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Autoreactive CD4+ T cells recognizing islet-derived antigens play a primary role in type 1 diabetes. Specific suppression of such cells therefore represents a strategic target for the cure of the disease. We have developed a methodology by which CD4+ T cells acquire apoptosis-inducing properties on antigen-presenting cells after cognate recognition of natural sequence epitopes. We describe here that inclusion of a thiol-disulfide oxidoreductase (thioreductase) motif within the flanking residues of a single MHC class II-restricted GAD65 epitope induces GAD65-specific cytolytic CD4+ T cells (cCD4+ T). The latter, obtained either in vitro or by active immunization, acquire an effector memory phenotype and lyse APCs by a Fas-FasL interaction. Further, cCD4+ T cells eliminate by apoptosis activated bystander CD4+ T cells recognizing alternative epitopes processed by the same APC. Active immunization with a GAD65 class II-restricted thioreductase-containing T cell epitope protects mice from diabetes and abrogates insulitis. Passive transfer of in vitro-elicited cCD4+ T cells establishes that such cells are efficient in suppressing autoimmunity. These findings provide strong evidence for a new vaccination strategy to prevent type 1 diabetes.

Abrogating an unwanted immune response toward a specific antigen without compromising the entire immune system is a hoped-for goal in immunotherapy. Instead of manipulating dendritic cells and suppressive regulatory T cells, depleting effector T cells or blocking their co-stimulatory pathways, we describe a method to specifically inhibit the presentation of an antigen eliciting an unwanted immune reaction. Inclusion of an oxidoreductase motif within the flanking residues of MHC class II epitopes polarizes CD4(+) T cells to cytolytic cells capable of inducing apoptosis in antigen presenting cells (APCs) displaying cognate peptides through MHC class II molecules. This novel function results from an increased synapse formation between both cells. Moreover, these cells eliminate by apoptosis bystander CD4(+) T cells activated at the surface of the APC. We hypothesize that they would thereby block the recruitment of cells of alternative specificity for the same autoantigen or cells specific for another antigen associated with the pathology, providing a system by which response against multiple antigens linked with the same disease can be suppressed. These findings open the way toward a novel form of antigen-specific immunosuppression.

Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2 d) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor mouse cationic amino acid transporter 1 (mCAT1) and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-B (NF-B) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination. © The American Society of Gene & Cell Therapy., SCOPUS: ar.j, info:eu-repo/semantics/published

L-HES is a rare disease characterized by symptoms linked with the persistence and proliferation of a CD4+ T-cell clone producing cytokines that induce polyclonal hypereosinophilia. This disease shares several characteristics with allergic diseases (allergic rhinitis, asthma, etc…), that exhibit hypereosinophilia driven by allergen specific CD4+ Th2 cells. The CD3-CD4+ T-cell clones detected in L-HES patients however are not directed to a specific antigen, thereby excluding the possibility of anti-idiotype or tolerization therapy. The work presented in this thesis addresses the question of what mechanisms are deregulated in the CD3-CD4+ T cells that allow them to persist and expand in L-HES patients, putting them at significant risk for the development of a T-cell lymphoma. The first aim of this study was to characterize the phenotypic and immunological traits of the CD3-CD4+ T cell-clone found in the majority of L-HES patients. Furthering our understanding of the nature of the underlying abnormal T-cell population provides a basis for improved diagnosis and ultimately tailoring treatment for L-HES patients. This objective was achieved by producing a complete profile of the CD3-CD4+ T-cells, including their immunophenotype, cytokine and chemokine secretion pattern, infectious status and T cell receptor rearrangement. The second aim of this work was to identify the genetic alterations linked with the sustained survival and expansion of the CD3-CD4+ T-cell clone. This goal was accomplished at the chromosomal, molecular and cellular levels by assessing the karyotype, the evolving molecular profiles as well as designing functional experiments to characterize mechanisms underlying the persistent expansion of the L-HES T-cell clones. Among the genetic aberrations detected, our ultimate aim was to identify potential suppressor genes and decipher the role they play in the normal apoptotic and proliferative mechanisms responsible for controlling CD4+ T cell growth. Understand, (SBIM 3) -- UCL, 2010

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 or 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-beta, 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. The Journal of Immunology, 2009, 183: 8232-8243.

Thèse de doctorat en sciences biomédicales (immunologie) -- UCL, 1997

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.

We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4 degrees C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30 degrees C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.

Complexes formed by antibodies and antigens have a considerable potential in terms of influencing immune response. Applied to allergy, these complexes have been used to reduce the production of specific IgE antibodies to certain allergens. Thus approximately 400 patients with either asthma or atopic dermatitis have been treated up to now by the injection of such complexes formed using their own antibodies. Such treatment has proved to be free of any adverse reactions and effective in the treatment of both these types of disorder, with a significant reduction in their inflammatory component. Biologically, the injection of these complexes results in a selective reduction in the production of antibodies specific to the allergen concerned, both IgG and IgE, and an increase in the production of corresponding anti-idiotype antibodies. The recently demonstrated efficacy of complexes formed of F (ab')2 antibody fragments has opened up the possibility of treatment in which antibodies are prepared from a pool of donors. The reduction of specific antibodies justifies envisaging the application of the same treatment concept to certain auto-immune diseases, e.g. myasthenia gravis.

A significant proportion of haemophilia A patients receiving transfusions of Factor VIII (FVIII) develop a specific antibody response towards FVIII. These antibodies are usually detected by assays in which they inhibit the function of the molecule, such as the Bethesda clotting test. We have prepared anti-FVIII antibodies by specific immunoadsorption from the plasma of four haemophiliacs with stable inhibitor levels. The isotypic distribution of such antibodies was determined and their capacity to bind to insolubilized FVIII was compared to their inhibitory activity in two functional assays, namely, the Bethesda assay and a chromogenic assay. In addition, the FVIII epitope specificity was determined by competition with monoclonal antibodies for the binding to insolubilized FVIII. We show here that: (i) anti-FVIII antibodies are not isotypically restricted; thus, a significant proportion of specific IgG2 was found; (ii) antibodies are frequently directed towards epitopes of FVIII that are not directly involved in the function of the molecule and therefore escape detection in the Bethesda method or chromogenic assay; (iii) each patient shows an unique pattern of FVIII epitope recognition. We conclude that evaluation of anti-FVIII antibodies by a functional method does to provide a complete evaluation of the antibody response. These findings have important implications for the comparison of the immunogenicity of FVIII molecules produced by different technologies and for the development of methods to control anti-FVIII antibody production, Thèse de doctorat en sciences biomédicales (immunologie) -- UCL, 1993

Delving through the literature on antigen-antibody complexes, one cannot help being astonished by the steadily increasing number of papers written on this subject over the last thirty years. Although this no doubt reflects the fundamental scientific interest if the subject matter, it may also be indicative of its intrinsic complexity. A great variety of antigen-antibody complexes have been studied through the years and they have been found to exert widely disparate – or even contradictory – effects on the immune system. Complexes have been used both to enhance or suppress an immune response, and so it was recognized early on that they could represent a powerful means of influencing the immune system. The interplay of numerous parameters determines the ultimate outcome on the immune system; the antigen to antibody ratio, the isotype of the antibody, the capacity to activate complement and the source of antibody – be it isologous or heterolous – are but a few of the factors capable of influencing profoundly the handling of complexes by the host. Several reviews have appeared in this subject, but there are so many variables involved that they tend other to focus on narrow aspects of the subjects (e.g., introduction of rheumatoid factor) or to remain at a general theoretical level. None of these reviews deal, event in a pragmatic fashion, with the practical application of antigen-antibody complexes, i.e., the potential of using complexes for therapeutic purposes. I venture to hope that this thesis may go some way to fill this void. During the last few years we have studied the use of antigen-antibody complexes for the treatment of human diseases that are characterized by an “inappropriate” production of antibodies. In this context, I mean by inappropriate the production of antibodies that is deemed to have a direct detrimental effect on the organism that produces them. For reasons which will be gone into a fully later, the first system my coworkers and I decided to, Thèse d'agrégation de l'enseignement supérieur (faculté de médecine) -- UCL, 1992