Biomedical subjects

A Cumano

Publications and source records attributed to A Cumano.

At least 37 records · Page 2Linked to original sources

PubMed1998

Extensive junctional diversity of Ig heavy chain rearrangements generated in the progeny of single fetal multipotent hematopoietic cells in the absence of selection.

We analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.

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PubMed1998

Ontogeny of the heavy chain immunoglobulin repertoire in fetal liver and bone marrow.

We studied the kinetics of maturation of B cell progenitors in the mouse embryo, from day 15 of development to birth, both in liver and bone marrow. The analysis of Ig heavy chain rearrangements at different time points of late fetal development shows that oligoclonal patterns of V(H)-D-J(H) rearrangements are detected by day 15 in fetal liver. The pattern is polyclonal and diverse by day 17; however, 80% of the rearrangements are nonproductive. In bone marrow, the pattern of rearrangements is less diverse at birth, although the percentage of nonproductive rearrangements approaches adult bone marrow levels (35-40%). After day 17 in fetal liver, there is a sudden reversal in the percentage of nonproductive rearrangements that reaches 33% at day 19 (birth). Maturation of B cells, as measured by the fraction of surface Ig+ in total B220+ cells and the presence of N sequence additions in V(H)-D-J(H) joints, occurs in the marrow before fetal liver. These results demonstrate that the lymphopoietic environment in fetal liver and bone marrow of animals at the same stage of development is functionally distinct.

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PubMed1998

Development of T cell precursor activity in the murine fetal liver.

The generation of T cell precursors in the liver of murine embryos was studied. The total number of T cell precursors in the liver was measured in thymic organ cultures by a limiting dilution assay. Sixty T cell precursors were detected in the liver at day 11 of gestation. By day 12 the number of precursors showed a 20-fold increase, half of which could be explained by in situ proliferation as ascertained by a fetal liver organ culture assay. By day 13 a further 2-3-fold increase was observed. Whereas the number of total liver cells continued to increase, that of T cell precursors declined in the following days, suggesting a massive exit of these cells after day 13. The capacity to generate a TCRB repertoire in the cells was evaluated by a PCR assay. T cell precursors in day 11 fetal liver developed a TCRB repertoire at day 8 of culture. The cells from days 12-15 developed an identically diverse repertoire by day 6, suggesting that day 11 precursors are more immature than those of later days. A mechanism for yielding a single wave of T cell precursors in the fetal liver is discussed with a proposed model.

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PubMed1997

TCR-beta repertoire development in the mouse embryo.

We studied the TCR beta-chain repertoire in thymocytes along embryonic development. At day 14 of gestation, complete V-D-J rearrangements of the TCR-beta locus were detected at the messenger RNA level, and a total of 56 different rearrangements were observed in one thymus. At this stage, thymocytes showed preferential usage of TCR-betaV1, TCR-betaV2, and TCR-betaV8 together with TCR-betaJ1S1, -J1S2, and -J2S7 segments. This frequent usage of V and J segments was similar to the usage seen in the adult thymus. Assuming that day 14 thymocytes were not subjected to specificity selection, we conclude that the expression of TCR-betaV and J segments is biased from the beginning of T cell repertoire creation. The first detectable transcripts of rearranged TCR-betas were different among individual fetuses and constituted a random representation of the ones seen at later stages of thymic ontogeny. The diversity of TCR-beta rearrangements increased with time and became indistinguishable from that of the adult by day 16. The first rearranged TCR-alphas were expressed at day 17 and their diversity increased thereafter. We conclude that the diversification of the beta-chain repertoire takes place in 3 days, before the alpha-chain rearrangement begins in transition from CD4/CD8 double negative to double positive cells.

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PubMed1997

Does positive selection determine the B cell repertoire?

To know whether each newly formed B cell has an equal chance of survival in the organism, we analyzed the composition of the B cell repertoire of extremely limited diversity by generating mu-transgenic kappa-knockout mice. Surprisingly, in both types of mice studied, the B cell repertoire is mainly composed of cells expressing the mu-transgene-encoded chain associated with only one out four available lambda types depending on the mu transgene. Moreover, B cell differentiation cultures in vitro show that newly formed B cells can express the various lambda types regardless of the presence or absence of the mu transgenes. These results show a drastic impact of the heavy chain on the lambda light chain repertoire expressed in the periphery. The overexpression of a unique heavy/light chain pairing therefore results from selective processes. The immature B cells may be positively selected to provide the immunocompetent B cells in the periphery.

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PubMed1997

Sox-4 facilitates thymocyte differentiation.

The mouse Sry-like transcription factor Sox-4 is expressed in thymus, bone marrow, and gonads of adult mice. Sox-4-deficient mice die at embryonic day E14 due to cardiac malformation. In transfer experiments to irradiated recipients, B cell development was shown to be severely impaired in Sox-4-deficient progenitor cells. However, no drastic effects on T lymphocyte development were noted, despite the high level expression of the Sox-4 gene in the thymus of normal mice. Here, we report a detailed analysis of T cell development from Sox-4-deficient progenitors. Explanted fetal thymic organ cultures (FTOC) of Sox-4-deficient thymi yielded 10-50-fold fewer CD4 CD8 double-positive and single-positive cells than FTOC of littermates. This effect was T cell-autonomous, since similar observations were made when FTOC were performed by culturing of Sox-4-deficient progenitors in wild-type thymus lobes. When Sox-4-deficient fetal liver cells were injected together with normal cells intrathymically, they did not compete efficiently for reconstitution. It is concluded that Sox-4 facilitates thymocyte development.

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PubMed1997

Maternal immunoglobulins have no effect on the rate of maturation of the B cell compartment of the offspring.

To elucidate a general role of maternal immunoglobulins (Ig) on the kinetics of B cell development of the offspring, we studied non-genetic influences of maternal Ig on the developing immune system of B cell-competent mice. These animals were the offsprings of either B cell-deprived microMT or of normal C57BL/6 females. In these mice, we have compared the kinetics of Ig production, the numbers of B cell progenitors, the expression of surface markers specific of the B lineage and the progression of Ig variable gene expression. We show that the absence of maternal Ig has no detectable effect on the kinetics of IgM and IgG production by the offspring's immune system. The number of B cell precursors, the kinetics of generation of B cells and their pattern of surface markers expression is identical in both types of mice. The acquisition of diversity in the B cell repertoire and the changes in the ratios of variable gene family expression are also indistinguishable. We conclude that maternally derived Ig has no influence on the rate of development and maturation of the B cell compartment of the offspring.

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PubMed1996

Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura.

Emergence of hemopoietic stem cells in the mammalian embryo has yet to be definitively allocated. Previously, we detected multipotent hemopoietic precursors in the region surrounding the dorsal aorta (paraaortic splanchnopleura) beginning at 8.5 days postcoitum (dpc). However, as circulation is already established, it remained unclear whether hemopoietic precursors arise in situ or are blood-delivered. By adding an organotypic step to our former culture system, we now detect lymphocyte and multipotent myeloid precursors from the intraembryonic splanchnopleura as early as 7.5 dpc. Under identical conditions, yolk sacs from the same embryos are unable to generate lymphoid progeny and have a reduced potential for myeloid differentiation and maintenance. Thus, if isolated before circulation, the yolk sac does not produce multipotent precursors and therefore does not contribute to definitive hemopoiesis in the mouse.

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PubMed1996

Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4.

A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro.

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PubMed1996

The expansion and selection of T cell receptor alpha beta intestinal intraepithelial T cell clones.

In conventional mice, the T cell receptor (TCR) alpha beta+ CD8 alpha alpha+ and CD8 alpha beta+ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Ep. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8 alpha alpha and CD8 alpha beta TCR alpha beta+ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCR alpha beta+ T cells than conventional mice. The TCR beta repertoire of the CD8 alpha alpha and the CD8 alpha beta IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show the intestinal microflora is not responsible for the repertoire oligoclonality of TCR alpha beta+ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8 alpha alpha and the CD8 alpha beta TCR alpha beta IEL clones have a heterogeneous size pattern, with clones containing from 3 x 10(3) cells up to 1.2 x 10(6) cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCR alpha beta IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.

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PubMed1996

Circulation of hematopoietic progenitors in the mouse embryo.

We used a sensitive in vitro culture system to follow, in embryonic blood, the number and state of commitment of B cell precursors along ontogeny. We describe a wave of circulating multipotent progenitors, first detectable at day 10 of gestation, and reaching a maximum in absolute numbers at day 12. They are undetectable by day 14 of gestation, when committed B cell precursors can be detected in fetal liver. Embryonic marrow contains B cell progenitors by day 15. We propose that fetal liver, thymus, and bone marrow are colonized by the same wave of multipotent hematopoietic cells and define embryonic blood at day 11 of gestation as an important source of multipotent hematopoietic cells, virtually deprived of committed and mature contaminants.

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PubMed1995

PCR-based analysis of the murine immunoglobulin heavy-chain repertoire.

The immunoglobulin heavy-chain repertoire has been mainly analysed by studying the proportion of genes belonging to each of the 14 described families, in terms of the expressed immunoglobulin molecules. Although the proportion of each variable gene family is kept stable throughout adult life and in different mice of the same strain, little information is available on the clonal representation in the repertoire of activated B cells. We describe here a new method that permits an approach to this question by separating the products of a polymerase chain reaction covering the VH-D-JH junction of the immunoglobulin heavy chain gene in a sequencing gel, thereby allowing discrimination of different rearrangements (according to their length) using a given JH and one of the member of a given VH family. Using this method, we show that it is possible to obtain a precise overview of the repertoire of activated B cells, at the mRNA level, as well as the potential repertoire, from a study of the DNA. We also show that this approach permits the detection of an induced immune B cell response and studies of emerging dominant specific B cell clones.

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PubMed1995

Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus.

We show by an in vitro approach that multipotent hemopoietic cells can be detected in the body of the mouse embryo between the stages of 10-25 somites (8.5-9.5 days of gestation)--i.e., prior to liver colonization (28-32 pairs of somites). Interestingly, hemopoietic cells appear in parallel in this location, the paraaortic splanchnopleura, and in the yolk sac, where they represent a new generation by reference to the primitive hemopoietic stem cells. Lymphoid cell clones, which could differentiate into mature B cells, were obtained from yolk sac and paraaortic splanchnopleura cell preparations but not from other tissues of the embryonic body. These B-cell precursors were first detected around the stage of 10 somites; thereafter, their initial minute numbers increased in parallel in the yolk sac and the paraaortic splanchnopleura, suggesting that their emergence in the two sites was simultaneous. By single cell manipulation, we show that these precursors can generate B and T lymphocytes and myeloid cells; these precursors can thus be defined as multipotent hemopoietic cells.

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PubMed1995

The TCR-beta chain repertoire of gut-derived T lymphocytes.

The gastrointestinal tract represents a natural barrier to environmental pathogens. The gut-associated lymphoid tissue (GALT) provides protection and interacts with the external environment, e.g. bacteria and food antigens. Large numbers of leukocytes are present as aggregates in the lymphoid follicles and as single cells in the lamina propria and epithelium. Intraepithelial T lymphocytes are localized within the tight junctions between epithelial cells. In conventional adult mice, they mainly express CD8, with half of the cells expressing the common CD8 molecule, the CD8 alpha beta heterodimer and the other half expressing a homodimer CD8 alpha alpha. The CD8 alpha beta IEL bear a TcR alpha beta while CD8 alpha alpha bear either a TcR gamma delta or a TcR alpha beta. Studies of repertoire with human IEL cell lines and with total human IEL population showed that both the alpha-chain and the beta-chain repertories are oligoclonal. Our own results of the analysis of repertoire diversity in genetically identical mice raised the same environment have shown that both CD8 alpha alpha and CD8 alpha beta TcR alpha beta IEL populations express a distinct oligoclonal repertoire that differs from one individual to the next. Compared to the lymph node lymphocytes, both IEL populations are composed of a very limited number of clones that undergo clonal expansion. Furthermore, the study of repertoire throughout the length of the small intestine has revealed that some of the clones are present all along the epithelium suggesting that the CD8 alpha alpha IEL and CD8 alpha beta IEL are capable of recirculating or migrating in the intestine. We propose that the repertoire of TcR alpha beta IEL is derived from a restricted number of T cells that either develop in situ or that arrive through circulation and can then undergo clonal expansion.

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PubMed1995

B-lymphoid potential in pre-liver mouse embryo.

Increasing interest in the initial steps of hemopoiesis in the embryo is prompted by the lack of information about the primordial origin and the nature of hemopoietic stem cells. It appears critical to understand the emergence, diversification and differentiation potential of hemopoietic cells in the preliver embryo. In-vitro studies of B-potential in the mouse embryo prior to liver colonization show that, contrary to interpretations prevalent 10 years ago, there are two sources of B-cell progenitors (and most probably stem cells) at these stages: the yolk sac and the paraaortic splanchnopleura within the embryo proper. The analysis of the phenotype and differentiation potential of precursors from both sources shows them to be very similar, while different from the fetal liver progenitors. Indications for an independent generation of precursors in both sites are reviewed.

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PubMed1995

[Intra-embryonic hematopoiesis in mice].

In vertebrate embryos, all hemopoietic tissues that are successively active during ontogeny (fetal liver, thymus, spleen and bone marrow) are colonized by extrinsic hemopoietic stem cells (HSC). The exception to this rule is the yolk sac (YS) whose progenitors arise in situ. Moreover, the YS is the first hemopoietic site to appear in the embryo. In consequence, it was considered as the primary source of the HSC that transfer from one site to the other until the demonstration that, in birds, the source of definitive HSC was located in the aortic region of the embryo. To assess a possible contribution of intraembryonic sites to mouse hemopoietic development, we developed an in vitro approach that permits the detection of multipotent hemopoietic progenitors in the region surrounding the dorsal aorta. This new site, the paraaortic splanchnopleura (P-Sp), is active from the 10 somite stage to the stage of fetal liver colonization by HSC. The in vitro analysis of the hemopoietic potential of the P-Sp revealed that: 1) progenitors present in this region are multipotent since a single micromanipulated P-Sp cell can give rise to mature B and T lymphocytes and various myeloid cells, when cultivated in appropriate conditions; 2) the P-Sp is the only intraembryonic site endowed with this potential; 3) numbering progenitors present in the YS and P-Sp indicated that precursors appear in both locations at the 10-12 somite stage reaching 15 in each site at 9.5 dpc (25 somites), shortly before the beginning of fetal liver colonisation. During a cytological study of the 9-11 dpc embryo aimed to disclose the cellular basis for our experimental results, two potential hemopoietic sites were uncovered. Cells clusters that resemble avian intraaortic hemopoietic clusters were found in the arteries located close to the coelomic cavity (dorsal aorta, umbilical and omphalomesenteric arteries). Moreover, groups of cells strikingly similar to YS blood islands were uncovered in the mesentery. All these sites are located in areas formed from the P-Sp and are fully developed at the onset of fetal liver colonization. These hemogenic sites most probably develop from the hemopoietic precursors present in the P-Sp.

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PubMed1994

Oligoclonal repertoire of the CD8 alpha alpha and the CD8 alpha beta TCR-alpha/beta murine intestinal intraepithelial T lymphocytes: evidence for the random emergence of T cells.

The epithelium of the small intestine in normal euthymic mice contains a large number of intraepithelial lymphocytes (IEL), some of which bear a T cell receptor alpha/beta (TCR-alpha/beta). About half of these TCR-alpha/beta IEL display the CD8 alpha alpha phenotype and the remaining have the CD8 alpha beta or the CD4 phenotypes. To examine whether TCR-alpha/beta IEL have a TCR-beta chain repertoire as diverse as that of TCR-alpha/beta lymph node lymphocytes (LNL), we used a recently described PCR technique that allows a global analysis of the TCR-beta chain repertoire. Within any given mouse, the repertoires expressed in both CD8 alpha alpha and CD8 alpha beta TCR-alpha/beta IEL populations are oligoclonal and nonoverlapping between the two subsets. The clones are largely conserved through the length of the small intestine of the same individual. However, genetically identical individuals raised under indistinguishable environmental conditions display distinct oligoclonal repertoires. Those findings indicate that few cells of CD8 alpha alpha or of the CD8 alpha beta phenotype are responsible for the repopulation of the intestinal epithelium.

Amino Acid Sequence↗

PubMed1994

Stromal cell independent growth of bipotent B cell--macrophage precursors from murine fetal liver.

We have established stromal cell independent culture conditions under which single murine hematopoietic progenitor cells give rise to B lymphocytes and macrophages. IL-11, mast cell growth factor, and IL-7 are sufficient for the growth of B lineage cells from AA4.1+B220-Mac-1-Ly6A+ fetal liver cells isolated at day 12 of gestation. These conditions are also sufficient for commitment to myeloid differentiation, although CSF-1 is required for substantial growth of macrophages. The progenitors responding under these culture conditions: (i) require 8 days in culture before they become competent to respond to B cell mitogens in the presence of stromal cells and (ii) undergo a minimum of three cell divisions prior to Ig heavy chain allotype restriction. The findings suggest that the stromal cell dependency of immature hematopoietic cells may reflect a requirement for stromal cell derived growth factors. Further, this provides an in vitro system in which commitment and differentiation can be studied in single cells.

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