Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

Terminally differentiated B cells are responsible for antibody generation, a key component of adaptive immunity. IgG antibodies play an important role in defence against infection but can be pathogenic in some autoimmune diseases and in solid organ transplantation. In addition to antibody generation, there is increasing interest in the antibody-independent functions of B cells, including their ability to regulate immune responses via the production of IL10. In this thesis I firstly explored the therapeutic potential of belimumab, an anti-BLyS antibody, in an experimental medicine study in kidney transplant recipients. The rationale for this study was based on published studies showing that B cells activate alloreactive T cells and secrete human leukocyte antigen (HLA) and non-HLA antibodies that negatively affect graft function and survival, but may also play a protective role by regulating alloimmune responses promoting transplant tolerance. B-Lymphocyte Stimulator (BLyS) is a cytokine that promotes B cell activation and survival. We performed the first randomized controlled trial using belimumab as early maintenance immunosuppression in kidney transplantation. In belimumab-treated subjects, we demonstrate a reduction in naïve and activated memory B cells, plasmablasts, IgG transcripts in peripheral blood and new antibody formation as well as evidence of reduced CD4 T cell activation and of a skewing of the residual B cell compartment towards an IL10-producing regulatory phenotype. This experimental medicine study highlights the potential of belimumab as a novel therapeutic agent in transplantation. In the second part of my project I performed a preclinical study investigating the potential efficacy of bromodomain inhibitors in reducing antibody-mediated immune cell activation. Immune complexed antigen can activate mononuclear phagocytes (MNP), comprising macrophages and dendritic cells (DCs), via ligation of Fc gamma receptors (FcγR), that bind the Fc region of IgG. FcγR-dependent MNP activation results in profound changes in gene expression that mediate antibody effector function in these cells. The resulting inflammatory response can be pathological in the setting of autoimmune diseases, such as systemic lupus erythematosus and in antibody-mediated rejection in transplantation. BET proteins are a family of histone modification 'readers' that bind acetylated lysine residues within histones and function as a scaffold for the assembly of complexes that regulate gene transcription. Bromodomain inhibitors (I-BET) selectively inhibit the transcription of a subset of inflammatory genes in macrophages following toll-like receptor stimulation. Since MNPs make a key contribution to antibody-mediated pathology, we sought to determine the extent to which I-BET inhibits macrophage and DC activation by IgG. We show that I-BET delays phagolysosome maturation associated with build-up of immune complex (IC) whilst selectively inhibiting IC induced cytokine production. I-BET changed MNP morphology, resulting in a less adherent phenotype, prompting an assessment of its impact on DC migration. In vitro, in a three-dimensional collagen matrix, IgG-IC induced augmentation of DC chemotaxis to chemokine (C-C motif) ligand 19 (CCL19) was abrogated by the addition of I-BET. In vivo, two photon imaging showed that systemic I-BET treatment reduced IC-induced dermal DC mobilisation. Tissue DCs and transferred DC also had reduced migration to draining lymph nodes following I-BET treatment. These observations provide mechanistic insight into the potential therapeutic benefit of I-BET in the setting of antibody-associated inflammation.

Human platelet antigens (HPA) is located on the platelet surface and they are inherited both from the mother and the father. If a mother who is homozygous for HPA-1b carries a child who has inherited HPA-1a from the father, the mother is in danger to form antibodies against HPA-1a on the fetal platelet. This may cause the child to suffer from neonatal alloimmune thrombocytopenia (NAIT) that could lead to death. This can be prevented by platelet transfusion. EVA Biosensor Technology is a new method for detection of HPA-1 that is currently only approved for scientific research. The aim of this study was to evaluate EVAreader R6 and find HPA-1a negative platelet donors that can donate platelets to children born with NAIT. The test material consisted of blood samples from 513 male blood donors with blood group 0. The blood was lysed and tested in EVA-reader R6 from Davos Diagnostics. The result was shown on the screen after 10 min. The results that came out negative or intermediate was analyzed a second time. In total, nine HPA-1a negative donors and 503 HPA-1a positive donors were found. Approximately 2 % of the population is HPA-1a negative, which was reflected in the result. To make sure that the results are correct, a validation with an already existing method has to be made. The conclusion is that the EVA Biosensor Technology could be used for typing of HPA in the future, as long as the results from the validation is correct.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody–antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to “shared” antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intralocus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new crossreactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second featurebased analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific crossreactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

L’hépatite allo-immune post-greffe de moelle osseuse (GMO) est très mal définie. Le premier objectif de notre thèse était d’identifier, par analyse sérologique du protéome, les antigènes (Ag) cibles d’auto-anticorps (Ac) présents dans le sérum de patients. Cinq patients, ayant reçu une GMO, ont développé des troubles hépatiques à l’arrêt du traitement immunosuppresseur, avec des signes histologiques ressemblant à ceux des hépatites auto-immunes (HAI). Avant et pendant l’épisode hépatique, des serums ont été testés sur des immunoblots réalisés avec des protéines nucléaires, cytosoliques, microsomiques et mitochondriales obtenues à partir d’homogénat de foie de rat, séparées par électrophorès bi-dimensionnelle et transférées sur membrane de nitro-cellulose. Après digestion trypsique, les protéines d’intérêt, marquées uniquement ou plus intensément par les sérums en phase d’hépatite, étaient identifiées par spectrométrie de masse MALDI-TOF/TOF et nanoHPLC LTQ-Orbitrap®. Un nombre total de 103 protéines antigéniques a été identifié, parmi lesquelles 12 sont reconnues par les sérums de 3 patients. A l’issue de cette première description immunologique des hépatites allo-imunes post-GMO, nous formulons des hypothèses physiopathologiques permettant d’expliquer l’évolution d’un mécanisme allo-immun vers une réaction auto-immune. En outre, nous recommandons la réduction très progressive des doses d’immunosuppresseur après GMO.La protéine hnRNP A2/B1 est une cible des anticorps anti-nucléaires (AAN) dans le lupus érythémateux disséminé (LED), la polyarthrite rhumatoïde (PR) et l’HAI. Dans la deuxième partie de la thèse, le but était de caractériser les interactions Ag-Ac en fonction de la pathologie, en utilisant la résonance plasmonique de surface par imagerie (SPRi). Trente-neuf peptides chevauchants de 17 acides aminés, couvrant l’ensemble de l’isoforme B1 de la protéine humaine, ont été immobilisés à la surface d’un prisme. Les interactions entre les peptides immobilisés et les sérums de 8 patients de chaque pathologie et de donneurs sains (D) ont été étudiées en temps réel. Les constantes de vitesse de dissociation koff, calculées pendant la phase de dissociation des complexes Ag-Ac formés, reflètent la stabilité de ces complexes.Plusieurs interactions significatives ont été observées : i) avec une stabilité élevée entre P55-70 et les sérums d’HAI par rapport aux contrôles (p= 0.003); ii) avec une faible stabilité entre P118-133 et P262-277 et les serums de LED, P145-160 et les sérums de PR par rapport aux serums D (p=0, 006; p=0,002; p=0,007). Le peptide P55-70 est donc un biomarqueur potentiel dans l’HAI. Les courbes d’interaction et les koff observés après la formation de complexes avec des anticorps anti-IgG et anti-IgM d’une part, et le traitement des sérums par des nucléases d’autre part, montrent que : i) les IgM sont majoritaires et ii) des acides nucléiques, présents ou non selon la maladie auto-immune, participent aux interactions entre les anti-hnRNP B1 et le peptide AA55-70 ainsi qu’entre les autres peptides et les sérums témoins, et contribuent à la stabilité des complexes Ag-Ac. Les résultats de nos travaux et les innovations technologiques en spectrométrie de masse et en SPR permettent d’envisager la mise au point de nouveaux tests pour le suivi des patients atteints d’hépatites auto et allo-immunes. / Alloimmune hepatitis following bone marrow transplantation (BMT) is poorly characterized. The first goal of this thesis was to identify antigens (Ag) targets of autoantibodies (auto-Ab) in sera from patients, using serological proteome analysis. Five patients who received an allogeneic BMT developed liver dysfunctions with histological features suggestive of autoimmune hepatitis (AIH) after the withdrawal of immunosuppressive therapy. Before and during the onset of hepatic dysfunction, sera were tested on immunoblots performed with cytosolic, microsomal, mitochondrial and nuclear proteins from rat liver homogenate, resolved by two-dimensional electrophoresis and transferred onto nitrocellulose membranes. After tryptic digestion, antigenic targets were identified by two tandem mass spectrometry techniques: MALDI-TOF/TOF and nanoHPLC LTQ Orbitrap®. A total of 103 different proteins were identified. Twelve of them were recognized by sera from three patients. This is the first immunological description of hepatitis occurring after BMT, enabling a discussion of the mechanisms that transform an alloimmune reaction into an autoimmune response. Any decision to withdraw immunosuppression after allogeneic BMT should be made with caution and hepatic parameters monitored systematically.Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is a target for antinuclear autoantibodies in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and autoimmune hepatitis (AIH). In the second part of the thesis, the goal was to characterize Ag-Ab interactions as a function of pathology, using surface plasmon resonance imagery (SPRi). Sera from 8 patients from each pathology and healthy donors (D) were passed across a SPRi surface containing 39 overlapping peptides of 17 mers covering the human hnRNP B1. Interactions involving the immobilised peptides were followed in real time and dissociation rate constants koff for each interaction were calculated. koff values reflect the stability of the complex. Several significant interactions were observed: i) high stability (lower koff values) between P55-70 and the AIH sera compared to controls (p= 0.003); ii) lower stability (higher koff values) between P118-133 and P262-277 and SLE sera, P145-160 and RA sera compared to controls (p=0.006, p=0.002, p=0.007). These results indicate that P55-70 of hnRNP B1 is a potential biomarker for AIH in immunological tests.The binding curves and koff values observed after the formation of complexes with anti-IgM and anti-IgG antibodies and after nuclease treatment of the serum indicate that i) IgM isotypes are prevalent and ii) circulating nucleic acids, present or absent according to the autoimmune disorders, participate in the interaction between anti-hnRNP B1 and P55-70 and also between controls and the peptides studied and are involved in antigen-antibody stability. Results from our work as well as promising innovations in mass spectrometry and SPR technologies lead us to consider the development of new tests, usable in monitoring patients with auto and alloimmune liver diseases.