Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing.

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.

Precursor B-cell lymphoblastic leukemia with surface immunoglobulin light chain expression in 2 chinese patients.

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.

Ig-free light chains play a crucial role in murine mast cell-dependent colitis and are associated with human inflammatory bowel diseases.

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.

E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification.

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.

Dendritic cell-based therapeutic vaccination against myeloma: vaccine formulation determines efficacy against light chain myeloma.

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.

Two distinct populations of H chain-edited B cells show differential surrogate L chain dependence.

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.

Primary cutaneous marginal zone lymphoma with subclinical cutaneous involvement and biclonality.

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.

Induction of lambda 1-immunoglobulin is determined by a regulatory gene (r lambda 1) linked (or identical) to the structural (c lambda 1) gene.

The cis-acting gene regulating specifically the inducibility of lambda 1-bearing B cells has been mapped within 2.9 cM of the structural gene. If the lambda 1lo-phenotype is due to the gly leads to val interchange in C lambda 1, then an argument can be made that (a) the lambda 1lo-phenotype is due to inefficient induction of lambda 1lo-bearing B cells and (b) B cell triggering is dependent upon a conformational change in the Ig receptor upon interaction with antigen. If the lambda 1lo-phenotype is due to a regulatory sequence linked to the structural C lambda 1-gene, then it must control the expression of the lambda 1-locus during development into adulthood, e.g., by an effect on methylation.

Control of immunoglobulin secretion in the murine plasmacytoma line MOPC 315.

Cells of the 315LV-1 (derived from NP1) variant line of MOPC 315 contain approximately 1% the normal intracellular level of the heavy (alpha) chain of IgA and no detectable light (lambda2) chain. The synthesis rate of alpha-chain in the variant, however, is similar to that in cells of the parent line. Moreover the relative amount of translatable alpha-chain mRNA that can be extracted from 315LV-1 cells is about the same as for parental cells. No light-chain synthesis can be detected either in vivo or in vitro in a wheat germ cell-free system. The 315LV-1 heavy chain synthesized in vivo or in vitro has slightly greater electrophoretic mobility than normal H chain and turns over rapidly intracellularly. The variant fails to secrete any of its heavy chain, despite the fact that its H chain mRNA is bound to membranes, as one would expect for a secretory protein message. Fusion of 315LV-1 cells with cells of a kappa-producing MPC 11 variant line leads to stabilization of the intracellular H chain and also to full recovery of secretion of the H chain as an H2L2 molecule.

Antibody response to the streptococcal group A-variant polysaccharide in BASILEA rabbits lacking kappa-polypeptide chains.

Rabbits from a variant strain called BASILEA, in which homozygotes express only lambda-type chains and heterozygotes have normal kappa/lambda ratios, were hyperimmunized with a streptococcal group A variant vaccine. Homozygotes (bas/bas) produced antibodies with lambda-chains, heterozygotes, however, produced predominantly antibodies with kappa-chains. The incidence of restricted high responders in the BASILEA strain was high; it was probably introduced by the original mutant rather than by the loss of kappa-chains (founder effect). The degree of heterogeneity of homozygotes is similar to the heterogeneity of heterozygotes, and to that of rabbits expressing kappa-chains. This suggests that in the rabbit, the repertoire of lambda-chain genes is of similar size to that of kappa-chain genes.

A common idiotype on SJL and C57BL/6 anti-(4-hydroxy-3-nitrophenyl) acetyl antibodies and its relationship with lambda chain production.

Hybridoma cell lines secreting antibodies specific to (3-nitro-4-hydroxyphenyl) acetyl (NP) were generated by fusion of NP-immunized SJL spleen cells with the SP2/0 cell line. One hybridoma (N-hybridoma) anti-NP antibody (mu, lambda2) was found to partially inhibit (35-40%) the binding of the predominant idiotype in primary C57BL/6 anti-NP antibodies (NPb). Iodinated hybridoma antibody could be completely bound with anti-idiotypic antiserum made against either specifically purified C57BL/6 anti-NP antibodies, SJL anti-NP antibodies, or N-hybridoma antibody. The idiotypic specificities defined with anti-idiotypic antiserum made against N-hybridoma antibody were termed NP-1 idiotype. Strain distribution and genetic mapping studies indicate that the gene(s) controlling the production of NP-1 idiotype is closely associated with Igh-1b and Igh-1e alleles and mapped within the same chromosomal segment that controls the synthesis of NPb idiotype. However, unlike NPb idiotype, the expression of NP-1 idiotype is not influenced by the gene(s) that control lambda1 chain synthesis. Thus, SJL mice that produce low or undetectable levels of NPb idiotype due to a defect in lambda1 chain production express high levels of NP-1 idiotype. Specifically purified C57BL/6 and SJL anti-NP antibodies fully express NP-1 idiotype, the level of which correlates with the level of lambda2 chain-bearing molecules. Nonetheless, further experiments indicate that lambda1-bearing anti-NP antibodies can express extremely weak NP-1 idiotypic cross-reactivity.

The omega/lambda 5 surrogate immunoglobulin light chain is expressed on the surface of transitional B lymphocytes in murine bone marrow.

The membrane immunoglobulin heavy chain (micron) plays a feedback role during the pre-B stage of B lymphocyte differentiation. In pre-B cell lines, micron associates with two surrogate light chain proteins. The omega chain is disulfide linked to mu and was predicted to be the product of the lambda 5 gene. The iota chain is noncovalently associated with micron. We demonstrate that the omega protein is indeed the product of the lambda 5 gene and that mu, omega, and iota are coassociated in the same complex. Antibodies against the omega/lambda 5 protein demonstrate the existence of a subpopulation of "transitional" bone marrow B cells that express micron and omega on the cell surface. The majority of these cells also express surface kappa light chains, indicating that in B lymphoid ontogeny the lambda 5 gene is inactivated after the onset of kappa light chain expression.

Lack of mature B cells in nude mice with X-linked immune deficiency.

Mice were bred that simultaneously expressed the mutations nude and x-linked immune deficiency (xid). These doubly deficient animals had less than 10% of normal serum immunoglobulin levels. Their spleen cells did not respond to thymus-independent antigens in vitro nor did they respond to lipopolysaccharide. There was a virtual absence of cells with surface mu, kappa, or lambda 1, as detected by fluorescence. Sections of lymphoid organs revealed an absence of primary B cell follicles. Taken together, these results indicate a lack of mature B cells in nude xid mice. The possibility is considered that mature B cells belong to two subpopulations representing two lineages, one controlled by alleles at the xid locus and the other by alleles at the nude locus.

Genomic structure of the human Ig lambda 1 gene suggests that it may be expressed as an Ig lambda 14.1-like protein or as a canonical B cell Ig lambda light chain: implications for Ig lambda gene evolution.

In pre-B cells, immunoglobulin mu (Ig mu) is associated with pre-B cell-specific proteins to form a multimeric complex that is found on the cell surface. One of these proteins is encoded by the three exon Ig lambda-like gene 14.1, whose expression is restricted to pre-B cells and occurs from an unrearranged gene. A comparison of the 14.1 gene structure to the seven-gene human Ig lambda locus revealed that the most 5' gene, Ig lambda 1, is organized in a three-exon structure very similar to the 14.1 gene. Transcription and splicing of these three-exon sequences would lead to an mRNA with an open reading frame which could encode a light (L) chain-like protein with a molecular weight of 23,045. Our analysis suggests that two transcripts may be produced from the Ig lambda 1 gene that share the same Ig lambda 1 constant region-containing third exon. One transcript would include all three 14.1-related exons and be expressed from the germline gene, and the second transcript would be produced after variable-joining (V-J) recombination has occurred to Ig lambda J1 and would encode a classic Ig lambda L chain protein. The conservation of the genomic organization of the human 14.1 and Ig lambda 1 genes and the mouse homolog, lambda 5, relative to the classic Ig lambda L chain genes provides insight into the evolution of Ig genes.

Biologic and genetic characterization of the novel amyloidogenic lambda light chain-secreting human cell lines, ALMC-1 and ALMC-2.

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge, no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach, we established the genetic relationship between the cell lines and the primary patient cells, and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly, we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity, the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover, these cell lines will provide an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology.

Multiple levels of selection responsive to immunoglobulin light chain and heavy chain structures impede the development of Dmu-expressing B cells.

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.

The in vivo association of BiP with newly synthesized proteins is dependent on the rate and stability of folding and not simply on the presence of sequences that can bind to BiP.

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine lambdaI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as "kinetic traps." Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.

Clinical features and differential diagnoses of solitary extramedullary plasmacytoma of the thyroid: a case report.

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.