In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: pathogenic autoreactivity requires permissive light chains

Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.     

Evidence by radioimmunoassay that mitogen-activated human blood mononuclear cells secrete significant amounts of light chain Ig unassociated with heavy chain

The culture supernatant (CS) Ig of PWM-activated human blood mononuclear cells was quantitatively determined using a panel of nanogram-sensitive radioimmunoassays (RIAs) that separately measured IgG, IgM, IgA, total k Ig, and total λ Ig. After initial RIA quantitation, separate CS aliquots were exposed to either a polyisotypic anti-heavy (H) chain or a nonimmune IgG solid-phase immunoabsorbent, and then reassayed for Ig content. The reassay results revealed that the anti-H chain-absorbed CS aliquots retained significant amounts of k and λ Ig, but yet had a virtual absence of isotypic IgG, IgM, and IgA. Comparisons of the absorbed CS aliquots suggested that as much as one-fourth to one-third of the total secreted L chain Ig in PWM-activated cultures lacked RIA-detectable associated H chain. This unexpected finding of significant amounts of unbound L chain in mitogen-stimulated cultures raises important theoretical issues relative to the functional role of secreted free L chain and the prospects that free L chain levels may represent useful quantitative markers of B-cell stimulation.     

B cell maturation in the chicken Harderian gland

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.     

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.     

Human B cells secrete predominantly lambda L chains in the absence of H chain expression

Ig H and L chains are independently assembled in B cells and then secreted together as a functional protein. H chains cannot be secreted without assembly to L chains; however, L chains can be secreted in the absence of H chains by both mice and human cells. To examine the influence of H chain expression on human L chain isotype selection (kappa or lambda), we compared the kappa/lambda ratio of L chains unassociated with H chains (free L chains) to the kappa/lambda ratio of L chains associated with H chains. Culture supernatants of human splenocytes were assayed for kappa and lambda L chains. Free L chains were the predominant form of L chains detected in unstimulated cultures, accounting for 68 to 70% of the total. This was in contrast to the minor proportion that free L chains represented (less than 20%) in cultures stimulated with PWM or LPS (p less than 0.01). Furthermore, the kappa/lambda ratio of light chains detected in unstimulated cultures was 0.5 as compared to 1.3 for PWM stimulated cultures (p = 0.0001). To demonstrate that the decreased kappa/lambda ratio of L chains in the supernatants of cultures of unstimulated B cells was due to free L chains, we measured the kappa/lambda ratio of IgG and IgM-associated L chains. In both the stimulated and unstimulated cultures, the kappa/lambda ratio of L chains associated with H chains was greater than the ratio determined for free L chains. Free L chains were shown to be predominantly lambda as compared to the predominantly kappa phenotype of L chains associated with H chains. Thus absence of H chain expression affects selection of L chain isotypes secreted by human B cells.     

DNA rearrangements affecting both variable and constant regions of Ig H chain genes in MPC11 mouse myeloma variants

We have examined the mechanisms that account for short Ig H chain production in two variants of the mouse myeloma cell line MPC11 (IgG2b, kappa) by mRNA sequencing and restriction enzyme mapping. One variant, F5.5, has a thymidine residue inserted into the (CH3) domain, of the Ig H chain, resulting in premature termination and translation of a gamma 2b H chain of 50,000 m.w. A second variant, E5.7A12, contains gamma 2a-derived sequences that extend from near the 3' end of the CH2 domain to the intervening sequence between the CH2 and CH3 domains, consistent with a microrecombination event (defined as either a double cross-over or gene conversion event). In this variant, the 5' end of the CH3 domain has been deleted, but the remainder of the gamma 2b(CH3) domain is present, resulting in the translation of a gamma 2b-gamma 2a-gamma 2b H chain of 52,000 m.w. Additional rearrangements affecting sequences in or adjacent to the variable region accompany H chain constant region alterations in these cell lines and subclones of these cell lines. In F5.5, novel sequences have recombined within one of two duplicated copies of the VH gene. In a sister clone of E5.7A12 that has ceased H chain production (E5.7A14), new sequences have recombined within 300 bp 5' of the enhancer element. Both F5.5 and E5.7A12, like their immediate unstable precursor cells, fail to assemble H-H dimers, halting the Ig assembly process at the heavy-light stage, and do not secrete H chains. We speculate that defects in H chain assembly and secretion, as exemplified by the parents of these variants (i.e., intermediates of these secondary variants), reactivate the Ig gene rearrangement machinery and result in the formation of these putatively equally unstable secondary variants.     

B cell abnormalities induced by a mu Ig transgene extend to L chain isotype usage

We have analyzed the phenotype of B cell populations from mice transgenic for a rearranged Ig mu H chain gene. We find a decrease in the number of B cells in the spleens of these mice. Transgenic B cells have decreased surface levels of both IgM and IgD. The circulating IgM in these mice is 3- to 10-fold enriched in lambda L chains, compared with that in non-transgenic mice. Analysis of IgM-producing hybridomas, from transgenic mice that express the transgene at high levels, demonstrates that this higher lambda frequency is observed in transgene-nonexpressing as well as transgene-expressing hybridomas. A partial loss of L chain isotype exclusion is also noted in these hybridomas, and a significant proportion of primary B cells expressing both kappa and lambda L chains on their surface can be demonstrated. These findings suggest an ability of the transgenic Ig H chain to affect events in B cell ontogeny beyond the H chain locus. Our results support a quantitative model of exclusion for both the H chain alleles and the L chain isotypes.     

DNase I sensitivity of immunoglobulin light chain genes in Abelson murine leukemia virus transformed pre-B cell lines.

We have used Abelson murine leukemia virus (A-MuLV) transformed pre-B cell lines to test the hypothesis that the rearrangement potential of a developing B-lymphocyte is dependent on an "opening" of the chromatin structure surrounding immunoglobulin (Ig) genes, thus allowing accessibility to an Ig gene recombinase. The chromatin structures surrounding heavy (H), kappa (kappa), and lambda (lambda) chain constant-region genes were assessed by DNase I sensitivity in A-MuLV transformed cell lines capable of H, kappa or lambda gene rearrangement. Our results indicate that DNase I-sensitive chromatin structures of these Ig constant-region genes correlate closely with the ability of the genes to undergo recombination. We also find that the chromatin structure of an Ig constant-region locus becomes DNase I sensitive before any DNA rearrangement events occur.     

Activity of multiple light chain genes in murine myeloma cells producing a single, functional light chain

Two cloned lambda 1-producing myelomas (HOPC-1, MOPC-104E) contain rearranged kappa genes and levels of mature-sized kappa RNA comparable to those found in kappa-producing myeloma cells. Another lambda 1-producing myeloma tumor line (HOPC-2020) and a lambda 1-containing B cell leukemia line (BCL1) also contain significant levels of kappa RNA. One lambda 11-producing line (MOPC-315) contains no detectable kappa RNA, but it also has no kappa genes in the embryonic configuration. kappa-related proteins are not detectable in the lambda 1-producing lines by standard procedures, but by sensitive methods at least two lines contain kappa protein fragments. The MOPC-104E line produces both a 14.5K kappa fragment that is not readily detectable because of its low rate of synthesis and short half-life (T 1/2 less than 5 min), and a major 16.5K protein that lacks kappa cross reactivity but is demonstrable by translation of purified MOPC-104E kappa RNA. The HOPC-1 kappa RNA also encodes a short-lived 14K kappa fragment. The MPC-11 line, which produces a mature kappa RNA and protein as well as an 800 base kappa fragment RNA and kappa protein fragment, has both kappa alleles rearranged, one apparently aberrantly between J and C kappa. Two different kappa RNA species, one the same size as the MPC-11 kappa fragment RNA, frequently are present in kappa RNA-containing Abelson murine leukemia virus-transformed lymphoid cells as well as in 18 and 19 day murine fetal liver. For light chains, neither allelic nor isotype exclusion is generally evident in myeloma and lymphoma cells; rather both produce only a single functional light chain. Models of light chain activation must explain restriction by considering the functional properties of the light chain rather than light chain gene expression.     

Idiotype variants emerging after anti-idiotype monoclonal antibody therapy of a murine B cell lymphoma

One of the difficulties encountered with the treatment of human B cell malignancies with anti-Id antibodies is the emergence of Id variants. The current study was designed to investigate this phenomenon further by using the murine B cell lymphoma model 38C13. Tumors were harvested that developed despite treatment with the anti-Id antibody S1C5 in mice inoculated with 38C13 cells and evaluated by immunofluorescence. Various phenotypes were found among escaping tumor cells. Some cells continued to react with S1C5 whereas others lost S1C5 reactivity. Among these latter cells, some continued to express surface IgM kappa, whereas others no longer expressed surface mu or kappa. After Id variant cell lines were established, immunofluorescence and ELISA of cell lysates from the surface IgM kappa- lines revealed persistent intracellular mu H chain but no detectable kappa. Surface IgM kappa+ lines were fused with myeloma cells and the Ig proteins secreted by the resultant hybridomas analyzed. The apparent m.w. of the mu-chains of these rescued Ig was the same as wild-type 38C13, whereas the kappa-chains were either the same or different in m.w. from the wild type. The IgM kappa of the variant line, T3C, weakly reacted with S1C5 and did not react with other anti-Id antibodies. The IgM kappa of the other variants were nonreactive with all the antibodies. Immunofluorescence of these surface Ig+ variants confirmed this finding. Some of the surface Ig+ and Ig- variant lines grew identically to wild-type tumor in vivo, but only the weakly S1C5-reactive variant T3C was inhibited in its growth by S1C5. Moreover, T3C was the only one of these lines capable of being lysed in vitro with S1C5 by antibody-dependent cellular cytotoxicity. Further studies revealed that surface Ig+ and Ig- variants emerge in escaping tumors with similar frequency and that these variants represent a major mode of tumor escape from anti-Id treatment in this model.     

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.     

Dissociation of κ- and λ-chains from reduced human immunoglobulins

1. The light chains of human immunoglobulin (Ig) exist in two forms, kappa (type K) and lambda (type L). The two types of chains can be partially separated by taking advantage of the fact that lambda-chains, for the most part, dissociate from reduced Ig at higher pH than do the kappa-chains. The same difference in dissociation of type K and L chains was observed with myeloma IgG and IgA proteins, but not with pathological IgM proteins. 2. When analysed in urea-glycine starch gels, pH7, both kappa- and lambda-chains show ten electrophoretic bands having the same mobilities as those of the whole light-chain subfractions. Normal kappa- and lambda-chains show similar differences in overall amino acid composition to those previously found with myeloma kappa- and lambda-chains and type K and L Bence-Jones proteins.     

Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease

The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells.     

Shared N-terminal sequences in monclonal IgMkappa and IgGkappa proteins from a patient with a complex multiple paraprotein disorder.

The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgM kappa and IgG kappa paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the kappa chains of the IgM and IgG were identical and belonged to the human V kappa III subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective mu and gamma heavy chains were also identical and belonged to the human VHIII subgroup. These identical VH sequences were unique with lysine residues at positions 13 and 19. This dual lysine substitution has not been seen in 37 other human VHIII sequences reported in the literature. This N-terminal sequence homology in the V-regions of Cam IgM kappa and IgG kappa paraproteins and the shared idiotypy expressed by Cam IgM, IgG, and IgA proteins strongly suggest the existence of complete structural homology in the variable regions of the and L chains of these Ig molecules of three separate Ig classes. At the cellular and genetic level, these results point toward a common clonal origin for the idiotypically related Ig molecules and suggest that identical V-region (VH and VL) genes were utilized by the Cam lymphoid clone in the biosynthesis of the respective IgM, IgC, and IgA proteins.     

Assessing the efficiency of free light chain assay in monitoring patients with multiple myeloma before and after autologous stem cell transplantation along with serum protein electrophoresis and serum protein immunofixation

Monoclonal gammopathies are a group of disorders, referred to as paraproteinaemias, dysproteinaemias or immunoglobulinopathies, associated with monoclonal proliferation of plasma cells. Monoclonal immunoglobulin secreted by these cells is an indicator of clonal proliferation. The aim of this study is to analyze the efficiency of three methods: serum protein electrophoresis (SPE), serum protein immunofixation (IFE) and FLC (free light chain) assay for the diagnosis and monitoring of the tumor burden in multiple myeloma. In this study we have presented the dynamic evolution of 7 patients with intact immunoglobulin multiple myeloma (IIMM) (2 IgG, kapa; 3 IgG, lambda; 1 IgA, kappa; 1 IgA, lambda) and 2 patients with light chain multiple myeloma before and after autologous peripheral blood stem cell transplantation (PBSCT). All 7 patients fulfilled the four criteria for the diagnosis of IIMM: bone marrow plasma cells exceeding 20%, lytic bone lesions, identification and quantification of M protein by scanning densitometry of electrophoresis gels, IFE (immunofixation protein electrophoresis) confirmed and typed the M protein. All patients had been given cytotoxic chemotherapy (VAD or VELCADE) before autologous (PBSCT). In two of the patients with IIMM both SPE and kappa/lambda ratio fell towards normal range after autologous PBSC and both reported a relapse of the disease after 23 months and 19 months respectively. SPE could not normalize after chemotherapy and transplantation in three patients with IIMM, the kappa/lambda ratio being the only marker used to monitor the tumor kill. In one patient the kappa/lambda ratio could not normalize even after PBSCT still indicating the presence of plasma cell disorder at the time when IFE was still negative. 16 months after PBSCT both SPE and FLC indicated a relapse of the disease. Classical SPE failed to demonstrate the presence of M-protein in light chain multiple myeloma, the diagnosis being established by using IFE and the FLC assay. Because IFE is a qualitative method and its interpretation may be sometimes subjective, FLC was the only method used to follow the disease course. The measurement of kappa/lambda ratio proved to be more sensitive than SPE, IFE and the levels of free light chains kappa or lambda individually indicating whether the treatment is effective or not.     

Immunoglobulin superantigen protein L induces IL-4 and IL-13 secretion from human Fc epsilon RI+ cells through interaction with the kappa light chains of IgE

Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1-B5) that interact with the variable domain of Ig kappa L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (r(s) = 0.82; p < 0.001) between the maximal percent IL-4 release induced by protein L and that induced by anti-IgE and between intact protein L and the B1-B4 fragment (r(s) = 0.90; p < 0.01). Removal of IgE bound to basophils markedly reduced the IL-4 release induced by anti-IgE, protein L, and B1-B4. Preincubation of basophils with protein L or anti-IgE caused complete cross-desensitization to subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda chains) blocked anti-IgE-induced IL-4 release, but not the releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with kappa L chains of the IgE isotype.     

Protein L. A novel bacterial cell wall protein with affinity for Ig L chains

A novel Ig-binding protein has been isolated from the surface of bacteria belonging to the anaerobic species Peptococcus magnus. To solubilize the protein, peptococci were treated with different proteolytic enzymes (papain, pepsin, and trypsin) or with mutanolysin, a bacteriolytic agent known to digest the cell walls of streptococci. Papain, trypsin, and mutanolysin all solubilized peptides showing affinity for radiolabeled human IgG in Western blot analysis. Compared with papain and trypsin, mutanolysin liberated a more homogeneous material, which also had a higher m.w. This mutanolysin-solubilized protein (Mr 95 kDa) was obtained highly purified by a single isolation step on IgG-Sepharose, and the molecule was found to exhibit unique Ig-binding properties. Thus, in dot blots and in Western blots, human IgG, F(ab')2 and Fab fragments of IgG, and human kappa and lambda L chains all showed affinity for the protein. Moreover, the molecule also bound human IgM and IgA, whereas no binding was recorded for IgG-Fc fragments or IgG H chains. Finally, the protein bound to human polyclonal Ig L chains immobilized on polyacrylamide beads. These different data demonstrate that the isolated peptococcal protein binds Ig through L chain interaction. The name protein L is therefore suggested for this novel Ig-binding bacterial cell wall protein.