Biological controls for standardization and interpretation of adaptive immune receptor repertoire profiling

Use of adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread, providing new insights into the immune system with potential broad clinical and diagnostic applications. However, like many high-throughput technologies, it comes with several problems, and the AIRR Community was established to understand and help solve them. We, the AIRR Community's Biological Resources Working Group, have surveyed scientists about the need for standards and controls in generating and annotating AIRR-seq data. Here, we review the current status of AIRR-seq, provide the results of our survey, and based on them, offer recommendations for developing AIRR-seq standards and controls, including future work. Keywords: B-cell Receptor (BCR); IG; T-cell Receptor (TCR); TR; antibody; immunoglobulin; immunology; inflammation; next generation sequencing (NGS)

TCR signatures of immune responses in autoimmune diseases and infections

Les lymphocytes T représentent les cellules immunitaires centrale du système immunitaire adaptatif se distinguant en deux populations majeures : les cellules T effectrices (Teffs), auxillaires ou cytotoxiques, ayant un rôle pro-inflammatoire, contribuant à l'élimination des pathogènes en contexte infectieux et responsables des atteintes tissulaires dans le contexte des maladies auto-immunes (MAI) ; les cellules T régulatrices (Tregs) ayant un rôle anti-inflammatoire sur la réponse Teffs, permettant le contrôle des désordres auto-immuns. Teffs et Tregs se caractérisent par la reconnaissance spécifique d'antigène, nécessaire à l'activation cellulaire, et portée par le récepteur des lymphocytes T, autrement appelé T-cell receptor (TCR). Chaque TCR résulte d'un mécanisme de recombinaison génique générant une collection très diverse de TCR, communément appelé répertoire. Véritable reflet de l'histoire immunitaire de chaque individu, l'implication directe de la reconnaissance par le TCR dans les fonctions antagonistes des Tregs et des Teffs, à la fois dans la genèse mais aussi dans la progression des pathologies immunes, soulève la nécessité d'analyser distinctement leurs répertoires TCR. Dans le cadre de ma thèse, j'avais pour objectif d'évaluer le caractère prédictif du répertoire TCR au regard de l'état physiopathologique et de son évolution de patients atteints de MAI, pour lesquelles les outils diagnostiques et/ou pronostiques demeurent limités ainsi que de patients atteints de la COVID-19, maladie infectieuse émergente caractérisée par une forte mortalité des individus immunodéprimés et touchant gravement également des individus considérés sains. Pour cela, j'ai fait appel aux technologies de séquençage à haut-débit offrant aujourd'hui précision et profondeur nécessaire à l'étude de cet objet biologique complexe. Dans ce contexte, la fiabilité des protocoles de biologie moléculaire ainsi que les méthodes bio-informatiques et de modélisations représentent un défi majeur. J'ai dans un premier temps comparé neufs protocoles publiés et/ou commercialisés parmi les plus utilisés dans la littérature. A l'aide d'analyses de la diversité et de modélisation statistique, j'ai élaboré un score de performance, à partir duquel j'ai mis en évidence les forces et faiblesses de chaque protocole. J'ai ainsi identifié la méthode la plus fiable, robuste et reproductible. Avec ce protocole, j'ai ensuite analysé les répertoires TCR des Teffs auxillaires et des Tregs issus de prélèvements sanguins de patients atteints de diabète de type 1 et de polyarthrite rhumatoïde, deux MAI. A l'aide d'outils de modélisation mathématiques et d'apprentissage machine, j'ai développé une stratégie d'identification de TCR discriminants des groupes d'individus. Cette méthode innovante a permis d'identifier des signatures TCR des différents MAI étudiées ainsi que des Teffs et des Tregs, soutenant l'idée que la composition du répertoire TCR signe l'état physiopathologique, y compris dans des pathologies chroniques établies. Appliquée au répertoire TCR de patients atteints de lupus érythémateux systémique sous traitement à l'interleukine-2 à faible dose, j'ai identifié des signatures TCR des Teffs et des Tregs définissant les patients traités répondeurs, pouvant être utilisées comme potentiels biomarqueurs prédictifs de l'efficacité du traitement. Finalement, j'ai également appliqué cette approche sur des répertoires de patients atteints de Sars-CoV-2 pour lesquels l'issue clinique était connue. J'ai ainsi identifié une signature TCR pronostique de la guérison et discriminant les patients décédés. Ensemble, ces résultats renforcent l'intérêt de l'étude du répertoire TCR en montrant que celui-ci contient une information clinique fine, capable non seulement de séparer des patients atteints de différentes maladies mais aussi de distinguer, de façon précoce, la réponse au traitement ainsi que le niveau de sévérité face à une infection.T cells represent the central immune cells of the adaptive immune system and are distinguished in two major populations. On one hand, effector T cells (Teffs), helper or cytotoxic, have a pro-inflammatory role and contribute to the elimination of pathogens in the context of infectious diseases as they can be responsible for tissue damage in the context of autoimmune diseases (AD). On the other hand, regulatory T cells (Tregs) have an anti-inflammatory role on the Teffs response, allowing the control of autoimmune disorders. Teffs and Tregs are characterized by antigen-specific recognition, a prerequisite for T cell activation, carried by the T-cell receptor (TCR). Each TCR results from a gene recombination mechanism generating a highly diverse collection of TCRs, commonly called repertoire. The direct implication of the TCR recognition in the antagonistic functions of Tregs and Teffs, both in the genesis and progression of immune diseases, raises the need to analyze their TCR repertoires separately. In the framework of my thesis, I aimed to evaluate the predictive character of the TCR repertoire with respect to the pathophysiological state and its evolution of patients. I focused on patients suffering from AD, for which diagnostic and/or prognostic tools remain limited, as well as of patients suffering from COVID-19, an emerging infectious disease characterized by a high mortality of immunocompromised individuals and also seriously affecting individuals considered healthy. For this purpose, I have used high-throughput sequencing technologies that now offer the precision and depth necessary to study this complex biological object. In this context, the reliability of molecular biology protocols as well as bioinformatics and modelling methods represent a major challenge. I first compared nine published and/or commercialized protocols among the most used in the literature. Using diversity analysis and statistical modeling, I developed a performance score, from which I highlighted the strengths and weaknesses of each protocol. I thus identified the most reliable, robust and reproducible method. With this protocol, I then analyzed the TCR repertoires of helper Teffs and Tregs from blood samples of patients with type 1 diabetes and rheumatoid arthritis, both ADs. Using mathematical modeling and machine learning tools, I developed a strategy to identify TCRs discriminating groups of individuals. This innovative method allowed identifying TCR signatures of the different AD studied as well as Teffs and Tregs, supporting the idea that the composition of the TCR repertoire signs the pathophysiological state, including in established chronic pathologies. Applied to the TCR repertoire of patients with systemic lupus erythematosus under low-dose interleukin-2 treatment, I identified TCR signatures of Teffs and Tregs defining responder treated patients, which can be used as potential predictive biomarkers of treatment efficacy. Finally, I also applied this approach on repertoires of Sars-CoV-2 patients for whom the clinical outcome was known. I thus identified a TCR signature that was prognostic of cure and discriminated against patients who died. Altogether, my work supports the interest of the TCR repertoire study by showing that it contains fine clinical information, capable not only of separating patients with different diseases but also of distinguishing, in an early stage, the response to treatment as well as the level of severity of an infection

Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?

Abstract Background The treatment gap for epilepsy is considerable in low and middle-income countries. In the Lao PDR it is estimated at over 90%. Health workers play a significant role in bridging the gap between people with epilepsy (PWE) and access to epilepsy care. In a national survey we assessed: 1) the knowledge and practices of health workers in the Lao PDR towards the disease, and, 2) patient attendance at health facilities. Methods We conducted a random three-stage sampling of health workers at the provincial, district and health center levels in 2009. Results Overall, 284 health workers were enrolled in 50 health facilities of 11 provinces: health centers 24.7%; district hospitals 23.2%; and province hospitals 52.1%. Only a minority of these (2.5%) recalled ever having received training or seeking information on epilepsy. Our survey showed a lack of knowledge in diagnosing and prescribing drugs for epilepsy, including phenobarbital, the first-line of treatment. The majority of respondents (59.9%) was unaware of the availability of antiepileptic drugs in health facilities. Only 10 (20%) health facilities, and no health centres, received people with epilepsy. It was estimated that one PWE per month receives medical attention. Traditional beliefs about PWE were common; such as the idea that epilepsy could be transmitted through saliva (63.2%). A higher attendance of PWE was observed in province hospitals where the knowledge of epilepsy care was higher. Global acceptance of people with epilepsy was low. Conclusions The low level of knowledge of epilepsy on the part of health workers may be contributing to the wide treatment gap in the Lao PDR. Improving knowledge of this disease and increasing the availability of antiepileptic drugs will reduce misconceptions about epilepsy, thus encouraging more PWE to seek treatment. Community-based educational programs and extensive advocacy for people with epilepsy only began in 2009.</p

Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?

International audienceBACKGROUND: The treatment gap for epilepsy is considerable in low and middle-income countries. In the Lao PDR it is estimated at over 90%. Health workers play a significant role in bridging the gap between people with epilepsy (PWE) and access to epilepsy care. In a national survey we assessed: 1) the knowledge and practices of health workers in the Lao PDR towards the disease, and, 2) patient attendance at health facilities. METHODS: We conducted a random three-stage sampling of health workers at the provincial, district and health center levels in 2009. RESULTS: Overall, 284 health workers were enrolled in 50 health facilities of 11 provinces: health centers 24.7%; district hospitals 23.2%; and province hospitals 52.1%. Only a minority of these (2.5%) recalled ever having received training or seeking information on epilepsy. Our survey showed a lack of knowledge in diagnosing and prescribing drugs for epilepsy, including phenobarbital, the first-line of treatment. The majority of respondents (59.9%) was unaware of the availability of antiepileptic drugs in health facilities. Only 10 (20%) health facilities, and no health centres, received people with epilepsy. It was estimated that one PWE per month receives medical attention. Traditional beliefs about PWE were common; such as the idea that epilepsy could be transmitted through saliva (63.2%). A higher attendance of PWE was observed in province hospitals where the knowledge of epilepsy care was higher. Global acceptance of people with epilepsy was low. CONCLUSIONS: The low level of knowledge of epilepsy on the part of health workers may be contributing to the wide treatment gap in the Lao PDR. Improving knowledge of this disease and increasing the availability of antiepileptic drugs will reduce misconceptions about epilepsy, thus encouraging more PWE to seek treatment. Community-based educational programs and extensive advocacy for people with epilepsy only began in 2009

The challenge of epilepsy in low-income countries - insights from Laos.

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Associated factors with adherence to antiepileptic drug in the capital city of Lao PDR.

International audienceAvailable medical care for epilepsy and antiepileptic drugs (AED) are provided in Vientiane Municipality by district hospitals supported by a non-governmental organization, which is referred to as a community-based intervention (CB), and reference hospital which is referred to as hospital-based interventions (HB). Identifying underlying factors of AED adherence is of public health interest. A community-based cross-sectional survey among randomly selected patients with epilepsy (PWE) who were being cared in Vientiane Municipality was undertaken in 2010. The Morisky scale was used to assess the level of adherence. Univariate and multivariate logistic regression analyses were performed to address predictive factors. Overall, 99 PWE were included in the study. Overall adherence was estimated at 57.6%, 57.1% and 58.0% for the HB and CB group, respectively. High level of adherence was related to illiteracy, being on monotherapy and experiencing fewer seizures. Implementing closer medical care at primary level to PWE improves the likelihood of reducing primary and secondary treatment gap which is related to PWEs' adherence. An active intervention through a downstream channel of training of medical health staff from reference level to community level enhances the prescription of adequate AED, the improvement of the quality of relation between PWE and medical staff

The challenge of epilepsy control in deprived settings: Low compliance and high fatality rates during a community-based phenobarbital program in rural Laos.

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Prevalence of epilepsy in a rural district of central Lao PDR.

International audiencePURPOSE: To assess the prevalence of epilepsy in a rural district of Lao PDR and to describe the clinical and epidemiological profile of the disease. METHODS: Door-to-door screening was performed on the entire population of 8 randomly selected villages in the Hinheub district, using an internationally validated and standardized questionnaire. Additional passive case detection was performed through village key informants. Suspected epilepsy patients identified by the questionnaire were revisited and examined by an experienced neurologist on two follow-up visits. The confirmation of epilepsy was based only on an in-depth clinical examination. Electroencephalograms were performed at the district health care center. RESULTS: In the 8 villages, 277 suspected cases of epilepsy were identified among 4,310 interviewed subjects; 194 of whom (70%) underwent a clinical examination by a neurologist during the first visit while 65 of 83 remaining suspected cases were seen on the second confirmation visit. Twenty-seven persons with epilepsy were identified. Six additional patients were diagnosed in 219 self-referred subjects. An overall prevalence of 7.7 cases of epilepsy per thousand inhabitants was calculated (95% CI 5.3-10.7). Generalized epilepsy (21 cases, 63.6%) was commoner than partial epilepsy (9 cases, 27.3%). The remaining 3 cases (9.1%) were not-classifiable as either generalized or partial. EEG abnormal findings were found in 12 of the 24 patients (50.0%) who had an EEG registration. CONCLUSIONS: This is the first study in Lao PDR to estimate the prevalence of epilepsy. Compared to Western countries it shows a pattern towards a higher prevalence

Adaptive immune receptor repertoire analysis

B cell and T cell receptor repertoires compose the adaptive immune receptor repertoire (AIRR) of an individual. The AIRR is a unique collection of antigen-specific receptors that drives adaptive immune responses, which in turn is imprinted in each individual AIRR. This supports the concept that the AIRR could determine disease outcomes, for example in autoimmunity, infectious disease and cancer. AIRR analysis could therefore assist the diagnosis, prognosis and treatment of human diseases towards personalized medicine. High-throughput sequencing, high-dimensional statistical analysis, computational structural biology and machine learning are currently employed to study the shaping and dynamics of the AIRR as a function of time and antigenic challenges. This Primer provides an overview of concepts and state-of-the-art methods that underlie experimental and computational AIRR analysis and illustrates the diversity of relevant applications. The Primer also addresses some of the outstanding challenges in AIRR analysis, such as sampling, sequencing depth, experimental variations and computational biases, while discussing prospects of future AIRR analysis applications for understanding and predicting adaptive immune responses