Immunofluorescent Evidence for Cellular Control of Synthesis of Variable Regions of Light and Heavy Chains of Immunoglobulins G and M by the Same Gene

Two distinct paraproteins (IgG(2)-K and IgM-K) from one patient shared identical light chains and significant portions of the variable regions of the heavy chains. Idiotypic determinants on the IgG and IgM molecules were shared. Earlier studies, using class-specific antisera, showed that these paraproteins were produced by two different populations of cells. The present study, using rhodamine and fluorescein conjugates of the anti-idiotype antisera, demonstrates that all plasma cells that contain immunoglobulin, whether IgG or IgM, stained with anti-idiotype antisera; this occurred irrespective of whether the antisera were made originally against the patient's IgG or IgM. This, plus previous data, indicates that both populations of cells share genetic information for the constant and variable regions of light chains and significant portions of the heavy-chain genes. These, and other cited data, strongly suggest the occurrence of a switch-over from IgM to IgG synthesis in the same cell during the course of the normal immune response.     

An unusual case of waldenström macroglobulinemia with half molecules of IgG in serum and urine

Summary Immunochemical studies are described in an unusual case of Waldenström's macroglobulinemia. Two monoclonal Igs (whole IgG₁/ and IgG₁/ half molecules) occurred in the serum in addition to the IgM monoclonal protein. Protein electrophoresis of the serum showed a monoclonal component in the region, and the immunoelectrophoresis allowed detection of a monoclonal IgM/ and another abnormality represented by a double precipitin line in serum and urine, observed when antiserum anti IgG was used. The abnormal proteins were purified and further analyzed. The IgG-related proteins were whole four chains IgG monoclonal molecules, 1/2 IgG monoclonal molecules, composed of one heavy and one light chain, and residual polyclonal IgG. The half molecules were antigenically deficient with respect to normal IgG. The idiotypic analysis showed that the three monoclonal proteins shared idiotypic determinants. This patient had clinical and morphological findings of Waldenström's macroglobulinemia and, as observed in other cases, the formation of half molecules was not associated with a distinct clinical syndrome.     

Demonstration of an idiotypic antigen on a monoclonal cold agglutinin and on its isolated heavy and light chains.

A potent anti-idiotype serum produced in a rabbit immunized with the isolated heavy chains of an IgM cold agglutinin "Col" was rendered specific by solid-state adsorptions. The anit-Col idiotype was shown to bind specifically to both isolated Col heavy (mu) and light (kappa) chains as well as to intact Col IgM by three methods: (i) reversal of anti-idiotype inhibition of Col cold agglutinin in an automated hemagglutination-inhibition assay system; (ii) adsorption of the anti-idiotype by affinity gels consisting of Col IgM, mu, or kappa chains covalently coupled to Sepharose 2B; (iii) binding of Col IgM and its isolated chains by an anti-idiotype affinity gel. Fragments of Col light chain lacking constant region determinants but still capable of inhibiting anti-idiotype were produced by limited pepsin digestion of the light chains. The finding of shared idiotypic determinants on isolated heavy and light chains of a monoclonal antibody suggests that these chains share a common sequence in a hypervariable region. As an extension of the gene insertion theory of Wu and Kabat, we postulate that genes coding for hypervariable regions may be available for insertion into the DNA for both heavy and light chains.     

Monoclonal anti-light chain idiotype as a tumor-specific probe for human neoplastic B lymphocytes

Tumor cells from patients with B cell neoplasms often secrete small amounts of free monoclonal light chains that can be found in the urine. Such tumor-derived light chains of the lambda type from a patient with typical chronic lymphocytic leukemia have been used to raise mouse monoclonal antibodies (MoAbs). A hybridoma-secreting antibody that recognized the idiotypic lambda chain but not normal lambda chains by a preliminary screen but which also reacted with idiotypic IgM from the patient's tumor cells was selected. This MoAb in fact recognized 1 in 20 X 10(3) molecules of pooled normal lambda chains, thus establishing its specificity for a private idiotypic determinant. It failed to give a detectable reaction with normal IgM, normal serum, or a panel of IgM paraproteins. The antibody bound to the patient's neoplastic B cells but not to normal tonsillar cells. The site of binding of the antibody to idiotypic IgM is clearly separate from that of another MoAb specific for idiotypic determinants on heavy plus light chains, since the two showed additive binding curves. The determinant also appeared to be less available in dimeric lambda chains than in monomeric lambda chains or in idiotypic IgM. Antibodies to idiotypic determinants on light chains show some technical advantages and should be useful for monitoring and possibly treating B cell tumors, either alone or together with the more conventional anti-idiotypic antibodies that usually recognize the heavy and light chain combination.     

Diclonal gammopathy (IgG kappa, IgA lambda) with nephrotic syndrome terminating into IgA lambda myeloma after three years--report of a case

A 43-year-old man was admitted to our hospital because of legs edema and periorbital edema in Dec. 1983. Laboratory findings showed massive proteinuria (3.7 g/day), Bence Jones protein (BJP) in urine, and hypoproteinemia. Peripheral blood examinations were normal and a bone marrow aspiration showed hypocellularity with slight increase of monocytes and plasma cells. Serum immunoelectrophoresis showed two M-components (IgG kappa, IgA lambda). Serum IgG was 1,690 mg/dl, IgA 379 mg/dl and IgM 160 mg/dl. No remarkable findings were obtained in bone survey, Ga-scintigraphy and rectal biopsy, and a diagnosis of diclonal gammopathy with nephrotic syndrome was made. In Aug. 1986, serum IgA started to increase rapidly with concomitant decrease IgG. He died of pneumonia due to pancytopenia in Dec. 1986, when serum IgG was 450 mg/dl, IgA 1,014 mg/dl, and IgM less than 39 mg/dl. Immunoelectrophoresis showed two M-components (IgG kappa, IgA lambda) in serum and BEP (kappa, lambda), IgG (kappa) and IgA (lambda) in urine. An autopsy showed massive infiltration of myeloma cells which were positive for lambda light chain in bone marrow, suggesting a development of myeloma from a diclonal gammopathy in about 3 years.     

Double myeloma. Production of both IgG type lambda and IgA type lambda myeloma proteins by a single plasma cell line

Monoclonal immunoglobulins G (IgG) and A (IgA) of the lambda light chain type were present in the serum of a patient with multiple myeloma. Three populations of myeloma cells were seen by immunofluorescence of bone marrow; those containing either IgG or IgA and those staining for both IgG and IgA. Idiotypic determinants present on the variable regions of the two myeloma proteins were immunologically identical and all the myeloma cells contained the same idiotypic determinant. The idiotypic specificity was related more closely to the variable region of the heavy chain than to the light chain. Structural and electrophoretic analysis confirmed that the light chains of the two myeloma proteins were identical. The myeloma in this patient appears to have arisen from a single clone of cells that was capable of synthesizing the constant portions of both the IgG and IgA heavy chain but only a single light and heavy chain variable region. These findings suggest that current concepts of antibody synthesis involving the sequential production of immunoglobulin M (IgM) and IgG antibodies may apply to certain cell lines synthesizing IgG and IgA antibodies as well.     

Genetic basis for the cross-reactive idiotypes on the light chains of human IgM anti-IgG autoantibodies.

The role of immunoglobulin structural genes in the generation of autoantibodies in humans has not been elucidated. Human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors, RFs) from unrelated people often share idiotypic antigens. Antibodies against synthetic peptides have localized two of the shared idiotypic determinants to the second and third complementarity-determining regions of the kappa light chain. The reported sequences of several human RF light chains are remarkably homologous in these regions. Animal studies have shown that some shared idiotypic antigens represent serological markers for immunoglobulin variable (V)-region genes. Therefore, we hypothesized that human RF light chains derived from a single germ-line gene, designated V kappa-(RF), or from a small family of very closely related genes. In the present experiments, we have isolated and sequenced two human V kappa germ-line genes that encode kappa light chains, which are identical or closely related to the light chains of human RF. The data indicate that the shared idiotypic antigens on RF are phenotypic markers for a kappa V-region gene that is highly conserved in the human population. The results also imply that the light chains of IgM anti-IgG autoantibodies can be encoded by germ-line genes without any somatic mutation.     

Diagnostic value of the concentration of M-component in initial classification of monoclonal gammopathy

The diagnostic value of the concentration of a serum M-component in initial classification of monoclonal gammopathy into malignant monoclonal gammopathy (MMG) and monoclonal gammopathy of undetermined significance (MGUS) was evaluated in 315 consecutive individuals with IgG, IgA or IgM type M-components. The final diagnosis was MMG in 84 and MGUS in 231 patients. Serum concentration was significantly highest in MMG, but for all 3 kinds of M-components, overlapping serum concentrations between MMG and MGUS were found. Highest efficiency was reached with a serum concentration of 30.2 g/l for IgG, 26.6 g/l for IgA and 22.7 g/l for IgM. Using these values, an initial correct classification was achieved in 0.90 of IgG M-components, in 0.91 of IgA M-components and in 0.79 of IgM M-components. Normal levels of uninvolved immunoglobulins were found to point strongly to MGUS.     

THE Ig CLASS AND LIGHT CHAIN TYPE OF THE LONG ACTING THYROID STIMULATOR

The Ig class and light chain type of LATS has been examined. Serum samples containing high levels of LATS were assayed before and after incubation with specific antisera to IgG, IgA and IgM, and kappa and lambda light chains. It was found that LATS activity was restricted to the IgG class of immunoglobulins and that the LATS activity was distributed approximately equally between the kappa and lambda type of IgG. These results are consistent with a polyclonal origin for LATS.     

Immunisation of guinea-pigs with circulating immune complexes from patients with rheumatoid arthritis.

Sixteen guinea-pigs were immunised with immune complexes isolated from serum of nine patients with rheumatoid arthritis. The resulting antisera were analysed by radioimmunoassays. All guinea-pig sera were extensively absorbed with normal human serum. After this absorption eight guinea-pig sera contained antibodies specific for immune complexes isolated from the sera of three patients. One of these antisera reacted not only with immune complexes (and serum) from the corresponding patient but also with immune complexes (and sera) from other patients with rheumatoid arthritis. The antigen(s) to which the guinea-pig antibodies were directed sedimented as IgM, and they bound to IgG Sepharose. Therefore the guinea-pig sera were absorbed with IgM-rheumatoid factors isolated from the serum of the corresponding patient. After this absorption, the guinea-pig sera had lost their reactivity with immune complexes. We conclude that these antisera did not detect an exogenous antigen in immune complexes from patients with rheumatoid arthritis. The positive reactions found were due to antibodies specific for (idiotypic?) antigenic determinants on IgM-rheumatoid factors.     

Normal maturation sequence of immunoglobulin light and heavy chains in hematogone stages 1, 2 and 3 in acute leukemia after treatment

The cellular diversity of bone marrow samples was studied by using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on acute leukemia blast cells and regeneration of normal B-cells, hematogones. This approach should enhance the ability to study normal hematopoiesis, and to identify and monitor hematopoietic disorders. The study was performed on a homogeneous group of patients (mainly children), all of them after finishing complete therapy for AL, mostly B-ALL. In all of these patients complete pattern of all three individual Hg stages was present. Maturation spectra of surface immunoglobulin kappa (sIgkappa) and lambda (sIglambda) light chains and IgM, IgA heavy chains in all three stages of Hgs are presented as reliable reports on sIgs as their incidence on Hgs are scarse and even contradictory. The Ig expression paralles CD20 expression. SIg of light (kappa,lambda) and heavy (IgM, IgA) chains were completally absent in stage 1 Hgs and their expression increased through stage 2 to 3; IgM was expressed similarly. Light Ig chains kappa/lambda were expressed in a polytypic way. The results completed information on normal maturation sequence of bone marrow stage 1, 2 and 3 hematogone regeneration in treated acute leukemia patients.     

A calcium binding IgG myeloma protein

A calcium binding IgG was isolated and purified by column chromatography from serum of a myeloma patient with asymptomatic hypercalcaemia. The myeloma IgG, characterized as an IgG kappa, revealed a normal sized heavy chain (56 000 dalton), and a light chain of 31 000 dalton. Another population of IgG separated and purified from the same patient's serum did not bind calcium and had a normal 26 000 dalton light chain. Calcium binding activity in vitro is optimal at pH 8.0, and reaches its maximum after 3 h of 45Ca myeloma IgG incubation. Cleavage of the purified IgG by trypsin yielded peptides which were further isolated by column chromatography and characterized as Fab and Fc fragments. Light and heavy chains were obtained by reacting the immunoglobulin with dithiothreitol and iodoacetamide followed by Sephadex G-100 chromatography. Calcium binding activity was proved to be associated with Fab IgG fragment. Preparates containing Fc, heavy or light chains did not bind calcium in vitro.     

Index of light kappa/lambda and lambda/kappa chains in monoclonal gammopathies

Concentrations of the light chains kappa and lambda were determined by simple radial immunodiffusion in the blood sera of 437 patients with monoclonal gammopathies. The kappa/lambda index was calculated in monoclonal gammopathies with the antigenic type of kappa light chains, while in monoclonal gammopathies with the antigenic type of lambda light chains the lambda/kappa index was calculated. The results obtained in malignant monoclonal gammopathies were compared with the results obtained in monoclonal gammopathies of undetermined significance for IgG and IgM paraproteinemias. Differences of high statistical significance were established (for IgG and IgA p less than 0.001, for IgM p less than 0.005) and thus the light-chain index can be used as another marker in differential diagnosis of monoclonal gammopathies.     

Complete amino acid sequence of variable domains from two monoclonal human anti-gamma globulins of the Wa cross-idiotypic group: suggestion that the J segments are involved in the structural correlate of the idiotype.

The complete amino acid sequences of the variable regions of two human IgM anti-gamma globulins are reported. The two proteins, Sie and Wol, share idiotypic determinants that are distinct from those shared by the two previously sequenced IgM anti-gamma globulins, Lay and Pom [Capra, J. D. & Kehoe, J. M. (1974) Proc. Natl. Acad. Sci. USA 71, 4032-4036; Klapper, D. G. & Capra, J. D. (1976) Ann. Immunol. (Inst. Pasteur) 127C, 261-271.] In contrast to Lay and Pom, Sie and Wol have very little homology in the complementarity-determining hypervariable regions of either their light or heavy polypeptide chains. However, there are only two amino acid differences in the framework portions of the light chains, and the J segments of both chains are homologous. These data suggest that the predominant idiotypic determinant in this cross-idiotypic system may be different than in the Po group and may be either related to their light chain framework structures or to the amino acid sequence in the J segments of these molecules.     

EVIDENCE FOR MULTIPLE GENE CONTROL OF A SINGLE POLYPEPTIDE CHAIN: THE HEAVY CHAIN OF RABBIT IMMUNOGLOBULIN

To determine the chemical basis for rabbit heavy chain allotypes, amino acid analyses were carried out on IgG and IgM antibodies isolated from rabbits which were homozygous a1 or a3 for the gamma chain locus and homozygous b4 for the light chain locus. The compositional differences between a1 and a3 IgM antibodies were found to be identical to those between their IgG counterparts. The identity of the allotypic amino acid replacements showed that the same genetic markers were present in the variable sequences of IgG and IgM heavy chains. Since the constant sequences of these heavy chains are controlled by different loci, these data demonstrated that rabbit heavy chains are coded by two separate germ-line genes, one producing the variable region and a second one of various genes producing the constant regions.The use of two antibodies, antiazophenylarsonate and antiazophenyl-beta-lactoside, permitted the compositions to be compared also on the basic of immunological specificity. The specificity amino acid replacements were found to be identical in the IgM and IgG heavy chains whether the two antibodies were isolated from and individual animal of a1 or a3 allotype. These results further support the conclusions of the allotype measurements that at least two genes code for the rabbit heavy chain.     

Antigen presentation by a B-cell line transfected with cloned immunoglobulin heavy- and light-chain genes specific for a defined hapten.

The rearranged genes encoding immunoglobulin heavy (mu) and light (kappa) chains specific for the hapten 2,4,6-trinitrophenyl (Tnp) were introduced into a B-lymphoma line that bears surface IgG with an unknown specificity and expresses surface Ia molecules. A transformant expressing surface IgM specific for Tnp was obtained. The transformant was found to present Tnp-proteins to antigen (protein)-specific T cells far more efficiently than the parenteral B-lymphoma line. This artificial system, utilizing recombinant DNA technology and gene transfer, provides several approaches for the study of T-cell-B-cell interactions.     

Triclonal gammopathy in an extranodal non-Hodgkin lymphoma patient

The case of a patient with an extranodal non-Hodgkin lymphoma and three M-components (IgGkappa + IgGlambda + IgMlambda) in the serum is described. Three separate populations of M-component producing cells have been identified. Since a kappa-->lambda chain switch is not demonstrated, the synthesis of IgGkappa and IgGlambda by two distinct clones is obvious. IgMlambda and IgGlambda M-components are synthesized by two different plasma cell populations that could represent two unrelated cell clones or, alternatively, two subclones originated by a common precursor. After chemotherapy the IgGlambda M-component is replaced by heavy gamma chains without evidence of lambda light chains.     

Structural bases for public idiotypic specificities of monoclonal antibodies directed against poly(Glu60Ala30Tyr10) and poly(Glu60Ala40) random copolymers.

NH2-terminal amino acid sequences of heavy and light chains of seven poly(Glu60Ala30Tyr10) (GAT) specific hybridoma products derived from DBA/2 and (DBA/2 X BALB/c)F1 hybrid mice and those of BALB/B polyclonal antibodies have been determined over the first 40 residues. Comparison of these sequences with those of nine other GAT or poly(Glu60Ala40) (GA) specific hybridoma products previously reported allowed the following conclusions. (i) Sequences of hybridoma H and L chains are present in the pool of polyclonal antibodies. (ii) The public CGAT (or pGAT) idiotypic specificities are strictly confined to antibodies exhibiting limited heterogeneity with regard to both the variable heavy (VH) and the variable kappa (V kappa) sequences that may be accounted for by one and two germ-line genes, respectively. (iii) The public idiotypic specificities GA-1, expressed by some anti-GAT and most anti-GA antibodies, make use of the same (or similar) VH germ-line genes as the CGAT or pGAT antibodies but possess a distinctive V kappa sequence. (iv) Antibodies expressing neither of the alternative public specificities mentioned above appear to be more heterogeneous and express VH and V kappa sequences that were found to differ from the basic structures defining the CGAT (pGAT) or GA-1 correlates. It is concluded that CGAT (or pGAT) and GA-1 public idiotypic specificities are germ-line markers of both VH and V kappa regions, an observation in agreement with previously reported serological data.     

Rearrangement and expression of endogenous immunoglobulin genes occur in many murine B cells expressing transgenic membrane IgM.

Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).