Immunoglobulin Heavy Chain Isotype Research Articles

Host immune signatures as predictors of response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC)

Abstract Background Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. However, clinical responses to systemic therapies in mRCC remain largely unpredictable and robust biomarkers are still lacking. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with ICI-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. T-cell receptor sequencing (TCR-seq) was performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-sequencing (RNA-seq) was performed on available pre-treatment PBMCs and primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts (186 VEGF-TT, 126 ICI and 74 ICI+VEGF-TT). Following quality-control, TCR-seq data were available for 367 patients (228 pre-treatment and 139 on-treatment), while RNA-seq data were available for 105 PBMC and 17 tumor-derived pre-treatment samples. In the TCR-seq analysis, responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06) (Fig. 1A), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). Therefore, responders to ICI-based regimens seem to have a more oligoclonal TCR repertoire (increased clonality) at baseline with no treatment-induced changes, whereas non-responders to ICI-based regimens seem to evolve from a more polyclonal to a more oligoclonal TCR repertoire in response to treatment. The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01) (Fig. 1B). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Furthermore, while shared clonotypes were detected between blood and tumor samples, there were no differences in the Jaccard similarity index between responders and non-responders to ICI-based or VEGF-TT regimens. Figure 1: (A) Evaluation of pre-treatment and post-treatment TCR clonality in responders vs. non-responders across treatment cohorts. (B) Differences in the fraction of IGH isotypes between responders and non-responders across treatment cohorts. Conclusions We were successfully able to characterize T-cell receptor and B-cell IgH repertoires in a large cohort of patients with mRCC, and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development. CDMRP DOD Funding: yes

Association of host immune signatures with response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC).

4550 Background: Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with immunotherapy-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods: We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. Bulk RNA-sequencing (RNA-seq) and T-cell receptor sequencing (TCR-seq) were performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-seq was additionally performed on available pre-treatment primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results: In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts. Following quality-control, TCR-seq and RNA-seq data were available for 367 and 134 patient samples, respectively. Responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Conclusions: We were successfully able to characterize T-cell receptor and IgH repertoires in a large cohort of patients with mRCC and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development.

Evolution of the Structure and Organization of the Mammalian Immunoglobulin Loci

Abstract There are three extant lineages of mammals: placentals (e.g. humans and mice), marsupials (e.g. kangaroos and opossums), and monotremes (e.g. platypus). Of the three lineages, the marsupials are unusually and uniformly limited in their immunoglobulin heavy chain (IgH) isotype content. All marsupials studied have only one IgM, IgG, IgE, and IgA and lack IgD. Comparatively, there is a greater complexity of IgH isotypes and subclasses in both placentals and monotremes. Marsupials also have a greater complexity in their light (L) chain V genes than IgH chains. Transposable elements (TEs), like retroelements, have been hypothesized to contribute to gene copy variation and genome evolution. Recently, these TEs have been found in the IgH loci. Here we investigate the impact these TEs have on the evolution of marsupial IgH and IgL loci. We have found that some of the TEs in the opossum IgH locus are short and non-functional, potentially contributing to the restriction seen in the locus. Conversely, the TEs in the L chain loci are longer and may explain the differences in complexity in both the V and C regions between the H and L chain loci. Supported by National Science Foundation Award IOS-2103367.

Association between Clinical Presentation of Multiple Myeloma, Immunoglobulin Isotypes, and Cytogenetic Risk Groups

Introduction: In multiple myeloma (MM), chromosomal abnormalities (CAs) are valuable in risk stratifying patients and predicting disease free survival. While immunoglobulin isotypes have historically contributed to MM staging, more recently established classifications of CAs have allowed for a revised staging system that better predicts disease behavior. In this retrospective study, we hypothesized that CAs correlate with disease characteristics including immunoglobulin heavy chain isotype, degree of bone marrow infiltration (PC%), and end-organ damage. Methods: MM patients diagnosed between 2013 to 2019 were included in this retrospective chart review using electronic records from two distinct sources: (1) 442 patients from an independent pathology database and (2) a validation cohort composed of 110 patients from our institution. CAs were stratified by Mayo mSMART 2.0 criteria into standard, intermediate, and high-risk groups (Mikhael et al. Mayo Clin Proc 2013). End-organ damage was defined as the presence of lytic bone lesions, anemia, hypercalcemia, or renal failure on clinical presentation. Within each cohort, associations between categorical variables were made using the chi-squared test or Fisher's exact test, as deemed appropriate. The Mann-Whitney test was used to compare between groups for continuous variables. A result was considered statistically significant at the p<0.05 level of significance. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC). Results: 552 MM patients were included in the study. Multi-variate analysis revealed that del(13q14), dup(1q21), t(4;14), trisomy (11q13), del(16q23), and hypodiploidy were associated with a significantly higher PC%, whereas kappa light chain only (LCO) disease was associated with a lower PC%. Higher median PC% was found when comparing the intermediate to standard CA risk group. IgA isotype was associated with intermediate risk CAs including del(13q) and standard risk CAs including t(11;14), while IgG isotype was associated with dup(1q21), and kappa LCO disease correlated with a higher rate of higher risk CAs including deletion of p53 at 17p13 and dup(1q21). With regard to clinical presentation, lytic lesions were more frequent in patients with normal cytogenetics and trisomy 11 and less frequent in IgA isotype, whereas the presence of anemia on presentation correlated with IgA isotype. Renal failure was associated with MAF translocations including t(14;16), a high-risk CA. Conclusions: We demonstrate that a relationship exists between specific CAs, immunoglobulin isotypes, and clinical presentations in MM. Our data indicate that IgA isotype is significantly associated with intermediate-risk cytogenetics including del(13q) and anemia on presentation, and that light chain disease and renal failure correlate with high risk CAs including del(17p13). These associations between biological and clinical features further support the concept of divergent cytogenetic evolution in MM as being an underlying factor leading to distinctive disease presentations. Disclosures Braunstein: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System.

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naïve pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed ≥8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

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.

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.

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.

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.

Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes

Teleosts possess three immunoglobulin (Ig) heavy chain isotypes viz., IgM, IgT and IgD and all three isotypes are reported in rainbow trout. The expression of these Ig isotypes in response to different immunization routes was investigated and results provide a better understanding of the role these Igs in different tissues. Rainbow trout (Oncorhynchus mykiss) were immunized with an attenuated Flavobacterium psychrophilum strain, 259-93-B.17 grown under iron limiting conditions, by intraperitoneal, anal intubation and immersion routes. Serum, gill mucus, skin mucus and intestinal mucus samples were collected at 0, 3, 7, 14, 28, 42 and 56 days post immunization by sacrificing four fish from each treatment group and the unimmunized control group, and the IgM levels were estimated by an enzyme linked immunosorbent assay (ELISA). In addition, blood, gill, skin and intestinal tissue samples were collected for Ig gene expression studies. The secretory IgM, IgD and IgT gene expression levels in these tissues were estimated by reverse transcription quantitative real time PCR (RT-qPCR). Levels of IgM in serum, gill and skin mucus increased significantly by 28 days after immunization in the intraperitoneally immunized group, while no significant increase in IgM level was observed in fish groups immunized by other routes. Secretory IgD and IgT expression levels were significantly upregulated in gills of fish immunized by the immersion route. Similarly, secretory IgT and IgD were upregulated in intestines of fish immunized by anal intubation route. The results confirm mucosal association of IgT and suggest that IgD may also be specialized in mucosal immunity and contribute to immediate protection to the fish at mucosal surfaces.

Chromatin Interactions in the Control of Immunoglobulin Heavy Chain Gene Assembly.

Expression of antibody heavy chain occurs via precisely timed developmental activation of the immunoglobulin heavy chain (IgH) gene locus during B cell development. IgH locus activation permits coordinated gene rearrangements that assemble variable (VH), diversity (DH), and joining (JH) gene segments into functional genes. Chromosomal conformation changes and epigenetic mechanisms play critical roles in ensuring rearrangement fidelity while minimizing hazardous consequences of broken DNA ends generated during recombination. In this review, we summarize the current status of regulatory mechanisms that underpin effective IgH gene assembly. For this, the germline locus is divided into two parts: a 2.4Mb 5' part that contains all VH gene segments and a 300kb 3' domain that contains DH and JH gene segments, as well as exons that encode IgH isotypes. Structural features of each part are discussed individually, followed by consideration of how the two parts come together to complete IgH recombination. Throughout we emphasize current insights, propose plausible mechanisms, and highlight key questions for future studies.

Detection of true IgE-expressing mouse B lineage cells.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

B Cell Receptor with Variant Surface Isotypes Transduce Functional Signals by Elevating Phospho-ERK1/2 Levels in Hairy Cell Leukemia with Mutant BRAF

A functional BCR is essential for survival of normal B-cells, in response to cognate antigen or tonic, antigen-independent stimuli. Cross-linking of BCR by antigen triggers key phosphorylation events in the early signalosome. Mammalian wild type BRAF, a member of the RAF cytosolic kinase family (A-RAF, B-RAF, C-RAF) is critical to BCR function, mediating RAS-RAF-MEK-ERK or mitogen-activated protein kinase (MAPK) pathway signal transduction, via phosphorylation of ERK1/2. The MAPK pathway is a central conduit for survival and proliferation. Importantly, use of inducible deletion models have shown that wild type BRAF is the predominant kinase that transduces BCR signals to activate ERK1/2, with C-RAF playing an accessory role and A-RAF displaying a x20-30 fold lower basal activity in ERK1/2 stimulation. Tonic BCR survival signals in normal B-cells are also transduced via phosphorylation of ERK1/2, but do not require antigen stimuli. Whether the BCR plays a comparable role in sustaining survival in malignant B-cells is less well characterized and remains a central question in understanding the origins and progression of mature B-cell neoplasms.Hairy cell leukaemia (HCL) is a rare B cell leukaemia with characteristic hair-like cytoplasmic projections on tumor cells, displaying distinctive activation markers. A striking feature of monoclonal HCL tumors is expression of multiple variant immunoglobulin heavy chain (sIgH) isotypes as components of the BCR on individual tumor cells, defining the major subset of disease (mult-HCL). Most mult-HCL tumors exhibit IGV somatic hypermutation with a low level of on-going mutations and AID expressed, implicating initial contact with antigen via the BCR. The functional relevance of the BCR to transformation and survival however has as yet not been mapped in HCL. Seminal exome sequencing data in typical HCL identified mutant BRAF V(600)E as almost universal in this tumor. Consistent with mutant BRAFV(600)E, levels of phosphorylated ERK (p-ERK) are raised in hairy cells. As there is an essential requirement for wild type BRAF in transducing BCR signals, the question arises how mutant BRAF may affect BCR function in HCL, given requirements for dimerization of wild type BRAF for ERK1/2 activation, and whether ERK1/2 activation can be enhanced by functional BCR signals for downstream effector signals in HCL.To examine this, we evaluated BCR signalling in mult-HCL (n=10), in cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ mult-HCL, IgD mediated persistent Ca2+ flux, which was also evident via >1 sIgH isotype, linked to increased ERK1/2 activation and BCR endocytosis (4/4 cases). In sIgD-vemult-HCL however (6/6 cases), BCR-mediated signals activating ERK1/2 for downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal a clear discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing mult-HCL (3/3 cases), only a single sIgL was fully functional.We next examined effects of anti-BCR stimuli on mult-HCL tumor cell survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both sets (4/4 cases) to establish a consistent outcome in these replicate cases. IgD stimuli, in marked contrast retained tumor viability with no evidence for pre-apoptotic effects (2 cases; 1 mult-HCL and 1 sIgD only-HCL).Despite mutant BRAF, BCR signals amplify ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL lacking IgD, a role for antigen in stimulating the BCR for tumor persistence appears unlikely. It remains feasible that mutant BRAF in these cases retains tonic signals through activated ERK1/2 for survival, but this remains speculative at present. Surface IgD emerges with potential to transduce BCR signals for tumor survival and persistence in-vivo, but given its enigmatic role even in normal B-cells, its relevance to HCL progression is unclear.These observations raise the possibility that mutant BRAF may be a mechanism to bypass any antigen-dependent BCR signalling constraints in mult-HCL. Disclosures:No relevant conflicts of interest to declare.

Serum immunoglobulin free light chain measurements and heavy chain isotype usage provide insight into disease biology in patients with POEMS syndrome

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome is a rare paraneoplastic syndrome in which nearly all patients have a monoclonal lambda restricted plasma cell disorder. We investigated whether patients with POEMS have abnormal serum immunoglobulin free light chain (FLC) ratios. Fifty patients with newly diagnosed POEMS syndrome were assessed. Cystatin C levels were measured to discern whether subclinical renal insufficiency could account for FLC elevations in the presence of a normal FLC ratio. Forty-five patients (90%) had elevated lambda FLC; however, only nine (18%) had abnormal FLC ratios. The rise in serum FLC of POEMS patients appeared to be multifactorial-both a function of subclinical renal insufficiency and polyclonal activation of medullary and extramedullary plasma cells. Those patients expressing a clonal IgA were more likely to have clonal plasmacytosis observed on iliac crest biopsy than those with IgG. In summary, serum immunoglobulin profiles are unique in POEMS syndrome as compared with other plasma cell disorders.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed 1 week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles.

Failure of IgG production due to a defect in the opening of the chromatin structure of I gamma 1 region in a patient with IgG and IgA deficiency.

Patients with common variable immunodeficiency (CVID) display reduced levels of two or all three of the major immunoglobulin isotypes, and the deficiency is characterized by failure of B cells to differentiate into plasma cells in many cases. A patient (14 years old, female) showed normal serum IgM levels and low serum IgG and IgA levels, including low levels of all IgG subclasses. Northern blot analysis suggested that the patient's B cells may be defective at the immunoglobulin heavy chain isotype switch. The germ-line C gamma 1 transcript was amplified from cDNA of healthy controls by the addition of recombinant IL-2 (rIL-2) to pokeweed mitogen-stimulated peripheral mononuclear cells or Staphylococcus aureus Cowan I (SAC)-stimulated IgM-producing lymphoblastoid cell lines (LCL) transformed by Epstein-Barr virus, while it was not amplified from cDNA of the patient. In the I gamma 1 region of LCL cultured with SAC plus rIL-2, the inner cytosine in the 5' C-C-G-G 3' sequence nearest the 3' site of the I gamma 1 region, at least, was not completely unmethylated in the patient. Moreover, the DNase I hypersensitive site was not induced in the patient's LCL by SAC plus rIL-2. These results indicate that the defects of the immunoglobulin heavy chain isotype switch in the patient's B cells are due to failure in the synthesis of germ-line C gamma transcripts, and this may be caused by defects in opening of the chromatin structures of specific switch regions.

Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.

Early cutaneous gene transcription changes in adult atopic dermatitis and potential clinical implications

Atopic dermatitis (AD) is a common pruritic dermatitis with macroscopically non-lesional skin that is often abnormal. Therefore, we used high-density oligonucleotide arrays to identify cutaneous gene transcription changes associated with early AD inflammation as potential disease control targets. Skin biopsy specimens analysed included normal skin from five healthy non-atopic adults and both minimally lesional skin and nearby or contralateral non-lesional skin from six adult AD patients. Data were analysed on an individual gene basis and to identify biologically relevant gene networks. Transcription levels of selected genes were also analysed by quantitative PCR. Differential transcription occurring early in AD skin was indicated for (i) individual genes such as C-C chemokine ligand (CCL)18, CCL13, and interferon-alpha2 (IFNalpha2), (ii) genes associated with peroxisome proliferator-activated receptor (PPAR)alpha- and PPARgamma-regulated transcription, and possibly for (iii) immunoglobulin J-chain and heavy chain isotype transcripts. These data suggest that local changes in immunoglobulin-associated transcription may favour IgE over secretory immunoglobulin (multimeric IgM and IgA) expression in AD skin. Decreased PPAR activity appears common to both AD and psoriasis, and reduced cutaneous IFNalpha2 transcription also appears characteristic of AD. Identification of these genes and pathways will direct future research towards controlling AD.

Protein Mapping and Characterization of Post-Translational Modifications of Ku86 Protein Variants in Multiple Myeloma Cell Lines.

A single biological event, immunoglobulin heavy chain (IgH) isotype class switch recombination (CSR) is thought to mark the onset of multiple myeloma (MM). The DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which consists of a heterodimeric regulatory subunit (Ku70/Ku86) and its catalytic subunit (DNA-PKcs), is the principal mediator of IgH isotype CSR. Studies in Ku70 and Ku86 knockout mice have suggested that Ku proteins could have a role to play in carcinogenesis. Since we have previously detected truncated variants of Ku86 (Ku86v) in 86% to 100% of freshly isolated patient MM cells, we therefore hypothesize that Ku86v could be a putative oncoprotein. Two variants of Ku86 are predominantly found in MM cells: a 69 kDa variant truncated at the C-terminus (Ku86v-N), which has lost its DNA repair function; and a 56 kDa N-terminus truncated variant (Ku86v-C), which is not translocated into the nucleus and aberrantly expressed on the cell membrane. Our prior studies using Northern blotting, whole cell polyacrylamide gel electrophoresis (PAGE) and electrophoretic mobility shift assays (EMSA) have demonstrated that Ku86vs in MM cells are not the result of alternative RNA splicing; or in vitro degradation, as has been described in human lymphocytes. Moreover, since many proteins in MM cells are known to be heavily glycosylated, and deglycosylation could lead to shorter forms of the protein, we now show using Endo H digestion that Ku86vs are also not the result of N-linked deglycosylation. Rather, Ku86vs are formed by in vivo proteolytic cleavage via a trypsin-like serine protease. Since the proteasome degradation pathway is highly active in MM cells; and protease digestion of DNA-PK holoenzyme is enhanced by proteasome inhibition (i.e. bortezomib treatment), these data support the notion that in vivo activation of putative Ku86v oncoproteins within MM cells could arise via a constitutively active post-translational proteolytic process. To define a functional role for Ku86v, we first demonstrate that Ku86v is physically associated with the most abundant anti-apoptotic protein in MM cells, BCL2. We then used cyanogen bromide immunoprecipitation to purify BCL2-bound Ku86vs followed by 2-dimensional gel electrophoresis (2DGE) to resolve these purified proteins, and demonstrate that only the 56 kDa Ku86v-C (pI 5.0) bound to BCL2. Both full length Ku86 and Ku86v-N were not detected. We then isolated Ku86v-C from the 2D gel and preliminary mass spectrometric analyses suggest that the N-terminus truncation retains the Ku autoantigen related protein 1 (KARP1) motif of Ku86. The significance of this is still under investigation. In conclusion, we have found that Ku86v-C (56 kDa pI 5.0) is formed by post-translational proteolytic cleavage. Since, Ku86v-C is significantly associated with BCL2 oncoprotein, we therefore speculate that Ku86v-C could possibly potentiate BCL2's anti-apoptotic function. Accordingly, Ku86v-C could be considered as a potential target for therapeutic intervention in MM.

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.