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1.
Sci Rep ; 7(1): 12713, 2017 10 05.
Article in English | MEDLINE | ID: mdl-28983085

ABSTRACT

In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Viral/biosynthesis , Feline Panleukopenia Virus/immunology , Feline Panleukopenia/therapy , Immunoglobulin A/genetics , Immunoglobulin G/genetics , RNA, Messenger/genetics , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibodies, Viral/therapeutic use , B-Lymphocytes/immunology , Cats , Immunoglobulin A/biosynthesis , Immunoglobulin G/biosynthesis , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Sequence Analysis, RNA
2.
J Immunol Methods ; 444: 1-6, 2017 05.
Article in English | MEDLINE | ID: mdl-28189705

ABSTRACT

To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines. This technique may also be applied to other types of cells as well as polyclonal Ab cell pools.


Subject(s)
Antibody-Producing Cells/metabolism , Cell Membrane/metabolism , Cloning, Molecular/methods , Immunoglobulin G/biosynthesis , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin lambda-Chains/biosynthesis , Immunomagnetic Separation/methods , Animals , Antibody-Producing Cells/immunology , CHO Cells , Cell Membrane/immunology , Cricetulus , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin lambda-Chains/genetics , Immunoglobulin lambda-Chains/immunology , Phenotype , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Transfection
3.
MAbs ; 9(2): 231-239, 2017.
Article in English | MEDLINE | ID: mdl-28001485

ABSTRACT

When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. κλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a κλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the κλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.


Subject(s)
Antibodies, Bispecific/biosynthesis , Protein Engineering/methods , Animals , Codon , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics
4.
PLoS One ; 11(11): e0166556, 2016.
Article in English | MEDLINE | ID: mdl-27846293

ABSTRACT

OBJECTIVES: We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. METHODS: We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. RESULTS: Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. CONCLUSIONS: Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.


Subject(s)
Demyelinating Diseases/diagnosis , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin lambda-Chains/biosynthesis , Multiple Sclerosis/diagnosis , Case-Control Studies , Demyelinating Diseases/blood , Demyelinating Diseases/cerebrospinal fluid , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoblotting/instrumentation , Immunoblotting/methods , Immunoglobulin kappa-Chains/blood , Immunoglobulin kappa-Chains/cerebrospinal fluid , Immunoglobulin lambda-Chains/blood , Immunoglobulin lambda-Chains/cerebrospinal fluid , Isoelectric Focusing/instrumentation , Isoelectric Focusing/methods , Multiple Sclerosis/blood , Multiple Sclerosis/cerebrospinal fluid , Nephelometry and Turbidimetry/instrumentation , Nephelometry and Turbidimetry/methods , Observer Variation , ROC Curve , Reproducibility of Results
5.
Ann Clin Biochem ; 53(Pt 1): 174-6, 2016 Jan.
Article in English | MEDLINE | ID: mdl-25753032

ABSTRACT

BACKGROUND: The results of free light chains quantitation in the cerebrospinal fluid were recently compared with the presence of cerebrospinal fluid-restricted oligoclonal IgG, but not oligoclonal free kappa light chains and oligoclonal free lambda light chains. We therefore aimed to compare the performance of the quantitative tests with the qualitative one for the same molecule. METHODS: Seventy-five paired cerebrospinal fluid and serum samples were analysed for oligoclonal IgG, oligoclonal free kappa light chains and oligoclonal free lambda light chains. Cerebrospinal fluid and serum free kappa and lambda light chains were quantified using Freelite™ kits on SPA Plus analyzer. ROC curves were analysed for the prediction of intrathecal synthesis and compared for cerebrospinal fluid concentration, cerebrospinal fluid/serum quotient (QfLC) and index (QfLC/QAlbumin). The presence of cerebrospinal fluid-restricted oligoclonal free kappa light chains and oligoclonal free lambda light chains bands was used as reference. RESULTS: No statistically significant differences were observed among cerebrospinal fluid concentration, QfLC and index for the prediction of free light chain intrathecal synthesis. Each parameter was able to predict the occurrence of cerebrospinal fluid-restricted oligoclonal free light chain bands (AUCs 0.932-0.999). However, we noted elevated cerebrospinal fluid free light chain concentrations in the absence of cerebrospinal fluid-restricted oligoclonal free light chain bands in two patients with very high serum free light chain values. CONCLUSIONS: Quantitation of cerebrospinal fluid free light chains reliably predicts their intrathecal synthesis. Yet, cerebrospinal fluid/serum quotient may still be preferred to correct for high serum free light chain concentrations. An appropriate formula should be sought to correct for blood-cerebrospinal fluid barrier status.


Subject(s)
Clinical Chemistry Tests/methods , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/cerebrospinal fluid , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/cerebrospinal fluid , Humans , Immunoglobulin kappa-Chains/blood , Immunoglobulin lambda-Chains/blood , Spinal Canal/metabolism
6.
Sci Rep ; 4: 5885, 2014 Jul 30.
Article in English | MEDLINE | ID: mdl-25073855

ABSTRACT

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.


Subject(s)
Cloning, Molecular/methods , Genetic Vectors/metabolism , Immunoglobulin E/biosynthesis , Reagent Kits, Diagnostic , Recombinant Fusion Proteins/biosynthesis , Animals , Antigens, Plant/biosynthesis , Antigens, Plant/genetics , Base Sequence , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , DNA Primers/chemical synthesis , Epitopes/chemistry , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Humans , Immunoglobulin E/genetics , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Molecular Sequence Data , Plant Proteins/biosynthesis , Plant Proteins/genetics , Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics
7.
Acta Haematol ; 130(3): 188-91, 2013.
Article in English | MEDLINE | ID: mdl-23774652

ABSTRACT

BACKGROUND: Precursor B-cell acute lymphocytic leukemia (ALL) with surface immunoglobulin light chain expression is a rare disease entity. The differential diagnosis is difficult but critical for disease management. AIMS: We report 2 cases (1 adult and 1 infant) of precursor B-cell ALL who presented at diagnosis with surface immunoglobulin light chain expression revealed by flow cytometric immunophenotyping and discuss its clinical significance. CASE REPORT: The 2 patients presented with nonspecific symptoms such as fever, pallor, fatigue or lymphadenopathy. Flow cytometric immunophenotyping showed that both patients expressed CD34/CD19/CD10/CD22/CD9/HLA-DR/CD38/CD123/CD13 (partial) and had unexpected single λ light chain expression. Cytogenetic analysis revealed t(9;22)(q34;q11) in the adult patient and normal karyotype in the infant. Both cases were diagnosed and managed as precursor B-ALL, and the patients showed good response to treatment regimens. CONCLUSION: We describe 2 cases of precursor B-ALL with unexpected surface light chain expression. The exceedingly rare immunophenotypes have diagnostic implication for immunophenotyping of this malignancy. Treatment regimens for precursor B-cell ALL are suitable for such cases.


Subject(s)
Gene Expression Regulation, Leukemic , Immunoglobulin lambda-Chains/biosynthesis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/blood , Adult , Antigens, CD/biosynthesis , Asian People , China , Female , Flow Cytometry , HLA-DR Antigens/metabolism , Humans , Infant , Male , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy
9.
Ugeskr Laeger ; 174(17): 1159-60, 2012 Apr 23.
Article in Danish | MEDLINE | ID: mdl-22533933

ABSTRACT

A 64 year-old woman with acute renal failure and cast nephropathy due to excessive production of lambda free light chains received chemotherapy (using bortezomib and dexamethason) and haemodialysis with a high cut off-filter. The concentration of free light chains was markedly reduced after a fortnight. Nine months after admission, the patient's kidney function had improved and dialysis was stopped. Three months later, she got an autologous stem cell transplantation. One year later, estimated glomerular filtration rate was 25 ml/min, and the production of free light chains was under control.


Subject(s)
Kidney Diseases/etiology , Multiple Myeloma/complications , Acute Kidney Injury/etiology , Acute Kidney Injury/pathology , Acute Kidney Injury/therapy , Antineoplastic Agents/therapeutic use , Boronic Acids/therapeutic use , Bortezomib , Dexamethasone/therapeutic use , Female , Humans , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin lambda-Chains/biosynthesis , Kidney Diseases/pathology , Kidney Diseases/therapy , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Paraproteinemias/etiology , Prognosis , Pyrazines/therapeutic use , Stem Cell Transplantation
10.
J Immunol ; 188(1): 47-56, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22131331

ABSTRACT

Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.


Subject(s)
Gene Expression Regulation/immunology , Immunoglobulin kappa-Chains/immunology , Immunoglobulin lambda-Chains/immunology , RNA Editing/immunology , RNA, Messenger/immunology , Receptors, Antigen, B-Cell/immunology , Animals , Cell Line , Gene Expression Regulation/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Mice , RNA Editing/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Antigen, B-Cell/biosynthesis , Receptors, Antigen, B-Cell/genetics
11.
J Cutan Pathol ; 38(9): 724-30, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21623870

ABSTRACT

Primary cutaneous extranodal marginal zone lymphoma (MZL) of mucosa-associated lymphoid tissue (MALT) represents a monoclonal B-cell neoplasm that typically presents with papules, plaques or nodules. We describe a patient with a primary cutaneous MALT lymphoma with unusual clinical features and an unusual immunophenotype. Conventional microscopy together with immunohistochemistry and in-situ hybridization showed the presence of lymphoma in normal-appearing and minimally erythematous skin as well as in clinically involved skin. Furthermore, at least two distinct clones were shown, one of which had κ-light chain restriction, and the other of which had λ-light chain restriction. This case represents a newly described clinical appearance of primary cutaneous MZL and shows that some patients may have more than one neoplastic clone.


Subject(s)
Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin lambda-Chains/biosynthesis , Lymphoma, B-Cell, Marginal Zone/metabolism , Lymphoma, B-Cell, Marginal Zone/pathology , Neoplasm Proteins/biosynthesis , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Adult , Humans , Immunohistochemistry , Male , Skin/metabolism , Skin/pathology
14.
J Immunol ; 185(1): 653-9, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20505143

ABSTRACT

Traditionally, mast cells were regarded as key cells orchestrating type I hypersensitivity responses. However, it is now recognized that mast cells are widely involved in nonallergic (non-IgE) chronic diseases. Also, in inflammatory bowel disease (IBD), a disease not associated with increased IgE concentrations, clear signs of activation of mast cells have been found. In this study, we investigated if Ig-free L chain-induced hypersensitivity-like responses through activation of mast cells could contribute to the pathophysiology of IBD. As a mast cell-dependent model for IBD, mice were skin-sensitized with dinitrofluorobenzene followed by intrarectal application of the hapten. In this murine IBD model, F991 prevented mast cell activation and also abrogated the development of diarrhea, cellular infiltration, and colonic lymphoid follicle hyperplasia. Furthermore, passive immunization with Ag-specific Ig-free L chains (IgLCs) and subsequent rectal hapten challenge elicited local mast cell activation and increased vascular permeability in the colon of mice. Clinical support is provided by the observation that serum concentrations of IgLCs of patients suffering from Crohn's disease are greatly increased. Moreover, increased presence of IgLCs was evident in tissue specimens from colon and ileum tissue of patients with IBD. Our data suggest that IgLCs may play a role in the pathogenesis of IBD, which provides novel therapeutic means to prevent or ameliorate the adverse gastrointestinal manifestations of IBD.


Subject(s)
Colitis/immunology , Colitis/metabolism , Immunoglobulin kappa-Chains/physiology , Immunoglobulin lambda-Chains/physiology , Inflammatory Bowel Diseases/immunology , Inflammatory Bowel Diseases/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Adult , Animals , Colitis/pathology , Crohn Disease/immunology , Crohn Disease/metabolism , Crohn Disease/pathology , Disease Models, Animal , Female , Humans , Immunization, Passive , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/blood , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/blood , Inflammatory Bowel Diseases/pathology , Male , Mast Cells/pathology , Mice , Mice, Inbred BALB C , Middle Aged , Up-Regulation/immunology , Young Adult
15.
Cancer Lett ; 284(2): 165-74, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19481340

ABSTRACT

The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.


Subject(s)
B-Lymphocytes/virology , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Viral , Herpesvirus 4, Human/physiology , Multiple Myeloma/pathology , Adaptor Proteins, Signal Transducing/biosynthesis , Adaptor Proteins, Signal Transducing/genetics , B-Lymphocytes/pathology , Cell Differentiation , Genes, Viral , Herpesvirus 4, Human/genetics , Humans , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Interferon Regulatory Factors/biosynthesis , Interferon Regulatory Factors/genetics , MicroRNAs/biosynthesis , MicroRNAs/genetics , Myeloma Proteins/biosynthesis , Myeloma Proteins/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , PAX5 Transcription Factor/biosynthesis , PAX5 Transcription Factor/genetics , Phenotype , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Syndecan-1/biosynthesis , Syndecan-1/genetics , Tumor Cells, Cultured/pathology , Tumor Cells, Cultured/virology , Virus Latency
16.
Ann Diagn Pathol ; 13(2): 119-23, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19302961

ABSTRACT

A 40-year-old woman presented with a rapidly enlarging palpable thyroid mass. The patient underwent a total thyroidectomy. The tumor fulfilled the criteria of primary solitary extramedullary plasmacytoma (SEP), including cellular expression of the CD138 and lambda light chain antibodies. Solitary extramedullary plasmacytoma of the thyroid occurs most commonly in patients with Hashimoto thyroiditis and must be distinguished from involvement of thyroid in multiple myeloma, inflammatory pseudotumor plasma cell variant, mucosa-associated lymphoid tissue lymphoma, and medullary carcinoma. The distinction is determined on the basis of histologic findings, immunohistochemical analysis, and other laboratory tests. Currently, no standard treatment exists for this entity. In this report, we discuss the differential diagnosis of SEP of the thyroid and the clinical features observed in this case.


Subject(s)
Plasmacytoma/pathology , Thyroid Neoplasms/pathology , Adult , Diagnosis, Differential , Female , Humans , Immunoglobulin lambda-Chains/biosynthesis , Immunohistochemistry , Lymphoma/pathology , Multiple Myeloma/pathology , Plasmacytoma/metabolism , Syndecan-1/biosynthesis , Thyroid Neoplasms/metabolism
17.
J Immunol ; 182(6): 3583-96, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19265137

ABSTRACT

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJkappa transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the lambda5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be lambda5 independent. We suggest that the unique CDR3 region of the tg-encoded muH chain is responsible for the lambda5 independence of tg-expressing B cells.


Subject(s)
B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Gene Rearrangement, B-Lymphocyte, Heavy Chain/genetics , Immunoglobulin Light Chains, Surrogate/genetics , RNA Editing/immunology , Animals , B-Lymphocyte Subsets/cytology , Cell Differentiation/genetics , Cell Differentiation/immunology , Complementarity Determining Regions/biosynthesis , Complementarity Determining Regions/genetics , Immunoglobulin Light Chains, Surrogate/biosynthesis , Immunoglobulin Light Chains, Surrogate/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Immunoglobulin mu-Chains/biosynthesis , Immunoglobulin mu-Chains/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , Mice, Transgenic , RNA Editing/genetics
18.
J Immunol ; 182(3): 1667-73, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-19155516

ABSTRACT

Multiple myeloma is an incurable plasma cell malignancy. Immunotherapy in myeloma patients had limited success to date. We have previously demonstrated that dendritic cells (DCs) pulsed with autologous Ig Id induced Id-reactive CD8(+) T cells and protection against a myeloma tumor challenge. In this work, we studied the therapeutic efficacy of chemotherapy combined with different formulations of DC-based vaccines in mice bearing large plasma cell tumors. The comparative study demonstrated that s.c. injection of DCs loaded with Id coupled to keyhole limpet hemocyanin, s.c. injection of DCs loaded with irradiated tumor cells, and intratumoral injection of naive DCs were similarly effective in mediating tumor regression and long-term survival. However, whereas the Id-keyhole limpet hemocyanin-DC vaccine was inefficient against myeloma cells that lost expression of the Ig H chain, intratumoral injection of naive DCs and s.c. injection of DCs loaded with irradiated tumor cells were highly effective against cells producing L chains only. This may be of particular importance for patients with L chain myeloma. Given that T cells respond primarily to peptides derived from H chain CDRs, attempts to treat L chain disease with myeloma protein-pulsed DCs may be futile. Vaccination with tumor cell-loaded DCs may, however, induce an effective antitumor response.


Subject(s)
Cancer Vaccines/administration & dosage , Dendritic Cells/transplantation , Immunoglobulin lambda-Chains/biosynthesis , Immunotherapy, Adoptive , Multiple Myeloma/immunology , Multiple Myeloma/prevention & control , Plasmacytoma/immunology , Plasmacytoma/prevention & control , Animals , Antibodies, Anti-Idiotypic/administration & dosage , Antibodies, Anti-Idiotypic/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Cyclophosphamide/administration & dosage , Dendritic Cells/immunology , Female , Hemocyanins/administration & dosage , Hemocyanins/immunology , Humans , Immunoglobulin lambda-Chains/metabolism , Injections, Intralesional , Injections, Subcutaneous , Mice , Mice, Inbred BALB C , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Plasmacytoma/drug therapy , Plasmacytoma/pathology
19.
J Immunol ; 182(1): 408-15, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19109172

ABSTRACT

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, activation-induced deaminase. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged Ig lambda(R) gene in the chicken DT40 B cell line, which performs constitutive Ig gene diversification. By analysis of a DT40 derivative in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that E2A must function in cis to promote diversification and that stimulation of diversification in cis depends on the E2A activation domains. By direct imaging, we show that lambda(R)/E2A colocalizations are most prominent in G(1). We further show that expression of the E2A antagonist Id1 prevents lambda(R)/E2A colocalizations in G(1) and impairs diversification but not transcription of lambda(R). Thus, E2A acts in cis to promote Ig gene diversification, and G(1) phase is the critical window for E2A action.


Subject(s)
Antibody Diversity/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , E-Box Elements/genetics , G1 Phase/genetics , Gene Rearrangement, B-Lymphocyte, Light Chain , Genes, Immunoglobulin , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Cell Line, Tumor , Chickens , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Inhibitor of Differentiation Proteins/biosynthesis , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/physiology , Isopropyl Thiogalactoside/analogs & derivatives , Isopropyl Thiogalactoside/physiology , TCF Transcription Factors/biosynthesis , TCF Transcription Factors/genetics , Transcription Factor 7-Like 1 Protein
20.
J Immunol ; 181(6): 4098-106, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18768866

ABSTRACT

The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.


Subject(s)
B-Lymphocyte Subsets/immunology , Cell Differentiation/immunology , Gene Rearrangement, B-Lymphocyte, Light Chain , Immunoglobulin Heavy Chains/physiology , Immunoglobulin Light Chains, Surrogate/physiology , Immunoglobulin kappa-Chains/physiology , Immunoglobulin lambda-Chains/physiology , Stem Cells/immunology , Alleles , Animals , B-Lymphocyte Subsets/metabolism , Cell Differentiation/genetics , Cell Line , Cell Line, Transformed , Cells, Cultured , Humans , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains, Surrogate/biosynthesis , Immunoglobulin Light Chains, Surrogate/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Mice , Mice, Knockout , Mutagenesis , Receptors, Antigen, B-Cell/biosynthesis , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/physiology , Stem Cells/metabolism
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