Expression of the chicken lysozyme locus in macrophages is regulated by at least six different positive and negative cis-regulatory elements. Chromatin of the chicken lysozyme locus is gradually reorganized during macrophage differentiation, indicating that each cis-regulatory element is activated at a different developmental stage. Irrespective of their differential developmental activation, individual cis-regulatory regions are capable of driving transcription of the lysozyme gene in mature macrophages of transgenic mice. In order to examine the role of different cis-regulatory regions in lysozyme locus activation, we analyzed the time course of transcriptional up-regulation of deletion mutants of the lysozyme locus in a new in vitro differentiation system based on enriched primary macrophage precursor cells from the bone marrow of transgenic mice. We show that constructs carrying cis-regulatory elements which are structurally reorganized early in development are also transcriptionally active at an early stage. A construct in which the early enhancer has been deleted shows a delay in transcriptional activation. The presence or absence of a negative regulatory element has no influence on the time course of transcriptional activation of the lysozyme locus.

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages, We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220(+) population generated a single transient wave of IgM(+) IgD(+) B cells but failed to generate T cells, In contrast, transfer of the B220(-) fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hematolymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.

In vitro differentiated embryonic stem (ES) cells contain a population which is similar to fetal liver pro/pre-B cells on the basis of cell surface antigens and cytoplasmic expression of immunoglobin heavy chain. This population was purified and transplanted into Rag-1 deficient recipients to characterize its developmental potential in vivo. Following intravenous transfer, these cells rapidly reconstituted the splenic B but not the T cell compartment. Reconstitution was transient, indicating the lack of long-term reconstituting capacity. Similar to fetal liver, B-1 type as well as conventional B cells were generated, accompanied by high serum IgM levels. Intraperitoneal injection generated high numbers of peritoneal B cells, predominately of the B-1a phenotype, with poor splenic repopulation and low serum IgM levels. These observations suggest the emergence of two different B lineage precursor populations during in vitro ES cell differentiation and define a possible role of the microenvironment in directing lymphoid development.

Thymocytes with a CD4hiCD8lo coreceptor‐skewed (CRS) phenotype have been shown to contain precursors for CD8 single‐positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4hiCD8lo cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I‐restricted P14 T cell receptor (TCR), on the H‐2bm13 background, generate a large number of CD4hiCD8lo CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4hiCD8lo population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up‐regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re‐expressed CD8 but failed to re‐express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide‐pulsed antigen‐presenting cells. Our data suggest that CD4hiCD8lo CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.

In several experimental systems analyzing the generation of single positive (SP) thymocytes from double positive (DP) thymocytes, CD4 SP cells have been shown to appear before CD8 SP cells. This apparent temporal asymmetry in the maturation of CD4 SP and CD8 SP thymocytes could either be due to divergent molecular differentiation programs of the two T cell lineages, or merely to slower degradation kinetics of the CD4 protein. To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells. We show that these TCRintCD69pos thymocytes represent a transitory stage in the mainstream αβ T cell lineage. The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells. Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

After productive rearrangement of a TCR beta chain gene, CD4(-)8(-) double negative (DN) thymocytes express TCR beta polypeptide chains on the cell surface together with pre-T alpha and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCR beta(+) DN thymocytes for further maturation to the CD4(+)8(+) double positive stage, whereas DN cells that fail to generate a productive TCR beta gene rearrangement do not continue in development. This process is termed TCR beta chain selection. Although it is likely that differences between proliferation dynamics of TCR beta(+) and TCR beta(-) cells may play a role, the exact mechanisms of TCR beta chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCR beta(+) and TCR beta(-) thymocytes during fetal development, i.e., when TCR beta chain selection takes place for the first time. We analyzed in situ accumulation of TCR beta(+) thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCR beta(+) and TCR beta(-) populations by flow cytometry. About 600 TCR beta(+) cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5. and 15.5. As of dg 14.5, most TCR beta(+) cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCR beta(+) cells has slowed down to less than 1/d. About three quarters of TCR beta(-) cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCR beta(-) cells, but not TCR beta(+) cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCR beta(-) cells. Positive selection of fetal TCR beta(+) cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.

We describe a novel mAb (F3) which reacts with a 65 kDa thymocyte surface protein, expressed on approximately 80% of thymocytes, referred to as F3Ag. In ontogeny, F3Ag expression begins in the CD4(-)CD8(-) double-negative (DN) CD25(+) population and is maintained through approximately 85% of the CD4(+)CD8(+) double-positive (DP) stage. DP cells with high TCR expression and CD4(+) single-positive (SP) cells are predominantly negative for F3Ag, whereas many CD8(+) SP thymocytes express F3Ag. F3Ag-DP thymocytes show a reduced expression of RAG-1 and RAG-2 compared with F3Ag+ DP cells. The shutdown of F3Ag expression during the DP stage is related to positive selection: mice deficient for MHC class I and class II molecules maintain F3Ag expression in almost all DP cells. Transgenic (tg) mice carrying TCR restricted for MHC class II show a more pronounced down-regulation of F3Ag in the DP compartment than normal mice, depending on the presence of a positively selecting MHC. The size of the F3Ag- DP subset is positively correlated with the efficacy of positive selection into the CD4(+) SP compartment. Because some CD8(+) SP cells express F3Ag, the relationship between F3Ag down-regulation and positive selection is less obvious in DP cells of mice carrying MHC class I-restricted tg TCR. However, in reaggregate thymic organ cultures, sorted F3Ag- DP cells differentiate into CD8(+) SP cells more rapidly than do F3Ag+ DP cells. Thus, after down-regulation in the DP stage, a proportion of CD8(+) SP cells appears to re-express F3Ag. In addition, the proportion of F3Ag-CD8(+) SP cells depends on the efficacy of positive selection into the CD8 lineage. Taken together, the regulation of the expression of the F3Ag appears to be associated with signals that control thymic repertoire selection.