Discordance in STING-Induced Activation and Cell Death Between Mouse and Human Dendritic Cell Populations.

Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes.

Human CD141+ dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model.

BACKGROUND: The conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141+ DC cDC1 equivalent in patients with melanoma. METHODS: We developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141+ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1). RESULTS: Blood CD141+ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141+ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141+ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment. CONCLUSIONS: These data illustrate quantitative and qualitative impairments in circulating CD141+ DCs in patients with advanced melanoma and that increasing CD141+ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.

Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141+ dendritic cells to activate naïve and memory NY-ESO-1-specific CD8+ T cells.

BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1-specific CD8+ T cells.

OBJECTIVES: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1-specific CD8+ T cells. METHODS: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or ß-galactosidase (untargeted control). Activation of WT1-specific CD8+ T-cell lines following cross-presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8+ T cells, were used to investigate naïve WT1-specific CD8+ T-cell priming. RESULTS: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines. CONCLUSIONS: Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141+ DCs is sufficient for effective CD8+ T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.

A reliable method for enrichment of neutrophils from peripheral blood in barramundi (Lates calcarifer).

Neutrophils are a short-lived, terminally differentiated, innate immune cell, that are critical first responders during infection. Research into neutrophil-pathogen interactions in fish has primarily employed cells derived from the pro-nephros and nephros. Since these sites are also the location of neutrophil and other immune cell development, there may be some ambiguity in maturation and functional ability of these cells, and difficulty in differentiating the effects of neutrophils from those of macrophages and monocytes. In contrast, peripheral blood circulating neutrophils are mature and ready to respond, thus it may be more physiologically relevant to use these cells for immune studies when evaluating interactions with blood-borne pathogens. The enrichment of tropical, euryhaline fish blood cells cannot follow classic mammalian enrichment methods for several reasons: Fish have nucleated red blood cells (RBC's), a high number of reticulocytes, a very low number of granulocytic leukocytes and an osmotic tolerance, rendering techniques such as water lysis ineffective. Enrichment of neutrophils, while minimizing RBC contamination, is imperative for studies where luminescence or fluorescence signals may be confounded by background from an overabundance of RBC's. We have optimized a method for enriching neutrophils from peripheral blood, with an initial settlement step employing 6% dextran (Mr 450,000-650,000), for 30-60 min at room temperature, followed by density separation on an 8-step Percoll density gradient. This method provides a cell suspension comprising 20-50% neutrophils, free of contamination from reticulocytes. These are then suitable for luminometric or fluorometric downstream analyses.

Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages.

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

Development of an automated, high-throughput bactericidal assay that measures cellular respiration as a survival readout for Neisseria meningitidis.

Complement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity against Neisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.

Characterization of the peptide-binding specificity of the chimpanzee class I alleles A 0301 and A 0401 using a combinatorial peptide library.

Chimpanzees represent important models for studying several human pathogens. In the present study, we utilized a combinatorial peptide library to characterize the binding specificities of the chimpanzee class I molecules Patr A 0301 and A 0401, both of which are present in about 17% of chimpanzees. Patr A 0301 was found to recognize peptides using the canonical position 2/C-terminus spacing, with the small residues S, T, and A being the most preferred in position 2, and the positively charged residues R and K preferred at the C terminus. Patr A 0401 was found to recognize a more complex motif where the C terminus and then the residue in positions 1 and/or 5 are the primary anchors. Like A 0301, the C-terminal preference of A 0401 is for positively charged residues. At positions 1 and 5, positively charged and large residues are the most preferred, respectively. Coefficient values derived from the combinatorial library proved to be an efficient means for predicting A 0301 and A 0401 binders. The present data provide detailed information to facilitate the identification of potential T cell epitopes recognized in the context of two common chimpanzee class I alleles, and further validate the combinatorial library approach as an efficient method to characterize class I binding specificities.

Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination.

Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 microg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines.

Reversal in the immunodominance hierarchy in secondary CD8+ T cell responses to influenza A virus: roles for cross-presentation and lysis-independent immunodomination.

Immunodominance is a central feature of CD8+ T cell (TCD8+) responses to pathogens, transplants, and tumors. Determinants occupy a stable position in an immunodominance hierarchy (alpha-, beta-, etc.) defined by the frequencies of responding TCD8+. In this paper, we study the mechanistic basis for place-swapping between alpha- (acid polymerase (PA)(224-233)) and beta-determinants (nuclear protein 366-374) in primary vs secondary anti-influenza A virus (IAV) responses in mice. This phenomena was recently correlated with the inability of IAV-infected nondendritic cells (DCs) to generate PA(224-233), and it was proposed that secondary TCD8+ are principally activated by IAV-infected epithelial cells, while primary TCD8+ are activated by IAV-infected DCs. In this study, we show that the inability of non-DCs to generate PA(224-232) is relative rather than absolute, and that the preferential use of cross-priming in secondary anti-IAV responses can also account for the revised hierarchy. We further show that immunodomination of PA(224-233)-specific TCD8+ by nucleoprotein 366-374-specific TCD8+ plays a critical role in the phenomena, and that this is unlikely to be mediated by TCD8+ lysis of APCs or other cells.

The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand.

Dendritic cells (DCs) show promise as adjuvants in anticancer immunotherapeutic strategies. Flt3 ligand (FL) is a hematopoietic growth factor that increases the number of immature DCs in the blood and other tissues. We treated 27 patients with metastatic or high-risk resected melanoma with s.c. FL daily for 14 d in three 28 d cycles. Eighteen of these patients also received vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides. To induce local DC maturation, 8 of the vaccinated patients had imiquimod, a Toll-like receptor-7 ligand (TLR7L), applied topically to their vaccine sites. Patients were monitored for clinical and hematological effects. Immune responses were assessed by cutaneous reactivity to vaccination and by the induction of peptide-specific CD8+ T-cells. Eight patients did not complete the protocol due to adverse events related to their cancer. The treatment was generally safe and well tolerated, although some patients developed clinically significant toxicities related to FL. FL induced increases in immature CD11c+ and CD123+ peripheral blood (PB) DCs. Other hematological effects included monocytosis, granulocytosis, and thrombocytosis, which were marked in some patients. Cutaneous reactions to peptide vaccination and circulating peptide-specific CD8+ T-cells were more frequent in imiquimod-treated patients. FL treatment of melanoma patients has pleiotropic clinical and hematological effects. In vivo maturation of FL-generated DCs using imiquimod may increase immune responses to tumor antigens.

Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO-1 formulated with ISCOMATRIX adjuvant.

There is increasing evidence showing the involvement of CD4(+) T cells in initiating and maintaining antitumor immune responses. NY-ESO-1 is expressed by various tumors but not normal tissues except testis. We conducted a cancer clinical trial by using full-length NY-ESO-1 protein formulated with ISCOMATRIX adjuvant and injected into patients intramuscularly. Autologous dendritic cells pulsed with NY-ESO-1 ISCOMATRIX in combination with overlapping synthetic peptides were used to identify immunodominant T cells from a vaccinated patient. We show here the identification and characterization of two novel CD4(+) T cell epitopes. T cells specific to these epitopes not only recognized autologous dendritic cells loaded with NY-ESO-1 but also NY-ESO-1-expressing tumor cell lines treated with IFN-gamma. One of the two responses identified was greater than the previously identified immunodominant HLA-DP4-restricted response and correlated with NY-ESO-1-specific CD8(+) T cell induction after vaccination. This T cell response was vaccinated in most patients who expressed HLA-DR2. This study has systematically surveyed patients vaccinated with full-length tumor antigen for a vaccinated CD4 helper T cell response.

NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ t-cell-mediated immunity and protection against NY-ESO-1+ tumors.

NY-ESO-1 is a 180 amino-acid human tumor antigen expressed by many different tumor types and belongs to the family of "cancer-testis" antigens. In humans, NY-ESO-1 is one of the most immunogenic tumor antigens and NY-ESO-1 peptides have been shown to induce NY-ESO-1-specific CD8(+) CTLs capable of altering the natural course of NY-ESO-1-expressing tumors in cancer patients. Here we describe the preclinical immunogenicity and efficacy of NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant (NY-ESO-1 vaccine). In vitro, the NY-ESO-1 vaccine was readily taken up by human monocyte-derived dendritic cells, and on maturation, these human monocyte-derived dendritic cells efficiently cross-presented HLA-A2-restricted epitopes to NY-ESO-1-specific CD8(+) T cells. In addition, epitopes of NY-ESO-1 protein were also presented on MHC class II molecules to NY-ESO-1-specific CD4(+) T cells. The NY-ESO-1 vaccine induced strong NY-ESO-1-specific IFN-gamma and IgG2a responses in C57BL/6 mice. Furthermore, the NY-ESO-1 vaccine induced NY-ESO-1-specific CD8(+) CTLs in HLA-A2 transgenic mice that were capable of lysing human HLA-A2(+) NY-ESO-1(+) tumor cells. Finally, C57BL/6 mice, immunized with the NY-ESO-1 vaccine, were protected against challenge with a B16 melanoma cell line expressing NY-ESO-1. These data illustrate that the NY-ESO-1 vaccine represents a potent therapeutic anticancer vaccine.

Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon.

"Cross-priming" refers to the activation of naive CD8+ T cells by antigen-presenting cells that have acquired nominal antigens from another cell. The biological relevance of cross-priming of CD8+ T cells has recently been challenged (Zinkernagel, R. M., Eur. J. Immunol. 2002. 32: 2385-2392), on the basis that responses are weak or poorly quantitated, and the determinants recognized are undefined. Here we show that cross-priming is a robust process that elicits vigorous primary responses to multiple peptides in two well-defined systems. Our findings support the relevance of cross-priming in CD8+ T cell responses to viruses and tumor cells, and demonstrate that cross-priming elicits CD8+ T cells to determinants generated by the endogenous processing pathway.

Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli.

Dendritic cells (DCs) are a family of leukocytes that initiate T- and B-cell immunity against pathogens. Migration of antigen-loaded DCs from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. In humans, the major peripheral blood DC (PBDC) types, CD1c+ DCs and interleukin 3 receptor-positive (IL-3R+) plasmacytoid DCs, are significantly expanded in vivo with the use of Flt3 ligand (FL). DC-like cells can also be generated from monocyte precursors (MoDCs). A detailed comparison of the functional potential of these types of DCs (in an autologous setting) has yet to be reported. Here, we compared the functional capacity of FL-expanded CD1c+ PBDCs with autologous MoDCs in response to 3 different classes of stimuli: (1) proinflammatory mediators, (2) soluble CD40 ligand trimer (CD40L), and (3) intact bacteria (Escherichia coli). Significant differences in functional capacities were found with respect to changes in phenotype, migratory capacity, cytokine secretion, and T-cell stimulation. MoDCs required specific stimuli for the expression of functions. They responded vigorously to CD40L or E coli, expressing cytokines known to regulate interferon-gamma (IFN-gamma) in T cells (IL-12p70, IL-18, and IL-23), but required prostaglandin E2 (PGE2) during stimulation to migrate to chemokines. In contrast, PBDCs matured in response to minimal stimulation, rapidly acquired migratory function in the absence of PGE2-containing stimuli, and were low cytokine producers. Interestingly, both types of DCs were equivalent with respect to stimulation of allogeneic T-cell proliferation and presentation of peptides to cytotoxic T lymphocyte (CTL) lines. These distinct differences are of particular importance when considering the choice of DC types for clinical applications.

Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E(2) regulates the migratory capacity of specific DC subsets.

Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to proinflammatory mediators, CD40L, and intact bacteria. All classes of stimuli induced DC phenotypic maturation. However, for MoDCs, only prostaglandin E(2) (PGE(2))-containing stimuli induced migratory-type DCs. Thus, immature MoDCs that encountered proinflammatory cytokines or CD40L or intact bacteria in the presence of PGE(2) acquired migratory capacity but secreted low levels of cytokines. Conversely, MoDCs that encountered pathogens or CD40L alone become nonmigratory cytokine-secreting cells (proinflammatory type). Interestingly, both migratory- and proinflammatory-type DCs expressed equivalent levels of chemokine receptors, suggesting that the role of PGE(2) was to switch on migratory function. We demonstrate that PGE(2) induces migration via the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN-gamma production. In contrast, CD1b/c(+) PBDC rapidly acquired migratory capacity irrespective of the class of stimulus encountered and secreted low levels of cytokines. This suggests that not all mature stages of DCs are destined to migrate to lymphoid organs and that the sequence in which stimuli are encountered significantly affects which functions are expressed. Thus, certain immature DC subsets recruited from the resting precursor pool may have multiple functional fates that play distinct roles during the induction and effector phases of the immune response. These findings have important implications for the clinical utility of DCs in immunotherapy.

IFN-alpha enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells.

Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte-derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)-induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up-regulated the expression of HLA-DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen-uptake function, increased the levels of IL-12p35 mRNA, and prolonged surface expression of peptide-MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co-factors for CD40L-mediated function. Here, they enhanced CD40L-mediated IL-6, IL-10 and IL-12p70 secretion. Furthermore, when combined with IL-1beta and/or IL-4, IFN-alpha2a type I IFN increased CD40L-mediated IL-12p70 production by 2- to 3-fold, and biased the IL-12 p40/p70 ratio towards the IFN-gamma inducing p70 heterodimer, this correlating with higher levels of IFN-gamma secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL-1beta at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L-mediated DC activation.

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1beta, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.