HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation

Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced

The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine

CD4+ Th1 type immunity is implicated in resistance to global infectious diseases. To improve the efficacy of T cell immunity induced by human immunodeficiency virus (HIV) vaccines, we are developing a protein-based approach that directly harnesses the function of dendritic cells (DCs) in intact lymphoid tissues. Vaccine proteins are selectively delivered to DCs by antibodies to DEC-205/CD205, a receptor for antigen presentation. We find that polyriboinosinic: polyribocytidylic acid (poly IC) independently serves as an adjuvant to allow a DC-targeted protein to induce protective CD4+ T cell responses at a mucosal surface, the airway. After two doses of DEC-targeted, HIV gag p24 along with poly IC, responder CD4+ T cells have qualitative features that have been correlated with protective function. The T cells simultaneously make IFN-γ, tumor necrosis factor (TNF)-α, and IL-2, and in high amounts for prolonged periods. The T cells also proliferate and continue to secrete IFN-γ in response to HIV gag p24. The adjuvant role of poly IC requires Toll-like receptor (TLR) 3 and melanoma differentiation-associated gene-5 (MDA5) receptors, but its analog poly IC 12U requires only TLR3. We suggest that poly IC be tested as an adjuvant with DC-targeted vaccines to induce numerous multifunctional CD4 + Th1 cells with proliferative capacity

Cell type-dependent retention and transmission of HIV-1 by DC-SIGN

DC-SIGN (CD209) is a C-type lectin expressed by several groups of dendritic cells (DC), including those derived from blood monocytes and DC found beneath genital epithelium. DC-SIGN binds the envelope glycoprotein of HIV-1 and facilitates transmission of infectious virus to permissive CD4+ T cells. We have compared the capacity of DC-SIGN in different cell types to bind, retain and transmit infectious HIV-1 to T cells. The analyzed cells included monocyte-derived DC, and three different DC-SIGN-expressing transfectants; termed THP, 293 and HOS. Our results show that DC-SIGN transfectants were able to bind HIV-1 virions comparably to DC. However, only the THP monocytic cell line shared with DC the capacity to retain for several days virus that was infectious for T cells. In both THP-DC-SIGN transfectants and DC, but not in 293 cells, HIV-1 was localized to intracellular compartments that did not double label for endosomal and lysosomal markers or for DC-SIGN itself. Virus remained detectable in these compartments for at least 2 days. Anti-DC-SIGN antibodies blocked the binding and transmission of HIV-1 in DC-SIGN transfectants, as monitored by PCR for HIV LTR/gag and p24 ELISA. However anti-DC-SIGN antibodies did not block virus binding and transmission to T cells as well in DC as in THP-DC-SIGN transfectants. Thus, the function of DC-SIGN in HIV-1 transmission depends on its cellular context, since only DC and the THP monocyte cell line, but not 293 and HOS, are able to use DC-SIGN to retain HIV-1 in a highly infectious state for several days

Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant

Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4+ T cell responses to a dendritic cell (DC)-targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205+ DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4+ immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow-derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity

DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes

Optimal HIV vaccines should elicit CD8+ T cells specific for HIV proteins presented on MHC class I products, because these T cells contribute to host resistance to viruses. We had previously found that the targeting of antigen to dendritic cells (DCs) in mice efficiently induces CD8+ T cell responses. To extend this finding to humans, we introduced the HIV p24 gag protein into a mAb that targets DEC-205/CD205, an endocytic receptor of DCs. We then assessed cross-presentation, which is the processing of nonreplicating internalized antigen onto MHC class I for recognition by CD8+ T cells. Low doses of αDEC-gag, but not control Ig-gag, stimulated proliferation and IFN-γ production by CD8+ T cells isolated from the blood of HIV-infected donors. αCD205 fusion mAb was more effective for cross-presentation than αD209/DC-SIGN, another abundant DC uptake receptor. Presentation was diverse, because we identified eight different gag peptides that were recognized via DEC-205 in 11 individuals studied consecutively. Our results, based on humans with highly polymorphic MHC products, reveal that DCs and DEC-205 can cross-present several different peptides from a single protein. Because of the consistency in eliciting CD8 + T cell responses, these data support the testing of αDEC-205 fusion mAb as a protein-based vaccine

Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8+ T cells

To improve the efficacy of T cell-based vaccination, we pursued the principle that CD4+ T cells provide help for functional CD8 + T cell immunity. To do so, we administered HIV gag to mice successively as protein and DNA vaccines. To achieve strong CD4+ T cell immunity, the protein vaccine was targeted selectively to DEC-205, a receptor for antigen presentation on dendritic cells. This targeting helped CD8+ T cell immunity develop to a subsequent DNA vaccine and improved protection to intranasal challenge with recombinant vaccinia gag virus, including more rapid accumulation of CD8+ T cells in the lung. The helper effect of dendritic cell-targeted protein vaccine wasmimicked by immunization with specificMHCII binding HIV gag peptides but not peptides from a disparate Yersinia pestis microbe. CD4+ helper cells upon adoptive transfer allowed wild-type, but not CD40-/-, recipient mice to respond better to the DNA vaccine. The transfer also enabled recipients to more rapidly accumulate gagspecific CD8+ T cells in the lung following challenge with vaccinia gag virus. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves plasmid DNA immunization, including mobilization of CD8+ T cells to sites of infection

Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-γ- and interleukin 2-producing CD4 + T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-γ-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities

The tumor-targeted CD40 agonist CEA-CD40 promotes T cell priming via a dual mode of action by increasing antigen delivery to dendritic cells and enhancing their activation

Tumor-targeted CD40 agonism represents an attractive strategy for cancer immunotherapy (CIT) as it promotes dendritic cell (DC) activation and concomitant tumor-specific T cell priming without causing systemic side effects. We developed the bispecific CD40 agonistic antibody CEA-CD40, which triggers CD40 stimulation exclusively in the presence of carcinoembryonic antigen (CEA), a glycoprotein specifically expressed on tumor cells. In this study, we demonstrate that CEA-CD40 can enable potent in vitro DC activation and consecutive T cell cross-priming in a CEA-specific manner. Furthermore, we provide evidence that CEA-CD40 increases colocalization of CEA$^{+}$ tumor material and DCs. Using CEA$^{+}$ tumor-derived extracellular vesicles (EVs), which are known to be an excellent tumor antigen source, we show that CEA-CD40 mediates delivery of CEA$^{+}$ EVs to DCs. Importantly, our data indicates that this fosters acquisition of tumor EV major histocompatibility complex I/peptide complexes by DCs, consequently improving CD8$^{+}$ T cell priming against EV-associated antigen in vitro. Thus, we provide mechanistic evidence for a dual mode of action of CEA-CD40 for CIT: we suggest that CEA-CD40 has the potential to activate DCs and in addition can promote their loading with tumor antigen derived from EVs to trigger tumor-specific T cell cross-priming

Lumazine synthase protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development

PURPOSE: Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. MATERIALS AND METHODS: Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. RESULTS: We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8(+)T cells and OT-2-specific CD4(+)T cells. CONCLUSION: Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.clos

An appreciation of Ralph Marvin Steinman (1943–2011)

Ralph Steinman, an editor at the Journal of Experimental Medicine since 1978, shared the 2011 Nobel Prize in Physiology or Medicine for his discovery of dendritic cells (DCs) and their role in immunity. Ralph never knew. He died of pancreatic cancer on September 30, 3 days before the Nobel announcement. Unaware of his death at the time of their announcement, the Nobel Committee made the unprecedented decision that his award would stand. Ralph was the consummate physician-scientist to the end. After his diagnosis, he actively participated in his 4.5 years of treatments, creating experimental therapies using his own DCs in conjunction with the therapies devised by his physicians, all the while traveling, lecturing, and most of all pursuing new investigations in his laboratory. For 38 years—from his discovery of DCs to his Nobel Prize—Ralph pioneered the criteria and methods used to identify, isolate, grow, and study DCs. He and his colleagues demonstrated that DCs are initiators of immunity and regulators of tolerance. In his most recent studies, Ralph was harnessing the specialized features of DCs to design improved vaccines. The following synopsis describes some of his seminal discoveries