Ig Repertoire Research Articles

A Comparative Analysis of the Immunoglobulin Repertoire in Leukemia Cells and B Cells in Chinese Acute Myeloid Leukemia by High-Throughput Sequencing.

It is common knowledge that immunoglobulin (Ig) is produced by B lymphocytes and mainly functions as an antibody. However, it has been shown recently that myeloblasts from acute myeloid leukemia (AML) could also express Ig and that AML-Ig played a role in leukemogenesis and AML progression. The difference between Ig from myeloblasts and B cells has not been explored. Studying the characteristics of the Ig repertoire in myeloblasts and B cells will be helpful to understand the function and significance of AML-Ig. We performed 5' RACE-related PCR coupled with PacBio sequencing to analyze the Ig repertoire in myeloblasts and B cells from Chinese AML patients. Myeloblasts expressed all five classes of IgH, especially Igγ, with a high expression frequency. Compared with B-Ig in the same patient, AML-Ig showed different biased V(D)J usages and mutation patterns. In addition, the CDR3 length distribution of AML-Ig was significantly different from those of B-Ig. More importantly, mutations of AML-IgH, especially Igμ, Igα, and Igδ, were different from that of B-IgH in each AML patient, and the mutations frequently occurred at the sites of post-translational modification. AML-Ig has distinct characteristics of variable regions and mutations, which may have implications for disease monitoring and personalized therapy.

Yclon: Ultrafast clustering of B cell clones from high-throughput immunoglobulin repertoire sequencing data

MotivationThe next-generation sequencing technologies have transformed our understanding of immunoglobulin (Ig) profiles in various immune states. Clonotyping, which groups Ig sequences into B cell clones, is crucial in investigating the diversity of repertoires and changes in antigen exposure. Despite its importance, there is no widely accepted method for clonotyping, and existing methods are computationally intensive for large sequencing datasets. ResultsTo address this challenge, we introduce YClon, a fast and efficient approach for clonotyping Ig repertoire data. YClon uses a hierarchical clustering approach, similar to other methods, to group Ig sequences into B cell clones in a highly sensitive and specific manner. Notably, our approach outperforms other methods by being more than 30 to 5000 times faster in processing the repertoires analyzed. Astonishingly, YClon can effortlessly handle up to 2 million Ig sequences on a standard laptop computer. This enables in-depth analysis of large and numerous antibody repertoires. Availability and implementationYClon was implemented in Python3 and is freely available on GitHub.

Phage Immunoprecipitation Sequencing (PhIP-Seq) for Analyzing Antibody Epitope Repertoires Against Food Antigens.

While thousands of food and environmental allergens have been reported, conventional methods for allergy testing typically rely on measuring immunoglobulin E (IgE) binding against panels of dozens to hundreds of antigens. Beyond IgE, also the specificity of other Ig (sub-)classes such as IgG4, has gained interest because of a potential protective role toward allergy.Phage immunoprecipitation sequencing (PhIP-Seq) allows to study hundreds of thousands of rationally selected peptide antigens and to resolve binding specificities of different Ig classes. This technology combines synthetic DNA libraries encoding antigens, with the display on the surface of T7 bacteriophages and next-generation sequencing (NGS) for quantitative readouts. Thereby binding of entire Ig repertoires can be measured to detect the exact epitopes of food allergens and to study potential cross-reactivity.In this chapter, we provide a summary of both the key experimental steps and various strategies for analyzing PhIP-Seq datasets, as well as comparing the advantages and disadvantages of this methodology for measuring antibody responses against food antigens.

Differentiation and traffic of IgM+ B cells between focal dark spots in skeletal muscle of Atlantic salmon, lymphoid and adipose tissues.

Focal dark spots (DS) in farmed Atlantic salmon fillets contain a significant number of B cells as revealed by the high abundance of immunoglobulin (Ig) transcripts in transcriptome data. The immune response in DS remains unknown while they represent a major problem in commercial aquaculture. Here, we characterized the diversity and clonal composition of B cells in DS. Sixteen gene markers of immune cells and antigen presentation were analyzed with RT-qPCR. All genes expression showed a positive correlation with DS area and intensity. The flatter the DS, the higher the expression of cd28, csfr, ctla, igt, and sigm, the lower expression of cd83 and btla, and the larger the cumulative frequency within DS. The expression of most of the analyzed immune genes, including three Ig types and markers of B cells was lower in DS than in the lymphatic organs, head kidney and spleen, but significantly higher compared to skeletal muscle. High levels of ctla4 and cd28 in DS might indicate the recruitment of T cells. Sequencing of IgM repertoire (Ig-seq) assessed migration of B cells by co-occurrence of identical CDR3 sequences in different tissues. The combination of gene expression and Ig-seq revealed the presence of several stages of B cell differentiation in DS. B cells at the earliest stage, with high ratio of membrane to secretory IgM (migm and sigm), showed minor Ig repertoire overlap with other tissues. Further differentiation stage (increased sigm to migm ratio and high expression of pax5 and cd79) was associated with active movement of B cells from DS towards lymphatic organs and visceral fat. Traffic and expression of immune genes decreased at later stages. These B cells could be involved in a response directed against viruses, pathogenic or opportunistic bacteria in DS. Seven of eight fish were positive for salmon alphavirus, and levels were higher in DS than in unstained muscle. PCR with universal primers to the 16S rRNA gene did not detect bacteria in DS. Although the evolution of DS most likely implies local exposure to antigens, neither this nor previous studies have found a necessary association between DS and pathogens or self-antigens.

N-Glycosylation of the Ig Receptors Shapes the Antigen Reactivity in Chronic Lymphocytic Leukemia Subset #201.

Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.

Plasmablast Ig repertoire dynamics through repeat Plasmodium falciparum challenges

Abstract The development of natural immunity against malaria requires multiple exposures and even after protection develops against clinical disease individuals continue to be susceptible to infections. To study the dynamics of the immune response to repetitive P. falciparum (PF) infected mosquito exposures, we used controlled human malaria challenges with the same strain of PF. The homologous strain was used to eliminate differences in immune responses due to strain polymorphisms and focus on the maturation of the human immune response to repeat exposures. Consistent with the gradual immune response observed in the field, after 4 sequential exposures over the course of 2 years, symptoms decreased and the time to parasite detection on a blood smear increased significantly, but all the volunteers continued to be susceptible to infections. Anti-PF antibody levels increased over time and were able to inhibit liver cell invasion in an in vitro assay. The Ig repertoire of plasmablasts collected 7 days after peak parasitemia demonstrated large clonal expansions in one or two sequential infections that decreased after subsequent exposures. This pattern suggests a marked difference in the lineage of plasmablast and memory cells populations. Naïve B cells stimulated by PF to proliferate and differentiate into plasmablasts may fail to differentiate into memory cells that can produce both plasmablasts and memory cells following subsequent exposures. This differentiation pattern would lead to new sets of expanded clones at each PF exposure, instead of further maturation of the same antibodies generated during the first response. Such a response could enhance the diversity of the Ig pool, but would delay the generation of a strong, durable memory response U0110852

Demultiplexing Ig repertoires by parallel mRNA/DNA sequencing shows major differential alterations in severe COVID-19

To understand the fine differential elements that can lead to or prevent acute respiratory distress syndrome (ARDS) in COVID-19 patients, it is crucial to investigate the immune response architecture. We herein dissected the multiple layers of B cell responses by flow cytometry and Ig repertoire analysis from acute phase to recovery. Flow cytometry with FlowSOM analysis showed major changes associated with COVID-19 inflammation such as an increase of double-negative B-cells and ongoing plasma cell differentiation. This paralleled COVID-19-driven expansion of two disconnected B-cell repertoires. Demultiplexing successive DNA and RNA Ig repertoire patterns characterized an early expansion of IgG1 clonotypes with atypically long and uncharged CDR3, the abundance of this inflammatory repertoire being correlated with ARDS and likely pejorative. A superimposed convergent response included convergent anti-SARS-CoV-2 clonotypes. It featured progressively increasing somatic hypermutation together with normal-length or short CDR3 and it persisted until a quiescent memory B-cell stage after recovery.

IgD shapes the pre-immune naïve B cell compartment in humans.

B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-β7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.

Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation.

The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.

Humoral Immune Reconstitution Following Therapy with Daratumumab, Carfilzomib, Lenalidomide and Dexamethasone (Dara-KRd), Autologous Hematopoietic Cell Transplantation (AHCT) and MRD-Response-Adapted Treatment Cessation

Introduction Quadruplet induction, AHCT, and measurable residual disease (MRD) response-adapted consolidation yields unprecedented depth of response in newly diagnosed multiple myeloma (NDMM). Immune reconstitution (IR) in patients (pts) receiving limited duration intensive therapy has not been well described. Methods NDMM pts treated on MASTER (NCT03224507) received induction with daratumumab, carfilzomib, lenalidomide, and dexamethasone (Dara-KRd; 4 cycles), followed by AHCT and 0, 4 or 8 cycles of Dara-KRd consolidation guided by serial assessments of bone marrow MRD by next-generation sequencing (NGS, ClonoSEQ®). Pts with two consecutive MRD <10-5 ceased therapy and entered active surveillance for MRD resurgence (MRD-SURE). The primary objective of this study is to evaluate humoral IR of MRD-SURE pts by characterizing quantitative changes in the repertoire of immunoglobulin (Ig) genes (IgH, IgK and IgL) by NGS and serum gamma globulin levels. Results After induction, the IgH repertoire was small and lacked diversity (median unique sequences (mus) 11,330/106, IQR 5,711-23,143). Of 63 evaluable pts who received AHCT and >=4 cycles of consolidation, a substantial expansion of the IgH repertoire was seen 90 days after AHCT (75,375 mus/106, IQR 49,361-113,305, P< 0.001) with a contraction after consolidation (15,795 mus/106, IQR 5,571-33,732, P< 0.001). Pts who received post-AHCT consolidation (N=27) had a smaller IgH repertoire at MRD-SURE entry compared to pts entering MRD-SURE after AHCT (N=28) (15,082 v 76,266 mus/106, p< 0.001), with near but incomplete recovery after 6 months (mo) of treatment cessation (83,954 v 107,715 mus/106, p=0.037) and no difference at 18 mo (109,989 v 115,246 mus/106, P=0.72) (Figure 1). Similar findings were seen for IgK and IgL. The serum protein gamma fraction was depressed (median 0.3 g/dL, IQR 0.23-0.41) in pts entering MRD-SURE post-AHCT, but rose off therapy reaching plateau after 12 months. Gamma globulin recovery was delayed in patients receiving consolidation (Figure 2). Risk of grade≥3 infection while on Dara-KRd was 3.8/100*patient*month during induction, and 2.2/100*patient*month during post-AHCT consolidation (overall 2.9/100*patient*month). Risk of grade≥3 infection reduced substantially to 0.22/100*patient*month during the first 6 months and 0.12/100*patient*month during subsequent 12 months of MRD-SURE (overall 0.15/100*patient*month). Conclusions Quadruplet, anti-CD38 mAb-containing therapy leads to profound hypogammaglobulinemia and reduction in the Ig gene repertoire. Pts who received post-AHCT consolidation had delayed IR as compared to those who did not, however the Ig repertoire steadily recovered, with no statistically significant difference 18 mo after treatment cessation. Grade ≥3 infection risk is very low after treatment cessation, as the Ig gene repertoire expands prior to recovery of immunoglobulin levels. Further evaluation of IR after MRD-adapted therapy is warranted as new immunotherapeutic agents are incorporated in therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P634: MHC-BASED LARGE-SCALE SCREENING FOR ANTI-TUMOR T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS CD8+ T CELLS WITH SPECIFICITY AGAINST THE CLONOTYPIC B-CELL RECEPTOR IMMUNOGLOBULIN

Background: Chronic lymphocytic leukemia (CLL) is currently incurable, indicating a need for novel strategies towards disease eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL appear selected by restricted antigens, with evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Aims: We previously performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. Here, we performed major histocompatibility complex (MHC)-based large-scale screening to identify autologous CD8+ T cells recognizing the predicted neoepitopes. Methods: We evaluated 653 peptides derived from the clonotypic BcR IG of 25 CLL patients (median 21 predicted neoepitopes/patient, across 13 MHC-I alleles). Considering the MHC-I typing of each patient, we constructed patient-specific peptide-MHC multimers labeled with a unique DNA barcode plus a fluorochrome (PE). We generated MHC-specific multimer libraries which we then mixed with respective autologous T-cell enriched peripheral blood mononuclear cells (PBMCs). Duplicate samples/patient were analyzed. In addition, known viral peptide-MHC multimers labeled with a different fluorochrome (APC) as well as three MHC-matched healthy donor PBMCs were used as controls. PE- and APC-positive multimer-binding CD8+ T cells were sorted for each cell sample; subsequently, DNA barcode amplification and sequencing was performed. Sequencing data were processed (Barracoda software) to obtain the number of clonally reduced barcode reads assigned to a given sample and peptide-MHC specificity. The number of clonally reduced reads for a given pMHC specificity was used to estimate the frequency of T cells specific for a given epitope, based on the average number of T cell receptor–MHC multimer interactions detected in the total MHC multimer-binding T cell pool in a given cell sample. Peptides with a log fold change >2 were considered as significant [log fold change: (number of DNA barcode reads associated with a specific pMHC)/3x (number of total barcode reads derived from the same sample)]. Results: Overall, 3 peptide-MHC multimers were recognized by CD8+ T cells: (i) VTVADTAVYY (peptide A) and (ii) INLNPSLKRR (peptide B), both within the context of the A03*01 allele, and (iii) YSFTSYWINW (peptide C) within the context of the A24*02 allele. Peptide A derived from the IGHV4-34 FR3 region of a somatically hypermutated clonotypic BcR IG, containing a single A to V somatic hypermutation (SHM) at position 96. Peptide B derived from the IGHV4-39 CDR2-FR3 junction of a somatically hypermutated clonotypic BcR IG, containing 3 SHMs (T to I at position 65, Y to L at position 67 and S to R at position 74). Peptide C derived from the IGHV5-10-1 CDR1-FR2 junction of a clonotypic BcR IG assigned to stereotyped subset #1, containing the sole SHM of the IG (S to N at position 40). The immunogenicity of these peptides was further corroborated by the fact that they were recognized not only by the autologous T cells of the patient from whom the peptide derived, but also by T cells of other patients as well as a healthy donor sharing the respective MHC allele. Summary/Conclusion: In conclusion, we offer proof of concept that the targeted SHM which shapes the CLL BcR IG repertoire may produce idiotypic targets for T cell-based therapy or peptide vaccine design. Characterization of the epitope-binding T cells is currently underway by our group, aiming to provide further insight on how these cells could be recruited into effective anti-tumor responses.

Activation-Induced Cytidine Deaminase Impacts the Primary Antibody Repertoire in Naive Mice.

Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.

B cell selection sites in the nurse shark spleen may represent evolutionary precursors of mammalian germinal centers

Abstract Sharks are members of the oldest extant taxonomic group (Chondrichthyes) to have immunoglobulin (Ig)-based adaptive immunity. While research has demonstrated that sharks can produce antigen-specific antibody responses, affinity mature these antibodies, and establish immunological memory, histological examination has so far failed to find evidence of germinal centers (GCs). To explore this apparent discrepancy, we immunized several nurse sharks (Ginglymostoma cirratum) with phycoerythrin and visualized the subsequent splenic response using a combination of single nuclei (sn)RNA sequencing and fluorescent microscopy. Our snRNA data reveal the presence of both centrocyte-like and centroblast-like B cells in sharks, as well as a T cell subset expressing several markers found in mammalian T follicular helper cells. RNA fluorescent in situ hybridization experiments show distinct regions of AID expression, polarization of CXCR4 and CXCR5 expression, and association of T cell aggregates with splenic B cell follicles. Fluorescent microscopy clearly demonstrates presentation of nondegraded antigen in the center of B cell follicles. Finally, analysis of Ig variable regions amplified from such B cell follicles reveals evidence of positive selection of clonotypes. Together, our data supports the presence of B cell selection sites in the cartilaginous fish spleen that functionally support the selection of antigen-specific B cells and affinity maturation of the shark Ig repertoire. Further, these sites potentially represent the primordial structures that gave rise to mammalian GCs. Supported by grants from NIH T32 AI095190, F31 AI147532

Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci

Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.

Single-Cell-Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques.

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired H and L chains of Igs and VDJ and VJ chains of TCRs from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single-cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. We used custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single-cell immune repertoire profiling. Using these rhesus-specific assays, we sequenced Ig and TCR repertoires in >60,000 cells from cryopreserved rhesus PBMCs, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single-cell level.

Next-Generation Sequencing Revealed a Distinct Immunoglobulin Repertoire with Specific Mutation Hotspots in Acute Myeloid Leukemia

Simple SummaryIdentifying new molecular targets is of great importance for prognosis prediction and target therapy of acute myeloid leukemia (AML). We previously reported on frequent expression of immunoglobulin (Ig) in myeloblasts. In this study, we investigated the clinical significance of Ig expression in sorted myeloblasts from 59 AML patients. We found that a higher level of AML-derived Ig expression correlated with a significantly shorter disease-free survival. Furthermore, we performed a comprehensive analysis of AML-derived Ig repertoire by next-generation sequencing (NGS) in 16 patients. The transcripts of AML-derived Ig shared some features with B cell-derived Ig, such as a typical V(D)J recombination and high mutation rates. However, they also showed distinct features. In contrast to the huge diversity of classical Ig, the VH-D-JH rearrangements used by AML-derived Ig were biased in each AML patient. In particularly, the Vκ-Jκ rearrangements were skewed in both AML blasts and normal peripheral blood mononucleated cells (PBMCs). However, AML-derived IGK showed high somatic mutation rates (>2%), while IGK in normal PBMCs rarely displayed hypermutation (<2%). More importantly, we identified five mutation hotspots at serine codons of IGKV3-20 in AML blasts, which may be involved in leukemogenesis and serve as a novel marker for disease monitoring and target therapy.Immunoglobulin (Ig) is known as a hallmark of B-lymphocytes exerting antibody functions. However, our previous studies demonstrated that myeloblasts from acute myeloid leukemia (AML) patients could also express Ig with distinct roles. Here, we quantified Ig (IGHG and IGK) transcripts by real-time PCR and performed a comprehensive analysis of Ig repertoire (both heavy chains and light chains) in AML blasts. We found that Ig was frequently expressed by AML blasts. A higher level of AML-derived IGHG expression correlated with a significantly shorter disease-free survival. Next-generation sequencing revealed dysregulated transcripts of all five Ig classes (IGHA, IGHD, IGHE, IGHG, and IGHM) and two Ig types (IGK and IGL) in AML. VH-D-JH rearrangements in myeloblasts were biased with individual specificity rather than generally diverse as in B-cells. Compared to AML-derived IgH, AML-derived IGK was more conserved among different AML samples. The frequently shared Vκ-Jκ patterns were IGKV3-20*01/IGKJ1*01, IGKV2D-28*01/IGKJ1*01, and IGKV4-1*01/IGKJ1*01. Moreover, AML-derived IGK was different from classical IGK in B-cells for the high mutation rates and special mutation hotspots at serine codons. Findings of the distinct Ig repertoire in myeloblasts may facilitate the discovery of a new molecular marker for disease monitoring and target therapy.

Demultiplexing Ig Repertoires by Parallel mRNA/DNA Sequencing Shows Major Differential Alterations in Severe COVID-19

Demultiplexing Ig Repertoires by Parallel mRNA/DNA Sequencing Shows Major Differential Alterations in Severe COVID-19

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Individualized VDJ recombination predisposes the available Ig sequence space.

The process of recombination between variable (V), diversity (D), and joining (J) immunoglobulin (Ig) gene segments determines an individual's naive Ig repertoire and, consequently, (auto)antigen recognition. VDJ recombination follows probabilistic rules that can be modeled statistically. So far, it remains unknown whether VDJ recombination rules differ between individuals. If these rules differed, identical (auto)antigen-specific Ig sequences would be generated with individual-specific probabilities, signifying that the available Ig sequence space is individual specific. We devised a sensitivity-tested distance measure that enables inter-individual comparison of VDJ recombination models. We discovered, accounting for several sources of noise as well as allelic variation in Ig sequencing data, that not only unrelated individuals but also human monozygotic twins and even inbred mice possess statistically distinguishable immunoglobulin recombination models. This suggests that, in addition to genetic, there is also nongenetic modulation of VDJ recombination. We demonstrate that population-wide individualized VDJ recombination can result in orders of magnitude of difference in the probability to generate (auto)antigen-specific Ig sequences. Our findings have implications for immune receptor–based individualized medicine approaches relevant to vaccination, infection, and autoimmunity.

Pro- and Anti- Effects of Immunoglobulin A- Producing B Cell in Tumors and Its Triggers.

B cells are well known as key mediators of humoral immune responses via the production of antibodies. Immunoglobulin A (IgA) is the most abundantly produced antibody isotype and provides the first line of immune protection at mucosal surfaces. However, IgA has long been a divisive molecule with respect to tumor progression. IgA exerts anti- or pro-tumor effect in different tumor types. In this review, we summarize emerging evidence regarding the production and effects of IgA and IgA+ cells in the tumor microenvironment (TME). Moreover, we discuss that the TME cytokines, host diet, microbiome, and metabolites play a pivotal role in controlling the class-switch recombination (CSR) of IgA. The analysis of intratumoral Ig repertoires and determination of metabolites that influence CSR may help establish novel therapeutic targets for the treatment of cancers.