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2.  Flt3L-dependence helps define an uncharacterized subset of murine cutaneous dendritic cells 
Skin-derived dendritic cells (DC) are potent antigen presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DC carry antigens and constitutively migrate to the skin draining lymph nodes (LN). In mice, Langerin-CD11b− dermal DC are a low-frequency, heterogeneous, migratory DC subset that traffic to LN (Langerin-CD11b-migDC). Here, we build on the observation that Langerin-CD11b− migDC are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice which accumulate migDC, demonstrate these cells are cutaneous residents. Langerin-CD11b-Flt3L responsive DC are largely CD24(+) and CX3CR1low and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11bmigDC present antigen with equal efficiency to other DC subsets ex vivo including classical CD8α cDC and Langerin+CD103+ dermal DC. Finally, transcriptome analysis suggests a close relationship to other skin DC, and a lineage relationship to other classical DC. This work demonstrates that Langerin- CD11b− dermal DC, a previously overlooked cell subset, may be an important player in the cutaneous immune environment.
PMCID: PMC3994898  PMID: 24288007
3.  Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross-tolerance 
EMBO Molecular Medicine  2014;6(9):1191-1204.
Skin dendritic cells (DCs) control the immunogenicity of cutaneously administered vaccines. Antigens targeted to DCs via the C-type lectin Langerin/CD207 are cross-presented to CD8+ T cells in vivo. We investigated the relative roles of Langerhans cells (LCs) and Langerin+ dermal DCs (dDCs) in different vaccination settings. Poly(I:C) and anti-CD40 agonist antibody promoted cytotoxic responses upon intradermal immunization with ovalbumin (OVA)-coupled anti-Langerin antibodies (Langerin/OVA). This correlated with CD70 upregulation in Langerin+ dDCs, but not LCs. In chimeric mice where Langerin targeting was restricted to dDCs, CD8+ T-cell memory was enhanced. Conversely, providing Langerin/OVA exclusively to LCs failed to prime cytotoxicity, despite initial antigen cross-presentation to CD8+ T cells. Langerin/OVA combined with imiquimod could not prime CD8+ T cells and resulted in poor cytotoxicity in subsequent responses. This tolerance induction required targeting and maturation of LCs. Altogether, Langerin+ dDCs prime long-lasting cytotoxic responses, while cross-presentation by LCs negatively influences CD8+ T-cell priming. Moreover, this highlights that DCs exposed to TLR agonists can still induce tolerance and supports the existence of qualitatively different DC maturation programs.
PMCID: PMC4197865  PMID: 25085878
CD8+ T-cell responses; dendritic cells; Langerhans cells; skin; tolerance
4.  Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice 
The Journal of Experimental Medicine  2011;208(12):2489-2496.
Regulatory T cells generated by allostimulation with dendritic cells, transforming growth factor β, and retinoic acid stably express Foxp3 and can suppress even ongoing GVHD in mice.
Foxp3+ regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor β (TGF-β) and retinoic acid. We found that the CD11chigh dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3+ cells. The induced CD4+CD25+Foxp3+ cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RBhi T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3+ T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease.
PMCID: PMC3256961  PMID: 22084406
5.  Induction of innate and adaptive immunity by delivery of poly dA:dT to dendritic cells 
Nature chemical biology  2013;9(4):250-256.
Targeted delivery of antigens to dendritic cells (DCs) is a promising vaccination strategy. However, to ensure immunity, the approach depends on coadministration of an adjuvant. Here we ask whether targeting of both adjuvant and antigen to DCs is sufficient to induce immunity. Using a protein ligation method, we develop a general approach for linking the immune stimulant, poly dA:dT (pdA:dT), to a monoclonal antibody (mAb) specific for DEC205 (DEC). We show that DEC-specific mAbs deliver pdA:dT to DCs for the efficient production of type I interferon in human monocyte-derived DCs and in mice. Notably, adaptive T-cell immunity is elicited when mAbs specific for DEC–pdA:dT are used as the activation stimuli and are administered together with a DC-targeted antigen. Collectively, our studies indicate that DCs can integrate innate and adaptive immunity in vivo and suggest that dual delivery of antigen and adjuvant to DCs might be an efficient approach to vaccine development.
PMCID: PMC3947612  PMID: 23416331
6.  Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract 
The Journal of Experimental Medicine  2013;210(9):1871-1888.
Lung DCs induce the expression of gut-homing molecules on T cells, resulting in their migration to the GI tract and protection against Salmonella infection after immunization
Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of α4β7 and CCR9 by Peyer’s patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid–dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103+ MLN DCs, up-regulate the gut-homing integrin α4β7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.
PMCID: PMC3754860  PMID: 23960190
7.  HIV gag protein is efficiently cross-presented when targeted with an antibody towards the DEC-205 receptor in Flt3 ligand-mobilized murine dendritic cells 
European journal of immunology  2010;40(1):36-46.
Dendritic cells present exogenous proteins to MHC class I restricted CD8+ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8+ T cell responses to immune complexes, inactivated microbes, dying cells and proteins like ovalbumin. In mice, the CD8+ or DEC-205+ DC are specialized for cross-presentation, and this subset can be increased 10 fold in numbers following Flt3L treatment in vivo. Therefore we studied cross-presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti-CD11c beads, cells from Flt3L mice are not only more abundant but are more highly enriched in CD11c high DC, particularly the DEC-205+ subset. DC cross-present HIV gag to primed CD8+ T cells, but when the antigen is delivered within an antibody to DEC-205 receptor, cross-presentation becomes 100 fold more efficient than non-targeted antigen. This finding requires gag to be engineered into anti-DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross-presentation, since their use overcomes the obstacle posed by the low number of cross-presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC-targeted vaccines.
PMCID: PMC2933266  PMID: 19830741
DC; Flt3L; poly IC
8.  Direct Type I IFN but Not MDA5/TLR3 Activation of Dendritic Cells Is Required for Maturation and Metabolic Shift to Glycolysis after Poly IC Stimulation 
PLoS Biology  2014;12(1):e1001759.
Type I IFN signaling is indispensable for the maturation of dendritic cells (DCs) that are required to elicit an immune response, and it also controls a shift in cellular metabolism to meet the increased energy demands of DC maturation.
Type I interferons (IFNs) play an important role in direct antiviral defense as well as linking the innate and adaptive immune responses. On dendritic cells (DCs), IFNs facilitate their activation and contribute to CD8+ and CD4+ T cell priming. However, the precise molecular mechanism by which IFNs regulate maturation and immunogenicity of DCs in vivo has not been studied in depth. Here we show that, after in vivo stimulation with the TLR ligand poly IC, IFNs dominate transcriptional changes in DCs. In contrast to direct TLR3/mda5 signaling, IFNs are required for upregulation of all pathways associated with DC immunogenicity. In addition, metabolic pathways, particularly the switch from oxidative phosphorylation to glycolysis, are also regulated by IFNs and required for DC maturation. These data provide evidence for a metabolic reprogramming concomitant with DC maturation and offer a novel mechanism by which IFNs modulate DC maturation.
Author Summary
Immune responses are orchestrated by a specialized cell type called dendritic cells (DCs). In order to achieve durable and robust immunity, DCs need to undergo an intricate differentiation process known as maturation. This process is poorly understood at the moment. Poly IC, which mimics viral RNA and is an agonist for the viral pattern recognition receptors (PRRs) that signal “danger” to the immune system, can induce full maturation of DCs and it has been shown that this requires type I IFN signaling. In this study we set out to examine the specific signals provided by direct PRR or IFN stimulation that are required for DC maturation. We found to our surprise that type I IFN can regulate almost all steps of the DC's maturation process without requiring direct PRR involvement. We also show that type I IFN regulates several metabolic switches essential for preservation of DC integrity: it stimulates the expression of the hypoxia-inducible factor 1α (Hif1α), which controls the metabolic shift from oxidative phosphorylation (used by resting cells to generate energy) to aerobic glycolysis, a less efficient but faster energy-producing process. This metabolic shift was required to meet increased energy demands of activated DCs and to prevent their premature death, thus sustaining an immune response to viral infection.
PMCID: PMC3883643  PMID: 24409099
9.  siRNA knockdown of PD-L1 and PD-L2 in monocyte-derived dendritic cells only modestly improves proliferative responses to Gag by CD8+ T cells from HIV-1 infected individuals 
Journal of clinical immunology  2009;29(5):637-645.
Due to their capacity to elicit and regulate immunity, dendritic cells (DCs) are important targets to improve vaccination. Knowing that PD-1 high virus-specific T cells become functionally exhausted during chronic exposure to HIV-1, the development of a therapeutic DC-based HIV-1 vaccine might include strategies that down regulate PD-L1 and PD-L2 counter-receptors. After showing that monocyte-derived DCs rapidly up regulated PD-L1 and PD-L2 expression upon maturation with a variety of stimuli e.g. TLR ligands and cytokines, we determined that PD-L1 and PD-L2 expression could be knocked down by electroporation of a single siRNA sequence twice at the monocyte and immature stages of DC development. This knockdown approached completion, and was specific and lasting for several days. We then added the PD-L1 and PD-L2 silenced monocyte-derived DCs to PBMCs from HIV-1 infected individuals along with pools of 15-mer HIV-1 Gag p24 peptides. However, in cultures from 6 patients, there was only a modest enhancing effect of PD-L1 and PD-L2 silencing on CD8+ T cell proliferative responses to the DCs. These findings suggest that in monocyte-derived DCs, additional strategies than PD-L1 or PD-L2 blockade will be needed to improve the function of PD-1 high T cells.
PMCID: PMC2933267  PMID: 19562472
Dendritic Cells; PD-L1; PD-L2; siRNA; HIV-1 Gag; CD8+ T cell
10.  Broad T cell immunity to the LcrV virulence protein is induced by targeted delivery to DEC-205/CD205 positive mouse dendritic cells 
European journal of immunology  2008;38(1):20-29.
There is a need for a more efficient vaccine against the bacterium Y. pestis, the agent of pneumonic plague. The F1-LcrV subunit vaccine in alhydrogel is known to induce humoral immunity. In this study, we utilized dendritic cells to investigate cellular immunity. We genetically engineered the LcrV virulence protein into the αDEC-205/CD205 monoclonal antibody and thereby targeted the conjugated protein directly to mouse DEC-205+ DCs in situ. We observed antigen-specific CD4+ T cell immunity measured by intracellular staining for interferon-γ in three different mouse strains (C57BL/6, BALB/c, and C3H/HeJ), while we could not observe such T cell responses with F1-V vaccine in alhydrogel. Using a peptide library for LcrV protein, we identified two or more distinct CD4+ T cell mimetopes in each MHC haplotype, consistent with the induction of broad immunity. When compared to nontargeted standard protein vaccine, DC targeting greatly increased the efficiency for inducing IFN-γ producing T cells. The targeted LcrV protein induced antibody responses to a similar extent as the F1-V subunit vaccine, but Th1-dependent IgG2a and IgG2c isotypes were observed only after αDEC-205:LcrV mAb immunization. This study sets the stage for the analysis of functional roles of IFN-γ producing T cells in Y. pestis infection.
PMCID: PMC2864640  PMID: 18081041
Dendritic cells; CD 205/DEC-205; Y. pestis; LcrV; Cellular immunity
11.  A new Trem family member, Treml4, binds to dead cells and is a DAP12 linked marker for subsets of mouse macrophages and dendritic cells1 
Dendritic cells (DCs) are professional antigen presenting cells that can control immune responses against self and altered self, typically foreign, determinants. DCs can be divided into several subsets including CD8α+ and CD8α− DCs. These subsets possess specific functions. For example, mouse splenic CD8α+, but not CD8α− DCs, selectively take up dying cells and cross-present cell-associated antigens to naïve T cells. In this study, we identified genes that were more expressed in CD8α+ than CD8α− DCs by microarray analysis. Only one of these genes, when the extracellular domains were linked to human IgG Fc domain, could bind to late apoptotic or necrotic cells. This gene was a new member of the Triggering receptor expressed on myeloid cells (Trem) family, Trem-like 4 (Treml4). Treml4 mRNA and protein, the latter detected with a new monoclonal antibody, were predominantly expressed in spleen. Treml4, like other Trem family members, could associate with the adaptor molecule DAP12, but neither DAP10 nor FcRγ. Consistent with the microarray data, we confirmed that Treml4 protein was more expressed on CD8α+ than CD8 α− DCs, and we also found that Treml4 was expressed at high levels on splenic macrophages in spleen particularly red pulp and marginal metallophilic macrophages. In addition, Treml4 expression on DCs was not changed after maturation induced by Toll-like receptor ligands. Thus, Treml4 is a new Trem family molecule that is abundantly expressed on CD8α+ DCs and subsets of splenic resident macrophages, and can recognize dead cells by different types of phagocytes in spleen.
PMCID: PMC2843158  PMID: 19155473
Dendritic cells; Monocytes/Macrophages; Cell surface molecules
12.  Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant 
The Journal of Experimental Medicine  2009;206(7):1589-1602.
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.
PMCID: PMC2715098  PMID: 19564349
13.  Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves 
Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP+ cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c+ cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8+ TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.
PMCID: PMC2699134  PMID: 19221394
14.  PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205 
Journal of immunological methods  2012;384(1-2):184-190.
DEC205/CD205, an endocytic receptor of C-type multilectin, is expressed highly in dendritic cells (DCs). DEC205 was shown to efficiently deliver vaccine antigens in surrogate ligands to the antigen processing and presentation machinery of DCs, which resulted in the development of DC-targeted vaccines employing anti-DC monoclonal antibodies (mAbs). During our studies to characterize a variety of anti-DC mAbs including anti-DEC205 by flow cytometric analysis, we discovered that a secondary anti-immunoglobulin antibody conjugated with PE-Cy5.5 bound strongly to the cells expressing mouse DEC205 (mDEC205) without incubation of a primary anti-mDEC205 mAb. In the present study we demonstrate that various antibodies and streptavidin conjugated with PE-Cy5.5 bind to the mDEC205-expressing cells including CHO, KIT6, and HEK293 cells. The interaction between the PE-Cy5.5 conjugates and the cells expressing mDEC205 appears distinctive, since none of PE-Cy5.5 conjugates bind to the cells that express human DEC205 on surface. Besides, only PE-Cy5.5 conjugates bind strongly to mDEC205-expressing cells; PerCP-Cy5.5, APC-Cy5.5, and Cy5.5 conjugates bind weakly; PE, PE-Cy5, Cy5, FITC, or Alexa488 conjugates do not bind. Therefore the use of PE-Cy5.5 conjugates, widely utilized in multicolor flow cytometry, requires precaution against nonspecific binding to mDEC205-positive cells.
PMCID: PMC3432670  PMID: 22841832
CD205; DEC205; PE-Cy5.5; Nonspecific binding; Dendritic Cells
15.  Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor 
Vaccine  2012;30(45):6359-6367.
It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Y. pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4+ T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4+ T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.
PMCID: PMC3461253  PMID: 22947140
Dendritic cells; CD205/DEC-205; Y. pestis; LcrV; Cellular immunity; Mucosal
16.  Correction: Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity 
PLoS ONE  2013;8(9):10.1371/annotation/5149bf8e-3843-4865-a726-0ca2820ee8f8.
PMCID: PMC3782404
17.  The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells 
The Journal of Clinical Investigation  2008;118(4):1427-1436.
DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41–scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-γ–producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41–scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.
PMCID: PMC2263146  PMID: 18324335
18.  Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin/CD209 Is Abundant on Macrophages in the Normal Human Lymph Node and Is Not Required for Dendritic Cell Stimulation of the Mixed Leukocyte Reaction1 
The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.
PMCID: PMC1378112  PMID: 16177066
20.  A subset of dendritic cells induces CD4+ T cells to produce IFN-γ by an IL-12–independent but CD70-dependent mechanism in vivo 
The Journal of Experimental Medicine  2007;204(5):1095-1106.
Interferon (IFN)-γ, a cytokine critical for resistance to infection and tumors, is produced by CD4+ helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12–independent pathway whereby DCs induce IFN-γ–secreting T helper (Th)1 CD4+ T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-γ in vivo but required IL-12, not CD70. Isolated CD205+ DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205+ DC function in vivo. Detection of the IL-12–independent IFN-γ pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205+ DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.
PMCID: PMC2118574  PMID: 17438065
21.  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.
PMCID: PMC2118242  PMID: 16505141
22.  Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells 
The Journal of Experimental Medicine  2005;202(11):1507-1516.
If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex–negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid α-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic αCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance.
PMCID: PMC2213343  PMID: 16330814
23.  Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity 
PLoS ONE  2013;8(6):e67453.
Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires development of type 1 T-helper (Th1) CD4+ T cell immunity. Because of their unique capacity to initiate and modulate immune responses, dendritic cells (DCs) are attractive targets for development of novel vaccines. In this study, for the first time, we investigated the capacity of a DC-targeted vaccine to induce protective responses against L. major. To this end, we genetically engineered the N-terminal portion of the stress-inducible 1 protein of L. major (LmSTI1a) into anti-DEC205/CD205 (DEC) monoclonal antibody (mAb) and thereby delivered the conjugated protein to DEC+ DCs in situ in the intact animal. Delivery of LmSTI1a to adjuvant-matured DCs increased the frequency of antigen-specific CD4+ T cells producing IFN-γ+, IL-2+, and TNF-α+ in two different strains of mice (C57BL/6 and Balb/c), while such responses were not observed with the same doses of a control Ig-LmSTI1a mAb without receptor affinity or with non-targeted LmSTI1a protein. Using a peptide library for LmSTI1a, we identified at least two distinct CD4+ T cell mimetopes in each MHC class II haplotype, consistent with the induction of broad immunity. When we compared T cell immune responses generated after targeting DCs with LmSTI1a or other L. major antigens, including LACK (Leishmania receptor for activated C kinase) and LeIF (Leishmania eukaryotic ribosomal elongation and initiation factor 4a), we found that LmSTI1a was superior for generation of IFN-γ-producing CD4+ T cells, which correlated with higher protection of susceptible Balb/c mice to a challenge with L. major. For the first time, this study demonstrates the potential of a DC-targeted vaccine as a novel approach for cutaneous leishmaniasis, an increasing public health concern that has no currently available effective treatment.
PMCID: PMC3694010  PMID: 23840706
24.  CD25+ CD4+ T Cells, Expanded with Dendritic Cells Presenting a Single Autoantigenic Peptide, Suppress Autoimmune Diabetes 
The Journal of Experimental Medicine  2004;199(11):1467-1477.
In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet β cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 105 polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.
PMCID: PMC2211787  PMID: 15184500
insulin-dependent diabetes mellitus; dendritic cells; CD25+ CD4+ regulatory; T cells; BDC2.5; autoimmunity
25.  In Vivo Targeting of Antigens to Maturing Dendritic Cells via the DEC-205 Receptor Improves T Cell Vaccination 
The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic α-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the αDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell–mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.
PMCID: PMC2212731  PMID: 15024047
dendritic cell; DEC-205 receptor; vaccination; CD8 T cell; immunotherapy

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