Functional and molecular analysis of hematopoietic progenitors derived from the aorta-gonad-mesonephros region of the mouse embryo.

Herein, we show that CD34, c-kit double-positive (CD34(+)c-kit(+)) cells from the aorta-gonad-mesonephros (AGM) region of the developing mouse are multipotent in vitro and can undergo both B-lymphoid and multimyeloid differentiation. Molecular analysis of individual CD34(+)c-kit(+) cells by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) shows coactivation of erythroid (beta-globin) and myeloid (myeloperoxidase [MPO]) but not lymphoid-affiliated (CD3, Thy-1, and lambda5) genes. Additionally, most cells coexpress the stem cell-associated transcriptional regulators AML-1, PU.1, GATA-2 and Lmo2, as well as the granulocyte colony-stimulating factor receptor (G-CSF-R). These results show that the CD34(+)c-kit(+) population from the AGM represents a highly enriched source of multipotent hematopoietic cells, and suggest that limited coactivation of distinct lineage-affiliated genes is an early event in the generation of hematopoietic stem and progenitor cells during ontogeny.

Stromal expression of Jagged 1 promotes colony formation by fetal hematopoietic progenitor cells.

The Notch signaling system regulates proliferation and differentiation in many tissues. Notch is a transmembrane receptor activated by ligands expressed on adjacent cells. Hematopoietic stem cells and early progenitors express Notch, making the stromal cells which form cell-cell contacts with progenitor cells candidate ligand-presenting cells in the hematopoietic microenvironment. Therefore, we examined primary stromal cell cultures for expression of Notch ligands. Using reverse transcription-polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting, we demonstrate expression of Jagged 1 in primary stromal cultures. To investigate if the stromal expression of Jagged 1 has functional effects on hematopoietic progenitors, we cultured CD34(+), c-kit+ hematopoietic progenitor cells derived from the aorto gonadal mesonephros region of day 11 mouse embryos on the Jagged 1(-) stromal cell line S17 and on S17 cells engineered to express Jagged 1. The presence of Jagged 1 increased the number of colonies formed in subsequent methylcellulose culture fourfold. Larger increases in colony numbers were observed under the same culture conditions with CD34(+), c-kit+ hematopoietic progenitor cells derived from d11 fetal liver. These results obtained in vitro table Jagged 1 as a candidate regulator of stem cell fate in the context of stromal microenvironments in vivo.

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.

Quantification of cytokine transcripts using polymerase chain reaction.

Quantitative PCR techniques are both attractive and daunting. Most users appreciate the need for accurate quantification, but achieving this is not always straightforward. It usually requires more technical equipment than classical PCR and common sense controls are no longer sufficient to generate confidence in the quantitative power of the assay. This review describes the principles that underlie the most commonly used quantitative PCR techniques and their different read-outs, and aims to provide the reader with the tools needed to discriminate between the different published techniques.

Clonal expansions of B lymphocytes in old mice.

The effect of age on the diversity of the murine Ig heavy chain repertoire has been studied in unimmunized C57BL/6 mice. We examined the heterogeneity of complementarity-determining region 3 (CDR3) sizes of Ig mRNA of the IgM and IgG isotypes using two VH families, VHJ558 and VHQ52, which together account for approximately 65% of the Ab repertoire. The broad and bell-shaped profiles representing the diversity of the VHJ558 family in the spleen of 2- to 6-mo-old C57BL/6 mice becomes significantly less diverse after 12 mo of age and by 18 mo of age, single CDR3 sizes that dominate the profiles can be observed in the spleens of > 85% of the mice. Readable sequences have been obtained from 40 dominant mRNA CDR3 size species indicating that they represent clonal populations of B lineage. There are no significant homologies among these sequences. Clones of B lymphocytes that express a dominant CDR3 mRNA species can also be found in the bone marrow, the mesenteric lymph nodes, and the thymus of C57BL/6 mice > 18 mo of age. Some clones of B cells can be detected in only one lymphoid compartment; others are found in two or more compartments. The splenic B cell clones in C57BL/6 mice > 18 mo of age are stable for at least 2 mo. The CDR3 mRNA species that dominate the splenic repertoire of Ig mRNA-expressing cells in vivo do not dominate the repertoire of splenic B cells activated in vitro by bacterial LPS, suggesting that they represent a modest population of B cells expressing high levels of Ig mRNA.

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.

Quantitative PCR with colorimetric detection.

We describe a quantitative polymerase chain reaction (PCR) technique with a colorimetric read-out, which enables quantitation of various sequences on a single microplate within one day. Using the quantitation of human IL10 and beta-actin transcripts, we show that this technique provides better reproducibility, as results are derived from a series of PCRs made with various amounts of standard. This also enables the control of the consistency of the data and elimination of some artifactual results.

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.

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.

Developmental shift in the patterns of interleukin production in early post-natal life.

In mice, immunological tolerance to self is established in the perinatal period, when tolerance susceptibility to allogenic tissues is higher than in adults. We have now investigated whether this could result from developmental regulation of effector functions of T cells exposed to specific antigens, by studying the "natural" or T cell receptor-induced expression of several interleukin genes. We used qualitative and quantitative polymerase chain reaction methods to study interleukin (IL)-2, IL-4, IL-10 and interferon-gamma mRNA expression by splenic cells at different ages. The results show that newborn peripheral cells (up to day 7), in contrast to the T lymphocytes of adult mice, express high levels of IL-4 and interferon-gamma, and very low levels of IL-2 messenger spontaneously and upon specific T cell activation. This characteristic phenotype depends on intrinsic T cell properties, as it is not due to the newborn environment.

Differential cytokine expression in maternal blood and placenta during murine gestation.

To evaluate the influence of gestation on peripheral blood T cells, we measured the mRNA production of several cytokines (IL-2, IL-4, IL-10, IFN-gamma) and the p55 subunit of IL-2R at different time points in the blood of pregnant mice and in the placenta. This was made possible by the use of a new PCR technique that is precise and quantitative. Our results show that pregnancy induces profound changes in the expression of these genes in peripheral blood cells. During the first week of gestation, there is an increase in the levels of all the cytokines, followed by a state of immunodepression characterized by levels of cytokines below normal (nonpregnant mice). In the placenta low levels of IL-2 and IL-10 are detected. IFN-gamma mRNA production is higher than the blood IFN-gamma mRNA in the last week of pregnancy. However, the main difference is found for IL-4 mRNA expression where the placenta levels are 5- to 10-fold higher than the blood mRNA expression. We discuss these results in the context of the placenta as a privileged immune site, where IL-4, being the main cytokine, may play a major regulatory role.

Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation.

In vitro enzymatic amplification of nucleic acids by PCR or other techniques is a very sensitive method to detect rare DNA segments. We present here a protocol that allows the rapid, sensitive and precise quantification of DNA molecules using PCR amplification run to saturation. The DNA (or cDNA) to be assayed is co-amplified with known amounts of an internal standard DNA. We show that the latter must be almost identical to the assayed DNA, otherwise quantification at the plateau is unreliable. The read-out of the amplification involves one or two additional oligonucleotides. Using fluorescent oligonucleotides as primers in run-off reactions together with an automated DNA sequencer, we could measure the level of expression of several genes, like the murine MHC class I H-2Kd or a specific T cell receptor beta chain transcript in the course of an immunization. mRNA levels were normalized by measuring in a similar manner the number of transcripts encoding the housekeeping gene HPRT. Finally, our procedure might allow the rapid analysis of a large number of samples at the same time, as illustrated by the simultaneous analysis of the mRNAs encoding the CD4 and CD8 murine T cell markers.

Nonhomogeneous distribution of human immunodeficiency virus type 1 proviruses in the spleen.

A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 proviruses in an infected spleen was observed. Antigenic stimulation of infected cells might explain this partition.

Absence of selection of HIV-1 variants in vivo based on transcription/transactivation during progression to AIDS.

The activity of the human immunodeficiency virus type 1 (HIV-1) transactivation protagonists tat and TAR has been analyzed from sequential primary material. The sequences were amplified from uncultured peripheral blood mononuclear cells. Despite fluctuations within the tat and TAR quasispecies there was no obvious selection for a variant encoding more powerful transactivation components either in vivo or ex vivo, indicating that this system is not exploited during disease progression. The basal levels of the natural promoters were, depending on the cell line, two- to fourfold higher than that of the reference promoter, itself derived from ex vivo adapted HIV-1 Lai.

LAV revisited: origins of the early HIV-1 isolates from Institut Pasteur.

Two of the first human immunodeficiency virus type-1 (HIV-1) strains isolated were authenticated by reanalyzing original cultured samples stored at the Collection Nationale de Culture des Microorganismes as well as uncultured primary material. Cloned polymerase chain reaction products were used to analyze coding sequences of the V3 loop in the gp120 glycoprotein. The original isolate HIV-1 Bru, formerly called LAV, was derived from patient BRU. HIV-1 Lai was derived from patient LAI and contaminated a HIV-1 Bru culture between 20 July and 3 August 1983. The culture became, in effect, HIV-1 Lai, identifiable by a unique motif in the V3 loop. Because of this contamination two, rather than one, HIV-1 isolates were sent to the Laboratory of Tumor Cell Biology at the National Cancer Institute on 23 September 1983. Original HIV-1 Bru was indeed present in the sample marked JBB/LAV. However the M2T-/B sample harbored HIV-1 Lai, a strain capable of growing on established cell lines. The striking similarity between HIV-1 Lai (formerly LAV-Bru) and HTLV-3B sequences remains.

Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro.

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A. Meyerhans, R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjö, and S. Wain-Hobson, Cell 58:901-910, 1989). Once again in vitro culture resulted in the selection of minor forms. Over a 4-year period in vivo, there was no obvious selection for, or outgrowth of, any particular nef or U3/R sequence. Few defective nef protein sequences were observed, which argues against nef acting as a negative regulatory factor. Although no functionally defective promoter/trans-activation-responsive elements were identified, the transactivation efficiencies varied between 0.2 and 2 times that of the control. The sequence encoding the most efficient trans-activation-responsive region did not outgrow others. The extreme genetic heterogeneity of the different samples of the locus, either in vivo or in vitro, indicates that there is no such thing as a single, distinct HIV sequence. It is suggested that different HIV-1 loci evolve independently, recombination being responsible for their uncoupling.

Genetic organization of gibbon ape leukemia virus.

Gibbon ape leukemia virus (GaLV) is a highly oncogenic C-type retrovirus capable of inducing myeloid leukemia in juvenile gibbons. GaLV is antigenically most closely related to a new world monkey virus, simian sarcoma associated virus (SSAV), and less to the murine and feline C-type leukemia viruses. To begin to understand how this virus induces leukemia at the molecular level, we have sequenced a GaLV genome and shown that it has a "minimal" genetic complement of 5'R-U5-gag-pol-env-U3-R3'. No additional genes could be identified. Such a genetic structure is identical to those of the murine and feline C-type leukemogenic viruses. Despite its suggested murine origin, the GaLV sequence is as closely related to the murine viruses as it is to the feline retroviruses. Finally the GaLV sequence is indistinguishable from that of the fragments of SSAV available indicating that, in fact, SSAV is of gibbon origin.