B-cell Receptor Repertoire Research Articles

The composition and variation of the BCR CDR3s in gastric cancer.

Gastric cancer (GC) is the fourth most common type of cancer and the second most common cause of cancer-associated mortality worldwide. B cell-associated autoantibodies against tumor-associated antigens are attractive biomarkers for the development of noninvasive serological tests for the early detection of cancer. This is due to their specificity and stability in the sera. In the present study multiplex polymerase chain reaction and Illumina high-throughput sequencing (HTS) was used to study the composition and variation of the B cell receptor (BCR) complimentary-determining region 3 (CDR3) in GC. The peripheral blood, cancer tissues and peri-cancer tissues were included from 7 patients with GC. On average there was a total of 403,959 CDR3 sequences, with 72,367 unique CDR3 nt sequences and 61,709 unique CDR3 aa sequences per sample identified, which are critical for further understanding the BCR repertoire in GC. The details of GC CDR3s may accelerate the screening process for possible new autoantigens and may provide additional information necessary for generating effective B cell targeted diagnosis and therapeutic strategies.

VDJServer: A Cloud-Based Analysis Portal and Data Commons for Immune Repertoire Sequences and Rearrangements.

Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation. VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provide access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene segment assignment, repertoire characterization, and repertoire comparison. VDJServer also provides sophisticated visualizations for exploratory analysis. It is accessible through a standard web browser via a graphical user interface designed for use by immunologists, clinicians, and bioinformatics researchers. VDJServer provides a data commons for public sharing of repertoire sequencing data, as well as private sharing of data between users. We describe the main functionality and architecture of VDJServer and demonstrate its capabilities with use cases from cancer immunology and autoimmunity. VDJServer provides a complete analysis suite for human and mouse T-cell and B-cell receptor repertoire sequencing data. The combination of its user-friendly interface and high-performance computing allows large immune repertoire sequencing projects to be analyzed with no programming or software installation required. VDJServer is a web-accessible cloud platform that provides access through a graphical user interface to a data management infrastructure, a collection of analysis tools covering all steps in an analysis, and an infrastructure for sharing data along with workflows, results, and computational provenance. VDJServer is a free, publicly available, and open-source licensed resource.

The contribution of autoantibody-inducing CD4+ T cell (aiCD4 T cell) help to the B cell maturation and possible autoantibody in Systemic Lupus Erythematosus

Abstract INTRODUCTION Autoantibody-inducing CD4 T (aiCD4 T) cell seems indispensable for the self-organized criticality theory that induces Systemic Lupus Erythematosus (SLE). We previously found that the aiCD4 T cell belongs to CD45RBlo122lo CD4 T cell subpopulation. We here analyze the B cell population and CD19+ splenic B cell receptor (BCR) repertoire, and test our contention whether aiCD4 T cell helps B cell maturation. METHODS BALB/c mice were immunized with OVA twelve times and SLE was induced. Cell surface markers of B cell were detected with flow cytometry. CD19+ splenic B cells were sorted and BCR repertoires of these cells were analyzed on next generation sequencer. RESULTS After twelve times immunization of mice with OVA, follicular B (Fo B) cell and germinal center B (GC B) cell were significantly increased(P<0.05), whereas marginal zone B cell was decreased. Activation markers of the B cell were increased and in particular, CD80 was significantly increased (P<0.05). In addition, the number of clonotypes of CD19+ splenic B cells in OVA immunized mice was increased. Among these repertoires, the same pairs of V and J usage were frequently found in OVA immunized mice. CONCLUSION Fo B cell and GC B cell and the activation markers of B cell, as well, were significantly increased in OVA immunized mice, and the result of BCR repertoire analysis suggested that somatic hyper mutation occurred frequently in OVA-stimulated mice, which is compatible with the contention that aiCD4 T cells helps the maturation of B cells into germinal center B cells to enhance autoantibody formation thereby inducing SLE.

Next-Generation Immune Repertoire Sequencing as a Clue to Elucidate the Landscape of Immune Modulation by Host-Gut Microbiome Interactions.

The human immune system is a fine network consisted of the innumerable numbers of functional cells that balance the immunity and tolerance against various endogenous and environmental challenges. Although advances in modern immunology have revealed a role of many unique immune cell subsets, technologies that enable us to capture the whole landscape of immune responses against specific antigens have been not available to date. Acquired immunity against various microorganisms including host microbiome is principally founded on T cell and B cell populations, each of which expresses antigen-specific receptors that define a unique clonotype. Over the past several years, high-throughput next-generation sequencing has been developed as a powerful tool to profile T- and B-cell receptor repertoires in a given individual at the single-cell level. Sophisticated immuno-bioinformatic analyses by use of this innovative methodology have been already implemented in clinical development of antibody engineering, vaccine design, and cellular immunotherapy. In this article, we aim to discuss the possible application of high-throughput immune receptor sequencing in the field of nutritional and intestinal immunology. Although there are still unsolved caveats, this emerging technology combined with single-cell transcriptomics/proteomics provides a critical tool to unveil the previously unrecognized principle of host–microbiome immune homeostasis. Accumulation of such knowledge will lead to the development of effective ways for personalized immune modulation through deeper understanding of the mechanisms by which the intestinal environment affects our immune ecosystem.

Tracking of long-term B-cell memory responses using B-cell receptor (BCR) sequencing in prostate cancer (PC) patients (pts) treated with sipuleucel-T (sip-T).

309 Background: Sip-T is an autologous cellular immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Sip-T’s induction of B-cell responses to PAP and other antigens correlates with improved survival (Sheikh 2013, GuhaThakurta 2015). Cancer vaccine-induced changes to BCR repertoire are unknown. To assess changes in BCR repertoire upon booster treatment, we compared patients retreated with sip-T (P10-1) to treatment-naïve patients (STRIDE). Methods: STRIDE pts (N = 52) received sip-T with concurrent or sequential enzalutamide for mCRPC (Petrylak 2015). P10-1 pts (N = 8) previously treated with sip-T for androgen-dependent PC were retreated with a booster course for mCRPC, after a median of 8.9 years (Beer 2017). Blood samples were collected at baseline (wk 0) and during (wk 2, 4)/post-sip-T (wk 6, 26, 52). Deep sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies). BCR diversity was assessed by clonality, and BCR dynamics by fold-change analysis (Zhang 2017). Results: BCR repertoire had significantly higher clonality in P10-1 vs STRIDE (wk 0: p = 0.003, wk 2: p < 0.001, wk 4: p < 0.001), suggestive of a more focused BCR repertoire. P10-1 also showed increased clonality from wk 0 to 4 (p = 0.063), whereas STRIDE showed a significant increase in clonality from wk 0 to 6 (p = 0.039), suggesting that BCR repertoire focused earlier in P10-1. Starting at wk 2, more clones remained in the repertoire in P10-1, indicating that sip-T stimulated immunologic memory early, after 1st retreatment (p < 0.05). There was less change over time (clonal shuffling) within the 100 most abundant baseline clones in P10-1 (p = 0.080), suggesting more relevant clones preexisted at baseline and enriched over time. After the first two sip-T infusions, more clones contracted in P10-1 (p = 0.027, p = 0.014), whereas more new clones were generated in STRIDE (p = 0.083, p = 0.003). Conclusions: Sip-T induces long-lasting changes in the BCR repertoire. Sip-T retreatment leads to quicker focusing of BCR repertoire than initial treatment. These results are consistent with sip-T inducing durable immunologic memory.

Biased IGH VDJ gene repertoire and clonal expansions in B cells of chronically hepatitis C virus–infected individuals

AbstractPatients chronically infected with hepatitis C virus (HCV) frequently develop mixed cryoglobulinemia (MC), a monoclonal expansion of immunoglobulin M (IgM)+ autoreactive B cells, and also have an increased B-cell lymphoma risk. Whether HCV infection also impacts the B-cell compartment and the B-cell receptor repertoire in patients not affected by MC or lymphomas is poorly understood. Flow cytometric analysis of peripheral blood B cells of 30 MC-negative HCV-infected patients and 15 healthy controls revealed that frequencies of class-switched memory B cells were increased in the patients, whereas frequencies of transitional and naive B cells were decreased. For 22 HCV+ patients and 7 healthy controls, we performed high-throughput sequencing of immunoglobulin heavy chain VDJ rearrangements of naive, mature CD5+, IgM+ memory, and class-switched memory B cells. An increased usage of several IGHV genes, including IGHV1-69 and IGHV4-59, which are closely linked to MC and HCV-associated lymphomas, was specifically seen among IgM+ memory B cells of the patients. Moreover, many, and partly very large, expanded clones were seen predominantly among IgM+ memory B cells of all HCV-infected patients analyzed. Thus, chronic HCV infection massively disturbs the B-cell compartment even in patients without clinically detectable B-cell lymphoproliferation and generates many large B-cell clones, especially among non–class-switched memory B cells. Because B-cell clones in MC and lymphomas derive from this B-cell subset, this establishes IgM+ memory B cells as a general target of lymphoproliferation in HCV+ patients, affecting apparently all patients.

B-cell receptor repertoire sequencing in patients with primary immunodeficiency: a review.

The advent of next-generation sequencing (NGS) now allows a detailed assessment of the adaptive immune system in health and disease. In particular, high-throughput B-cell receptor (BCR) repertoire sequencing provides detailed information about the functionality and abnormalities of the B-cell system. However, it is mostly unknown how the BCR repertoire is altered in the context of primary immunodeficiencies (PID) and whether findings are consistent throughout phenotypes and genotypes. We have performed an extensive literature search of the published work on BCR repertoire sequencing in PID patients, including several forms of predominantly antibody disorders and combined immunodeficiencies. It is somewhat surprising that BCR repertoires, even from severe clinical phenotypes, often show only mild abnormalities and that diversity or immunoglobulin gene segment usage is generally preserved to some extent. Despite the great variety of wet laboratory and analytical methods that were used in the different studies, several findings are common to most investigated PIDs, such as the increased usage of gene segments that are associated with self-reactivity. These findings suggest that BCR repertoire characteristics may be used to assess the functionality of the B-cell compartment irrespective of the underlying defect. With the use of NGS approaches, there is now the opportunity to apply BCR repertoire sequencing to multiple patients and explore the PID BCR repertoire in more detail. Ultimately, using BCR repertoire sequencing in translational research could aid the management of PID patients by improving diagnosis, estimating functionality of the immune system and improving assessment of prognosis.

Characterization of the B-cell receptor repertoires in peanut allergic subjects undergoing oral immunotherapy

B-cell receptors (BCRs) play a critical role in adaptive immunity as they generate highly diverse immunoglobulin repertoires to recognize a wide variety of antigens. To better understand immune responses, it is critically important to establish a quantitative and rapid method to analyze BCR repertoire comprehensively. Here, we developed “Bcrip”, a novel approach to characterize BCR repertoire by sequencing millions of BCR cDNA using next-generation sequencer. Using this method and quantitative real-time PCR, we analyzed expression levels and repertoires of BCRs in a total of 17 peanut allergic subjects’ peripheral blood samples before and after receiving oral immunotherapy (OIT) or placebo. By our methods, we successfully identified all of variable (V), joining (J), and constant (C) regions, in an average of 79.1% of total reads and 99.6% of these VJC-mapped reads contained the C region corresponding to the isotypes that we aimed to analyze. In the 17 peanut allergic subjects’ peripheral blood samples, we observed an oligoclonal enrichment of certain immunoglobulin heavy chain alpha (IGHA) and IGH gamma (IGHG) clones (P = 0.034 and P = 0.027, respectively) in peanut allergic subjects after OIT. This newly developed BCR sequencing and analysis method can be applied to investigate B-cell repertoires in various research areas, including food allergies as well as autoimmune and infectious diseases.

Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses.

Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103–105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox’ robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including IGHV-family usage, SHM percentage, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature—enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.

T-dependent B cell responses to Plasmodium induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein.

The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.

Understanding the reconstitution of the B-cell compartment in bone marrow and blood after treatment for B-cell precursor acute lymphoblastic leukaemia.

A better understanding of the reconstitution of the B-cell compartment during and after treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) will help to assess the immunological status and needs of post-treatment BCP-ALL patients. Using 8-colour flow cytometry and proliferation-assays, we studied the composition and proliferation of both the B-cell precursor (BCP) population in the bone marrow (BM) and mature B-cell population in peripheral blood (PB) during and after BCP-ALL therapy. We found a normal BCP differentiation pattern and a delayed formation of classical CD38dim -naive mature B-cells, natural effector B-cells and memory B-cells in patients after chemotherapy. This B-cell differentiation/maturation pattern was strikingly similar to that during initial B-cell development in healthy infants. Tissue-resident plasma cells appeared to be partly protected from chemotherapy. Also, we found that the fast recovery of naive mature B-cell numbers after chemotherapy was the result of increased de novo BCP generation, rather than enhanced B-cell proliferation in BM or PB. These results indicate that post-treatment BCP-ALL patients will eventually re-establish a B-cell compartment with a composition and B-cell receptor repertoire similar to that in healthy children. Additionally, the formation of a new memory B-cell compartment suggests that revaccination might be beneficial after BCP-ALL therapy.

Targeting Bruton’s tyrosine kinase expression levels through microRNAs in chronic lymphocytic leukemia treatment

CLL is the most common leukemia in the western world and is characterized by the accumulation of monoclonal mature circulating CD5+ B cells that generally express low levels of surface immunoglobulin (Ig). Many lines of evidence indicate that chronic signaling through the B-cell receptor (BCR) plays a key role in CLL pathogenesis. CLL prognosis is correlated with the BCR somatic hypermutation status and the CLL BCR repertoire is highly restricted. Often, CLL cells show constitutive activation of several kinases that are activated immediately downstream of the BCR. Thus, the BCR signaling pathway is aberrantly active in CLL and may play a role in disease development. One of the signal transduction molecules downstream of the BCR is BTK, a Tec family non-receptor kinase that is primarily expressed in most hematopoietic lineages, but not in T cells. BTK has been shown to be essential for several constitutively active pathways implicated in CLL cell survival, including the AKT, ERK and NF-κB pathway. Importantly, small molecule inhibitors of BTK have shown impressive anti-tumor activity in clinical studies, with high response rates in various B cell malignancies, in particular CLL. [...]

Altered B cell signalling in autoimmunity.

Recent work has provided new insights into how altered B cell-intrinsic signals - through the B cell receptor (BCR) and key co-receptors - function together to promote the pathogenesis of autoimmunity. These combined signals affect B cells at two distinct stages: first, in the selection of the naive repertoire; and second, during extrafollicular or germinal centre activation responses. Thus, dysregulated signalling can lead to both an altered naive BCR repertoire and the generation of autoantibody-producing B cells. Strikingly, high-affinity autoantibodies predate and predict disease in several autoimmune disorders, including type 1 diabetes and systemic lupus erythematosus. This Review summarizes how, rather than being a downstream consequence of autoreactive T cell activation, dysregulated B cell signalling can function as a primary driver of many human autoimmune diseases.

Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role of the transcription factor Bhlhe41, with a lesser contribution of Bhlhe40, in controlling late stages of B-1a cell differentiation. Bhlhe41–/–Bhlhe40–/– B-1a cells were severely reduced as compared to their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B-cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12/Vκ4 BCR. Expression of a pre-rearranged VH12/Vκ4 BCR failed to rescue the mutant phenotype and revealed enhanced proliferation accompanied with increased cell death. Bhlhe41 directly repressed the expression of cell cycle regulators and inhibitors of BCR signaling, while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Phylogenetic analysis of the human antibody repertoire reveals quantitative signatures of immune senescence and aging

The elderly have reduced humoral immunity, as manifested by increased susceptibility to infections and impaired vaccine responses. To investigate the effects of aging on B-cell receptor (BCR) repertoire evolution during an immunological challenge, we used a phylogenetic distance metric to analyze Ig heavy-chain transcript sequences in both young and elderly individuals before and after influenza vaccination. We determined that BCR repertoires become increasingly specialized over a span of decades, but less plastic. In 50% of the elderly individuals, a large space in the repertoire was occupied by a small number of recall lineages that did not decline during vaccine response and contained hypermutated IgD+ B cells. Relative to their younger counterparts, older subjects demonstrated a contracted naive repertoire and diminished intralineage diversification, signifying a reduced substrate for mounting novel responses and decreased fine-tuning of BCR specificities by somatic hypermutation. Furthermore, a larger proportion of the repertoire exhibited premature stop codons in some elderly subjects, indicating that aging may negatively affect the ability of B cells to discriminate between functional and nonfunctional receptors. Finally, we observed a decreased incidence of radical mutations compared with conservative mutations in elderly subjects' vaccine responses, which suggests that accumulating original antigenic sin may be limiting the accessible space for paratope evolution. Our findings shed light on the complex interplay of environmental and gerontological factors affecting immune senescence, and provide direct molecular characterization of the effects of senescence on the immune repertoire.

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA’s utility in B-cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B-cell affinity maturation process through immunosequencing methods.

Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires.

For investigations of human B-cell receptor (BCR) repertoires, we have developed a protocol for large-scale surveys of human antibody heavy chain (VH) rearrangements. Here we study IgA repertoires, as more IgA antibodies are synthesized in the human body on a daily level than all other isotypes combined. In fact, IgA is secreted at all mucosal surfaces, and it is also secreted in the perspiration that coats our cutaneous surfaces. In these studies we can characterize the IgA clonal diversity of B-cell populations obtained from any donor. To recover representative repertoire libraries, we make our libraries from antibody gene transcript templates (i.e., cDNA), as these are closer reflections of the immune repertoire expressed at the antibody protein level. To avoid biases potentially introduced by upstream oligonucleotide primers that hybridize to variable region framework regions, our approach also uses rapid amplification of cDNA ends (RACE) of antibody transcripts. For exploration of human IgA responses, we have designed a duplexing antisense constant region primer that efficiently amplifies, side-by-side, heavy chain transcripts of both the IgA1 and IgA2 subclasses. By these methods we have begun to define the molecular differences in the IgA1 and IgA2 responses occurring simultaneously in different donors. These methods will be used to investigate the effects of microbial virulence factors on host defenses, during autoimmune responses, and in B-cell malignancies.

How to Monitor Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation: A Survey from the EBMT- Cellular Therapy & Immunobiology Working Party

Background:Post transplant immune reconstitution plays a major role in determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is currently monitored with different techniques in different Centers, with the aim of identifying clinically relevant immunological biomarkers. However, it is unclear which and how many of these immunological tests are currently performed on a routine basis, and which ones have the potential to predict patient outcome, and possibly guide patient care after allo-HSCT.Methods:The EBMT Cellular Therapy & Immunobiology Working Party (CTIWP) conducted a survey to identify current policies to monitor immune reconstitution in patients undergoing allo-HSCT and possibly reach a general consensus.This study followed the EBMT study guidelines. All EBMT Centers were invited to participate. Each participating Center received a questionnaire on the availability of specific immunomonitoring assays, specifying the use in clinical practice and/or within investigational trials. Assays were based on relatively simple and readily available parameters such as absolute lymphocyte counts (ALC) to more complex cellular and molecular tests. Moreover, the Centers were asked to define the transplant platform (HLA-identical sibling, matched unrelated donor, haploidentical and/or cord blood) on which each test is generally performed.Results:Policies for post-transplant immunomonitoring have been reported by 35 participating EBMT Centers active in 14 Countries and performing allo-HSCT from HLA identical related (35 centers), matched unrelated (33), haploidentical (34), unrelated cord blood (27).Complete blood counts and immunoglobulins are routinely tested for patients' care by all centers. Relative proportions of T cell subsets are currently tested by flow-cytometry as "standard of care" or "investigational" by 82% and 17% of centers respectively. B cell and NK cell counts are quantified routinely by 46% and 23% of Centers, and investigationally by 40% of Centers.The availability of molecular tests (STR, qPCR, Fish) to measure post-transplant engraftment are reported by all Centers, except two, as a standard of care measure.T cell receptor-expressing circles (TRECs) and/or K-deleting recombination excision circles (KRECs) are quantified within selected clinical trials by 37% of Centers. Interestingly, 60% of Centers evaluate, mostly as an investigational measure, antigen specific T cell responses by: proliferation assays (49%), interferon-gamma enzyme-linked immunospot-Elispot (49%), intracellular cytokine staining (46%) and tetramer/dextramer staining (37%). Most of these Centers test responses to Cytomegalovirus and Epstein Barr Virus, and 5 Centers use at least one of these assays on a routine basis.About half of the participating Centers (43%) commonly test antigen-specific antibodies, mainly as responses to vaccines, and not routinely.T-cell receptors (TCR) and B-cell receptors (BCR) repertoires are measured by spectratyping in 14 out of 35 Centers (4 as clinical practice and 10 in selected trials), or, in selected trials, by next generation sequencing (in 11 out of 35 the participating Centers).Conclusions:Results of this survey indicate that country- and center expertise are associated with heterogeneous and distinct protocols, and underline the clinical need to harmonize methods and to provide practical recommendations for monitoring post-transplant immune reconstitution, both for routine purposes and investigational studies. Adequate reporting and connection between individual Centers exploiting these data will foster collaborative and comparative research studies, with the ultimate goals of improving patient care and refining our understanding of the immunological correlates to clinical outcome.Acknowledgments:R. Ram, M. A. Diaz, G. McQuaker, D. Russo, E. Faber, P. Chiusolo, C. Rössig, S. M. Martin, A. Anagnostopoulos, M. Stelljes, K. Orchard, P. Jindra, A. Sampol, K. Patrick, M. A. Bekadja, J. Gayoso, A. Olivieri, J. Passweg, E. Jost, H Labussiere-Wallet, Y Koc, A. Lange, I. Garcia Cadenas, N. Kröger, A. Biondi, N. Milpied, D. Olive, E. Lanino, G. Stuhler, J.H. Dalle, J.R. Cabrera Marín, F. Ciceri, D. Uckan-Cetinkaya, R. Parody Porras, G. Kriván. DisclosuresCiceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

IMPre: An Accurate and Efficient Software for Prediction of T- and B-Cell Receptor Germline Genes and Alleles from Rearranged Repertoire Data.

Large-scale study of the properties of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires through next-generation sequencing is providing excellent insights into the understanding of adaptive immune responses. Variable(Diversity)Joining [V(D)J] germline genes and alleles must be characterized in detail to facilitate repertoire analyses. However, most species do not have well-characterized TCR/BCR germline genes because of their high homology. Also, more germline alleles are required for humans and other species, which limits the capacity for studying immune repertoires. Herein, we developed “Immune Germline Prediction” (IMPre), a tool for predicting germline V/J genes and alleles using deep-sequencing data derived from TCR/BCR repertoires. We developed a new algorithm, “Seed_Clust,” for clustering, produced a multiway tree for assembly and optimized the sequence according to the characteristics of rearrangement. We trained IMPre on human samples of T-cell receptor beta (TRB) and immunoglobulin heavy chain and then tested it on additional human samples. Accuracy of 97.7, 100, 92.9, and 100% was obtained for TRBV, TRBJ, IGHV, and IGHJ, respectively. Analyses of subsampling performance for these samples showed IMPre to be robust using different data quantities. Subsequently, IMPre was tested on samples from rhesus monkeys and human long sequences: the highly accurate results demonstrated IMPre to be stable with animal and multiple data types. With rapid accumulation of high-throughput sequence data for TCR and BCR repertoires, IMPre can be applied broadly for obtaining novel genes and a large number of novel alleles. IMPre is available at https://github.com/zhangwei2015/IMPre.

Evaluation of the Antigen-Experienced B-Cell Receptor Repertoire in Healthy Children and Adults.

Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1–CDR3 sequence, but different subclasses. Together, these data suggest that a single antigen can provoke a B-cell response with BR of different subclasses and that, during the course of an immune response, some B cells change their isotype without acquiring additional SHM or can directly switch to different isotypes.