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Objectives: Immune dysregulation contributes to the development of RA. Altered surface expression patterns of integrin adhesion receptors by immune cells is one mechanism by which this may occur. We investigated the role of β2 integrin subunits CD11a and CD11b in dendritic cell (DC) subsets of RA patients., Methods: Total β2 integrin subunit expression and its conformation ('active' vs 'inactive' state) were quantified in DC subsets from peripheral blood (PB) and SF of RA patients as well as PB from healthy controls. Ex vivo stimulation of PB DC subsets and in vitro-generated mature and tolerogenic monocyte-derived DCs (moDCs) were utilized to model the clinical findings. Integrin subunit contribution to DC function was tested by analysing clustering and adhesion, and in co-cultures to assess T cell activation., Results: A significant reduction in total and active CD11a expression in DCs in RA SF compared with PB and, conversely, a significant increase in CD11b expression was found. These findings were modelled in vitro using moDCs: tolerogenic moDCs showed higher expression of active CD11a and reduced levels of active CD11b compared with mature moDCs. Finally, blockade of CD11b impaired T cell activation in DC-T cell co-cultures., Conclusion: For the first time in RA, we show opposing expression of CD11a and CD11b in DCs in environments of inflammation (CD11alow/CD11bhigh) and steady state/tolerance (CD11ahigh/CD11blow), as well as a T cell stimulatory role for CD11b. These findings highlight DC integrins as potential novel targets for intervention in RA., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Osteoarthritis (OA) is a leading cause of disability, globally. Despite an emerging role for synovial inflammation in OA pathogenesis, attempts to target inflammation therapeutically have had limited success. A better understanding of the cellular and molecular processes occurring in the OA synovium is needed to develop novel therapeutics. We investigated macrophage phenotype and gene expression in synovial tissue of OA and inflammatory-arthritis (IA) patients. Compared with IA, OA synovial tissue contained higher but variable proportions of macrophages (P < 0.001). These macrophages exhibited an activated phenotype, expressing folate receptor-2 and CD86, and displayed high phagocytic capacity. RNA sequencing of synovial macrophages revealed 2 OA subgroups. Inflammatory-like OA (iOA) macrophages are closely aligned to IA macrophages and are characterized by a cell proliferation signature. In contrast, classical OA (cOA) macrophages display cartilage remodeling features. Supporting these findings, when compared with cOA, iOA synovial tissue contained higher proportions of macrophages (P < 0.01), expressing higher levels of the proliferation marker Ki67 (P < 0.01). These data provide new insight into the heterogeneity of OA synovial tissue and suggest distinct roles of macrophages in pathogenesis. Our findings could lead to the stratification of OA patients for suitable disease-modifying treatments and the identification of novel therapeutic targets.

Emerging evidence suggests that the β 2 integrin family of adhesion molecules have an important role in suppressing immune activation and inflammation. β 2 integrins are important adhesion and signaling molecules that are exclusively expressed on leukocytes. The four β 2 integrins (CD11a, CD11b, CD11c, and CD11d paired with the β 2 chain CD18) play important roles in regulating three key aspects of immune cell function: recruitment to sites of inflammation; cell-cell contact formation; and downstream effects on cellular signaling. Through these three processes, β 2 integrins both contribute to and regulate immune responses. This review explores the pro- and anti-inflammatory effects of β 2 integrins in monocytes, macrophages, and dendritic cells and how they influence the outcome of immune responses. We furthermore discuss how imbalances in β 2 integrin function can have far-reaching effects on mounting appropriate immune responses, potentially influencing the development and progression of autoimmune and inflammatory diseases. Therapeutic targeting of β 2 integrins, therefore, holds enormous potential in exploring treatment options for a variety of inflammatory conditions.

Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte-derived DC. These tolDC exert potent pro-tolerogenic actions on CD4 + T cells. Lack of interleukin (IL)-12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)-β1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte-derived DC. By inhibiting TGF-β1 signalling we demonstrate that tolDC regulate CD4 + T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF-βRII on CD4 + T cells from RA patients and healthy controls, RA patient CD4 + T cells are measurably less responsive to TGF-β1 than healthy control CD4 + T cells [reduced TGF-β-induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)-γ secretion]. However, CD4 + T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF-β1-dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA., (© 2016 British Society for Immunology.)

Tolerogenic dendritic cells (TolDCs) are promising tools for therapy of autoimmune diseases, such as rheumatoid arthritis (RA). Here, we characterize monocyte-derived TolDCs from RA patients modulated with dexamethasone and activated with monophosphoryl lipid A (MPLA), referred to as MPLA-tDCs, in terms of gene expression, phenotype, cytokine profile, migratory properties, and T cell-stimulatory capacity in order to explore their suitability for cellular therapy. MPLA-tDCs derived from RA patients displayed an anti-inflammatory profile with reduced expression of co-stimulatory molecules and high IL-10/IL-12 ratio, but were capable of migrating toward the lymphoid chemokines CXCL12 and CCL19. These MPLA-tDCs induced hyporesponsiveness of autologous CD4+ T cells specific for synovial antigens in vitro . Global transcriptome analysis confirmed a unique transcriptional profile of MPLA-tDCs and revealed that RA-associated genes, which were upregulated in untreated DCs from RA patients, returned to expression levels of healthy donor-derived DCs after treatment with dexamethasone and MPLA. Thus, monocyte-derived DCs from RA patients have the capacity to develop tolerogenic features at transcriptional as well as at translational level, when modulated with dexamethasone and MPLA, overcoming disease-related effects. Furthermore, the ability of MPLA-tDCs to impair T cell responses to synovial antigens validates their potential as cellular treatment for RA.

Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A (MPLA)-activated DCs [MPLA-tolerogenic DCs (tDCs)] to exert immunomodulatory effects on naive and memory CD4 + T cells in an antigen-specific manner. For this purpose, MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naive or memory CD4 + T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naive CD4 + T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4 + effector T cells and PPD-pulsed mDCs. We detected that memory CD4 + T cells primed by MPLA-tDCs presented reduced proliferation and proinflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naive CD4 + T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific restimulation and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion, MPLA-tDCs are able to modulate antigen-specific responses of both naive and memory CD4 + T cells and might be a promising strategy to "turn off" self-reactive CD4 + effector T cells in autoimmunity.

The number of patients with autoimmune diseases and severe allergies and recipients of transplants increases worldwide. Currently, these patients require lifelong administration of immunomodulatory drugs. Often, these drugs are expensive and show immediate or late-occurring severe side effects. Treatment would be greatly improved by targeting the cause of autoimmunity, that is, loss of tolerance to self-antigens. Accumulating knowledge on immune mechanisms has led to the development of tolerogenic dendritic cells (tolDC), with the specific objective to restrain unwanted immune reactions in the long term. The first clinical trials with tolDC have recently been conducted and more tolDC trials are underway. Although the safety trials have been encouraging, many questions relating to tolDC, for example, cell-manufacturing protocols, administration route, amount and frequency, or mechanism of action, remain to be answered. Aiming to join efforts in translating tolDC and other tolerogenic cellular products (e.g., Tregs and macrophages) to the clinic, a European COST (European Cooperation in Science and Technology) network has been initiated-A FACTT (action to focus and accelerate cell-based tolerance-inducing therapies). A FACTT aims to minimize overlap and maximize comparison of tolDC approaches through establishment of minimum information models and consensus monitoring parameters, ensuring that progress will be in an efficient, safe, and cost-effective way.

Novel treatments in development for rheumatoid arthritis target 3 broad areas: cytokines, cells, and signaling pathways. Therapies from each domain share common advantages (for example previously demonstrated efficacy, potential long-term immunomodulation, and oral administration respectively) that have stimulated research in each area but also common obstacles to their development. In this review recent progress in each area will be discussed alongside the factors that have impeded their path to clinical use.

Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24(+)CD64(-) DCs and contaminating CSF-1R-dependent CD24(-)CD64(+) macrophages. Functionally, loss of CD24(+)CD11b(+) DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24(+)CD11b(+) DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Dendritic cells with tolerogenic function (tolDC) have become a promising immunotherapeutic tool for reinstating immune tolerance in rheumatoid arthritis (RA) and other autoimmune diseases. The concept underpinning tolDC therapy is that it specifically targets the pathogenic autoimmune response while leaving protective immunity intact. Findings from human in-vitro and mouse in-vivo studies have been translated into the development of clinical grade tolDC for the treatment of autoimmune disorders. Recently, two tolDC trials in RA and type I diabetes have been carried out and other trials are in progress or are imminent. In this review, we provide an update on tolDC therapy, in particular in relation to the treatment of RA, and discuss the challenges and the future perspectives of this new experimental immunotherapy., (© 2012 British Society for Immunology.)

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) -dependent disease that is induced with antigen in complete Freund's adjuvant, whereas PGIA is Th1-mediated and is induced using antigen in dimethyldioctadecyl-ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T-cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan-T-cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin-17 (IL-17) produced by T cells isolated from CFA-immunized mice after antigen-specific stimulation were not significantly different from those found in DDA-immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL-17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity., (© 2012 Blackwell Publishing Ltd.)

Objectives: Lymphocyte depleting therapies have been used to treat refractory autoimmune disease, including RA, but treatment may be associated with long-term lymphopenia. It is unclear whether delayed reconstitution preferentially affects lymphocyte subsets, how this modulates immune challenges and whether thymic function influences the outcome. These questions are now addressed in a detailed analysis of RA patients 12 years after alemtuzumab (anti-CD52) treatment., Methods: Blood was obtained from 20 RA patients 12 years after alemtuzumab treatment. Lymphocyte subsets were enumerated by flow cytometry. T-cell receptor excision circles (TRECs)/ml were determined to quantify thymic function, and serological responses to neoantigens and recall antigens were assessed., Results: RA patients remained lymphopenic 12 years after their first dose of alemtuzumab. CD5(+) B cells, which may be associated with autoantibody production, were significantly reduced in alemtuzumab-treated patients compared with age-matched disease controls. In addition, naïve and memory CD4(+) T-cell subsets were present in altered proportions in patients who had received alemtuzumab, with increased effector memory CD4(+) T cells, and decreased naïve and central memory CD4(+) T cells. TRECs were detectable in alemtuzumab-treated patients and correlated with CD4(+) lymphocyte counts. Vaccine responses to neoantigens and recall antigens fell within the normal range for an ageing population., Conclusions: Alemtuzumab therapy resulted in long-term alterations in lymphocyte subsets. The significance of these changes remains uncertain but patients respond normally to antigenic challenges. Thymic function remains an important determinant of T-cell reconstitution even several years after lymphocytotoxic therapy.

Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells. Immunohistochemistry was performed on explanted CF lungs and compared with the non-suppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs. Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p=0.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells·mm(-1) compared with 9 cells·mm(-1) basement membrane, p=0.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor. In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

We show that the strength of T-cell stimulation determines the capability of human CD4(+) T cells to become interleukin-17 (IL-17) producers. CD4(+) T cells received either high- (THi) or low (TLo)-strength stimulation via anti-CD3/CD28 beads or dendritic cells pulsed with superantigen in the presence of pro-Th17 cytokines IL-1β, transforming growth factor β, and IL-23. We found that TLo, but not THi, stimulation profoundly promoted Th17 responses by enhancing both the relative proportion and total number of Th17 cells. Titration of anti-CD3 revealed that low TCR signaling promoted Th17 cells, but only in the presence of anti-CD28. Impaired IL-17 production in THi cells could not be explained by high levels of Foxp3 or transforming growth factor β-latency-associated peptide expressed by THi cells. Nuclear factor of activated T cells was translocated to the nucleus in both THi and TLo cells, but only bound to the proximal region of the IL-17 promoter in TLo cells. The addition of a Ca(2+) ionophore under TLo conditions reversed the pro-Th17 effect, suggesting that high Ca(2+) signaling impairs Th17 development. Although our data do not distinguish between priming of naive T cells versus expansion/differentiation of memory T cells, our results clearly establish an important role for the strength of T-cell activation in regulating Th17 responses.

Objective: Tolerogenic dendritic cells (DCs) are antigen-presenting cells with an immunosuppressive function. They are a promising immunotherapeutic tool for the attenuation of pathogenic T cell responses in autoimmune arthritis. The aims of this study were to determine the therapeutic action of tolerogenic DCs in a type II collagen-induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets in mice with established arthritis., Methods: Tolerogenic DCs were generated by treating bone marrow-derived DCs with dexamethasone and vitamin D(3) during lipopolysaccharide-induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or saline. Arthritis severity was monitored for up to 4 weeks after treatment. Fluorescence-labeled tolerogenic DCs were used for in vivo trafficking studies. The in vivo effect of tolerogenic DCs on splenic T cell populations was determined by intracellular cytokine staining and flow cytometry., Results: Tolerogenic DCs displayed a semi-mature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen-pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin-10-producing CD4+ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin-17-producing γ/δ T cells., Conclusion: Treatment with type II collagen-pulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis., (Copyright © 2010 by the American College of Rheumatology.)

Objective: Dendritic cells (DCs) are enriched in RA synovium and have been implicated in the pathogenesis of RA primarily through their ability to present autoantigen and activate T cells. However, whether DCs play an effector role in cartilage destruction is unknown. The aim of this study was to investigate whether DCs can induce collagen release from cartilage and the mechanism involved., Methods: Human monocyte-derived DCs (mDCs) were activated with CD40 ligand (CD40L) to mimic DC-T-cell interaction, and supernatants were incubated with cartilage explants. Hydroxyproline was assessed as a measure of collagen release and collagenolytic activity was measured by a bioassay using tritiated collagen. TNF-alpha in DC supernatants was measured by specific ELISA., Results: Supernatants from CD40L-activated mDCs, but not unstimulated mDCs, strongly induced the destruction of cartilage collagen. mDC supernatants did not contain collagenases but did induce collagenolytic activity in cartilage explants. Neutralization of TNF-alpha in mDC supernatants completely abolished collagenolysis., Conclusions: This study shows that mDCs, upon CD40-ligation, induce cartilage collagen degradation through an indirect mechanism via the production of TNF-alpha. Our data suggest a potential important role for mDC-derived TNF-alpha in RA, which is in line with the previously reported observations that DCs are a major source of TNF-alpha in early autoimmune lesions and that anti-TNF-alpha therapeutics effectively suppress joint damage in RA patients. We propose that DCs can act as effectors in cartilage destruction, adding a new aspect to the functional role of DCs in RA pathogenesis.

Autoimmune pathologies are caused by a breakdown in self-tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self-tolerance in an antigen-specific manner. Studies about tolDC have focused largely on generating stable maturation-resistant DC, but few have fully addressed questions about the antigen-presenting and migratory capacities of these cells, prerequisites for successful immunotherapy. Here, we investigated whether human tolDC, generated with dexamethasone and the active form of vitamin D3, maintained their tolerogenic function upon activation with LPS (LPS-tolDC), while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS-tolDC, but not tolDC, expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore, LPS-tolDC were superior to tolDC in their ability to present type II collagen, a candidate autoantigen in rheumatoid arthritis. tolDC and LPS-tolDC had low stimulatory capacity for allogeneic, naïve T cells and skewed T cell polarization toward an anti-inflammatory phenotype, although LPS-tolDC induced significantly higher levels of IL-10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen-presenting activity in tolDC is important for optimizing their therapeutic potential.

Promising immunotherapeutic tools for T cell-mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As naïve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of naïve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by naïve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Naïve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN-gamma/high IL-10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL-2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL-12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in naïve T cells, indicating that the lack of IL-12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate naïve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII259-273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII259-273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.

Using the principle of functional polarization of dendritic cells (DCs), we have developed a novel protocol to generate human DCs combining the three features critical for the induction of type-1 immunity: (a) fully mature status; (b) responsiveness to secondary lymphoid organ chemokines; and (c) high interleukin-12p70 (IL-12p70)-producing ability. We show that IFN-alpha and polyinosinic:polycytidylic acid (p-I:C) synergize with the "classical" type-1-polarizing cytokine cocktail [tumor necrosis factor alpha (TNFalpha)/IL-1beta/IFNgamma], allowing for serum-free generation of fully mature type-1-polarized DCs (DC1). Such "alpha-type-1-polarized DC(s)" (alphaDC1) show high migratory responses to the CCR7 ligand, 6C-kine but produce much higher levels of IL-12p70 as compared to TNFalpha/IL-1beta/IL-6/prostaglandin E2 (PGE2)-matured DCs (sDC), the current "gold standard" in DC-based cancer vaccination. A single round of in vitro sensitization with alphaDC1 (versus sDCs) induces up to 40-fold higher numbers of long-lived CTLs against melanoma-associated antigens: MART-1, gp100, and tyrosinase. Serum-free generation of alphaDC1 allows, for the first time, the clinical application of DCs that combine the key three features important for their efficacy as anticancer vaccines.

A sensitive, specific, reproducible, robust, and cost-effective customized cDNA array system based on established nylon membrane technology has been developed for convenient multisample expression profiling for several hundred genes of choice. The genes represented are easily adjusted (depending on the availability of corresponding cDNAs) and the method is accordingly readily applicable to a wide variety of systems. Here we have focused on the expression profiles for interferon-alpha2a, the most widely used interferon for the treatment of viral hepatitis and malignancies, in primary cells (peripheral blood mononuclear cells, T cells, and dendritic cells) and cell lines (Kit255, HT1080, HepG2, and HuH7). Of 150 genes studied, only six were consistently induced in all cell types and donors, whereas 74 genes were induced in at least one cell type. IRF-7 was identified as the only gene exclusively induced in the hematopoietic cells. No gene was exclusively induced in the nonhematopoietic cell lines. In T cells 12, and in dendritic cells, 25 genes were induced in all donors whereas 45 and 42 genes, respectively, were induced in at least one donor. The data suggest that signaling through IFN-alpha2 can be substantially modulated to yield significant cell-type and donor-specific qualitative and quantitative differences in gene expression in response to this cytokine under highly standardized conditions.

The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.

IL-12 is a key cytokine in the development of Th1 responses. IL-12 production by antigen-presenting cells (APC) can be induced by the interaction between CD40 on the APC and CD40 ligand (CD40L) expressed on T cells after activation. Our previous study indicated that in dendritic cells (DC), the only APC that can activate naive T(h) cells efficiently, the mere CD40 engagement is insufficient to induce IL-12 production. The aim of the present study was to dissect the conditions for efficient IL-12 production by DC further. Using populations of naive and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-gamma production and CD40L expression levels, we here show that T cell-induced IL-12 production by DC results from the action of two signals, mediated by CD40L and IFN-gamma, and that the inability of naive T(h) cells to induce IL-12 production resides in their inability to produce IFN-(gamma). Other factors than CD40L and IFN-gamma can provide the required signals for IL-12 production by DC, as either factor could be replaced by lipopolysaccharide (LPS). The two-signal requirement proved unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of tumor necrosis factor-alpha, IL-8 and the p40 subunit of IL-12, and may be considered as a safety mechanism for optimal control of potentially harmful T(h)1 responses.

Dendritic cells (DC) are important initiators of specific primary immune responses because they are the only APC that can efficiently activate naive Th cells. DC have the capacity to produce interleukin-12 (IL-12), a cytokine that plays a pivotal role in the development of Th1-mediated cellular immune responses. The present study focuses on the conditions under which human DC produce bioactive IL-12 p70 and, consequently, direct the development of naive T helper (Th) cells toward the Th1 phenotype. Bacteria or bacterial compounds such as Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS) induced substantial IL-12 levels in DC, which could be further upregulated by interferon-gamma (IFN-gamma), whereas induction of IL-12 production via CD40 ligation required IFN-gamma as an obligatory, complementary signal. Also, activated naive Th cells were poor inducers of IL-12 production, unless exogenous IFN-gamma was present, whereas activated memory Th cells were effective inducers of IL-12 production and did not require exogenous IFN-gamma. Next, the cytokine profiles of matured Th cells that were primed by DC under different conditions were examined. DC promoted the development of naive Th cells into memory Th0 cells that produced both the type 1 cytokine IFN-gamma and the type 2 cytokine IL-4. In contrast, after activation with SAC, DC efficiently directed the development of Th1 cells through the release of IL-12. An APC-independent Th cell maturation model, using either recombinant IL-12 or supernatants of SAC-activated DC and neutralizing anti-IL-12 antibodies, confirmed that DC-derived IL-12 was the major Th1 skewing factor. Together, these data indicate that the contact between DC and naive Th cells during the initiation of specific immune responses does not result in the efficient induction of IL-12 production and that, consequently, exogenous IL-12-inducing factors are required to promote primary Th1-mediated cellular immune responses.