Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals

Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells.

In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection.     

Acquisition of MHC:Peptide Complexes by Dendritic Cells Contributes to the Generation of Antiviral CD8+ T Cell Immunity In Vivo

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8(+) T cells in host defense. However, although it has been shown that memory CD8(+) T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8(+) T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8(+) T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α(-) DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8(+) T cell effectors with cytolytic function. As CD8α(-) DCs are poor cross-presenters, this may represent the main mechanism by which CD8α(-) DCs present exogenously encountered Ag to CD8(+) T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.     

Preferential induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-type lectin-1

Targeting of Ags and therapeutics to dendritic cells (DCs) has immense potential for immunotherapy and vaccination. Because DCs are heterogeneous, optimal targeting strategies will require knowledge about functional specialization among DC subpopulations and identification of molecules for targeting appropriate DCs. We characterized the expression of a fungal recognition receptor, DC-associated C-type lectin-1 (Dectin-1), on mouse DC subpopulations and investigated the ability of an anti-Dectin-1 Ab to deliver Ag for the stimulation of immune responses. Dectin-1 was shown to be expressed on CD8alpha-CD4-CD11b+ DCs found in spleen and lymph nodes and dermal DCs present in skin and s.c. lymph nodes. Injection of Ag-anti-Dectin-1 conjugates induced CD4+ and CD8+ T cell and Ab responses at low doses where free Ag failed to elicit a response. Notably, qualitatively different immune responses were generated by targeting Ag to Dectin-1 vs CD205, a molecule expressed on CD8alpha+CD4-CD11b- DCs, dermal DCs, and Langerhans cells. Unlike anti-Dectin-1, anti-CD205 conjugates failed to elicit an Ab response. Moreover, when conjugates were injected i.v., anti-Dectin-1 stimulated a much stronger CD4+ T cell response and a much weaker CD8+ T cell response than anti-CD205. The results reveal Dectin-1 as a potential targeting molecule for immunization and have implications for the specialization of DC subpopulations.     

Selection of an antibody library identifies a pathway to induce immunity by targeting CD36 on steady-state CD8 alpha+ dendritic cells

Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8alpha+ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4+ and CD8+ Ag-specific T lymphocytes and the differentiation of primed CD8+ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8alpha+ via CD36 or DEC205 was qualitatively different. Anti-DEC205-OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8+ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8alpha+ DCs.     

Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram-positive bacteria to CD4+ T lymphocytes.

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.     

Influenza A infection enhances cross-priming of CD8+ T cells to cell-associated antigens in a TLR7- and type I IFN-dependent fashion

The initiation of antitumor immunity relies on dendritic cells (DCs) to cross-present cell-associated tumor Ag to CD8(+) T cells (T(CD8+)) due to a lack of costimulatory molecules on tumor cells. Innate danger signals have been demonstrated to enhance cross-priming of T(CD8+) to soluble as well as virally encoded Ags; however, their effect on enhancing T(CD8+) cross-priming to cell genome-encoded Ags remains unknown. Furthermore, influenza A virus (IAV) has not been shown to enhance antitumor immunity. Using influenza-infected allogeneic cell lines, we show in this study that T(CD8+) responses to cell-associated Ags can be dramatically enhanced due to enhanced T(CD8+) expansion. This enhanced cross-priming in part involves TLR7- but not TLR3-mediated sensing of IAV and is entirely dependent on MyD88 and IFN signaling pathways. We also showed that the inflammasome-induced IL-1 and IFN-γ did not play a role in enhancing cross-priming in our system. We further demonstrated in our ex vivo system that CD8(+) DCs are the only APCs able to prime TCR-transgenic T(CD8+). Importantly, plasmacytoid DCs and CD8(-) DCs were both able to enhance such priming when provided in coculture. These observations suggest that IAV infection of tumor cells may facilitate improved cross-presentation of tumor Ags and may be used to augment clinical vaccine efficacy.     

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

Lymph node resident rather than skin-derived dendritic cells initiate specific T cell responses after Leishmania major infection

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11c(high)CD11b(high)Gr-1-CD8-langerin- DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11c(high) CD11b(high) DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.     

Nonsecreted bacterial proteins induce recall CD8 T cell responses but do not serve as protective antigens

Secreted or nonsecreted Ag expressed by recombinant Listeria monocytogenes can prime CD8 T cells. However, Ag-specific memory CD8 T cells confer protection against bacteria secreting Ag, but not against bacteria expressing the nonsecreted form of the same Ag. This dichotomy may be explained by a long-standing hypothesis that nonsecreted Ags are less effective than secreted Ags at inducing a protective immune response at the onset of infection. We tested this hypothesis by examining whether these two different forms of Ag induce different primary and secondary CD8 T cell responses. The primary responses to secreted and nonsecreted Ags expanded and contracted almost synchronously, although the responses to nonsecreted Ags were of lower magnitude. These results demonstrate that the kinetics of the CD8 T cell response are similar regardless of whether Ag is accessible to the endogenous MHC class I pathway or can only be presented through cross-presentation. No differences were detected in the CD8 T cell recall response to L. monocytogenes expressing secreted or nonsecreted Ags. Nonsecreted Ags are as effective as secreted Ags at the induction of a rapid recall response by memory CD8 T cells. Thus, the inability of nonsecreted bacterial proteins to serve as protective Ags cannot be attributed to a defective CD8 T cell response.     

TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.     

Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcgammaR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in "license to kill." These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.     

Hematopoietic cells are required to initiate a Chlamydia trachomatis-specific CD8+ T cell response

Chlamydia trachomatis is a global human pathogen causing diseases ranging from blinding trachoma to pelvic inflammatory disease. To explore how innate and adaptive immune responses cooperate to protect against systemic infection with C. trachomatis L2, we investigated the role of macrophages (Mphi) and dendritic cells (DCs) in the stimulation of C. trachomatis-specific CD8(+) T cells. We found that C. trachomatis infection of Mphi and DCs is far less productive than infection of nonprofessional APCs, the typical targets of infection. However, despite the limited replication of C. trachomatis within Mphi and DCs, infected Mphi and DCs process and present C. trachomatis CD8(+) T cell Ag in a proteasome-dependent manner. These findings suggest that although C. trachomatis is a vacuolar pathogen, some Ags expressed in infected Mphi and DCs are processed in the host cell cytosol for presentation to CD8(+) T cells. We also show that even though C. trachomatis replicates efficiently within nonprofessional APCs both in vitro and in vivo, Ag presentation by hematopoietic cells is essential for initial stimulation of C. trachomatis-specific CD8(+) T cells. However, when DCs infected with C. trachomatis ex vivo were adoptively transferred into naive mice, they failed to prime C. trachomatis-specific CD8(+) T cells. We propose a model for priming C. trachomatis-specific CD8(+) T cells whereby DCs acquire C. trachomatis Ag by engulfing productively infected nonprofessional APCs and then present the Ag to T cells via a mechanism of cross-presentation.     

Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells

The activation of naive CD8+ T cells has been attributed to two mechanisms: cross-priming and direct priming. Cross-priming and direct priming differ in the source of Ag and in the cell that presents the Ag to the responding CD8+ T cells. In cross-priming, exogenous Ag is acquired by professional APCs, such as dendritic cells (DC), which process the Ag into peptides that are subsequently presented. In direct priming, the APCs, which may or may not be DC, synthesize and process the Ag and present it themselves to CD8+ T cells. In this study, we demonstrate that naive CD8+ T cells are activated by a third mechanism, called cross-dressing. In cross-dressing, DC directly acquire MHC class I-peptide complexes from dead, but not live, donor cells by a cell contact-mediated mechanism, and present the intact complexes to naive CD8+ T cells. Such DC are cross-dressed because they are wearing peptide-MHC complexes generated by other cells. CD8+ T cells activated by cross-dressing are restricted to the MHC class I genotype of the donor cells and are specific for peptides generated by the donor cells. In vivo studies demonstrate that optimal priming of CD8+ T cells requires both cross-priming and cross-dressing. Thus, cross-dressing may be an important mechanism by which DC prime naive CD8+ T cells and may explain how CD8+ T cells are primed to Ags that are inefficiently cross-presented.     

CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake

Dendritic cell (DC) Ag cross-presentation is generally associated with immune responses to tumors and viral Ags, and enhancement of this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8(+) murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA Ag, although development, maturation, and Ag processing and presentation of DCs are normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of Ags such as OVA and transferrin but not fluid-phase or phagocytic Ag uptake. CD13 and Ag are cointernalized in DCs, but CD13 did not coimmunoprecipitate with Ag receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs, and blocking of these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance Ag uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine

Dendritic cells (DCs) play an important role in the induction of T cell responses. Fc gammaRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual Fc gammaRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that Fc gammaRI and Fc gammaRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating Fc gammaRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating Fc gammaRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.