Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation.

Candida albicans is a dimorphic fungus responsible for chronic mucocutaneous and systemic infections. Mucocutaneous immunity to C. albicans requires T helper 17 (Th17) cell differentiation that is thought to depend on recognition of filamentous C. albicans. Systemic immunity is considered T cell independent. Using a murine skin infection model, we compared T helper cell responses to yeast and filamentous C. albicans. We found that only yeast induced Th17 cell responses through a mechanism that required Dectin-1-mediated expression of interleukin-6 (IL-6) by Langerhans cells. Filamentous forms induced Th1 without Th17 cell responses due to the absence of Dectin-1 ligation. Notably, Th17 cell responses provided protection against cutaneous infection while Th1 cell responses provided protection against systemic infection. Thus, C. albicans morphology drives distinct T helper cell responses that provide tissue-specific protection. These findings provide insight into compartmentalization of Th cell responses and C. albicans pathogenesis and have critical implications for vaccine strategies.

Targeting human langerin promotes HIV-1 specific humoral immune responses.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.

Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties.

CD40 is a potent activating receptor within the TNFR family expressed on APCs of the immune system, and it regulates many aspects of B and T cell immunity via interaction with CD40 ligand (CD40L; CD154) expressed on the surface of activated T cells. Soluble CD40L and agonistic mAbs directed to CD40 are being explored as adjuvants in therapeutic or vaccination settings. Some anti-CD40 Abs can synergize with soluble monomeric CD40L. We show that direct fusion of CD40L to certain agonistic anti-CD40 Abs confers superagonist properties, reducing the dose required for efficacy, notably greatly increasing total cytokine secretion by human dendritic cells. The tetravalent configuration of anti-CD40-CD40L Abs promotes CD40 cell surface clustering and internalization and is the likely mechanism of increased receptor activation. CD40L fused to either the L or H chain C termini, with or without flexible linkers, were all superagonists with greater potency than CD40L trimer. The increased anti-CD40-CD40L Ab potency was independent of higher order aggregation. Moreover, the anti-CD40-CD40L Ab showed higher potency in vivo in human CD40 transgenic mice compared with the parental anti-CD40 Ab. To broaden the concept of fusing agonistic Ab to natural ligand, we fused OX40L to an agonistic OX40 Ab, and this resulted in dramatically increased efficacy for proliferation and cytokine production of activated human CD4+ T cells as well as releasing the Ab from dependency on cross-linking. This work shows that directly fusing antireceptor Abs to ligand is a useful strategy to dramatically increase agonist potency.

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice.

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG+ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS+ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.

Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines.

Systems immunology approaches were employed to investigate innate and adaptive immune responses to influenza and pneumococcal vaccines. These two non-live vaccines show different magnitudes of transcriptional responses at different time points after vaccination. Software solutions were developed to explore correlates of vaccine efficacy measured as antibody titers at day 28. These enabled a further dissection of transcriptional responses. Thus, the innate response, measured within hours in the peripheral blood, was dominated by an interferon transcriptional signature after influenza vaccination and by an inflammation signature after pneumococcal vaccination. Day 7 plasmablast responses induced by both vaccines was more pronounced after pneumococcal vaccination. Together, these results suggest that comparing global immune responses elicited by different vaccines will be critical to our understanding of the immune mechanisms underpinning successful vaccination.

Signaling Cascade through DC-ASGPR Induces Transcriptionally Active CREB for IL-10 Induction and Immune Regulation.

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However, how DCs display functional plasticity and control host immune responses have not been fully understood. In this study, we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR), a C-type lectin receptor (CLR) expressed on human DCs, resulted in rapid activation of Syk, followed by PLCγ2 and PKCδ engagements. However, different from other Syk-coupled CLRs, including Dectin-1, signaling cascade through DC-ASGPR did not trigger NF-κB activation. Instead, it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB, which consequently bound to IL10 promoter and initiated cytokine expression. In addition, DC-ASGPR ligation activated Akt, which differentially regulated the activities of GSK-3α/ß and ß-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway, which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses.

Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific T helper cell responses.

Skin-resident dendritic cells (DCs) are well positioned to encounter cutaneous pathogens and are required for the initiation of adaptive immune responses. There are at least three subsets of skin DC- Langerhans cells (LC), Langerin(+) dermal DCs (dDCs), and classic dDCs. Whether these subsets have distinct or redundant function in vivo is poorly understood. Using a Candida albicans skin infection model, we have shown that direct presentation of antigen by LC is necessary and sufficient for the generation of antigen-specific T helper-17 (Th17) cells but not for the generation of cytotoxic lymphocytes (CTLs). In contrast, Langerin(+) dDCs are required for the generation of antigen specific CTL and Th1 cells. Langerin(+) dDCs also inhibited the ability of LCs and classic DCs to promote Th17 cell responses. This work demonstrates that skin-resident DC subsets promote distinct and opposing antigen-specific responses.

Superiority in Rhesus Macaques of Targeting HIV-1 Env gp140 to CD40 versus LOX-1 in Combination with Replication-Competent NYVAC-KC for Induction of Env-Specific Antibody and T Cell Responses.

We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly-ICLC adjuvant either alone or coadministered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly-ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to a P value of 0.01) than in a group without poly-ICLC. The responses were robust and cross-reactive and contained antibodies specific to multiple epitopes within gp140, including the C1, C2, V1, V2, and V3, C4, C5, and gp41 immunodominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to tier 1 viruses and antibody-dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4+ and CD8+ T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines plus poly-ICLC. Together, these results indicate that prime-boost immunization via NYVAC-KC and either anti-CD40.Env gp140/poly-ICLC or anti-LOX-1.Env gp140/poly-ICLC induced balanced antibody and T cell responses against HIV-1 Env. Coadministration of NYVAC-KC with the DC-targeting vaccines increased T cell responses but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability than with LOX-1.IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells, which are now known to be the key cell type for controlling immunity. In this study, we have tested in nonhuman primates two prototype vaccines engineered to direct the HIV-1 coat protein Env to dendritic cells. These vaccines bind to either CD40 or LOX-1, two dendritic cell surface receptors with different functions and tissue distributions. We tested the vaccines described above in combination with attenuated virus vectors that express Env. Both vaccines, but especially that delivered via CD40, raised robust immunity against HIV-1 as measured by monitoring potentially protective antibody and T cell responses in the blood. The safety and efficacy of the CD40-targeted vaccine justify further development for future human clinical trials.

Human dendritic cells induce the differentiation of interleukin-21-producing T follicular helper-like cells through interleukin-12.

T follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4(+) T cells to become IL-21-producing Tfh-like cells through IL-12. CD4(+) T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21(+)IFN-gamma(+)T-bet(+) Th1 cells and IL-21(+)IFN-gamma(-)T-bet(-) non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4(+) T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4(+) T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development.

Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848.

Opposing Roles of Dectin-1 Expressed on Human Plasmacytoid Dendritic Cells and Myeloid Dendritic Cells in Th2 Polarization.

Dendritic cells (DCs) can induce and control host immune responses. DC subset-dependent functional specialties and their ability to display functional plasticity, which is mainly driven by signals via pattern recognition receptors, identify DCs as immune orchestrators. A pattern recognition receptor, Dectin-1, is expressed on myeloid DCs and known to play important roles in Th17 induction and activation during fungal and certain bacterial infections. In this study, we first demonstrate that human plasmacytoid DCs express Dectin-1 in both mRNA and protein levels. More interestingly, Dectin-1-activated plasmacytoid DCs promote Th2-type T cell responses, whereas Dectin-1-activated myeloid DCs decrease Th2-type T cell responses. Such contrasting outcomes of Th2-type T cell responses by the two DC subsets are mainly due to their distinct abilities to control surface OX40L expression in response to ß-glucan. This study provides new insights for the regulation of host immune responses by Dectin-1 expressed on DCs.

Induction and activation of human Th17 by targeting antigens to dendritic cells via dectin-1.

Recent compelling evidence indicates that Th17 confer host immunity against a variety of microbes, including extracellular and intracellular pathogens. Therefore, understanding mechanisms for the induction and activation of Ag-specific Th17 is important for the rational design of vaccines against pathogens. To study this, we employed an in vitro system in which influenza hemagglutinin (HA) 1 was delivered to dendritic cells (DCs) via Dectin-1 using anti-human Dectin-1 (hDectin-1)-HA1 recombinant fusion proteins. We found that healthy individuals maintained broad ranges of HA1-specific memory Th17 that were efficiently activated by DCs targeted with anti-hDectin-1-HA1. Nonetheless, these DCs were not able to induce a significant level of HA1-specific Th17 responses even in the presence of the Th17-promoting cytokines IL-1ß and IL-6. We further found that the induction of surface IL-1R1 expression by signals via TCRs and common γ-chain receptors was essential for naive CD4(+) T cell differentiation into HA1-specific Th17. This process was dependent on MyD88, but not IL-1R-associated kinase 1/4. Thus, interruptions in STAT3 or MyD88 signaling led to substantially diminished HA1-specific Th17 induction. Taken together, the de novo generation of pathogen-specific human Th17 requires complex, but complementary, actions of multiple signals. Data from this study will help us design a new and effective vaccine strategy that can promote Th17-mediated immunity against microbial pathogens.

Macrophage- and neutrophil-derived TNF-α instructs skin langerhans cells to prime antiviral immune responses.

Dendritic cells are major APCs that can efficiently prime immune responses. However, the roles of skin-resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. In this study, we demonstrate for the first time, to our knowledge, that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin Ags to skin LCs using recombinant fusion proteins of anti-Langerin Ab and Ags resulted in the induction of the viral Ag-specific responses. We further demonstrated that such Ag-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands, polyriboinosinic polyribocytidylic acid, and R848. These enhancements were not due to the direct actions of TLR ligands on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens.

Skin dendritic cells induce follicular helper T cells and protective humoral immune responses.

BACKGROUND: The contribution of individual subsets of dendritic cells (DCs) to generation of adaptive immunity is central to understanding immune homeostasis and protective immune responses. OBJECTIVE: We sought to define functions for steady-state skin DCs. METHODS: We present an approach in which we restrict antigen presentation to individual DC subsets in the skin and monitor the effects on endogenous antigen-specific CD4(+) T- and B-cell responses. RESULTS: Presentation of foreign antigen by Langerhans cells (LC) in the absence of exogenous adjuvant led to a large expansion of T follicular helper (TFH) cells. This was accompanied by B-cell activation, germinal center formation, and protective antibody responses against influenza. The expansion of TFH cells and antibody responses could be elicited by both systemic and topical skin immunization. TFH cell induction was not restricted to LCs and occurred in response to antigen presentation by CD103(+) dermal DCs. CD103(+) DCs, despite inducing similar TFH responses as LCs, were less efficient in induction of germinal center B cells and humoral immune responses. We also found that skin DCs are sufficient to expand CXCR5(+) TFH cells through an IL-6- and IFN-α/ß receptor-independent mechanism, but B cells were required for sustained Bcl-6(+) expression. CONCLUSIONS: These data demonstrate that a major unappreciated function of skin DCs is their promotion of TFH cells and humoral immune responses that potentially represent an efficient approach for vaccination.

Dendritic cell-based therapeutic vaccine elicits polyfunctional HIV-specific T-cell immunity associated with control of viral load.

Efforts aimed at restoring robust immune responses limiting human immunodeficiency virus (HIV)-1 replication therapeutically are warranted. We report that vaccination with dendritic cells generated ex vivo and loaded with HIV lipopeptides in patients (n = 19) on antiretroviral therapy was well tolerated and immunogenic. Vaccination increased: (i) the breadth of the immune response from 1 (1-3) to 4 (2-5) peptide-pool responses/patient (p = 0.009); (ii) the frequency of functional T cells (producing at least two cytokines among IFN-γ, TNF-α, and IL-2) from 0.026 to 0.32% (p = 0.002) and from 0.26 to 0.35% (p = 0.005) for CD4(+) and CD8(+) T cells, respectively; and (iii) the breadth of cytokines secreted by PBMCs upon antigen exposure, including IL-2, IFN-γ, IL-21, IL-17, and IL-13. Fifty percent of patients experienced a maximum of viral load (VL) 1 log10 lower than the other half following antiretroviral treatment interruption. An inverse correlation was found between the maximum of VL and the frequency of polyfunctional CD4(+) T cells (p = 0.007), production of IL-2 (p = 0.006), IFN-γ (p = 0.01), IL-21 (p = 0.006), and IL-13 (p = 0.001). These results suggest an association between vaccine responses and a better control of viral replication. These findings will help in the development of strategies for a functional cure for HIV infection.

Targeting human dendritic cell subsets for improved vaccines.

Dendritic cells (DCs) were discovered in 1973 by Ralph Steinman as a previously undefined cell type in the mouse spleen and are now recognized as a group of related cell populations that induce and regulate adaptive immune responses. Studies of the past decade show that, both in mice and humans, DCs are composed of subsets that differ in their localization, phenotype, and functions. These progresses in our understanding of DC biology provide a new framework for improving human health. In this review, we discuss human DC subsets in the context of their medical applications, with a particular focus on DC targeting.

Immunoglobulin-like transcript receptors on human dermal CD14+ dendritic cells act as a CD8-antagonist to control cytotoxic T cell priming.

Human Langerhans cells (LCs) are highly efficient at priming cytolytic CD8(+) T cells compared with dermal CD14(+) dendritic cells (DCs). Here we show that dermal CD14(+) DCs instead prime a fraction of naïve CD8(+) T cells into cells sharing the properties of type 2 cytokine-secreting CD8(+) T cells (TC2). Differential expression of the CD8-antagonist receptors on dermal CD14(+) DCs, the Ig-like transcript (ILT) inhibitory receptors, explains the difference between the two types of DCs. Inhibition of CD8 function on LCs inhibited cytotoxic T lymphocytes (CTLs) and enhanced TC2 generation. In addition, blocking ILT2 or ILT4 on dermal CD14(+) DCs enhanced the generation of CTLs and inhibited TC2 cytokine production. Lastly, addition of soluble ILT2 and ILT4 receptors inhibited CTL priming by LCs. Thus, ILT receptor expression explains the polarization of CD8(+) T-cell responses by LCs vs. dermal CD14(+) DCs.

The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming.

We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by ∼ 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-ß1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.

Noncovalent assembly of anti-dendritic cell antibodies and antigens for evoking immune responses in vitro and in vivo.

Targeting of Ags directly to dendritic cells (DCs) through anti-DC receptor Ab fused to Ag proteins is a promising approach to vaccine development. However, not all Ags can be expressed as a rAb directly fused to a protein Ag. In this study, we show that noncovalent assembly of Ab-Ag complexes, mediated by interaction between dockerin and cohesin domains from cellulose-degrading bacteria, can greatly expand the range of Ags for this DC-targeting vaccine technology. rAbs with a dockerin domain fused to the rAb H chain C terminus are efficiently secreted by mammalian cells, and many Ags not secreted as rAb fusion proteins are readily expressed as cohesin directly fused to Ag either via secretion from mammalian cells or as soluble cytoplasmic Escherichia coli products. These form very stable and homogeneous complexes with rAb fused to dockerin. In vitro, these complexes can efficiently bind to human DC receptors followed by presentation to Ag-specific CD4⁺ and CD8⁺ T cells. Low doses of the HA1 subunit of influenza hemagglutinin conjugated through this means to anti-Langerin rAbs elicited Flu HA1-specific Ab and T cell responses in mice. Thus, the noncovalent assembly of rAb and Ag through dockerin and cohesin interaction provides a useful modular strategy for development and testing of prototype vaccines for elicitation of Ag-specific T and B cell responses, particularly when direct rAb fusions to Ag cannot be expressed.

CD34-derived dendritic cells transfected ex vivo with HIV-Gag mRNA induce polyfunctional T-cell responses in nonhuman primates.

The pivotal role of DCs in initiating immune responses led to their use as vaccine vectors. However, the relationship between DC subsets involved in antigen presentation and the type of elicited immune responses underlined the need for the characterization of the DCs generated in vitro. The phenotypes of tissue-derived APCs from a cynomolgus macaque model for human vaccine development were compared with ex vivo-derived DCs. Monocyte/macrophages predominated in bone marrow (BM) and blood. Myeloid DCs (mDCs) were present in all tested tissues and were more highly represented than plasmacytoid DCs (pDCs). As in human skin, Langerhans cells (LCs) resided exclusively in the macaque epidermis, expressing CD11c, high levels of CD1a and langerin (CD207). Most DC subsets were endowed with tissue-specific combinations of PRRs. DCs generated from CD34(+) BM cells (CD34-DCs) were heterogeneous in phenotype. CD34-DCs shared properties (differentiation and PRR) of dermal and epidermal DCs. After injection into macaques, CD34-DCs expressing HIV-Gag induced Gag-specific CD4(+) and CD8(+) T cells producing IFN-γ, TNF-α, MIP-1ß, or IL-2. In high responding animals, the numbers of polyfunctional CD8(+) T cells increased with the number of booster injections. This DC-based vaccine strategy elicited immune responses relevant to the DC subsets generated in vitro.