Immunoglobulin Heavy Chain Gene Research Articles

Comparative transcriptomic profiling in HPV-associated cervical carcinogenesis: Implication of MHC class II and immunoglobulin heavy chain genes

AimWe aimed to determine the biological processes and pathways involved in cervical carcinogenesis associated with high-risk human papillomavirus (HPV) infection. Materials and methodsTotal RNA was extracted from three formalin-fixed paraffin-embedded (FFPE) samples each of normal cervix, HPV-infected low-grade squamous intraepithelial lesion (LSIL), high-grade SIL (HSIL) and squamous cell carcinoma (SCC). Transcriptomic profiling by microarrays was conducted followed by downstream Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. ResultsWe examined the difference in GOs enriched for each transition stage from normal cervix to LSIL, HSIL, and SCC, and found 307 genes to be differentially expressed. In the transition from normal cervix to LSIL, the extracellular matrix (ECM) genes were significantly downregulated. The MHC class II genes were significantly upregulated in the LSIL to HSIL transition. In the final transition from HSIL to SCC, the immunoglobulin heavy locus genes were significantly upregulated and the ECM pathway was implicated. ConclusionDeregulation of the immune-related genes including MHC II and immunoglobulin heavy chain genes were involved in the transitions from LSIL to HSIL and SCC, suggesting immune escape from host anti-tumour response. The extracellular matrix plays an important role during the early and late stages of cervical carcinogenesis.

A Novel Synthetic Dual Agonistic Liposomal TLR4/7 Adjuvant Promotes Broad Immune Responses in an Influenza Vaccine With Minimal Reactogenicity.

The limited efficacy of seasonal influenza vaccines is usually attributed to ongoing variation in the major antigenic targets for protective antibody responses including hemagglutinin (HA) and neuraminidase (NA). Hence, vaccine development has largely focused on broadening antigenic epitopes to generate cross-reactive protection. However, the vaccine adjuvant components which can accelerate, enhance and prolong antigenic immune responses, can also increase the breadth of these responses. We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus HA, inducing rapid, and sustained antibody responses that are protective against influenza viruses in homologous and heterologous murine challenge models. To further enhance adjuvant efficacy, we performed a structure-activity relationship study for the TLR4 ligand, N-cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)thio)acetamide (C25H26N4O2S; 1Z105), and identified the 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog (C29H28N4O3S; 2B182C) as a derivative with higher potency in activating both human and mouse TLR4-NF-κB reporter cells and primary cells. In a prime-boost immunization model using inactivated influenza A virus [IIAV; A/California/04/2009 (H1N1)pdm09], 2B182C used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to 1Z105, and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, 1V270, 2B182C induced equivalent levels of anti-NA and anti-HA IgG1 to 1V270+1Z105. However, the combination of 1V270+2B182C induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to 1V270+1Z105. A stable liposomal formulation of 1V270+2B182C was developed, which synergistically enhanced anti-HA and anti-NA IgG1 and IgG2a responses without demonstrable reactogenicity after intramuscular injection. Notably, vaccination with IIAV plus the liposomal formulation of 1V270+2B182C protected mice against lethal homologous influenza virus (H1N1)pdm09 challenge and reduced lung viral titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Unsupervised machine learning and prognostic factors of survival in chronic lymphocytic leukemia.

ObjectiveUnsupervised machine learning approaches hold promise for large-scale clinical data. However, the heterogeneity of clinical data raises new methodological challenges in feature selection, choosing a distance metric that captures biological meaning, and visualization. We hypothesized that clustering could discover prognostic groups from patients with chronic lymphocytic leukemia, a disease that provides biological validation through well-understood outcomes.MethodsTo address this challenge, we applied k-medoids clustering with 10 distance metrics to 2 experiments (“A” and “B”) with mixed clinical features collapsed to binary vectors and visualized with both multidimensional scaling and t-stochastic neighbor embedding. To assess prognostic utility, we performed survival analysis using a Cox proportional hazard model, log-rank test, and Kaplan-Meier curves.ResultsIn both experiments, survival analysis revealed a statistically significant association between clusters and survival outcomes (A: overall survival, P = .0164; B: time from diagnosis to treatment, P = .0039). Multidimensional scaling separated clusters along a gradient mirroring the order of overall survival. Longer survival was associated with mutated immunoglobulin heavy-chain variable region gene (IGHV) status, absent Zap 70 expression, female sex, and younger age.ConclusionsThis approach to mixed-type data handling and selection of distance metric captured well-understood, binary, prognostic markers in chronic lymphocytic leukemia (sex, IGHV mutation status, ZAP70 expression status) with high fidelity.

Evaluation of Somatic Hypermutation Status in Chronic Lymphocytic Leukemia (CLL) in the Era of Next Generation Sequencing.

Somatic hypermutation (SHM) status provides an important prognostic indicator for chronic lymphocytic leukemia (CLL), a very common type of mature B-cell leukemia. Owing to the adverse prognosis associated with an unmutated immunoglobulin heavy chain variable (IGHV) status, SHM testing is performed as a standard of care in CLL. Conventionally, SHM testing has been performed using labor intensive and primarily analog Sanger sequencing method following PCR amplification of the clonal immunoglobulin heavy chain gene rearrangements in CLL cells. In comparison, recent availability of next generation sequencing (NGS) allows more versatile detection and direct identification of clonal immunoglobulin gene rearrangements in neoplastic B-cell populations. The ability to identify specific clonal IGHV signature(s) in both baseline (diagnostic) and post-treatment settings enables unique clinical applications of NGS such as determination of SHM status, minimal residual disease (MRD) monitoring, clonal heterogeneity and B cell receptor IG stereotypy. We provide a review of current practices and recommendations for SHM determination using NGS including examples of difficult cases.

Extreme genomic volatility characterizes the evolution of the immunoglobulin heavy chain locus in cyprinodontiform fishes.

The evolution of the adaptive immune system has provided vertebrates with a uniquely sophisticated immune toolkit, enabling them to mount precise immune responses against a staggeringly diverse range of antigens. Like other vertebrates, teleost fishes possess a complex and functional adaptive immune system; however, our knowledge of the complex antigen-receptor genes underlying its functionality has been restricted to a small number of experimental and agricultural species, preventing systematic investigation into how these crucial gene loci evolve. Here, we analyse the genomic structure of the immunoglobulin heavy chain (IGH) gene loci in the cyprinodontiforms, a diverse and important group of teleosts present in many different habitats across the world. We reconstruct the complete IGH loci of the turquoise killifish (Nothobranchius furzeri) and the southern platyfish (Xiphophorus maculatus) and analyse their in vivo gene expression, revealing the presence of species-specific splice isoforms of transmembrane IGHM. We further characterize the IGH constant regions of 10 additional cyprinodontiform species, including guppy, Amazon molly, mummichog and mangrove killifish. Phylogenetic analysis of these constant regions suggests multiple independent rounds of duplication and deletion of the teleost-specific antibody class IGHZ in the cyprinodontiform lineage, demonstrating the extreme volatility of IGH evolution. Focusing on the cyprinodontiforms as a model taxon for comparative evolutionary immunology, this work provides novel genomic resources for studying adaptive immunity and sheds light on the evolutionary history of the adaptive immune system.

Elucidating the role of ATM in BER and MMR during B cell CSR

Abstract Secondary immunoglobulin isotypes are produced by class switch recombination (CSR), which requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates staggered DNA breaks that promote recombination between S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with a BER protein, suggesting that ATM regulates CSR through BER. However, the mechanism by which ATM participates in BER remains unknown. ATM−/− mice were bred to knockout mice of the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/−MSH2−/− adult mice were not obtained and preliminary data indicate a late embryonic lethal phenotype. To bypass the observed lethality, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression. These ATMd/dMSH2−/− mice and B cells display a CSR phenotype comparable to ATM−/− B cells, which suggest that ATM may function genetically upstream of MSH2 and implicate a role for ATM in both MMR and BER.

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

Primary hepatic extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), is one of the specific type of low-grade B-cell lymphoma not infrequently found worldwide. It typically involves mucosal sites such as stomach and conjunctiva; however, primary hepatic MALT lymphoma has been extremely rarely reported. We describe a case of hepatic MALT lymphoma in a 70-year-old male patient who underwent left hepatectomy due to the incidentally detected liver masses at a medical checkup. The resected specimen revealed multinodular masses consisting of small-to-intermediate-sized lymphoid cells with serpentine pattern and focal lymphoepithelial lesions. The tumor cells were diffusely positive for CD20 and Bcl-2 but negative for CD3, CD10, CD5, CD23, CD43, and cyclinD1. The Ki-67 labeling index was 10% and immunoglobulin heavy chain gene rearrangement study confirmed monoclonal proliferation. In this paper, we discuss several unique clinicopathologic characteristics which will be helpful to the differential diagnosis of hepatic MALT lymphoma.

Evolution in the management of chronic lymphocytic leukemia in Japan: should MRD negativity be the goal?

Advances in the molecular biology of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and development of molecularly targeted therapies have resulted in treatment innovations. Therapeutic approaches for previously untreated CLL/SLL patients are changing from chemoimmunotherapy (CIT) to molecularly targeted drugs. The aim of therapy for CLL patients has been to control the disease; however, FCR (fludarabine, cyclophosphamide, rituximab) has improved outcomes and reduced the high incidence of undetectable minimum/measurable residual disease (MRD) in previously untreated CLL patients with no 17p deletion/TP53 disruption and mutated immunoglobulin heavy chain gene (IGHV). Patients achieving undetectable MRD in the bone marrow are expected to be cured. BTK inhibitors and BCL-2 inhibitors are effective for CLL/SLL patients. However, atrial fibrillation and bleeding are associated with the BTK inhibitor, ibrutinib, while tumor lysis syndrome is an adverse event (AE) of the BCL-2 inhibitor, venetoclax. Although these novel targeted drugs are very useful, they are also expensive. Emergence of resistant clones of CLL cells must also be addressed. Therefore, treatments of indefinite duration until progression have been replaced by fixed-duration treatments. This review introduces advances in the treatment of previously untreated CLL/SLL patients in Europe and the United States.

Aryl hydrocarbon receptor-induced activation of the human IGH hs1.2 enhancer: Mutational analysis of putative regulatory binding motifs

The human hs1.2 enhancer within the Ig heavy chain gene (IGH) is polymorphic and associated with a number of autoimmune diseases. The polymorphic region is characterized by tandem repeats of an ∼53-bp invariant sequence containing possible binding sites for several transcription factors. Our previous studies suggest the human hs1.2 enhancer is sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant and high affinity ligand of the aryl hydrocarbon receptor (AhR). TCDD induced hs1.2 enhancer activity in an AhR-dependent manner and the number of invariant sequences influenced the magnitude of activity. To better understand the regulation of human hs1.2 enhancer activity, the objective of the current study was to utilize mutational analysis and luciferase reporter constructs to evaluate the contribution of putative transcription factor binding sites to overall hs1.2 enhancer activity and modulation by TCDD. Basal and LPS-induced activity of the hs1.2 enhancer appeared to be most affected by mutation of sites outside of the invariant sequence or deletion of the entire invariant sequence; whereas sites influencing the effect of TCDD were dependent on the cellular activation state (i.e. unstimulated vs. LPS stimulation) and relatively independent of the putative AhR binding site within the invariant sequence. These results suggest that AhR activation affects human hs1.2 activity through an as yet undetermined non-canonical pathway. A better understanding regarding the role of the hs1.2 enhancer in human Ig expression and how AhR ligands modulate its activity may lead to insights into overall Ig regulation and mechanisms of dysfunction.

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

BackgroundWhile achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence...

Localised pulmonary crystal-storing histiocytosis with underlying extranodal marginal zone lymphoma: A case report

Crystal-storing histiocytosis (CSH) is a rare entity, composed of a proliferation of non-neoplastic histiocytes with an accumulation of intracytoplasmic crystalline material. It is often associated with an underlying lymphoproliferative disorder, most often those that express monoclonal immunoglobulins, such as multiple myeloma and lymphoplasmacytic lymphoma. Here we report a case of localised pulmonary CSH in an asymptomatic 54-year-old man, found to have an incidental solid lesion in the right lung on computed tomography (CT). The lesion was composed of sheets of CD68 positive histiocytes that contained refractile eosinophilic crystalline inclusions, scattered lymphoid follicles of CD20 positive B lymphocytes, and occasional plasma cells. PCR for immunoglobulin heavy chain gene rearrangement confirmed the presence of a monoclonal population of B cells. These findings were supportive of a diagnosis of CSH with an underlying B cell/ plasma cell lymphoproliferative disorder, which was favoured to be an extranodal marginal zone lymphoma after clinical and radiological workup. Recognition of this rare entity is important as it may obscure an underlying lymphoproliferative disorder. A diagnosis of CSH should prompt careful exclusion of neoplasia, including clinical workup for systemic disease. In this case report we also discuss clinical features and diagnostic pitfalls.

Novel genes exhibiting DNA methylation alterations in Korean patients with chronic lymphocytic leukaemia: a methyl-CpG-binding domain sequencing study

Chronic lymphocytic leukaemia (CLL) exhibits differences between Asians and Caucasians in terms of incidence rate, age at onset, immunophenotype, and genetic profile. We performed genome-wide methylation profiling of CLL in an Asian cohort for the first time. Eight Korean patients without somatic immunoglobulin heavy chain gene hypermutations underwent methyl-CpG-binding domain sequencing (MBD-seq), as did five control subjects. Gene Ontology, pathway analysis, and network-based prioritization of differentially methylated genes were also performed. More regions were hypomethylated (2,062 windows) than were hypermethylated (777 windows). Promoters contained the highest proportion of differentially methylated regions (0.08%), while distal intergenic and intron regions contained the largest number of differentially methylated regions. Protein-coding genes were the most abundant, followed by long noncoding and short noncoding genes. The most significantly over-represented signalling pathways in the differentially methylated gene list included immune/cancer-related pathways and B-cell receptor signalling. Among the top 10 hub genes identified via network-based prioritization, four (UBC, GRB2, CREBBP, and GAB2) had no known relevance to CLL, while the other six (STAT3, PTPN6, SYK, STAT5B, XPO1, and ABL1) have previously been linked to CLL in Caucasians. As such, our analysis identified four novel candidate genes of potential significance to Asian patients with CLL.

Concerted variation of the 3' regulatory region of Ig heavy chain and Gm haplotypes across human continental populations.

The 3' regulatory region of the immunoglobulin heavy chain gene (IGH) includes the HS1.2 enhancer displaying length polymorphism with four known variants. The goal of the research was to provide an overview of this variability and of its evolutionary significance across human populations. We compiled published and original data on HS1.2 polymorphism in 3,100 subjects from 26 human populations. Moreover, we imputed the haplotypic arrangement of the HS1.2 region in the 1000 Genomes Project (1KGP). In this dataset, imputation could also be obtained for the G1m-G3m allotype by virtue of the precise correspondence between serological types and amino acid (and DNA) substitutions in IGHG1 and IGHG3. HS1.2 variant frequencies displayed similar patterns of continental partitioning as those reported in the literature for the physically neighboring IGHG1-IGHG3 system. The 1KGP data revealed that linkage disequilibrium (LD) can explain the spread of joint HS1.2-IGHG1-IGHG3 associations across continents and within continental populations, with stronger LD out of Africa and the features of an evolutionarily stable genomic block with differential expression in lymphoblastoid cell lines. Strong population structuring involves at least the entire 70 kb genomic region here considered, due to the tight LD which maintained HS1.2, IGHG1, and IGHG3 in nonrandom arrangements. This might be key to better understand the evolutionary path of the entire genomic region driven by immune response capabilities, during the formation of continental gene pools.

Diffuse Large B-Cell Lymphoma during Complete Remission of Acute Myeloid Leukemia (M4Eo) Exhibiting Immunoglobulin Heavy Chain Gene Rearrangement and Inv(16)

In previous studies, it has been reported that 10-20% of acute myeloid leukemia (AML) cases showed immunoglobulin heavy chain gene (IGH) rearrangements, a genetic hallmark of B-cell differentiation. However, the clinical significance of this is uncertain. Here, we report a case of diffuse large B-cell lymphoma (DLBCL) after complete remission (CR) from AML that exhibited an IGH rearrangement.

The Yin and Yang of RNA surveillance in B lymphocytes and antibody-secreting plasma cells

The random V(D)J recombination process ensures the diversity of the primary immunoglobulin (Ig) repertoire. In two thirds of cases, imprecise recombination between variable (V), diversity (D), and joining (J) segments induces a frameshift in the open reading frame that leads to the appearance of premature termination codons (PTCs). Thus, many B lineage cells harbour biallelic V(D)J-rearrangements of Ig heavy or light chain genes, with a productively-recombined allele encoding the functional Ig chain and a nonproductive allele potentially encoding truncated Ig polypeptides. Since the pattern of Ig gene expression is mostly biallelic, transcription initiated from nonproductive Ig alleles generates considerable amounts of primary transcripts with out-of-frame V(D)J junctions. How RNA surveillance pathways cooperate to control the noise from nonproductive Ig genes will be discussed in this review, focusing on the benefits of nonsense-mediated mRNA decay (NMD) activation during B-cell development and detrimental effects of nonsense-associated altered splicing (NAS) in terminally differentiated plasma cells.

The Immunoglobulins: New Insights, Implications, and Applications.

Immunoglobulins (Igs), as one of the hallmarks of adaptive immunity, first arose approximately 500 million years ago with the emergence of jawed vertebrates. Two events stand out in the evolutionary history of Igs from cartilaginous fish to mammals: (a) the diversification of Ig heavy chain (IgH) genes, resulting in Ig isotypes or subclasses associated with novel functions, and (b) the diversification of genetic and structural strategies, leading to the creation of the antibody repertoire we know today. This review first gives an overview of the IgH isotypes identified in jawed vertebrates to date and then highlights the implications or applications of five new recent discoveries arising from comparative studies of Igs derived from different vertebrate species.

Clonality testing in the lymph nodes from dogs with lymphadenomegaly due to Leishmania infantum infection.

IntroductionIn southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis.Materials and methods31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR.ResultsAll 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background.ConclusionInfections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings.

Molecular diagnosis of bovine B-cell lymphoma using polymerase chain reaction for immunoglobulin heavy chain gene.

We performed a clonality analysis using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) gene rearrangement, specifically with regard to its utility as a method to diagnose bovine B-cell lymphoma. PCR for IgH gene rearrangement indicated monoclonal proliferation of B-cells in 24 of 35 cattle with B-cell lymphoma. In contrast, PCR for IgH gene rearrangement in lymph nodes and tumor tissues from 65 cattle diagnosed with tumors other than B-cell lymphoma and non-tumors revealed polyclonal population of B-cells. Sensitivity, specificity, positive predictive value, and negative predictive value for PCR for IgH gene rearrangement for bovine B-cell lymphoma were 68.6%, 100%, 100%, and 85.5%, respectively. Clonality analysis using PCR for IgH gene rearrangement may be useful for adjunctive diagnosis of bovine B-cell lymphoma.