Tolerogenic dendritic cells (TDC) offer a promising therapeutic potential to ameliorate autoimmune diseases. Reported to inhibit adaptive immune responses, little is known about their innate immunity ...receptor repertoire. In this study, we compared three types of human TDC (IL-10-DC, dexamethasone (DX)-DC, and 1,25(OH)(2)D(3)-DC) by their TLR expression and response to a set of TLR ligands. TDC are endowed with the same TLR set as standard monocyte-derived dendritic cells but respond differentially to the TLR stimuli Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and flagellin. TDC expressed low or no IL-12-related cytokines and remarkably elevated IL-10 levels. Interestingly, only TDC up-regulated the expression of TLR2 upon stimulation. This boosted the tolerogenic potential of these cells, because IL-10 production was up-regulated in TLR2-stimulated, LPS-primed DX-DC, whereas IL-12 and TNF-alpha secretion remained low. When comparing the TDC subsets, DX-DC and 1,25(OH)(2)D(3)-DC up-regulated TLR2 irrespective of the TLR triggered, whereas in IL-10-DC this effect was only mediated by LPS. Likewise, DX-DC and 1,25(OH)(2)D(3)-DC exhibited impaired ability to mature, reduced allostimulatory properties, and hampered capacity to induce Th1 differentiation. Therefore, both DX-DC and 1,25(OH)(2)D(3)-DC display the strongest tolerogenic and anti-inflammatory features and might be most suitable tools for the treatment of autoimmune diseases.
The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell ...subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8^＋ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells （LCs） were able to cross-present a synthetic long peptide （SLP） to CD8^＋ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1 ^＋ early endosomes. The potency of LCs to enhance CD8^＋ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic：polycytidylic acid （phC）. Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Langerhans cells (LCs) are antigen-presenting cells that reside in the skin. They uniquely express high levels of the C-type lectin receptor Langerin (CD207), which is an attractive target for ...antigen delivery in immunotherapeutic vaccination strategies against cancer. We here assess a library of 20 synthetic, well-defined mannoside clusters, built up from one, two, and three of six monomannosides, dimannosides, or trimannosides, appended to an oligopeptide backbone, for binding with Langerin using surface plasmon resonance and flow cytometric quantification. It is found that Langerin binding affinity increases with increasing number of mannosides. Hexavalent presentation of the mannosides resulted in binding affinities ranging from 3 to 12 μM. Trivalent presentation of the dimannosides and trimannosides led to Langerin affinity in the same range. The model melanoma gp100 antigenic peptide was subsequently equipped with a hexavalent cluster of the dimannosides and trimannosides as targeting moieties. Surprisingly, although the bifunctional conjugates were taken up in LCs in a Langerin-dependent manner, limited antigen presentation to cytotoxic T cells was observed. These results indicate that targeting glycan moieties on immunotherapeutic vaccines should not only be validated for target binding, but also on the continued effects on biology, such as antigen presentation to both CD8+ and CD4+ T cells.
The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post‐translational N‐glycosylation of Asn31 of MOG seems ...to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis‐type glycan structures in the N‐glycan of MOG with the DC‐SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (LeX)‐containing Fmoc‐SPPS‐compatible asparagine building block (SPPS=solid‐phase peptide synthesis), as well as asparagine building blocks containing two LeX‐derived oligosaccharides: LacNAc and Fucα1‐3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG31‐55) and analyzed with respect to their ability to bind to DC‐SIGN in different biological setups, as well as their ability to inhibit the citrullination‐induced aggregation of MOG31‐55. Finally, a cytokine secretion assay was carried out on human monocyte‐derived DCs, which showed the ability of the neoglycopeptide decorated with a single LeX to alter the balance of pro‐ and anti‐inflammatory cytokines, inducing a tolerogenic response.
Immunomodulation. A novel asparagine solid‐phase peptide synthesis building block harboring a Lewis X type DC‐SIGN ligand has been synthesized (see scheme) and incorporated into the immunodominant portion of myelin oligodendrocyte glycoprotein (MOG31‐55). This glycopeptide was evaluated for its ability to inhibit citrullination‐induced aggregation of MOG35‐55, to bind to DC‐SIGN in vitro and for its immunomodulatory effects on human dendritic cells, with the Lewis X yielding a tolerogenic response.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that ...are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
P.69 pertactin (P.69 Prn), an adhesion molecule from the causative agent of pertussis, Bordetella pertussis, is present in cellular and most acellular vaccines that are currently used worldwide. ...Although both humoral immunity and cellular immunity directed against P.69 Prn have been implicated in protective immune mechanisms, the identities of CD4⁺ T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-Ad-restricted B. pertussis conserved CD4⁺ T-cell epitope at the N terminus of P.69 Prn was identified by using a BALB/c T-cell hybridoma. The epitope appeared immunodominant among four other minor strain-conserved P.69 Prn epitopes recognized after vaccination and B. pertussis infection, and it was capable of evoking a Th1/Th17-type cytokine response. B. pertussis P.69 Prn immune splenocytes did not cross-react with natural variants of the epitope as present in Bordetella parapertussis and Bordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4⁺ T cells in an HLA-DQ-restricted manner. During B. pertussis infection, the epitope was associated with a Th1-type CD4⁺ T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4⁺ T-cell immunity after B. pertussis vaccination and infection in mice and, more importantly, in humans. Thus, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance of B. pertussis-specific CD4⁺ T-cell mechanisms in preclinical and clinical vaccine studies.
TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell-mediated immune response. With a dense network of dendritic cells ...(DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell-priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C-stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C-induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines.
In schistosomiasis, a major human parasitic disease caused by helminths, different life-stages of the parasite contribute to the developing host immune response. To increase our understanding of the ...mechanisms that play a role in shaping the host immune responses, we have investigated the effects of schistosome glycoconjugates on the phenotype of dendritic cells (DCs), which form a crucial link between the innate and the adaptive immunity. We show here that
Schistosoma mansoni worm glycolipids induce DC activation as indicated by upregulation of the maturation markers CD80, CD86 and MHC-II, as well as the production of the cytokines interleukin-12 p40 (IL-12 p40), IL-10, IL-1β, IL-6, IL-8 and tumor necrosis factor-α (TNF-α). Co-culture of glycolipid-primed DCs with naïve T cells results in skewing of the T cell response towards a Th1 profile. Remarkably, the DC activation is dependent on fucosylated glycan moieties of the glycolipids. On the DCs, the C-type lectin DC-SIGN and TLR4 are both critically involved in the induced activation, as was demonstrated by using monoclonal antibodies that block interaction of these receptors with the glycolipids. Furthermore, whereas the worm glycolipids were not able to activate HEK 293 cells expressing TLR4, they did show TLR4 activation after introduction of DC-SIGN in the HEK 293-TLR4 cells. Our data provide evidence for a novel function of DC-SIGN as an essential co-receptor for TLR4-induced activation of human DCs. This mechanism of TLR4 activation by worm glycolipids may contribute to eliciting Th1 immune responses in schistosome infection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Toll‐like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by ...dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra‐)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara‐treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro‐inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara‐stimulated DCs showed enhanced cross‐presentation of the melanoma antigen MART‐1, which resulted in increased priming and activation of MART‐1‐specific CD8+ T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Dendritic cells (DCs) have an important role in tumor control via the induction of tumor-specific T-cell responses and are therefore an ideal target for immunotherapy. The human skin is an attractive ...site for tumor vaccination as it contains various DC subsets. The simultaneous delivery of tumor antigen with an adjuvant is beneficial for cross-presentation and the induction of tumor-specific T-cell responses. We therefore developed liposomes that contain the melanoma-associated antigen glycoprotein 100280-288 peptide and Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) as adjuvant. These liposomes are efficiently taken up by monocyte-derived DCs, and antigen presentation to CD8+ T cells was significantly higher with MPLA-modified liposomes as compared with non-modified liposomes or the co-administration of soluble MPLA. We used a human skin explant model to evaluate the efficiency of intradermal delivery of liposomes. Liposomes were efficiently taken up by CD1a+ and especially CD14+ dermal DCs. Induction of CD8+ T–cell responses by emigrated dermal DCs was significantly higher when MPLA was incorporated into the liposomes as compared with non-modified liposomes or co-administration of soluble MPLA. Thus, the modification of antigen-carrying liposomes with TLR ligand MPLA significantly enhances tumor-specific T-cell responses by dermal DCs and is an attractive vaccination strategy in human skin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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