Immunoglobulin Light Chain Variable Region Research Articles

Serum Proteomic Analysis by Nanoflow LC-MS/MS-Based Proteomics in IgA Chronic Kidney Disease.

IgA nephropathy (IgAN) is the most frequently occurring primary glomerulonephritis. A lack of specific biomarkers hinders the early diagnosis and treatment of this disease. This study analyzes and validates potential serum biomarkers using mass spectrometry proteomics. Global proteomics profiles of serum from 60 patients with IgAN and 43 healthy control subjects were compared to identify significantly changed proteins. These proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in an independent validation set consisting of samples from 67 different stage IgAN patients and 60 healthy controls. A total of 37 significantly changed proteins were found in the discovery set, among which 18 proteins were identified as potential biomarkers for IgAN through PRM assays in the validation set. Of these 18 proteins, IgGFc-binding protein, MS-A1 light chain variable region, transthyretin, ficolin-3, and myosin-reactive immunoglobulin light chain variable region were up-regulated in different IgAN stages, B cell receptor heavy chain variable region, rheumatoid factor RF-ET6, heavy chain Fab, cryocrystalglobulin CC1 heavy chain variable region, FLJ94213, lumican, and Q68CN4 (uncharacterized protein) were down-regulated in different IgAN stages. These proteins support previous findings that CKD is accompanied by altered immune response. This study lays the groundwork for additional research using biomarkers to clinically diagnose IgAN. These proteins are potential molecular markers that could help us understand the potential molecular mechanism of IgAN.

Hyper N-Glycosylated SEL1L3 As B-Cell Receptor Autoantigen of Primary Vitreoretinal Lymphoma

Chronic antigenic stimulation of B lymphocytes through their B-cell receptor (BCR) is thought to play an important role in the genesis of B-cell lymphomas. SAMD14/neurabin-I has been described as auto-antigenic target of primary central nervous system lymphoma (PCNSL) BCRs. Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress to PCNSL. Besides manifestation in an immunologically privileged site, PVRL and PCNSL share frequent recurrent genetic alterations like MYD88 and CD79B mutations. Both PVRL and PCNSL are considered being related to the MCD subtype of DLBCLs and may depend on chronic BCR signaling through antigenic stimulation. To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed BCRs from PVRL patients as antibody fragments (Fabs) and tested for specific binding against common antigens. All cases with corresponding BCR sequences were previously reported by Belhouachi et al. (Blood Adv. 2020;4(7):1357-1366). For 20 of 55 PVRL BCR sequences both immunoglobulin heavy and light chain variable region (V) genes were available. DNA clones comprising an ApaLI restriction site, the complete light chain V gene, a κ- or λ-constant region gene, a ribosome binding site, a signal sequence, the heavy chain V gene and a BstEII restriction site were ordered from GeneCust® (GeneCust, 5690 Ellange, Luxembourg) in a pUC57 vector. DNA fragments were cloned in front of a γ-1 constant region gene and a Histidine-tag into a modified pCES-1 vector for expression as Fabs. PVRL-BCRs were screened on protein macroarrays representing clones of UniPEx human cDNA expression libraries expressed in E. coli (Bioscience, Dublin, Ireland). SEL1L3 was identified as the auto-antigenic target of the B-cell receptors in 3 out of 20 PVRL cases. None of the PVRL Fabs bound to LRPAP1 (common BCR antigen of mantle cell lymphomas), RpoC (Moraxella catarrhalis bacterial antigen and target of nodular lymphocyte-predominant Hodgkin lymphoma cells) or galectin-3 (member of the lectin family). SEL1L3 is a 1132 amino acid long protein with multiple glycosylation and phosphorylation sites (uniprot.org). This is especially important since previously identified BCR antigens from different lymphoma entities showed post-translational modifications such as hyper-N-glycosylation or hypo-phosphorylation. Furthermore, SEL1L3 is highly expressed in the pigmented layer of the retina. Western Blot analysis of the blood of a PVRL patient with serum antibodies against SEL1L3 showed an increased molecular weight of SEL1L3 as compared to SEL1L3 of controls. After treatment with N-acetyl-β-d-glucosaminidase or PNGase F, this difference in molecular weight disappeared, indicating a hyper N-glycosylation of SEL1L3 in PVRL patients with SEL1L3-reactive BCRs as trigger for its' immunogenicity. SEL1L3 induced proliferation of aggressive lymphoma cell lines transfected with SEL1L3-reactive BCRs. Moreover, the BCR binding epitope of SEL1L3 (aa 560 - 585) conjugated to a truncated form of Pseudomonasaeruginosa exotoxin A, killed exclusively lymphoma cells expressing BCRs with the respective reactivity. Additionally, BCRs of a locally treated PVRL patient with serum antibodies against SEL1L3 were cloned from biopsies of the vitreous body at initial diagnosis and of a lymphoma manifestation in the breast at relapse. Both BCRs showed binding to SEL1L3 suggesting a continued dependence of the BCR on antigen stimulation. These results suggest an important role of chronic antigenic stimulation by the post-translationally modified auto-antigen SEL1L3 in the genesis of a subgroup of patients with PVRL, might give an explanation for the selective tropism of PVRLs and provide the basis for a new treatment approach with high specificity.

Proteomic profiling of sporadic late-onset nemaline myopathy.

ObjectiveTo define the proteomic profile of sporadic late‐onset nemaline myopathy (SLONM) and explore its pathogenesis.MethodsWe performed mass spectrometry on laser‐dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL).ResultsThere were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T‐cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers.ConclusionOur findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.

IGVL gene region usage correlates with distinct clinical presentation in IgM vs non-IgM light chain amyloidosis

AbstractPatients with immunoglobulin M (IgM) light chain (AL) amyloidosis have a distinct clinical presentation compared with those with non-IgM amyloidosis. We hypothesized that differential immunoglobulin light-chain variable region (IGVL) gene usage may explain the differences in organ involvement, because IGVL usage correlates with organ tropism. IGVL usage was evaluated by mass spectrometry of amyloid deposits (IgM, n = 45; non-IgM, n = 391) and differed across the 2 groups. In the λ family, LV2-08 (13% vs 2%; P < .001) and LV2-14 (36% vs 10%; P < .001) usage was more common in IgM vs non-IgM amyloidosis, whereas LV1-44 (0% vs 10%; P = .02) and LV6-57 (2% vs 18%; P = .004) usage was less common. In the κ family, there was a trend toward higher KV4-01 (11% vs 4%; P = .06) usage in IgM amyloidosis. IGVL usage correlated with disease characteristics/organ tropism. LV2-14 (more common in IgM amyloidosis) has historically been associated with peripheral nerve involvement and lower light chain burden, which were more frequent in IgM amyloidosis. LV1-44 (less common in IgM), associated with cardiac involvement, was less frequent in IgM patients. LV6-57 (less common in IgM) is associated with t(11;14), which was less frequent in IgM patients. In conclusion, IGVL gene usage differs in patients with IgM vs non-IgM amyloidosis and may explain the distinct clinical presentation.

Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody

Single-chain variable fragments (scFvs) are small-sized artificial constructs composed of the immunoglobulin heavy and light chain variable regions connected by a peptide linker. We have previously described an anti-fibroblast growth factor 2 (FGF2) immunoglobulin G (IgG) monoclonal antibody (mAb), named 3F12E7, with notable antitumor potential revealed by preclinical assays. FGF2 is a known angiogenesis-associated molecule implicated in tumor progression. In this report, we describe a recombinant scFv format for the 3F12E7 mAb. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Overall, our experimental and prediction dataset describes an scFv scaffold for the 3F12E7 mAb and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes.

The analysis of organization and diversity mechanism in goat immunoglobulin light chain gene loci.

The genomic organization of goat immunoglobulin light chains (Igλ and Igκ) loci were annotated based on the goat genome database. The goat Igλ chain located on chromosome 17 contains at least 35 Vλ gene fragments (seven potential functional genes, one ORF and 27 pseudogenes), two Jλ-Cλ clusters arranged in a Vλ(35)-Jλ2-Cλ1-Jλ1-Cλ2 pattern, with another Cλ3 on scaffold. The Igκ locus included 11 Vκ (five potential functional genes, two ORFs and four pseudogene fragments), three Jκ genes and a single Cκ gene ordered in Vκ(35)-Jκ(3)-Cκ pattern on chromosome 11. By analyzing the clonies of Igλ and Igκ, we further found Vλ2 (26.23 %) &Vλ3 (73.11 %), Vκ2 (52.07 %) &Vκ4 (46.15 %) were predominately used in the expression of λ and κ chains respectively. λ chain showed more abundance in connective diversity than κ chain. Besides, somatic hypermutation with higher frequency in both immunoglobulin light chains was the major mechanism for the goat repertoire diversity. These results demonstrated goat immunoglobulin light chain variable region genome loci and repertoire diversity.

Immunoglobulin Variable Gene Region (IGVL) Usage Correlates with Distinct Clinical Presentation in IgM Versus Non-IgM Light Chain Amyloidosis

Introduction: The clinical presentation and frequency of organ involvement are quite distinct in patients with IgM amyloidosis compared to patients with non-IgM amyloidosis, with less heart involvement and more nerve, soft tissue and lung involvement in IgM patients. It has been observed that immunoglobulin light chain variable region (IGVL) gene and gene family of the plasma cell clone impact phenotypic manifestations in AL amyloidosis, particularly organ tropism. (Kourelis et al. Blood. 2017;129). Given the variability in clinical features amongst patients with IgM vs. non IgM AL amyloidosis, we hypothesized that IGVL gene usage would be different between these two groups. Methods: Patients with newly diagnosed IgM and non-IgM amyloidosis seen at our institution from 01/2006 to 12/2015 were identified. IGVL gene usage was assessed by liquid chromatography tandem mass spectrometry (LC/MS/MS) as previously described. (Vrana et al Blood 2009;Dasari et al J of Proteome Res; Kourelis et al. Blood. 2017). Mass spectrometry data for IGVL analysis was obtained from clinically available data. LC/MS/MS was performed on available archival specimens when such data was not available clinically. Results: Patients with newly diagnosed amyloidosis who had sufficient mass spectrometry data on fat aspirate or tissue biopsy for IGVL gene usage identification were included (IgM AL, N=44 and Non-IgM AL, N=391). Clinical Features: As expected, differences were noted in baseline characteristics of patients with IgM vs. non-IgM AL amyloidosis. Patients with IgM amyloidosis were older (68 vs. 64 years, p=0.04) and more likely to have kappa as the involved light chain (34% vs. 25%, p=0.2), though the difference did not reach statistical significance. IgM amyloidosis patients had lower difference in involved and uninvolved free light chains (dFLC) (15.5 vs. 25.8 mg/dL, p=0.01) and lower NTProBNP levels (median: 1987 vs. 2655 pg/mL, p=0.02) compared with non-IgM amyloid patients. Presence of t(11;14) was less common by FISH (30% vs. 48%, p=0.1), though the difference did not reach statistical significance. Patterns of organ involvement were different, with heart involvement being less frequent in IgM patients (64% vs. 76%), while peripheral nerve (30% vs. 18%, p=0.05), soft tissue (41% vs. 21%, p=0.005) and lung involvement (7% vs. 3%, p=0.1) were more common in patients with IgM vs. non-IgM AL amyloidosis. There was no difference in liver (9% vs. 15%), kidney (50% vs. 51%) GI (23% vs. 25%) or autonomic nervous system involvement (18% vs. 14%). IGVL Gene Usage: As shown in Figure 1, IGVL usage differed across the two groups. In the lambda group, LV2-08 (14% vs. 2%, p&lt;0.001) and LV2-14 (36% vs. 10%, p&lt;0.001) were more common in IgM amyloid patients, while LV1-44 (0% vs. 10%, p=0.02) and LV6-57 (2% vs. 18%, p=0.004) were less common in the IgM group. There was also a trend towards less frequent involvement of LV3-01 (2% vs. 12%, p=0.07) in IgM amyloid patients. In the kappa group, KV4-01 (11% vs. 4%, p=0.04) was more common in patients with IgM amyloidosis. Correlation Between IGVL Gene usage and Clinical Features: IGVL gene usage correlated with disease characteristics and differences in the clinical presentation of patients across the two groups. LV2-14 was more commonly seen in IgM amyloid patients (above). It has been previously associated with a higher frequency of peripheral nerve involvement (Kourelis et al. Blood 2017), which was more frequent in IgM group. LV2-14 is also associated with low dFLC levels, which were noted to be lower in the IgM AL cohort. LV1-44 has been associated with higher likelihood of cardiac involvement. LV1-44 was seen less frequently in the IgM amyloid patients, who also had lower rates of cardiac involvement. LV6-57 has been historically associated with higher rates of t(11;14). It was less common in IgM AL patients, who also had lower rates of t(11;14). Conclusions: IGVL gene usage differed significantly in patients with IgM vs. non-IgM amyloidosis, correlating with differences in disease characteristics and organ involvement. This is the first study to our knowledge to report IGVL gene usage differences in IgM vs. non-IgM AL amyloidosis. Differences in IGVL gene usage may explain the distinct clinical presentation seen in IgM amyloidosis, the biology of which has so far remained poorly understood. *SS and SD contributed equally Disclosures Dasari: The Binding Site: Patents &amp; Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Dispenzieri:Alnylam: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Pfizer: Research Funding; Janssen: Consultancy; Akcea: Consultancy; Intellia: Consultancy. Murray:The Binding Site: Patents &amp; Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding. Gertz:Prothena: Honoraria; Alnylam: Honoraria; Ionis: Honoraria; Janssen: Honoraria; Spectrum: Honoraria, Research Funding; Celgene: Honoraria.

Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential implications for immune checkpoint blockade and neoantigen-based immunotherapies

BackgroundDiffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma.MethodsTo determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens’ immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry.ResultsSHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell–like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell–like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM.ConclusionsThese results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

Long-term and high dose dexamethasone injection decreases the expression of Immunoglobulin Heavy (Light) Chain Variable Region Genes (IGH(L)Vs) in the mouse spleen

Glucocorticoid hormones have been widely used in clinical practice as potent anti-inflammatory and immunosuppressive agents. However, the underlying mechanisms of how they work remain unaddressed. Here, we used RNA-set to profile spleen gene expression in adult mice after consistent intraperitoneal injection of dexamethasone. We identified 13565 genes in control (injected with 0.9% NaCl) and 13702 genes in dexamethasone-injected group, 12920 genes were expressed in both, but 645 genes were identified only in control and 782 genes were identified only in the dexamethasone group. In the dexamethasone-injected group 101 and 67 genes were down-and up-regulated, respectively. Among these, 129 were coding genes, 19 were identified as non-coding genes or pseudogenes, and the remaining 20 were TEC (to be experimentally confirmed) genes. Gene ontology (GO) and KEGG pathway analysis revealed that the cytokine-cytokine receptor interaction pathway was highly enriched in these differentially expressed genes, and that a majority of the 129 identified coding genes were involved in immune system and cell adhesion-associated processes. Moreover, systemic lupus erythematosus, renin-angiotensin system, fat digestion and absorption, and glycerolipid metabolism pathways were significantly affected in the dexamethasone-treatment group. No obvious signaling pathway was enriched in the control group. Additionally, 20 immunoglobulin heavy or light chain variable region genes (IGH(L)Vs) were down regulated in the dexamethasone-injected group. IGH(L)Vs encode the variable region of immunoglobulin heavy chain and determine the diversity and specificity of antibodies. We were unable to determine the function of the 19 non-coding genes with differential expression following dexamethasone treatment. Our findings indicate that the expression of IGH(L)Vs and non-coding genes play an important role in the anti-inflammatory and immunosuppressive effects of dexamethasone and could be developed as potential agents in clinical practice.

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

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

JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells.

Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by re-searching only unmatched high-quality spectra and reusing amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then, the JUMPg program is applied to process a label-free mass spectrometry data set of Alzheimer's disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and "non-coding gene" translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled data set of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multiomics data integration.

Characterization of recombinant human monoclonal antibodies against the Bile Salt Export Pump

Background: Inhibitory, Bile Salt Export Pump (BSEP)-reactive IgG-class antibodies cause phenotypic recurrence of BSEP deficiency in some patients after liver transplantation for Progressive Familial Intrahepatic Cholestasis Type 2 (PFIC-2). A detailed characterization of these disease-causing antibodies is key to elucidate the molecular mechanism of this emerging disease. Methods: BSEP-reactive B cells were isolated from peripheral blood of a patient with a high anti-BSEP titer by fluorescence-activated single cell sorting. Corresponding full-length immunoglobulin heavy and light chain variable region gene transcripts were amplified by single cell RT-PCR and cloned into eukaryotic expression vectors to enable the in vitro production of the respective monoclonal antibodies. These monoclonal antibodies were characterized by immunofluorescence studies of rat liver slices and transiently transfected cells, Western blot, FACS analysis, in situ rat liver perfusion and in vitro BSEP transport assays. Results: Immunofluorescent staining of fixed, transiently transfected human embryonal kidney (HEK293) cells identified several BSEP-reactive antibodies that also stained canalicular patterns on fixed rat liver slices. In contrast, only a few recognized denatured rat BSEP on Western blots, while all antibodies detected its denatured human homologue. FACS analysis of antibody-treated, unpermeabilized cells transiently expressing BSEP revealed BSEP-specific staining that was absent on empty control cells. BSEP-reactive antibodies reached the canalicular space of in situ perfused rat livers within two hours. Finally, in vitro vesicular transport assays showed clone-dependent influence on transport activity. Conclusion: We provide characterization of a novel set of patient-derived, monoclonal antibodies directed against extracellular features of BSEP. These include species that are BSEP-inhibitory, -neutral, and, strikingly, -stimulatory in vitro. The antibodies and their monovalent Fab derivatives may have great potential in applied research as well as in future clinical applications.

Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10.

BackgroundSemi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P.falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described.MethodsB lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A.ResultsSupernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10−7 M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10−9 M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10−9 M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6–6.6 mg/ml], 6.9 mg/ml (CI 5.5–8.6 mg/ml) and 9.5 mg/ml (CI 5.5–16.4 mg/ml), respectively.ConclusionThis report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

Serum proteomic-based analysis by iTRAQ of damp-heat impeding syndrome of rheumatoid arthritis

IntroductionIn this study we aim to investigate the characteristic proteins in rheumatoid arthritis (RA) by using iTRAQ-based proteomics approach to explore the possible inherent molecular mechanisms, disease monitoring and therapeutic targets of damp-heat impeding syndrome. MethodsThe iTRAQ analyses combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) was performed to compare serum protein expression patterns of damp-heat impeding syndrome of RA with that of cold-damp impeding syndrome of RA, and healthy volunteers. Gene-ontology (GO) analysis of these differentially expressed proteins were performed using WEB based bioinformatics tools (DAVID v6.7, UniProtKB/Swiss-Prot, and Ingenuity Pathway Analysis). Ingenuity Pathways Analysis (IPA) was also used to interpret the interaction network and predominant canonical pathways involved in these differentially expressed proteins. ResultsBy iTRAQ analyses and mass spectroscopic identification, 6 proteins including FLJ00382, Ig lambda chain V-I region WAH, Myosin-reactive immunoglobulin light chain variable region, C-reactive protein, S100A9, and Cathepsin G were found significantly overexpressed in damp-heat impeding syndrome. Bioinformatics analyses showed that these proteins were involved in inflammatory response, suggesting that RA patients with damp-heat impeding syndrome have severe inflammatory responses and high RA inflammatory activity. ConclusionThis preliminary work provides a better understanding of the proteomic alterations of serum from RA patients with damp-heat impeding syndrome and may provide us insights into the pathophysiology and potential diagnostic biomarkers for early detection, disease monitoring and therapeutic targets for damp-heat impeding syndrome.

Role of Microbes in B-Cell Lymphopoiesis and Early Ig Repertoire Development

Progenitor (pro-) and precursor (pre-) B cells express the recombination-activating gene (RAG1/RAG2) endonuclease, which initiates the V(D)J recombination reaction that assembles Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments. Expression of productively assembled IgH μ chains and IgL (Igκ or Igλ) chains generates IgM molecules that form the B-cell antigen binding receptor (BCR) with a diverse primary repertoire of binding specificities. As the newly assembled and expressed IgM interacts with local antigens, RAG expression can continue, allowing continued Igκ V(D)J recombination that can replace the previously assembled VκJκ exon with one that generates a new specificity. This "receptor editing" process provides an antigen-mediated regulatory checkpoint that helps shape the pre-immune BCR repertoire. As the principal location of early B-cell development, the bone marrow antigenic environment is the main force affecting receptor editing. However, the extent to which self-antigens outside of the bone marrow — as well as environmental antigens — may shape the primary Ig repertoire is not known. Here we show that early B-cell development takes place in peripheral sites, including the small intestinal mucosa, in young mice. Cells in these sites co-express RAG with cytoplasmic Igκ and Igμ, indicating active receptor editing; and, correspondingly, they have significant differences in Vκ usage compared to RAG2-expressing bone marrow B-lineage cells and other RAG2-expressing B-lineage cells from other peripheral sites. In addition, the luminal microbial environment appears to also affect Igλ/Igκ ratios. We conclude that early B-cell development can occur in the periphery, including the intestinal mucosa, where commensal microbes may influence BCR diversification and gut pre-immune Ig repertoires. DisclosuresNo relevant conflicts of interest to declare.

Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis (HUM1P.314)

Abstract In lupus nephritis (LuN), severity of tubulointerstitial inflammation (TII) on renal biopsy predicts subsequent renal failure. In severe TII, infiltrating B and T cells have features of tertiary lymphoid neogenesis including in situ antigen-driven clonal B cell selection. To identify the autoantigen(s) driving selection, we laser captured single CD38+ or Ki-67+ B cell mRNA from seven LuN biopsies. Twenty clonally expanded immunoglobulin heavy and light chain variable region pairs were cloned and expressed as antibodies. Seven more were obtained from an eighth biopsy by flow sorting of CD38+ cells. Immunofluorescence indicated that 13 of 27 antibodies had immunoreactivity with cytoplasmic structures. Only 4 reacted with nuclei and none with dsDNA, Sm or RNP. Interrogation of protein arrays with TII antibodies and immunoprecipitations followed by mass spectroscopy identified a limited number of self-antigens with only vimentin common to both approaches. Eleven of 13 cytoplasmic TII antibodies directly bound vimentin, vimentin was highly expressed by tubulointerstitial infiltrating cells and tested TII antibodies preferentially bound inflamed tubulointerstitum. Finally, high-titers of serum anti-vimentin antibodies were strongly associated with severe TII. These finding suggest that in situ adaptive immunity to vimentin, an antigenic feature of inflammation, feeds forward to worsen TII and renal damage.

Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis

In lupus nephritis (LuN), severity of tubulointerstitial inflammation (TII) on renal biopsy predicts subsequent renal failure. In severe TII, infiltrating B and T cells have features of tertiary lymphoid neogenesis including in situ antigen-driven clonal B cell selection. To identify the autoantigen(s) driving selection, we laser captured single CD38+ or Ki-67+ B cell mRNA from seven LuN biopsies. Twenty clonally expanded immunoglobulin heavy and light chain variable region pairs were cloned and expressed as antibodies. Seven more were obtained from an eighth biopsy by flow sorting of CD38+ cells. Immunofluorescence indicated that 13 of 27 antibodies had immunoreactivity with cytoplasmic structures. Only 4 reacted with nuclei and none with dsDNA, Sm or RNP. Interrogation of protein arrays with TII antibodies and immunoprecipitations followed by mass spectroscopy identified a limited number of self-antigens with only vimentin common to both approaches. Eleven of 13 cytoplasmic TII antibodies directly bound vimentin, vimentin was highly expressed by tubulointerstitial infiltrating cells and tested TII antibodies preferentially bound inflamed tubulointerstitum. Finally, high-titers of serum anti-vimentin antibodies were strongly associated with severe TII. These finding suggest that in situ adaptive immunity to vimentin, an antigenic feature of inflammation, feeds forward to worsen TII and renal damage.

Comparative in vitro and in vivo assessment of toxin neutralization by anti-tetanus toxin monoclonal antibodies

Tetanus is caused by the tetanus neurotoxin (TeNT), a 150 kDa single polypeptide molecule which is cleaved into an active two-chain molecule composed of a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chains. Recently, extensive effort has focused on characterization of TeNT binding receptors and toxin neutralization by monoclonal antibodies (mAbs). Toxin binding inhibition and neutralization is routinely assessed either in vitro by the ganglioside GT1b binding inhibition assay or in vivo using an animal model. These two assay systems have never been compared. In the present study, we report characterization of eleven mAbs against different parts of TeNT. The toxin inhibitory and neutralization activity of the mAbs was assessed in vitro and in vivo respectively. Our data demonstrated that seven mAbs bind to fragment C of the heavy chain, two mAbs react with the light chain, one mAb recognizes both chains and one mAb reacts with neither light chain nor fragment C. Six fragment C specific mAbs were able to inhibit TeNT binding to GT1b ganglioside in vitro but three failed to neutralize the toxin in vivo. One in vitro inhibitory mAb (1F3E3) was found to synergize with the in vivo neutralizing mAbs to reduce toxin lethal activity in vivo. Sequencing of the immunoglobulin heavy and light chain variable region genes revealed that the three in vivo neutralizing mAbs were derived from a common origin. Altogether, our data suggests that fragment C specific mAbs contribute to toxin neutralization in both systems, though some of the GT1b binding inhibitory mAbs may not be able to neutralize TeNT in vivo.

Microbial colonization influences early B-lineage development in the gut lamina propria

The RAG1/RAG2 endonuclease ("RAG") initiates the V(D)J recombination reaction that assembles Ig heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B cell antigen-binding receptor ("BCR"). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3–5. This “receptor editing” process, which also can lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen-encounter at the Rag-expressing immature B cell stage helps shape pre-immune BCR repertoires. As the major site of post-natal B cell development, the bone marrow is the principal location of primary Ig repertoire diversification in mice. Here, we report that early B cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP Ig repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B lineage cells that harbor intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B, and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing BM counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B cell development occurs in the intestinal mucosa, where it is regulated by extra-cellular signals from commensal microbes that influence gut Ig repertoires.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.

BackgroundThe outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).Methodology/Principal FindingsThe genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.Conclusions/SignificanceOur study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.