Activated Ras signals developmental progression of recombinase-activating gene (RAG)-deficient pro-B lymphocytes.

To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras-RAG). Similar to the effects of an immunoglobulin (Ig) mu heavy chain (HC) transgene, activated Ras caused progression of RAG1-deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of lambda5, RAG2, and germline kappa locus transcripts. However, these Ras-RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig mu HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.

Allelic exclusion of the T cell receptor beta locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adaptor protein.

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

MHC class I expression in mice lacking the proteasome subunit LMP-7.

Proteasomes degrade endogenous proteins. Two subunits, LMP-2 and LMP-7, are encoded in a region of the major histocompatibility complex (MHC) that is critical for class I-restricted antigen presentation. Mice with a targeted deletion of the gene encoding LMP-7 have reduced levels of MHC class I cell-surface expression and present the endogenous antigen HY inefficiently; addition of peptides to splenocytes deficient in LMP-7 restores wild-type class I expression levels. This demonstrates the involvement of LMP-7 in the MHC class I presentation pathway and suggests that LMP-7 functions as an integral part of the peptide supply machinery.

Developmental regulation of V(D)J recombination and lymphocyte differentiation.

Recent insights into the mechanism of V(D)J recombination have clarified the direct role of the products of the recombination-activating genes Rag-1 and Rag-2 in site-specific DNA cleavage at recombination signal sequences and have identified components of the general DNA double-strand break repair pathway that participate in the rejoining of the Rag-1 and Rag-2-cut receptor gene segments. The V(D)J reaction is restricted to particular antigen receptor loci in a lineage-specific and stage-specific manner. This specificity appears to involve cis-regulatory elements, some of which also regulate transcription of the germline antigen receptor loci. Early developmental steps in the T and B lineages - including phenotypic differentiation, expansion of precursors, and selection processes - are effected in a stepwise fashion by signals generated, at least in part, by the products of the functionally rearranged antigen receptor genes themselves.

Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction.

BACKGROUND: Antigen-receptor interactions on lymphocytes result in local clustering of actin, receptors and signaling molecules into an asymmetric membrane structure termed a cap. Although actin polymerization is known to be required, the mechanisms underlying cap formation are unclear. We have studied the events underlying cap formation using mice bearing a null mutation in vav (vav-/-), a gene that encodes a guanine-nucleotide exchange factor for the GTPase Rac. RESULTS: Lymphocytes from vav-/- mice failed to form T-cell receptor caps following activation and had a defective actin cytoskeleton. The vav-/- T cells were deficient in interleukin-2 (IL-2) production and proliferation, and the peak of Ca2+ mobilization was reduced although of normal duration. Activation of Jun N-terminal kinase or stress-activated kinase (JNK or SAPK) and mitogen-activated protein kinase (MAPK) and the induction of the transcription factor NF-ATc1 and egr-1 genes was normal. Despite the reduced Ca2+ mobilization, translocation of cytoplasmic NF-ATc to the nucleus was normal, reflecting that the lower levels of Ca2+ in vav-/- cells were still sufficient to activate calcineurin. Treatment of lymphocytes with cytochalasin D, which blocks actin polymerization, inhibited cap formation and produced defects in signaling and IL-2 transcriptional induction in response to antigen-receptor signaling that were nearly identical to those seen in vav-/- cells. In transfection studies, either constitutively active Vav or Rac could complement constitutively active calcineurin to activate NF-AT-dependent transcription. CONCLUSIONS: These results indicate that Vav is required for cap formation in lymphocytes. Furthermore, the correlation between cap formation, IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals.

Vav family proteins couple to diverse cell surface receptors.

Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFkappaB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.

Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry.

Apoptosis, i.e., programmed cell death, may be the mechanism by which both autoreactive and unselected immature CD4+8+ thymocytes are eliminated in the thymus. In the present paper we describe a simple and rapid flow cytometric method which permits one to study the induction and kinetics of apoptosis of CD4+8+ thymocytes using in vivo and in vitro suspension cultures. Analysing the level of surface expression of CD4 and CD8 molecules, forward light scatter and side (90 degrees) scatter as well as staining with ethidium bromide, three distinct stages of apoptosis of CD4+8+ thymocytes were defined. By counting cells passing through different stages of apoptosis one can attempt to quantitate this process. This method should be useful for in vitro studies on the mechanisms of negative and positive selection of CD4+8+ thymocytes, i.e., induction and inhibition of apoptosis respectively.

A protein cross-reacting with anti-spectrin antibodies is present in higher plant cells.

Proteins that react with anti-human spectrin antibodies raised in rabbit were found in pea seedlings and leaves. The immunoreactive proteins seem to be associated with the membranes and can be extracted with low ionic strength solutions.

CD69 expression during selection and maturation of CD4+8+ thymocytes.

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4+8+ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca++ flux. In contrast, a small proportion of CD4+8+TCRlow and most TCRhigh (CD4+8+ and CD4-8+) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate--a direct activator of PKC--induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4+8+ thymocytes results on coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4+8+ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells.

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.

Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo.

To investigate the role of the receptor-type tyrosine kinase, c-kit and its ligand, stem cell factor (SCF) in T cell development, we analyzed c-kit (W/W) and SCF (SI/SI) deficient mice. We also engrafted wild-type or SCF-deficient fetal thymi onto wild-type recipient mice and analyzed the rate of proliferation by in vivo bromodeoxyuridine labeling. The results show that the most immature thymocyte compartment defined as CD3-CD4-CD8- is significantly reduced in SI/SI grafts and W/W thymi compared with wild-type counterparts. Also, the expansion rate of these immature thymocytes in SI/SI graft is reduced by -50%. These experiments provide direct evidence for an important role for c-kit-SCF interactions in expansion of very early thymocytes.

Small CD4+8+TCRlow thymocytes contain precursors of mature T cells.

To evaluate directly the developmental potential of cortical CD4+8+ thymocytes, highly purified populations of small, nondividing CD4+8+TCRlow and large, dividing CD4+8+TCRhigh thymocytes from H-2d mice expressing a transgenic T cell receptor restricted by H-2Db (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H-2b recipient mice. The results show that both populations generate CD4-8+ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a 'dead end' but an important intermediate stage in T cell development.

Central tolerance: clonal deletion or clonal arrest?

Studies in various experimental animals have shown that developing T cells with specificity for self antigens can be prevented from maturation at an early stage of development. While several in vitro and in vivo experiments have shown that the mechanism of silencing autospecific T cells is the deletion of immature CD4+8+ thymocytes other experiments were interpreted to indicate that tolerance could also result from developmental arrest of more immature CD4-8+ thymocytes not involving cell death. Here we show that immature CD4-8+ cells when confronted with T cell receptor ligands in vitro neither survive nor differentiate into cells which cannot be deleted, indicating that clonal elimination rather than developmental arrest is the mechanism of central tolerance of all immature T cells.

Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene.

The product of the vav proto-oncogene, p95vav or Vav, is tyrosine phosphorylated upon stimulation of T and B cells by antigen and other receptors, and contains motifs associated with signal transduction. To determine its role in vivo, we used vav-gene-targeted embryonic stem cells and RAG-2-/- blastocyst complementation. The vav(-/-)-RAG-2-/- chimaeras displayed thymic atrophy with reduced numbers of peripheral T cells. Whereas the total number of B cells was normal, the subset of peritoneal B-1 (CD5+) cells was missing. The vav-/- T and B cells were hyporeactive when stimulated through antigen receptors, but vav-/- T cells proliferated on exposure to phorbol ester and calcium ionophore, whereas B cells responded normally to bacterial mitogen, lipopolysaccharide or the CD40 ligand. Thus, we have established here a functional role for vav in the control of T- and B-cell development and activation.

Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.

Phenotypic changes accompanying positive selection of CD4+CD8+ thymocytes.

We know little about the way mature CD4 (helper) and CD8 (killer) T cells develop from thymic CD4+CD8+ precursors. Here we show that small but not large CD4+CD8+ cells with high levels of the alpha beta T cell receptor (TcRhigh) result from positive selection. Neither CD4+CD8+ cells with low TcR levels nor large CD4+CD8+ thymocytes with high TcR levels differentiate in vitro. However, small CD4+CD8+ cells with high TcR levels develop in vitro into mature cells by gradually decreasing the surface levels of one or the other co-receptor and acquiring the potential to respond with proliferation to ligation of the TcR. Small CD4+CD8+ cells with high levels of a major histocompatibility complex (MHC) class I-restricted transgenic TcR develop in vitro exclusively into CD4-CD8+ cells while small CD4+CD8+ TcRhigh cells with heterogeneous TcR from various mice yield both CD4 and CD8 T cells. While these experiments are consistent with an instructive model of CD4/CD8 lineage commitment they do not rule out other mechanisms which require multiple TcR-MHC ligand interactions in the generation of mature alpha beta T cells.

Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras.

During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement.

The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages.

The T cell leukemia oncoprotein SCL/tal-1, a basic-helix-loop-helix transcription factor, is required for production of embryonic red blood cells in the mouse yolk sac. To define roles in other lineages, we studied the hematopoietic potential of homozygous mutant SCL/tal-1 -/- embryonic stem cells upon in vitro differentiation and in vivo in chimeric mice. Here we show that in the absence of SCL/tal-1, hematopoiesis, Including the generation of red cells, myeloid cells, megakaryocytes, mast cells, and both T and B lymphoid cells, is undetectable. These findings suggest that SCL/tal-1 functions very early in hematopoietic development, either in specification of ventral mesoderm to a blood cell fate, or in formation or maintenance of immature progenitors.