Tethering Innate Surface Receptors on Dendritic Cells: A New Avenue for Immune Tolerance Induction?

Dendritic cells (DCs) play a key role in immunity and are highly potent at presenting antigens and orienting the immune response. Depending on the environmental signals, DCs could turn the immune response toward immunity or immune tolerance. Several subsets of DCs have been described, with each expressing various surface receptors and all participating in DC-associated immune functions according to their specific skills. DC subsets could also contribute to the vicious circle of inflammation in immune diseases and establishment of immune tolerance in cancer. They appear to be appropriate targets in the control of inflammatory diseases or regulation of autoimmune responses. For all these reasons, in situ DC targeting with therapeutic antibodies seems to be a suitable way of modulating the entire immune system. At present, the field of antibody-based therapies has mainly been developed in oncology, but it is undergoing remarkable expansion thanks to a wide variety of antibody formats and their related functions. Moreover, current knowledge of DC biology may open new avenues for targeting and modulating the different DC subsets. Based on an update of pathogen recognition receptor expression profiles in human DC subsets, this review evaluates the possibility of inducing tolerant DCs using antibody-based therapeutic agents.

Hypoxia/Reoxygenation Inhibits P2Y11 Receptor Expression and Its Immunosuppressive Activity in Human Dendritic Cells.

High concentrations of extracellular ATP (eATP) resulting from cell damage may be found during an ischemia/reperfusion (I/R) episode at the site of injury. eATP activates purinergic receptors in dendritic cells (DCs) and may inhibit inflammation. This immunosuppressive activity could be of interest in the field of I/R, which is an inflammatory condition involved in myocardial infarction, stroke, and solid organ transplantation. However, the specific purinergic receptor responsible for this effect remains to be identified. In this study, we report that eATP induced maturation of human monocyte-derived DCs. Additionally, eATP inhibited IL-12 production whereas IL-10 levels remained unchanged in activated DCs. These effects were prevented by the P2Y11R antagonist NF340. Interestingly, a 5-h hypoxia prevented the effects of eATP on cytokine production whereas a 1-h hypoxia did not affect the eATP-mediated decrease of IL-12 and IL-6. We showed a time-dependent downregulation of P2Y11R at both mRNA and protein levels that was prevented by knocking down hypoxia-inducible factor-1α. In this study, we showed an immunosuppressive role of P2Y11R in human DCs. Additionally, we demonstrated that the time-dependent downregulation of P2Y11R by hypoxia orientates DCs toward a proinflammatory phenotype that may be involved in post-I/R injuries as observed after organ transplantation.

Unexpected impairment of TNF-α-induced maturation of human dendritic cells in vitro by IL-4.

BACKGROUND: An efficient strategy for programming dendritic cells (DCs) for cancer immunotherapy is the optimization of their maturation so that they can efficiently stimulate cancer-specific T cell responses. Interleukin (IL)-4 has appeared as an essential cytokine, widely used in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate monocytes into immature DCs (iDC) and to prevent macrophage formation. Conflicting data have been published regarding the effect of IL-4 on functional DC maturation. To further understand IL-4's effects on DC maturation and function in vitro, we choose the most commonly used maturation factor tumor necrosis factor (TNF)-α. METHODS: Human monocyte-derived iDC were treated for 48 h with GM-CSF and TNF-α in the presence (IL-4(+)-DC) or absence (IL-4(-)-DC) of IL-4 and functions of both DC populations were compared. RESULTS: On mixed lymphocyte reaction assay, IL-4(+)-DC were less potent than IL-4(-)-DC at inducing the proliferation of allogeneic CD4(+) T cells and the proportion of activated T cells expressing CD69 and/or CD25 was smaller. Interleukin-4 reduced the cell-surface expression of TNF-α-induced DC maturation markers CD83, CD86, HLA-DR and CD25 and generated a heterogeneous population of DCs. IL-4(+)-DC secreted less IL-12 and more IL-10 than IL-4(-)-DC following activation by soluble CD40L, and IL-4(+)-DC-activated T cells secreted lesser amounts of T helper (Th) 1 cytokines (IL-2 and interferon-γ). Importantly, IL-4 impaired the in vitro migratory capacity of DCs in response to CCL21 and CCL19 chemokines. This effect was related to reduced expression of CCR7 at both mRNA and protein levels. CONCLUSION: Interleukin-4 used with GM-CSF and TNF-α during the maturation of DCs in vitro impaired DC functions and disturbed the maturation effect of TNF-α. Finally, our study reinforces the view that the quality of the DC maturation stimulus, which regulates DC migration and cytokine production, may be a decisive feature of the immunogenicity of DCs.

IL-2 phosphorylates STAT5 to drive IFN-γ production and activation of human dendritic cells.

Human dendritic cells (hDCs) produce IL-2 and express IL-2R α-chain (CD25), but the role of IL-2 in DC functions is not well defined. A recent study suggested that the main function of CD25 on hDCs was to transpresent IL-2 to activate T lymphocytes. Our results demonstrate the expression of the three chains of the IL-2R on hDCs and that IL-2 induces STAT5 phosphorylation. Interestingly, use of inhibitors of p-STAT5 revealed that IL-2 increases LPS-induced IFN-γ through STAT5 phosphorylation. Finally, we report that IL-2 increases the ability of hDCs to activate helpless CD8(+) T cells, most likely because of IL-2-triggered IFN-γ synthesis, as we previously described. For the first time, to our knowledge, we disclose that IL-2 induces monocyte-derived hDC's functional maturation and activation through IL-2R binding. Interestingly, our study suggests a direct effect of anti-CD25 mAbs on hDCs that may contribute to their clinical efficacy.

Mycophenolic acid-treated dendritic cells generate regulatory CD4+ T cells that suppress CD8+ T cells' allocytotoxicity.

Regulatory T cells (Treg) play a crucial role in controlling immunity and transplant rejection. Two main groups of Treg have been described: antigen-induced Treg (iTreg) and natural Treg (nTreg). The ways to induce and the mechanisms of action of Treg subsets remained ill defined, particularly for their effects on CD8(+) T cells. CD8(+) T cells are major agents in the rejection of allografts; the aim of this study is to investigate the effects exerted on CD8(+) T cells by human CD4(+) iTreg induced by mycophenolic acid-treated dendritic cells. iTreg suppress the proliferation of CD8(+) T cells by allogeneic cell-cell interaction with mature dendritic cells and irrespectively of the TCR specificity of the CD8(+) T cells and cell-cell contact of iTreg with CD8(+) T cells. In our model, this suppression is independent of the action of IL-10 and TGF-ß1. iTreg were able to modify phenotype and inhibited IFN-γ and TNF-α secretion by CD8(+) T cells. Most interestingly, iTreg inhibit the synthesis of perforin and of granzymes A and B by CD8(+) T cells and impaired their cytotoxicity against allogeneic targets. In summary, our study showed the involvement of iTreg in the down-regulation of cytotoxic responses mediated by CD8(+) T cells in an allospecific context. Following studies that have shown the existence of a regulation control exerted by iTreg on CD4(+) T cells and dendritic cells, this work ultimately shows that this regulation can reach CD8(+) T-cell functions.

Bispecific antibodies tethering innate receptors induce human tolerant-dendritic cells and regulatory T cells.

There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor ß1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogen-recognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance.

Interferon gamma licensing of human dendritic cells in T-helper-independent CD8+ alloimmunity.

The high frequency of allogeneic reactive CD8(+) T cells in human and their resistance to immunosuppression might be one of the reasons why successful tolerance-inducing strategies in rodents have failed in primates. Studies on the requirement for T-helper cells in priming CD8(+) T-cell responses have led to disparate findings. Recent studies have reported CD8(+)-mediated allograft rejection independently of T-helper cells; however, the mechanisms that govern the activation of these T cells are far from being elucidated. In this study, we demonstrated that lipopolysaccharide-treated dendritic cells (DCs) were able to induce proliferation and cytotoxic activity of allogeneic CD8(+) T cells independently of CD4(+) T cells, while adding mycophenolic acid (MPA) to LPS abolished this capacity and resulted in anergic CD8(+) T cells that secreted high levels of interleukin-4 (IL-4), IL-5, IL-10, and transforming growth factor-ß. Interestingly, we demonstrated that MPA inhibited the LPS-induced synthesis of tumor necrosis factor-α, IL-12, and interferon-γ (IFN-γ) in DCs. Importantly, we found that adding exogenous IFN-γ to MPA restored both the synthesis of cytokines and the ability to activate CD8(+) T cells. However, adding IL-12 or tumor necrosis factor-α had no effect. These results suggest that IFN-γ has an important role in licensing DCs to prime CD4-independent CD8 allogeneic T cells via an autocrine loop.

Anticalin N- or C-Terminal on a Monoclonal Antibody Affects Both Production and In Vitro Functionality.

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

Association between a polymorphism in the human programmed death-1 (PD-1) gene and cytomegalovirus infection after kidney transplantation.

BACKGROUND: Cytomegalovirus (CMV) infection is the most frequent infectious disease following organ transplantation. Strategies to prevent this infection remain a matter for debate, and discovering genetic risk factors might assist in adapting preventive strategies. By inhibiting IFNgamma production, programmed death 1 (PD-1) has a crucial role in anti-CMV immune response. A single nucleotide polymorphism (SNP) within intron 4 of the gene (rs11568821), called PD-1.3, has recently been reported to be clinically relevant in several immune disorders. However, its association with CMV infection has never been reported. METHODS: In this study, the risk of CMV infection according to PD-1.3 genotype was investigated in 469 kidney graft recipients transplanted between 1995 and 2005. RESULTS: It was found that the A allele was associated with the risk of CMV infection in seropositive patients who did not receive CMV prophylaxis (OR=2.60, p=0.006). Multivariate analysis including other risk factors for CMV infection showed that this allele was independently associated with CMV infection (OR=2.54; p=0.010). Interestingly, combined analysis of PD-1.3 with the IL12B 3'UTR SNPs (previously shown to be associated with CMV infection) revealed that patients with the PD-1.3 A allele had a much higher risk of CMV infection compared to those having neither risk allele (OR=3.76; p=0.0003). CONCLUSION: This study identified a new genetic risk factor for CMV infection after kidney transplantation and suggests that an adjustment of CMV prophylaxis based on genetic markers would merit further investigation.

CD40 engagement strongly induces CD25 expression on porcine dendritic cells and polarizes the T cell immune response toward Th1.

Orientation of the immune response toward Th1, Th2, Th17 or Treg plays an important role in self-tolerance and defence against pathogens and tumors. However, this orientation has not been fully characterised in the pig and little is known about the influence of maturation stimulus on the capacity of dendritic cells selectively to direct different types of Th cell responses. Dendritic cell (DC) maturation can be induced by different agents such as inflammatory cytokines, TLR ligands and CD40L. However, the role of the latter in the maturation of pig DC has never been reported. In this study we analysed how different maturation agents influence the capacity of DC to skew the immune response. Monocyte-derived porcine DCs were matured with human CD40L-transfected L-cells, Lipopolysaccharide (LPS) alone or LPS in combination with Tumor necrosis factor-alpha (TNFalpha) and interferon-alpha (IFNalpha). We found that human CD40L induced DC maturation characterised by increased expression of co-stimulatory CD80/86 molecules, high production of IL-12p40 in DC and induction of IFNgamma and t-bet mRNA in T cells, suggesting a Th1 orientation. Moreover we report for the first time the appearance of CD25 after activation of porcine DC. Furthermore, DC activated with TNF+LPS+IFN showed the highest allo-stimulatory capacity of allogeneic lymphocytes and induced IL-17 mRNA in T lymphocytes, suggesting a Th17 orientation that has never been previously reported in the pig. We also showed that immature DCs did not produce any IL-10 or IL-12 and induced both GATA-3 and IL-13 transcription in allogeneic MLR suggesting a Th2 orientation. This study therefore underlines that the nature of the stimulus strongly influences the capacity of DC to steer the immune response in the pig.

Contribution of Intrinsic Fluorescence to the Design of a New 3D-Printed Implant for Releasing SDABS.

Single-domain antibodies (sdAbs) offer great features such as increased stability but are hampered by a limited serum half-life. Many strategies have been developed to improve the sdAb half-life, such as protein engineering and controlled release systems (CRS). In our study, we designed a new product that combined a hydrogel with a 3D-printed implant. The results demonstrate the implant's ability to sustain sdAb release up to 13 days through a reduced initial burst release followed by a continuous release. Furthermore, formulation screening helped to identify the best sdAb formulation conditions and improved our understanding of our CRS. Through the screening step, we gained knowledge about the influence of the choice of polymer and about potential interactions between the sdAb and the polymer. To conclude, this feasibility study confirmed the ability of our CRS to extend sdAb release and established the fundamental role of formulation screening for maximizing knowledge about our CRS.

Neospora caninum: a new class of biopharmaceuticals in the therapeutic arsenal against cancer.

BACKGROUND: Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus-T-VEC-has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent. METHODS/RESULTS: We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-γ secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-γ secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model. CONCLUSION: These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

Induction of human CD4+ regulatory T cells by mycophenolic acid-treated dendritic cells.

Depending on their degree of maturation, costimulatory molecule expression, and cytokine secretion, dendritic cells (DC) can induce immunity or tolerance. DC treated with mycophenolic acid during their maturation (MPA-DC) have a regulatory phenotype and may therefore provide a new approach to induce allograft tolerance. Purified CD4(+) T cells stimulated in a human in vitro model of mixed culture by allogeneic MPA-DC displayed much weaker proliferation than T cells activated by mature DC and were anergic. This hyporesponsiveness was alloantigen-specific. Interestingly, T cells stimulated by MPA-DC during long-term coculture in four 7-day cycles displayed potent, suppressive activity, as revealed by marked inhibition of the proliferation of naive and preactivated control T cells. These regulatory T cells (Tregs) appeared to have antigen specificity and were contact-dependent. Tregs induced by MPA-DC were CD25(+)glucocorticoid-induced TNFR(+)CTLA-4(+)CD95(+), secreted IL-5 and large amounts of IL-10 and TGF-beta, and displayed enhanced forkhead box p3 expression. These results obtained in vitro demonstrate that human MPA-DC can induce allospecific Tregs that may be exploited in cell therapy to induce allograft tolerance.

Association between a polymorphism in the IL-12p40 gene and cytomegalovirus reactivation after kidney transplantation.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with a significant rate of morbidity after organ transplantation. The genetic factors influencing its occurrence have been little investigated. IL-12 plays a crucial role in anti-infectious immune responses, especially by stimulating IFNgamma production. An A-to-C single nucleotide polymorphism (SNP) within the 3'-untranslated region of the IL-12p40 gene has been characterized and was reported to be both functionally and clinically relevant. However, the impact of this single nucleotide polymorphism on events after organ transplantation has never been reported. METHODS: In this study, we investigated the impact of the 3'-untranslated region polymorphism on the occurrence of CMV infection in 469 kidney recipients transplanted at the University Hospital of Tours between 1995 and 2005. The polymorphism was genotyped using the restriction fragment length polymorphism method and CMV infection was determined by pp65 antigenemia. RESULTS: Multifactorial Cox regression analysis demonstrated that the presence of the C allele was an independent risk factor for CMV infection (OR=1.52, P=0.043), the risk being even higher when study was restricted to patients with positive CMV serological status before the graft and who did not receive any CMV prophylaxis (OR=1.88, P=0.028). CONCLUSIONS: This study identified a new genetic risk factor for CMV reactivation after kidney transplantation. The results of our study suggest that C carriers might especially benefit from CMV prophylaxis.

Regulation of human dendritic cell immune functions by ion channels.

Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) able to induce both specific immunity and immune tolerance. Using information gathered from the tissue where they reside, DCs adjust their functional activity to ensure that protective immunity is favoured while unwanted or exaggerated immune responses are prevented. The remarkable ability of these cells to induce, enhance and orient the immune response, while at the same time maintaining self-tolerance, makes them key players in the immune system. Despite the fact that the role of Ca2+ has been clearly established in human DC functions, the link between ion homeostasis, mainly Ca2+, and DC functions is not fully understood. After all, a growing number of works clearly show the role of SOCE and associated channels in the maturation step, and those of K+ channels in migration. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation.

Tocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14+ monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients.

Immature human dendritic cells enhance their migration through KCa3.1 channel activation.

Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14(+)-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca(2+) entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.

The Orai-1 and STIM-1 complex controls human dendritic cell maturation.

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

Mycophenolic acid differentially affects dendritic cell maturation induced by tumor necrosis factor-alpha and lipopolysaccharide through a different modulation of MAPK signaling.

Mycophenolic acid (MPA) is an immunosuppressive drug which induces resistance to several maturation signals in human dendritic cells (DC) by unknown mechanisms. As mitogen-activated protein kinases (MAPK) are involved in the maturation process, we studied whether MPA affected p38MAPK and extracellular signal-regulated kinase (ERK1/2) in human DC. We first showed that MPA reduced TNFalpha-induced phenotype maturation, whereas it had no effect after LPS activation, suggesting that MPA preferentially affects the signaling pathway used by TNFalpha. We found that TNFalpha preferentially used p38MAPK to induce phenotype maturation in DC, whereas LPS preferentially activated NF-kappaB. Importantly, we showed that MPA more strongly inhibited p38MAPK phosphorylation induced by TNFalpha than by LPS. This difference in inhibition may therefore explain its different effect on DC phenotype. Interestingly, MPA inhibited the inflammatory cytokine synthesis and allostimulatory capacity induced by both stimuli. Exogenous guanosine antagonized the effect of MPA on the phenotype of TNFalpha-matured-DC as well as the IL-12p70 and IFN gamma secretion induced by both stimuli, without affecting p38MAPK phosphorylation. The action of MPA on human DC phenotype maturation appears mainly to be due to its ability to inhibit p38MAPK. Furthermore, the difference between LPS and TNFalpha emphasizes that the DC microenvironment strongly influences DC sensitivity to MPA.