery. Our in vivo studies demonstrate that autoantigen-specific B cells, when present in the repertoire, are the first subset of APCs to capture and present self-proteins for activating T cells. Thereafter, DCs acquire self-Ag and become effective APCs for stimulating the same subsets of autoreactive T cells. This mechanism provides one explanation of how early steps in autoimmunity can focus responses, via BCR, at a small group of self-proteins among the total milieu of intracellular self-proteins. Subsequently, DCs and other professional APCs may then amplify and perpetuate the autoimmune T cell response.     

Endosomal sorting of MHC class II determines antigen presentation by dendritic cells

Dendritic cells (DCs) initiate primary immune responses by presenting pathogen-derived antigens in association with major histocompatibility Class II molecules (MHC II) to T cells. In DCs, MHC II is constitutively synthesized and loaded at endosomes with peptides from hydrolyzed endogenous proteins or exogenously acquired antigens. Whether peptide loaded MHC II (MHC II-p) is subsequently recruited to and stably expressed at the plasma membrane or degraded in lysosomes is determined by the status of the DC. In immature DCs, MHC II-p is ubiquitinated after peptide loading, driving its sorting to the luminal vesicles of multivesicular bodies. These luminal vesicles, and the MHC II-p they carry, are delivered to lysosomes for degradation. MHC II-p is inefficiently ubiquitinated in DCs that are activated by pathogens or inflammatory stimuli, thus allowing its transfer to and stable expression at the plasma membrane.     

AKAPs in lipid rafts are required for optimal antigen presentation by dendritic cells

Dendritic cell (DC) maturation and antigen presentation are regulated by activation of protein kinase A (PKA) signaling pathways, through unknown mechanisms. We have recently shown that interfering with PKA signaling through the use of anchoring inhibitor peptides hinders antigen presentation and DC maturation. These experiments provide evidence that DC maturation and antigen presentation are regulated by A-kinase anchoring proteins (AKAPs). Herein, we determine that the presence of AKAPs and PKA in lipid rafts regulates antigen presentation. Using a combination of western blotting and immuno-cytochemistry, we illustrate the presence of AKAP149, AKAP79, Ezrin and the regulatory subunits of PKA in DC lipid rafts. Incubation of DCs with the type II anchoring inhibitor, AKAP-in silico (AKAP-IS), removes Ezrin and RII from the lipid raft without disrupting raft formation. Addition of a lipid raft disruptor, methyl-β-cyclodextrin, blocks the efficacy of AKAP-IS, suggesting that the lipid raft must be intact for AKAP-IS to inhibit antigen presentation. Ezrin and AKAP79 are present in the lipid raft of stimulated KG1 cells, but Ezrin is not present in the lipid raft of unstimulated KG1 cells and AKAP79 levels are greatly diminished, suggesting that Ezrin and AKAP79 may be the key AKAPs responsible for regulating antigen presentation.     

Selective serotonin reuptake inhibitors attenuate the antigen presentation from dendritic cells to effector T lymphocytes

Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been found to possess immune modulation effects, in addition to its antidepressant effects. However, it remains unclear whether SSRIs can suppress the antigen-presenting function of dendritic cells (DCs). Therefore, Fluoxetine was applied to a co-culture of Aggregatibacter actinomycetemcomitans (Aa)-reactive T cells (×Aa-T) isolated from Aa-immunized mice and DCs. This resulted in the suppressed proliferation of ×Aa-T stimulated with Aa-antigen presentation by DCs. Specifically, Fluoxetine increased the extracellular 5-hydroxytryptamine (5-HT) in the ×Aa-T/DC co-culture, whereas exogenously applied 5-HT promoted T-cell proliferation in the ×Aa-T/DC co-culture, indicating that Fluoxetine-mediated suppression of ×Aa-T/DC responses cannot be attributed to extracellular 5-HT. Instead, Fluoxetine remarkably suppressed the expression of costimulatory molecule ICOS-L on DCs. Fluoxetine also promoted a greater proportion of CD86(Low) immature DCs than CD86(High) mature DCs, while maintaining the expression levels of CD80, MHC-class-II and PD-L1. These results suggested that Fluoxetine suppressed the ability of DCs to present bacterial antigens to T cells, and the resulting T-cell proliferation, in a SERT/5-HT-independent manner and that diminished expression of ICOS-L on DCs and increase of CD86(Low) immature DCs caused by Fluoxetine might be partially associated with Fluoxetine-mediated suppression of DC/T-cell responses.     

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.     

Antigen presentation by liver sinusoidal lining cells after antigen exposure in vivo

The ability of liver sinusoidal lining cells (LSLC), a mixture of Kupffer cells and endothelial cells, to function as antigen-presenting cells (APC) was examined. Guinea pig LSLC were found to present antigen in vitro, albeit somewhat less effectively than a reference population of peritoneal exudate macrophages. The difference in APC function could not be explained by a deficiency of interleukin 1 (IL 1), as LSLC secreted IL 1 and expressed membrane-bound thymocyte stimulatory activity. The ability of LSLC to take up antigen from the portal blood in vivo and present it to primed T lymphocytes in vitro was also investigated. Trinitrophenyl-ovalbumin was injected intraportally into either strain 13 or strain 2 guinea pigs. The LSLC were subsequently isolated by collagenase digestion and density separation and assessed for the ability to induce proliferation of antigen-primed accessory cell-depleted syngeneic peritoneal exudate T lymphocytes in vitro. The in vivo antigen-pulsed LSLC were found to present antigen in vitro to primed T cells in an antigen-specific and genetically restricted manner. T cell DNA synthesis induced by antigen-bearing LSLC could be augmented by coculture with additional accessory cells, but not IL 1-containing macrophage supernatants. Enhancement of responsiveness was not genetically restricted. The demonstration that LSLC can take up, process, and retain antigen in vivo and present it to primed T cells in vitro suggests that LSLC are capable of contributing to the immune response to antigens appearing in portal blood.     

Lysosomal cysteine proteases regulate antigen presentation

Antigen presentation by both classical MHC class II molecules and the non-classical MHC class I-like molecule CD1D requires their entry into the endosomal/lysosomal compartment. Lysosomal cysteine proteases constitute an important subset of the enzymes that are present in this compartment and, here, we discuss the role of these proteases in regulating antigen presentation by both MHC class II and CD1D molecules.     

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.     

Ionizing radiation affects human MART-1 melanoma antigen processing and presentation by dendritic cells

Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-1(27-35) that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-1(27-35) also protected mice against growth of B16-A2/K(b) tumors in HLA-A2.1/K(b) transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.     

Silencing of SOCS1 enhances antigen presentation by dendritic cells and antigen-specific anti-tumor immunity

Tumor vaccines represent a promising therapeutic approach, but thus far have achieved only limited success in the clinic. The major challenge is to find a means of overcoming inhibitory immune regulatory mechanisms and eliciting effective T-cell responses to antigens preferentially expressed by tumor cells. Here we show that the stimulatory capacity of dendritic cells (DCs) and the magnitude of adaptive immunity are critically regulated by the suppressor of cytokine signaling (SOCS) 1 in DCs. Silencing SOCS1 in antigen-presenting DCs strongly enhances antigen-specific anti-tumor immunity. Our findings indicate that SOCS1 represents an inhibitory mechanism for qualitatively and quantitatively controlling antigen presentation by DCs and the magnitude of adaptive immunity. This study has implications for understanding the regulation of antigen presentation and for developing more effective tumor vaccines by silencing the critical brake in antigen presentation.