Antibodies internalization mechanisms by dendritic cells and their role in therapeutic antibody immunogenicity.

Internalization and processing by antigen-presenting cells such as dendritic cells (DCs) are critical steps for initiating a T-cell response to therapeutic antibodies. Consequences are the production of neutralizing antidrug antibodies altering the clinical response, the presence of immune complexes, and, in some rare cases, hypersensitivity reactions. In recent years, significant progress has been made in the knowledge of cellular uptake mechanisms of antibodies in DCs. The uptake of antibodies could be directly related to their immunogenicity by regulating the quantity of materials entering the DCs in relation to antibody structure. Here, we summarize the latest insights into cellular uptake mechanisms and pathways in DCs. We highlight the approaches to study endocytosis, the impact of endocytosis routes on T-cell response, and discuss the link between how DCs internalize therapeutic antibodies and the potential mechanisms that could give rise to immunogenicity. Understanding these processes could help in developing assays to evaluate the immunogenicity potential of biotherapeutics.

Human dendritic cell maturation induced by amorphous silica nanoparticles is Syk-dependent and triggered by lipid raft aggregation.

BACKGROUND: Synthetic amorphous silica nanoparticles (SAS-NPs) are widely employed in pharmaceutics, cosmetics, food and concretes. Workers and the general population are exposed daily via diverse routes of exposure. SAS-NPs are generally recognized as safe (GRAS) by the Food and Drug Administration, but because of their nanoscale size and extensive uses, a better assessment of their immunotoxicity is required. In the presence of immune "danger signals", dendritic cells (DCs) undergo a maturation process resulting in their migration to regional lymph nodes where they activate naive T-cells. We have previously shown that fumed silica pyrogenic SAS-NPs promote the two first steps of the adaptative immune response by triggering DC maturation and T-lymphocyte response, suggesting that SAS-NPs could behave as immune "danger signals". The present work aims to identify the mechanism and the signalling pathways involved in DC phenotype modifications provoked by pyrogenic SAS-NPs. As a pivotal intracellular signalling molecule whose phosphorylation is associated with DC maturation, we hypothesized that Spleen tyrosine kinase (Syk) may play a central role in SAS-NPs-induced DC response. RESULTS: In human monocyte-derived dendritic cells (moDCs) exposed to SAS-NPs, Syk inhibition prevented the induction of CD83 and CD86 marker expression. A significant decrease in T-cell proliferation and IFN-γ, IL-17F and IL-9 production was found in an allogeneic moDC:T-cell co-culture model. These results suggested that the activation of Syk was necessary for optimal co-stimulation of T-cells. Moreover, Syk phosphorylation, observed 30 min after SAS-NP exposure, occurred upstream of the c-Jun N-terminal kinase (JNK) Mitogen-activated protein kinases (MAPK) and was elicited by the Src family of protein tyrosine kinases. Our results also showed for the first time that SAS-NPs provoked aggregation of lipid rafts in moDCs and that MßCD-mediated raft destabilisation altered Syk activation. CONCLUSIONS: We showed that SAS-NPs could act as an immune danger signal in DCs through a Syk-dependent pathway. Our findings revealed an original mechanism whereby the interaction of SAS-NPs with DC membranes promoted aggregation of lipid rafts, leading to a Src kinase-initiated activation loop triggering Syk activation and functional DC maturation.

The FcγRIIa-Syk Axis Controls Human Dendritic Cell Activation and T Cell Response Induced by Infliximab Aggregates.

The development of anti-drug Abs in response to biological products (BP) is a major drawback in the treatment of patients. Factors related to the patient, the treatment, and the product can influence BP immunogenicity. Among these factors, BP aggregates have been suggested to promote immunogenicity by acting as danger signals recognized by dendritic cells (DC) facilitating the establishment of an anti-BP CD4 T cell-dependent adaptive immune response leading to anti-drug Abs production. To date, little is known on the mechanism supporting the effect of aggregates on DCs and consequently on the T cell response. The aim of this work was to identify key signaling pathways involved in BP aggregate DC activation and T cell response. We generated aggregates by submitting infliximab (IFX), an immunogenic anti-TNF-α chimeric Ab, to heat stress. Our results showed that IFX aggregates were able to induce human monocyte-derived DC (moDC) maturation in a concentration-dependent manner. Aggregate-treated moDCs enhanced allogeneic T cell proliferation and IL-5, IL-9, and IL-13 production compared with native Ab-treated moDCs. We then investigated the implication of FcγRIIa and spleen tyrosine kinase (Syk) in DC activation and showed that they were both strongly implicated in moDC maturation induced by IFX aggregates. Indeed, we found that neutralization of FcγRIIa inhibited DC activation, and consequently, Syk inhibition led to a decrease in T cell proliferation and cytokine production in response to IFX aggregates. Taken together, our results bring new insight, to our knowledge, on how protein aggregates could induce DC and T cell activation via the FcγRIIa-Syk signaling pathway.

Cutting Edge: Nrf2 Regulates Neutrophil Recruitment and Accumulation in Skin during Contact Hypersensitivity.

Neutrophils are essential during contact hypersensitivity (CHS), a common skin allergic disease. NF-E2-related factor-2 (Nrf2) is a key regulator of redox balance and skin homeostasis playing a protective role in CHS. In this study, we investigated Nrf2 role in neutrophil recruitment during the sensitization phase of CHS. Comparing wild-type and Nrf2 knockout mice, we demonstrated that Nrf2 regulated dinitrochlorobenzene-induced xenoinflammation, notably neutrophil recruitment to sensitized skin. Nrf2 protective role was associated with high expression of antioxidant genes (ho-1, gclc, nqo1…) and decreased chemokine production (CCL2, CCL4, CCL11). Interestingly, skin sensitization induced CD36 upregulation in skin-resident macrophages. In vitro results confirmed that the transcription of cd36 gene in macrophages was dependent on Nrf2 and led to an improved capacity to phagocyte-damaged neutrophils by efferocytosis. Nrf2 emerges as a critical target in the sensitization phase of CHS regulating neutrophil recruitment and accumulation in the skin through antioxidant-dependent and -independent mechanisms.

Skin immune cell characterization in juvenile and adult Göttingen Minipigs.

The skin hosts a sophisticated immune system involving responses from both innate and adaptive immune cell populations. Swine skin is close to human skin by its structure, composition and function. In addition, the minipig is considered the model of choice in toxicology studies for drugs applied by the dermal route and developed for both the adult and paediatric indications. However, knowledge on the skin immune system in minipigs, particularly in Göttingen Minipigs, is still limited. The objective of our work was first to characterize the main skin immune populations (Langerhans cells, dermal dendritic cells, macrophages and T lymphocytes) in Göttingen Minipigs. In parallel, we compared the skin immune populations from healthy and immunocompromised piglets following oral treatment with cyclosporin A (CsA) at 10 mg/kg/day. We also explored other pathological conditions using a contact dermatitis model in minipigs challenged with a sensitizer, 2,4-dinitrochlorobenzene (DNCB). Langerhans cells and dermal MHCIIlowCD163+ cells were increased one month after oral treatment with CsA at 10 mg/kg/day. The contact dermatitis model in Göttingen Minipigs challenged by DNCB suggested migration of Langerhans cells and dermal dendritic cells as well as T cell recruitment into the skin. These data bring new information in skin immunotoxicology in Göttingen Minipigs and could contribute to a better understanding of the effects of new therapeutic drugs on the developing immune system.

Treatment- and population-specific genetic risk factors for anti-drug antibodies against interferon-beta: a GWAS.

BACKGROUND: Upon treatment with biopharmaceuticals, the immune system may produce anti-drug antibodies (ADA) that inhibit the therapy. Up to 40% of multiple sclerosis patients treated with interferon ß (IFNß) develop ADA, for which a genetic predisposition exists. Here, we present a genome-wide association study on ADA and predict the occurrence of antibodies in multiple sclerosis patients treated with different interferon ß preparations. METHODS: We analyzed a large sample of 2757 genotyped and imputed patients from two cohorts (Sweden and Germany), split between a discovery and a replication dataset. Binding ADA (bADA) levels were measured by capture-ELISA, neutralizing ADA (nADA) titers using a bioassay. Genome-wide association analyses were conducted stratified by cohort and treatment preparation, followed by fixed-effects meta-analysis. RESULTS: Binding ADA levels and nADA titers were correlated and showed a significant heritability (47% and 50%, respectively). The risk factors differed strongly by treatment preparation: The top-associated and replicated variants for nADA presence were the HLA-associated variants rs77278603 in IFNß-1a s.c.- (odds ratio (OR) = 3.55 (95% confidence interval = 2.81-4.48), p = 2.1 × 10-26) and rs28366299 in IFNß-1b s.c.-treated patients (OR = 3.56 (2.69-4.72), p = 6.6 × 10-19). The rs77278603-correlated HLA haplotype DR15-DQ6 conferred risk specifically for IFNß-1a s.c. (OR = 2.88 (2.29-3.61), p = 7.4 × 10-20) while DR3-DQ2 was protective (OR = 0.37 (0.27-0.52), p = 3.7 × 10-09). The haplotype DR4-DQ3 was the major risk haplotype for IFNß-1b s.c. (OR = 7.35 (4.33-12.47), p = 1.5 × 10-13). These haplotypes exhibit large population-specific frequency differences. The best prediction models were achieved for ADA in IFNß-1a s.c.-treated patients. Here, the prediction in the Swedish cohort showed AUC = 0.91 (0.85-0.95), sensitivity = 0.78, and specificity = 0.90; patients with the top 30% of genetic risk had, compared to patients in the bottom 30%, an OR = 73.9 (11.8-463.6, p = 4.4 × 10-6) of developing nADA. In the German cohort, the AUC of the same model was 0.83 (0.71-0.92), sensitivity = 0.80, specificity = 0.76, with an OR = 13.8 (3.0-63.3, p = 7.5 × 10-4). CONCLUSIONS: We identified several HLA-associated genetic risk factors for ADA against interferon ß, which were specific for treatment preparations and population backgrounds. Genetic prediction models could robustly identify patients at risk for developing ADA and might be used for personalized therapy recommendations and stratified ADA screening in clinical practice. These analyses serve as a roadmap for genetic characterizations of ADA against other biopharmaceutical compounds.

Assessing the effect of genetic markers on drug immunogenicity from a mechanistic model-based approach.

BACKGROUND: With the growth in use of biotherapic drugs in various medical fields, the occurrence of anti-drug antibodies represents nowadays a serious issue. This immune response against a drug can be due either to pre-existing antibodies or to the novel production of antibodies from B-cell clones by a fraction of the exposed subjects. Identifying genetic markers associated with the immunogenicity of biotherapeutic drugs may provide new opportunities for risk stratification before the introduction of the drug. However, real-world investigations should take into account that the population under study is a mixture of pre-immune, immune-reactive and immune-tolerant subjects. METHOD: In this work, we propose a novel test for assessing the effect of genetic markers on drug immunogenicity taking into account that the population under study is a mixed one. This test statistic is derived from a novel two-part semiparametric improper survival model which relies on immunological mechanistic considerations. RESULTS: Simulation results show the good behavior of the proposed statistic as compared to a two-part logrank test. In a study on drug immunogenicity, our results highlighted findings that would have been discarded when considering classical tests. CONCLUSION: We propose a novel test that can be used for analyzing drug immunogenicity and is easy to implement with standard softwares. This test is also applicable for situations where one wants to test the equality of improper survival distributions of semi-continuous outcomes between two or more independent groups.

Identification and characterization of a naïve CD8+ T cell repertoire for benzylpenicillin.

BACKGROUND: Beta-lactams allergy is the most commonly reported drug allergy and constitutes an important health problem. We previously showed the pre-existence of a naïve CD4+ T cell repertoire for benzylpenicillin (BP) coupled to human serum albumin (HSA) but little is known about the naïve CD8+ T cell repertoire specific for BP. OBJECTIVE: The purpose of this work was to identify naïve CD8+ T cells specific for BP and to explore mechanisms dictating their activation. METHODS: Co-cultures were established with naïve CD8+ T cells and autologous dendritic cells (DCs) loaded with HSA-BP or free BP. T cells were restimulated once a week with autologous DCs loaded with HSA-BP or BP. The specific CD8+ T cell response was measured using an IFN-γ ELISpot assay. RESULTS: When using free BP, we were able to detect a naïve CD8+ T cell repertoire for BP in the 6 out of 7 tested healthy donors. However, our results showed that HSA-BP was recognized by naïve CD8+ T cells in only one donor out of five tested healthy donors. Using free BP, we evidenced its binding to cellular proteins in DCs that was concentration dependent and was correlated with BP-specific CD8+ T cell activation. Moreover, the BP-specific CD8+ cell response was MHC class I-dependent and required intracellular processing and proteasome activity. CONCLUSION AND CLINICAL RELEVANCE: This work showed the existence of a naïve CD8+ T cell repertoire for BP when DCs were treated with free BP suggesting that patients could be immunized by haptenated peptides from cellular proteins generated in antigen-presenting cells.

Prediction of natalizumab anti-drug antibodies persistency.

BACKGROUND: Anti-drug antibodies (ADA) against natalizumab develop early during treatment. ADA persistency is defined by two consecutive positive results as performed by the current qualitative ELISA assay (positive/negative). Very little is known about the magnitude of the natalizumab ADA response and persistency. DESIGN/METHODS: We developed a highly sensitive natalizumab ADA titration assay on the Meso Scale Discovery (MSD) platform and a pharmacokinetic (PK) assay. We included 43 patients with a positive ELISA-ADA result within 6 months of treatment initiation (baseline) of whom a follow-up serum sample was available 12-30 months after treatment start. MSD-ADA titres and drug levels were measured. RESULTS: Median MSD-ADA titre at baseline was 4881 and 303 at follow-up. A titre of >400 at baseline had a 94% sensitivity and 89% specificity to predict ADA persistency. Reversion to ADA negativity occurred in 10 patients with mean drug levels of 10.8 µg/mL. The median trough drug level in ADA-positive samples was 0 µg/mL. PK levels and ADA titres correlated strongly negatively ( r = -0.67). CONCLUSION: High baseline natalizumab ADA titres accurately predict persistency. Despite continuous treatment, the majority of patients with persistent ADA had no detectable drug levels indicating loss of efficacy in line with phase 3 study results.

The THP-1 cell toolbox: a new concept integrating the key events of skin sensitization.

According to the current scientific consensus, one in vitro test is insufficient to cover the key events (KE) defined by the adverse outcome pathway (AOP) for skin sensitization. To address this issue we combined different end points in the same cell line to cover all KEs defined by the skin sensitization AOP. Since dendritic cells (DC) play a key role in the sensitization phase leading to the development of allergic contact dermatitis (ACD), we used THP-1 cells as a surrogate for DC. We measured ROS production and GSH depletion for KE1 (binding to proteins), Nrf2 activation pathway and gene expressions for KE2 (keratinocyte response), phenotype modifications using cell-surface markers and cytokine production for KE3 (DC activation), and T-cell proliferation for KE4 (T-cell activation). These measurements were performed using the THP-1 cell line and an original THP-1/T-cell co-culture system following exposure to a variety of chemicals, including irritant, non-sensitizers, and chemicals sensitizers (pro/prehaptens). Results showed that treatment with sensitizers such as cinnamaldehyde (100 µM) or methylisothiazolinone (150 µM) was able to trigger the three main key events (KE1, KE2, and KE3) of the sensitization phase of ACD in THP-1 cells. In addition, all sensitizers were able to induce T lymphocyte proliferation (KE4), while non-sensitizers and irritants did not. Our study shows for the first time that addressing the four main KE of skin sensitization AOP in a single cell line is an achievable task.

Methotrexate and BAFF interaction prevents immunization against TNF inhibitors.

OBJECTIVES: TNF inhibitors (TNFi) can induce anti-drug antibodies (ADA) in patients with autoimmune diseases (AID) leading to clinical resistance. We explored a new way of using methotrexate (MTX) to decrease this risk of immunisation. METHODS: We treated BAFF transgenic (BAFFtg) mice, a model of AID in which immunisation against biologic drugs is high, with different TNFi. We investigated the effect of a single course of MTX during the first exposure to TNFi. Wild-type (WT) and BAFFtg mice were compared for B-Cell surface markers involved in MTX-related purinergic metabolism, adenosine production and regulatory B-cells (Bregs).We translated the study to macaques and patients with rheumatoid arthritis from the ABIRISK cohort to determine if there was an interaction between serum BAFF levels and MTX that prevented immuniation. RESULTS: In BAFFtg but not in WT mice or macaques, a single course of MTX prevented immunisation against TNFi and maintained drug concentration for over 52 weeks. BAFFtg mice B-cells expressed more CD73 and CD39 compared to WT mice. MTX induced adenosine release from B cells and increased Bregs and precursors. Use of CD73 blocking antibodies reversed MTX-induced tolerance. In patients from the ABIRISK cohort treated with TNFi for chronic inflammatory diseases, high BAFF serum level correlated with absence of ADA to TNFi only in patients cotreated with MTX but not in patients on TNFi monotherapy. CONCLUSION: MTX and BAFF interact in mice where CD73, adenosine and regulatory B cells were identified as key actors in this phenomenon. MTX and BAFF also interact in patients to prevent ADA formation.

IL-27 Production and Regulation in Human Dendritic Cells Treated with the Chemical Sensitizer NiSO4.

Allergic contact dermatitis (ACD) is a major cause of occupational skin disease, and nickel is among the most prevalent contact allergens. Dendritic cells (DC) play an important role in ACD and in the type of the ensuing immune response through differential phenotypes and cytokine production. The interleukin (IL)-12 cytokine family is composed of heterodimeric cytokines sharing homology at the subunit, receptors and signaling levels. We previously showed that nickel can upregulate the production of IL-12p40 and IL-23, both known to be pro-inflammatory. In this work, we aimed to extend our knowledge on nickel regulation of the IL-12 cytokine family by focusing on IL-27, a recently identified immunomodulatory cytokine from this family. We showed that nickel induced the production of IL-27 in human monocyte-derived DC (MoDC), regulating IL-22 production by human CD4+ T cells. We also showed that nickel was able to induce the expression of the two subunits of IL-27: il-27p28 and ebi3. Furthermore, we demonstrated that the production of IL-27 was dependent on the TLR4, p38 MAPK, NF-κB, and Jak-STAT signaling. However, IL-27 subunits were differentially regulated by these pathways. Indeed, both subunits were positively regulated by the TLR4 and NF-κB pathways, but only il-27p28 was also dependent on p38 MAPK and Jak-STAT pathways. Our results contribute to a better understanding of nickel-induced ACD by focusing on the IL-12 cytokine family and elucidating the mechanism of IL-27 regulation in human dendritic cells.

Glucocorticoid-Induced Leucine Zipper Protein Controls Macropinocytosis in Dendritic Cells.

Ag sampling is a key process in dendritic cell (DC) biology. DCs use constitutive macropinocytosis, receptor-mediated endocytosis, and phagocytosis to capture exogenous Ags for presentation to T cells. We investigated the mechanisms that regulate Ag uptake by DCs in the steady-state and after a short-term LPS exposure in vitro and in vivo. We show that the glucocorticoid-induced leucine zipper protein (GILZ), already known to regulate effector versus regulatory T cell activation by DCs, selectively limits macropinocytosis, but not receptor-mediated phagocytosis, in immature and recently activated DCs. In vivo, the GILZ-mediated inhibition of Ag uptake is restricted to the CD8α+ DC subset, which expresses the highest GILZ level among splenic DC subsets. In recently activated DCs, we further establish that GILZ limits p38 MAPK phosphorylation, providing a possible mechanism for GILZ-mediated macropinocytosis control. Finally, our results demonstrate that the modulation of Ag uptake by GILZ does not result in altered Ag presentation to CD4 T cells but impacts the efficiency of cross-presentation to CD8 T cells. Altogether, our results identify GILZ as an endogenous inhibitor of macropinocytosis in DCs, the action of which contributes to the fine-tuning of Ag cross-presentation.

Effect of growth hormone and IgG aggregates on dendritic cells activation and T-cells polarization.

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' death induced by contact sensitizers is controlled by Nrf2 and depends on glutathione levels.

Dendritic cells (DC) are known to play a major role during contact allergy induced by contact sensitizers (CS). Our previous studies showed that Nrf2 was induced in DC and controlled allergic skin inflammation in mice in response to chemicals. In this work, we raised the question of the role of Nrf2 in response to a stress provoked by chemical sensitizers in DC. We used two well-described chemical sensitizers, dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA), known to have different chemical reactivity and mechanism of action. First, we performed a RT-qPCR array showing that CinA was a higher inducer of immune and detoxification genes compared to DNCB. Interestingly, in the absence of Nrf2, gene expression was dramatically affected in response to DNCB but was slightly affected in response to CinA. These observations prompted us to study DC's cell death in response to both chemicals. DNCB and CinA increased apoptotic cells and decreased living cells in the absence of Nrf2. The characterization of DC apoptosis induced by both CS involved the mitochondrial-dependent caspase pathway and was regulated via Nrf2 in response to both chemicals. Oxidative stress induced by DNCB, and leading to cell death, was regulated by Nrf2. Unlike CinA, DNCB treatment provoked a significant reduction of intracellular GSH levels and up-regulated bcl-2 gene expression, under the control of Nrf2. This work underlies that chemical reactivity may control Nrf2-dependent gene expression leading to different cytoprotective mechanisms in DC.

Changes in markers associated with dendritic cells driving the differentiation of either TH2 cells or regulatory T cells correlate with clinical benefit during allergen immunotherapy.

BACKGROUND: Regulatory dendritic cell (DC) markers, such as C1Q, are upregulated in PBMCs of patients with grass pollen allergy exhibiting clinical benefit during allergen immunotherapy (AIT). OBJECTIVES: We sought to define markers differentially expressed in human monocyte-derived DCs differentiated toward a proallergic (DCs driving the differentiation of TH2 cells [DC2s]) phenotype and investigate whether changes in such markers in the blood correlate with AIT efficacy. METHODS: Transcriptomes and proteomes of monocyte-derived DCs polarized toward DCs driving the differentiation of TH1 cells (DC1s), DC2s, or DCs driving the differentiation of regulatory T cells (DCreg cells) profiles were compared by using genome-wide cDNA microarrays and label-free quantitative proteomics, respectively. Markers differentially regulated in DC2s and DCreg cells were assessed by means of quantitative PCR in PBMCs from 80 patients with grass pollen allergy before and after 2 or 4 months of sublingual AIT in parallel with rhinoconjunctivitis symptom scores. RESULTS: We identified 20 and 26 new genes/proteins overexpressed in DC2s and DCreg cells, respectively. At an individual patient level, DC2-associated markers, such as CD141, GATA3, OX40 ligand, and receptor-interacting serine/threonine-protein kinase 4 (RIPK4), were downregulated after a 4-month sublingual AIT course concomitantly with an upregulation of DCreg cell-associated markers, including complement C1q subcomponent subunit A (C1QA), FcγRIIIA, ferritin light chain (FTL), and solute carrier organic anion transporter family member 2B1 (SLCO2B1), in the blood of clinical responders as opposed to nonresponders. Changes in such markers were better correlated with clinical benefit than alterations of allergen-specific CD4(+) T-cell or IgG responses. CONCLUSIONS: A combination of 5 markers predominantly expressed by blood DCs (ie, C1Q and CD141) or shared with lymphoid cells (ie, FcγRIIIA, GATA3, and RIPK4) reflecting changes in the balance of regulatory/proallergic responses in peripheral blood can be used as early as after 2 months to monitor the early onset of AIT efficacy.

TSC-22 Promotes Interleukin-2-Deprivation Induced Apoptosis in T-Lymphocytes.

Originally described as a TGF-ß-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-ß. 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.

Immunogenicity of long-lasting recombinant factor VIII products.

Replacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs.

Bioinspired Design and Oriented Synthesis of Immunogenic Site-Specifically Penicilloylated Peptides.

ß-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4+ T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naïve CD4+ T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naïve CD4+ T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naïve CD4+ T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.