Mice lacking p27(Kip1) have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs ...exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27(-/-) thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similar to mice with the Rb mutation, the p27(-/-) mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27(Kip1) acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFbeta, rapamycin, or contact inhibition remained intact in p27(-/-) cells, suggesting that p27(Kip1) is not required in these pathways.
bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic ...cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.
During thymic development, T cells that can recognize foreign antigen in association with self major histocompatibility complex (MHC) are selected for survival (positive selection) and autoreactive T ...cells are eliminated (negative selection). Both of these selective events are mediated by interaction between the T-cell receptor (TCR) and the peptide-MHC complex. But the signalling pathways that lead to cell survival or to cell death are still unclear. ZAP-70 is a protein tyrosine kinase (PTK) that is associated with the TCR signalling subunits (CD3 and zeta) and is expressed in T cells and natural killer cells. It has been shown that ZAP-70 plays a crucial role in T-cell activation and development. Here we show that mice lacking ZAP-70 had neither CD4 nor CD8 single-positive T cells, but human ZAP-70 reconstituted both CD4 and CD8 single-positive populations. Moreover, ZAP-70-/- thymocytes were not deleted by peptide antigens. Natural killer cell function was intact in the absence of ZAP-70. These data suggest that ZAP-70 is a central signalling molecule during thymic selection for CD4 and CD8 lineage.
The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into ...phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and γ-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth.
Introduction of TCRα transgene, TCRβ transgene, or both into RAG-2$^{-/-}$ mice differentially rescues T cell development. RAG-2$^{-/-}$ mice have small numbers of TCR$^-$CD4$^-$CD8$^-$ (double ...negative, DN) thymocytes that express CD3γδε and ζ proteins intracellularly. Introduction of a TCRβ transgene, but not a TCRα transgene, into the RAG-2$^{-/-}$ background restored normal numbers of thymocytes. These cells were CD4$^+$CD8$^+$ (double positive, DP) and expressed small amounts of surface TCRβ chain dimers in association with CD3γδε but not ζ. RAG-2$^{-/-}$ mice that expressed α and β TCR transgenes developed both DP and single positive thymocytes. Thus, the TCRβ subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.
An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of ...CD4-CD8-double negative (DN) cells to CD4+CD8+double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.
The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells ...and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.
SATB1 is expressed primarily in thymocytes and can act as a transcriptional repressor. SATB1 binds in vivo to the matrix attachment regions (MARs) of DNA, which are implicated in the loop domain ...organization of chromatin. The role of MAR-binding proteins in specific cell lineages is unknown. We generated SATB1-null mice to determine how SATB1 functions in the T-cell lineage. SATB1-null mice are small in size, have disproportionately small thymi and spleens, and die at 3 weeks of age. At the cellular level, multiple defects in T-cell development were observed. Immature CD3(-)CD4(-)CD8(-) triple negative (TN) thymocytes were greatly reduced in number, and thymocyte development was blocked mainly at the DP stage. The few peripheral CD4(+) single positive (SP) cells underwent apoptosis and failed to proliferate in response to activating stimuli. At the molecular level, among 589 genes examined, at least 2% of genes including a proto-oncogene, cytokine receptor genes, and apoptosis-related genes were derepressed at inappropriate stages of T-cell development in SATB1-null mice. For example, IL-2Ralpha and IL-7Ralpha genes were ectopically transcribed in CD4(+)CD8(+) double positive (DP) thymocytes. SATB1 appears to orchestrate the temporal and spatial expression of genes during T-cell development, thereby ensuring the proper development of this lineage. Our data provide the first evidence that MAR-binding proteins can act as global regulators of cell function in specific cell lineages.
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