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.     

Fc gamma receptor IIB on dendritic cells enforces peripheral tolerance by inhibiting effector T cell responses

The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c+ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the consequence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcgammaRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8+ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcgammaRIIB, resulting in a high incidence of diabetes. FcgammaRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcgammaRs. Further FcgammaRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcgammaRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcgammaRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcgammaRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of exogenously acquired Ag.     

Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells

The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

Antigen presentation by an immature myeloid dendritic cell line does not cause CTL deletion in vivo, but generates CD8+ central memory-like T cells that can be rescued for full effector function

Immature dendritic cells (DC), in contrast to their mature counterparts, are incapable of mobilizing a CD8+ CTL response, and, instead, have been reported to induce CTL tolerance. We directly addressed the impact of immature vs mature DC on CTL responses by infusing adenovirus peptide-loaded DC (of the D1 cell line) into mice that had received adenovirus-specific naive TCR-transgenic CD8+ T cells. Whereas i.v. injection of mature DC triggered vigorous CTL expansion, immature DC elicited little proliferation involving only a minority of the TCR-transgenic CTL. Even though the latter CTL developed effector functions, including cytolytic activity and proinflammatory cytokine secretion, these cells differed significantly from CTL primed by mature DC in that they did not exhibit down-regulation of CD62L and CCR7, receptors involved in trapping of T cells in the lymphoid organs. Interestingly, adoptive transfer of CTL effector cells harvested after priming by either mature or immature DC into naive recipient mice, followed by exposure to adenovirus, yielded quantitatively and qualitatively indistinguishable CTL memory responses. Therefore, in vivo priming of naive CD8+ T cells by immature DC, although failing to induce a full-blown, systemic CTL response, resulted in the formation of central memory-like T cells that were able to expand and produce IFN-gamma upon secondary antigenic stimulation.     

Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.

MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.     

Liposomal delivery of CTL epitopes to dendritic cells

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein.

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II(+) epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8(+) CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-gamma-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-gamma-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8(+) CTL.     

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.     

Natural splice variant of MHC class I cytoplasmic tail enhances dendritic cell-induced CD8+ T-cell responses and boosts anti-tumor immunity

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-K(b) generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-D(b)-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-D(b) DCs were superior to WT-D(b) DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.     

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.     

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.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

CD8alpha+ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.     

Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells

The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.