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.     

Comparative Analysis of Transduced Primary Human Dendritic Cells Generated by the Use of Three Different Lentiviral Vector Systems

Lentiviral gene transfer vectors are suitable for genetically modifying non-cycling primary human cells. In this study, we analyzed transduced human dendritic cells (DC) generated by the use of three different GFP-encoding lentiviral vectors, HIV-2 ROD A Δenv-GFP (ROD A), SIVsmm PBj ΔE EGFP (PBj), and SIVmac ΔE EGFP (SIVmac). CD14⁺ monocytes were isolated from buffy coat, transduced, and differentiated to immature and mature DC. Cytofluometric analysis of DC revealed high transduction efficiencies at MOI 1 for simian immunodeficiency virus (SIV)-derived vectors PBj and SIVmac ranging between 80–90 and 70–90%, respectively. In contrast, transduction with ROD A resulted only in approximately 30%-positive DC at the same MOI. Of note, none of the analyzed vectors affected expression of maturation and/or activation markers. Moreover, transduction with PBj or SIVmac did not induce significant cytokine responses whereas ROD A transduction stimulated weak interferon-alpha responses. SIVmac transduced DC showed normal phagocytosis of antigen and normal allo T cell stimulatory capacity when compared with untreated DC. Thus, the SIVmac lentiviral transduction vector is suitable for efficient genetic modification of human DC without affecting phenotype or function and thus qualifies this vector as a versatile tool for use in basic research.     

Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.     

Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity

Dendritic cell (DC) therapies are currently being evaluated for the treatment of cancer. The majority of ongoing clinical trials use DCs loaded with defined antigenic peptides or proteins, or tumor-derived products, such as lysates or apoptotic cells, as sources of Ag. Although several theoretical considerations suggest that DCs expressing transgenic protein Ags may be more effective immunogens than protein-loaded cells, methods for efficiently transfecting DCs are only now being developed. In this study we directly compare the immunogenicity of peptide/protein-pulsed DCs with lentiviral vector-transduced DCs, and their comparative efficacy in tumor immunotherapy. Maturing, bone marrow-derived DCs can be efficiently transduced with lentiviral vectors, and transduction does not affect DC maturation, plasticity, or Ag presentation function. Transduced DCs efficiently process and present both MHC class I- and II-restricted epitopes from the expressed transgenic Ag OVA. Compared with peptide- or protein-pulsed DCs, lentiviral vector-transduced DCs elicit stronger and longer-lasting T cell responses in vivo, as measured by both in vivo killing assays and intracellular production of IFN-gamma by Ag-specific T cells. In the B16-OVA tumor therapy model, the growth of established tumors was significantly inhibited by a single immunization using lentiviral vector-transduced DCs, resulting in significantly longer survival of immunized animals. These results suggest that compared with Ag-pulsed DCs, vaccination with lentiviral vector-transduced DCs may achieve more potent antitumor immunity. These data support the further development of lentiviral vectors to transduce DCs with genes encoding Ags or immunomodulatory adjuvants to generate and control systemic immune responses.     

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy

Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC.This review considers the use of HIV-1-derived lentiviral vectors to transduce human and murine DC for cancer immunotherapy. Lentivirally transduced DC have been shown to present antigenic peptides, prime transgene-specific T cells in vitro and elicit a protective cytotoxic T-lymphocyte (CTL) response in animal models. Different parameters determining the efficacy of transduction are considered. The influence of lentiviral transduction on the DC phenotype and function is described and the induction of immune responses by lentivirally transduced DC in vitro and in vivo is discussed in detail. In addition, direct in vivo administration of lentiviral vectors aiming at the induction of antigen-specific immunity is reviewed. This strategy might overcome the need for ex vivo generation and antigen loading of DC. Finally, future perspectives towards the use of lentiviral vectors in cancer immunotherapy are presented.     

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.     

Early adenoviral gene expression mediates immunosuppression by transduced dendritic cell (DC): implications for immunotherapy using genetically modified DC

Long-lasting, high-level gene expression in the absence of a toxic or inflammatory response to viral Ags is necessary for the successful application of genetically modified dendritic cell (DC). We previously demonstrated that efficient transduction of mature DC using DeltaE1DeltaE3 adenoviruses suppressed their stimulatory capacity for T cells. The current study was designed to investigate in more detail the suppressive effect of Ad-DC. We demonstrate that immunosuppression is not mediated by alterations in the T cell phenotype or cytokine profiles released by stimulated T cells. Also DC phenotypes are not affected. However, we demonstrate a cell cycle arrest of the T cell population stimulated by adenovirally transduced DC. Surprisingly, only freshly transduced DC are perturbed in their stimulatory capacity. Experiments using cycloheximide to block early intracellular viral gene expression showed that viral genes expressed in DC are responsible for this transient immunosuppression. In agreement with these findings, high-capacity (gutless) Ad-vectors that differ in viral gene expression from conventional DeltaE1DeltaE3 adenovirus are suitable for an efficient transduction of human DC. DC transduced with gutless Ad-vectors showed a high allostimulatory capacity for CD4(+) and CD8(+) T cells. Thus, the immunosuppressive effect of DeltaE1DeltaE3 Ad-transduced mature DC seems to be the result of early viral gene expression in DC that can be prevented using gutless Ad-vectors for transduction. These results have important implications for the use of genetically modified DC for therapeutic application.     

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B (RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.     

HMGB1, an alarmin promoting HIV dissemination and latency in dendritic cells

Dendritic cells (DCs) initiate immune responses by transporting antigens and migrating to lymphoid tissues to initiate T-cell responses. DCs are located in the mucosal surfaces that are involved in human immunodeficiency virus (HIV) transmission and they are probably among the earliest targets of HIV-1 infection. DCs have an important role in viral transmission and dissemination, and HIV-1 has evolved different strategies to evade DC antiviral activity. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can act as an alarmin, a danger signal to alert the innate immune system for the initiation of host defense. It is the prototypic damage-associated molecular pattern molecule, and it can be secreted by innate cells, including DCs and natural killer (NK) cells. The fate of DCs is dependent on a cognate interaction with NK cells, which involves HMGB1 expressed at NK–DC synapse. HMGB1 is essential for DC maturation, migration to lymphoid tissues and functional type-1 polarization of naïve T cells. This review highlights the latest advances in our understanding of the impact of HIV on the interactions between HMGB1 and DCs, focusing on the mechanisms of HMGB1-dependent viral dissemination and persistence in DCs, and discussing the consequences on antiviral innate immunity, immune activation and HIV pathogenesis.     

Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin explants after in situ delivery of CD40-targeted adenoviral vectors

Therapeutic tumor vaccination with viral vectors or naked DNA, carrying the genetic code for tumor-associated Ags, critically depends on the in vivo transduction of dendritic cells (DC). Transfection of predominantly nonprofessional APC and only small numbers of DC may hamper proper T cell activation. Aim of this study was, therefore, the targeted, selective, and enhanced in situ transduction of DC. A human skin explant model was used to explore targeted transduction of cutaneous DC after intradermal injection of a bispecific Ab conjugate to link adenoviral (Ad) vectors directly to CD40 on the DC surface. A significantly enhanced transduction efficiency and selectivity, and an increased activation state of migrating DC were thus achieved. Moreover, DC transduced by CD40-targeted Ad maintained their Ag-specific CTL-stimulatory ability for up to 1 wk after the start of migration, in contrast to DC transduced by untargeted Ad, which had lost this capacity by that time. Because DC targeting in vivo might obviate the need for the in vitro culture of autologous DC for adoptive transfer, CD40-targeted Ad vectors constitute a promising new vaccine modality for tumor immunotherapy.     

Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans.     

Severe impairment of dendritic cell allostimulatory activity by Sendai virus vectors is overcome by matrix protein gene deletion

Delivery of Ags to dendritic cells (DCs) plays a pivotal role in the induction of efficient immune responses ranging from immunity to tolerance. The observation that certain viral pathogens are able to infect DCs has led to a concept in which applications of recombinant viruses are used for Ag delivery with the potential benefit of inducing potent Ag-specific T cell responses directed against multiple epitopes. As a prerequisite for such an application, the infection of DCs by recombinant viruses should not interfere with their stimulatory capacity. In this context, we could show that an emerging negative-strand RNA viral vector system based on the Sendai virus (SeV) is able to efficiently infect monocyte-derived human DCs (moDCs). However, after infection with SeV wild type, both the response of DCs to bacterial LPS as a powerful mediator of DC maturation and the allostimulatory activity were severely impaired. Interestingly, using various recombinant SeV vectors that were devoid of single viral genes, we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, use of M-deficient SeV vectors preserved the allostimulatory activity in infected moDCs despite an efficient expression of all other virally encoded genes, thereby identifying M-deficient vectors as a highly potent tool for the genetic manipulation of DCs.     

Enrichment of an antigen-specific T cell response by retrovirally transduced human dendritic cells.

The superior ability of dendritic cells (DC) in triggering antigen-specific T cell responses makes these cells attractive tools for the generation of antitumor or antiviral immunity. We report here an efficient retroviral transduction system for the introduction of antigens into DC. A retroviral vector encoding several CTL epitopes in a string-of-beads fashion in combination with the marker gene green fluorescence protein (GFP) was generated. Polyepitope transduced EBV-LCL could be isolated on the basis of GFP expression and were found to be sensitive to lysis by antigen-specific cytotoxic T cells, demonstrating that antigens encoded by the retroviral construct were stably expressed, processed, and presented in the context of HLA class I molecules. CD34(+) cells isolated from G-CSF mobilized peripheral blood were transduced with high efficiency (40-60%) with this retroviral construct. These cells could be considerably expanded in vitro and differentiated into mature DC without loss of the transduced antigen. DC transduced with the polyepitope constructs were able to mount a CTL response against an influenza epitope in the context of HLA-A2, demonstrating the antigen-specific CTL priming capacity of retrovirally transduced DC. Staining of the T cells with tetramers of HLA-A2 and the influenza virus peptide demonstrated a marked antigen-specific CTL enrichment after 2 in vitro stimulations using DC transduced with the polyepitope. However, additional in vitro stimulations of the T cells with transduced DC did not result in a further enrichment of tetramer staining cells.     

Potential of equine herpesvirus 1 as a vector for immunization

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells.     

A novel viral system for generating antigen-specific T cells

Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with approximately 50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-alpha treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-alpha, and IFN-alpha. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-alpha-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.     

Dendritic cells in innate immune responses against HIV

Dendritic cells (DCs) were recently found to be innate immunity effectors against tumoral cells and viruses. (i) In response to most viruses, including HIV, plasmacytoid DCs are responsible for most of the type I IFN secretion, which is strongly anti-viral and induces TH1 type responses. Myeloid DCs secrete IL-12, which is also important for TH1-type and cytotoxic responses. In HIV patient blood, both DC population numbers decrease as early as the primary stage. Plasmacytoid DC numbers correlate with type I IFN secretion, which is a prognosis predictor, particularly under treatment. IL-12 secretion is also defective. Immunotherapies to replace the defective cytokines or to restore a potentially defective DC-T lymphocyte feed-back might help patients restore their immune responses under antiviral therapy. (ii) After measles and other viral infections, or incubation with dsRNA, DCs become cytotoxic and consequently exhibit natural killer function, through upregulation of type I IFN secretion which enhances TRAIL expression. In HIV infection, this mechanism was not demonstrated yet, but it might a) be responsible for the massive apoptosis of uninfected lymphocytes, and b) increase specific immunity through cross-presentation of antigens from infected cells killed by DCs. (iii) DCs direct expansion and effector functions of NK cells in the absence of adaptive-type cytokines and modulate NKT cell IFN-gamma production. Reciprocally, NK activation triggers DC maturation. HIV-1 Tat inhibits NK cell cytotoxicity directly and probably through inhibition of IL-12 secretion by DC. Therefore, understanding the functions of DCs in innate immune responses and in pathogenesis will help obtain better HIV replication control.     

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.