Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Platelets present antigen in the context of MHC class I

Platelets are most recognized for their vital role as the cellular mediator of thrombosis, but platelets also have important immune functions. Platelets initiate and sustain vascular inflammation in many disease conditions, including arthritis, atherosclerosis, transplant rejection, and severe malaria. We now demonstrate that platelets express T cell costimulatory molecules, process and present Ag in MHC class I, and directly activate naive T cells in a platelet MHC class I-dependent manner. Using an experimental cerebral malaria mouse model, we also demonstrate that platelets present pathogen-derived Ag to promote T cell responses in vivo, and that platelets can be used in a cell-based vaccine model to induce protective immune responses. Our study demonstrates a novel Ag presentation role for platelets.     

Induction of vigorous helper and cytotoxic T cell as well as B cell responses by dendritic cells expressing a modified antigen targeting receptor-mediated internalization pathway

Efficient Ag presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of current immunotherapy and vaccine development is to enhance Ag presentation to induce potent and broad immune responses. Here, a novel Ag presentation strategy is developed by transducing dendritic cells (DCs) to produce an Ag for presentation as an exogenous Ag to efficiently induce both humoral and cellular immunity. The principle of this strategy is illustrated by genetically modifying DCs to secrete a model hepatitis B virus Ag fused with a cell-binding domain and to process the fusion Ag as an exogenous Ag after receptor-mediated internalization for MHC class I and II presentation. Vigorous Ag-specific CD4(+) helper and CD8(+) cytotoxic T cell, as well as B cell, responses were induced by the transduced DCs in mouse models. Thus, this novel strategy uses a receptor-mediated internalization process to efficiently induce all arms of the adaptive immunity and may provide a powerful means to develop potent vaccines and immunotherapies.     

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.     

Efficient in vivo presentation of Listeria monocytogenes- derived CD4 and CD8 T cell epitopes in the absence of IFN-gamma

IFN-gamma is an essential component of the early Listeria monocytogenes-specific immune response, and is also an important regulator of Ag processing and presentation. Ag presentation is required for the induction and also the effector function of antimicrobial T cells. To evaluate the effect of IFN-gamma on bacterial Ag presentation in vivo, macrophages and dendritic cells were separated from L. monocytogenes-infected tissues and analyzed with peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based ex vivo Ag presentation assay. The comparison of professional APCs isolated from infected IFN-gamma-deficient and wild-type mice revealed different peptide presentation patterns of L. monocytogenes-derived CD8 T cell epitopes, while the presentation pattern of CD4 T cell epitopes remained unchanged. The further in vitro analysis of the generation of CD8 T cell epitopes revealed a peptide-specific effect of IFN-gamma on MHC class I-restricted Ag presentation. These results show that despite this modulation of the Ag presentation pattern of CD8 T cell epitopes, IFN-gamma is not generally required for the MHC class I- and MHC class II-restricted presentation of L. monocytogenes-derived antigenic peptides by professional APCs in vivo.     

Impaired ability of MHC class II-/- dendritic cells to provide tumor protection is rescued by CD40 ligation.

The contribution of CD4+ T cells to dendritic cell (DC) activation and to the induction of CD8+ T cell responses in vivo was investigated using a model of antitumor immune responses. Immunization with peptide-loaded MHC class II-deficient (MHC class II-/-) DC induced the activation of Ag-specific CD8+ T cells and their accumulation in the lymph nodes and spleens of immunized mice. The accumulation induced by MHC class II-/- DC immunization was lower than the accumulation observed after immunization with MHC class II+/+ DC. Similarly, immunization with peptide-loaded, MHC class II-/- DC induced some degree of protection against tumor challenge, but this protection was lower than the protection achieved after immunization with MHC class II+/+ DC. Incubation with a membrane-associated form of CD40 ligand resulted in the up-regulation of costimulatory molecules on MHC class II-/- DC and fully rescued their ability to induce antitumor immunity. We conclude that CD4+ T cells play a critical role in the generation of antitumor immune responses through their capacity to induce the activation of DC via CD40/CD40 ligand interaction, and thus maximize CD8+ T cell responses.     

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.     

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.     

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.     

Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.     

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.     

Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.     

Disruption of MHC class II-restricted antigen presentation by vaccinia virus

Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.     

Design of immunogenic and effective multi-epitope DNA vaccines for melanoma

Plasmid DNA vaccination is an attractive way to elicit T cell responses against infectious agents and tumor cells. DNA constructs can be designed to contain multiple T cell epitopes to generate a diverse immune response to incorporate numerous antigens and to reduce limitations due to MHC restriction into a single entity. We have prepared cDNA plasmid constructs containing several mouse T cell epitopes connected by either furin-sensitive or furin-resistant linkers and studied the effects of a cationic cell-penetrating sequence from HIV-tat. Significant CD8 T cell responses were obtained with multi-epitope DNA vaccines followed by in vivo electroporation regardless of the type of linker used and whether the construct had the HIV-tat sequence. The magnitude of immune responses was very similar to all CD8 T cell epitopes contained within each vaccine construct, indicating the absence of immunodominance. Incorporating a T helper epitope into the constructs increased the T cell responses. Prophylactic and therapeutic antitumor responses against B16 melanoma were obtained using a construct containing epitopes from melanosomal proteins, indicating that this vaccination was successful in generating responses to self-antigens that potentially may be subjected to immune tolerance. These findings are useful for designing DNA vaccines for a multitude of diseases where T lymphocytes play a protective or therapeutic role.     

Dendritic cells acquire antigens from live cells for cross-presentation to CTL

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 microm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.     

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.     

Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.     

Delivery of antigen to CD40 induces protective immune responses against tumors

Ligation of CD40 induces maturation of dendritic cells (DC) and could be a useful target for vaccines. In this study, we have constructed two types of Ab-based vaccine constructs that target mouse CD40. One type is a recombinant Ab with V regions specific for CD40 and has defined T cell epitopes inserted into its C region. The other type is a homodimer, each chain of which is composed of a targeting unit (single-chain fragment variable targeting CD40), a dimerization motif, and an antigenic unit. Such proteins bound CD40, stimulated maturation of DC, and enhanced primary and memory T cell responses. When delivered i.m. as naked DNA followed by electroporation, the vaccines induced T cell responses against MHC class II-restricted epitopes, Ab responses, and protection in two tumor models (myeloma and lymphoma). Two factors apparently contributed to these results: 1) agonistic ligation of CD40 and induction of DC maturation, and 2) delivery of Ag to APC and presentation on MHC class II molecules. These results highlight the importance of agonistic targeting of Ag to CD40 for induction of long-lasting and protective immune responses.