Immunoglobulin Heavy Chain Repertoire Research Articles

IGH repertoire analysis at scale: deciphering the complexity of B cell infiltration and migration in esophageal squamous cell carcinoma.

Tumor-infiltrating B-lineage cells have become predictors of prognosis and immunotherapy responses in various cancers. However, limited knowledge about their infiltration and migration patterns has hindered the understanding of their anti-tumor functions. Here, we examined the immunoglobulin heavy chain (IGH) repertoires in 496 multi-regional tumor, 107 normal tissue, and 48 metastatic lymph node samples obtained from 107 patients with esophageal squamous cell carcinoma (ESCC). Our study revealed higher IgG-type B-lineage cells infiltration in tumors than in healthy tissue, which was associated with improved patient outcomes. Genes such as ACTN1, COL6A5, and pathways like focal adhesion, which shapes the physical structure of tumors, could affect B-lineage cell infiltration. Notably, the IGH sequence was used as an identity-tag to monitor B cell migration, and their infiltration schema within the tumor were depicted based on our multi-regional tumor specimens. This analysis revealed an escalation in B cell clones overlapped between metastatic lymph nodes and tumors. Therefore, the Lymph Node Activation Index was defined, which could predict the outcomes of patients with lymph node metastasis. This research introduces a novel framework for probing B cell infiltration and migration within the tumor microenvironment using large-scale transcriptome data, while simultaneously providing fresh perspectives on B cell immunology within ESCC.

Host immune signatures as predictors of response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC)

Abstract Background Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. However, clinical responses to systemic therapies in mRCC remain largely unpredictable and robust biomarkers are still lacking. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with ICI-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. T-cell receptor sequencing (TCR-seq) was performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-sequencing (RNA-seq) was performed on available pre-treatment PBMCs and primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts (186 VEGF-TT, 126 ICI and 74 ICI+VEGF-TT). Following quality-control, TCR-seq data were available for 367 patients (228 pre-treatment and 139 on-treatment), while RNA-seq data were available for 105 PBMC and 17 tumor-derived pre-treatment samples. In the TCR-seq analysis, responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06) (Fig. 1A), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). Therefore, responders to ICI-based regimens seem to have a more oligoclonal TCR repertoire (increased clonality) at baseline with no treatment-induced changes, whereas non-responders to ICI-based regimens seem to evolve from a more polyclonal to a more oligoclonal TCR repertoire in response to treatment. The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01) (Fig. 1B). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Furthermore, while shared clonotypes were detected between blood and tumor samples, there were no differences in the Jaccard similarity index between responders and non-responders to ICI-based or VEGF-TT regimens. Figure 1: (A) Evaluation of pre-treatment and post-treatment TCR clonality in responders vs. non-responders across treatment cohorts. (B) Differences in the fraction of IGH isotypes between responders and non-responders across treatment cohorts. Conclusions We were successfully able to characterize T-cell receptor and B-cell IgH repertoires in a large cohort of patients with mRCC, and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development. CDMRP DOD Funding: yes

Association of host immune signatures with response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC).

4550 Background: Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with immunotherapy-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods: We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. Bulk RNA-sequencing (RNA-seq) and T-cell receptor sequencing (TCR-seq) were performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-seq was additionally performed on available pre-treatment primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results: In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts. Following quality-control, TCR-seq and RNA-seq data were available for 367 and 134 patient samples, respectively. Responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Conclusions: We were successfully able to characterize T-cell receptor and IgH repertoires in a large cohort of patients with mRCC and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development.

Vaccination of SARS-CoV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages

Vaccination of SARS-CoV-2 convalescent individuals generates broad and potent antibody responses. Here, we isolate 459 spike-specific monoclonal antibodies (mAbs) from two individuals who were infected with the index variant of SARS-CoV-2 and later boosted with mRNA-1273. We characterize mAb genetic features by sequence assignments to the donors’ personal immunoglobulin genotypes and assess antibody neutralizing activities against index SARS-CoV-2, Beta, Delta, and Omicron variants. The mAbs used a broad range of immunoglobulin heavy chain (IGH) V genes in the response to all sub-determinants of the spike examined, with similar characteristics observed in both donors. IGH repertoire sequencing and B cell lineage tracing at longitudinal time points reveals extensive evolution of SARS-CoV-2 spike-binding antibodies from acute infection until vaccination five months later. These results demonstrate that highly polyclonal repertoires of affinity-matured memory B cells are efficiently recalled by vaccination, providing a basis for the potent antibody responses observed in convalescent persons following vaccination.

T-cell and B-cell repertoire diversity are selectively skewed in children with idiopathic nephrotic syndrome revealed by high-throughput sequencing.

Clinical studies suggest that the dysfunction of T cells and B cells may play an essential role in the pathogenesis of idiopathic nephrotic syndrome (INS), but laboratory evidence is lacking. Therefore, this study explored T-cell receptor (TCR) and B-cell receptor (BCR) profiling in children with idiopathic nephrotic syndrome. High-throughput sequencing technology was used to profile the TCR and BCR repertoires in children with INS. Peripheral blood was collected from ten INS patients, including five vinculin autoantibody-positive patients and five vinculin autoantibody-negative patients, before and after treatment. TCR and BCR libraries were constructed by 5'-RACE and sequenced by a DNBSEQ-T7 sequencer, and sequence analyses were performed using ReSeqTools, FastP, MiXCR, and VDJtools. The TRA (T-cell receptor α), TRG (T-cell receptor γ), and IGH (immunoglobulin heavy chain) repertoires of the INS group were occupied by highly abundant clonotypes, whereas small clonotypes occupied the healthy group, especially TRA. A significant increase in the Shannon-Weaver index was observed for the TRA and TRG repertoires after treatment in vinculin autoantibody-negative patients, but a significant increase in the IGH repertoire after treatment was observed in vinculin autoantibody-positive patients. The frequency of some V-J pairs was significantly enriched in steroid-sensitive nephrotic syndrome patients. The usage frequency of the V and J genes was skewed in patients, which seemed not related to immunosuppressive therapy. However, after effective treatment, dynamic changes in the size of the individual clonotype were observed. T-cell and B-cell immunity contribute to the pathogenesis of different INSs. Video: (MP4 99,786KB).

Angioimmunoblastic T-cell lymphoma with predominant CD8+ tumor-infiltrating T-cells is a distinct immune pattern with an immunosuppressive microenvironment.

Angioimmunoblastic T-cell lymphoma (AITL) has a rich tumor microenvironment (TME) that typically harbors plenty of CD4+tumor infiltrating lymphocytes, (TIL)-T-cells (so called common AITL). Nonetheless, AITL with large numbers of CD8+TIL-Ts that outnumber CD4+cells have been observed (CD8-predominant AITL). However, detailed comparison of CD8-predominant AITL and common AITL are still lacking. We compared clinicopathological features, TIL subsets, TME T cell receptor-β (TRB), and immunoglobulin heavy chain (IGH) repertoires, and gene expression profiles in six CD8-predominant and 12 common AITLs using case-control matching (2014 to 2019). Comparing with common AITLs, CD8-predominant AITLs showed more frequent edema (P = 0.011), effusion (P = 0.026), high elevated plasma EBV-DNA (P = 0.008), and shorter survival (P = 0.034). Moreover, they had more pronounced eosinophil increase (P = 0.004) and a higher Ki67 index (P = 0.041). Flow cytometry revealed an inverted CD4/CD8 ratio in TIL-Ts and lower TIL-B proportions (P = 0.041). TRB repertoire metrics deteriorated, including lower productive clones (P = 0.014) and higher clonality score (P = 0.019). The IGH repertoire was also narrowed, showing a higher proportion of the top 10 clones (P = 0.002) and lower entropy (P = 0.027). Gene expression analysis showed significant enrichment for upregulated negative regulation of immune system processes and downregulated T-cell activation and immune cell differentiation. Our findings demonstrated that CD8-predominant AITL is a distinct immune pattern of AITL characterized by anti-tumor immunity impairment and an immunosuppressive microenvironment. These characteristics can interpret its severe clinical manifestations and poor outcomes.

Myeloablative autologous haematopoietic stem cell transplantation resets the B cell repertoire to a more naïve state in patients with systemic sclerosis

ObjectivesMyeloablative autologous haematopoietic stem cell transplant (HSCT) was recently demonstrated to provide significant benefit over cyclophosphamide (CYC) in the treatment of diffuse cutaneous systemic sclerosis (dcSSc) in the Scleroderma: Cyclophosphamide...

Memory persistence and differentiation into antibody-secreting cells accompanied by positive selection in longitudinal BCR repertoires.

The stability and plasticity of B cell-mediated immune memory ensures the ability to respond to the repeated challenges. We have analyzed the longitudinal dynamics of immunoglobulin heavy chain repertoires from memory B cells, plasmablasts, and plasma cells from the peripheral blood of generally healthy volunteers. We reveal a high degree of clonal persistence in individual memory B cell subsets, with inter-individual convergence in memory and antibody-secreting cells (ASCs). ASC clonotypes demonstrate clonal relatedness to memory B cells, and are transient in peripheral blood. We identify two clusters of expanded clonal lineages with differing prevalence of memory B cells, isotypes, and persistence. Phylogenetic analysis revealed signs of reactivation of persisting memory B cell-enriched clonal lineages, accompanied by new rounds of affinity maturation during proliferation and differentiation into ASCs. Negative selection contributes to both persisting and reactivated lineages, preserving the functionality and specificity of B cell receptors (BCRs) to protect against current and future pathogens.

Inadequate Activation of γδT- and B-cells in Patient with Wiskott-Aldrich Syndrome (WAS) Portrayed by TRG and IGH Repertoire Analyses

Patients with Wiskott-Aldrich syndrome (WAS) harbor mutations in the WAS gene and suffer from immunodeficiency, microthrombocytopenia, and eczema. T-cells play an important role in immune response in the skin and the γδT-cells have an important role in skin homeostasis. Since WAS patients often present with eczema, we wanted to examine whether the T-cell receptor gamma (TRG) repertoire of the γδT-cells is affected in these patients. In addition, the immunoglobulin heavy chain (IGH) repertoire from genomic DNA of WAS patients was not yet studied. Thus, we sought to determine the effects that specific WAS mutations from our patients have in shaping the TRG and IGH immune repertoires. We collected clinical and genetic data on four WAS patients, each harboring a different mutation in the WAS gene. Using next-generation sequencing (NGS), we analyzed their TRG and IGH repertoires using genomic DNA isolated from their peripheral blood. We analyzed the TRG and IGH repertoire sequences to show repertoire restriction, clonal expansions, preferential utilization of specific V genes, and unique characteristics of the antigen binding region in WAS patients with eczema compared to healthy controls. Both the TRG and IGH repertoire showed diverse repertoire comparable to healthy controls on one the hand, and on the other hand, the IGH repertoire showed increased diversity, more evenly distributed repertoire and immaturity of the antigen binding region. Thus, we demonstrate by analyzing the repertoire based on genomic DNA, the various effect that WAS mutations have in shaping the TRG and IGH adaptive immune repertoires.

P1274: EARLY DETECTION OF NON-HODGKIN LYMPHOMA IN PREDISPOSED PATIENT GROUPS THROUGH IMMUNOGENETIC SEQUENCING

Background: Since chronic lymphocytic leukemia development can be detected up to 16 years prior to diagnosis through immunogenetic sequencing, we aim to evaluate whether early detection through immunogenetic sequencing translates to other types of non-Hodgkin lymphoma (NHL). A particularly interesting group with potential clinical utility consists of patients with underlying conditions that confer an increased risk of lymphoma and leukemia. Generally, this group is characterized by dysregulation of the immune system, in the form of autoimmune diseases, primary immunodeficiencies, immunosuppressive treatment and several infectious diseases. Aims: To evaluate the capacity of immunogenetic sequencing for early detection of NHL and leukemia in patient groups at increased risk. Methods: We selected two patient groups with an increased risk of NHL and leukemia: solid organ transplant recipients and patients with auto-immune disease. For each group, we selected patients who had developed NHL and controls with the same underlying condition matched on sex, age and sampling window. Currently, we have included 16 cardiac and 10 renal transplant recipients and 10 primary Sjögren’s syndrome (SjS) patients (in total 18 cases and 18 controls). A total of 114 longitudinal pre-diagnostic samples were included in the study, taken a median of 1.2 years before diagnosis (IQR 3.5 years). Genomic DNA was isolated from the PBMC samples of the participants and the immunoglobulin heavy chain (IGH) was sequenced on the Illumina Miseq platform. All PBMC samples were taken after SjS diagnosis or solid organ transplantation and prior to NHL diagnosis. Results: In 7 out of 18 NHL patients we identified clonotypes in prediagnostic PBMC samples that were present at an abnormal frequency compared to the healthy background clonotypes. Notably, distribution of the IGH gene repertoire in controls was normal. Prevalence of IGH gene repertoire skewing was particularly pronounced among SjS patients (3 out of 5 patients) and cardiac transplant recipients (4 out of 8 patients) compared to renal transplant recipients (1 out of 5 patients). IGH gene repertoire skewing was restricted to samples taken within 5 months to two years before diagnosis for the majority of the cases. In solid organ transplant recipients with an abnormal clonotype, the degree of skewing of the IGH gene repertoire increased over time towards the moment of NHL diagnosis or remained stable at elevated levels. For the SjS patients, two out of the three abnormal clonotypes utilized the IGHV1-69/IGHJ4 or IGHV4-59/IGHJ5 rearrangements stereotypic for rheumatoid factors, which have previously been associated with MALT lymphoma development in SjS. Unbiased marker identification has yielded putative prediagnostic lymphoma markers for 7 out of 18 patients. Image:Summary/Conclusion: We conducted a pilot study investigating a novel method of early detection of NHL development in 36 patients at increased risk of hematological malignancy. In total, we observed an abnormal distribution of the IGH repertoire between 5 months to 2 years prior to NHL diagnosis in 7 out of 18 patients, compared to 0 out of 18 of the matched controls. While our results suggest potential merit for early detection of NHL development through immunogenetic sequencing, the current cohort size does not yet permit drawing definitive conclusions. Therefore, inclusion of additional patients will be a priority during the continuation of the study.

P1245: CLINICAL IMPACT OF IMMUNOGLOBULIN HEAVY CHAIN REPERTOIRE IN MANTLE CELL LYMPHOMA: A STUDY FROM THE FONDAZIONE ITALIANA LINFOMI (FIL) PHASE III MCL0208 TRIAL.

Background: Mantle cell lymphoma (MCL) is characterized by a highly restricted immunoglobulin heavy chain (IGH) gene repertoire with stereotyped IGHV CDR3s, strongly implying a role for antigen-driven selection. Nonetheless, differently from chronic lymphocytic leukemia, where the prognostic role of IGH parameters is known since decades, published data are less consistent in MCL. Recently, LRPAP1 autoantibodies were detected in 13% of patients from the European MCL Network trials and were associated with superior outcome, while the autoreactivity of IGHV 4-34 antibodies is largely known. Aims: We aimed at characterizing the IGH VDJ repertoire and its putative clinical relevance in a large, prospective, trial of the Fondazione Italiana Linfomi (FIL) enrolling younger MCL patients. Methods: IGH clonal rearrangements detection was performed by Sanger sequencing on bone marrow or peripheral blood samples collected for minimal residual disease purposes in FIL MCL0208 trial (NCT02354313). This is a prospective, randomized phase III trial comparing lenalidomide maintenance vs observation after an intensive cytarabine containing chemo-immunotherapy followed by autologous transplantation in frontline <66 year-old MCL patients. IMGT/V-QUEST tool was used to annotate IGHV-D-J and mutational status (cut-off 98%) according to ERIC guidelines. Finally, these biological data were integrated with published clinical, pathological, and mutational data from the trial (current median follow-up 38 months). Results: A clonal IGH productive rearrangement was detected in 209/300 patients (70%), resulting in 159 (76%) unmutated (UM) and in 50 (24%) mutated (M) cases, as expected. Overall, VDJ usage included 29 IGHV families, with IGHV 3-21 (#45, 22%), 4-34 (#33, 16%), 3-7 (#15, 7%), 3-23 and 5-51 (#12 each, 6%), 4-39 and 4-59 (#10 each, 5%) as the most frequent groups, together representing the 66% of the analyzed series (Figure 1A). Interestingly, patients carrying IGHV 3-21 were often characterized by low Ki-67 index (Ki-67 <30%: 86% vs 64% of other cases, p<0.01) and low risk MIPI score (73% vs 43%, p=0.001), while no case of blastoid morphology was registered among the IGHV 4-34 patients (vs 89%, p<0.05). Moreover, by grouping together these two rearrangements, statistically significant associations were found with several biomarkers of favorable prognosis (low Ki-67, p<0.01; non-blastoid, p<0.05; absence of TP53 aberrations, p<0.05, or KMT2D mutations, p<0.05, and low-risk MIPI, p<0.0001). Accordingly, both IGHV 3-21 and 4-34 patients showed better long-term outcomes, with longer PFS and OS if compared with all the other cases, both singularly and when grouped together (3 years PFS: 75% vs 53%, p<0.05; 3 years OS: 91% vs 76%, p<0.01, Figure 1B, C). Finally, these results were independent from IGHV mutational status (p=0.09) and lenalidomide maintenance (p=0.46). Image:Summary/Conclusion: This is the first study assessing the clinical impact of VDJ IGH repertoire in a large prospective trial in MCL. As expected, 76% of patients resulted UM, with 3-21 and 4-34 representing more than one third of all the IGHV rearrangements. Notably, in our series, no significant correlation between mutational status and outcome was recorded. On the other hand, patients carrying either IGHV 3-21 or 4-34 rearrangements were characterized by favorable clinical and biological known prognosticators and were associated with both PFS and OS improvement, if compared to other patients. Deeper translational insights are needed to clarify the biological (autoreactive?) basis of this observation.

Different B cell subpopulations show distinct patterns in their IgH repertoire metrics.

Several human B cell subpopulations are recognised in the peripheral blood, which play distinct roles in the humoral immune response. These cells undergo developmental and maturational changes involving VDJ recombination, somatic hypermutation and class switch recombination, altogether shaping their immunoglobulin heavy chain (IgH) repertoire. Here, we sequenced the IgH repertoire of naïve, marginal zone, switched and plasma cells from 10 healthy adults along with matched unsorted and in silico separated CD19+ bulk B cells. Using advanced bioinformatic analysis and machine learning, we show that sorted B cell subpopulations are characterised by distinct repertoire characteristics on both the individual sequence and the repertoire level. Sorted subpopulations shared similar repertoire characteristics with their corresponding in silico separated subsets. Furthermore, certain IgH repertoire characteristics correlated with the position of the constant region on the IgH locus. Overall, this study provides unprecedented insight over mechanisms of B cell repertoire control in peripherally circulating B cell subpopulations.

Age-related changes in the TRB and IGH repertoires in healthy adult males and females

A diverse immune repertoire is capable of recognizing the enormous universe of foreign antigens encountered over life. Aging has a profound impact on the immune repertoires. However, whether continuous age-related changes in the immune repertoires differ between sexes is unclear. In this study, the characteristics of immune repertoires stratified by sex during aging are deciphered by analyzing T-cell receptor β-chain (TRB) and immunoglobulin heavy chain (IGH) sequences in 361 healthy adults. A similar change was observed between males and females across their lifespan, whereas age-subgroup analysis revealed sex-specific signatures in TRB and IGH repertoires. In regard to TRB, in males, repertoire richness and evenness increases slightly before the age of 32 years and 45 years respectively, and decreases sharply thereafter. Intriguingly, in females, they decrease significantly until around the age 57 years old, and subsequently undergo a stable stage up to the age of 83 years. Although IGH repertoire evenness increases significantly with age in both sexes, richness decreases significantly with age in males but remains stable in females. Moreover, average length of IGH CDR3 increases with age. In conclusion, these findings provide fundamental insights into the mechanisms underlying sex differences in adaptive immunity.

Human transitional and IgMlow mature naïve B cells preserve permissive B-cell receptors.

The level of immunoglobulin M (IgM) displayed on the surface of peripheral blood B cells exhibits a broad dynamic range and has been associated with both development and selection. To determine whether IgM surface expression associates with distinct immunoglobulin heavy‐chain (IGH) repertoire properties, we performed deep IgM sequencing of peripheral blood transitional and mature naïve B cells in the upper and lower quartiles of surface IgM expression for 12 healthy donors. Mature naïve B cells within the lowest quartile for surface IgM expression displayed more diverse IGH features including increased complementarity‐determining region 3 length, IGHJ6 segment usage and aromatic amino acids compared with mature naïve B cells with high surface IgM. There were no differences between IGH repertoires for transitional B cells with high or low surface IgM. These findings suggest that a selection checkpoint during progression of transitional to mature naïve B cells reduces the breadth of the IGH repertoire among high surface IgM B cells but that diversity is preserved in B cells expressing low levels of surface IgM.

The Diagnostic and Prognostic Potential of the B-Cell Repertoire in Membranous Nephropathy.

Membranous nephropathy (MN), an autoimmune glomerular disease, is one of the most common causes of nephrotic syndrome in adults. In current clinical practice, the diagnosis is dependent on renal tissue biopsy. A new method for diagnosis and prognosis surveillance is urgently needed for patients. In the present study, we recruited 66 MN patients before any treatment and 11 healthy control (HC) and analyzed multiple aspects of the immunoglobulin heavy chain (IGH) repertoire of these samples using high-throughput sequencing. We found that the abnormalities of CDR-H3 length, hydrophobicity, somatic hypermutation (SHM), and germ line index were progressively more prominent in patients with MN, and the frequency of IGHV3-66 in post-therapy patients was significantly lower than that in pre-therapy patients. Moreover, we found that the IGHV3-38 gene was significantly related to PLA2R, which is the most commonly used biomarker. The most important discovery was that several IGHV, IGHD transcripts, CDR-H3 length, and SHM rate in pre-therapy patients had the potential to predict the therapeutic effect. Our study further demonstrated that the IGH repertoire could be a potential biomarker for prognosis prediction of MN. The landscape of circulating B-lymphocyte repertoires sheds new light on the detection and surveillance of MN.

Maternal and Infant Immune Repertoire Sequencing Analysis Identifies Distinct Immunoglobulin and T Cell Receptor Development in Term and Preterm Infants

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. While many studies have focused on investigating the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. Here, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries and hypothesized that the high incidence of infection in patients with PTL may result in specific changes in the TCR and BCR repertoires. We analyzed T-cell receptor beta chain (TCR-β) and immunoglobulin heavy chain (IgH) diversity, CDR3 lengths, clonal sharing, and preferential usage of variable (V), diversity (D), and joining (J) gene segments. Additionally, we studied the rates of somatic hypermutation (SHM) in IgH. Overall, the cord blood IgH repertoires had significantly (padj < 0.05) lower rates of SHM and both TCR-β and IgH repertoires had shorter CDR3s compared to maternal blood. When comparing term and PPROM cord blood samples, we found that CDR3 lengths correlated with gestational age, with the shorter CDR3s in preterm neonates suggesting a ‘less developed’ repertoire. Preterm cord blood displayed preferential usage of a number of V genes and J genes. Furthermore, the term maternal repertoires displayed significant preferential usage of TRBV7-8 compared to preterm maternal repertoires. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features yet preferential use of particular genes and convergence with maternal TCR-β clones compared to term infants. While the higher TCR-β diversity might reflect less clonal expansion, the higher clonal convergence between mothers and infants in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients, and contribute to a better understanding of neonate immune repertoire development and potential changes associated with preterm labor.

Preferential Usage of Specific Immunoglobulin Heavy Chain Variable Region Genes with Unmutated Profile and Advanced Stage at Presentation Are Common Features in Patients with Chronic Lymphocytic Leukemia from Senegal

Introduction : Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with Causasien patients. In this study, we aimed to identify genetic factors that may account for this difference Methods: We collected peripheral blood mononuclear cells (PBMCs) from a total of 75 patients with CLL, 25 from Senegal (Africa), and 50 from Siena. Since it is well known that there are differences in germline IGH repertoires between different populations, we also collected PBMCs from five healthy Senegalese individuals as control. We analyzed immunoglobulin heavy chain (IGH) genes mutational status by performing next-generation sequencing in these 2 groups of patients. Results: We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series. Conclusion: The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background. Disclosures Gozzetti: Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding.

The Diagnostic and Prognosis Potential of B Cell Repertoire in Membranous Nephropathy

Background: Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, which is an autoimmune glomerular disease. In current clinical practice, the diagnosis is dependent on renal tissue biopsy. A non-invasive method for diagnosis and prognosis surveillance is urgent need for patients. Methods: From 2017 to 2018 we recruited 66 MN patients before any treatment and 11 healthy controls. We analyzed multiple aspects of immunoglobulin heavy chain (IGH) repertoire between MN patients and healthy controls using high-throughput sequencing. Findings: We found that the frequency of IGHV3-66 in post-therapy patients is significantly lower than pre-therapy remarkably. Moreover, we found that IGHV3-38 gene is significantly related to PLA2R, which is the common used biomarker. The most important discovery was that in the patient before treatment we identified 24 IGHV genes had the potential to predict the therapeutic effect. Interpretation: Our study further demonstrates IGH repertoire can be a non-invasive biomarkers potentially for prognosis prediction of MN. The landscape of circulating B-lymphocyte repertoires shed new light on MN detection and surveillance. Funding Statement: This work was funded by grants from the National Natural Science Foundation of China (81972335) and Guangdong Provincial Science and Technology Plan (2017A020215185) and Guangdong Province Natural Science Funds for Distinguished Young Scholar (2016A030306050) and Science and Technology Innovation Platform in Foshan City (FS0AA-KJ218-1301-0007) and “Guangdong Te Zhi Program” youth science and technology talent project (2015TQ01R462) and Medical Scientific Research Foundation of Guangdong Province of China (B2017006). Declaration of Interests: The authors declare no financial or commercial conflicts of interest. Ethics Approval Statement: This study was approved by the ethics committee of the Affiliated Foshan Hospital of Sun Yat-Sen University.

Maturation of the Human Immunoglobulin Heavy Chain Repertoire With Age.

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced immunoglobulin heavy chain (IgH) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream IgH constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing IgH repertoires and stress the importance of using well-selected, age-appropriate controls in IgH studies.

Longitudinal analysis of the antibody repertoire of a Zika virus-infected patient revealed dynamic changes in antibody response

ABSTRACT The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neonatal abnormalities and other disorders. Antibodies to the ZIKV envelope (E) protein can block infection. In this study, next-generation sequencing (NGS) of immunoglobulin heavy chain (IgH) mRNA transcripts was combined with single-cell PCR cloning of E-binding monoclonal antibodies for analysing antibody response in a patient from the early stages of infection to more than one year after the clearance of the virus. The patient's IgH repertoire 14 and 64 days after symptom onset showed dramatic dominant clonal expansion but low clonal diversity. IgH repertoire 6 months after disease-free status had few dominant clones but increased diversity. E-binding antibodies appeared abundantly in the repertoire during the early stages of infection but quickly declined after clearance of the virus. Certain VH genes such as VH5-10-1 and VH4-39 appeared to be preferentially enlisted for a rapid antibody response to ZIKV infection. Most of these antibodies require relatively few somatic hypermutations to acquire the ability to bind to the E protein, pointing to a possible mechanism for rapid defence against ZIKV infection. This study provides a unique and holistic view of the dynamic changes and characteristics of the antibody response to ZIKV infection.