Ig Heavy Chain Variable Region Research Articles

Proximally Biased V(D)J Recombination and Evolution of Non-Productive Clones in B-Cell Precursor Acute Lymphocytic Leukemia with KMT2A::AFF1 Fusion Genes

Background: B-cell precursor acute lymphocytic leukemia (BCP-ALL) carrying KMT2A::AFF1 rearrangements is an aggressive disease with unsatisfactory response to treatment and poor prognosis. KMT2A is a histone methyl transferase involved in the establishment of epigenetic memory. In the fusion protein encoded by the KMT2A::AFF1 fusion gene, the catalytic SET domain of KMT2A mediating H3K4 trimethylation is lost. Instead, the fusion protein interacts with DOT1L and mediates H3K79 methylation. The subversion of the catalytic activity of KMT2A arrests affected cells in an immature stage of development that may precede V(D)J recombination. Aim: Investigate the immune repertoire of KMT2A::AFF1 rearranged BCP-ALL and evaluate the feasibility of measurable residual disease (MRD) detection using immunoglobulin (Ig) gene rearrangements. Patients and Methods: The cohort includes 290 adult and 89 pediatric BCP-ALL patients. The diagnosis was established following WHO classification. WTS (5x107 reads, 2x101bp) data were generated on HiSeq and NovaSeq instruments. Ig gene rearrangements were analyzed using MiXCR and NCBI-IgBlast. Results: We analyzed Ig heavy-chain variable region (IGHV) gene rearrangements in BCP-ALL samples. We observed that the immune repertoire of leukemic cells carrying KMT2A::AFF1 is less diverse than that of other BCP-ALL samples. We investigated the expression levels of IGHV genes using WTS data. We found that IGHV genes located proximally to the IGHD/IGHJ cluster of genes on chr14 were more likely to be expressed in samples carrying KMT2A::AFF1 (Fig 1A). This difference was particularly significant (p<10-18) for IGHV6-1, the IGHV gene closest to the IGHD/IGHJ cluster (Fig 1B). Proximally biased V(D)J recombination is indicative of incomplete Ig locus contraction. Locus contraction is regulated by PAX5, which suppresses WAPL, a negative regulator of chromatin loop extrusion. Chromatin looping of IGHV genes ensures a balanced immune repertoire by bringing distant IGHV genes in proximity to the V(D)J recombination center. We found that in samples with KMT2A::AFF1 fusion genes WAPL expression levels are significantly (p<10-10) higher than in other subtypes of BCP-ALL. Analysis of the Ig gene rearrangements in KMT2A::AFF1 samples revealed abundant expression of alleles encoding non-productive V(D)J rearrangements or IGHV alleles in germline configuration. Only 35% of complementarity determining region 3 covering reads encoded productive rearrangements in KMT2A::AFF1 samples while in other types of BCP-ALL 67% of reads are productive (p<10-6). In samples with chronic lymphocytic leukemia, a tumor of post-germinal center B cells, this percentage is generally 90% or more. Among the non-productive V(D)J rearranged alleles, in-frame stop codons were equally overrepresented as out-of-frame V-J fusions when compared to their abundance in other types of BCP-ALL (p<10-15 in both cases). As light chain rearrangements require signaling from a productive heavy chain, the significantly (p<9x10-7) lower expression of light chain alleles detectable in KMT2A::AFF1 samples supports the notion that the abundant expression of non-productive alleles is not an artefact. Clonal expansion as measured by the Gini index for the number of reads per clonotype is distributed uniformly between 0 (polyclonality) and 1 (monoclonality). Monoclonality was observed in only 10% of cases. Furthermore, some DJ-rearrangements with identical non-templated linker sequences were found to be recombined with different IGHV genes, indicating ongoing V(D)J recombination and clonal evolution. Conclusions: Our data suggest that the KMT2A::AFF1 rearrangement locks the cell of origin at a point of ongoing V(D)J recombination. V(D)J recombination appears to be induced prematurely in these cells. Poor locus contraction could explain the proximally biased V(D)J recombination pattern. The non-sense mediated decay pathway involved in down regulating the expression of non-productive alleles seems to be poorly functional. Consequently, the cells express unusually high amounts of non-productive alleles or alleles in germline configuration that are clonally evolving and derived frequently from IGHV6-1. These features distinguish KMT2A::AFF1 BCP-ALL from other BCP-ALL subtypes and make MRD detection in KMT2A::AFF1 BCP-ALL based on specific Ig gene rearrangements highly error prone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gastric DLBCL clonal evolution as function of patient age.

Diffuse large B cell lymphoma (DLBCL) is the most common type of NHL, accounting for about 40% of NHL cases, and is one of the most aggressive lymphomas. DLBCL is widespread in individuals aged more than 50 years old, with a maximum incidence in the seventh decade, but it may also occur in younger patients. DLBCL may occur in any immune system tissue, including those around the gastrointestinal tract, and even in the stomach, though gastric DLBCL has yet to be sufficiently investigated. This study aimed to understand changes in gastric Diffuse Large B cell lymphoma (gastric DLBCL) development with age. Immunoglobulin (Ig) heavy chain variable region genes were amplified from sections of nine preserved biopsies, from patients whose age varied between 25 and 89 years, sequenced and analyzed. We show first that identification of the malignant clone based on the biopsies is much less certain than was previously assumed; and second that, contrary to expectations, the repertoire of gastric B cell clones is more diverse among the elderly DLBCL patients than among the young.

Analysis of expressed immunoglobulin heavy chain genes in familial B-CLL

In this study, we wished to determine whether familial chronic lymphocytic leukemia of B-cell phenotype (CLL) shares with sporadic B-CLL the same immunoglobulin (Ig) heavy chain variable region (VH) gene usage and occurrence of somatic mutation, to gain insight into the pathogenetic relatedness of these epidemiologically distinct forms of CLL. We therefore analyzed the expressed Ig heavy chain genes in 23 cases (11 families) of familial CLL, and compared these results with data previously reported for sporadic CLL. In addition, we assessed the relationship of the occurrence of somatic mutation to several clinical and phenotypic features. The distribution of V genes among these cases was similar to that observed in sporadic CLL: VH3 > VH1 > VH4. Thirteen of the 23 cases (57%) showed germ line VH gene sequences, whereas somatic mutations were detected in 10 cases (43%). The average mutation frequency of these latter 10 cases of was 6.7% (ranging from 1.7% to 8.8%), and evidence of antigen selection was noted in 6. Intraclonal variation, followed by clonal evolution and the appearance of a second clone over a 20-year period was observed in 1 case, suggesting that mutations can continue to accumulate after neoplastic transformation. The presence of somatic mutations correlated with age at presentation, low white blood cell (WBC) count, and low fluorescence intensity of surface CD5, and the potential significance of these relationships is discussed. Our data indicate that familial and sporadic B-CLL display a similar pattern of immunoglobulin gene usage and frequency of somatic mutation, and are consistent with a common ontogeny and immunogenetic origin for these 2 epidemiologically distinct forms of CLL. (Blood. 2000;95:1413-1419)

Longitudinally persistent cerebrospinal fluid B cells can resist treatment in multiple sclerosis.

B cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B cells exist in the cerebrospinal fluid (CSF), meninges, and CNS parenchyma of MS patients. We sought to investigate the presence of clonally related B cells over time by performing Ig heavy chain variable region repertoire sequencing on B cells from longitudinally collected blood and CSF samples of MS patients (n = 10). All patients were untreated at the time of the initial sampling; the majority (n = 7) were treated with immune-modulating therapies 1.2 (±0.3 SD) years later during the second sampling. We found clonal persistence of B cells in the CSF of 5 patients; these B cells were frequently Ig class-switched and CD27+. Specific blood B cell subsets appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B cell persistence in CSF and blood, even in patients on immune-modulating therapy. Our findings support the concept that peripheral B cell activation and CNS-compartmentalized immune mechanisms can in part be therapy resistant.

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Prostate adenocarcinoma (by Nephron, Wikimedia Commons).A chimeric mouse model with reliable formation of prostate tumors was used to test therapies for advanced prostate cancer. Checkpoint blockade alone was ineffective, but, by also targeting MDSCs (by inhibiting tyrosine kinases and the PI3K pathway), IL1 receptor antagonist was upregulated, suppressive Gr-MDSCs and the supporting cytokines were reduced, infiltration of CD8+ T cells into tumors increased, and growth of tumors and metastases were inhibited.Lu X, …, DePinho RA. Nature 2017 Mar 20; doi: 10.1038/nature21676.DCs and peacock flounder have signatures responsive to their locales (by B. Inaglory, Wikimedia).Organ donor tissue from 78 people of different ages was used to assess DC populations in mucosal tissues and lymph nodes during the human lifespan. cDC2s were the dominant cDCs in lymph nodes from an early age. DC subset composition varied by location, with DCs expressing tissue-specific signatures. cDC2s also appear to function as the primary guardians at mucosal surfaces.Granot T, …, Farber DL. Immunity 2017;46:504–15.Strengthening macrophage antitumor responses (from PublicDomainPictures.net).Tumor-associated macrophages are often immunosuppressive. The class IIa HDAC inhibitor TMP195 was found to activate macrophages at the tumor site and normalize the vasculature without directly altering lymphocytes. Administering the drug reduced tumor burdens and decreased metastasis. When combined with checkpoint blockade, TMP195 induced a durable antitumor response in a mouse MMTV-PyMT breast cancer model.Guerriero JL., …, Letai A. Nature 2016;543:428–32.SPADE tree of V δ2 subsets (Lo Presti et al. Cancer Immunol Res 2017. DOI: 10.1158/2326-6066.CIR-16-0348).Knowledge of the human γδ TCR repertoire during development or after infection is scant. Next-gen sequencing revealed that repertoires of rearranged genes encoding γδ TCRs in the peripheral blood of healthy adults were stable over time. Although γδ T cells were quickly reconstituted after hematopoietic stem cell transplantation, the TCR repertoires were profoundly altered. CMV infection resulted in clonal expansion of distinct γδ T cell responses, underscoring the clinical association between increased γδ T cells and improved long-term disease-free survival, and providing further evidence for adaptive anti-viral γδ T cell immunity.Ravens S, …, Prinz I. Nat Immunol 2017 Apr; 18:393–401.Mantle cell lymphoma of the terminal ileum (by Nephron, Wikimedia Commons).Analysis by liquid chromatography and tandem mass spectrometry of 17 human mantle cell lymphomas revealed that all presented neoepitopes were derived from the lymphoma's unique immunoglobulin. The neoantigenic peptides were exclusively derived from the Ig heavy and light chain variable regions, and presented by MHC class II. Circulating CD4+ T cells specific for these neoantigens were isolated from patients and could kill autologous lymphoma cells. Peptides from other genes were not recovered from MHC, supporting a focus for immunotherapy on tumor-expressed immunoglobulins.Khodadoust MS, …, Alizadeh AA. Nature 2017;543:723–7.PD-1 blocks signal flow from CD28 (from Siena College on Flickr)..Two papers in SCIENCE looked at the interplay between PD-1 and CD28 signaling, in different systems. Kamphorst and colleagues found that blockade of PD-1 will not restore T cell responsiveness unless CD28 can signal. A biochemical analysis by Hui and colleagues surprisingly revealed that CD28 is more sensitive than the TCR to PD-1 and SHP-2 dephosphorylation. These studies suggest costimulatory pathways play key roles in regulating responses to anti-PD-L1/PD-1 therapy, and imply a distinct early memory population may be the target of rescue.Kamphorst AO, …, Ahmed R. Science 2017 Mar 31;355:1423–7.Hui E, …, Vale RD. Science 2017 Mar 31;355:1428–33.

Antigen presentation profiling reveals T-cell recognition of lymphoma immunoglobulin neoantigens

Abstract Presentation of novel antigenic peptides (neoantigens) that distinguish malignant from normal cells by major histocompatibility complexes (MHC) can serve as potent substrates for specific anti-tumor immune responses. We sought to identify mantle cell lymphoma (MCL) neoantigens by taking an integrated genomic and proteomic strategy that interrogates antigen peptides presented by MHC-class I and class II. Peptides bound to MHC were purified via immunoprecipitation followed by identification using mass spectrometry. Mass spectra were searched against patient-specific proteome databases generated by whole exome sequencing and targeted immunoglobulin gene sequencing. This approach was applied to systematically characterize over 36,000 immunopeptides from 17 patients’ tumor specimens. Interestingly, 52 neoantigenic peptides were derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we identified MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. Furthermore, somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC-II. T-cells specific for Ig-derived neoantigens were found in two patients. Following ex vivo activation and expansion, the T-cells were remarkably able to mediate killing of autologous lymphoma cells. These results demonstrate that combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to MCL implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

Change in IgHV Mutational Status of CLL Suggests Origin From Multiple Clones

Fluorescence in situ hybridization and immunoglobulin (Ig) heavy-chain variable-region (IgHV) mutational status are used to predict outcome in chronic lymphocytic leukemia (CLL). Although DNA aberrations change over time, IgHV sequences and mutational status are considered stable. In a retrospective review, 409 CLL patients, between 2008 and 2015, had IgHV analysis: 56 patients had multiple analyses performed. Seven patients' IgHV results changed: 2 from unmutated to mutated and 5 from mutated to unmutated IgHV sequence. Three concurrently changed their variable heavy-chain sequence. Secondary to allelic exclusion, 2 of the new variable heavy chains produced were biologically nonplausible. The existence of these new nonplausible heavy-chain variable regions suggests either the CLL cancer stem-cell maintains the ability to rearrange a previously silenced IgH allele or more likely that the cancer stem-cell produced at least 2 subclones, suggesting that the CLL cancer stem cell exists before the process of allelic exclusion occurs.

The active intrathecal B-cell response in LGI1-antibody encephalitis

BackgroundLeucine-rich glioma inactivated 1 (LGI1) is a component of the voltage-gated potassium channel complex. IgG antibodies against LGI1 are associated with immunotherapy-responsive encephalitis and epilepsies. LGI1-antibody concentrations are 10–100 times greater in serum than in cerebrospinal fluid (CSF). Oligoclonal IgG bands are rarely found in patients with LGI1-antibody encephalitis or epilepsy. These observations raise questions about the sources of the B cells that result in production of LGI1 antibodies and how the IgGs reach the brain. We aimed to investigate the migration and expansions of peripheral and central B cells to the production of LGI1-specific IgG. MethodsWe performed PCR amplification and next generation deep immune repertoire sequencing of immunoglobulin (Ig) heavy chain variable regions (VH) from CSF and subsorted peripheral blood B-cell populations from two patients with limbic encephalitis and faciobrachial dystonic seizures associated with LGI1 antibodies. Bioinformatics clustering of related IgM-VH or IgG-VH transcripts was used to determine whether active B-cell diversification could be observed, and whether intrathecal B-cell repertoires, if present, were related to peripheral B cells. FindingsWe identified clusters of related Ig-VH transcripts in the CSF of both patients. Within these clusters there was a range of somatic hypermutations along the IGHV germline segment-derived portion. In addition, we identified a large number of closely related Ig-VH clusters that were common to both CSF and peripheral blood, including a small number of dominating Ig-VH clusters that might represent the most active clonally related B-cell populations. InterpretationOur data suggest that some B-cell affinity maturation occurs inside the CNS compartment in LGI1-antibody encephalitis. Somatic hypermutation rates point to a CSF antigen-driven activation of clonally related B cells that shape the intrathecal immune repertoire. The target antigen or antigens of these clonally related B cells remain unknown; our work continues to determine the relative contribution of intrathecally activated and peripheral LGI1-specific B cells in this autoimmune CNS disease. FundingWellcome Trust Intermediate Fellowship to SRI, Fulbright-MS Society, Epilepsy Research UK, BMA Vera Down Research Grant.

Limbic encephalitis with antibodies to LGI1 is associated with an active intrathecal B cell response

Background: Leucine-rich glioma inactivated 1 (LGI1) is a component of the voltage-gated potassium channel complex (VGKC). Antibodies against LGI1 are associated with limbic encephalitis and related central nervous system (CNS) disorders. In most patients, anti-LGI1 antibodies are found in the serum, while their occurrence in the cerebrospinal fluid (CSF) is much less common. Oligoclonal IgG (oligoclonal bands, OCB), a prominent indicator for intrathecal B cell activation, are rarely found in patients with LGI1 encephalitis. Overall, the immune pathology resulting in LGI1 autoimmunity is unclear. However, partially due to the absence of OCB, peripheral immune processes are believed to be the principal disease drivers. Objective: To investigate whether an active intrathecal B cell immune response is present in patients with LGI1 antibodies. Methods: We applied PCR amplification and next generation deep immune repertoire sequencing (DIRS) of B cell receptor immunoglobulin (Ig) heavy chain variable regions (VH) from CSF and peripheral blood B cells of 2 patients with LGI1 encephalitis. Extensive bioinformatics clustering of related IgM-VH or IgG-VH transcripts was employed to determine 1) whether active B cell immune mechanisms operate in LGI1 encephalitis, and 2) whether intrathecal B cell repertoires, if present, are related to peripheral B cells. Results: We identified numerous clusters of related Ig-VH transcripts in the CSF of both patients; within these clusters we found a range of somatic hypermutations (SHM) along the IGHV germline segmentderived portion suggesting that local antigen-driven activation of clonally related B cells was involved in shaping the intrathecal immune repertoire. Furthermore, we identified a large number of closely related Ig-VH clusters that were common to both, CSF and periphery. Interestingly, in each patient we identified a small number of dominating Ig-VH clusters that may represent the most active clonally related B cell populations. Conclusion: Our data suggest that in LGI1 encephalitis B cell affinity maturation occurs inside the CNS compartment. The target antigen(s) of the clonally related B cells described here remain unknown; ongoing work will determine whether intrathecally activated B cells and/or B cells that apparently target identical antigens in the periphery and CNS compartments are involved in LGI1 autoimmunity.

‘Cytokine defined’ B cell subsets in multiple sclerosis

Background: Leucine-rich glioma inactivated 1 (LGI1) is a component of the voltage-gated potassium channel complex (VGKC). Antibodies against LGI1 are associated with limbic encephalitis and related central nervous system (CNS) disorders. In most patients, anti-LGI1 antibodies are found in the serum, while their occurrence in the cerebrospinal fluid (CSF) is much less common. Oligoclonal IgG (oligoclonal bands, OCB), a prominent indicator for intrathecal B cell activation, are rarely found in patients with LGI1 encephalitis. Overall, the immune pathology resulting in LGI1 autoimmunity is unclear. However, partially due to the absence of OCB, peripheral immune processes are believed to be the principal disease drivers. Objective: To investigate whether an active intrathecal B cell immune response is present in patients with LGI1 antibodies. Methods: We applied PCR amplification and next generation deep immune repertoire sequencing (DIRS) of B cell receptor immunoglobulin (Ig) heavy chain variable regions (VH) from CSF and peripheral blood B cells of 2 patients with LGI1 encephalitis. Extensive bioinformatics clustering of related IgM-VH or IgG-VH transcripts was employed to determine 1) whether active B cell immune mechanisms operate in LGI1 encephalitis, and 2) whether intrathecal B cell repertoires, if present, are related to peripheral B cells. Results: We identified numerous clusters of related Ig-VH transcripts in the CSF of both patients; within these clusters we found a range of somatic hypermutations (SHM) along the IGHV germline segmentderived portion suggesting that local antigen-driven activation of clonally related B cells was involved in shaping the intrathecal immune repertoire. Furthermore, we identified a large number of closely related Ig-VH clusters that were common to both, CSF and periphery. Interestingly, in each patient we identified a small number of dominating Ig-VH clusters that may represent the most active clonally related B cell populations. Conclusion: Our data suggest that in LGI1 encephalitis B cell affinity maturation occurs inside the CNS compartment. The target antigen(s) of the clonally related B cells described here remain unknown; ongoing work will determine whether intrathecally activated B cells and/or B cells that apparently target identical antigens in the periphery and CNS compartments are involved in LGI1 autoimmunity.

Immunoglobulin heavy chain variable region and major histocompatibility region genes are linked to induced graves' disease in females from two very large families of recombinant inbred mice.

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans.

Genome complexity in the coelacanth is reflected in its adaptive immune system

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

Developmental progression of equine immunoglobulin heavy chain variable region diversity

Humoral immunity is a critical component of the immune system that is established during fetal life and expands upon exposure to pathogens. The extensive humoral immune response repertoire is generated in large part via immunoglobulin (Ig) heavy chain variable region diversity. The horse is a useful model to study the development of humoral diversity because the placenta does not transfer maternal antibodies; therefore, Igs detected in the fetus and pre-suckle neonate were generated in utero. The goal of this study was to compare the equine fetal Ig VDJ repertoire to that of neonatal, foal, and adult horse stages of life. We found similar profiles of IGHV, IGHD, and IGHJ gene usage throughout life, including predominant usage of IGHV2S3, IGHD18S1, and IGHJ1S5. CDR3H lengths were also comparable throughout life. Unexpectedly, Ig sequence diversity significantly increased between the fetal and neonatal age, and, as expected, between the foal and adult age.

Molecular characterization of immunoglobulin (Ig) genes reveals that plasmablasts generated during heterologous secondary dengue infections and circulating memory B cells after recovery are of distinct origin

Event Abstract Back to Event Molecular characterization of immunoglobulin (Ig) genes reveals that plasmablasts generated during heterologous secondary dengue infections and circulating memory B cells after recovery are of distinct origin Ramapraba Appanna1, Xu Mei Hui1, Toh Ying Xiu1, Thavamalar Balakrishnan1, Srinivasan Kandhadayar Gopalan1, Francesca Zolezzi1, Michael Poidinger1, Leo Yee Sin2, Wang Cheng-I1 and Katja Fink1* 1 Singapore Immunology Network, Singapore 2 Tan Tock Seng Hospital, Department of Infectious Diseases, Singapore Memory B cells generated after exposure to dengue viral (DENV) infection are a central component in shaping immune memory. However, re-exposure to dengue virus of a different serotype is often associated with an increased disease pathogenesis. During repeated infection, the rapid activation of memory B cells leads to the generation of massive amounts of cross-reactive antibodies. In the acute phase of a secondary infection, this B cell memory pool probably outcompetes the newly generated virus specific B cells. To characterize the specificity of plasmablasts and memory B cells generated during and after acute infection, we performed single cell RT-PCR for sequence analysis of Ig variable regions. Surprisingly, we found that memory B cells isolated one month after disease were of different genetic composition than acute phase plasmablasts. Our data reveal preferential usage of the Ig heavy chain variable region gene VH1 in DENV binding plasmablasts. In contrast, VH3 family repertoires were noted in specific long lasting circulating memory cells isolated over a month after infection. The antibodies isolated from these memory B cells had a broad reactivity, with only 30% being specific to DENV envelope (E) protein, whereas 70-80% plasmablasts were specific to E protein. Whether VH gene usage differs in primary and secondary infected patients and reflects a specific antigen/epitope selection is currently under investigation. This study can help to understand immune mechanism of protection for potential application in vaccination. Keywords: Dengue, memory B cell, plasmablast, immunoglobulin, repertoire Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Appanna R, Mei Hui X, Ying Xiu T, Balakrishnan T, Kandhadayar Gopalan S, Zolezzi F, Poidinger M, Yee Sin L, Cheng-I W and Fink K (2013). Molecular characterization of immunoglobulin (Ig) genes reveals that plasmablasts generated during heterologous secondary dengue infections and circulating memory B cells after recovery are of distinct origin. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00905 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Katja Fink, Singapore Immunology Network, SINGAPORE, Singapore, katjafink3@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ramapraba Appanna Xu Mei Hui Toh Ying Xiu Thavamalar Balakrishnan Srinivasan Kandhadayar Gopalan Francesca Zolezzi Michael Poidinger Leo Yee Sin Wang Cheng-I Katja Fink Google Ramapraba Appanna Xu Mei Hui Toh Ying Xiu Thavamalar Balakrishnan Srinivasan Kandhadayar Gopalan Francesca Zolezzi Michael Poidinger Leo Yee Sin Wang Cheng-I Katja Fink Google Scholar Ramapraba Appanna Xu Mei Hui Toh Ying Xiu Thavamalar Balakrishnan Srinivasan Kandhadayar Gopalan Francesca Zolezzi Michael Poidinger Leo Yee Sin Wang Cheng-I Katja Fink PubMed Ramapraba Appanna Xu Mei Hui Toh Ying Xiu Thavamalar Balakrishnan Srinivasan Kandhadayar Gopalan Francesca Zolezzi Michael Poidinger Leo Yee Sin Wang Cheng-I Katja Fink Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The clonality of immunoglobulin repertoire in prostate cancer-infiltrating B cells.

e15168 Background: There is emerging evidence that inflammation plays a role in prostate carcinogenesis. To better understand the cellular biology of the immune response in prostate cancer, we investigated the immunoglobulin (Ig) repertoire of tumor-infiltrating B cells. Methods: The Ig heavy chain variable region (VH) usage reflects the antigenic experience of tumor-infiltrating B cells and may help to identify potential therapeutic targets. We analyzed IgM-VH and IgG-VH transcripts of tumor-infiltrating B cells in four tumor samples of localized prostate cancer. Immunohistochemical staining revealed groups of CD20+ B cells surrounding neoplastic prostate glands. To exclude PCR amplification bias, two cDNA librarys were prepared from separate tissue sections of each tumor. IgM-VH and IgG-VH transcripts were amplified by RT-PCR from each cDNA library, cloned and sequenced. Results: Sequencing of a total of 80 IgM-VH and 80 IgG-VH PCR products (10 IgM-VH and 10 IgG-VH PCR products from each cDNA library) revealed an oligoclonal Ig repertoire in 3 of 4 and evidence of antigen experience of B cells in 4 of 4 prostate cancer samples. IgM and IgG B cell clones, defined by the same complementarity-determining region 3 (CDR3) and detection in two separate cDNA librarys, were widely distributed in the prostate. Prostate cancer-infiltrating B cells showed somatic hypermutation, Ig class switch and insertions or deletions indicating antigen experience. Conclusions: Clonally related B cells are widely distributed in the neoplastic prostate gland suggesting the recognition of a limited number of epitopes. Future studies will identify the antigens eliciting this immune response.

Regulation of the Mutation Threshold During Immune Diversification by Activation Induced Cytidine Deaminase,

Abstract 3244Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. AID converts cytosine to uracil and resulting U-G mismatches are subsequently processed by low-fidelity base excision and mismatch repair pathways to yield point mutations (for SHM) and DNA strand breaks (for CSR). Under normal conditions, genotoxic stressors activate DNA damage response pathways that result in DNA repair or cell cycle arrest and apoptosis. However, in normal germinal center B cells, key DNA damage checkpoint factors such as ATR, p53 and p21 are repressed by the germinal center transcriptional repressor BCL6. BCL6 thus creates a permissive environment to allow the accumulation of mutations within the Ig loci. However, in addition to this fundamental role in immune diversification, aberrant targeting of AID contributes to point mutations and translocations of proto-oncogenes associated with B cell malignancy. Indeed, the combined effect of BCL6 and AID poses a direct threat to genomic integrity but the mechanism responsible for regulating the mutation threshold in germinal center B cells is not understood. To determine if B cells have a mechanism for down-regulating AID activity in response to genotoxic stress, we subjected the Ramos-A23 cell line to continuous low-dose exposure to several genotoxic agents. Ramos-A23 is a Burkitt lymphoma line that constitutively expresses AID and mutates the Ig heavy chain variable region with high frequency. Mutation frequency can be monitored by flow cytometry through loss of surface expression of IgM (due to AID-dependent nonsense and missense mutations) and by direct sequencing of the variable region. IgM+ Ramos-A23 cells were maintained in continuous culture in the presence or absence of etoposide (100 nM), cytarabine (5 nM), 5-azacytidine (10 nM), or trichostatin A (15 nM). Drug concentrations were titrated to the highest dose that would minimally affect proliferation or survival when compared with untreated cells. After 4 weeks in culture, untreated clones were 20–22% IgM-, consistent with ongoing SHM. In contrast, clones treated with the DNA damaging agents etoposide or cytarabine were only 4–6% IgM- (p<0.0001). Cells treated with the epigenetic modifiers 5-azacytidine or trichostatin A were 15–19% IgM-. Sequence analysis of the Ig variable region (3 clones from each group) demonstrated 6.2 and 3.8-fold reductions in mutation frequency in cells treated with etoposide and cytarabine, respectively, when compared with untreated cells. The mutation frequency in the cells treated with epigenetic modifiers did not differ significantly from untreated controls. This suggests that while epigenetic stress has little effect on SHM, genotoxic stress leads to a significant reduction in AID activity. It has previously been demonstrated that genotoxic stress leads to an ATM-dependent down-regulation of BCL6, a finding that we also observed in our model. In addition, we found that genotoxic stress causes a dose and time-dependent decrease in AID protein and transcript levels, thus explaining the decrease in SHM in our system. Like BCL6, repression of AID by genotoxic stress was inhibited by pre-treating the cells with the ATM inhibitor KU-55933. Further analysis of markers of B cell differentiation revealed up-regulation of IRF4 and Blimp1 with complete down-regulation of PAX5 (a transcriptional regulator of AID) in response to genotoxic stress. Lentiviral expression of BCL6 shRNA in Ramos cells also lead to down-regulation of AID and SHM, consistent with the observation that BCL6-deficient B cells do not undergo SHM. It has also recently been demonstrated that DNA damage in germinal center B cells leads to ATM-dependent inactivation of the CREB transcriptional co-activator CRTC2 with subsequent down-regulation of CRTC2 target genes, including AID. Altogether, these data suggest that, during the germinal center response, a mutation threshold recognized by ATM leads to repression of the SHM machinery and reprogramming to facilitate B cell maturation. Additional investigation is needed to further define the critical steps in this regulatory pathway and how it might breakdown during the pathogenesis of B cell malignancy. Disclosures:No relevant conflicts of interest to declare.

Chlamydia Psittaci negative MALT Lymphomas of the Ocular Adnexa Are Characterized by Biased Use of the VH4 Family and Evidence for Antigen Selection

Abstract 2644Mucosa-associated lymphoid tissue (MALT) lymphomas are the most common lymphomas in the ocular adnexa. Similar to extranodular MALT lymphomas in other tissues, ocular adnexa MALT lymphomas (OAMALTL) are frequently characterized by an indolent clinical course and often remain localized for many years. While chronic infection (e. g. Helicobacter pylori) or autoimmune diseases (e.g. Hashimoto’s thyroiditis and Sjogren’s syndrome) frequently precede and predispose patients to develop MALT lymphomas in the stomach, thyroid and salivary glands, respectively, the etiology and pathogenesis of OAMALTL are still controversial. An Italian group and investigators from several other geographic regions demonstrated Chlamydia psittaci (C. psittaci) DNA present in OAMALTL, suggesting that this pathogen may be implicated in the development of these lymphomas. However, similar studies performed by us and other investigators on US-based patients have failed to corroborate this finding. Furthermore, DNA from other bacteria was also not detected by us, thus not supporting a bacterial etiology in US-based patients. Since non-bacterial antigens may predispose patients to OAMALTL, we have examined immunoglubulin (Ig) heavy chain variable region (VH) usage and mutations in the largest to date cohort of C psittaci negative OAMALTL originating from a single institution. DNA was extracted by standard techniques from 68 fresh OAMALTL tissues and used for direct PCR or PCR followed by cloning and sequencing using family specific VH leader and JH primers. To control for potential PCR error, all patient samples were evaluated by 2 independent PCR reactions and sequencing and only cases with identical CDR3 regions in the independent PCR runs were defined as clonal. Using this definition, clonal rearranged VHDJH sequences were identified in 44 (64.7%) tumors originating from the orbit (19), conjunctivae (19) and lacrimal gland (6). Forty seven clonal VH gene sequences were detected (3 of the patients had 2 clonal sequences) with 46 potentially functional and 1 harboring an out-of-frame junction with a stop codon. In 14 (31.8%) cases the PCR product could be sequenced directly, whereas in 30 (68.2%) cases PCR amplicons had to be subcloned to identify the VH gene. The 46 potentially functional VH were derived from 4 of the 7 human VH gene families with the following distribution: VH1, 13.1%; VH2, 2.2%; VH3, 39.1%; VH4, 45.6%, demonstrating a biased overrepresentation of the VH4 gene family (p=0.001). The most frequently encountered genes were VH4-34 (n=8), VH3-30 (n=6), VH3-23 (n=5) and VH4-30 (n=4). VH4-34 represented 17% of all the potentially functional VH genes identified in this study in contrast to its usage in 4–7% in adult peripheral B lymphocytes. Only 3 VH gene sequences were unmutated. The average percent homology to the germ line sequence in the 43 functional mutated sequences was 93.2% (range 71.5–99.6). Multinomial algorithm for antigen selection revealed evidence for positive selection in the CDR in 15 sequences, negative selection in the FR in 17 sequences, and selection in both CDR and FR (in the same sequence) in 7 sequences. Selection analysis using the focused binomial test demonstrated evidence of selection in the FR in 12 sequences and in the CDR in only 4 sequences. Selection was detected in 5 of the 8 functional VH4-34 sequences. Analysis of the tumor-derived CDR3 sequences revealed low similarity and an absence of stereotyped sequences with no homology to antibacterial and other previously published antibodies. The average CDR3 isoelectric point was 5.91±1.89 (SD). The average CDR3 length was 15.73±13.62 (SD) amino acids, with 19 sequences harboring 15–19 amino acids and 7 CDR3 sequences longer than 19 amino acids. Intraclonal variation was assessed by extensive molecular cloning in 8 potentially functional VH gene isolates from 8 randomly selected tumor specimens. Confirmed ongoing mutations were detected in the 6 of the 8 analyzed sequences. Overall our findings demonstrate that C. psittaci negative OAMALTL exhibit biased usage of VH families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating immunological stimulation in the pathogenesis of these lymphomas. The nature of the antigens potentially playing role in these processes is currently unknown and requires further studies. Disclosures:No relevant conflicts of interest to declare.

Recurrent Fevers, Cough, and Pulmonary Opacities in a Middle-Aged Man

A 55-year-old man was initially given a diagnosis of chronic lymphocytic leukemia (CLL) in April 1994 at age 44 years. After a period of observation, he was treated for progressive disease with chlorambucil in 1996 and again in 2002. In 2003, a prognostic marker evaluation showed that his CLL cells displayed “high-risk” biologic factors (unmutated Ig heavy chain variable region, and expression of CD38 and ζ-chain-associated protein 70) for progressive disease.1 The patient then developed cytopenia and was treated for progressive CLL in October 2003 with one cycle of high-dose dexamethasone (200 mg m−2 day−1 × 5 days) and rituximab (375 mg m−2 week−1 × 4 weeks) (Rituxan; Biogen Idec, Inc.

Autoreactivity in an HIV-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance

We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (V(H)DJ(H)) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 V(H)DJ(H) insertion supported development of large- and small pre-B cells that expressed the chimeric human/mouse Igmu chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 V(H)DJ(H) knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 V(H)DJ(H) knock-in mice support reduced numbers of residual splenic B cells with low surface IgM density, severely diminished serum IgM levels, but normal to elevated quantities of serum IgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery.

Somatic Hyperrepair: A Novel Tumor Suppression Mechanism for Germinal Center B Cells.

Abstract 92 BACKGROUND:All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells. HYPOTHESIS:We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation. METHODS:Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells. RESULTS:Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment. Disclosures:No relevant conflicts of interest to declare.