BackgroundAutoimmune demyelinating diseases (ADD) are a major cause of neurological disability due to autoreactive cellular and humoral immune responses against brain antigens. A cure for chronic ADD could be obtained by appropriate immunomodulation.MethodsWe implemented a preclinical scheme to foster immune tolerance to myelin oligodendrocyte glycoprotein (MOG), in a cynomolgus-macaque model of experimental autoimmune encephalomyelitis (EAE), in which administration of recombinant human MOG (rhMOG) elicits brain inflammation mediated by MOG-autoreactive CD4+ lymphocytes and anti-MOG IgG. For immunotherapy, we used a recombinant antibody (Ab) directed against the dendritic cell-asialoglycoprotein receptor (DC-ASGPR) fused either to MOG or a control antigen PSA (prostate-specific antigen).FindingsrhMOG and the anti-DC-ASGPR-MOG were respectively detected in CD1a+ DCs or CD163+ cells in the skin of macaques. Intradermal administration of anti-DC-ASGPR-MOG, but not control anti-DC-ASGPR-PSA, was protective against EAE. The treatment prevented the CD4+ T cell activation and proinflammatory cytokine production observed in controls. Moreover, the administration of anti-DC-ASGPR-MOG induced MOG-specific CD4+CD25+FOXP3+CD39+ regulatory lymphocytes and favoured an upsurge in systemic TGFβ and IL-8 upon rhMOG re-administration in vivo.InterpretationWe show that the delivery of an anti-DC-ASGPR-MOG allows antigen-specific adaptive immune modulation to prevent the breach of immune tolerance to MOG. Our findings pave the way for therapeutic vaccines for long-lasting remission to grave encephalomyelitis with identified autoantigens, such as ADD associated with anti-MOG autoantibodies.FundWork supported by the French ANR (ANR-11-INBS-0008 and ANR-10-EQPX-02-01), NIH (NIH 1 R01 AI 105066), the Baylor Scott and White Healthcare System funding and Roche Research Collaborative grants.

To study the effect of vaccination on tolerization to the myelin antigen MOG we used a macaque model of experimental autoimmune encephalitis (EAE) in which immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) elicits brain inflammation and demyelination mediated by MOG-specific autoreactive CD4+ T lymphocytes and anti-MOG IgG. For antigen targeting to tolerizing antigen presenting cells, we used a recombinant antibody directed to the Dendritic Cells (DC)-Asialoglycoprotein receptor (DC-ASGPR). The intradermal administration of an anti-DC-ASGPR-MOG fusion protein, but not a control anti-DC-ASGPR-PSA (prostate specific antigen) protein, protected monkeys committed to develop EAE. Although effective treatment did not modify anti-MOG IgG production, it prevented the CD4+ T lymphocyte activation and pro-inflammatory cytokine production. Moreover, animals treated with anti-DC-ASGPR-MOG experienced a rise of MOG-specific CD4+CD25+FOXP3+CD39+ regulatory T cells as well as a TGF β 1, TGF β 2 and IL-8 upsurge after rhMOG re-immunization. Our results indicate that the pathogenicity of autoantibodies directed to MOG is mitigated in the presence of MOG-specific regulatory lymphocytes. This vaccination scheme appears suitable to treat relapsing autoimmune diseases with identified autoantigens such as that harboring anti-MOG or anti-AQP4 autoantibodies.

ABSTRACT 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.

Improved antigenicity against HIV-1 envelope (Env) protein is needed to elicit vaccine-induced protective immunity in humans. Here we describe the first tests in non-human primates (NHPs) of Env gp140 protein fused to a humanized anti-LOX-1 recombinant antibody for delivering Env directly to LOX-1-bearing antigen presenting cells, especially dendritic cells (DC). LOX-1, or 1ectin-like oxidized low-density lipoprotein (LDL) receptor-1, is expressed on various antigen presenting cells and endothelial cells, and is involved in promoting humoral immune responses. The anti-LOX-1 Env gp140 fusion protein was tested for priming immune responses and boosting responses in animals primed with replication competent NYVAC-KC Env gp140 vaccinia virus. Anti-LOX-1 Env gp140 vaccination elicited robust cellular and humoral responses when used for either priming or boosting immunity. Co-administration with Poly ICLC, a TLR3 agonist, was superior to GLA, a TLR4 agonist. Both CD4+ and CD8+ Env-specific T cell responses were elicited by anti-LOX-1 Env gp140, but in particular the CD4+ T cells were multifunctional and directed to multiple epitopes. Serum IgG and IgA antibody responses induced by anti-LOX-1 Env gp140 against various gp140 domains were cross-reactive across HIV-1 clades; however, the sera neutralized only HIV-1 bearing sequences most similar to the clade C 96ZM651 Env gp140 carried by the anti-LOX-1 vehicle. These data, as well as the safety of this protein vaccine, justify further exploration of this DC-targeting vaccine approach for protective immunity against HIV-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-1β 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.

ABSTRACTSmoking is associated with increased susceptibility to tuberculosis and influenza. However, little information is available on the mechanisms underlying this increased susceptibility. Mice were left unexposed or were exposed to cigarette smoke and then infected withMycobacterium tuberculosisby aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenicM. tuberculosisprotein. Gamma interferon (IFN-γ) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-γ during stimulationin vitrowith anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection withM. tuberculosisand influenza A virusin vivo. Reduced IFN-γ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-γ expression. Cigarette smoke exposure increased the bacterial burden in mice infected withM. tuberculosisand increased weight loss and mortality in mice infected with influenza virus. This study provides the first demonstration that cigarette smoke exposure directly inhibits the pulmonary T-cell response toM. tuberculosisand influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens.

We evaluated human CD8(+) T-cell responses generated by targeting antigens to dendritic cells (DCs) through various lectin receptors. We found the immunoreceptor tyrosine-based inhibitory motif-containing DC immunoreceptor (DCIR) to mediate potent cross-presentation. A single exposure to a low dose of anti-DCIR-antigen conjugate initiated antigen-specific CD8(+) T-cell immunity by all human DC subsets including ex vivo-generated DCs, skin-isolated Langerhans cells, and blood myeloid DCs and plasmacytoid DCs. The delivery of influenza matrix protein (FluMP) through DCIR resulted in expansion of FluMP-specific memory CD8(+) T cells. Enhanced specific CD8(+) T-cell responses were observed when an antigen was delivered to the DCs via DCIR, compared with those induced by a free antigen, or antigen conjugated to a control monoclonal antibody or delivered via DC-SIGN, another lectin receptor. DCIR targeting also induced primary CD8(+) T-cell responses against self (MART-1) and viral (HIV gag) antigens. Addition of Toll-like receptor (TLR) 7/8 agonist enhanced DCIR-mediated cross-presentation as well as cross-priming, particularly when combined with a CD40 signal. TLR7/8 activation was associated with increased expansion of the primed CD8(+) T cells, high production of interferon-γ and tumor necrosis factor-α, and reduced levels of type 2-associated cytokines. Thus, antigen targeting via the human DCIR receptor allows activation of specific CD8(+) T-cell immunity.

Dectin-1 recognizing fungal pathogens and mycobacteria, delivers intracellular signals to activate DCs. Here we report that Dectin-1 expressed on human DCs possesses unique immunological function that potentiates antigen-specific CD8+ T cell responses. We first generated an agonistic anti-Dectin-1 monoclonal antibody (mAb), which recognized Leu192-Asn202 in Dectin-1 and efficiently bound to both in vitro monocyte-derived DCs and in vivo CD1c+ skin dermal DCs, but not Langerin cells. Syk-dependent activation by anti-Dectin-1 mAb induced DCs to upregulate T cell costimulatory molecules, particularly CD70 and 4-1BBL, as well as cytokines and chemokines. This signaling via Dectin-1 allowed DCs to potentiate both memory and naïve CD8+ T cell responses by promoting their expansions and functional activities. Furthermore, both neo- and non-self-antigens delivered to DCs via Dectin-1 resulted in potent antigen-specific CD8+ T cell responses. This was largely dependent on CD70 and 4-1BBL upregulated by the signaling via Dectin-1. We also demonstrated that Dectin-1-mediated activation of DCs did not promote Foxp3+ and IL-10-producing regulatory CD4+ T cell responses, but potentiated CD4+ helper T cell function for inducing effector CD8+ T cells. We conclude that Dectin-1 on human DCs has unique function for eliciting antigen-specific CD8+ T cell responses by taking up antigens and delivering intracellular signals favoring effector CD8+ T cell responses.

To determine the immunogenicity and protective efficacy of the Mycobacterium tuberculosis 10 kD culture filtrate protein (CFP10), and to evaluate strategies that enhance local immunity, we used C57Bl/6 DR4 mice that were transgenic for human HLA DRB1 0401, because CFP10 contains epitopes for DRB1 0401 but not for C57Bl/6 mice. Intramuscular immunization with a DNA vaccine encoding CFP10 elicited production of IFN-gamma by systemic CD4+ T cells, and one intravenous dose of the CFP10-based DNA vaccine coated with polyethylenimine (PEI) stimulated IFN-gamma production by lung CD4+ cells and reduced the pulmonary bacillary burden. We conclude that CFP10 is a potential vaccine candidate and that coating vaccines with PEI enhances local protective immunity to tuberculosis

IFN-γ production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-γ, −73 to −48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-γ in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-γ upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-γ proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-γ and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-γ proximal promoter and that they up-regulate IFN-γ transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

CD4+ T cells are believed to play a dominant role in human defenses against Mycobacterium tuberculosis through production of interferon (IFN)-gamma, cytolytic T-cell (CTL) activity, and inhibition of intracellular mycobacterial growth. Most functional studies of CD4+ cells have used bulk T-cells that recognize crude mycobacterial antigens, and the functional capacity of individual human T cells is not well defined. We studied the functional capacity of human CD4+ T-cell clones that recognize a specific mycobacterial peptide. Clone B9 produced high concentrations of IFN-gamma and exhibited potent CTL activity, whereas clone D3 produced IFN-gamma but showed poor CTL activity. The CTL activity of clone B9 was inhibited by SrCl(2) and concanamycin A but not by anti-Fas antibodies. Clone B9 also reduced the mycobacterial burden in dendritic cells by more than 90%, and this antimycobacterial activity was inhibited by SrCl(2) and concanamycin A. We conclude that: (1) individual human peptide-specific CD4+ T-cell clones have differential capacity to produce Th1 cytokines and to lyse M tuberculosis-infected target cells; and (2) both granulysin and perforin contribute to the capacity of human CD4+ T-cells to lyse infected targets and to inhibit intracellular mycobacterial growth.

IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.

RATIONALE Diagnosis of latent tuberculosis infection (LTBI) is currently based on the tuberculin skin test. The enzyme-linked immunospot assay (ELISPOT) is a new blood test to diagnose LTBI. OBJECTIVE To compare the ELISPOT and the tuberculin skin test for detecting LTBI in contacts of patients with tuberculosis. METHODS Prospective study of 413 contacts of patients with tuberculosis. MEASUREMENTS AND MAIN RESULTS Because there is no gold standard for LTBI, the sensitivity and specificity of the ELISPOT and tuberculin skin test cannot be directly measured. For each contact, we therefore estimated the likelihood of having LTBI by calculating a contact score that quantified exposure to and infectiousness of the index case. We analyzed the relationship of contact score to ELISPOT and tuberculin skin test results. The likelihood of a positive ELISPOT (p = 0.0005) and a tuberculin skin test (p = 0.01) increased significantly with rising contact scores. The contact score was more strongly related to the ELISPOT than to the tuberculin skin test results, although this difference was not statistically significant. Among U.S.-born persons and those who were not vaccinated with bacille Calmette-Guérin, approximately 30% had positive ELISPOT or tuberculin skin test results. Foreign-born, bacille Calmette-Guérin-vaccinated persons were significantly more likely to have a positive tuberculin skin test than a positive ELISPOT result (p < 0.0001). CONCLUSIONS Compared with the tuberculin skin test, the ELISPOT appears to be at least as sensitive for diagnosis of LTBI in contacts of patients with tuberculosis.

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.