Unique type I interferon responses determine the functional fate of migratory lung dendritic cells during influenza virus infection

Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103(+) DCs and CD11b(high) DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103(+) DCs allow the virus to replicate to significantly higher levels than do the CD11b(high) DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11b(high) DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103(+) DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11b(high) DCs. The attenuated IFNAR signaling by CD103(+) DCs correlates with their described superior antigen presentation capacity for na?ve CD8(+) T cells when compared to CD11b(high) DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The "interferon-resistant" CD103(+) DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination.     

Immunotherapy of established tumors using bone marrow transplantation with antigen gene--modified hematopoietic stem cells

A major focus of cancer immunotherapy is to develop strategies to induce T-cell responses through presentation of tumor antigens by dendritic cells (DCs). Current vaccines are limited in their ability to efficiently transfer antigens to DCs in vivo. Ex vivo-generated DCs can be efficiently loaded with antigen but after reinjection, few DCs traffic to secondary lymphoid organs, the critical sites for antigen presentation. To enhance efficiency and durability of antigen presentation by DCs, we transduced hematopoietic stem-progenitor cells (HSCs) with a model tumor antigen and then transplanted the gene-modified cells into irradiated recipient mice, which resulted in efficient expression of the transgene in a large proportion of donor derived DCs in lymphoid organs. The combination of bone marrow transplantation (BMT) using transduced HSCs, systemic agents that generate and activate DCs, and mature T-cell infusion resulted in substantial expansion and activation of antigen-specific T cells. This tripartite strategy provided potent antigen-specific immunotherapy for an aggressive established tumor.     

Interleukin 10 (IL-10)-mediated Immunosuppression: MARCH-I INDUCTION REGULATES ANTIGEN PRESENTATION BY MACROPHAGES BUT NOT DENDRITIC CELLS*

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen-specific CD4 T cells; however, the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of antigen presenting cell function in macrophages is March-I-dependent, whereas in DCs, suppression is March- I-independent.     

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.     

Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.     

Label-free proteomics and systems biology analysis of mycobacterial phagosomes in dendritic cells and macrophages

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.     

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8⁺ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8⁺ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8⁺ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8⁺ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8⁺ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.     

Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis

Immunity to tumors as well as to viral and bacterial pathogens is often mediated by cytotoxic T lymphocytes (CTLs). Thus, the ability to induce a strong cell-mediated immune response is an important requirement of novel immunotherapies. Antigen-presenting cells (APCs), including dendritic cells (DCs), are specialized in initiating T-cell immunity. Harnessing this innate ability of these cells to acquire and present antigens, we sought to improve antigen presentation by targeting antigens directly to DCs in vivo through apoptosis. We engineered Fas-mediated apoptotic death of antigen-bearing cells in vivo by co-expressing the immunogen and Fas in the same cell. We then observed that the death of antigen-bearing cells results in increased antigen acquisition by APCs including DCs. This in vivo strategy led to enhanced antigen-specific CTLs, and the elaboration of T helper-1 (Th1) type cytokines and chemokines. This adjuvant approach has important implications for viral and nonviral delivery strategies for vaccines or gene therapies.     

Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis

Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.     

Phenotypic and functional alterations of dendritic cells induced by human herpesvirus 6 infection

Human herpesvirus 6 (HHV-6) has a tropism for T lymphocytes and monocytes/macrophages, suggesting that HHV-6 infection affects the immunosurveillance system. In the present study, we investigated the HHV-6-induced phenotypic and functional alterations of dendritic cells (DCs), which are professional antigen-presenting cells. HHV-6 infection of monocyte-derived immature DCs appeared to induce the up-regulation of CD80, CD83, CD86, and HLA class I and class II molecules, suggesting that HHV-6 infection induces the maturation of DCs. In addition, the antigen capture capacity of DCs was found to decrease following infection with HHV-6. In contrast to up-regulation of mature-DC-associated surface molecules on HHV-6-infected DCs, their capacity for presentation of alloantigens and exogenous virus antigens to T lymphocytes decreased significantly from that of uninfected DCs. In contrast, there appeared to be no reduction in the capacity for presentation of an HLA class II-binding peptide to the peptide-specific CD4(+) T lymphocytes. These data indicate that HHV-6 infection induces phenotypic alterations and impairs the antigen presentation capacity of DCs. The present data also suggest that the dysfunction of HHV-6-infected DCs is attributable mainly to impairment of the antigen capture and intracellular antigen-processing pathways.     

Effect of interleukin-15 on depressed splenic dendritic cell functions following trauma-hemorrhage

Although trauma-hemorrhage (T-H) induces suppressed splenic dendritic cell (DC) maturation and antigen presentation capacity, it remains unclear whether IL-15 modulates splenic DC functions. The aim of this study therefore was to investigate the effect of IL-15 on splenic DC functions after T-H. Male C3H/HeN mice (6-8 wk old) were randomly assigned to T-H or sham operation. T-H was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were killed, splenic DCs were isolated, and the effects of exogenous IL-15 on their costimulatory factors, major histocompatibility class II expression, ability to produce cytokines, and antigen presentation were measured. The results indicate that IL-15 production capacity of splenic DCs was reduced following T-H. Ex vivo exposure to IL-15 attenuated the suppressed production of TNF-alpha, IL-6, and IFN-gamma from splenic DCs following T-H. In addition, expression of surface antigen studies demonstrate that exogenous IL-15 attenuated T-H-induced downregulation of the activation of DC. The suppressed splenic DC antigen presentation function following T-H was also attenuated by IL-15 treatment. Moreover, IL-15 enhanced IL-12-induced IFN-gamma production and antigen presentation by splenic DCs. These data suggest that ex vivo treatment with IL-15 following T-H provides beneficial effects on splenic DCs. The depression in IL-15 production by splenic DCs could contribute to the host's enhanced susceptibility to infections following T-H.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Immortalized dendritic cell line with efficient cross-priming ability established from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene

The uptake of an antigen and its presentation to specific T cells by dendritic cells (DCs) is a primary event in initiation of humoral and cellular immune responses as well as the induction of cytotoxic T cells (CTLs). DCs are induced by culturing bone marrow cells in the presence of GM-CSF. However, the resulting DCs are short-lived and the culture usually contains CD11c-negative non-DC cells, which adversely affects reproducibility and makes interpretation of the experimental results difficult. Therefore, it would be useful if DCs could be readily immortalized with their functions being retained. In this study we established a novel, immortalized murine DC line with antigen-presenting capacity in vitro as well as an augmenting effect on humoral and cellular immune responses in vivo, utilizing bone marrow cells from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene. In the presence of GM-CSF, the resulting DC line, termed SVDC, could be continuously subcultured for more than 12 months. When pulsed with OVA alone or OVA-IgG immune complexes via Fcgamma receptors, SVDC augmented OVA-specific T cell proliferation efficiently in vitro, and elicited OVA-specific IgG production in vivo on the adoptive transfer of pulsed SVDC into naive mice. Interestingly, SVDC exhibited significantly high cross-priming ability compared to DCs in a short-term culture, thus leading to their extremely high effectiveness in inducing anti-tumor immunity in vivo. Thus, SVDC is useful for the detailed characterization of antigen presentation, and for research on the various therapeutic benefits of DC vaccination to elicit specific immune responses in immunodeficiencies, infectious diseases and cancer.     

Immunogenic cell death in cancer therapy: Present and emerging inducers

In the tumour microenvironment (TME), immunogenic cell death (ICD) plays a major role in stimulating the dysfunctional antitumour immune system. Chronic exposure of damage‐associated molecular patterns (DAMPs) attracts receptors and ligands on dendritic cells (DCs) and activates immature DCs to transition to a mature phenotype, which promotes the processing of phagocytic cargo in DCs and accelerates the engulfment of antigenic components by DCs. Consequently, via antigen presentation, DCs stimulate specific T cell responses that kill more cancer cells. The induction of ICD eventually results in long‐lasting protective antitumour immunity. Through the exploration of ICD inducers, recent studies have shown that there are many novel modalities with the ability to induce immunogenic cancer cell death. In this review, we mainly discussed and summarized the emerging methods for inducing immunogenic cancer cell death. Concepts and molecular mechanisms relevant to antitumour effects of ICD are also briefly discussed.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin

The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103⁺ dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3⁺ dermal DCs (thought to be equivalent to murine CD103⁺ dermal DCs) and DC-SIGN⁺ DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3⁺ dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3⁺ dermal DCs and DC-SIGN⁺ cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.     

Macrophage-colony stimulating factor enhances MHC-restricted presentation of exogenous antigen in dendritic cells

Previous studies have shown that dendritic cells (DCs) can phagocytize, process and present a microencapsulated form of ovalbumin (OVA) in the context of class I MHC as well as class II MHC. In the present study, we examined the effects of recombinant human macrophage-colony stimulating factor (M-CSF) on the MHC-restricted presentation of microencapsulated OVA by DCs. Two types of DCs were generated from mouse bone marrow (BM) cells, one type with granulocyte/macrophage-colony stimulating factor (GM-CSF) alone, the other type with GM-CSF and interleukin (IL)-4. Pretreatment with M-CSF significantly enhanced both class I MHC and class II MHC-restricted presentation of exogenous OVA by both types of DCs. The enhancing activity of M-CSF on antigen presentation was more potent in DCs generated with GM-CSF alone compared to DCs generated with both GM-CSF and IL-4. Pretreatment of the DCs with M-CSF did not increase phagocytic activity or total level of expression of class I MHC (H-2K(b)) molecules, but increased expression of OVA peptide-H-2K(b) complexes upon phagocytosis of microencapsulated OVA. These results demonstrate that M-CSF increases intracellular processing events of phagocytized antigen in DCs.     

Involvement of aquaporin-7 in the cutaneous primary immune response through modulation of antigen uptake and migration in dendritic cells

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.     

The role of dendritic cells in tumor microenvironments and their uses as therapeutic targets

Dendritic cells (DC), which consist of several different subsets, specialize in antigen presentation and are critical for mediating the innate and adaptive immune responses. DC subsets can be classified into conventional, plasmacytoid, and monocyte-derived DC in the tumor microenvironment, and each subset plays a different role. Because of the role of intratumoral DCs in initiating antitumor immune responses with tumor-derived antigen presentation to T cells, DCs have been targeted in the treatment of cancer. By regulating the functionality of DCs, several DC-based immunotherapies have been developed, including administration of tumor-derived antigens and DC vaccines. In addition, DCs participate in the mechanisms of classical cancer therapies, such as radiation therapy and chemotherapy. Thus, regulating DCs is also important in improving current cancer therapies. Here, we will discuss the role of each DC subset in antitumor immune responses, and the current status of DC-related cancer therapies.     

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.