Chain Locus Research Articles

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype

Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. Using a flow cytometry-based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. Abelson virus-transformed Rag2-/- pro-B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild-type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP-expressing cells was evaluated by using flow cytometry, and high-throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild-type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. Our data support genotype-phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease-causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype.

Anti-insulin B cells are poised for antigen-presentation in pro-inflammatory subsets in type 1 diabetes

Abstract Type 1 diabetes (T1D) results from T-cell mediated autoimmune destruction of insulin-producing beta cells. Early breaches in B cell tolerance are central to T1D progression in mouse and man. Conventional transgenic mouse models (VH125.Tg NOD) suggested that B cell specificity is a powerful driver of disease. However, the precise staging of B cell development is limited in the conventional IgM-only model by the inability to class-switch. To investigate the developmental fate and functional status of anti-insulin B cells in NOD mice, we generated a new model (VH125SD.NOD) in which anti-insulin VDJH125 is targeted to the IgH chain locus and recombines with endogenous Vk chains to produce fully functional BCRs. This single copy VH transgene generates a small (1–2%) population of class-switch-competent anti-insulin B cells. Tracking their developmental fate reveals that anti-insulin B cells are preferentially skewed into marginal and late transitional zones which are known to have increased sensitivity to innate signals and enhanced capacity for antigen-driven expansion. Functional studies demonstrate that VH125SD.NOD anti-insulin B cells are anergic to antibody production; however, proliferation in response to B cell mitogens remains intact, diabetes is accelerated, and autoaggressive T cell responses to islet epitopes are increased. These findings validate the power of BCR specificity to drive T1D in a new mouse model and highlight the pathogenic potential of coupling a critical beta cell autoepitope to pro-inflammatory B cell subsets. We believe these findings may have implications for the discovery of new therapeutic targets in sources of innate signals that enhance autoreactivity in these B cell subsets, such as gut microbiota.

Small molecule inhibitors of Activation-Induced Deaminase

Abstract Activation-Induced Deaminase (AID) is a cytosine deaminase that converts cytosine into uracil in DNA, which initiates a cascade of mutagenic DNA repair to introduce point mutations and double-strand breaks. Specific targeting of AID to the immunoglobulin heavy chain locus promotes somatic hypermutation in antibody variable genes for affinity maturation, and breaks in switch regions for class switch recombination (CSR). However, mis-targeting of AID to other loci could initiate tumor development and lead to greater drug resistance among cancer cells when continually expressed. To identify a small molecule inhibitor of AID, we screened ~400,000 compounds in conjunction with the NCATS core facility at NIH. Using FRET based analysis of cytosine deamination, we identified 150 potential inhibitors of AID catalytic activity. To confirm biological function, we examined their effects on CSR in an in vitro murine B-cell activation assay using CH12 cells, wherein 30 were confirmed inhibitor candidates. We then selected the top seven molecules to proceed with further characterization in wild type primary splenocytes, and found two near-identical compounds that had inhibitory activity. From these structures, we tested commercially available analogues and identified two molecules with inhibitory efficacy in the nanomolar range. Using these approaches, we hope to identify a small molecule with great efficacy and low toxicity for use as a molecular probe to further characterize AID’s intricacies, or even as a therapeutic agent.

The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V segment locus synteny and chain-specific evolution

Abstract Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent order Afrotheria. We have previously described the immunoglobulin heavy chain locus of the Florida manatee (Trichechus manatus latirostris) and found limited antigen receptor diversity in this humoral arm of the adaptive immune system. We therefore investigated the antigen receptor loci of the cellular arm: the T cell receptor. Manatees are the first species from either Afrotheria or Xenarthra, the most basal eutherian taxonomic groups, to have an in-depth characterization of all T cell receptor loci and expressed repertoires. We manually annotated the three TR loci on the T. m. latirostris genomic scaffolds using BLAST and RSSsite, and compared the segments and overall genomic organization to the human TR loci. We identified the human V segment subgroups that are conserved in T. m. latirostris and found that TRADV are most conserved, TRBV are moderately conserved, and TRGV are the least conserved. The V segment order in the TRAD and TRB loci also showed synteny between the two species. The TRAD locus had five V segment subgroup synteny blocks that showed long-range duplications in the T. m. latirostris locus. The TRB locus had three V segment subgroup synteny blocks that showed short-range duplications. The conservation of V segment sequence and order correlated to the locus complexity, emphasizing the role of genomic organization on gene evolution. We also identified evidence for a VHδ pseudogene for the first time in a eutherian mammal. These novel findings underline the value of including species within the basal radiation of eutherian evolution in comparative studies.

Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates.

Analysis of TCRβ and TCRγ genes in Chinese alligator provides insights into the evolution of TCR genes in jawed vertebrates

All jawed vertebrates have four T cell receptor (TCR) chains that are expressed by thymus-derived lymphocytes and play a major role in animal immune defence. However, few studies have investigated the TCR chains of crocodilians compared with those of birds and mammals, despite their key evolutionary position linking amphibians, reptiles, birds and mammals. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization, evolution and expression of TRB and TRG loci in Alligator sinensis. According to the sequencing data, the Alligator sinensis TRB locus spans approximately 500 Kb of genomic DNA containing two D-J-C clusters and 43 V gene segments and is organized as Vβ(39)-pJβ1-pCβ1-pDβ1-Dβ2- Jβ2(12)-Cβ2-Vβ(4), whereas the TRG locus spans 115 Kb of DNA genomic sequence consisting of 18 V gene segments, nine J gene segments and one C gene segment and is organized in a classical translocon pattern as Vγ(18)–Jγ(9)-Cγ. Moreover, syntenic analysis of TRB and TRG chain loci suggested a high degree of conserved synteny in the genomic regions across mammals, birds and Alligator sinensis. By analysing the cloned TRB/TRG cDNA, we identified the usage pattern of V families in the expressed TRB and TRG. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed TRB and TRG sequences. Phylogenetic analysis revealed that TRB and TRG loci possess distinct evolutionary patterns. Most Alligator sinensis V subgroups have closely related orthologues in chicken and duck, and a small number of Alligator sinensis V subgroups have orthologues in mammals, which supports the hypothesis that crocodiles are the closest relatives of birds and mammals. Collectively, these data provide insights into TCR gene evolution in vertebrates and improve our understanding of the Alligator sinensis immune system.

Genetic Determinants of Antibody Levels in Cerebrospinal Fluid in Multiple Sclerosis: Possible Links to Endogenous Retroviruses.

The pathogenesis of multiple sclerosis (MS) has not been clarified. In addition to environmental factors; genetic determinants have been implicated in the pathogenesis of MS. Furthermore, endogenous retroviruses (ERV) might play a role in MS. The presence of oligoclonal immunoglobulin in cerebrospinal fluid (CSF) is a typical feature of MS. Recently, genetic polymorphisms in loci on human chromosomes 6, 14 and 18 have been identified as major determinants of CSF antibody levels in MS. The functional relevance of these single nucleotide polymorphisms (SNPs) remains unclear and none of them is located in an open reading frame. In previous studies, we identified ERV sequences in the vicinity of MS associated SNPs. Here, we describe the identification of ERV sequences in the neighborhood of SNPs associated with CSF antibody levels. All of the identified SNPs are located in the vicinity of ERV sequences. One of these sequences has very high homology to a sequence derived from the so-called MS-associated retrovirus (MSRV). Another cluster of three ERV sequences from the immunoglobulin heavy chain locus has retained the typical organization of retroviral genomes. These observations might shed new light on a possible association between ERVs and MS pathogenesis.

ELDA qASO-PCR for High Sensitivity Detection of Tumor Cells in Bone Marrow and Peripheral Blood.

A quantitative allele-specific polymerase chain reaction in combination with an extreme limiting dilution approach (ELDA qASO-PCR) enables the detection of tumor cells in patients with multiple myeloma (MM) in bone marrow (BM) samples and in peripheral blood (PB) with a sensitivity of <10-6. The two-step procedure of patient-specific tumor cell identification via the immunoglobulin heavy chain (IgH) and kappa/lambda light chain (k/λ LC) locus, followed by tumor cells quantification by ELDA qASO-PCR allows for the application of this method to the majority of MM patients, including those with Bence Jones proteinuria.

RAG Chromatin Scanning During V(D)J Recombination and Chromatin Loop Extrusion are Related Processes.

An effective adaptive immune system depends on the ability of developing B and T cells to generate diverse immunoglobulin (Ig) and T cell receptor repertoires, respectively. Such diversity is achieved through a programmed somatic recombination process whereby germline V, D, and J segments of antigen receptor loci are assembled to form the variable region V(D)J exons of Ig and TCRs. Studies of this process, termed V(D)J recombination, have provided key insights into our understanding of a variety of general gene regulatory and DNA repair processes over the last several decades. V(D)J recombination is initiated by the RAG endonuclease which generates DNA double-stranded breaks at the borders of V, D, and J segments. In this review, we cover recent work that has elucidated RAG structure and work that revealed that RAG has a novel chromatin scanning activity, likely mediated by chromatin loop extrusion, that contributes to its ability to locate V, D, J gene segment substrates within large chromosomal loop domains bounded by CTCF-binding elements (CBEs). This latter function, coupled with the role CBE-based chromatin loop domains and subdomains within them play in focusing V(D)J recombination activity within antigen receptor loci, provide mechanistic explanations for long-standing questions regarding V(D)J segment usage diversification and in limiting potentially deleterious off-target RAG-initiated recombination events genome-wide. This review will focus mainly on studies of the mouse Ig heavy chain locus, but the principles described also apply to other Ig loci and to TCR loci in mice and humans.

Abstract IA22: Targeting CARs to the TRAC locus enhances T-cell potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. The most successful CARs used to date are those targeting CD19, which offer the prospect of complete remissions in patients with chemorefractory/relapsed B cell malignancies. CARs are typically transduced into patient T cells using γ-retroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific modifications in human primary cells, including site-specific transgene integration into the CCR5 and AAVS1 loci in T lymphocytes. Using CRISPR/Cas9 to edit human T cells, we have established conditions yielding efficient target gene disruption and CAR insertion in a single step. We have found that directing a CD19 CAR to the human T-cell receptor (TCR) alpha chain (TRAC) locus not only results in efficient and uniform CAR expression in human peripheral blood T cells, but, remarkably, also enhances T-cell potency, vastly outperforming that of conventionally generated CAR T cells. We further show that CAR gene expression under the control of the TCR alpha promoter minimizes tonic signaling and allows effective regulation of CAR expression. These findings provide important insights into CAR immunobiology and highlight the potential of CRISPR/Cas9 genome editing to advance immunotherapies. Citation Format: Michel Sadelain, Justin Eyquem. Targeting CARs to the TRAC locus enhances T-cell potency [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA22.

BCL2 and miR-15/16: from gene discovery to treatment

In 1984, we investigated the t(14;18) chromosomal translocations that frequently occur in patients with follicular lymphoma. We first identified a locus on chromosome 18 involved in these translocations with the chromosome 14 containing the immunoglobulin heavy chain locus. Within this region on chromosome 18, we then discovered a gene that we called BCL2, which was activated by the translocations. Since that time, many studies determined that BCL2 is one of the most important oncogenes involved in cancer by inhibiting apoptosis. In 2002, we studied 13q deletions in chronic lymphocytic leukemia (CLL) and found that the microRNA cluster miR-15a/miR-16-1 (miR-15/16) is deleted by 13q deletions. In 2005, we discovered that miR-15/16 function as tumor suppressors by directly targeting BCL2. Thus the loss of two negative regulators of BCL2 expression results in overexpression of BCL2. Very recently, a specific BCL2 inhibitor ABT-199 (Venetoclax) was developed and approved by FDA for CLL treatment. Thus it took 32 years from fundamental discovery of a critical oncogene to the development of a drug capable to cure CLL. In this review, we discuss the discovery, functions and clinical relevance of miR-15/16 and BCL2.

PRAME Gene Copy Number Variation Is Related to Its Expression in Multiple Myeloma.

Multiple myeloma (MM) patients commonly present abnormal expression of cancer-testis antigens, which may serve as immunotherapeutic targets and prognostic factors. We previously reported that preferentially expressed antigen of melanoma (PRAME) overexpression in bone marrow mononuclear cells is related to progression in MM patients treated with non-bortezomib-containing regimens. The mechanism underlying variations in PRAME expression remains unknown. To investigate the impact of gene copy number variation (CNV) on PRAME expression, plasma cells were sorted from 50 newly diagnosed patients and 8 healthy volunteers to measure PRAME transcript levels and gene copy numbers by real-time quantitative polymerase chain reaction. A total of 14 (28.0%), 7 (14.0%), and 29 (58.0%) patients exhibited overexpression, expression within the normal range, and low expression, respectively. PRAME overexpression was significantly related to a lower 1-year progression-free survival rate compared with PRAME low expression (20.0% vs. 88.9%, p = 0.043). The mean PRAME gene copy number relative to albumin (ALB) in normal samples was ∼1.0, whereas 4.0%, 24.0%, 70.0%, and 2.0% of patients had PRAME gene relative copy numbers of approximately 0, 0.5, 1.0, and 2.0, respectively. Patients with PRAME gene deletion (relative copy number of 0 or 0.5) had significantly higher frequency of PRAME nonoverexpression and lambda light chain expression than those with no deletion (p = 0.011 and 0.003). Thus, PRAME gene CNV occurs in MM. Gene deletion may be one mechanism leading to PRAME nonoverexpression and related to immunoglobulin lambda light chain locus rearrangement. PRAME overexpression in plasma cells might be an adverse prognostic factor for progression in MM.

A genome-wide association study identifies a novel susceptibility locus for the immunogenicity of polyethylene glycol

Conjugation of polyethylene glycol (PEG) to therapeutic molecules can improve bioavailability and therapeutic efficacy. However, some healthy individuals have pre-existing anti-PEG antibodies and certain patients develop anti-PEG antibody during treatment with PEGylated medicines, suggesting that genetics might play a role in PEG immunogenicity. Here we perform genome-wide association studies for anti-PEG IgM and IgG responses in Han Chinese with 177 and 140 individuals, defined as positive for anti-PEG IgM and IgG responses, respectively, and with 492 subjects without either anti-PEG IgM or IgG as controls. We validate the association results in the replication cohort, consisting of 211 and 192 subjects with anti-PEG IgM and anti-PEG IgG, respectively, and 596 controls. We identify the immunoglobulin heavy chain (IGH) locus to be associated with anti-PEG IgM response at genome-wide significance (P = 2.23 × 10−22). Our findings may provide novel genetic markers for predicting the immunogenicity of PEG and efficacy of PEGylated therapeutics.

Conserved Vδ1 Binding Geometry in a Setting of Locus-Disparate pHLA Recognition by δ/αβ T Cell Receptors (TCRs): Insight into Recognition of HIV Peptides by TCRs.

Given the limited set of T cell receptor (TCR) V genes that are used to create TCRs that are reactive to different ligands, such as major histocompatibility complex (MHC) class I, MHC class II, and MHC-like proteins (for example, MIC molecules and CD1 molecules), the Vδ1 segment can be rearranged with Dδ-Jδ-Cδ or Jα-Cα segments to form classical γδTCRs or uncommon αβTCRs using a Vδ1 segment (δ/αβTCR). Here we have determined two complex structures of the δ/αβTCRs (S19-2 and TU55) bound to different locus-disparate MHC class I molecules with HIV peptides (HLA-A*2402-Nef138-10 and HLA-B*3501-Pol448-9). The overall binding modes resemble those of classical αβTCRs but display a strong tilt binding geometry of the Vδ1 domain toward the HLA α1 helix, due to a conserved extensive interaction between the CDR1δ loop and the N-terminal region of the α1 helix (mainly in position 62). The aromatic amino acids of the CDR1δ loop exploit different conformations ("aromatic ladder" or "aromatic hairpin") to accommodate distinct MHC helical scaffolds. This tolerance helps to explain how a particular TCR V region can similarly dock onto multiple MHC molecules and thus may potentially explain the nature of TCR cross-reactivity. In addition, the length of the CDR3δ loop could affect the extent of tilt binding of the Vδ1 domain, and adaptively, the pairing Vβ domains adjust their mass centers to generate differential MHC contacts, hence probably ensuring TCR specificity for a certain peptide-MHC class I (pMHC-I). Our data have provided further structural insights into the TCR recognition of classical pMHC-I molecules, unifying cross-reactivity and specificity.IMPORTANCE The specificity of αβ T cell recognition is determined by the CDR loops of the αβTCR, and the general mode of binding of αβTCRs to pMHC has been established over the last decade. Due to the intrinsic genomic structure of the TCR α/δ chain locus, some Vδ segments can rearrange with the Cα segment, forming a hybrid VδCαVβCβ TCR, the δ/αβTCR. However, the basis for the molecular recognition of such TCRs of their ligands is elusive. Here an αβTCR using the Vδ1 segment, S19-2, was isolated from an HIV-infected patient in an HLA-A*24:02-restricted manner. We then solved the crystal structures of the S19-2 TCR and another δ/αβTCR, TU55, bound to their respective ligands, revealing a conserved Vδ1 binding feature. Further binding kinetics analysis revealed that the S19-2 and TU55 TCRs bind pHLA very tightly and in a long-lasting manner. Our results illustrate the mode of binding of a TCR using the Vδ1 segment to its ligand, virus-derived pHLA.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/-Mb1-Cre+/- mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/- bone marrow. For Hdac3Δ/- B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/- progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells.

Abstract 499: IgH enhancers hs3b/hs4 are dispensable for c-Myc deregulation in mouse plasmacytomas with T(12;15) translocations

Abstract c-Myc deregulating t(12;15) chromosomal translocation is the hallmark cytogenetic abnormality of murine plasmacytomas (PCTs). In the great majority of PCTs, the immunoglobulin heavy chain (IgH) locus is broken between Eμ enhancer and the 3’ regulatory region (3’RR), making the latter the major candidate for orchestrating c-Myc deregulation. To elucidate the role of 3’RR in tumorigenesis, we induced PCTs in mice deficient for the major 3’RR enhancer elements, namely hs3b and hs4 (3’KO). Unexpectedly, and contrary to the previous observations made in a mouse lymphoma model, Hs3b/Hs4 3’KO mice did develop t(12;15)-positive PCTs, although with a lower incidence than wild-type controls. In heterozygous mice, there was no allelic bias in targeting IgH for the t(12;15). Molecular analysis of IgH/Myc junctions revealed dominance of Sμ region breakpoints compared to the prevalence of hits into Sγ or Sα in the controls. Analysis of c-Myc expression as well as Ig secretion in 3’KO PCT cell lines revealed no significant differences from the controls. Our current findings highlight the complexity of the 3’RR and the potential of its components to compensate for each other in the context of differentiation from B-lymphocyte to plasma cell. We next extended our observations to include a PCT model where Cre-mediated deletion can remove all four 3'IgH enhancers: hs3a, hs1,2, hs3b and hs4. In PCT cell lines where c-Myc is translocated to an IgH BAC transgene with a floxed 3’IgH enhancer region, induction of retrovirally-expressed Cre-recombinase causes gradual Myc downregulation and complete cell death within 96 hours. The later observation confirms the requirement of the full 3’RR for c-Myc deregulation by T(12;15). Citation Format: Alexander L. Kovalchuk, Herbert C. Morse. IgH enhancers hs3b/hs4 are dispensable for c-Myc deregulation in mouse plasmacytomas with T(12;15) translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 499. doi:10.1158/1538-7445.AM2017-499

Abstract 4318: Role of Bcl-2 in hematological cancer formation

Abstract B cell leukemia/lymphoma 2 (BCL-2) is a 26 kilodalton, amphipathic, anti-apoptotic protein located on the outer mitochondrial membrane. BCL-2 interacts with other anti-apoptotic and pro-apoptotic factors in order to regulate apoptosis. If a structural change occurs on the BCL-2 gene, overexpression of the BCL-2 protein can result. This causes a change in the ratio of BCL-2 to its BCL-2 Homologous 3 (BH3) ligands, preventing the release of the apoptotic signal. The primary change associated with BCL-2 overexpression in lymphomas is a t(14;18) chromosomal translocation, which causes an increase in BCL-2 transcription. The upregulation of the oncogene allows for the survival of the mutated B cells by raising the apoptotic threshold, which then dislodges regular lymphocytes in the bone marrow or lymph nodes. Lack of cell death due to overexpression of BCL-2 has been linked to hematological malignancies, specifically lymphomas and myelomas. Both B-lymphocytic cancers can result from alterations of the immunoglobulin heavy chain locus on chromosome 14. While BCL2 translocation can cause Follicular Lymphoma, BCL2 over-expression is observed in many other tumor types and likely contributes to cancer progression and drug resistance in these tumors. Treatments to inhibit BCL-2 include attempts at activation and overexpression of the pro apoptotic BH3 proteins, BCL-2 Associated X Protein (BAX) and BCL-2 Associated K protein (BAK), and creation of BH3 mimetics. Indeed, the FDA approved the BCL2 inhibitor Venetoclax (ABT-199) earlier this year for the treatment of Chronic Lymphocytic Leukemia. These treatments could help cause cancer cell death by binding to targeted anti-apoptotic proteins and causing the signal for apoptosis to be released. This could neutralize the cancerous effects of the BCL-2 oncogene upregulation. Walton High School SMART (Students Modeling A Research Topic) Team used the computer program JMOL to create a 3D printed model of BCL-2 and composed a review poster of our secondary research to investigate and demonstrate the relationship between the structure and function of this molecule. Citation Format: Abigail V. Fortier, Ally Koh, Lisa Stanovski, Alisa Kepner, Supriya Jain, Misha Ul-Islam, Ellie Walter, Lawrence Boise. Role of Bcl-2 in hematological cancer formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4318. doi:10.1158/1538-7445.AM2017-4318

Immunoglobulin M gene association with autoantibody reactivity and type 1 diabetes

Several lines of evidence show that autoimmune responses evolving in type 1 diabetes (T1D) patients include the generation of multi-reactive autoantibody (AutoAb) repertoires, but their role in T1D pathogenesis remains elusive. We tested the hypothesis that variants at the immunoglobulin heavy chain (IGH) locus are genetic determinants of AutoAbs against pancreatic antigens and contribute to T1D susceptibility. With this aim, two independent study designs were used: a case-control study and a family-based cohort comprising a total of 240 T1D patients, 172 first-degree relatives (mother and/or father), and 130 unrelated healthy controls living in Portugal. We found that three SNPs in the IGH locus show suggestive association with T1D with the highest nominal association at rs1950942 (in the IGHM-IGHJ gene region) in both the case-control study (P = 9.35E−03) and the family-based cohort (P = 3.08E−03). These SNPs were also associated with IgG AutoAbs against pancreatic antigens and with AutoAb multi-reactivity in T1D patients. Notably, we found that the SNP with the highest association with T1D susceptibility and IgG autoantibody reactivity (rs1950942) was also associated with anti-GAD IgM reactivity in T1D patients (P = 5.98E−03) and in non-affected parents (P = 4.17E−03). This finding implies that IGH association with autoreactive IgM is detectable irrespective of disease status.These results suggest that genetic variants at the IgM gene region of the IGH locus contribute to antibody autoreactivity and are associated with T1D. We propose that the control of autoantibody generation by IGH polymorphisms is a component of the complex architecture of T1D genetic susceptibility.

The pathogenesis of follicular lymphoma, beyond apoptosis resistance

Follicular lymphoma (FL) is the second most common indolent B-cell lymphoma (1). FL is characterized by a heterogeneous clinical evolution and remains incurable despite recent progress. The accepted founding event of FL pathogenesis is the acquisition of the t(14;18)(q32;q21) during primary VDJ recombination in the bone marrow (2). This chromosomal translocation juxtaposes the antiapoptotic proto-oncogene BCL2 to the immunoglobulin heavy chain locus during VDJ recombination (3). The resulting dysregulation of BCL2 expression provides a survival advantage through activation of antiapoptotic programs. The consequences of this early genetic event become manifest much later during B-cell maturation, when FL cells clonally expand and adopt malignant growth in germinal centers (GC) (2). The precise mechanisms involved in the initiation of malignant growth of apoptosisresistant t(14:18)-positive B-cells remain unclear.