AIRR Community Guide to Planning and Performing AIRR-Seq Experiments

The development of high-throughput sequencing of adaptive immune receptor repertoires (AIRR-seq of IG and TR rearrangements) has provided a new frontier for in-depth analysis of the immune system. The last decade has witnessed an explosion in protocols, experimental methodologies, and computational tools. In this chapter, we discuss the major considerations in planning a successful AIRR-seq experiment together with basic strategies for controlling and evaluating the outcome of the experiment. Members of the AIRR Community have authored several chapters in this edition, which cover step-by-step instructions to successfully conduct, analyze, and share an AIRR-seq project

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)

CANDIDATURE A L'HABILITATION A DIRIGER DES RECHERCHES Spécialité : IMMUNOLOGIE

Since the beginning of my career in academic research, my work has focused on one of the fundamental characteristics of the adaptive immune system, namely the extraordinary diversity of antigen receptors, in particular T-cells, also called TCR (for T-cell Receptor). Indeed, this diversity gives the immune system the potential to recognize the antigenic universe of the "non-self", including pathogens and autologous tumors, even though these antigens are not a priori present in the body. T cells differentiate in the thymus, where they acquire surface expression of the TCR, a heterodimer consisting of two chains, through mechanisms of random somatic rearrangements between several dozen genes present at the genomic level in germline form. Selection steps shape the diversity of differentiated cells, avoiding in particular the export to peripheral sites of cells ineffective in recognizing an antigen, but also and above all eliminating cells, which at random rearrangements could have acquired a TCR capable of recognizing with a very high affinity antigens of the self, and thus contributing to the establishment of a pathological autoimmunity The potential diversity of the repertoire was 10^19 different TCRs (modeled to reach up to 10^61 different TCRs very recently!). Obviously, only a small fraction of the potential repertoire can actually be used, due to the limited number of cells an individual can contain in the body (in humans ~ 10^12 and in mice ~ 10^8 αβ T cells). Once in the periphery, the set of T cells selected in the thymus will constitute the repertoire available for the maintenance of the homeostasis of the organism, involving both cells capable of responding to pathogen infections, allergen intrusion, tumor transformations but also cells capable of avoiding pathological autoimmune manifestations by maintaining tolerance for the self in the periphery. During these immune responses, the available T lymphocyte repertoire will be engaged in the control of the agent to be circumscribed, potentially leading to a modulation, more or less marked, of the diversity of the TCR repertoire. Characterizing these modulations could allow (i) a better understanding of pathological states (e.g. for the development of anti-infective vaccines or tumor cell targeting) and (ii) the identification of biological markers of the pathophysiological state of an individual (e.g. for a better therapeutic management).This diversity of TCRs cannot be dissociated from the diversity of T cell populations now identified on the basis of their respective functions, inflammatory vs. regulatory, intracellular vs. extracellular anti-pathogens, involved in the regulation of antibody production.... Thus, the study of the TCR repertoire only makes sense if we take into account this additional dimension, and consider the immune system as a whole and not simply the sum of its subparts. This approach, now called systems immunology or integrative immunology, has been the foundation of all the research work I have done so far. Thus, my work has focused on (i) the characterization of TCR repertoire modulations under pathological conditions (infection or autoimmune pathology), (ii) the adaptation and improvement of methods for investigating TCR repertoire diversity using molecular biology tools, (iii) the development of statistical modeling strategies of this diversity. This work is based on a systems immunology approach, supported by time and technological developments. My perspectives for the coming years aim at extracting relevant information from the TCR repertoire in the context of autoimmune diseases in particular, by targeting functionally characterized subpopulations, in order to describe their diversity but also to identify TCR signatures associated with pathological situations (in response or not to a treatment). My interest is mainly focused on the balance between regulatory T cells and effector T cells, which is critical for the maintenance of the homeostasis of the organism

Regulatory T Cells As Supporters of Psychoimmune Resilience: Toward Immunotherapy of Major Depressive Disorder

International audienceThere is growing evidence that inflammation plays a role in major depressive disorder (MDD). As the main role of regulatory T cells (Tregs) is to control inflammation, this might denote a Treg insufficiency in MDD. However, neither a qualitative nor a quantitative defect of Tregs has been ascertained and no causality direction between inflammation and depression has been established. Here, after reviewing the evidence supporting a relation between Treg insufficiency and MDD, we conclude that a novel therapeutic approach based on Treg stimulation could be valuable in at least the subset of patients with inflammatory MDD. Low-dose interleukin-2 appears to be a good candidate as it is not only a safe stimulator of Tregs in humans but also an inhibitor of pro-inflammatory Th17 lymphocytes. Here, we discuss that a thorough immune investigation as well as immunotherapy will be heuristic for deciphering the pathophysiology of MDD

RepSeq Data Representativeness and Robustness Assessment by Shannon Entropy

High-throughput sequencing (HTS) has the potential to decipher the diversity of T cell repertoires and their dynamics during immune responses. Applied to T cell subsets such as T effector and T regulatory cells, it should help identify novel biomarkers of diseases. However, given the extreme diversity of TCR repertoires, understanding how the sequencing conditions, including cell numbers, biological and technical sampling and sequencing depth, impact the experimental outcome is critical to proper use of these data. Here, we assessed the representativeness and robustness of TCR repertoire diversity assessment according to experimental conditions. By comparative analyses of experimental datasets and computer simulations, we found that (i) for small samples, the number of clonotypes recovered is often higher than the number of cells per sample, even after removing the singletons; (ii) high-sequencing depth for small samples alters the clonotype distributions, which can be corrected by filtering the datasets using Shannon entropy as a threshold; and (iii) a single sequencing run at high depth does not ensure a good coverage of the clonotype richness in highly polyclonal populations, which can be better covered using multiple sequencing. Altogether, our results warrant better understanding and awareness of the limitation of TCR diversity analyses by HTS and justify the development of novel computational tools for improved modeling of the highly complex nature of TCR repertoires

Regulatory T Cells As Supporters of Psychoimmune Resilience: Toward Immunotherapy of Major Depressive Disorder

There is growing evidence that inflammation plays a role in major depressive disorder (MDD). As the main role of regulatory T cells (Tregs) is to control inflammation, this might denote a Treg insufficiency in MDD. However, neither a qualitative nor a quantitative defect of Tregs has been ascertained and no causality direction between inflammation and depression has been established. Here, after reviewing the evidence supporting a relation between Treg insufficiency and MDD, we conclude that a novel therapeutic approach based on Treg stimulation could be valuable in at least the subset of patients with inflammatory MDD. Low-dose interleukin-2 appears to be a good candidate as it is not only a safe stimulator of Tregs in humans but also an inhibitor of pro-inflammatory Th17 lymphocytes. Here, we discuss that a thorough immune investigation as well as immunotherapy will be heuristic for deciphering the pathophysiology of MDD

RepSeq Data Representativeness and Robustness Assessment by Shannon Entropy

International audienceHigh-throughput sequencing (HTS) has the potential to decipher the diversity of T cell repertoires and their dynamics during immune responses. Applied to T cell subsets such as T effector and T regulatory cells, it should help identify novel biomarkers of diseases. However, given the extreme diversity of TCR repertoires, understanding how the sequencing conditions, including cell numbers, biological and technical sampling and sequencing depth, impact the experimental outcome is critical to proper use of these data. Here, we assessed the representativeness and robustness of TCR repertoire diversity assessment according to experimental conditions. By comparative analyses of experimental datasets and computer simulations, we found that (i) for small samples, the number of clonotypes recovered is often higher than the number of cells per sample, even after removing the singletons; (ii) high-sequencing depth for small samples alters the clonotype distributions, which can be corrected by filtering the datasets using Shannon entropy as a threshold; and (iii) a single sequencing run at high depth does not ensure a good coverage of the clonotype richness in highly polyclonal populations, which can be better covered using multiple sequencing. Altogether, our results warrant better understanding and awareness of the limitation of TCR diversity analyses by HTS and justify the development of novel computational tools for improved modeling of the highly complex nature of TCR repertoires

Heterogeneity of natural Foxp3+ T cells: A committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity

Natural regulatory T cells (Treg) represent a distinct lineage of T lymphocytes committed to suppressive functions, and expression of the transcription factor Foxp3 is thought to identify this lineage specifically. Here we report that, whereas the majority of natural CD4+Foxp3+ T cells maintain stable Foxp3 expression after adoptive transfer to lymphopenic or lymphoreplete recipients, a minor fraction enriched within the CD25− subset actually lose it. Some of those Foxp3− T cells adopt effector helper T cell (Th) functions, whereas some retain “memory” of previous Foxp3 expression, reacquiring Foxp3 upon activation. This minority “unstable” population exhibits flexible responses to cytokine signals, relying on transforming growth factor-β to maintain Foxp3 expression and responding to other cytokines by differentiating into effector Th in vitro. In contrast, CD4+Foxp3+CD25high T cells are resistant to such conversion to effector Th even after many rounds of cell division. These results demonstrate that natural Foxp3+ T cells are a heterogeneous population consisting of a committed Treg lineage and an uncommitted subpopulation with developmental plasticity

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

Regulatory T lymphocytes/Th17 lymphocytes imbalance in autism spectrum disorders: evidence from a meta-analysis

International audienceBackground: Immune system dysfunction has been proposed to play a critical role in the pathophysiology of autism spectrum disorders (ASD). Conflicting reports of lymphocyte subpopulation abnormalities have been described in numerous studies of patients with ASD. To better define lymphocytes abnormalities in ASD, we performed a metaanalysis of the lymphocyte profiles from subjects with ASD. Methods: We used the PRISMA recommendations to query PubMed, Embase, PsychoINFO, BIOSIS, Science Direct, Cochrane CENTRAL, and Clinicaltrials.gov for terms related to clinical diagnosis of ASD and to lymphocytes' populations. We selected studies exploring lymphocyte subpopulations in children with ASD. The search protocol has been registered in the international Prospective Register of Systematic Reviews (CRD42019121473). Results: We selected 13 studies gathering 388 ASD patients and 326 healthy controls. A significant decrease in the CD4+ lymphocyte was found in ASD patients compared to controls [− 1.51 (95% CI − 2.99; − 0.04) p = 0.04] (I 2 = 96% [95% CI 94.6, 97.7], p < 0.01). No significant difference was found for the CD8+ T, B and natural killer lymphocytes. Considering the CD4+ subpopulation, there was a significant decrease in regulatory T lymphocytes (Tregs) in ASD patients (n = 114) compared to controls (n = 107) [− 3.09 (95% CI − 4.41; − 1.76) p = 0.0001]; (I 2 = 90.9%, [95% CI 76.2, 96.5], p < 0.0001) associated with an increase oin the Th17 lymphocytes (ASD; n = 147 controls; n = 128) [2.23 (95% CI 0.79; 3.66) p = 0,002] (I 2 = 95.1% [95% CI 90.4, 97.5], p < 0.0001). Limitations: Several factors inducing heterogeneity should be considered. First, differences in the staining method may be responsible for a part in the heterogeneity of results. Second, ASD population is also by itself heterogeneous, underlying the need of studying subgroups that are more homogeneous. Conclusion: Our meta-analysis indicates defects in CD4+ lymphocytes, specifically decrease oin Tregs and increase in Th17 in ASD patients and supports the development of targeted immunotherapies in the field of ASD