Jean-Marie Saint-Remy

CD4+CD25+ T Cells Lyse Antigen-Presenting B Cells by Fas-Fas Ligand Interaction in an Epitope-Specific Manner

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4+CD25+ population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4+ cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4+ T cell line derived from BALB/c mice immunized with peptide 21–35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4+CD25+ T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.

A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes

Several therapeutic self-proteins elicit immune responses when administered to patients. Such adverse immune responses reduce drug efficacy. To induce an immune response, a protein must interact with different immune cells, including antigen-presenting cells, T cells, and B cells. Each cell type recognizes distinct immunogenic patterns on antigens. Mannose-terminating glycans have been identified as pathogen-associated molecular patterns that are essential for internalization of microbes by antigen-presenting cells, leading to presentation. Here, we have investigated the importance of exposed mannosylation on an immunogenic therapeutic self-protein, procoagulant human factor VIII (FVIII). Administration of therapeutic FVIII to hemophilia A patients induces inhibitory anti-FVIII antibodies in up to 30% of the cases. We demonstrate that entry of FVIII into human dendritic cells (DC) leading to T cell activation, is mediated by mannose-terminating glycans on FVIII. Further, we identified macrophage mannose receptor (CD206) as a candidate endocytic receptor for FVIII on DC. Saturation of mannose receptors on DC with mannan, and enzymatic removal of mannosylated glycans from FVIII lead to reduced T cell activation. The interaction between FVIII and CD206 was blocked by VWF, suggesting that, under physiological conditions, the intrinsic mannose-dependent immunogenicity of FVIII is quenched by endogenous immunochaperones. These data provide a link between the mannosylation of therapeutic self-proteins and their iatrogenic immunogenicity. Such a link would be of special relevance in the context of replacement therapy where mechanisms of central and peripheral tolerance have not been established during ontogeny because of the absence of the antigen.

Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies.

Anti-Factor VIII (FVIII) antibodies were prepared by a combination of salt precipitation, gel filtration chromatography, and specific adsorption over insolubilized FVIII from the serum of 10 healthy subjects with normal levels of FVIII. Antibody specificity was confirmed by the capacity to recognize soluble and insolubilized FVIII and to neutralize FVIII cofactor activity in FX activation. Epitope mapping was carried out using a competition ELISA in which affinity-purified human antibodies inhibited the binding of labeled monoclonal antibodies. In most cases, a single region of the A3 domain of the FVIII light chain was recognized by the antibodies, while the reactivity toward heavy chain epitopes differed from one antibody preparation to the other. Sera or IgG fractions of the serum before immunoadsorption over insolubilized FVIII did not bind to FVIII. The IgG fraction that was not retained on the FVIII immunosorbent contained IgG that bound to the variable part of anti-FVIII mouse monoclonal antibodies and inhibited the binding of labeled FVIII; in addition, the IgG fraction inhibited the binding of affinity-purified human antibodies to FVIII, thereby strongly suggesting the presence of anti-idiotypic antibodies. These findings indicate that the presence of anti-FVIII antibodies is a more universal phenomenon than previously thought and that anti-idiotypic antibodies capable of inhibiting the binding of anti-FVIII antibodies to FVIII are produced spontaneously.

Allergic Bronchial-asthma Due To Dermatophagoides-pteronyssinus Hypersensitivity Can Be Efficiently Treated By Inoculation of Allergen-antibody Complexes

Antigen-antibody complexes were made from allergens of the common house dust mite, Dermatophagoides pteronyssinus (Dpt) and an excess of purified autologous specific antibodies. These complexes have been used to treat Dpt-hypersensitive patients who suffered from chronic bronchial asthma. Clinical symptoms and medication intake were followed by filling in diary cards. Peak expiratory flow, measured four times a day, was also followed. Intradermal skin tests and bronchial challenge tests were performed with allergen together with an evaluation of nonspecific bronchial reactivity. Specific IgE and IgG antibodies were assayed after separation from the bulk of serum immunoglobulins by immunoadsorption. The study was carried out over two years according to a double-blind protocol. Intradermal inoculation of antigen-antibody complexes resulted in a marked reduction of both clinical and medication scores. No systemic side-effects were observed and only mild wheal and flare reactions were noted at the injection site. The treatment showed a drastic reduction of specific skin and bronchial reactivities with only marginal effects on nonspecific bronchial reactivity. Concentrations of specific IgE antibodies decreased significantly during the first weeks of treatment and remained at these lower values throughout the study. Specific IgG antibodies actually decreased in the majority of treated patients. The total amount of allergen used in this study was less than 1% of the amount currently used for conventional hyposensitization with the same allergen. These findings show that antigen-antibody complex inoculation is an efficient and safe means of treating allergic bronchial asthma and that the mechanism of action is likely to differ from conventional hyposensitization.

Inhibitors in haemophilia: pathophysiology.

Summary.  Development of inhibitors to coagulation factors is one of the major problems faced by people with haemophilia. Up to a third of patients, following treatment with factor concentrates, will develop an antibody (inhibitor) to that factor, rendering it inactive, and leaving the patient at risk from life‐threatening bleeding. Evidence shows that this immune response is T‐cell‐dependent, but as yet, the epitopes responsible have not been identified. Risk for inhibitor development is highest within the first 50 days of treatment, with reactions being rare after 200 days. The risk is mediated by the major histocompatibility complex class of the patient, and by mutations in the factor VIII genotype, with large deletions conferring greatest risk.

Activation of human endothelial cells from specific vascular beds induces the release of a FVIII storage pool

Although the liver is known to be the main site of factor VIII (FVIII) production, other organs are probably also important for the regulation of FVIII secretion. However, the study of the regulation of extrahepatic FVIII production has been hampered by the lack of definitive identification of human tissues able to secrete FVIII. Recent studies have shown that lung endothelial cells can synthesize FVIII. We therefore studied the production of FVIII by endothelial cells purified from other vascular beds. Because physiologic stress results in a rapid elevation of FVIII, we also investigated whether endothelial cells can store FVIII and secrete it after treatment with agonists. Microvascular endothelial cells from lung, heart, intestine, and skin as well as endothelial cells from pulmonary artery constitutively secreted FVIII and released it after treatment with phorbol-myristate acetate and epinephrine. By contrast, endothelial cells from the aorta, umbilical artery and umbilical vein did not constitutively secrete FVIII or release it after treatment with agonists, probably because of a lack of FVIII synthesis. Extrahepatic endothelial cells from certain vascular beds therefore appear to be an important FVIII production and storage site with the potential to regulate FVIII secretion in chronic and acute conditions.

The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high‐titre inhibitors: association of clinical outcome with inhibitor epitope profile

Summary.  A retrospective chart review of 11 subjects with severe haemophilia A and high‐titre inhibitors who received a von Willebrand factor‐containing FVIII concentrate (VWF/FVIII) for immune tolerance induction (ITI) was accompanied by B cell inhibitor epitope mapping during 10/11 treatment courses. ITI was successful or partially successful in all seven subjects who received VWF/FVIII for initial ITI, and failed in all four subjects whose ITI with this product was initiated following treatment failure using recombinant factor VIII. Variables including age at inhibitor development and age at ITI initiation, interval between inhibitor detection and ITI initiation, titre at start of ITI, and peak historical titres prior to and during ITI were not statistically significant outcome predictors in this cohort. However, the B cell epitope specificity in all four successful and in one of two partially successful ITI subjects for whom information was available included the C2 and excluded the A2 domains. Conversely, FVIII B cell epitopes in one partially successful ITI and in all three failed ITI subjects for whom data were available mapped to both the C2 and the A2 domains. The FVIII B cell epitope profile was associated with ITI outcome in this VWF/FVIII‐treated cohort. Its role in predicting ITI outcome and guiding choice of FVIII product for ITI requires further study.

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody.

Summary.  Background: N‐glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood. Objectives: We investigated the function of N‐glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb‐LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation. Methods and results: Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N‐glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N‐glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies. Conclusions: Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.

Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation

Alloimmunization against exogenous factor VIII represents the major hurdle of hemophilia A treatment. This study shows that CD91 and other members of the LDL receptor family are not involved in factor VIII internalization by monocyte-derived dendritic cells. Background The development of factor VIII (FVIII) inhibitors remains the major hurdle in the clinical management of patients with hemophilia A. FVIII uptake by professional antigen-presenting cells (APC) is the first step involved in initiation of immune responses to FVIII. Studies on FVIII catabolism have highlighted the role played by CD91/LRP as a potential target for increasing FVIII half-life in patients and prolonging treatment efficiency. We investigated the involvement of CD91 in FVIII endocytosis by human dendritic cells (DC), a model of professional APC. Design and Methods Immature DC were generated from circulating monocytes from healthy donors. Surface expression of CD91 was assessed by flow cytometry. Uptake of fluoroscein isothiocyanate-conjugated ligands by immature DC was studied in the presence of various blocking agents. Results CD91 was expressed on approximately 20% of DC and mediated the internalization of its model ligand, α2-macroglobulin. DC internalized FVIII and activated a human FVIII-specific T-cell clone in a dose-dependent manner. FVIII uptake by DC and subsequent T-cell activation were not inhibited by receptor-associated protein. Conclusions Our results indicate that CD91 and other members of the LDL receptor family are not strongly implicated in FVIII internalization by monocyte-derived DC, and suggest the involvement of alternative divalent ion-dependent endocytic receptors.

In Vivo Induction of Type 1-Like Regulatory T Cells Using Genetically Modified B Cells Confers Long-Term IL-10-Dependent Antigen-Specific Unresponsiveness

Regulatory T cells (Tregs) hold much promise for the therapy of allergy and autoimmunity, but their use is hampered by lack of Ag specificity (natural Tregs) and difficulty to expand in vitro or in vivo (adaptive Tregs). We designed a method for in vivo induction of Ag-specific Tregs, in BALB/c H-2d, that share characteristics with type 1 Tregs (Tr1). A retroviral vector was constructed encoding a major T cell epitope of a common allergen, Der p 2, fused to an endosomal targeting sequence (gp75) for efficient MHC class II presentation. B cells transduced with such construct were adoptively transferred to BALB/c mice before or after peptide immunization. Long-lasting Ag-specific immune tolerance was achieved in both cases. Genetically modified B cells constitutively expressed the transgene for at least 3 mo. B cells from IL-10−/− mice were unable to induce tolerance. Upon transfer, B cells induced Foxp3−CD4+ T cells showing phenotypic and functional characteristics comparable to Tr1-cells, including production of IL-10 but not of TGF-β, and high expression of CTLA-4. Adoptive transfer of such T cells conferred unresponsiveness to allergen immunization and prevented the development of Der p 2-induced asthma. Functional Tr1-like cells can therefore be induced in vivo using retrovirally transduced B cells.