Serologically defined V region subgroups of human lambda light chains

The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease.     

Antibody domain mutants demonstrate autonomy of the antigen binding site.

We have constructed derivatives of a lambda I light chain-bearing anti-(4-hydroxy,3-nitrophenyl)acetyl (NP) antibody which have the V regions exchanged between heavy chains and light chains of the kappa or lambda I type. These antibodies are assembled and secreted normally, and bind haptenic and macromolecular ligands like the wild-type; similar results are obtained for monovalent heterodimers of VHCL and lambda I light chains. The observed independence of the binding site from the constant region context argues against a role of longitudinal interactions between constant and variable domains in antigen recognition, and therefore against cooperativity between binding sites.     

Primary structure of the variable region of a human lambda VI light chain: Bence Jones protein SUT

To ascertain if lambda VI light chains have unique structural features that account for the preferential association of these proteins with primary or multiple myeloma-related amyloidosis (amyloidosis AL) we have determined the complete amino acid sequence of the variable (V) region of the lambda VI Bence Jones protein SUT. This protein, obtained from a patient with amyloidosis AL, represents a complete light chain consisting of 216 residues and it has structural and serologic properties characteristic for lambda VI light chains. The sequence of the joining segment (J) (positions 100 to 111) of protein SUT is identical to that of the J lambda I segment of the mouse IG lambda light chain gene. V region SUT is closely homologous in sequence to that of another lambda VI amyloid fibrillar protein, AR, differing by 21 residues. The V regions of proteins SUT and AR contain a two-residue insertion at positions 68 and 69 that has also been found in two other lambda VI human light chains but not in the lambda-chains of other V region subgroups.     

Immunoglobulin light and heavy chain isotypes in skin diseases: restricted distribution in bullous pemphigoid and linear IgA bullous dermatosis

To assess the heterogeneity of immunoglobulins involved in various skin diseases, direct and indirect immunofluorescence studies of skin biopsies and sera, respectively, for kappa and lambda light chains, were performed. The anti-basement membrane zone (anti-BMZ) antibodies of patients with bullous pemphigoid showed a predominance of kappa light chains, and patients with linear IgA bullous dermatosis showed a predominance of one light chain that was sometimes kappa and sometimes lambda. The bullous pemphigoid autoantibodies were then studied for IgG subclass distribution; a predominance of IgG4 was found. Although other explanations are possible, the light chain restriction in bullous pemphigoid most likely reflects heavy chain restriction and preferential association of heavy and light chain isotypes. The basis of the heavy chain restriction is not apparent. The light chain restriction in linear IgA bullous dermatosis may represent a restricted idiotypic repertoire.     

Determination of the subclasses of monoclonal human immunoglobulins G and of the light chain type with the aid of specific antisera]

Sera of 86 patients suffering from G-myeloma were studied for the purpose of determination of subclasses of monoclone IgG. Investigations were carried out by means of antisera to subclasses IgG by the double diffusion method in gel after Ouchterlony. The following distribution of myeloma Ig was revealed: G1--70%, G2--17%, G3--11%,and G4--2%. In typing of the light igG chains by the method of immunoelectrophoresis, using antisera to the light chains of immunoglobulins of the chi and lambda type it was found that IgG1 chi was encountered more frequently than IgG1 lambda (3:1 ratio). The amount of the sera with the IgG2, IgG3, and IgG4 was insufficient for the reliable conclusion of their distribution by the type of light chains.     

Thermodynamic instability of human lambda 6 light chains: correlation with fibrillogenicity.

Certain types of human light chains have the propensity to deposit pathologically as amyloid fibrils as evidenced by the preferential association of monoclonal lambda 6 proteins with AL amyloidosis. However, the molecular features that render such proteins amyloidogenic have not been elucidated. Based upon the demonstrated relationship between the thermodynamic stability of light chains and their propensity to aggregate in vitro, we have initiated studies where the thermodynamic properties and fibrillogenic potential of two recombinant (r) V lambda 6 molecules were compared. The first protein was generated from cDNA cloned from marrow-derived plasma cells from a patient (Wil) who had AL amyloidosis and renal amyloid deposits; the second was from a patient (Jto) with multiple myeloma in whom the lambda 6 protein was deposited not as amyloid but in the form of renal tubular casts. The thermodynamic stabilities of rV lambda 6Wil and -Jto were determined from chaotropic and thermal denaturation studies. Based upon the Delta GH2O, Delta H, Delta G25 degrees C, Tm, and Cm values, the rV lambda 6Wil was less stable than its nonamyloidogenic counterpart, rV lambda 6Jto. Measurement of fibril formation using a novel in vitro fibril forming assay demonstrated that although both rV lambda 6 proteins formed fibrils in vitro, Wil had a shorter lag time and exhibited faster kinetics under physiologic conditions. Comparative amino acid sequence analyses of these two components and other lambda 6 amyloid-associated light chains revealed that the Jto protein had certain primary structural features that we posit contributed to its increased stability and thus rendered this protein nonamyloidogenic. Our studies provide the first evidence that stabilizing interactions within the V L domain can influence the kinetics of light chain fibrillogenicity.     

Steroid-carrier proteins in patients with multiple myeloma

Patients with multiple myeloma have transcortin levels lower than normal. This is due in essence to a subgroup of patients producing IGG heavy chains with lambda light chains. Patients producing IGG with predominantly kappa light chains have almost normal transcortin levels. On the other hand, the binding activity of the steroid binding beta globulin (SB beta G) of the kappa type of multiple myeloma is significantly higher than the steroid binding of the lambda type of multiple myeloma. The serum levels of vitamin D binding protein (DBP) fall in the normal range.     

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.     

Localization of monoclonal B-cell populations through the use of Komogorov-Smirnov D-value and reduced chi-square contours

In this study three different flow cytometric analysis techniques are woven together into a single system that permits improved detection of small percentages of monoclonal B cells in a milieu of either normal blood leukocytes or bone marrow cells. This analysis is an extension of the concept of clonal excess, which is used to detect the presence of a tumor that is a clonal expansion of B cells expressing either kappa or lambda light chains. The technique also utilizes "multiple listmode processing," which is defined in this context as the simultaneous analysis of two or more listmode files that share one or more common parameters. This type of data structure enables the segmentation of two parameter light scatter displays into regions from which numerous kappa and lambda histograms subsequently can be analyzed for their respective Komogorov-Smirnov D-values or R-values (reduced chi-square value). The final technique makes use of a calculated parameter display system. Superimposed on the light scatter dot density plot are D-value or R-value contours. The contours target the location of the population that is abnormal, thus providing information for setting optimal bitmap gates for clonal excess studies, other phenotypic analyses, or cell sorting. In experiments using model systems, the sensitivity of this assay is estimated to be between 0.25% and 2.5%. The technique's distribution information and sensitivity may prove useful for staging, treatment monitoring, and relapse detection of B-cell leukemia and lymphoma. This application illustrates the potential of combining multiple listmode processing and calculated parameter display to expand the effective dimensionality of listmode data.     

Expression of immunoglobulin lambda light chain by the promyelocytic cell line HL-60

The HL-60 cell line, established from a patient with acute promyelocytic leukemia, can be induced to undergo differentiation along the granulocyte or monocyte/macrophage line, depending on the particular inducer that is used. In this communication we provide evidence that HL-60 cells also have B lymphoid characteristics because by flow cytometry and clonal excess calculations, these cells are found to express immunoglobulin (Ig) lambda light chains on their surface. Furthermore, HL-60 cells contain poly(A)+ RNA that hybridizes with a DNA fragment encoding the constant region of Ig lambda chains and comigrates with lambda mRNA on RNA blots. Treatment of HL-60 cells with a phorbol ester that induces monocyte/macrophage differentiation resulted in the loss of surface Ig lambda chains and lambda RNA.     

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.     

Value of lymphocyte marker studies in diagnostic cytopathology

The separation of lymphoreticular neoplasms from poorly differentiated epithelial and mesenchymal tumors and reactive processes can be a difficult problem in cytopathology. Immunodiagnostic techniques can be applied to cytologic specimens to detect cellular antigens, which may aid in their proper identification. We have reviewed 67 cytologic specimens in which immunoperoxidase techniques were employed using antibodies to common leukocyte antigen (HLE1), B-cell markers (B1, Leu 12 and kappa and lambda light chains), T-cell markers (Leu 1, OKT11, Leu 12 and kappa and lambda light chains), T-cell markers (Leu 1, OKT11, OKT4 and OKT8) and monocytes (OKM1 and LeuM1). These specimens included 33 body cavity fluids (21 pleural, 8 ascitic and 4 pericardial), 22 cerebrospinal fluids (CSF) and 12 fine needle aspirates (4 brain, 1 adrenal, 2 liver, 1 kidney, 3 retroperitoneal masses and 1 lymph node). The marker studies confirmed the initial cytomorphologic diagnoses in 31 specimens and modified the final diagnoses in 16 specimens. Markers in 20 specimens were noncontributory due to low cellularity or technical difficulties. Two problems may limit the usefulness of these procedures. First, many of the CSF specimens contained too few cells for adequate processing. Second, the mesothelial cells from pleural specimens often stained with HLE1. Our findings indicate that marker studies are of value in the diagnosis of problematic cases presenting as undifferentiated tumors in cytopathology.     

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.     

Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells

The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive.     

Antibody elbow angles are influenced by their light chain class

We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195 degrees ) elbow angles. This apparent hyperflexibility of lambda chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is described.     

Primary structure of the variable region of an amyloidogenic Bence Jones protein NIG-77

The complete amino acid sequence of the variable region of a Bence Jones protein NIG-77 from an individual with myeloma-associated systemic amyloidosis has been determined. This protein represents a complete light chain consisting of 216 residues and it has a sequence characteristic of V lambda I subgroup, which is closely homologous to that of another amyloidogenic V lambda I Bence Jones protein NIG-51, differing by 20 of 111 residues (82% homology). In contrast, it differs by 29 residues (74% homology) to that of non-amyloidogenic V lambda I light chain NIG-64. This finding shows that, in accordance with our previous report(1), the V lambda I-related light chains can further be divided into two distinct subsubgroups, V lambda I-1 and V lambda I-2, and the latter property seems to be more prone in association with the amyloid process.