B-cell Receptor Repertoire Research Articles

Prediction of immunotherapy response in deficient mismatch repair colorectal cancers using an immune-enhanced exome and transcriptome platform.

270 Background: While immune checkpoint blockade (ICB) is used to treat metastatic deficient mismatch repair (dMMR) colorectal cancers (CRCs), such tumors are heterogeneous and resistance to ICB is frequent. To date, biomarkers that can consistently predict patient response to ICB are lacking. We determined whether biomarkers that integrate tumor and immune-related molecular mechanisms could predict ICB response. Methods: Consecutive patients with metastatic dMMR CRCs (N=32) who had been treated with anti-PD-1 therapy were profiled using a validated immune-enhanced exome and transcriptome platform (ImmunoID NeXT, Personalis) using tumor and paired normal tissue. This platform provides antigen-specificity of T or B cells, indicated by the TCR or BCR respectively, neoantigen prediction, tumor mutation burden (TMB), immune profile, HLA allele-specific loss of heterogeneity (LOH) and a Neoantigen Presentation Score (NEOPS) that combines a neoantigen prediction tool with mechanisms of immune evasion. Tumor variables were analyzed in relationship to objective tumor response, and a Cox proportional hazards model was fit to predict progression-free survival (PFS) or overall survival (OS). Results: In CRCs, TCR and BCR repertoire diversity were each significantly increased in tumors from responders versus those with stable or progressive disease after anti-PD-1 treatment (p=0.015 and p=0.048, respectively). Neoantigen burden score, TMB, and HLA allele-specific LOH (reflects loss of antigen presentation machinery) were each unable to distinguish responders from nonresponders. An immune checkpoint gene expression signature was increased in nonresponders relative to responders (P= 0.005). Neoantigen presentation score (NEOPS) was prognostic when adjusted for tumor grade only for OS (p=0.048). Conclusions: The T-cell and B-cell receptor (TCR/BCR) repertoire, as well as NEOPS, were each significantly predictive of favorable response to anti-PD-1 therapy in dMMR tumors. In addition, an immune checkpoint gene expression signature was predictive of resistance to ICB.

Anti-retinal immune response in sarcoid uveitis: A potential role for PCLO as an antigenic target.

To explore the autoimmune component of sarcoid uveitis (SU) by analyzing serum anti-retinal antibodies (ARAs), identifying targeted retinal proteins, T- and B-cell receptor repertoires and HLA genotype. Material from 45 sarcoidosis patients with no presenting uveitis (SNPU) and 46 with SU was analyzed. Serum ARAs and targeted retinal layers were assessed using indirect immunofluorescence staining. HuScan analysis identified autoantibody-targeted linear epitopes. Validation included a bead-based assay for anti-Piccolo Presynaptic Cytomatrix Protein (PCLO) antibodies and an ELISpot assay for PCLO-reactive T-lymphocytes. T cell receptor beta (TCRB) and B cell receptor heavy (BCRH) repertoire analyses were performed using next-generation sequencing and HLA class II genotypes were determined by sequence-specific primer analysis. ARAs were more prevalent in SU patients than in SNPU patients (52 vs. 22%, p=0.003), with significant more reactivity against the nuclear retinal layer (32 vs. 7%, p=0.005). HuScan identified autoantibodies against three retinal proteins, including PCLO. Bead-based analysis showed higher anti-PCLO autoantibody levels in ARA-positive patients (median: 913.3 vs. 544.5, p=0.035), and PCLO-directed T-lymphocytes were present in ARA-positive SU patients. Two TCRB clusters were identified in four unique ARA positive patients, while absent in ARA negative patients. No HLA allele association with ARA status could be detected. Our findings reveal an association between serum ARA-positivity and SU, suggesting a link to autoimmune processes. An humoral and cellular response against the retinal protein PCLO was identified, highlighting PCLO as a potential autoimmune target in ARA-positive patients. Additionally, specific TCRB clusters were found to correlate with ARA status.

Systematic evaluation of intratumoral and peripheral BCR repertoires in three cancers

The current understanding of humoral immune response in cancer patients suggests that tumors may be infiltrated with diffuse B cells of extra-tumoral origin or may develop organized lymphoid structures, where somatic hypermutation and antigen-driven selection occur locally. These processes are believed to be significantly influenced by the tumor microenvironment through secretory factors and biased cell-cell interactions. To explore the manifestation of this influence, we used deep unbiased immunoglobulin profiling and systematically characterized the relationships between B cells in circulation, draining lymph nodes (draining LNs), and tumors in 14 patients with three human cancers. We demonstrated that draining LNs are differentially involved in the interaction with the tumor site, and that significant heterogeneity exists even between different parts of a single lymph node (LN). Next, we confirmed and elaborated upon previous observations regarding intratumoral immunoglobulin heterogeneity. We identified B cell receptor (BCR) clonotypes that were expanded in tumors relative to draining LNs and blood and observed that these tumor-expanded clonotypes were less hypermutated than non-expanded (ubiquitous) clonotypes. Furthermore, we observed a shift in the properties of complementarity-determining region 3 of the BCR heavy chain (CDR-H3) towards less mature and less specific BCR repertoire in tumor-infiltrating B-cells compared to circulating B-cells, which may indicate less stringent control for antibody-producing B cell development in tumor microenvironment (TME). In addition, we found repertoire-level evidence that B-cells may be selected according to their CDR-H3 physicochemical properties before they activate somatic hypermutation (SHM). Altogether, our work outlines a broad picture of the differences in the tumor BCR repertoire relative to non-tumor tissues and points to the unexpected features of the SHM process.

Systematic evaluation of intratumoral and peripheral BCR repertoires in three cancers.

The current understanding of humoral immune response in cancer patients suggests that tumors may be infiltrated with diffuse B cells of extra-tumoral origin or may develop organized lymphoid structures, where somatic hypermutation and antigen-driven selection occur locally. These processes are believed to be significantly influenced by the tumor microenvironment through secretory factors and biased cell-cell interactions. To explore the manifestation of this influence, we used deep unbiased immunoglobulin profiling and systematically characterized the relationships between B cells in circulation, draining lymph nodes (draining LNs), and tumors in 14 patients with three human cancers. We demonstrated that draining LNs are differentially involved in the interaction with the tumor site, and that significant heterogeneity exists even between different parts of a single lymph node (LN). Next, we confirmed and elaborated upon previous observations regarding intratumoral immunoglobulin heterogeneity. We identified B cell receptor (BCR) clonotypes that were expanded in tumors relative to draining LNs and blood and observed that these tumor-expanded clonotypes were less hypermutated than non-expanded (ubiquitous) clonotypes. Furthermore, we observed a shift in the properties of complementarity-determining region 3 of the BCR heavy chain (CDR-H3) towards less mature and less specific BCR repertoire in tumor-infiltrating B-cells compared to circulating B-cells, which may indicate less stringent control for antibody-producing B cell development in tumor microenvironment (TME). In addition, we found repertoire-level evidence that B-cells may be selected according to their CDR-H3 physicochemical properties before they activate somatic hypermutation (SHM). Altogether, our work outlines a broad picture of the differences in the tumor BCR repertoire relative to non-tumor tissues and points to the unexpected features of the SHM process.

Learning the language of antibody hypervariability

Protein language models (PLMs) have demonstrated impressive success in modeling proteins. However, general-purpose "foundational" PLMs have limited performance in modeling antibodies due to the latter's hypervariable regions, which do not conform to the evolutionary conservation principles that such models rely on. In this study, we propose a transfer learning framework called Antibody Mutagenesis-Augmented Processing (AbMAP), which fine-tunes foundational models for antibody-sequence inputs by supervising on antibody structure and binding specificity examples. Our learned feature representations accurately predict mutational effects on antigen binding, paratope identification, and other key antibody properties. We experimentally validate AbMAP for antibody optimization by applying it to refine a set of antibodies that bind to a SARS-CoV-2 peptide, and obtain an 82% hit-rate and up to 22-fold increase in binding affinity. AbMAP also unlocks large-scale analyses of immune repertoires, revealing that B-cell receptor repertoires of individuals, while remarkably different in sequence, converge toward similar structural and functional coverage. Importantly, AbMAP's transfer learning approach can be readily adapted to advances in foundational PLMs. We anticipate AbMAP will accelerate the efficient design and modeling of antibodies, expedite the discovery of antibody-based therapeutics, and deepen our understanding of humoral immunity.

EXPRESSION OF IMMUNOGLOBULIN LIGHT CHAIN GENES IN STEREOTYPED CASES FROM UKRAINIAN COHORT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS.

Analysis of immunoglobulin heavy chain gene (IGHV) rearrangements expressed in chronic lymphocytic leukemia (CLL) cells has provided insights into the B-cell receptor (BCR) repertoire in CLL. In more than 40% of CLL patients, (quasi)identical or stereotyped BCR is expressed. The recent data point at the non-stochastic expression of immunoglobulin light lambda (IGLV) or kappa (IGKV) chains as well. Several pairs of IGHV and IGK/LV have been described for some major stereotyped subsets, but most subsets have not been characterized. To study the IGK/LV gene expression in stereotyped CLL cases. Analysis was performed in a group of 105 CLL patients with stereotyped BCR. The cases with stereotyped BCRs were identified according to Agathangelidis et al. (2021). The IGHV and IGK/LV gene expressions were studied by a polymerase chain reaction followed by direct sequencing. The expression of the IGK/LV genes in the most presented major stereotyped subsets (#1, #2, #3C3, #4, #6, #28а) was in agreement with the data reported by other authors. For the cases of subsets #5b, #9D1, #9D4, #16, #50, #59, and #77, differences were found. The new data on the IGK/LV gene expression in 55 minor clusters were presented. A number of patterns of the IGK/LV gene expression depending on the phylogenetic clan and mutational status of the IGHV genes have been described. The non-stochastic distribution of the IGKV/LV gene expression in the individual stereotyped subsets was confirmed. Taking into account the complementary role of the light chains in antigen recognition by the clonotypic BCRs, it was suggested that the subsets with the heterogeneous IGK/LV expression might be reclassified and divided into separate subgroups based on the IGHV and IGK/LV association.

Donor HLA-DQ reactive B cells clonally expand under chronic immunosuppression and include atypical CD21 low CD27 - B cells with high-avidity germline B-cell receptors.

Long-term allograft survival is limited by humoral-associated chronic allograft rejection, suggesting inadequate constraint of humoral alloimmunity by contemporary immunosuppression. Heterogeneity in alloreactive B cells and the incomplete definition of which B cells participate in chronic rejection in immunosuppressed transplant recipients limits our ability to develop effective therapies. Using a double-fluorochrome single-HLA tetramer approach combined with single-cell in vitro culture, we investigated the B-cell receptor (BCR) repertoire characteristics, avidity, and phenotype of donor HLA-DQ reactive B cells in a transplant recipient with end-stage donor specific antibody (DSA)-associated cardiac allograft vasculopathy while receiving maintenance immunosuppression (tacrolimus, mycophenolate mofetil, prednisone). Donor DQB1*03:02/DQA1*03:01 (DQ8)-reactive IgG+ B cells were enriched for minimally mutated and germline encoded high avidity BCRs (median K D 4.26×10 -09 ) with an atypical, antigen-experienced and proliferative phenotype (CD27 - CD21 low CD71 + CD11c +/- ). These B cells coexisted with a smaller subset of more highly mutated, affinity matured IgG+CD27+ B cells. Circulating donor-reactive B cells and DSA remained detectable after rituximab, contrasting with the marked reduction in DSA after allograft explant and retransplant. Together, these findings define the persistence of germline high-avidity HLA-DQ alloreactive B cells and their co-existence with affinity matured clones that were both driven by the allograft despite conventional immunosuppression.

T-cell receptor and B-cell receptor repertoires profiling in pleural tuberculosis.

Tuberculosis (TB) is a leading cause of death worldwide from a single infectious agent. In China the most common extra-pulmonary TB (EPTB) is pleural tuberculosis (PLTB). An important clinical feature of PLTB is that the lymphocytes associated with TB will accumulate in the pleural fluid. The adaptive immune repertoires play important roles in Mycobacterium tuberculosis (Mtb) infection. In this study, 10 PLTB patients were enrolled, and their Peripheral Blood Mononuclear Cells(PBMCs) and Pleural Effusion Mononuclear Cells(PEMCs) were collected. After T cells were purified from PBMCs and PEMCs, high-throughput immunosequencing of the TCRβ chain (TRB), TCRγ chain(TRG), and B cell receptor(BCR) immunoglobulin heavy chain (IGH) were conducted on these samples. The TRB, TRG, and BCR IGH repertoires were characterized between the pleural effusion and blood in PLTB patients, and the shared clones were analyzed and collected. The binding activity of antibodies in plasma and pleural effusion to Mtb antigens was tested which indicates that different antibodies responses to Mtb antigens in plasma and pleural effusion in PLTB patients. Moreover, GLIPH2 was used to identify the specificity groups of TRB clusters and Mtb-specific TRB sequences were analyzed and collected by VJ mapping. We characterize the adaptive immune repertoires and identify the shared clones and Mtb-specific clones in pleural effusion and blood in PLTB patients which can give important clues for TB diagnosis, treatment, and vaccine development.

Investigating the Role of B Cell Receptors in Pathogen Recognition and Immune Response Activation: Implications for Infection Control and Therapeutic Development

B cell receptors (BCRs) are critical components of the adaptive immune system, enabling precise pathogen recognition and the activation of immune responses. This research examines the molecular and genetic mechanisms underlying BCR function, focusing on their role in detecting, binding, and neutralizing infectious agents. By exploring antigen-BCR interactions, we aim to elucidate how BCRs achieve their remarkable specificity and affinity, ultimately shaping the strength and duration of immune responses. Through advanced immunological assays, flow cytometry, and bioinformatics, this study characterizes the diversity of BCR expression across various infectious models. Genetic processes such as somatic hypermutation and clonal selection are analyzed to understand how BCR repertoires adapt to rapidly evolving pathogens. Predictive models developed using machine learning identify biomarkers within BCR pathways, providing insights into acute infections, chronic diseases, and autoimmune conditions. The findings highlight variations in BCR function that influence immune resilience and susceptibility to infections. These insights inform vaccine development, antibody engineering, and therapeutic strategies targeting BCR pathways. This research underscores the potential of BCRs as biomarkers and therapeutic targets, paving the way for innovative approaches in infection control and immune modulation, ultimately advancing precision medicine and adaptive immunity research.

High throughput sequencing reveals alterations in B cell receptor repertoires associated with the progression of hepatic cirrhosis to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is developed as a consequence of chronic liver cirrhosis, and both diseases are difficult to diagnose and differentiate. Accurate noninvasive biomarkers for HCC and liver cirrhosis are urgently needed. Here we used high-throughput sequencing to characterize the B cell receptor (BCR) repertoires from 36 HCC tumor samples and 10 liver cirrhosis (LC) tissue biopsies to understand the immune alterations during hepatic carcinogenesis. The principal components analysis (PCA) showed that the pattern of BCR in HCC was distinct from that in LC. As measured by Clonality and Shannon indexes, the diversity of BCR repertoire was significantly lower in HCC than in LC (P < 0.01). Our results corroborated that the BCR diversity and composition could be closely correlated with hepatic carcinogenesis. And BCR repertoire may be used to predict the progression of HCC and design targeting immunotherapy in the near future.

BCR, not TCR, repertoire diversity is associated with favorable COVID-19 prognosis.

The SARS-CoV-2 pandemic has had a widespread and severe impact on society, yet there have also been instances of remarkable recovery, even in critically ill patients. In this study, we used single-cell RNA sequencing to analyze the immune responses in recovered and deceased COVID-19 patients during moderate and critical stages. Expanded T cell receptor (TCR) clones were predominantly SARS-CoV-2-specific, but represented only a small fraction of the total repertoire in all patients. In contrast, while deceased patients exhibited monoclonal B cell receptor (BCR) expansions without COVID-19 specificity, survivors demonstrated diverse and specific BCR clones. These findings suggest that neither TCR diversity nor BCR monoclonal expansions are sufficient for viral clearance and subsequent recovery. Differential gene expression analysis revealed that protein biosynthetic processes were enriched in survivors, but that potentially damaging mitochondrial ATP metabolism was activated in the deceased. This study underscores that BCR repertoire diversity, but not TCR diversity, correlates with favorable outcomes in COVID-19.

Single-Cell Antigen Receptor Sequencing in Pigs with Influenza.

Understanding the pulmonary adaptive immune system of pigs is important as respiratory pathogens present a major challenge for swine producers and pigs are increasingly used to model human pulmonary diseases. Single-cell RNA sequencing (scRNAseq) has accelerated the characterization of cellular phenotypes in the pig respiratory tract under both healthy and diseased conditions. However, combining scRNAseq with recovery of paired T cell receptor (TCR) α and β chains as well as B cell receptor (BCR) heavy and light chains to interrogate their repertoires has not to our knowledge been demonstrated for pigs. Here, we developed primers to enrich porcine TCR α and β chains along with BCR κ and λ light chains and IgM, IgA, and IgG heavy chains that are compatible with the 10x Genomics VDJ sequencing protocol. Using these pig-specific assays, we sequenced the T and B cell receptors of cryopreserved lung cells from CD1D-expressing and -deficient pigs after one or two infections with influenza A virus (IAV) to examine whether natural killer T (NKT) cells alter pulmonary TCR and BCR repertoire selection. We also performed paired single-cell RNA and receptor sequencing of FACS-sorted T cells longitudinally sampled from the lungs of IAV-vaccinated and -infected pigs to track clonal expansion in response to IAV exposure. All pigs presented highly diverse repertoires. Pigs re-exposed to influenza antigens from either vaccination or infection exhibited higher numbers of expanded CD4 and CD8 T cell clonotypes with activated phenotypes, suggesting potential IAV reactive T cell populations. Our results demonstrate the utility of high throughput single-cell TCR and BCR sequencing in pigs.

Analysis of B-cell receptor repertoire to evaluate the immunogenicity of SARS-CoV-2 RBD mRNA vaccine: MAFB-7256a (DS-5670d).

A monovalent Omicron XBB.1.5 mRNA RBD analogue vaccine, MAFB-7256a (DS-5670d), was newly developed and approved in Japan in the Spring of 2024 for the prevention of COVID-19. However, clinical efficacy data for this vaccine are currently lacking. We previously established the Quantification of Antigen-specific Antibody Sequence (QASAS) method to assess the response to SARS-CoV-2 vaccination at the mRNA level using B-cell receptor (BCR) repertoire assays and the Coronavirus Antibody Database (CoV-AbDab). Here, we used this method to evaluate the immunogenicity of MAFB-7256a. We analyzed repeated blood samples using the QASAS method from three healthy volunteers before and after MAFB-7256a vaccination. BCR response increased rapidly one week post-vaccination and then decreased, as with conventional vaccine. Notably, the matched sequences after MAFB-7256a vaccination specifically bound to the receptor-binding domain (RBD), with no sequences binding to other epitopes. These results validate that MAFB-7256a is an effective vaccine that exclusively induces antibodies specific for the RBD, demonstrating its targeted immunogenic effect.

APRIL/BAFF upregulation is associated with clonal B-cell expansion in Hunner-type interstitial cystitis.

Hunner-type interstitial cystitis (HIC) is a chronic inflammatory disease of the urinary bladder with an unknown etiology. We conducted comprehensive immunogenomic profiling of bladder specimens obtained by biopsy and cystectomy from 37 patients with HIC. Next-generation RNA sequencing demonstrated abundant plasma cell infiltration with frequent light chain restriction in HIC-affected bladder tissue. Subsequent analysis of the B-cell receptor repertoire revealed spatial and temporal expansion of B-cell clones. The extent of B-cell clonal expansion was significantly correlated with the gene expression levels of TNFSF13 and TNFSF13B, which encode APRIL and BAFF, respectively. These findings indicate that APRIL and BAFF are the key regulators of clonal B-cell expansion in HIC and might serve as therapeutic targets in this debilitating disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BcRflow: a Nextflow pipeline for characterizing B cell receptor repertoires from non-targeted transcriptomic data.

B cells play a critical role in the adaptive recognition of foreign antigens through diverse receptor generation. While targeted immune sequencing methods are commonly used to profile B cell receptors (BCRs), they have limitations in cost and tissue availability. Analyzing B cell receptor profiling from non-targeted transcriptomics data is a promising alternative, but a systematic pipeline integrating tools for accurate immune repertoire extraction is lacking. Here, we present bcRflow, a Nextflow pipeline designed to characterize BCR repertoires from non-targeted transcriptomics data, with functional modules for alignment, processing, and visualization. bcRflow is a comprehensive, reproducible, and scalable pipeline that can run on high-performance computing clusters, cloud-based computing resources like Amazon Web Services (AWS), the Open OnDemand framework, or even local desktops. bcRflow utilizes institutional configurations provided by nf-core to ensure maximum portability and accessibility. To demonstrate the functionality of the bcRflow pipeline, we analyzed a public dataset of bulk transcriptomic samples from COVID-19 patients and healthy controls. We have shown that bcRflow streamlines the analysis of BCR repertoires from non-targeted transcriptomics data, providing valuable insights into the B cell immune response for biological and clinical research. bcRflow is available at https://github.com/Bioinformatics-Core-at-Childrens/bcRflow.

Identification of primary mediastinal B-cell lymphomas with higher clonal dominance and poorer outcome using 5′RACE

AbstractThere is a scarcity of data on the tumor B-cell receptor (BCR) repertoire and lymphoid microenvironment in primary mediastinal B-cell lymphoma (PMBL). We applied 5ʹ rapid amplification of complimentary DNA ends (5′RACE) to tumor RNA samples from 137 patients with PMBL with available gene expression profiling and next-generation sequencing data. We obtained 5′RACE results for 75 of the 137 (54.7%) patients with the following clinical characteristics: median age (range), 33 years (18-64); female, 53.3%; performance status score 0 to 1, 86.7%; stage I to II, 57.3%; first-line treatment with anti-CD20 plus doxorubicin-based chemotherapy, 100%. Among the 60 biopsies that expressed a productive BCR, we highlighted a strong somatic hypermutation profile, defined as <98% identity to the germ line sequence, with 58 (96.7%) patients carrying mutated IgVH. We then identified a subgroup of 12 of the 75 patients (16%) with a worse prognosis (progression-free survival [PFS]: hazard ratio [HR], 17; overall survival [OS]: HR, 21) that was associated with the highest clonal dominance (HCD) status, defined as the dominant clonotype representing >81.1% and >78.6% of all complementarity-determining region 3 sequences for IgVH and IgVL, respectively. When compared with other patients, this subgroup had similar clinical characteristics but a greater median allele frequency for all somatic variants, a decreased BCR diversity, and greater expression of PDL1/PDL2 and MS4A1 genes, suggesting greater tumoral infiltration. We confirmed this poorer prognosis in a multivariate model and in an independent validation cohort in which 6 of 37 (16%) PMBL patients exhibited HCD (PFS: HR, 12; OS: HR, 17).

Immunotherapy shapes B-cell receptor repertoire to induce anti-tumor antibodies production in colon and lung cancer

Immunotherapy shapes B-cell receptor repertoire to induce anti-tumor antibodies production in colon and lung cancer

Computational detection of antigen-specific B cell receptors following immunization

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.