Function of B cells expressing a human immunoglobulin M rheumatoid factor autoantibody in transgenic mice

We have generated transgenic mice that express the immunoglobulin (Ig)M heavy chain and kappa light chain genes coding for a human IgM rheumatoid factor (RF), Les. Transgenic B cells expressing human IgM RF show striking similarities to their counterparts in normal humans. They comprise a significant proportion of the adult B cell population, but secrete only low levels of RF into the serum. The RF transgene- expressing B cells localize to primary B cell follicles and the mantle zone regions of secondary follicles in the spleen. Using these mice we have been able to show that one of the central functions of normal RF- expressing B cells may be to act as highly efficient antigen-presenting cells for low concentrations of immune-complexed antigen. High levels of secretion of IgM RF can not be induced under normal circumstances, although RF-expressing B cells proliferate well in vitro to both aggregated human IgG and anti-human IgM antibodies. However, these mice are not intrinsically secretion deficient. By crossing the RF transgenic mice with the autoimmune MRL/lpr background, we find a dramatic increase, > 200-fold, in levels of serum RF. The results strongly suggest that a major function of normal resting RF B cells is unrelated to antibody secretion. Rather, the RF B cells in the follicles may play a role in antigen presentation and regulation of immune responses to antibody-bound nonself-, and possibly self- antigens. This physiologic role of RF B cells may be disrupted in RF- associated autoimmune disease.     

Cardiac resistance to adriamycin in transgenic mice expressing a rat alpha-cardiac myosin heavy chain/human multiple drug resistance 1 fusion gene.

Cardiac toxicity is a major factor that limits the use of anthracyclines in cancer chemotherapy. Heart failure frequently develops in patients treated with doxorubicin (Adriamycin), when they receive a cumulative dose greater than 500 mg/m2. To make a mouse model for gene therapy designed to prevent this toxic effect, we have produced transgenic mice overexpressing the human cDNA for the multiple drug resistance (h-mdr1) gene driven by 2.12 kb of the 5' flanking region of the rat alpha-cardiac myosin (aCM) heavy chain gene. Two lines of transgenic mice expressed the transgene at a high level in heart muscle. Transgenic and control animals were treated with Adriamycin intravenously at either a single dose of 10 mg/kg or a cumulative dose of 30 mg/kg in three injections. Subsequent light and electron microscopic examination of heart tissue demonstrated degenerative changes in control mice that were absent in transgenic animals at both doses. These results show that expression of the alphaCM/h-mdr1 transgene in heart confers protection from the toxic effect of Adriamycin and suggest that such constructs, if employed effectively in cardiac gene therapy protocols, could allow a more aggressive use of anthracyclines in the treatment of cancer.     

Strain dependency of B and T lymphoma development in immunoglobulin heavy chain enhancer (E mu)-myc transgenic mice

The transgenic mice were produced by injecting eggs of B6 and C3H/HeJ mice with the human E mu-myc gene. Preferential development of B lymphomas was observed in the B6 transgenic mice, whereas the C3H/HeJ transgenic mice developed mostly T lymphomas. The phenotypic activation of B lineage cells but not of T lineage cells was detected in the prelymphomatous transgenic mice of both strains. The transgene was similarly expressed in B and T cells of the transgenic mice of both strains. These results suggest that a high incidence of T lymphomas in the C3H/HeJ transgenic mice may not be due to the preferential activation of or the preferential E mu-myc expression in T lymphocytes. When the bone marrow or fetal liver cells from the prelymphomatous transgenic mice of both strains were transferred into irradiated normal C3H/HeJ mice, most of the recipients developed T lymphomas. Moreover, even when irradiated B6 mice received the hematopoietic stem cells from the prelymphomatous B6 transgenic mice, the incidence of T lymphoma increased up to 50%. These findings suggest that B6 and C3H/HeJ mice might provide the environment that supports the development or growth of B and T lymphomas, respectively, and that such an environment could be modified by irradiation of the mice.     

B cell tolerance in mice transgenic for anti-CD8 immunoglobulin mu chain

To analyze in vivo the induction of B cell tolerance against a T cell surface antigen, we generated transgenic mice expressing an anti-CD8.2 mu heavy chain gene. We show that self-specific B cells are efficiently tolerized if they express the membrane-bound form of the transgenic mu chain on their surface but that they can escape tolerization if they express only the secreted form. In the latter, we find an enhanced expression of anti-CD8.2 antibodies after polyclonal B cell activation. As a result, transgenic anti-CD8.2 antibodies bind to the CD8+ T cells but they did not induce their elimination. Furthermore, we observed the preferential expression of a limited subset of endogenous light chains with the transgenic mu chain. This suggests a positive or negative selection for particular heavy and light chain combinations in B lymphocytes.     

Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes.

cDNA libraries for IgM heavy chain variable regions were prepared from unmanipulated peripheral blood lymphocytes of two healthy people. Partial sequencing of 103 clones revealed VH gene family use and complete CDR3 and JH sequences. The libraries differed in the two subjects. In one person's cDNA the VH5 family was overexpressed and the VH3 family underexpressed relative to genomic complexity. In the second person's cDNA, VH3 was most frequently expressed. In both libraries, JH4 was most frequent. VH segments of several clones were closely related to those in fetal repertoires. However, there was also evidence of mutation in many cDNAs. Three clones differed from the single nonpolymorphic VH6 germline gene by 7-13 bases. Clones with several differences from VH5 germline gene VH251 were identified. CDR3 segments were highly diverse. JH portions of several CDR3's differed from germline JH sequences. 44% of the clones had DH genes related to the DLR and DXP families, most with differences from germline sequences. In 11 DLR2-related sequences, several base substitutions could not be accounted for by polymorphism. Thus, circulating IgM-producing B cell populations include selected clones, some of which are encoded by variable region gene segments that have mutated from the germline form.     

Tolerance in transgenic mice expressing class II major histocompatibility complex on pancreatic acinar cells

To study the nature of tolerance to antigens not expressed by cells of the lymphoid system, expression of class II MHC I-E was targeted to the acinar cells of the exocrine pancreas in transgenic mice (elastase [EL]-I-E). Despite the absence of detectable I-E in the thymus of EL-I-E transgenic mice, both thymocytes and peripheral T lymphocytes were tolerant to I-E, and the pancreas was free of autoimmune infiltrates. Nontolerant T cells adoptively transferred into irradiated or T-depleted transgenic mice rapidly destroy the I-E+ components of the pancreas; however, adoptive transfer of nontolerant T lymphocytes into nonirradiated transgenic mice do not. These results suggest that tolerance in transgenic mice is maintained by some peripheral tolerance mechanism. However, further studies indicate that tolerance in transgenic mice is not maintained by specific Ts cells. For example, cell mixing experiments both in vitro and in vivo fail to reveal dominant unresponsiveness. Furthermore, nontolerant T cells injected into otherwise unmanipulated EL-I-E mice can be primed in situ (by injections of I-E+ spleen cells) to destroy the I-E+ acinar cells.     

Secondary heavy chain rearrangement: a mechanism for generating anti-double-stranded DNA B cells

The chronic graft-versus-host (cGVH) reaction results in a syndrome that closely resembles systemic lupus erythematosus (SLE). It is induced in nonautoimmune mice by the transfer of alloreactive T cells. The availability of anti-DNA transgenes allows us to study the genetic origins of autoantibodies in this model. We induced cGVH in two anti-DNA H chain site-directed transgenic mouse strains. This resulted in clonal expansion and selection of specific mutations in the anti-double-stranded (ds) DNA B cell population. These data, together with a high frequency of anti-dsDNA B cell clones recovered as hybridomas, suggested that anti-dsDNAs are the product of an antigen-driven immune response. Genetic analysis associated this response with the generation of anti-dsDNA B cells through secondary rearrangements that replaced the site-directed transgene (sd-tg) with endogenous VH genes.     

Context-dependent perturbation of neural systems in transgenic mice expressing a cytosolic prion protein

We analyzed the relationship between pathogenic protein expression and perturbations to brain anatomy and physiology in a genetic model of prion disease. In this model, the mouse line 1D4, neuropathology is promoted by accumulation of a cytosolic form of the prion protein (cyPrP). CyPrP distribution was determined and compared with anatomical magnetic resonance imaging (MRI) data, a form of functional MRI based on manganese labeling, and immediate early gene mapping with an antibody to c-Fos. Significant discrepancies between 1D4 and control mice became apparent well in advance of overt behavioral pathology in the mutant mice. Alterations to brain structure and function in the mutants varied among brain regions, however, and differed strikingly even among regions with the highest levels of cyPrP expression. In the cerebellum, gross neurodegeneration was accompanied by increased Mn²⁺-enhanced MRI signal, raising the possibility that compensatory mechanisms act to preserve cerebellar function in the face of massive atrophy. In the hippocampus of 1D4 mice, no significant structural alterations were observed, but both Mn²⁺-enhanced MRI and c-Fos data indicated perturbations to neurophysiology. In the neocortex, there were no clear neural activity differences between 1D4 and control animals, but mutant mice showed significant reduction in cortical thickness. Our finding that distinct combinations of anatomical and functional abnormalities accompanied cyPrP overexpression in different parts of the brain indicates the importance of context in conditioning effects of protein pathogens, and exemplifies the notion that neurodegenerative phenotypes extend beyond cell death and the immediate consequences of atrophy for particular neural systems.     

Antiangiogenic gene therapy of myeloproliferative disease developed in transgenic mice expressing P230 bcr/abl

Antiangiogenic gene therapy offers an attractive approach to the treatment of a variety of malignancies, including those of the hematological system. However, evaluation of this approach has been hampered by the lack of appropriate animal models. We have recently produced transgenic mice expressing P230 bcr/abl that develop myeloproliferative disease (MPD) closely resembling human chronic myelogenous leukemia. Using this MPD murine model, we examined the feasibility of systemic antiangiogenic gene therapy for hematological malignancy. An adenoviral vector containing the secretable endostatin gene was injected into the right quadriceps muscle of the MPD mice. The increased endostatin level was detected for at least 6 months. Hematological parameters including platelet counts, granulocyte counts, and the hemoglobin concentration were improved by this gene therapy. Infiltration of megakaryocytes was also significantly inhibited in treated MPD mice. Reduction of the microvessel density was confirmed by histological examination. These results demonstrated, for the first time, that antiangiogenic gene therapy is effective to inhibit leukemogenesis caused by expression of the chimeric bcr/abl gene.     

Oral insulin treatment suppresses virus-induced antigen-specific destruction of beta cells and prevents autoimmune diabetes in transgenic mice.

Oral administration of self-antigens has been proposed as a therapy to prevent and treat autoimmune diseases. Here we report that oral treatment with insulin prevents virus-induced insulin-dependent diabetes mellitus (IDDM) in a transgenic (tg) mouse model. Such mice express the viral nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter in their pancreatic beta cells and < 2% spontaneously develop diabetes. However, 2 mo after challenge with LCMV, IDDM occurs in > 95% of tg mice but not in controls. Oral treatment with 1 mg of insulin twice per week for 2 mo starting either 1 wk before or 10 d after initiating LCMV infection prevents IDDM in > 50% of the tg mice (observation time 8 mo). Thus, insulin therapy is effective in preventing progression to overt IDDM in prediabetic tg mice with ongoing islet infiltration. Oral administration of insulin does not affect the generation of LCMV-NP-specific anti-self cytotoxic T lymphocytes nor the infiltration of lymphocytes into the pancreas. However, less beta cells are destroyed in insulin-treated mice, upregulation of MHC class I and II molecules does not occur, and antiviral (self) cytotoxic T lymphocytes are not found in the islets, events present in tg mice developing IDDM. The majority of lymphocytes in the islets of insulin-treated tg mice without IDDM produces IL-4, IL-10, and TGF-beta. In contrast, lymphocytes from islets of tg mice developing IDDM mainly make gamma-IFN.     

Extended duration of DH-JH rearrangement in immunoglobulin heavy chain transgenic mice: implications for regulation of allelic exclusion

Here we show that suppression of VH-DJH rearrangement in mice bearing a mu heavy (H) chain transgene (mu-tg mice) is associated with an extended period of DH-JH rearrangement, the first step of Immunoglobulin H chain gene rearrangement. Whereas DH-JH rearrangement is normally initiated and completed at the pro-B cell stage, in mu-tg mice it continues beyond this stage and occurs most frequently at the small (late) pre-B stage. Despite ongoing DH-JH rearrangement in late pre-B cells of mu-tg mice, VH-DJH rearrangement is not detectable in these cells. We infer that the lack of VH-DJH rearrangement primarily reflects tg-induced acceleration of B cell differentiation past the stage at which rearrangement of VH elements is permissible. In support of this inference, we find that the normal representation of early B lineage subsets is markedly altered in mu-tg mice. We suggest that the effect of a productive VH-DJH rearrangement at an endogenous H chain allele may be similar to that of a mu-tg; i.e., cells that make a productive VH-DJH rearrangement on the first attempt rapidly progress to a developmental stage that precludes VH-DJH rearrangement at the other allele (allelic exclusion).     

Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy.

Missense mutations in the beta-myosin heavy chain (beta-MHC) gene cause hypertrophic cardiomyopathy (HCM). As normal and mutant beta-MHCs are expressed in slow-twitch skeletal muscle of HCM patients, we compared the contractile properties of single slow-twitch muscle fibers from patients with three distinct beta-MHC gene mutations and normal controls. Fibers with the 741Gly-->Arg mutation (near the binding site of essential light chain) demonstrated decreased maximum velocity of shortening (39% of normal) and decreased isometric force generation (42% of normal). Fibers with the 403Arg-->Gln mutation (at the actin interface of myosin) showed lowered force/stiffness ratio (56% of normal) and depressed velocity of shortening (50% of normal). Both the 741Gly-->Arg and 403Arg-->Gln mutation-containing fibers displayed abnormal force-velocity relationships and reduced power output. Fibers with the 256Gly-->Glu mutation (end of ATP-binding pocket) had contractile properties that were indistinguishable from normal. Thus there is variability in the nature and extent of functional impairments in skeletal fibers containing different beta-MHC gene mutations, which may correlate with the severity and penetrance of the disease that results from each mutation. These functional alterations likely constitute the primary stimulus for the cardiac hypertrophy that is characteristic of this disease.     

Paucity of phosphorylcholine-specific clones in B cells expressing the VHT15 gene product

The aim of this work was to study the cellular basis of the phenomenon of clonal dominance. To this end we analyzed two collections of BALB/c and C.B20 hybridomas that we selected on the basis of the expression of the VHT15 gene product independently from their antigen specificity. Our study demonstrates that none of the 28 BALB/c and only 2 of the 29 C.B20 hybridomas obtained have variable regions that bind PC. We conclude therefore that the domination of the immune response to PC by particular variable regions cannot be due to the establishment of clonal dominance prior to immunization.     

Activation of antigen-specific suppressor T cells by B cells from mice immunized with type III pneumococcal polysaccharide

The transfer of B lymphocytes from mice immunized with type III pneumococcal polysaccharide (SSS-III) results in antigen-specific suppression of the antibody response of recipients immunized with SSS-III. Such suppression shares many features associated with low-dose paralysis, a phenomenon mediated by suppressor T cells; it reaches maximal levels 3 d after the transfer of viable or irradiated immune B cells and can be eliminated by the depletion of SSS-III-binding cells from spleen cell suspensions before transfer. In a two-step cell transfer experiment, purified T lymphocytes, isolated from recipients previously given immune B cells, caused suppression upon transfer to other mice immunized with SSS-III. Also, B-cell-induced suppression could be abrogated in a competitive manner by the infusion of amplifier T lymphocytes, as was previously demonstrated in the case of low-dose paralysis. These findings suggest that B cell surface components, presumably the idiotypic determinants of cell-associated antibody specific for SSS-III, are instrumental in activating suppressor T cells involved in regulating the magnitude of the antibody response to SSS-III.     

Regulation of azophenylarsonate-specific repertoire expression. 1. Frequency of cross-reactive idiotype-positive B cells in A/J and BALB/c mice

A large proportion of p-azophenylarsonate (ARS)-specific antibodies from A/J mice share a cross-reactive idiotype (CRIA) that comprises a family of closely related but nonidentical clonotypes. I determined that only 2.6 % (7 out of 267) A/J ARS-specific monoclonal antibodies generated in the splenic focus system possess the predominant CRIA. Because ARS-specific B cells are present at a frequency of 1/68,000 B cells, the frequency of the entire idiotype family is 1 per 2.8 X 10(6) splenic B cells. Thus, there is a striking discrepancy between the representation of this idiotype at the clonal precursor cell level and the serum antibody response. In addition, BALB/c mice have the potential to generate CRIA-positive precursor cells within their nonimmune repertoire. When A/J mice are immunized with ARS-protein conjugates, the serum antibody response and precursor cell population are both dominated by CRIA. The frequency of CRIA-positive B cells increases over 100-fold after immunization, whereas CRIA-negative precursor cells may initially decrease, followed by a later rise in frequency. Finally, although ARS-specific precursor cells are present in high frequency at birth, CRIA-positive monoclonal anti-ARS antibodies are not observed during the early neonatal period. These data provide evidence to suggest that complex regulatory networks influence precursor cell and serum antibody expression.     

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.     

Immunoglobulin heavy chain gene rearrangement in heavy chain deposition disease suggests it is a plasma cell disease: a case report

Heavy chain deposition disease (HCDD) is characterized by the deposition of truncated monoclonal immunoglobulin heavy chains along glomerular basement membranes. Truncated heavy chains are thought to be associated with plasma cell disease (PCD), but previous bone marrow cytology tests showed that only 30% of HCDD cases are related to PCDs. We report the first known use of immunoglobulin heavy chain (IGH) gene rearrangement to diagnose a patient with γ3-HCDD, although bone marrow morphology test identified no abnormalities. Our findings provide strong evidence for a correlation between PCDs and HCDD, which could help understand the genetic background underlying abnormal heavy chains and assess disease prognosis. Further, concordant with previous findings, bortezomib-based chemotherapy had a good therapeutic effect in our patient. We summarize the experience of diagnosing and treating a case of HCDD, and combine this with a literature review to further explore the correlation between PCDs and HCDD, which has important clinical value.     

Immunoglobulin gamma 2b transgenes inhibit heavy chain gene rearrangement, but cannot promote B cell development

Transgenic mice with a gamma 2b transgene were produced to investigate whether gamma 2b can replace mu in the development of B lymphocytes. Transgenic gamma 2b is present on the surface of B cells. Young transgenic mice have a dramatic decrease in B cell numbers, however, older mice have almost normal B cell numbers. Strikingly, all gamma 2b- expressing B cells in the spleen also express mu. The same is true for mice with a hybrid transgene in which the mu transmembrane and intracytoplasmic sequences replace those of gamma 2b (gamma 2b-mumem). The B cell defect is not due to toxicity of gamma 2b since crosses between gamma 2b transgenic and mu transgenic mice have normal numbers of B cells. Presence of the gamma 2b transgene strongly enhances the feedback inhibition of endogenous heavy chain gene rearrangement. Light chain genes are expressed normally, and the early expression of transgenic light chains does not improve B cell maturation. When the endogenous mu locus is inactivated, B cells do not develop at all in gamma 2b transgenic mice. The data suggest that gamma 2b cannot replace mu in promoting the developmental maturation of B cells, but that it can cause feedback inhibition of heavy chain gene rearrangement. Thus, the signals for heavy chain feedback and B cell maturation appear to be different.