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Innate immune cells such as dendritic cells (DCs) sense and engulf nanomaterials potentially leading to an adverse immune response. Indeed, as described for combustion-derived particles, nanomaterials could be sensed as danger signals, enabling DCs to undergo a maturation process, migrate to regional lymph nodes and activate naive T lymphocytes. Synthetic amorphous silica nanoparticles (SAS-NPs) are widely used as food additives, cosmetics, and construction materials. This work aimed to evaluate in vitro the effects of manufactured SAS-NPs, produced by thermal or wet routes, on human DCs functions and T-cell activation. Human monocyte-derived DCs (moDCs) were exposed for 16 h to 3 endotoxin-free test materials: fumed silica NPs from Sigma-Aldrich (no. S5505) or the JRC Nanomaterial Repository (NM-202) and colloidal LudoxTMA NPs. Cell viability, phenotypical changes, cytokines production, internalization, and allogeneic CD4+ T-cells proliferation were evaluated. Our results showed that all SAS-NPs significantly upregulated the surface expression of CD86 and CD83 activation markers. Secretions of pro-inflammatory cytokines (CXCL-8 and CXCL-12) were significantly enhanced in a dose-dependent manner in the moDCs culture supernatants by all SAS-NPs tested. In an allogeneic coculture, fumed silica-activated moDCs significantly increased T-lymphocyte proliferation at all T-cell: DC ratios compared with unloaded moDCs. Moreover, analysis of coculture supernatants regarding the production of T-cell-derived cytokines showed a significant increase of IL-9 and IL-17A and F, as well as an upregulation of IL-5, consistent with the pro-inflammatory phenotype of treated moDCs. Taken together, these results suggest that SAS-NPs could induce functional moDCs maturation and play a role in the immunization process against environmental antigens.

Background Biopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited. Methods and findings The European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn’s disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253–0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437–0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616–4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319–3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923–5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139–6.764], p < 1 × 10−5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106–4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings. Conclusion In our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies., In a multicohort prospective study of patients from 12 countries, Signe Hässler and colleagues investigate clinical and genetic factors associated with development of anti-biopharmaceutical drug antibodies in patients with autoimmune diseases., Author summary Why was this study done? Biopharmaceutical products such as monoclonal antibodies are widely used to treat autoimmune diseases. Biopharmaceutical products may induce the development of antidrug antibodies, which often lead to therapy failure. Patient-related factors that influence the development of antidrug antibodies need to be characterized. What did the researchers do and find? We set up a European multicohort prospective study on 4 autoimmune diseases (multiple sclerosis, rheumatoid arthritis, Crohn’s disease, and ulcerative colitis) treated with 8 different biopharmaceutical products. We collected demographic and clinical data and tested antidrug antibodies in longitudinal serum samples from 560 patients. For 457 patients who gave consent, we also collected genetic data. We identified antibiotics and immunosuppressants as negatively associated risk factors and heavy smoking, infections during the study, the HLA-DQA1*05 allele and a minor variant in the CXCL12 chemokine gene associated with increased protein expression as risk factors of antidrug antibody development. What do these findings mean? Our findings suggest that the combination of immunosuppressant and biopharmaceutical therapy could be associated with decreased risk of antidrug antibody occurrence, which has implications for rheumatoid arthritis and inflammatory bowel diseases, for which immunosuppressants are often, but not always, given together with biopharmaceuticals. Patients heterozygotes or homozygotes for the HLA-DQA1*05 allele may have an increased risk of antidrug antibody occurrence associated with biopharmaceutical therapy. The small study size warrants a validation through independent studies, in particular for the genetic findings.

Originally described as a TGF-β-inducible gene, tsc-22 (Transforming growth factor-beta Stimulated Clone 22) encodes a transcriptional regulator affecting biological processes such as cell growth, differentiation, or apoptosis. Along with GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 belongs to the evolutionary conserved TSC-22 Domain family. We previously showed that, in T-lymphocytes, GILZ expression was induced upon IL-2 withdrawal, delaying apoptosis through down-regulation of the pro-apoptotic protein BIM expression. The aim of this work was then to elucidate the respective roles of GILZ and TSC-22 upon IL-2 deprivation-induced apoptosis. We report here that these two highly homologous genes are concomitantly expressed in most human tissues and in primary T-lymphocytes and that expression of TSC-22 promotes T-lymphocytes apoptosis by inhibiting GILZ functions. Indeed, we demonstrated that TSC-22 expression in the murine lymphoid CTLL-2 cell line promoted IL-2 deprivation-induced apoptosis. BIM expression and caspases-9 and -3 activities were markedly increased in TSC-22 expressing clones compared to control clones. Analysis of GILZ expression revealed that TSC-22 prevented the induction of the GILZ protein upon IL-2 deprivation, by inhibiting gilz mRNA transcription. These results suggested that TSC-22 could counteract the protective effect of GILZ on IL-2-deprivation-induced apoptosis. Moreover, TSC-22-induced inhibition of GILZ expression was also found in CTLL-2 cells treated with glucocorticoids or TGF-β. In the human NKL cell line deprived of IL-2, TSC-22 showed the same effect and thus may represent a potent repressor of GILZ expression in IL-2-dependent cells, independently of the cell type, or the stimulus, leading to an increase of IL-2-deprived T-cells apoptosis. J. Cell. Biochem. 117: 1855-1868, 2016. © 2016 Wiley Periodicals, Inc.

Background:Anti-Drug Antibodies (ADA) to adalimumab increase drug clearance in rheumatoid arthritis (RA).Objectives:To study the ability of drug concentration or estimating clearance to identify ADA to adalimumab.Methods:Adalimumab concentration was measured with a validated ELISA. ADA was measured using a capture ELISA (Theradiag®) and the Meso scale discovery (MSD) platform. Using a bayesian PK model, adalimumab clearance was estimated at 1, 3, 6 and 12 months. Predictions for ADA presence were calculated, and the correlation between ADA and adalimumab clearance was analysed.Results:We analyzed 108 samples from 53 RA patients. Serum concentrations and clearance estimates showed good prediction performance for ADA presence (Table 1). There was a correlation between adalimumab clearance and ADA (Figure 1).Table 1.Immunogenicity prediction of adalimumab, using trough concentration or estimated clearanceTime of visitADA methodAdalimumab trough concentrationAdalimumab estimated clearanceAUC ROCp-valueAUC ROCp-valueMonth 1THER.55.6411.52.8358MSD.65.0821.61.1872Month 3THER.89.0006.91.0003MSD.73.0096.72.0131Month 6THER.95.0035.95.0035MSD.85.0004.84.0006Month 12THER.87.0045.86.0057MSD.88.0002.88.0002Figure 1.correlation between adalimumab estimated clearance and ADA as provided by the Meso scale discovery (MSD) plateformConclusion:Adalimumab concentration and clearance should be considered as reliable predictors for ADA presence in RA patients.Acknowledgments:Measurement of adalimumab serum concentrations was performed within the ‘Centre pilote de suivi biologique des anticorps thérapeutiques’ (CePiBAc)– Pilot centre for therapeutic antibodies monitoring platform of Tours University Hospital, which was cofinanced by the European Regional Development Fund (ERDF). We thank Oscar Knight, Delphine Delord and Fabien Giannoni (ABIRISK lab technician), Caroline Brochon and Anne Claire Duveau (CePIBAc), Aliette Decock-Giraudaud (Centre de ressource-Biobank), Sophie Tourdot (ABRISIK Project manager), Aline Doublet (Assistance Publique Hopitaux de Paris, Agnès Hincelin-Méry (Sanofi, Chilly-Mazarin, France). This work has received support from the Innovative Medicines Initiative Joint Undertaking (IMI JU) under grant agreement no. 115303, the resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies’ in-kind contributions.Disclosure of Interests:David Ternant Consultant of: Sanofi and Amgen., Jamal Elhasnaoui: None declared, Natacha Szely: None declared, Salima Hacein-Bey: None declared, Aude Gleizes: None declared, Christophe Richez Consultant of: Abbvie, Amgen, Mylan, Pfizer, Sandoz and UCB., Jessica Manson: None declared, Martin SOUBRIER: None declared, Olilvier Brocq: None declared, Jérôme Avouac: None declared, Anna Fogdell-Hahn Grant/research support from: Biogen Idec and Pfizer., Consultant of: Pfizer, Biogen, Merck-Serono, and Sanofi-Genzyme., Pierre Dönnes: None declared, Gilles Paintaud Grant/research support from: Amgen, Genzyme (Sanofi), Lilly, Merck, Novartis, and Roche Pharma., Consultant of: Chugai, Novartis and Shire (Takeda), with remunerations received by his institution., Céline Desvignes: None declared, Florian Deisenhammer: None declared, Sebastian Spindeldreher Employee of: Novartis, Marc Pallardy: None declared, Xavier Mariette Consultant of: BMS, Gilead, Medimmune, Novartis, Pfizer, Servier, UCB, Denis Mulleman Grant/research support from: Non-governmental organisation Lions Club Tours Val de France, French Society for Rheumatology., Consultant of: Pfizer, Novartis.

Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response. This work focuses on the development of predictive in vitro assays that can monitor DC maturation, in order to determine whether drug products have direct DC stimulatory capabilities. To this end, four independent laboratories aligned a common protocol to differentiate human monocyte-derived DC (moDC) that were treated with either native or aggregated preparations of infliximab, natalizumab, adalimumab, or rituximab. These drug products were subjected to different forms of physical stress, heat and shear, resulting in aggregation and the formation of subvisible particles. Each partner developed and optimized assays to monitor diverse end-points of moDC maturation: measuring the upregulation of DC activation markers via flow cytometry, analyzing cytokine, and chemokine production via mRNA and protein quantification and identifying cell signaling pathways via quantification of protein phosphorylation. These study results indicated that infliximab, with the highest propensity to form aggregates when heat-stressed, induced a marked activation of moDC as measured by an increase in CD83 and CD86 surface expression, IL-1β, IL-6, IL-8, IL-12, TNFα, CCL3, and CCL4 transcript upregulation and release of respective proteins, and phosphorylation of the intracellular signaling proteins Syk, ERK1/2, and Akt. In contrast, natalizumab, which does not aggregate under these stress conditions, induced no DC activation in any assay system, whereas adalimumab or rituximab aggregates induced only slight parameter variation. Importantly, the data generated in the different assay systems by each partner site correlated and supported the use of these assays to monitor drug-intrinsic propensities to drive maturation of DC. This moDC assay is also a valuable tool as an in vitro model to assess the intracellular mechanisms that drive DC activation by aggregated therapeutic proteins.

Neutrophil extracellular traps (NETs) are extracellular DNA filaments formed during neutrophil activation. This process, called netosis, was originally associated with neutrophil antibacterial properties. However, several lines of evidence now suggest a major role for netosis in thrombosis, autoimmune diseases, and cancer. We demonstrate here that highly purified human blood monocytes are also capable of extracellular trap (ET) release in response to several stimuli. Monocyte ETs display a morphology analogous to NETs and are associated with myeloperoxidase (MPO), lactoferrin (LF), citrullinated histones, and elastase. Monocyte ET release depends on oxidative burst but not on MPO activity, in contrast to neutrophils. Moreover, we demonstrate procoagulant activity for monocyte ETs, a feature that could be relevant to monocyte thrombogenic properties. This new cellular mechanism is likely to have implications in the multiple pathologic contexts where monocytes are implicated, such as inflammatory disorders, infection, or thrombosis.

Patients treated with therapeutic biological products (BP) frequently develop anti-drug antibodies (ADA) with potential neutralizing capacities leading to loss of clinical response or serious side effects. BP aggregates have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are key effectors in T-cell and B-cell fates, and the subsequent generation of immunogenicity. The objective of this work was to determine if BP aggregates can participate to DC maturation and T-cell activation. We compared aggregates from three different proteins: human growth hormone (hGH), Rituximab, a chimeric anti-CD20 antibody and a serum-purified human IgG1. All three proteins underwent a stir stress, generating comparable populations of aggregated particles. Maturation of human monocyte-derived DC (moDC) upon exposure to native BPs or aggregates was evaluated in vitro. Results showed that hGH aggregates induced an increased expression of moDC co-stimulation markers, and augmented levels of IL-6, IL-8, IL-12p40, CCL2, CCL3, CCL4 and CXCL10. Both antibodies aggregates were also able to modify DC phenotype, but cytokine and chemokine productions were seen only with IL-6, IL-8, IL-12p40 and CXCL10. Aggregates-treated moDC enhanced allogenic T-cell proliferation and cytokines production, suggesting Th1 polarization with hGH, and mixed T-cell responses with antibodies aggregates. These results showed that BP aggregates provoked DC maturation, thus driving adaptive T-cell responses and polarization.

Dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self-antigens. Here, we demonstrate for the first time that mouse plasmacytoid DCs (pDCs) can be segregated into three distinct populations, exhibiting phenotypic and functional differences, according to their surface expression of CD8α or CD8β as CD8α⁻β⁻, CD8α⁺β⁻, or CD8α⁺β⁺. In a mouse model of lung inflammation, adoptive transfer of CD8α⁺β⁻ or CD8α⁺β⁺ pDCs prevents the development of airway hyper-reactivity. The tolerogenic features of these subsets are associated with increased production of retinoic acid, which leads to the enhanced induction of Foxp3⁺ regulatory T cells compared with CD8α⁻β⁻ pDCs. Our data thus identify subsets of pDCs with potent tolerogenic functions that may contribute to the maintenance of tolerance in mucosal sites such as the lungs.

The angiogenic factor Midkine (MDK) is overexpressed in various human malignant tumors, although its expression is low or undetectable in normal adult tissues. Its expression in tumors and its detection in plasma have been associated with poor disease outcome, whereas its blockade was found to contribute to tumor regression. By weekly stimulation of T lymphocytes harvested in HLA-A2 healthy donors, we derived CD8 T cell lines specific for several MDK peptides. The T cell response was mostly dominated by two nonamer peptides localized in the signal peptide and in the C-terminal part of the protein, as assessed by IFN-γ ELISPOT and HLA-A2 tetramer labeling. Peptide-specific T cell lines recognized cells transfected with an MDK-encoded plasmid and tumor cell lines naturally expressing the MDK protein, but not untransfected cells. T cell presentation of the two MDK epitopes was found to be TAP dependent. Experiments performed in HLA-A2 transgenic mice demonstrated the capacity of the two identified CD8 T cell epitopes to elicit a cytotoxic response. Altogether, our data show that the secreted MDK protein is a candidate vaccine for multiple cancers.

Glucocorticoid-induced leucine zipper (GILZ) is a potent anti-inflammatory protein, the expression of which is mainly induced by glucocorticoids (GCs) in haematopoietic cells. GILZ regulates signal transduction pathways of inflammation and plays a role in cell survival. The objective of this study was to evaluate the expression and mechanisms of action of GILZ in the apoptosis of human neutrophils. GILZ expression was induced by GCs in human neutrophils, enhanced upon phosphatidylinositol 3-kinase inhibition and resulted in apoptosis amplification. We then stably transfected PLB-985 cells with the human gilz gene and differentiated both control and GILZ-overexpressing clones in neutrophil-like cells. GILZ overexpression in PLB-985 cells led to an exacerbated apoptosis, associated with caspase-3, caspase-9 and caspase-8 activations, and a loss of mitochondrial potential, suggesting that GILZ-induced apoptosis used the mitochondrial pathway. The expression of BH3 interacting domain death agonist, Bcl-2 interacting mediator of cell death, annexin-A1 and Bcl-2-associated X was not affected in PLB-985-GILZ clones, but phosphorylation and subsequent proteasomal degradation of myeloid cell leukemia-1 (Mcl-1) were observed. Noteworthy, Mcl-1 phosphorylation was related to a significant and sustained activation of c-Jun N-terminal kinase (JNK) in PLB-985-GILZ clones. These results reveal GILZ to be a new actor in apoptosis regulation in neutrophil-like cells involving JNK and Mcl-1.

Originally described as a TGF-β-inducible gene, tsc-22 (Transforming growth factor-beta Stimulated Clone 22) encodes a transcriptional regulator affecting biological processes such as cell growth, differentiation, or apoptosis. Along with GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 belongs to the evolutionary conserved TSC-22 Domain family. We previously showed that, in T-lymphocytes, GILZ expression was induced upon IL-2 withdrawal, delaying apoptosis through down-regulation of the pro-apoptotic protein BIM expression. The aim of this work was then to elucidate the respective roles of GILZ and TSC-22 upon IL-2 deprivation-induced apoptosis. We report here that these two highly homologous genes are concomitantly expressed in most human tissues and in primary T-lymphocytes and that expression of TSC-22 promotes T-lymphocytes apoptosis by inhibiting GILZ functions. Indeed, we demonstrated that TSC-22 expression in the murine lymphoid CTLL-2 cell line promoted IL-2 deprivation-induced apoptosis. BIM expression and caspases-9 and -3 activities were markedly increased in TSC-22 expressing clones compared to control clones. Analysis of GILZ expression revealed that TSC-22 prevented the induction of the GILZ protein upon IL-2 deprivation, by inhibiting gilz mRNA transcription. These results suggested that TSC-22 could counteract the protective effect of GILZ on IL-2-deprivation-induced apoptosis. Moreover, TSC-22-induced inhibition of GILZ expression was also found in CTLL-2 cells treated with glucocorticoids or TGF-β. In the human NKL cell line deprived of IL-2, TSC-22 showed the same effect and thus may represent a potent repressor of GILZ expression in IL-2-dependent cells, independently of the cell type, or the stimulus, leading to an increase of IL-2-deprived T-cells apoptosis. J. Cell. Biochem. 117: 1855-1868, 2016. © 2016 Wiley Periodicals, Inc.

The Agence National de Recherche sur le SIDA et les hepatitis Lipo5 vaccine is composed by five long fragments of HIV proteins and was recently shown to induce in seronegative volunteers a CD4 T cell response largely dominated by the G2 fragment. To understand this response profile, we submitted the five HIV fragments to HLA-DR–binding assays and evaluated the frequency of naive Lipo5-specific CD4 T lymphocytes in the blood of 22 healthy individuals. We enumerated the Lipo5-specific T cell lines induced in vitro by weekly rounds of specific stimulation. Four peptides and hence not only G2 exhibited a broad specificity for HLA-DR molecules. In contrast, most of the T cell lines specific for Lipo5 reacted with G2, revealing a G2-specific T cell repertoire superior to 2 cells per million, whereas it is close to 0.4 for the other peptides. We also found good cross-reactivity of all the peptides with clade B and C variants and that G2 and P1 are able to recruit T cells that recognize HIV-infected cells. We therefore mainly observed very good concordance between the frequency to individual Lipo5 peptides among vaccinees in a large-scale vaccine trial and the distribution of peptide specificity of the in vitro induced T cell lines. These findings underline the role of the size of the epitope-specific naive repertoire in shaping the CD4 T cell response after vaccination and highlight the value of evaluating the naive repertoire to predict vaccine immunogenicity.

Respiratory exposure to allergen induces T cell tolerance and protection against the development of airway hyperactivity in animal models of asthma. Whereas systemic administration of dexamethasone during the delivery of respiratory Ag has been suggested to prevent the development of mucosal tolerance, the effects of local administration of corticosteroids, first-line treatment for patients with bronchial asthma, on mucosal tolerance remain unknown. To analyze the effects of systemic versus local administration of different types of corticosteroids on the development of mucosal tolerance, mice were exposed to respiratory allergen to induce mucosal tolerance with or without systemic or intranasal application of different doses of dexamethasone or prednisolone. After the induction of mucosal tolerance, proliferation of T cells was inhibited in tolerized mice, whereas systemic applications of corticosteroids restored T cell proliferation and secretion of Th2 cytokines. In contrast, inhaled corticosteroids showed no effect on both T cell proliferation and cytokine secretion. In addition, mice systemically treated with corticosteroids showed an increased airway hyperactivity with a significant lung inflammation, but also an increased T effector cells/regulatory T cells ratio in the second lymphoid organs when compared with mice that receive corticosteroids by inhalation. These results demonstrate that local administration of corticosteroids has no effect on the development of immune tolerance in contrast to systemically applied corticosteroids. Furthermore, although different concentrations of corticosteroids are administered to patients, our results demonstrated that the route of administration rather than the doses affects the effect of corticosteroids on respiratory tolerance induction. Considering the broad application of corticosteroids in patients with allergic disease and asthma, the route of administration of steroid substances seems crucial in terms of treatment and potential side effects. These findings may help elucidate the apparently contradicting results of corticosteroid treatment in allergic diseases.

International audience; Recent studies have suggested including nonstructural proteins as Tat and Vpr in HIV vaccines. However, little is known about the CD4(+) T-cell response that these small proteins induce in humans. We have therefore evaluated these responses by in vitro priming experiments of CD4(+) T lymphocytes harvested in healthy donors. In the Tat protein, only one peptide primed CD4(+) T cells of eight HLA unrelated healthy donors. T cells induced by this peptide recognized immature DC loaded with the native Tat protein and are restricted by multiple HLA-DR molecules, in agreement with its binding capacity. This peptide was therefore processed in an appropriate manner and was highly immunoprevalent. CD4(+) T-cell response to Vpr peptides was more disperse and involved six different peptides depending on the HLA-DR molecules of the donors. Two overlapping peptides were T-cell stimulating in at least half of the donors. T-cell response to Vpr in multiple donors is the result of a combination of several CD4(+) T-cell epitopes with good to moderate immunoprevalence. Altogether, our results show that the frequency of responders to HIV Tat or Vpr proteins relies on one or multiple CD4(+) T-cell epitopes, respectively.

Subunit vaccine candidates against poxvirus infection induced protective humoral and cellular response in animal models but their immunogenicity in human remains unknown. We have therefore evaluated in vitro the CD4 T cell response of the major antigens B5R and A33R and characterized their CD4 T cell epitopes. Twelve peptides selected on the basis of their binding capacity to HLA-DR molecules, induced CD4 T lymphocytes harvested in healthy donors. In the A33R proteins two peptides are T cell stimulating for at least half of the donors and are restricted to multiple HLA-DR molecules in agreement with their broad specificity for HLA-DR molecules. In B5R, two peptides exhibited a good immunoprevalence but only one is a good binder to multiple HLA-DR molecules. One peptide was a moderate binder for multiple HLA-DR molecules, although it was efficiently presented to peptide-specific T cell lines. Altogether, our data demonstrated the capacity of B5R and A33R peptides to elicit a T cell response in multiple healthy donors and showed that promiscuity and immunoprevalence of CD4 T cell epitopes are not necessarily associated.

Asthma is defined as a chronic inflammatory disease of the airways; however, the underlying physiologic and immunologic processes are not fully understood. The aim of this study was to determine whether Th9 cells develop in vivo in a model of chronic airway hyperreactivity (AHR) and what factors control this development. We have developed a novel chronic allergen exposure model using the clinically relevant antigen Aspergillus fumigatus to determine the time kinetics of Th9 development in vivo. Th9 cells were detectable in the lungs after chronic allergen exposure. The number of Th9 cells directly correlated with the severity of AHR, and anti-IL-9 treatment decreased airway inflammation. Moreover, we have identified programmed cell death ligand (PD-L) 2 as a negative regulator of Th9 cell differentiation. Lack of PD-L2 was associated with significantly increased TGF-β and IL-1α levels in the lungs, enhanced pulmonary Th9 differentiation, and higher morbidity in the sensitized mice. Our findings suggest that PD-L2 plays a pivotal role in the regulation of Th9 cell development in chronic AHR, providing novel strategies for modulating adaptive immunity during chronic allergic responses.

BACKGROUND: Asthma is defined as a chronic inflammatory disease of the airways; however, the underlying physiologic and immunologic processes are not fully understood. OBJECTIVE: The aim of this study was to determine whether T(H)9 cells develop in vivo in a model of chronic airway hyperreactivity (AHR) and what factors control this development. METHOD: We have developed a novel chronic allergen exposure model using the clinically relevant antigen Aspergillus fumigatus to determine the time kinetics of T(H)9 development in vivo. RESULTS: T(H)9 cells were detectable in the lungs after chronic allergen exposure. The number of T(H)9 cells directly correlated with the severity of AHR, and anti-IL-9 treatment decreased airway inflammation. Moreover, we have identified programmed cell death ligand (PD-L) 2 as a negative regulator of T(H)9 cell differentiation. Lack of PD-L2 was associated with significantly increased TGF-β and IL-1α levels in the lungs, enhanced pulmonary T(H)9 differentiation, and higher morbidity in the sensitized mice. CONCLUSION: Our findings suggest that PD-L2 plays a pivotal role in the regulation of T(H)9 cell development in chronic AHR, providing novel strategies for modulating adaptive immunity during chronic allergic responses.

Recent findings suggest that different subsets of dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self antigens. Latest data propose that plasmacytoid DCs (pDCs) act as antigen-presenting cells during adaptive immune responses and promote peripheral tolerance. In this study, we demonstrate for the first time that mouse pDCs can be segregated into three distinct populations according to their surface expression of CD8α alone or combined with CD8β. These subsets not only differ in phenotype but are also functionally distinct since CD8α+ CD8β− and CD8α+ CD8β+ pDCs are more potent inducers of CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) compared to CD8α− CD8β− pDCs. Our results indicate that, in a mouse model of allergic asthma, adoptive transfer of CD8α+ CD8β− or CD8α+ CD8β+ prevents the development of airway hyperreactivity. By comparing the gene expression profile of the three subsets of pDCs described in this study, we established that the expression of CD98hc, a receptor for the Galectin-3, was significantly up-regulated in both CD8α+ CD8β− and CD8α+ CD8β+ subsets. Adding Galectin-3 to sorted CD8α+ CD8β− or CD8α+ CD8β+ pDCs in vitro, enhances the conversion of naive CD4+ T cells into Tregs. Thus, our data unveil for the first time, subsets of pDCs with the capacity to induce regulatory functions that may contribute to the establishment of immunological tolerance.