Fetal Organ Culture Research Articles

Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations, thymic hypoplasia, hypoparathyroidism, and/or cardiac defects. Thymic hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymic hypoplasia in 22q11.2DS remain unknown. Comparison of embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed proportions of mesenchymal, epithelial, and hematopoietic cell types similar to those of control thymuses. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymic mesenchymal cells with normal ones restored tissue growth. Comparative single-cell RNA-Seq of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 cell line. The transcripts affected included those for extracellular matrix proteins, consistent with the increased collagen deposition we observed in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymic tissue expansion for hypoplastic lobes. In colony-forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, in contrast to the normal growth of thymic epithelial cells. These findings suggest that mesenchymal cells were causal to the small embryonic thymuses in the 22q11.2DS mouse models, which was correctable by substitution with normal mesenchyme.

Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days.

Regenerative medicine requires better pre-clinical tools in order to increase the efficiency of novel therapies transitioning to the clinic. Current monolayer cell culture methods are suboptimal for effectively testing new therapies and live mouse models are expensive, time consuming and require invasive procedures. Fetal organ culture, organoids, microfluidics and culture of thick sections of adult organs all aim to fill the knowledge gap between monolayer culture and live mouse studies. Here we report on an ex vivo organ perfusion system that can support whole adult mouse organs. Ex vivo perfusion of healthy and diseased mouse organs allows for real-time analysis that provides immediate feedback and accurate data collection throughout the experiment. Having a suitable normothermic ex vivo perfusion system for mouse organs provides a tool that will help contribute to our understanding of kidney physiology and disease and can take advantage of the many mouse models of human disease that already exist. Furthermore, an ex vivo kidney perfusion system can be used for testing novel cell therapies, drug screening, drug validation and for the detection of nephrotoxic substances. Critical to the success of mouse ex vivo organ perfusion is having a suitable bioreactor to maintain the organ. Here we have focused on the mouse kidney and mathematically modeled, built and validated a bioreactor that can maintain a kidney for 7 days. The long duration of the ex vivo perfusion will help to advance studies on kidney disease and can rapidly test for new regenerative medicine therapies compared to whole animal studies.

Structural and Functional Thymic Biomarkers Are Involved in the Pathogenesis of Thymic Epithelial Tumors: An Overview

The normal human thymus originates from the third branchial cleft as two paired anlages that descend into the thorax and fuse on the midline of the anterior–superior mediastinum. Alongside the epithelial and lymphoid components, different types of lymphoid accessory cells, stromal mesenchymal and endothelial cells migrate to, or develop in, the thymus. After reaching maximum development during early postnatal life, the human thymus decreases in size and lymphocyte output drops with age. However, thymic immunological functions persist, although they deteriorate progressively. Several major techniques were fundamental to increasing the knowledge of thymic development and function during embryogenesis, postnatal and adult life; these include immunohistochemistry, immunofluorescence, flow cytometry, in vitro colony assays, transplantation in mice models, fetal organ cultures (FTOC), re-aggregated thymic organ cultures (RTOC), and whole-organ thymic scaffolds. The thymic morphological and functional characterization, first performed in the mouse, was then extended to humans. The purpose of this overview is to provide a report on selected structural and functional biomarkers of thymic epithelial cells (TEC) involved in thymus development and lymphoid cell maturation, and on the historical aspects of their characterization, with particular attention being paid to biomarkers also involved in Thymic Epithelial Tumor (TET) pathogenesis. Moreover, a short overview of targeted therapies in TET, based on currently available experimental and clinical data and on potential future advances will be proposed.

Paracrine regulation of fetal lung morphogenesis using human placenta-derived mesenchymal stromal cells

BackgroundRecent experimental work suggests the therapeutic role of mesenchymal stromal cells (MSC) during perinatal lung morphogenesis. The purpose of this study was to investigate the potential paracrine effects of human placenta-derived mesenchymal stromal cells (PL-MSCs) on pulmonary development. MethodsHuman MSCs were isolated from preterm placental chorion. Normal E14.5–15.5 fetal rat lungs were subsequently harvested and cultured ex vivo in the presence of conditioned media from PL-MSCs for 72 h. The lungs were analyzed morphometrically and by quantitative DNA, protein, and gene expression. Postnatal human bone marrow-derived mesenchymal stromal cells and neonatal foreskin fibroblasts (FF) were used as controls. ResultsThe MSC phenotype of the isolated placental cells was confirmed. Compared with lungs cultured in the absence of PL-MSCs, fetal lung growth was markedly accelerated on exposure to PL-MSC conditioned media as demonstrated by increases in Δlung surface area, terminal bud formation, and Δterminal bud formation. Pulmonary growth was predominantly impacted by enhanced branching morphogenesis, as shown by 73.5 ± 6.1 terminal buds after stimulation with PL-MSCs compared with 46.7 ± 5.7 terminal buds in control unconditioned media (P < 0.05). Significant differences were noted favoring PL-MSCs over FFs based on terminal bud formation and Δterminal bud formation (P < 0.05). There was significant upregulation of club cell secretory protein in lungs exposed to PL-MSCs compared with all other groups. ConclusionsThese data suggest that human PL-MSCs are potent paracrine stimulators of pulmonary morphogenesis in a fetal organ culture model. Cell therapies based on autologous or donor-derived PL-MSCs may represent a novel strategy for enhancing perinatal lung growth.

Thymic epithelial cell culture.

Two dimensional monolayer culture of thymic epithelial cells has been used for more than two decades for evaluation of the nature of these cells. Both cells from infant thymi and from thymi from a variety of laboratory animals have been used. The main reason for the broad interest in culture of these epithelial cells is the documented importance of thymic stromal cells, and among them, especially, epithelial cells in the selection of T-lymphocyte precursors and their differentiation to functionally mature T lymphocytes (-). However, differentiation of T-lymphocyte precursors, which is dependent on correct spatial organization of subtypes of epithelial and mesenchymal cells, may be better studied in murine fetal organ cultures or reaggregate cultures ( ), whereas the culture of thymic epithelial cells in 2-dimensional monolayer cultures, as described here, is useful for characterization of the epithelial cells per se, their morphology, subtypes, surface characteristics, secretion, antigen presentation, and direct interaction with T-lymphocyte precursors added to the cultures, as well as for establishment of epithelial cell lines.

Fetal and Adult Hematopoietic Stem Cells Require β1 Integrin Function for Colonizing Fetal Liver, Spleen, and Bone Marrow

Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that β1 integrin–deficient HSCs from the para-aortic splanchnopleura and the fetal blood had hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult β1 integrin null HSCs isolated from mice carrying loxP-tagged β1 integrin alleles and ablated for β1 integrin expression by retroviral cre transduction failed to engraft irradiated recipient mice. Moreover, absence of β1 integrin resulted in sequestration of HSCs in the circulation and their reduced adhesion to endothelioma cells. These findings define β1 integrin as an essential adhesion receptor for the homing of HSCs.

Epidermal growth factor modulates fetal thymocyte growth and differentiation.

In the present study, we used the fetal organ culture (FTOC) technique in order to study a putative effect of epidermal growth factor (EGF) on the thymus ontogeny. Functional EGF receptors and more recently the EGF molecule itself, respectively, on the membrane of epithelial components of thymic stroma and on a few thymocytes in adult thymus, had been reported in the literature. We could observe a dose-dependent decrease in cellularity and a progressive retention of thymocytes in the double-negative (CD4-/CD8-) stage of differentiation when exogenous EGF was added. Epidermal growth factor interfered with both fetal stroma growth and thymocyte development at a precise moment, that is, in the passage from double-negative to the double-positive (CD4+/CD8+) stage. After a 7-day FTOC in the presence of EGF, most cells recovered were Thy-1.2+, c-kit+, TSA1-/int, CD3-, and one of CD44high/CD25int, CD44-/CD25int, or CD44-/CD25-. Some developed into γδTCR+ cells with a mature (CD3+) phenotype, but not into αβTCR+ thymocytes. It seems that EGF addition makes the cultures "nonpermissible" for αβTCR+ thymocyte generation. We report here the presence of a high Mr "EGF-like" molecule on the membrane of fetal thymocytes, which role in the observed effects is under investigation. Further biochemical characterization of this molecule is still required, because its presence was only evidenced on the basis of its antigenicity.

Cytodynamics of in vitro developing airways and interaction with extracellular matrix proteins.

We studied epithelial and mesenchymal cell kinetics during lung branching and the possible importance of proteoglycan and collagen in rat fetal organ culture for 5 days, starting from 15 days gestation. The lungs were randomly divided into three groups: (1) basal medium (controls); (2) basal medium + beta-D-xyloside (beta-XYL); and (3) basal medium+cis-4-hydroxy-L-proline (cis-HYP). The labeling index of incorporation of 5-bromo-2-deoxyuridine (BrdU) was used as an indicator of DNA synthesis and assessed in the parent airways and in the tips and sides of peripheral buds. In controls, BrdU incorporation was highest in bud tips, followed by bud sides and then parent airway for both epithelium and mesenchyme. These data suggest that preferential cell proliferation occurs in normal airway branching. In parent airways, labeling of epithelium and mesenchyme was consistently diminished throughout the culture period in beta-XYL, whereas in cis-HYP this was not the case. In the peripheral buds, the DNA synthetic activity in both epithelium and mesenchyme was impaired in beta-XYL and cis-HYP, but the effect was more marked in beta-XYL. We conclude that although collagen may be required in peripheral bud morphogenesis, the proteoglycans appear to be essential in growth and development of both parent airways and peripheral buds in the lung.

A role for CD81 in early T cell development.

Early stages of T cell development are thought to include a series of coordinated interactions between thymocytes and other cells of the thymus. A monoclonal antibody specific for mouse CD81 was identified that blocked the appearance of alpha beta but not gamma delta T cells in fetal organ cultures initiated with day 14.5 thymus lobes. In reaggregation cultures with CD81-transfected fibroblasts, CD4-CD8- thymocytes differentiated into CD4+CD8+ T cells. Thus, interactions between immature thymocytes and stromal cells expressing CD81 are required and may be sufficient to induce early events associated with T cell development.

Identification of Kv1.1 Expression by Murine CD4−CD8− Thymocytes: A ROLE FOR VOLTAGE-DEPENDENT K+ CHANNELS IN MURINE THYMOCYTE DEVELOPMENT

The patch-clamp recording technique and RNA-polymerase chain reaction were used to identify the voltage-dependent K+ channels expressed by murine fetal and adult CD4-CD8- thymocytes. Two distinct currents, encoded by the genes Kv1.1 and Kv1.3 were identified based upon their biophysical and pharmacologic characteristics and confirmed with RNA-polymerase chain reaction. Peptide blockers of Kv1.1 and Kv1.3 gene products were also applied to a murine fetal thymic organ culture system to investigate the developmental role of these K+ channels. Dendrotoxin (DTX) and charybdotoxin (CTX), antagonists of Kv1.1 and Kv1.3 channels, respectively, decreased thymocyte yields in organ culture without affecting thymocyte viability. DTX-treated thymi contained 56 +/- 8% (n = 8 experiments), and CTX-treated thymi contained 74 +/- 4% (n = 7 experiments) as many thymocytes as untreated lobes. DTX and CTX also altered the developmental progression of thymocytes in fetal organ culture. These data provide the first evidence of Kv1.1 expression in a lymphoid cell and indicate that thymocyte voltage-dependent K+ channels are critical to thymocyte preclonal expansion and/or maturation.

Regenerative healing of incisional wounds in murine fetal lungs maintained in organ culture

Although fetal dermal repair is known to be fundamentally different from adult healing, the response to wounding in other organs is less well characterized. Scarless repair in mid-gestation dermis with a transition to adult-type healing at term has been shown in fetal organ culture. A lung explant culture system was used to investigate whether wound repair in the fetal lung shows characteristics similar to those found in fetal dermis. Lungs from 14-day and 18-day CD-1 murine fetuses and 2-day-old newborns, (term = 20 days, n = 24) were wounded by linear incision and incubated at 37°C, in a 21% O 2, 5% CO 2 environment, in BGJb supplemented with vitamin C and antibiotics. Medium was changed daily. Samples were fixed at 7 days and embedded in paraffin. Sections were stained with hematoxalyn-eosin and Masson Trichrome. Additional 14-day and 18-day samples were frozen in freon and immunohistochemical staining for TGF-β performed. Other frozen tissues from each time point were homogenized and used to assay for endogenous TGF-β levels by Western blot analysis. Histology showed reconstitution of tissue architecture across the wound in 14-day and 18-day specimens. In representative histological sections, intact bronchial architecture developed across the previous wound site. No cellular inflammatory response was observed, and collagen deposition was undetectable at the site of the wound by Trichrome staining. By 22 days the lung explants showed a much less ordered repair, including disorganized collagen deposition. Immunohistochemistry showed TGF-β throughout the lung parenchyma of 14-day and 18-day lungs without recruitment at the wound site. Western blotting showed a marked increase in signal for TGF-β in 18-day compared to 14-day explants. A smaller increase was seen from 18 days to 22 days. The authors conclude that the murine fetal lung shows scarless repair even late into gestation. This implies a plasticity in lung not seen in the integument, as well as an ability of tissues to exert a strong degree of local control over the healing response.

In Vitro Developmental Toxicity of Aspirin and Its Major Metabolites on Cultured Fetal Mouse Palates

AbstractPalatal primordia of day‐12.5 ICR mouse fetuses were cultured in a chemically‐defined serumless medium by a suspension culture technique, and the developmental toxicity of aspirin and its metabolites on in vitro palatogenesis was studied. Explanted fetal palates were exposed in vitro for 72 hr to 0.5‐2 mM aspirin (ASP), 0.25‐2 mM salicylic acid (SA), 0.5‐2 mM salicyluric acid (SUA), 1–2 mM 2,3‐dihydroxybenzoic acid (3DHB), or 1–2 mM 2,5‐dihydroxybenzoic acid (5DHB). After 72 hr culture, ASP at 2 mM and SA at 0.25 mM inhibited the growth and fusion of palatal shelves, and SUA at 1 mM prevented palatal fusion. On the other hand, 3DHB and 5DHB did not exert any significant toxic effects on cultured palates at concentrations up to 2 mM. Judging from the 50% inhibitory concentration (IC50), SA (IC50= 0.9 mM) was the most toxic of the 5 compounds tested, with a decreasing order of ASP (IC50= 1.5 mM), SUA (IC50= 1.6 mM), and DHBs (IC50= over 2 mM for both 3DHB and 5DHB). With respect to developmental toxicity, cultured fetal mouse palates showed the susceptibility to aspirin and its metabolites which is intermediate between the susceptibility of rat embryos in vivo and that of postimplantation rat embryos cultured in vitro. The significance of fetal organ culture for evaluating developmental toxicity of chemicals is also discussed.

Lymphoid and myeloid differentiation of fetal liver CD34+lineage- cells in human thymic organ culture.

In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.

CD4+/CD8- thymocytes dominate the fetal thymus treated with a combination of anti-T cell receptor-beta and anti-CD4 antibodies.

Two major phenotypic changes characterize the development of a mature thymocyte from its CD4+/CD8+ (double positive or DP) precursor: the loss of expression of either CD4 or CD8 and the increase in the level of surface TCR. The specific surface interactions responsible for these changes are unknown, but studies using the fetal thymus as an experimental system have provided clues by identifying conditions that alter these maturational events. Development to the CD4+/CD8-/TCR-alpha beta high (single positive) phenotype is inhibited when thymocytes in fetal organ culture are exposed to antibodies directed against the CD4 molecule, the CD3 complex or the TCR-alpha/beta heterodimer. We show in this study, however, that treatment of fetal thymic lobes with a combination of anti-CD4 and anti-TCR-beta antibodies results in a marked increase in the proportion of mature CD4+/CD8-/TCR-alpha beta+ thymocytes and a decrease in the proportion of DP thymocytes. Although treatment of lobes with a combination of anti-CD4 and anti-CD3 epsilon antibodies also depletes cultures of DP thymocytes, the CD4+/CD8-/TCR+ population does not develop. Our results are consistent with the hypothesis that coengagement of CD4 and TCR biases development to the CD4 single positive phenotype and with observations that TCR engagement and CD3 engagement have different developmental consequences.

Exogenous IL-7 promotes the growth of CD3-CD4-CD8-CD44+CD25+/- precursor cells and blocks the differentiation pathway of TCR-alpha beta cells in fetal thymus organ culture.

Addition of human rIL-7 to fetal thymic organ culture started at day 13, 14, or 15 did not influence the number of cells generated during a 12-day culture period. However, the IL-7 treatment resulted in a preferential expansion of cells with a phenotype characteristic for cells at an early step of differentiation. The cells were CD4-CD8-CD3-CD2- and SCA-1+. Analysis of the coordinate expression of CD44 and CD25 on these cells showed that the majority of the cells were either CD44+CD25- or CD44+CD25intermediate. TCR-alpha beta cells were present but in a significantly lower number as compared to the control cultures. The cell number of TCR-gamma delta cells was increased. All these effects were moderate after 6 days, but unequivocal after 12 days of culture. Treatment of the fetal organ culture with mAb-neutralizing murine IL-7 resulted in an inhibition of the proliferation of the fetal thymocytes. No particular subset studied was preferentially inhibited. By using a model of reconstitution of 14-day embryonic thymuses depleted of thymocytes by deoxyguanosine and reconstituted with fetal day 13 liver cells and set up in organ culture with or without IL-7, it was shown in a clear cut way that IL-7 indeed promotes expansion of the early precursor cells and TCR-gamma delta cells, but prevents the generation of TCR-alpha beta cells. In addition, reconstitution experiments were set up in the presence of mAb-neutralizing murine IL-7. This treatment resulted in the inhibition of the growth of the fetal thymocytes without inhibiting preferentially a particular subset. These data indicate that IL-7 acts at an early step of T cell differentiation and plays a role to expand precursor cells, but prevents this population from additional differentiation towards the TCR-alpha beta pathways, whereas the differentiation towards TCR-gamma delta cells is not influenced or even enhanced.

CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation

Peripheral CD4+ and CD8+ T lymphocytes carry out different functions during immune reactions, partly as a result of the distinct patterns of lymphokines that they secrete upon stimulation. Using thymic cells from adult and newborn mice as well as from fetal organ cultures, we show here that this functional differentiation occurs inside the thymus and is completed during the single positive stage by the time the T-cell receptor becomes fully coupled to the intracellular activation pathways leading to lymphokine secretion. Surprisingly, CD4+8- thymocytes differ from their immediate progeny, naive peripheral CD4+ cells, in that they secrete a broader range of lymphokines, including interleukins 4, 5 and 10 and gamma-interferon, and more closely resemble immunologically experienced (activated or memory) CD4+ lymphocytes.

Effect of hydrocortisone on sialyltransferase activity in the rat small intestine during maturation Changes along the villus-crypt axis and in fetal organ culture

Sialyltransferase activity was assayed in rat intestinal cells isolated as fractions reflecting the villus-crypt axis of differentiation. In 13-day-old rats both endo- and exogenous sialyltransferase activity reached their maximum in undifferentiated crypt cells and their peaks overlapped. In contrast, sialyltransferase of the adult intestine was 4-fold lower than that of sucklings in the crypts, with slight tendency to be transferred to the villus cells. Hydrocortisone applied to 10-day-old rats caused three days later a precocious drop of sialyltransferase activity in the crypt cells. Unlike in vivo, glucocorticoid responsiveness was accompanied by increased sialyltransferase activity in fetal small intestine cultivated for 17 days.

Effect of growth and differentiation stimuli on the development of antigen-responsive B cells in fetal liver

The development of the B cell immune repertoire was studied using an in vitro fetal organ culture system. In order to analyze the mechanism by which B cell precursors clonally expand and diversify, fetal lymphoid tissues were incubated in the presence of several factors known to influence B cell differentiation: IL-1, IL-2, WEHI-3 culture supernatant containing IL-3, and a factor from a cyclic neutropenia patient (CNF). By analyzing the effect of exogenous factors on the frequency of antigen-responsive B cells, the ability of the factor to either inhibit or enhance clonal expansion was determined. It was found that the addition of IL-1, WEHI-3 supernatant, or CNF increased the frequency of DNP-responsive B cells suggesting an enhancement of clonal expansion. IL-2, on the other hand, did not alter the frequency of antigen-responsive B cells. The effect of added factors on the kinetics of appearance of phosphorylcholine (PC)-responsive B cells, which are known to be acquired in ontogeny about 2 weeks later than DNP-responsive B cells, was also analyzed. The data indicate that CNF, unlike IL-1, IL-2, and WEHI-3 culture supernatant, results in an earlier appearance of PC-responsive B cells. These results suggest that soluble factors may play a role in the generation of the B cell repertoire.

Differentiation of thymocytes in fetal organ culture: lack of evidence for the functional role of the interleukin 2 receptor expressed by prothymocytes.

Thymic rudiments of 14-day-old mouse embryos were put into organ culture in the presence of interleukin 2 (IL2) and PC.61, a monoclonal antibody that binds to the IL2 receptor (IL2R). After 5 and 7 days of culture, we found no influence of PC.61 on the growth of the thymus and the composition of the thymocyte subpopulations as studied with a panel of monoclonal antibodies. High-dose IL2 treatment of the organ culture resulted in a reduction of the number of thymocytes and a decrease in the single CD4+ and CD4+CD8+ thymocytes, whereas the number of CD8+Ly-1+ thymocytes and the number of CD4-CD8-Ly-1- IL2R+ thymocytes increased. The effect of high-dose IL2 treatment was ascribed to the induction of a nonspecific LAK activity. Our findings argue against a functional role of IL2R on prothymocytes during early T cell development.

B cell immune repertoire diversifies in a predictable temporal order in vitro.

The appearance of functional, antigen-specific B cells was studied in an in vitro fetal organ culture system in the absence of environmental influences associated with circulation and cell migration. In this way the B cell diversification process could be analyzed when genetic influences were dominating. By using this system in combination with the splenic focus assay, the frequency of developing B cells responsive to a number of hapten probes was measured. The results indicate that B cell diversification in vitro, in the apparent absence of many environmental influences, results in the appearance of antigen-responsive B cells in a predictable, temporal order. The results suggest that the acquisition of the expressed repertoire to a large extent is genetically controlled.