Heavy Chain Isotype Research Articles

Abstract B046: Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment

Abstract The study of complex immunologic diseases and tumor microenvironment has progressed through recent developments that enable the sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from both B cell antibodies (Igs) and T-cell receptors (TCRs). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from tissue and PBMCs were used to generate immune sequencing libraries. Using a unique molecule index (UMI) to discretely barcode each mRNA molecule, PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. The B cell genes were enriched during library preparation by IGH, IGK and IGL primes, including isotype-specific primers (IgA, IgD, IgE, IgG and IgM). The T-cell genes were enriched by TCRα and TCRβ specific primers. To investigate the applicability of minimal residual disease assessment, we obtained Jurkat RNA, a homogenous population of leukemic Jurkat T-cells and spiked it into a PBMC RNA sample at varying proportions of Jurkat RNA (10%, 1%, 0.1%, 0.01% and 0.001%). The RNA mixtures were made into TCR libraries, sequenced on both Illumina MiSeq and Oxford Nanopore MinION, and analyzed to assess the TCR repertoire. Utilization of UMIs enabled absolute quantification of the B cell antibody/TCR clones and accurate ranking of their abundance. For B cell repertoire sequencing, the use of isotype-specific primers enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody analysis enabled measurement of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. For TCR repertoire sequencing, distinct and shared clonal sequences were quantitatively detected in PBMC samples. The method also accurately and sensitively detected the control TCR clone spiked into both Illumina and Oxford Nanopore libraries at levels appropriate for minimal residual disease assessment.This immune repertoire sequencing approach allows accurate clonal determination for both Igs and TCRs. This technique is applicable for a variety of applications including design of antibody chains for in vitro synthesis, investigation of T-cell infiltration of tumor microenvironments, and monitoring of minimal residual disease. Citation Format: Chen Song, Luo Sun, Pingfang Liu, Bradley Langhorst, Andrew Barry, Theodore B. Davis, Eileen T. Dimalanta. Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B046.

Understanding the Cellular Origin and Pathogenic Transcriptional Programs in Multiple Myeloma (MM) and Light-Chain Amyloidosis (AL) through the Dissection of the Normal Plasma Cell (PC) Development

Understanding the Cellular Origin and Pathogenic Transcriptional Programs in Multiple Myeloma (MM) and Light-Chain Amyloidosis (AL) through the Dissection of the Normal Plasma Cell (PC) Development

PL-012 Effect of hypoxic resistance training on the regulation of muscle mass and phenotype

Objective Hypoxia is a state of lowered oxygen tension in tissue that can be created by environmental or pathological conditions. Whatever the origin of hypoxia, different tissues will adapt acutely and/or chronically to deal with this reduction in oxygen availability. Hypoxia has recently emerged as a particularly efficient stimulus to stimulate muscle cell proliferation and accretion of muscle mass and hypoxic resistance training has become popular amongst athletes as it is thought to favor muscle accretion. However, the molecular mechanisms are largely unknown. 
 Methods To determine those molecular mechanisms, 19 volunteers participated to 12 sessions of resistance training spread over 4 weeks whether in normoxia (n=9) or in hypoxia (n=10, FiO213.5% corresponding to 3500m altitude). Each session consisted in 6 sets of 10 repetitions of a one-leg extension exercise at 80% of one repetition maximum (1-RM). Blood and muscle samples in each leg were taken before and after the 4-week training period. Fiber types were determined by immunohistochemistry based on myosin heavy chain isotypes. Blood saturation (SpO2, pulsoximetry) and tissue saturation index (TSI, near-infrared spectroscopy) were monitored during the exercise sessions. 
 Results Muscle thickness determined by ultrasound was increased by 7% in normoxia only (p=0.04). The 1-RM was increased in both groups but the increase was higher in hypoxia (+34%) than in normoxia (+24%) (p=0.02). In average, SpO2stayed around 98-99% in normoxia and around 93-94% in hypoxia during each set of contractions. No difference in TSI between normoxia and hypoxia was measured, which averaged 60% before starting muscle contractions and 40% during muscle contractions. A trend towards a shift in fiber type from type I to type IIa was observed in normoxia (p<0.09) but not in hypoxia. Fiber area was not modified by any condition. 
 Conclusions In summary, 4 weeks of hypoxic resistance training induced a larger increase in 1-RM compared to normoxic resistance training, independently of muscle hypertrophy or any change in fiber type. Further investigation should determine whether metabolic or molecular changes may explain this potentiation of maximal muscle force by hypoxia.

Abstract 5761: Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions

Abstract Several agonistic antibodies against human CD40 are currently being explored as immune therapy for cancer patients. Previously, anti-CD40 antibody therapy showed some clinical efficacy, but significant toxicity at the efficacious dose limited further development. To develop an anti-CD40 agonistic antibody with an improved clinical therapeutic index, we focused on modifications to the heavy chain isotype that would impact interactions with specific Fc receptors. In mouse models, anti-CD40 murine IgG1 demonstrated stronger immune stimulatory activities than murine IgG2a. The CD40 murine IgG2a antibody induced acute cytokine release within hours of dosing and led to mortality weeks later. To generate human antibodies with an Fc structure that functions similarly to murine IgG1, human IgG1 Fc mutations with reduced binding on human CD16, enhanced binding on CD32A/B, and maintained binding on CD64 were identified through a proprietary mammalian cell surface display technology. A panel of anti-human CD40 antibodies carrying different Fc regions were created. In a CD40 transfected HEK293 NFκB reporter cell assay, where agonistic activity was not related to Fcγ receptor interactions, anti-CD40 human IgG2 demonstrated stronger agonistic activity than human IgG1 antibodies with either wild type or mutated Fc. This result confirmed the reported role of human IgG2 hinge in impacting the agonistic activity of anti-CD40. In assays using primary B cells or monocyte-derived dendritic cells, which constitutively express Fcγ receptors, a subset of anti-CD40 human IgG1 antibodies showed stronger potency than the corresponding human IgG2, due to the better Fcγ receptor crosslinking for IgG1 antibodies. Swapping the human IgG2 hinge into IgG1 Fc did not further enhance the agonistic activity of anti-CD40 antibodies in the primary cell assays, suggesting that the Fc-crosslinking plays a dominant role in mediating the agonistic activity of these anti-CD40 antibodies in biological systems with cells expressing Fcγ receptor. Engineering the proprietary human IgG1 Fc mutations to these anti-CD40 antibodies further enhanced agonistic activity compared to wild type IgG1 in primary cell assays. In addition to the potent agonistic activity, anti-CD40 with the IgG1 Fc mutations demonstrated lower ADCC and reduced cytokine production driven by Fc receptor activation in a co-culture system. Finally, an Fc modified anti-CD40 agonistic antibody demonstrated an acceptable safety profile in cynomolgous monkey toxicity studies. These data suggest anti-CD40 with the proprietary IgG1 Fc mutations represents a newer generation of CD40 agonists for oncology immune therapy. Citation Format: Shiming Ye, Diane Cohen, Donghee Choi, Siu sze Tan, Joanna Xiong, Yoshiko Akamatsu, Debra Chao, Diane Hollenbaugh, Fiona Harding. Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5761.

Abstract 4675: B-cell and T-cell repertoire sequencing enables somatic hypermutation and minimal residual disease assessment

Abstract The study of complex immunological diseases has progressed through recent developments that enable the sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from both B-cell antibodies and T-cell Receptors (TCR). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from PBMCs were used to generate immune sequencing libraries in duplicate from a single patient. Using a unique barcoding scheme specifically designed to discretely barcode each mRNA molecule with a unique molecule index (UMI), PCR copies of each mRNA fragment can be collapsed into a single consensus sequence, improving accuracy through the resolution of PCR bias and sequencing errors. Utilization of the UMIs enabled absolute quantification of input RNA molecules and accurate ranking of the antibody/TCR clone abundance. This approach was also applied to the analysis of tumor samples for abundance of expanded clones through clustering based on V gene, J gene and CDR3 similarity and ranking by mRNA abundance. The use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody sequences generated using this method were aligned to germline genes from reference databases, enabling quantitation of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. Additionally, this approach was capable of detecting minimal residual diseases by sensitive quantification of TCR rearrangement. This novel method allows for exhaustive somatic mutation profiling across complete V, D and J segments and full isotype information analysis. The information obtained enables the possibility for synthesis and expression of complete antibody chains and T-cell receptor chains for downstream immunological assays. Citation Format: Chen Song, Pingfang Liu, Andrew Barry, Eileen T. Dimalanta, Fiona J. Stewart, Salvatore Russello, Theodore B. Davis. B-cell and T-cell repertoire sequencing enables somatic hypermutation and minimal residual disease assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4675.

Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood

Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n=19) and peripheral blood (n=215) from healthy donors aged 0 to 90years by using flow cytometry and nephelometry, respectively. IgH-switched MBCs expressing IgG1, IgG2, IgG3, IgA1, and IgA2 were already detected in cord blood and newborns at very low counts, whereas CD27+IgM++IgD+ MBCs only became detectable at 1 to 5months and remained stable until 2 to 4years, and IgD MBCs peaked at 2 to 4years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG1, IgG3, and IgA1) peaked later during childhood (2-4years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG2, IgG4, and IgA2) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11months for IgM, IgG1, IgG2, IgG3, IgA1, and IgA2; until 2 to 4years for IgD; and until 5 to 9years for IgG4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG4), maximum plasma levels were reached after PC and MBC counts peaked. PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of IgH isotype switch recombination through life.

The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding

Current therapeutic antibodies such as Trastuzumab, are typically of the blood circulatory IgG1 class (Cκ/ CHγ1). Due to the binding to Her2 also present on normal cell surfaces, side effects such as cardiac failure can sometimes be associated with such targeted therapy. Using antibody isotype swapping, it may be possible to reduce systemic circulation through increased tissue localization, thereby minimising unwanted side effects. However, the effects of such modifications have yet to be fully characterized, particularly with regards to their biophysical properties in antigen binding. To do this, we produced all light and heavy chain human isotypes/subtypes recombinant versions of Trastuzumab and Pertuzumab, and studied them with respect to recombinant production and Her2 binding. Our findings show that while the light chain constant region changes have no major effects on production or Her2 binding, some heavy chain isotypes, in particularly, IgM and IgD isotypes, can modulate antigen binding. This study thus provides the groundwork for such isotype modifications to be performed in the future to yield therapeutics of higher efficacy and efficiency.

Isotype Determination of Rodent-Derived Monoclonal Antibodies Using Sandwich ELISA

For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody.

IgH isotype-specific B cell receptor expression influences B cell fate

Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgμ > mIgγ1 > mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells-which essentially do not exist under normal conditions-to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

IgH isotype-specific B cell receptor dosage regulates B cell fate

Abstract Immunoglobulin heavy chain (IgH) isotypes (e.g. IgM, IgG and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCR) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is unclear. We examined IgH isotype-specific BCR function by engineering polyclonal Ighγ1/γ1 and Ighɛ/ɛ mice, which initially produce IgG1 or IgE from their respective native genomic configurations. Transcripts producing IgM, IgG1, and IgE are produced in an alternative splice form bias hierarchy, where mIgμ>mIgγ1>mIgɛ—influencing respective BCR densities and fitness for populating the periphery—a pattern replicated in activated and memory B cells from wild-type mice. Restrained B cell development from Ighγ1/γ1 and Ighɛ/ɛ mice was proportional to sIg/mIg ratios, and was rescued by enforced expression of the respective mIgs. In addition, enhanced BCR signaling by way of PTEN deletion in IgE B cells generated functional IgE memory responses from IgE memory B cells themselves, which are normally absent under normal conditions, indicating that weak signal strength normally restrains IgE responses. Thus, IgH isotype-specific mIg dosage regulates B cell fate by influencing quantitative baseline BCR signal strength.

Three IgH isotypes, IgM, IgA and IgY are expressed in Gentoo penguin and zebra finch.

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

A transcriptional serenAID: the role of noncoding RNAs in class switch recombination.

During an immune response, activated B cells may undergo class switch recombination (CSR), a molecular rearrangement that allows B cells to switch from expressing IgM and IgD to a secondary antibody heavy chain isotype such as IgG, IgA or IgE. Secondary antibody isotypes provide the adaptive immune system with distinct effector functions to optimally combat various pathogens. CSR occurs between repetitive DNA elements within the immunoglobulin heavy chain (Igh) locus, termed switch (S) regions and requires the DNA-modifying enzyme activation-induced cytidine deaminase (AID). AID-mediated DNA deamination within S regions initiates the formation of DNA double-strand breaks, which serve as biochemical beacons for downstream DNA repair pathways that coordinate the ligation of DNA breaks. Myriad factors contribute to optimal AID targeting; however, many of these factors also localize to genomic regions outside of the Igh locus. Thus, a current challenge is to explain the specific targeting of AID to the Igh locus. Recent studies have implicated noncoding RNAs in CSR, suggesting a provocative mechanism that incorporates Igh-specific factors to enable precise AID targeting. Here, we chronologically recount the rich history of noncoding RNAs functioning in CSR to provide a comprehensive context for recent and future discoveries. We present a model for the RNA-guided targeting of AID that attempts to integrate historical and recent findings, and highlight potential caveats. Lastly, we discuss testable hypotheses ripe for current experimentation, and explore promising ideas for future investigations.

Myasthenia gravis: a pathologic approach

Our immune system protects us from all kinds of pathogens our body faces every day. However, the immune system can also initiate an immune response against our own proteins. This is called autoimmunity. Myasthenia gravis (MG) is a rare autoimmune disease in which patients show severe muscle weakness, due to antibody production against proteins of the neuromuscular junction, which is the connection between nerve and muscle. At this moment, three different forms of myasthenia gravis are identified. Muscle-specific tyrosine kinase (MuSK)-MG is a form of MG in which patients produce antibodies, predominantly immunoglobulin (Ig)G4, against MuSK. This paper is a summary of a B cell immortalization project, in which antibodies produced by MuSK-MG patients are investigated. The aim of this study was to sequence and generate antibodies produced by these patients in vitro to learn more about the pathological mechanism of MuSK-MG. Blood-derived B cells of MuSK-MG patients were immortalized by the Epstein Bar Virus and stimulated to grow and produce antibodies in culture. Genes of these antibodies were amplified and analyzed. These amplification products were cloned in vectors and will be transfected in a human embryonic kidney (HEK)-cell line. This study showed that the B cell immortalization method worked and that MuSK-MG patients predominantly produce IgM antibodies against MuSK. This is very striking as literature states that these patients predominantly produce IgG4 against MuSK. It is suggested that this high production of IgM is due to the absence of the heavy chain isotype switch from IgM to IgG. Moreover, as the pathologic mechanism of MuSK-MG is still unknown, IgM may play an important pathological role.

Distinct Immunogenetic Signatures in IgA Versus IgG Multiple Myeloma

Immunogenetic analysis of MM has proven instrumental in elucidating disease ontogeny e.g. by revealing the clonal relationship between switch variants expressed by the bone marrow plasma cells and myeloma progenitors in the marrow and blood; demonstrating the marked under-representation of the inherently autoreactive IGHV4-34 gene; and, identifying patterns of somatic hypermutation (SHM) indicative of post-germinal center derivation. Yet, limited information exists about the composition of the immunoglobulin (IG) gene repertoire in MM cases expressing different heavy chain isotype, in particular A versus G. This is relevant in light of studies showing an overall higher SHM impact in CD27+IgA+ compared to CD27+IgG+ normal memory B cells, perhaps reflecting a distinct location of the immune response, especially considering that IgA class switching mostly occurs in mucosa-associated lymphoid tissues. From a clinical perspective, it is also relevant to note that IgA patients exhibit a higher incidence of the t(4;14) translocation, shorter progression-free survival and worse median overall survival compared to IgG patients. Here, we explored potential differences in the immunoprofiles of IgA versus IgG MM focusing on IG gene repertoire and SHM characteristics. In total, 428 patients with a diagnosis of MM following the IMWG criteria from collaborating institutions in Greece, Italy and Spain (n=355) or retrieved from the LIGM-DB (n=73) were included in the study. Of these, 135 and 293 belonged to IgA and IgG MM groups, respectively. Amongst the evaluated productive IG rearrangements, IGHV3 subgroup genes predominated in both groups (IgA: 58.5%; IgG: 52.2%). However, at the individual gene level, major asymmetries were noted, since only 7 IGHV genes accounted for 41.6% of the IgA and 46.7% of the IgG cases, respectively. Of these, 3 genes were shared between IgA and IgG MM cases: IGHV3-30 (IgA: 11.9% - IgG: 13.3%), IGHV3-23 (IgA: 5.2% - IgG: 6.8%) and IGHV3-9 (IgA: 6.7% - IgG: 4.4%), whereas the remaining 4 of the 7 most frequent genes were specific for each group with significant (p<0.05) differences regarding the IGHV3-7 (5.2% in IgA versus 1.7% in IgG) and IGHV3-21 gene (0.7% in IgA 4.1% in IgG). IGHD3 predominated in both groups (IgA: 37% - IgG: 39.6%) followed by IGHD2 in IgG MM (18.4%) and IGHD6 in IgA MM (20.7%). IGHJ4 and IGHJ6 were the most frequent IGHJ genes with no significant differences in relative frequency. Searching for restricted IGHV-IGHJ combinations, we noted that the IGHV3-9 gene preferentially paired with the IGHJ6 gene in IgA MM versus the IGHJ4 gene in IgG MM (66.7% and 46.2% of all IGHV3-9 rearrangements, respectively). The median complementarity-determining region 3 (CDR3) length was identical in both IgA and IgG MM (15 amino acids, aa), yet differences were identified for specific CDR3 lengths as in the case of 19 aa, concerning 10.4% of all IgA versus 4.8% of all IgG rearrangements (p<0.05). Turning to SHM, the vast majority of rearrangements (IgA: 90.4%, IgG: 85%) were heavily mutated (IGHV germline identity (GI) <95%) with median GI of 91.8% for IgA and 92.2% for IgG. To study the topology of SHM, we compared the ratios of replacement (R) to silent (S) mutations in the framework (FR) and complementarity determining regions (CDRs) in cases expressing common frequent IGHV genes, namely IGHV3-23, IGHV3-30 and IGHV3-9 and identified distinct SHM patterns in all 3 instances: (i) IGHV3-23: the highest R/S ratios in IgA versus IgG MM were observed in FR2 (3.88) and CDR1 (3.9), respectively; (ii) IGHV3-30: overall "normal" SHM topology with higher R/S in CDRs rather than FRs, however, compared to IgG, IgA cases also showed a very high R/S in FR2 (5.3 versus 1.4); and, (iii) IGHV3-9: significantly (p<0.05) higher R/S ratios in CDR1 and CDR2 in IgG versus IgA cases (10.2 and 8.3 versus 1 and 2.9, respectively). Overall, in-depth immunogenetic analysis in the largest to-date series of IgA MM and IgG MM patients reveals differences regarding IGH gene repertoires, CDR3 characteristics and the topology of SHM. These findings suggest distinct antigen exposure histories and/or affinity maturation processes for IgA versus IgG MM, further highlighting the importance of microenvironmental stimuli in disease pathogenesis. DisclosuresTerpos:Celgene: Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Bone lesions in Chinese POEMS syndrome patients: imaging characteristics and clinical implications.

Objective. Bone lesion is crucial for diagnosing and management of polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin change (POEMS) syndrome, a rare plasma cell disorder. This study is to compare the effectiveness of X-ray skeletal survey (SS) and computed tomography (CT) for detecting bone lesions in Chinese POEMS syndrome patients, and to investigate the relationship between bone lesion features and serum markers.Methods. SS and chest/abdomen/pelvic CT images of 38 Chinese patients (26 males, 12 females, aged 21–70 years) with POEMS syndrome recruited at our medical center between January 2013 and January 2015 were retrospectively analyzed. Bone lesions identified by CT were further categorized according to the size (<5 mm, 5–10 mm, >10 mm) and appearance (osteosclerotic, lytic, mixed). The percentage of plasma cells in bone marrow smears, type of immunoglobulin, platelet (Plt), and levels of serum bone metabolic markers and inflammatory factors including alkaline phosphatase (ALP), calcium, phosphate, parathyroid hormone (PTH), beta-isomerized C-telopeptide (β-CTx), vascular endothelial growth factor (VEGF), and interleukin (IL)-6 levels were also recorded.Results. Of the 38 POEMS syndrome patients, the immunoglobulin heavy chain isotypes were IgA in 25 patients (65.8%; 25/38) and IgG in 13 patients (34.2%; 13/38), and the light chain isotypes were λ in 35 patients (92.1%; 35/38) and κ in 3 patients (7.9%; 3/38). There were 23 patients with thrombocytosis. More patients with bone lesions were detected by CT than by SS (97.4% vs. 86.8%). The most commonly affected location was the pelvis (89.5%), followed by the spine, clavicle/scapula/sternum/ribs, skull, and long bones. Of the 38 POEMS syndrome patients, 35 (94.6%) had osteosclerotic and 32 (86.5%) had mixed lesions. Osteosclerotic lesions were typically scattered, variable in size, and plaque-like, whereas mixed lesions were pouch-shaped or soup bubble-like with a clear sclerotic margin and were generally larger. Although the majority of bone lesions were small in size, 23 (62.2%) had at least one lesion >10 mm. There was no correlation between serum marker levels and bone lesion patterns after Bonferroni correction (all P > 0.001).Conclusions. CT is more sensitive and accurate than SS in detecting bone lesions in POEMS syndrome.

Molecular cloning of IgZ heavy chain isotype in Catla catla and comparative expression profile of IgZ and IgM following pathogenic infection.

Immunoglobulins serve as a crucial arm of the adaptive immune system against detrimental pathogenic threats in teleosts. However, whether the novel Ig isotype IgZ is present in the Indian major carp, Catla catla, has not yet been elucidated. The present study reports the presence of IgZ ortholog in C. catla (CcIgZ) and further demonstrates its comparative tissue specific expression with IgM (CcIgM) in response to bacterial and parasitic stimulation. The putative 139 amino acid sequence of IgZ heavy chain cDNA of C. catla showed homology with IgZ constant domains of other teleosts. Phylogenetic analysis of the predicted IgZ transcript sequence clustered with previously identified IgZ heavy chain sequences of Cyprinidae family members. The inductive expression profiles of IgZ and IgM genes were evaluated in immunologically relevant tissues at 24, 48 and 72 hr post infection with Aeromonas hydrophila, Streptococcus uberis and Argulus sp. Both CcIgZ and CcIgM were expressed most strongly in the kidneys of healthy fish. Basal expression of CcIgM transcript was higher than that of CcIgZ in all the examined tissues. Stimulation with bacteria triggered significant increase of IgZ in the intestine (P < 0.001) and spleen (P < 0.01), whereas IgM was relatively up-regulated in blood (P < 0.001) after stimulation with each of the three pathogens assessed. The study is the first to report identification of IgZ in C. catla. Further, it provides insights into the differential expression profiles of IgZ and IgM isotypes against various pathogenic infection in C. catla, which may facilitate better prophylaxis again such infections.

High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.

The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season. Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA. Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73- CD25+ CD71+ BR 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected. This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses.

Abstract 3998: Sequencing the B-cell and T-cell repertoire

Abstract Immune sequencing allows for the study of complex immunological diseases by sequencing millions of V(D)J combinations from B-cell antibody and T-cell receptors. The popularity of this technique has increased due to recent throughput and read length improvements in next-generation sequencing technologies. However, structural and sequence complexities of antibody genes have made reliable targeting approaches challenging. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. The method uses a unique barcoding scheme specifically designed to tag every mRNA molecule with a unique identifier (UID) so that all PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. This makes the assay extremely accurate, by resolving PCR bias and sequencing errors as well as allowing quantitative digital molecule counting. Immune sequencing libraries were generated from total RNA extracted from Peripheral Blood Mononuclear Cells in duplicate from a single patient. The use of UIDs enabled absolute quantification of starting RNA molecules present in the original sample and therefore accurate ranking of the antibody clone abundance, by avoiding the bias incorporated by PCR or sequencing when total reads only were measured. Using the same sequencing method, tumor samples were analyzed for abundance of expanded clones via grouping clones by V gene, J gene and CDR3 similarity and ranking by mRNA abundance. Additionally, the use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Further, alignment of full-length heavy chain antibody sequences generated using this method to germline genes from reference databases enabled quantitation of the mutation level of each antibody sequence, thereby providing information on the overall maturity and mutational profile of the sample repertoire. Citation Format: Fiona J. Stewart, Mehmet Karaca, Kris Adams, Chris Clouser, Bonny Patel, Sonia Timberlake, William Donahue, Lynne Apone, Salvatore Russello, Eileen T. Dimalanta, Theodore B. Davis, Francois Vigneault. Sequencing the B-cell and T-cell repertoire. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3998.

HLA Class I Components Increase Pancreatic Cancer Proliferation and Migration

Abstract Recent studies in transplantation models have implicated HLA class I molecules as important factors in activating downstream pathways that lead to cell proliferation and migration. In a few cancers, β2m alone has been proposed to be high in expression and to increase proliferation and migration. No research to date has reported if β2m and/or the HLA class I heavy chain promote the migration and proliferation of pancreatic cancer cells and, if so, whether β2m and the HLA class I heavy chain work together or as separate entities within the signaling pathways to promote these processes. Using pancreatic cancer cells, we demonstrate that siRNA knockdown of β2m causes a decrease in cell migration and proliferation without altering viability. This decrease of β2m also leads to a reduction in the expression of the HLA class I heavy chain as well as downstream signaling molecules, pCREB and pFAK. Downregulation of HLA-A leads to decreased pCREB while HLA-B knockdown does not and neither heavy chain isotype has an effect on pFAK expression. Overall, this study demonstrates the importance of β2m in cancer cell migration and proliferation through signaling pathways involving pCREB and pFAK. We show that HLA class I heavy chains are expressed by pancreatic cancer cells and that HLA-A heavy chains may be more influential in cell migration and proliferation than HLA-B heavy chains. Loss of β2m expression causes a decrease in HLA class I heavy chains, suggesting that β2m may exert some of its effects on growth and migration via the heavy chain. However, the fact that some downstream molecules are altered in cells with β2m reduction, yet no change is observed with the downregulation of either HLA heavy chain isoform, indicates that each may also work in divergent pathways.

Preferential combination between the light and heavy chain isotypes of fish immunoglobulins

Immunoglobulin light chain (IgL) is necessary for the assembly of an Ig molecule, which plays important roles in the immune response. IgL genes were identified in various teleost species, but the basic functions of different IgL isotypes and the preferential combination between IgL and IgH (Ig heavy chain) isotypes remain unclear. In the current study, by EST database searching and cDNA cloning in rainbow trout, 8 IgL sequences were obtained, which could be classified into the IgLκF, IgLκG, IgLσ and IgLλ isotypes, respectively. Trout IgL isotypes were highly expressed in the immune-related tissues, and participated in the immune responses in spleen and gut by stimulation with LPS and poly (I:C). The results of FACS and LC-MS/MS indicated that the IgLκG and IgLσ isotypes preferentially bonded with the heavy chains of IgM and IgT, respectively, in trout B cells and serum. In addition, the genomic organization of trout IgL isotypes and the utilization of recombination signal sequences were studied.