Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification.

Genetic and molecular evidence indicates that visceral endoderm, an extraembryonic cell lineage, is required for gastrulation, early anterior neural patterning, cell death and specification of posterior mesodermal cell fates. We show that variant Hepatocyte Nuclear Factor 1 (vHNF1), a homeodomain-containing transcription factor first expressed in the primitive endoderm, is required for the specification of visceral endoderm. vHnf1-deficient mouse embryos develop normally to the blastocyst stage, start implantation, but die soon afterwards, with abnormal or absent extraembryonic region, poorly organised ectoderm and no discernible visceral or parietal endoderm. However, immunostaining analysis of E5.5 nullizygous mutant embryos revealed the presence of parietal endoderm-like cells lying on an abnormal basal membrane. Homozygous mutant blastocyst outgrowths or differentiated embryonic stem cells do not express early or late visceral endoderm markers. In addition, in vHnf1 null embryoid bodies there is no activation of the transcription factors HNF-4alpha1, HNF1alpha and HNF-3gamma. Aggregation of vHnf1-deficient embryonic stem cells with wild-type tetraploid embryos, which contribute exclusively to extraembryonic tissues, rescues periimplantation lethality and allows development to progress to early organogenesis. Our results place vHNF1 in a preeminent position in the regulatory network that specifies the visceral endoderm and highlight the importance of this cell lineage for proper growth and differentiation of primitive ectoderm in pregastrulating embryos.

Functional gene transfer from intracellular bacteria to mammalian cells.

We provide evidence of direct transfer of functional DNA from bacteria to mammalian cells. An Escherichia coli K12 diaminopimelate auxotroph made invasive by cloning the invasin gene from Yersinia pseudotuberculosis transfers DNA after simple co-incubation, into a variety of mammalian cell lines. Transfer efficiency was enhanced in some cells by coexpression of the gene for listeriolysin from Listeria monocytogenes. Expression of the acquired genes occurs in both dividing and quiescent cells. The only requirement for bacteria to transfer genetic material into nonprofessional phagocytic cells and macrophages is the ability to invade the host cell.

Maternal IgG stimulates B lineage cell development in the progeny.

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous muMT/muMT or heterozygous muMT/+ females to muMT/muMT or muMT/+ males, respectively. We could thus compare normal or B cell-deficient mice born from Ig-deprived (Ig-) or phenotypically normal mothers (Ig+). B cell-deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7-21 days of age, decay after weaning with a half-life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, muMT/+ progeny born of Ig+ mothers contain two- to threefold higher numbers of bone marrow (BM) pre-B and B cells, and of splenic B cells, compared to mice of the same age born from Ig mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig-secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG-dependent stimulation of BM B cell precursors is also observed in muMT/muMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.

Targeted disruption of the V(H) 81X gene: influence on the B cell repertoire.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

The perinatal presence of antigen (p-azophenylarsonate) or anti-mu antibodies lead to the loss of the recurrent idiotype (CRIA) in A/J mice.

The immune response of A/J mice against p-azophenylarsonate (Ars)-keyhole limpet hemocyanin (KLH) is characterized by the dominance, late in primary and during the secondary, of a recurrent idiotype called CRIA, encoded by a canonical combination of Ig gene segments. In this study, A/J mice were given Ars coupled to deaggregated human gamma globulins (dHGG) within 24 h after delivery. The offsprings from these mice were then exposed as adults to Ars-KLH. These animals developed an unusual immune response. The level of anti-Ars antibodies was nearly normal but a dramatic shift in repertoire was observed: the cross-reactive idiotype which is the hallmark of the anti-Ars response in A/J mice was completely absent. The idiotype could be recovered by injection of anti-idiotypic antibodies alone, with no need of lipopolysaccharide coupling. Therefore the presence of antigen at birth can lead to a strong perturbation of idiotype selection. Similar results were obtained with neonatal treatment using anti-IgM antibodies. After recovery of suppression, A/J mice can mount an anti-arsonate response of normal level but devoid of the dominant idiotype.

VH-gene family dominance in ageing mice.

The cellular composition and VH-gene family repertoire were compared in different B-cell compartments from young adult (8-12 weeks) and old (18-24 months) C57BL/6 and BALB/c mice. Ageing mice were found to have a higher frequency of peripheral mature B cells utilizing genes from a single VH-gene family. While in each individual old C57BL/6 mice cells expressing the VH J558 gene family consistently were over-represented, a marked individual variation was observed in old BALB/c mice with increased frequency of either the VH J558, Q52 or J606 families. Aged mice were found also to have a reduced number of bone-marrow pre-B cells and an augmented number of splenic Ig-secreting cells. These results suggest that old mice express less diversified antibody repertoires possibly as a consequence of reduced input from precursors and increased peripheral selection, which may be responsible for the progressive establishment of immunodeficiency.

V-region directed selection in differentiating B lymphocytes.

We here analyse the repertoire of VH7183 rearrangements isolated from different stages of B cell differentiation in adult mice. The nucleotide sequence analyses of VH7183-D-JH rearrangements derived from large pre-B cells (B220+, mu-), small pre-B cells (B220+, mu-) and mature B cells (B220+, mu+) isolated from adult bone marrow revealed a sequential accumulation, among functional rearrangements, of D segments of the FL16 family and a depletion of D segments using the second and the third reading frame (RF). One member (VH7183.1) of the VH7183 gene family was utilized in 60-80% of the rearrangements of all populations analysed. In neonates the majority of the rearrangements utilizing this gene was found to be functional. In contrast, > 96% of the VH7183 rearrangements isolated from adult spleen were non-functional. These data provide evidence for cellular selection of VH regions acting at different points of the B cell differentiation pathway and at the transition of B cells from the bone marrow to the periphery.

Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells.

The bulk of natural IgM secretion is currently attributed to peritoneal CD5+ B cells and their progeny, believed to be independent of adult bone marrow precursors. We have compared the capacity of peritoneal or splenic cells from normal adult mice to generate serum IgM after transfer into allotype-congenic, irradiated and bone marrow-protected mice. Recipients of either cell population produced donor-allotype IgM-secreting cells in the spleen, and had donor-derived serum IgM. In both cases as well, recipient IgM secretion recovered to control levels. Since the spleen cell-derived natural IgM production could result from expansion of CD5+ B cells present in the inoculum, we next investigated the ability of Ig- bone marrow (BM) cells (Ig- BM) to reconstitute natural IgM secretion in irradiated mice. This cell population was most efficient in reconstituting donor-derived IgM secretion. The origin and phenotype (IgM, CD5) of B cells present in spleen and peritoneum of recipient mice were also analyzed. In agreement with the high level of donor IgM-secreting cells, transfers of splenic and Ig- BM cells fully reconstitute donor B cells in spleen and peritoneum and inhibit reconstitution from host origin. In contrast, donor peritoneal cells reconstitute B cells very poorly in spleen and allow for reconstitution by host cells. Furthermore, Ig- BM cells as well as splenic or peritoneal donor cells, all reconstitute CD5+ B cells in the peritoneum of recipient mice. Interestingly, the fraction of IgM+ cells of each allotype that differentiate to IgM secretion varies widely, but normal levels of IgM are established even when the number of donor B cells present in the animal is very limited.

Biased VH gene expression in murine CD5 B cells results from age-dependent cellular selection.

Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single-stranded DNA, trimethyl ammonium and bovine gamma-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms.

Normal serum immunoglobulins influence the numbers of bone marrow pre-B and B cells.

The homeostatic mechanisms controlling B lymphocyte output from bone marrow are not well understood. The present experiments evaluated putative influences of circulating immunoglobulins (Ig) on bone marrow (BM) pre-B and B cell populations. Injections into normal mice of Ig isolated from normal mouse serum, resulted in a dose-dependent and reversible reduction in numbers of BM B lineage cells, in particular of small B220+ surface IgM- cells. Maximal effects were observed upon injection of isologous polyclonal Ig and were independent of mature T cells. These results suggest a feedback modulation of peripheral Ig on cellular activities in BM B lineage compartments, mediated by mechanisms that seem to involve the variable regions of the Ig molecule.

Stimulation of B and T cells by in vivo high dose immunoglobulin administration in normal mice.

Adult BALB/c mice were injected intravenously with a preparation of pooled normal murine IgG (400 mg/kg/day, on five consecutive days) and studied 8, 15, and 60 days later. High dose IgG administration increased the total numbers of splenic activated B and CD4+ (but not CD8+) T cells, as well as the numbers of splenic Ig-secreting cells, particularly in the IgG isotypes. Reactivities to some autoantigens, but not to bacterial or other heteroantigens, were selectively amplified amongst IgM-secreting cells. IgG administration did not alter the specific primary immune response to heterologous erythrocytes or bacterial dextran. No cellular alterations were detected in the lymph nodes or peritoneal cavity of treated animals. Most of these effects subsided with time, but some autoantibody reactivities remained elevated 60 days later. The present results suggest that the therapeutic effects of high dose IgG administration which have been reported in human diseases might be associated with the immunostimulatory activities of such treatment.

All VH11 genes expressed in peritoneal lymphocytes encode anti-bromelain-treated mouse red blood cell autoantibodies but other VH gene families contribute to this specificity.

We have studied the relationship between B cell reactivity to bromelain-treated autologous mouse erythrocytes (BrMRBC) and expression of the VH11 gene family in splenic, peritoneal and pleuropericardial cell populations from normal C57BL/6 mice. B lymphocytes producing antibodies to BrMRBC were selectively enriched or depleted from normal populations by rosette formation with BrMRBC, followed by centrifugation over density gradients. This selection method, based on the presence of functional receptors (membrane IgM), is harmless for the cells and allowed subsequent cloning in agar (colony-forming unit-B). The utilization of the 10 VH gene families was then scored in mRNA colony blot assays. The analysis of greater than 650 anti-BrMRBC clones and greater than 350 VH11-expressing colonies indicates that about half of those antibody reactivities are encoded by VH11 genes. Furthermore, it appears that all VH11-expressing B cells in the peritoneal cavity produce anti-BrMRBC antibodies.

Cellular basis for the age-associated increase in autoimmune reactions.

The mechanisms that lead to the increased expression of autoantibodies with age are poorly understood. We have studied the number, size, and density of spleen and peritoneal cells from young and old BALB/c and C57BL/6 mice as well as the frequency of clonal precursors for antibodies to mouse erythrocytes, thyroglobulin, and IgG in these lymphoid preparations. Old mice have a 6-fold increase in the number of resident peritoneal cells and a 2-fold increase in the absolute number of Ly1-bearing B cells in this population. Furthermore, old mice have twice as many large, low density splenic B cells as young mice. The frequencies of B cell clonal precursors for anti-BrMRBC and anti-thyroglobulin antibody-forming cells in old mice were 3-10 times greater than in young mice. In the same cultures, however, no increase in the frequencies of B cell clonal precursors for anti-IgG or anti-DNA antibody forming cells was detected in old compared to young mice. These findings and other data suggest that there are at least two families of B cell autoantibody precursors, one including anti-BrMRBC and anti-thyroglobulin autoantibodies, the other including anti-IgG and anti-DNA antibodies. Studies of the differential regulation of these two families of autoantibody precursors might contribute to a greater understanding of autoimmune phenomena in age and disease.

All T15 Id-positive antibodies (but not the majority of VHT15+ antibodies) are produced by peritoneal CD5+ B lymphocytes.

After adult irradiation and reconstitution with autologous bone marrow, BALB/c and C.B20 mice no longer utilize the T15 Id in response to phosphorylcholine. T15 Id expression can be restored by transfers of peritoneal B cells or by FACS-purified CD5+ IgM+ lymphocytes (but not by T lymphocytes) from syngeneic donors. Using bone marrow and peritoneal cell donors that are congeneic for heavy and light chain allotypes, the exclusive origin of the T15 Id in peritoneal B cells was ascertained. These conclusions have been essentially confirmed by immunization with either anti-T15 Id or anti-VHT15 antibodies conjugated to lipopolysaccharide. Thus, the production of VHT15-positive antibodies continues at control levels in bone marrow-reconstituted animals while no T15 Id production can be stimulated even in this protocol of direct B cell stimulation. These results constitute the first formal demonstration of the exclusive production of and Id by CD5+ B-cells.

Further evidence for coelomic-associated B lymphocytes.

Previous observations indicate that "CD5 B lymphocytes" are preferentially clustered in gut-related mesenchymal areas, such as peritoneum, thymus and tonsils. We have now found that pleuropericardial spaces contain an homogeneous population of large-sized, noncycling, nonsecretory B cells, expressing very high levels of surface IgM, little or no IgD, Mac-1 and low levels of B220. This phenotype and the over-representation of some antibody clonotypes suggest that the pleuropericardial cavity contains a pure "CD5 B cell" population. In all mouse strains analyzed, however, many of these cells are CD5-. These findings, together with the common origin of peritoneum and pleural layers in the primitive coelomic cavity, suggest that such B cells differentiate locally from intraembryonic precursors; we propose to designate them as "coelomic", to distinguish them from "stromal", bone marrow-derived B cells.

Ontogenic development of autoantibody repertoires in spleen and peritoneal cavity of normal mice: examples of T cell-dependent and -independent reactivities.

The ontogenic development of B cell clonal precursors (BCP) reactive to bromelain-treated, syngeneic erythrocytes (BrMRC) and to single-stranded DNA has been studied by limiting dilution of both spleen and peritoneal cells. It was found that the frequency of anti-BrMRC BCP in the spleen is very low up to 4 weeks of age and slowly increases thereafter, to reach adult levels by 6-10 weeks. In the peritoneal cavity, no such BCP can be found before 2 weeks, but they occur at a very high frequency already by 3 weeks of age. Injection of adult, normal syngeneic T cells at birth has no apparent effect on the representation of anti-BrMRC BCP in the peritoneal cavity, but brings these to adult levels or even higher in the spleen already at 3 weeks of age. Accordingly, adult athymic (nude) mice contain normal frequencies of BrMRC-specific BCP in the peritoneal cavity but are devoid of such clones in the spleen. In contrast, the frequency of anti-DNA BCP is very high throughout postnatal development in both spleen and peritoneal cavity, of normal and athymic mice, in both resting and naturally activated splenic B cell compartments, and it is independent of T cell transfers into nude animals. These results indicate the role of T cells in the establishment of some clonal specificities in the adult, splenic autoreactive B cell repertoire.

Immunoglobulin VH gene expression in Ly-1+ and conventional B lymphocytes.

Lymphocyte populations in which Ly-1 B cells are differentially represented were studied for the expression of ten VH gene families, either by an RNA colony blot assay or by in situ hybridization of single cells, in BALB/c and C57BL/6 mice. The comparisons of cells from lymph nodes, Peyer's patches and adult spleen (poor in Ly-1 B cells) with cells from peritoneal cavity and neonatal spleen (rich in Ly-1 B cells) were confirmed by the analysis of adult peritoneal Ly-1- and Ly-1+ B cells sorted on the fluorescence-activated cell sorter. The results indicate that the peritoneal Ly-1+ B subset uses the whole spectrum of known VH gene families, and shows a preferential utilization of CP12 VH genes, most likely as a result of a selective process during life.