Affinity-dependent alterations of mouse B cell development by noninherited maternal antigen

We have examined the passage of maternal cells into the fetus during the gestation and postpartum in mice. Using enhanced green fluorescent protein (EGFP)-transgenic females, we showed that maternal cells frequently gain access to the fetus, mostly in syngeneic pregnancies, but also in allogeneic and outbred crosses. EGFP-transgenic cells, including B, T, and natural killer cells, can persist until adulthood, primarily in bone marrow and thymus. We then asked whether maternal cells, bearing antigens not inherited by the fetus, influence the development of fetal and neonatal B lymphocytes. We have used the B cell receptor 3-83 mu/delta transgenic mouse model, whose B cells recognize the major histocompatibility complex class I molecules H-2Kk and H-2Kb, with a high or moderate affinity, respectively. The fate of transgenic B cells in animals exposed to noninherited H-2Kk or H-2Kb maternal antigens (NIMA) during gestation and lactation was compared with those of nonexposed controls. In H-2Kk-exposed fetuses, NIMA-specific transgenic B cells are partially deleted during late gestation. Nondeleted cells have downmodulated their B cell receptor. In contrast, in NIMA H-2Kb-exposed neonates, transgenic B cells present an activated phenotype, including proliferation, upregulation of surface CD69, and preferential localization in the T cell zone of splenic follicles. This state of activation is still clearly detectable up to 3 wk of age. Thus, we show that fetal and neonatal B cell development is affected by maternal cells bearing antigens noninherited by the fetus and that this phenomenon is highly dependent on the affinity of the B cell receptor for the NIMA.     

Cytolytic T cells recognize a chimeric MHC class I antigen expressed in influenza A infected transgenic mice

A chimeric H-2Kd/Kk gene, called pC31, contains the extracellular alpha 1 domain of Kd origin whereas the rest of the molecule is of Kk origin. Disruption of the syngeneic alpha 1-alpha 2 structure results in a total abrogation of the function of the C31 protein as a restriction element for H-2Kd and Kk restricted T cells during virus infection. In an attempt to obtain information on the functional polymorphism of MHC class I antigens as restriction elements, we have introduced the pC31 gene into the germ line of C3H/He mice (H-2k). The pC31 gene was transcribed in all tissues examined and the expression pattern paralleled the endogenous H-2Kk gene. However, the mRNA for the transgene was approximately 10-times more abundant, which was reflected in an elevated expression of the C31 protein in transgenic splenocytes. Most of the C31 antigen was found intracellularly. The C31 antigen could condition transgenic cytotoxic T lymphocytes in a specific manner during influenza A virus infection and functioned as the restricting element during T cell lysis of the infected cells. These results suggest that entire exons may be exchanged between MHC class I genes and that this exchange can generate novel and functional restriction elements.     

Effect of upstream RNA processing on selection of mu S versus mu M poly(A) sites.

All of the regulatory factors responsible for augmenting microseconds mRNA levels preceding the dramatic increase in secretory IgM production upon B cell activation has not been totally elucidated. Whereas previous experiments have centered on the region of the gene specifying the choice between splicing to mu M exons versus selection of the mu S poly(A) site, we have found that upstream sequences within the Cmu gene, specifically the Cmu 4 acceptor splice site together with intronic sequences between the Cmu 3++ and Cmu 4 exons, play an important role in dictating the precision or the extent of splicing to the mu M exons even under conditions in which functional polyadenylation factors should be in excess. Therefore, splicing of upstream exons can affect remotely located downstream exons. These findings suggest that regulation of differential mu S/mu M mRNA expression may involve general processing enzymes that recognize specific cis -regulatory sequences residing within the body of the mu gene and account for the unique ability of activated B cells to secrete copious amounts of IgM.     

Inability of IL-2 and IL-10 to counteract B cell clonal deletion.

The B cell antigen receptor (BCR) delivers inhibitory signals in nascent B cells leading to the establishment of tolerance via clonal deletion or clonal anergy depending upon the type of antigen to which the B cells are exposed. In previous work, it has been demonstrated that activated Th2 cells, as well as some recombinant lymphokines, prevent the inhibition of growth and subsequent cell death induced through the BCR in model B cell lymphomas. Herein, we extend this work to another Th2 lymphokine, IL-10, that in contrast to IL-4 does not interfere with the deletion promoted by IgM crosslinking. The effect of individual lymphokines has also begun to be analyzed in a transgenic model of B cell clonal deletion. To this end, we have administered a recombinant vaccinia virus producing human IL-2 to mice expressing an autoreactive H-2Kk,b-specific transgenic IgMk and found that IL-2 does not abrogate B cell deletion in vivo.     

Interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. II. Levels of expression of H-2K, H-2D, and H-2L in different mouse strains

The numbers of MHC class I molecules expressed by spleen cells from various mouse strains were determined by using MHC-specific monoclonal antibodies and a radioactive binding assay. Although small differences were found to exist in some cases, our general conclusion is that different mice of the same strain, congenic mice of different haplotypes, and syngeneic mice of varying background all express similar numbers of class I antigens. B10.A mice (8 to 10 wk old), for example, express 5.3 X 10(4) Kk molecules/cell, 5.4 X 10(4) Dd molecules/cell, and 2.2 X 10(4) Ld molecules/cell. Some of the differences observed in class I antigen expression included: 1) the level of Kk expression increased to a small but significant extent with age in B10.A mice; 2) female B10.A mice expressed slightly higher amounts of Kk than male mice; and 3) B10.A(2R) and B10.A(4R) recombinant strains expressed elevated levels of K-end antigens and slightly decreased levels of D-end antigens when compared with the unrecombinant B10.A strain. In several strains, F1 mice express approximately 50% as many copies of each parental antigen as do the homozygous parents. B10 mice, which are negative for the L antigen, nevertheless express the same total number of D-end molecules as do B10.A mice. The data suggest that the levels of expression of MHC class I molecules are controlled by at least two factors: gene dosage and another factor(s) that gives rise to the small variations in class I antigen expression seen with age, sex, and strain, and to the low expression of Ld relative to Dd and Kk.     

Elimination of CD8+ thymocytes in transgenic mice expressing an anti-Lyt2.2 immunoglobulin heavy chain gene.

Individual T cell populations are characterized by specific surface proteins, namely by the T cell receptor complex (TCR) and by two accessory molecules, CD8 (Lyt2) and CD4 (L3T4). CD8 and CD4 are required for T cell interactions with class I or class II major histocompatibility complex molecules. In the thymus, immature CD8(-4)-TCR- cells differentiate, possibly via a short stage of CD8+4- thymocytes, into CD8+4+ TCR+ T cells and mature further into the main T cell populations, the CD8+4- TCR+ cytotoxic T lymphocytes and the CD4+8- TCR+ T helper cells. In order to analyse the differentiation steps involving CD8, we generated transgenic mice expressing mu heavy chain genes from an anti-Lyt2.2 hybridoma. Transgenic lines expressing either the complete (mu sm) or only the secreted mu protein (mu s) suffer from a severe depletion of their CD8+4+ thymocytes affecting also the mature CD8+4- and CD4+8- populations. The depletion is correlated to the expression of transgenic mu-chain proteins within thymocytes. This intrathymocyte expression of the mu chain prevents CD8-4- thymocytes from further differentiation, most probably via intracellular interactions between mu heavy chain and CD8 proteins. These results show that CD8 plays an important role during thymocyte maturation.     

A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells.

We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.     

HLA-B27 in inbred and non-inbred transgenic mice. Cell surface expression and recognition as an alloantigen in the absence of human beta 2-microglobulin

A gene encoding the H chain of the human class I MHC Ag HLA-B27 was introduced into the germ lines of inbred C57BL/6 (B6) and non-inbred (B6 X SJL/J) F2 mice. By immunofluorescence and flow cytometry, the HLA-B27 gene product was expressed on lymphoid cells at levels comparable to the endogenous H-2b and H-2s class I MHC molecules. In both primary and secondary MLC between responder spleen cells from non-transgenic (B6 X SJL/J) F1 mice and transgenic stimulator cells, CTL were generated that specifically lysed mouse L cell (H-2k) or human B cell targets expressing HLA-B27, and this lysis thus appeared largely unrestricted by H-2. These results indicate that transgenic mice express a functional HLA-B27 gene product on cell surfaces in the absence of the human beta 2-microglobulin gene. These transgenic mice promise to be a valuable resource in the investigation of the unique role of HLA-B27 in inflammatory human disease.     

Nonimmune thyroid destruction results from transgenic overexpression of an allogeneic major histocompatibility complex class I protein.

The overexpression of major histocompatibility complex (MHC) class I molecules in endocrine epithelial cells is an early feature of autoimmune thyroid disease and insulin-dependent diabetes mellitus, which may reflect a cellular response, e.g., to viruses or toxins. Evidence from a transgenic model in pancreatic beta cells suggests that MHC class I overexpression could play an independent role in endocrine cell destruction. We demonstrate in this study that the transgenic overexpression of an allogeneic MHC class I protein (H-2Kb) linked to the rat thyroglobulin promoter, in H-2Kk mice homozygous for the transgene, leads to thyrocyte atrophy, hypothyroidism, growth retardation, and death. Thyrocyte atrophy occurred in the absence of lymphocytic infiltration. Tolerance to allogeneic class I was revealed by the reduced ability of primed lymphocytes from transgenic mice to lyse H-2Kb target cells in vitro. This nonimmune form of thyrocyte destruction and hypothyroidism recapitulates the beta-cell destruction and diabetes that results from transgenic overexpression of MHC class I molecules in pancreatic beta cells. Thus, we conclude that overexpression of MHC class I molecules may be a general mechanism that directly impairs endocrine epithelial cell viability.     

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in target cells

Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.     

Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.     

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

Specificity for molecules of the major histocompatibility complex mediated by a hybrid immunoglobulin-T cell receptor.

A chimeric T-cell receptor (TCR) alpha-chain gene was produced by shuffling the immunoglobulin VDJH from a 40-140 digoxin-specific hybridoma onto alpha-chain constant region (C alpha) exons. This hybrid immunoglobulin-TCR gene was used to produce transgenic mice. Previous results indicated that this chimeric gene encoded a polypeptide that associated with endogenously encoded beta chains to form a hybrid TCR. T cells expressing this receptor could be stimulated with antibodies specific for CD3 or the 40-140 idiotype (Id40-140), and also with digoxin coupled to bovine serum albumin (digoxin-BSA). We were interested in determining whether a hybrid receptor such as this could also recognize the natural ligand of T cells, namely allelic variants of major histocompatibility complex (MHC) molecules. A T-cell hybridoma was produced that expressed a hybrid receptor with specificity for an IAk-encoded determinant, digoxin-BSA, or staphyloccocal enterotoxin B. Transfection experiments showed that the specificity for MHC determinants was dependent on both the hybrid alpha chain and a particular beta chain. These results indicate that a V beta domain combined with a VH domain can produce a receptor capable of reacting with MHC molecules, and at the same time retain specificities mediated by the beta chain and alpha chain alone. A conclusion is that the pervasive MHC specificity of the TCR is not unique to the family of TCR heterodimers, but is selected, and can be mediated by immunoglobulin domains.     

Precursors of both conventional and Ly-1 B cells can escape feedback inhibition of Ig gene rearrangement

Experiments with transgenic mice carrying rearranged Ig transgenes have shown that membrane bound Ig molecules cause feedback inhibition of endogenous Ig gene rearrangement. However, this inhibition is never complete. It has been postulated that escape from feedback may be a property of the Ly-1 B cell subset, whereas rearrangement of endogenous Ig genes may be completely inhibited in conventional B cells. This possibility was investigated in transgenic mice carrying a lambda transgene under the control of the H chain enhancer. It was found that kappa producing B cells in these lambda transgenic mice were for the most part, although not exclusively, of the conventional B cell phenotype. Examination of peritoneal exudate cells revealed that a large proportion of Ly-1 B cells also express kappa. Adoptive transfer of bone marrow from adult lambda transgenic mice, a source of conventional B cell precursors, resulted in the production of relatively high levels of serum kappa 2 to 3 mo after transfer into recipient SCID mice. A high proportion of donor B cells in the spleen produced endogenous kappa protein with or without co-production of lambda. It is concluded that precursors of both conventional and Ly-1 B cells can escape feedback inhibition of L chain gene rearrangement.