alphabeta T cell receptors as predictors of health and disease

The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR \u27signatures\u27 raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome

NGS based studies on primary immunodeficiencies (PIDs) : causative gene identification, tool development and application

Primary immunodeficiency diseases (PIDs) are composed by a group of highly heterogeneous immune system diseases, of which approximately 350 forms of PID have been described so far. The causative gene of around 60% of patients with PIDs has yet unknown. In recent years, Next Generation Sequencing (NGS) has been increasingly adopted for gene identification and molecular diagnosis of rare diseases, including PIDs. An overview of the genetic makeup that underlies PID using NGS has been suggested as a promising approach to elucidate the etiology of PIDs, which could yield diagnostic and, possibly, provide new treatment advances for PID. To approach this goal, we performed either whole exome sequencing (WES, 454 samples) or targeted region sequencing (TRS, 217 samples) on 602 samples of 500 PID pedigrees. We have summarized the practical suggestions for the interpretation of NGS data and the techniques that can be used to search disease-causative PID genes in Paper I. This work aims to improve data annotation, interpretation, and application of NGS data in PIDs, which also facilitates a wide range of application of NGS data analysis in other Mendelian disorders. The genetic approach together with immunological investigations have identified potential pathogenic variants in 86 primary antibody deficiency (PAD) patients (68.2%), and a correct diagnosis can guide/change treatment plan in around half of the patients with PAD (Paper II). We identified potentially disease-causing variants (including variants classified as VUS (variants of unknown clinical significance)) in around 34% of genetically unidentified PID samples, which had been subjected to TRS using a panel of 219 common PID genes. Notably, the genetic diagnosis of a specific atypical ITK deficiency case adds to the growing amount of evidence supporting the importance of genetic investigations initiated at an early stage of the patient´s disease (Paper III). Altogether, around 60% of PID patients have a possible diagnosis via WES/TRS. Copy number variation defects were identified in 16 patients (4 genes were involved, LRBA, ATM, DOCK8 and PMS2). Beyond the identification of the monogenic causal gene based on pedigree analysis, mutation frequency analysis has been used to identify genes with rare functional variants in the higher proportion of patients in specific patient group compared to control samples, which have discovered several potential novel PID genes (TNFRSF18, PIK3CG, LILRB1, EPHB2, TXNIP, CD5 and NLRP5). Other possible models beyond the monogenic scenario were also explored, and 16 severe combined immunodeficiency (SCID) or common variable immunodeficiency (CVID) patients might be due to an accumulation of rare amino acid substitution variants in genes related to the same function or pathway (RAG1 & RAG2, RAG1 & ATM, C3 & ITGB2, PRKDC & ATM, C5 & NIPBL, LRBA & CR2, CR2 & NFKB1, UNC93B1 & NIPBL, PLCG2 & NOD2 and IGLL1 & ATM). These findings indicate that NGS, together with a large sample size, is powerful in decoding the genetic characteristics of PID and provide insight into molecular mechanisms that cause the disease. Existing variants impact prediction software/algorithms still have a challenge to evaluate the pathological consequences of the prioritized variants or genes. We thus developed a Random Forest-based discriminator, Variant Impact Predictor for PIDs (VIPPID), to refine the prediction algorithms, which utilized the features of pathogenic variants and benign mutations, integrated with other 24 predictive softwares currently used. Evaluation of VIPPID showed that it had superior performance (AUC=0.95) over existing tools, we also showed the gene-specific model outperformed the non-gene-specific model and provided a possibility to explore the underlying molecular mechanism based on our gene-specific model in Paper IV. Specific mutations of PID causative genes may exert different effects on TCR repertoire diversity and composition, which ultimately lead to heterogeneous phenotypes. DNA damage response/methylation is an essential process during antigen receptor recombination. To investigate the effect of mutations in DNA repair genes on adaptive immunity, 19 patients with DNA repair/methylation defects were selected and subdivided into several groups based on their causative genes, we then performed deep immune repertoire sequencing and comparison with 14 age-matched healthy controls. Patients with different molecular diagnosis exhibited distinct repertoire diversity, clonality and V-J pairing patterns. Aberrant complementarity-determining region 3 (CDR3) length distribution was observed both in unproductive and productive TCRs in all patients, suggesting that it predominantly arose before thymic selection. Shorter CDR3 lengths in AT patients resulted from a decreased number of insertions, led to an increase in the number of shared clonotypes, whereas patients with DNMT3B and ZBTB24 mutations presented longer CDR3 lengths and reduced specificity for pathogen-associated CDR3 sequences (Paper V). This study revealed the role of DNA repair/methylation machinery in patients with ATM, DNMT3B and ZBTB24 deficiency, and shed light on the mechanistic etiology of their T cell dysfunction

Studies of different clinical manifestations of Sarcoidosis and the role of genetic factors

Sarcoidosis is a systemic disease of unknown etiology characterized by the formation of non-necrotizing granulomas in the affected organs. Engagement of the lungs and/or thoracic lymph nodes (LN) are found in more than 90 % of all cases, but almost any organ such as the eyes, skin, heart and nervous system can be involved. Genetic factors influence the risk for disease as well as the clinical picture seen in sarcoidosis and especially the genes localized to the human leukocyte antigen (HLA) region on chromosome six are believed to be of importance. For example, the HLA-DRB1*0301 allele is found to be strongly associated with Löfgren’s syndrome (LS). Characteristic for LS is an acute onset usually with fever, bilateral ankle arthritis and/or erythema nodosum and bilateral hilar lymphadenopathy with in some cases parenchymal infiltrates. The HLA-DRB1*0301 allele is also associated with an accumulation of T cells expressing the T cell receptor variable gene segment AV2S3 in bronchoalveolar lavage fluid (BALF) of sarcoidosis patients. The aim of this thesis has been to identify risk factors for different clinical manifestations in sarcoidosis as well as markers of importance for the inflammatory cell response seen in sarcoidosis. The results show that HLA-DRB1*04 positive sarcoidosis patients had an increased risk for the three organ engagements associated with Heerfordt´s syndrome. Heerfordt´s syndrome is a phenotype of sarcoidosis that in its complete form consists of uveitis, parotid and/or salivary gland enlargement and cranial nerve palsy. In comparison to BALF where a high CD4/CD8-ratio is strongly associated with sarcoidosis, the CD4/CD8-ratio in the affected LNs of sarcoidosis patients had no diagnostic value. Further, in HLA-DRB1*03 positive patients the associated accumulation of AV2S3+ T cells was strictly compartmentalized in BALF. This finding indicates an airborne antigen as the triggering factor in sarcoidosis. The risk for cardiac sarcoidosis (CS) was significantly higher in patients with an abnormal electrocardiography (ECG) compared to those with a normal ECG. The risk for CS was highest in patients who had a pathologic ECG in combination with cardiac related symptoms. Further, non-LS was associated with an increased risk for CS. In LS patients was the absence of HLA-DRB1*03 a risk factor for extra-pulmonary manifestations (erythema nodosum and ankle arthritis excluded). Another risk marker in all patients was HLA-DRB1*04/*15 where half of the patients had extra-pulmonary manifestations. In conclusion, the HLA-DRB1*04 allele is associated with an increased risk for involvement of the eyes, parotid and/or salivary glands and cranial nerves in sarcoidosis patients. Moreover, an increased CD4/CD8-ratio in enlarged LNs is not diagnostic for sarcoidosis in comparison to BALF where a high ratio is strongly associated with sarcoidosis. Further, a pathologic ECG is a risk marker for CS in sarcoidosis patients. Finally, not only the single HLA-DRB1 alleles are of importance for the risk of extra-pulmonary manifestations in sarcoidosis, but also the allele combinations and where especially the combination HLA-DRB1*04/*15 calls for an increased awareness and a more intensive follow-up

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Detection of minimal residual disease in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in childhood. Last decades brought enormous progress in ALL treatment and in the understanding of ALL biology (see Chapter 1.1 ), but still 20 to 30% of children suffer from relapse and many of them will ultimately die of disease progression. The currently used cytomorphological (microscopic) techniques can only detect 1 to 5% of malignant cells, which is not sufficiently sensitive for identification of patients who are prone to relapse and who might be rescued by treatment intensification. During the past 15 years several approaches have been developed for detection of much lower numbers of malignant cells, i.e. for detection of minimal residual disease (MRD) in various hematopoietic malignancies (see Chapter 1.2). Monitoring of MRD with sensitivities of 1 Q-4 to 1 o-6 (i.e. one malignant cell within the background of 104 to 106 normal cells) has significantly higher prognostic value than conventional cytomorphological techniques and other clinical parameters at diagnosis and is therefore currently implemented into clinical practice in several hematopoietic malignancies, including ALL. In childhood ALL, detection of MRD most frequently relies on patient-specific immunoglobulin (lg) and T-cell receptor (TCR) gene rearrangements as molecular markers for PCR studies. The junctional regions of rearranged lg and TCR genes are unique "fingerprint-like" sequences, which are assumed to be different in each lymphoid cell and thus also in each lymphoid malignancy. They can be easily identified and characterized for instance by using heteroduplex PCR analysis (see Chapter 2.2) and direct sequencing. This thesis aimed at detailed evaluation of lg and TCR gene rearrangements in ALL with regard to the following aspects: -characterization of lg/TCR gene rearrangements patterns in precursor-BALL and T-ALL; - immunobiological differences between malignant and normal lymphoid cells; -stability of clonal lg/TCR gene rearrangements at relapse of ALL; -applicability of lg/TCR gene rearrangements as PCR targets for detection of MRD. Virtually all precursor-B-ALL (96%) have rearranged lg heavy chain (/GH) genes. In most cases (80-90%) this concerns complete VH-DH-JH rearrangements on at least one allele. Incomplete DH-JH rearrangements could be identified in 22% of patients, being the sole /GH gene rearrangements in only 5% of patients (see Chapter 2.3). Most precursor-B-ALL contain lg kappa (/GK) light chain gene rearrangements (30%) or deletions (50%); 20% of precursor-B-ALL cases even have lg lambda (IGL) gene rearrangements. Deletions in the IGK genes are predominantly mediated via the IGK deleting element (Kde) sequence. Such Kde rearrangements occur in 50% of precursor-B-ALL case

Extensive reshaping of the T cell repertoire following autologous haematopoietic stem cell transplantation in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory condition of the central nervous system (CNS) that affects over 25 000 Australians. It is established that a significant proportion of the disability acquired in patients with MS results from inflammation mediated by an antigen specific immune response. Genetic studies have consistently confirmed that the MHC class II allele HLA-DRB1 15:01 is the most important risk factor for the development of MS and T lymphocytes are believed to be a major pathogenic mediator of disease. Despite the proposition of numerous potential targets, the antigenic driver(s) of peripherally activated, clonally expanded lymphocytes in MS remains unknown. Advances over the last 3 decades in immunotherapy for MS have demonstrated that early suppression of CNS inflammation alters the trajectory of disability in patients with aggressive disease. Immunomodulation and/or suppression have been shown to decrease relapses and retard disability progression, yet a proportion of patients will fail to adequately respond to currently available pharmacotherapy. Furthermore, the risks associated with long term immunosuppression have triggered increasing interest in immune reconstitution therapies for MS. Autologous haematopoietic stem cell transplant (AHSCT), a prototypical immune reconstitution therapy, has been demonstrated to induce sustained disease remission in patients with highly active/aggressive disease. AHSCT has been performed in a clinical trial setting in Australia since 2010. Here, the results of an ongoing phase 2 clinical trial of myeloablative AHSCT in MS are presented, confirming that durable disease remission can be achieved in the vast majority of relapsing-MS patients. Relapses and MRI activity occur at a low frequency in a small proportion of individuals following AHSCT, whilst many patients remain free from recurrent inflammation, disability progression and CNS atrophy for periods of follow-up that extend beyond six years. Corresponding immune reconstitution studies presented herein demonstrate that dynamic changes in the clonal T cell repertoire occur not only with myeloablation, but in the months and years post-AHSCT. This thesis outlines that clones surviving or regenerated following chemotherapy undergo (i) lymphopenia induced homeostatic proliferation and (ii) clonal expansion, attrition and subsequent proliferative exhaustion, and explores in detail the factors that govern clonal growth and decline. Furthermore, the regeneration of a diverse, thymic derived T cell repertoire is fostered by the creation of clonal space. Finally, with the application of high-throughput T cell receptor (TCR) repertoire sequencing and innovative bioinformatics pipelines, the public lymphocyte response to AHSCT is characterised. In a cohort of MS patients expressing HLA DRB1*15:01 public clones are depleted following AHSCT. Notably, public clones detected pre- AHSCT, with the potential for autoreactivity are not detected/regenerated post-AHSCT. This synopsis of the dynamic changes in T cell immunobiology following AHSCT in MS provides potential insights into disease pathogenesis and identifies T cell clones which may serve as diagnostic biomarkers and future treatment targets in MS

L'immunité innée dans le diabète sucré

Le diabète de type 1 (T1D) est une maladie auto-immune caractérisée par la destruction des cellules b du pancréas par les lymphocytes T auto-réactifs. Durant ma thèse, nous nous sommes intéressés au rôle des cellules de l immunité innée dans le T1D à l aide d un modèle murin de la maladie : la souris NOD. Au contraire des cellules du système adaptatif (lymphocytes T et B), les cellules de l immunité innée constituent la première ligne de défense de l organisme lors d une infection. Cette population est constituée entre autre de neutrophiles, cellules dendritiques plasmacytoïdes (pDC), macrophages, mais aussi de lymphocytes T et B non conventionnels tel que les cellules iNKT et B-1a. Précédemment, notre laboratoire a mis en lumière le rôle des lymphocytes iNKT dans le développement du T1D. Durant la première partie de ma thèse, nous avons démontré que les lymphocytes iNKT17, une sous-population des lymphocytes iNKT, ont un rôle délétère dans le T1D chez la souris NOD. Ces cellules infiltrent le pancréas et y produisent de l IL-17, une cytokine pro-inflammatoire. Grâce à des expériences de transferts, nous avons mis en évidence que les lymphocytes iNKT17 exacerbent la maladie via la production d IL-17. Dans la deuxième partie de ma thèse, nous nous sommes intéressés aux mécanismes qui induisent l activation des lymphocytes T auto-réactifs. Nous avons observé chez la souris NOD, que la mort physiologique des cellules b conduit à l activation de cellules de l immunité innée : les neutrophiles, les lymphocytes B-1a et les pDC. La coopération entre ces cellules conduit à l activation des pDC qui produisent de l IFNa. Cette cytokine active les lymphocytes T auto-réactifs qui vont détruire les cellules b du pancréas. Nos résultats montrent que l immunité innée est un acteur important dans la physiopathologie du diabète sucré.The type 1 diabetes ( T1D ) is an autoimmune disease characterized by the destruction of b cells in the pancreas by autoreactive T lymphocytes. During my thesis, we are interested in the role of cells of innate immunity in T1D using a mouse model of the disease: NOD mice. In contrast to cells of the adaptive system (T and B lymphocytes ) cells of innate immunity is the first line of defense of the body during infection . This population consists of neutrophils , among other , plasmacytoid dendritic cells ( pDC ) , macrophages , T lymphocytes but not conventional B as iNKT cells and B -1a.Previously, our laboratory has highlighted the role of iNKT cells in the development of T1D . During the first part of my thesis , we demonstrated that iNKT17 cells, a subpopulation of iNKT cells, have a deleterious role in T1D in NOD mice . These cells infiltrate the pancreas and there produce IL -17 , a proinflammatory cytokine. Through transfer experiments , we demonstrated that lymphocytes iNKT17 exacerbate disease through the production of IL-17 . In the second part of my thesis , we investigated the mechanisms that induce the activation of autoreactive T lymphocytes. We observed in NOD mice , the physiological death of b cells leads to activation of innate immunity cells : neutrophils, lymphocytes B- 1a and pDCs . The cooperation between these cells leads to activation of pDC that produce IFNa . This cytokine activates autoreactive T cells which will destroy the b cells of the pancreas. Our results show that innate immunity is an important player in the pathogenesis of diabetes mellitus.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF