Dendritic Cells Maturation Signals Research Articles

Priming CD8+ T cells with dendritic cells matured using TLR4 and TLR7/8 ligands together enhances generation of CD8+ T cells retaining CD28

AbstractTLRs expressed on dendritic cells (DCs) differentially activate DCs when activated alone or in combination, inducing distinct cytokines and costimulatory molecules that influence T-cell responses. Defining the requirements of DCs to program T cells during priming to become memory rather than effector cells could enhance vaccine development. We used an in vitro system to assess the influence of DC maturation signals on priming naive human CD8+ T cells. Maturation of DCs with lipopolysaccharide (LPS; TLR4) concurrently with R848 (TLR7/8) induced a heterogeneous population of DCs that produced high levels of IL12 p70. Compared with DCs matured with LPS or R848 alone, the DC population matured with both adjuvants primed CD8+ T-cell responses containing an increased proportion of antigen-specific T cells retaining CD28 expression. Priming with a homogenous subpopulation of LPS/R848–matured DCs that were CD83Hi/CD80+/CD86+ reduced this CD28+ subpopulation and induced T cells with an effector cytokine signature, whereas priming with the less mature subpopulations of DCs resulted in minimal T-cell expansion. These results suggest that TLR4 and TLR7/8 signals together induce DCs with fully mature and less mature phenotypes that are both required to more efficiently prime CD8+ T cells with qualities associated with memory T cells.

Drug Antigenicity, Immunogenicity, and Costimulatory Signaling: Evidence for Formation of a Functional Antigen through Immune Cell Metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Liposomal Ag engrafted with peptides of sequence derived from HMGB1 induce potent Ag-specific and anti-tumour immunity

High-mobility group box 1 (HMGB1) protein is a nuclear binding protein which is released by monocytes and macrophages and is a potent maturation signal for dendritic cells (DCs). Synthetic HMGB1-related peptides are reported to be potent DC stimulants. Two HMGB1-related peptides, denoted as pHMGB-89 and pHMGB-106, were explored for their ability to enhance the immunogenicity of Ag-containing liposomes. pHMGB-engrafted liposomes targeted murine CD11c + and CD11b + cells in vitro and in vivo. Vaccination of mice with OVA-containing liposomes engrafted with pHMGB-89 and pHMGB-106 induced OVA-specific T cell priming and production of IgG 1, IgG 2a and IgG 2b antibodies. Importantly, vaccination of mice with B16-OVA-derived plasma membrane vesicles (PMVs) engrafted with pHMGB-89 and pHMGB-106 inhibited tumour growth and metastasis, in syngeneic mice challenged with highly metastatic B16-OVA melanoma. The results show that vaccination with Ag-containing liposomes/PMVs engrafted with HMGB1 peptides could be an effective approach for developing novel vaccines and cancer immunotherapies.

Redundant Mechanisms for Dendritic Cell Activation in GVHD Induction: Signalings Via TLRs, TNF-α, IL-1 and CD40 Are Not Required

Graft-versus-host disease (GVHD) is initiated by antigen-presenting cells (APCs) that prime alloreactive donor T cells. Amongst APCs, dendritic cells (DCs) are the most potent at priming naïve T cells. A paradigm of DC function holds that instructive maturation signals transform “immature” DCs into “mature” DCs which are optimized for T cell activation. Signaling via Toll-like receptors (TLRs) has emerged as a central mechanism for inducing this maturation in anti-pathogen responses. However, in allogeneic stem cell transplantation, there is no specific pathogen and the signals that induce DC maturation in GVH responses have not been defined. To study potential maturation stimuli we performed GVHD experiments in model systems wherein host APCs are essential for GVHD induction (C3H.SW (H-2b)→B6 and B6bm12→B6). We then compared GVHD in wild type (wt) recipients to that in hosts genetically unable to respond to various DC maturation signals. We first used as hosts mice deficient in TLR4, CD40, IL1-R or TNFR1/R2 and found that in C3H.SW→B6 model, GVHD in each of these was similar. Likewise, GVHD was similar in wt and mice deficient in MyD88, essential for signaling via all TLRs except TLR3 and TLR4. To focus our studies on APCs, we used as hosts radiation bone marrow (BM) chimeras in which wt B6 (CD45.1) mice were reconstituted with BM from CD45.2 wt, MyD88−/−, TRIF−/− (required for signaling via TLR3 and, in-part, TLR4), and MyD88/TRIF−/− mice, in which signaling by all TLRs, IL-1 and IL-18 is completely blocked. When retransplanted with C3H.SW BM and CD8 cells, all chimeras developed similar clinical and histologic GVHD. To exclude that T cells were being primed by residual wt host APCs in BM chimeric recipients, we used as hosts B6bm12 mice that were reconstituted with BM from B6 wt, MyD88−/−, TRIF−/− or MyD88/TRIF−/− mice. These chimeras were then retransplanted with B6bm12 BM and CD4 cells. Because residual host APCs in these BM chimeric hosts were B6bm12 and therefore syngeneic with the donor cells, T cell priming could only occur on B6 background APCs derived from the first transplant. As in the prior experiments, all chimeras developed similar clinical GVHD; pathology scoring is underway. Therefore in contrast to nearly all adaptive T cell responses, GVHD induction in both MHC-matched and MHC-mismatched systems did not require APC maturation signals mediated by TLRs. Absent any phenotype when APC maturation signals were blocked, we used the same approach in the C3H.SW→B6 model to study the roles of IL12 and type I IFNs, which are produced as a consequence of DC maturation. B6 p35−/− and B6 wt mice developed similar GVHD, excluding an essential role for host IL12. Type I IFN receptor-deficient (IFNIR−/−) mice developed similar GVHD as did wt controls, except that IFNIR−/− CD8 recipients lost more weight but had less histologic liver GVHD (P=0.0024). To focus on the activity of Type I IFNs on host hematopoietic cells, we used as recipients IFNIR−/− → B6 chimeras. Both clinical and histologic GVHD were similar in IFNIR−/−→B6 and wt→B6 control chimeras, suggesting that the differences in weight loss and hepatic GVHD observed in IFNIR−/− hosts, were due to parenchymal expression of IFNIR−/−. In sum, these data indicate that DC maturation signals in GVHD are at a minimum redundant, and independent of TLR ligands such as LPS, which are certainly available to the host immune system. This may be a consequence of the ubiquitous expression of alloantigen, such that any maturation signal will create APCs primed to activate donor T cells. If so, strategies for preventing GVHD by blocking APC maturation may need to target final common APC maturation pathways. Alternatively, instructive APC maturation may not be required for alloreactive T cell activation in GVHD.

An adjuvant role of in situ dendritic cells (DCs) in linking innate and adaptive immunity

Dendritic cells (DCs) work as a natural adjuvant to elicit T cell immunity. Though DCs have been widely used in immunotherapy, little is known about their number and function in patients with cancer or autoimmune disease. In recent studies, antigen has been targeted to DCs through DC-specific receptors, such as DEC205, the mannose receptor and dying cell receptors. However, antigen captured by DCs in the absence of danger signals induces tolerance. Therefore, the duration and/or magnitude of danger signals plays a crucial role in generating an immunogeneic response. Various danger signals, i.e., pathogen-associated molecular pattern (PAMP), damage-associated molecular pattern (DAMP) and the activation of innate lymphocytes, serve as maturation signals for DCs. An immunotherapeutic approach which delivers both maturation signals and antigen to DCs would link the innate and adaptive arms of the immune system for a more effective and global immune response. It is therefore crucial to determine optimal conditions for antigen delivery to DCs in an environment suited to maximally stimulate the immune system.

Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8+ T lymphocytes

BackgroundIn the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by γ irradiation (Apo-Nec cells).MethodsWe evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.ResultsApo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 ± 10.5%) at 12 hs of coculture. By 12–24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC – Dextran uptake (iDC: 81 ± 5%; DC/Apo-Nec 33 ± 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3β. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-γ secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.ConclusionWe conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.

Functional inactivation of immature dendritic cells by the intracellular parasite Toxoplasma gondii.

Despite its noted ability to induce strong cellular immunity, and its known susceptibility to IFN-gamma-dependent immune effector mechanisms, the protozoan Toxoplasma gondii is a highly successful parasite, able to replicate, disseminate, and either kill the host or, more commonly, establish resistant encysted life forms before the emergence of protective immune responses. We sought to understand how the parasite gains the advantage. Using transgenic clonal parasite lines engineered to express fluorescent markers in combination with dendritic cells (DC) grown from the bone marrow of wild-type mice or transgenic mice expressing fluorescent protein-tagged MHC class II molecules, we used flow cytometry and fluorescence microscopy to analyze the responses of infected DC to both invasion by the parasite and subsequent DC maturation signals. We found that T. gondii preferentially invades immature dendritic cells but fails to activate them in the process, and renders them resistant to subsequent activation by TLR ligands or the immune-system-intrinsic maturation signal CD40L. The functional consequences of T. gondii-mediated suppression of DC activation are manifested in a relative inability of infected immature DC to activate naive CD4(+) Th lymphocytes, or to secrete cytokines, such IL-12 and TNF-alpha, that play important roles in innate and/or adaptive immunity. The findings reveal that T. gondii suppresses the ability of immature DC to participate in innate immunity and to induce adaptive immune responses. The ability of T. gondii to temporarily evade recognition could provide a selective advantage that permits dissemination and establishment before adaptive immune response initiation.

Local delivery of recombinant vaccinia virus expressing secondary lymphoid chemokine (SLC) results in a CD4 T-cell dependent antitumor response

Secondary lymphoid chemokine (SLC) attracts mature dendritic cells (DCs) and naı̈ve T cells. Co-localization of these cells within local tumor environments may enhance the induction of tumor-specific T cells. However, the presence of danger signals or other DC maturation signals are required to optimize T-cell priming. We hypothesized that expression of SLC in vaccinia virus would provide local chemokine delivery and adjuvant factors. A recombinant vaccinia virus expressing murine SLC (rVmSLC) was constructed and characterized. SLC expression was confirmed by Western blot analysis and functional activity was determined by in vitro chemotaxis assay. Supernatants from rVmSLC-infected cells attracted CD4 T cells, and also induced the migration of CD8 T cells and DCs. Although poxviruses are known to express several chemokine-binding proteins, systemic injection of rVmSLC was well tolerated in mice up to a dose of 1×10 7 pfu and did not significantly alter vaccinia-specific T-cell immunity. Local injection of rVmSLC into established tumors derived from the murine colon cancer line, CT26, resulted in enhanced infiltration of CD4 T cells, which correlated with inhibition of tumor growth. The central role of CD4 T cells was further demonstrated by loss of anti-tumor activity in CD4 T-cell depleted mice. Intratumoral delivery of SLC using a poxviral vaccine extends the use of SLC in anti-tumor therapies and may present an effective alternative for improving the immunotherapy of cancer alone or in combination with other anti-tumor agents for clinical therapy.

IL-1R-associated kinase 4 is required for lipopolysaccharide-induced activation of APC.

The bacterial product LPS is a critical stimulus for the host immune system in the response against the corresponding bacterial infection. LPS provides an activation stimulus for macrophages and a maturation signal for dendritic cells to set up innate and adaptive immune responses, respectively. The signaling cascade of myeloid differentiation factor 88-->IL-1R-associated kinase (IRAK)-->TNFR-associated factor 6 has been implicated in mediating LPS signaling. In this report, we studied the function of IRAK-4 in various LPS-induced signals. We found that IRAK-4-deficient cells were severely impaired in producing some IFN-regulated genes as well as inflammatory cytokines in response to LPS. Among the critical downstream signaling pathways induced by LPS, NF-kappaB activation but not IFN regulatory factor 3 or STAT1 activation was defective in cells lacking IRAK-4. IRAK-4 was also required for the proper maturation of dendritic cells by LPS stimulation, particularly in terms of cytokine production and the ability to stimulate Th cell differentiation. Our results demonstrate that IRAK-4 is critical for the LPS-induced activations of APCs.

Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.

Heat shock protein 70: role in antigen presentation and immune stimulation

Heat shock proteins (HSP) when released into the extracellular milieu can act simultaneously as a source of antigen due to their ability to chaperone peptides and as a maturation signal for dendritic cells, thereby inducing DCs to cross-present antigens to CD8+ T-cells. HSP can also act independently from associated peptides, stimulating the innate immune system. Previous results regarding the activation of NK cells by HSP70 cell surface expression on tumour cells and soluble HSP70 will be further covered elsewhere within this issue. For cross-presentation, HSP70-peptide complexes (HSP70-PC) were used from two human melanoma cell lines that differ in the expression of the tumour-associated antigen tyrosinase. Purified HSP70-PC consists of both the constitutively expressed HSC70 and the inducible HSP70. HSP70-peptide complexes purified from tyrosinase positive (HSP70-PC/tyr + ) human melanoma cells, incubated with immature DCs, results in the activation of HLA-*A0201-restricted tyrosinase peptide-specific T-cells. Receptor-mediated uptake of HSP70-PC by DCs and intracellular transport are required for efficient MHC class I restricted cross-presentation of chaperoned peptides. Demonstration of HSP70-PC mediated cross-presentation of such non-mutated naturally expressed tumour antigens is of special clinical interest with regard to hyperthermia. Tumour regression and improved local control have been shown within clinical phase II/III trials integrating regional hyperthermia combined with radiation and/or chemotherapy in multimodal treatment strategies. According to the proposed concept, local necrosis induced by hyperthermic treatment induces the release of HSPs, followed by uptake, processing and presentation of associated peptides by DCs. By acting as chaperone and a signal for DC maturation, HSP70-PC might efficiently prime circulating T-cells. Therefore, upregulating HSP70 and causing local necrosis in tumour tissue by hyperthermia offers great potential as a new approach to directly activate the immune system.

Cytotoxic Activity of Human Dendritic Cells Is Differentially Regulated by Double-Stranded RNA and CD40 Ligand

The main function of dendritic cells (DCs) is to induce adaptive immune response through Ag presentation and specific T lymphocyte activation. However, IFN-alpha- or IFN-gamma-stimulated CD11c+ blood DCs and IFN-beta-stimulated monocyte-derived DCs were recently reported to express functional TNF-related apoptosis-inducing ligand (TRAIL), suggesting that DCs may become cytotoxic effector cells of innate immunity upon appropriate stimulation. In this study, we investigate whether dsRNA and CD40 ligand (CD40L), that were characterized as potent inducers of DC maturation, could also stimulate or modulate DC cytotoxicity toward tumoral cells. We observed that dsRNA, but not CD40L, is a potent inducer of TRAIL expression in human monocyte-derived DCs. As revealed by cytotoxicity assays, DCs acquire the ability to kill tumoral cells via the TRAIL pathway when treated with dsRNA. More precisely, dsRNA is shown to induce IFN-beta synthesis that consecutively mediates TRAIL expression by the DCs. In contrast, we demonstrate that TRAIL expression in dsRNA- or IFN-alpha-treated DCs is potently inhibited after CD40L stimulation. Unexpectedly, CD40L-activated DCs still developed cytotoxicity toward tumoral cells. This latter appeared to be partly mediated by TNF-alpha induction and a yet unidentified pathway. Altogether, these results demonstrate that dsRNA and CD40L, that were originally characterized as maturation signals for DCs, also stimulate their cytotoxicity that is mediated through TRAIL-dependent or -independent mechanisms.