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M Malissen

Publications and source records attributed to M Malissen.

At least 37 records · Page 2Linked to original sources
PubMed1997

Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals, respectively, to thymic medullary stromal cells.

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3 epsilon-deficient mice where thymocyte maturation is arrested at the CD4- CD8- stage, few scattered 95+ and 29+ epithelial cells are found. When few mature thymocytes develop as in CD3- zeta/eta mice, expansion and organization of 95+ but not 29+ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29+ epithelial cells are absent and 95+ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1+ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95+ and 29+ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29+ medullary epithelial cells.

Animals↗
PubMed1996

Germline genomic structure of the B10.A mouse Tcra-V2 gene subfamily.

The number of mouse Tcra-V gene segments varies from one individual to another and is estimated to be about 100. Southern blot analysis revealed that most of the Tcra-V are organized in clusters composed of copies of Tcra-V belonging to different subfamilies. We analyzed in detail a Tcra-V subfamily and looked for new Tcra-V in order to improve the knowledge of the mouse Tcra locus organization. A series of genomic clones derived from the B10.A mouse strain enclosing these clusters was used to determined the structure of all the Tcra-V2. We were able to identify ten Tcra-V2. This study showed that the Tcra-V2 can be organized into three structural subgroups. The distribution of the genes along the Tcra locus, plus their structural organization, indicates that successive duplications occurred during the processes of expansion and contraction of the Tcra-V gene subfamilies. Several Tcra-V2 are also identical, indicating recent duplications. The most divergent Tcra-V2 differ by 7.4% nucleotides, leading to 5.2% differences in amino acid contents.

Amino Acid Sequence↗
PubMed1996

Functions of TCR and pre-TCR subunits: lessons from gene ablation.

Recent gene-targeting experiments have highlighted the existence of checkpoints that ensure that alpha beta T cells do not complete intrathymic differentiation if they have not attained certain landmark events. These 'proofreading' mechanisms operate by way of the pre-TCR and TCR complexes, which are sequentially expressed during T-cell development. These complexes are likely to signal via their associated CD3 subunits. By activating intracellular effectors, the CD3 subunits probably modulate gene expression profiles and drive the maturing alpha beta T cells through a precise developmental sequence.

Animals↗
PubMed1995

Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene.

To determine which CD3 components are required for early T cell development, we generated mice with a targeted mutation of the CD3-epsilon gene and characterized their T cell populations relative to those found in CD3-zeta/eta-and recombinase activating gene (RAG)-deficient mice. In the absence of intact CD3-epsilon subunit, thymocytes do not progress beyond the CD44-/lowCD25+ triple-negative stage and appear to be arrested at the very same developmental control point as RAG-deficient thymocytes. In contrast, the disruption of the CD3-epsilon/eta gene does not totally abrogate the progression through this control point. CD3-epsilon-deficient thymocytes do rearrange their T cell receptor (TCR) beta gene segments and produce low levels of full-length TCR beta transcripts. Taken together, these results establish an essential role for the CD3-epsilon gene products during T cell development and further suggest that the CD3-epsilon polypeptides start to exert their function as part of a pre-TCR through which CD44-/lowCD25+ triple-negative cells monitor the occurrence of productive TCR beta gene rearrangements. Finally, the absence of intact CD3-epsilon polypeptides had no discernible effect on the completion of TCR gamma and TCR delta gene rearrangements, emphasizing that they are probably not subjected to the same epigenetic controls as those operating on the expression of TCR alpha and beta genes.

Animals↗
PubMed1995

Normal development and function of natural killer cells in CD3 epsilon delta 5/delta 5 mutant mice.

The CD3 epsilon polypeptide contributes to the cell surface display as well as to the signal transduction properties of the T-cell antigen receptor complex. Intriguingly, the distribution of CD3 epsilon is not restricted to T cells, since activated mouse, human, and avian natural killer (NK) cells do express intracytoplasmic CD3 epsilon polypeptides. CD3 epsilon is also present in the cytoplasm of fetal thymic T/NK bipotential progenitor cells, suggesting that it constitutes a component of the NK differentiation program. We report here that the genetic disruption of CD3 epsilon exon 5 alters neither NK cell development nor in vitro and in vivo NK functions, although it profoundly blocked T-cell development. These results support the notion that CD3 epsilon is dispensable for mouse NK cell ontogeny and function and further suggest that the common NK/T-cell progenitor cell utilizes CD3 epsilon as a mandatory component only when differentiating toward the T-cell lineage.

Amino Acid Sequence↗
PubMed1994

Reconstitution of CD3 zeta coupling to calcium mobilization via genetic complementation.

The integrity of the T cell receptor complex (CD3-TCR) transduction machinery is central to T cell development and to T cell effector function. Molecular dissection of the multimeric CD3-TCR complex revealed that at least two associated polypeptides, CD3 zeta and CD3 epsilon, autonomously couple antigenic recognition event to early and late events of the intracytoplasmic activation cascade. A 18-amino acid motif based on a tandem YXXL stretch, the activation receptor homology sequence 1 (ARH-1) motif, is necessary and sufficient to the transducing properties of both CD3 zeta and CD3 epsilon. Stimulation of chimeric molecules made of ecto- and transmembrane domains of various cell surface proteins and intracytoplasmic domains of CD3 epsilon or CD3 zeta leads to an increase in the intracellular Ca2+ concentration ([Ca2+]i) in Jurkat cells. We describe here that a similar CD25/zeta chimeric molecule was unable to induce a detectable [Ca2+]i rise upon CD25 cross-linking once expressed in the murine thymoma BW-. A Ca2+ influx could, however, be triggered in BW- cells by thapsigargin, i.e. following depletion of Ca2+ stores. Somatic cell hybrids made from BW- and either thymocytes or mature lymph node T cells reconstituted the coupling of CD3 zeta to the Ca2+ signal via an ARH-1 motif-dependent pathway. However, pervanadate-induced Ca2+ mobilization, a phenomenon attributed to tyrosine phosphorylation, was impaired in BW-cells and reconstituted in hybridomas. In contrast to the Ca2+ response, IL-2 production was induced in both BW- and hybrids cells, which questions the functional relevance of [Ca2+]i augmentation in T cell activation. In conclusion, the properties of the BW- thymoma, which define a novel group of CD3 zeta transduction cell mutants, as well as its complementation by somatic cell fusion demonstrate that this cell line represents a useful model to dissect the signaling pathway that couples CD3 zeta to Ca2+ mobilization by genetic reconstitution.

Animals↗
PubMed1993

T cell development in mice lacking the CD3-zeta/eta gene.

The CD3-zeta and CD3-eta polypeptides are two of the components of the T cell antigen receptor (TCR) which contribute to its efficient cell surface expression and account for part of its transducing capability. CD3-zeta and CD3-eta result from the alternative splicing of a single gene designated CD3-zeta/eta. To evaluate the role of these subunits during T cell development, we have produced mice with a disrupted CD3-zeta/eta gene. The analysis of thymocyte populations from the CD3-zeta/eta-/- homozygous mutant mice revealed that they have a profound reduction in the surface levels of TCR complexes and that the products of the CD3-zeta/eta gene appear to be needed for the efficient generation and/or survival of CD4+CD8+ thymocytes. Despite the almost total absence of mature single positive thymocytes, the lymph nodes from zeta/eta-/- mice were found to contain unusual CD4+CD8- and CD4-CD8+ single positive cells which were CD3-. In contrast to the situation observed in the thymus, the thymus-independent gut intraepithelial lymphocytes present in zeta/eta-/- mice do express TCR complexes on their surface and these are associated with Fc epsilon RI gamma homodimers. These results establish an essential role for the CD3-zeta/eta gene products during intrathymic T cell differentiation and further emphasize the difference between conventional T cells and thymus-independent gut intraepithelial lymphocytes.

Animals↗
PubMed1992

Regulation of TCR alpha and beta gene allelic exclusion during T-cell development.

Early in their development, most T cells become committed to the expression of one, and only one, TCR alpha beta combination. How do T cells achieve this TCR allelic exclusion? This article discusses the configuration and expression of TCR alpha and beta genes in mature T-cell lines and TCR alpha beta transgenic mice, and proposes three nonexclusive models to account for the significant occurrence of T cells with two productive alpha gene rearrangements.

Alleles↗
PubMed1992

T cell activation and thymic tolerance induction require different adhesion intensities of the CD8 co-receptor.

Activation of mature lymphocytes requires in addition to the TCR contact with the corresponding antigen the binding of the CD8 or CD4 co-receptors to MHC class I or class II proteins respectively. To investigate the contribution of the CD8-class I interaction to the elimination of autoreactive T cells during negative selection in the thymus we generated two types of transgenic mice. One set expressed a modified Kb molecule which contained a human HLA-A2 alpha 3 domain, thereby missing the binding residues for the murine CD8 molecules. The second set of mice expressed an anti-Kb specific TCR. Both lines were crossed and in the resulting double transgenic mice the development of Kb-reactive T cells was followed with an anti-clonotypic antibody. Surprisingly, efficient clonal deletion in the thymus was still observed, although the reduced CD8-class I adhesion abrogated effector functions in vivo and in vitro. These results imply that even T cells with intermediate affinity for self are negatively selected in the thymus despite the fact that they are not able to react against self antigens in the periphery. Thus a safety window is created which decreases the risk of autoaggression.

Animals↗
PubMed1992

Influence of antigen density on degree of clonal deletion in T cell receptor transgenic mice.

The extent of peripheral clonal deletion of the T cell antigen receptor (TCR) from a CD8-dependent cytotoxic T lymphocyte of H-2k origin with alloreactivity for H-2Kb was measured in a TCR transgenic (Tg) model by use of a clonotype-specific mAb (anti-Ti mAb). Deletion varied depending on whether the TCR Tg was expressed in mice heterozygous (H-2k x b) or homozygous (H-2b x b) for the H-2Kb antigen. CD8 surface staining and functional analyses of peripheral T cells stimulated with polyclonal activators in bulk culture or by limiting dilution confirmed that in the H-2b x b mice few Ti+ cells were generated as measured by anti-Ti mAb-dependent target cell killing; while such cells could readily be detected in the H-2k x b mice. The latter maintained non-responsiveness to H-2Kb, as measured by cytolysis of H-2Kb-expressing tumor target cells, due to their down-regulation of surface CD8 expression. The question of whether the difference between H-2b x b and H-2k x b mice was due to the influence of positive selection imposed by the k haplotype in the H-2k x b hybrid, or to the lower antigen density in heterozygous than homozygous mice was addressed by analysis of H-2b x d Tg mice, H-2d being a non-selecting haplotype. Results obtained were similar for H-2b x d and H-2k x b Tg mice, suggesting that the density of the H-2Kb antigen may be one of the parameters controlling the extent of clonal deletion in the thymus.

Animals↗
PubMed1992

Distinct mechanisms of extrathymic T cell tolerance due to differential expression of self antigen.

Self-reactive T lymphocytes escaping thymic tolerance induction can be rendered non-responsive by contact with antigens in the periphery. In order to determine the parameters controlling peripheral tolerance induction we followed the fate of one well-defined self-reactive T cell population in three different mice expressing the self antigen in various nonlymphoid tissues outside the thymus. This was achieved by crossing anti-Kb T cell receptor (TCR) transgenic mice with transgenic animals expressing the Kb antigen exclusively on hepatocytes or keratinocytes or neuroectodermal cells. Due to this differential expression clonotype+, anti-Kb reactive T cells were found to exist at three different levels of tolerance. These levels were distinct with regard to downregulation of TCR and CD8 molecules, and the requirements for reexpression of TCR in vitro. This differential induction of peripheral tolerance suggest that some tissues are more likely to be affected in autoimmune diseases than others.

Animals↗
PubMed1991

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction.

By generating two types of transgenic mice we have investigated how extrathymic events can contribute to self tolerance. The major histocompatibility complex class I gene Kb was expressed under the control of the glial fibrillary acidic protein promoter in cells of neuroectodermal origin outside the thymus. These mice were tolerant to Kb. When crossed to transgenic mice expressing a Kb-specific T cell receptor (TCR), clonotype+, CD8+CD4- mature T cells could be detected in normal numbers in the thymus of the double-transgenic mice but were strongly reduced in spleen and lymph nodes in comparison with TCR single-transgenic mice. After isolation of clonotype negative splenic T cells and activation in vitro, reappearance of the clonotype+, CD8+CD4- cells was observed. These results indicate that down-regulation of TCR and CD8 molecules on the antigen-specific T cells is a novel mechanism, by which peripheral tolerance to this antigen can occur.

Animals↗
PubMed1991

Each of the two productive T cell receptor alpha-gene rearrangements found in both the A10 and BM 3.3 T cell clones give rise to an alpha chain which can contribute to the constitution of a surface-expressed alpha beta dimer.

The H-2 Kb specific cytotoxic T cell clone BM3.3 carries two productive TCR alpha gene rearrangements but expresses a single detectable TCR alpha beta chain combination on its surface. However, when separately analyzed by gene transfer, both productive alpha gene rearrangements are translated into alpha chains capable of pairing with the BM3.3 beta chain and of cell surface expression. Similar observations have been made with the products of the two productive alpha gene rearrangements previously identified in the A10 T cell clone. These observations contrast with those made for the two TCR alpha chains expressed in the MS202 T cell clone. In the latter instance, when analyzed by gene transfer, one of the alpha chains was unable to make a pair with the beta chain. Consequently, when considered with respect to the mechanisms of TCR allelic exclusion, our data suggest that the mere expression of a TCR alpha beta dimer on the surface of immature CD4+ CD8+ thymocytes may be necessary but not sufficient to shutdown further alpha gene rearrangements. Rather, the ability of a TCR alpha beta dimer to be positively selected may be needed to trigger the maturation of thymocytes to a stage devoid of recombinase activity. Alternatively, if non-dividing CD4+ CD8+ CD3+ small thymocytes do not experience secondary alpha-gene rearrangement, our data suggest that TCR alpha gene rearrangements are attempted quasi-simultaneously on both alpha alleles.

Alleles↗
PubMed1991

Non-deletional mechanisms of peripheral and central tolerance: studies with transgenic mice with tissue-specific expression of a foreign MHC class I antigen.

The studies described here reveal a surprising variety of non-deletional manifestations of tolerance (anergy and unresponsiveness) in both the thymus and peripheral lymphoid organs. This can be observed in anti-Kb TCR transgenic mice crossed with normal Kb positive mice, and in TCR mice crossed with transgenic mice expressing Kb in restricted tissues, such as liver, epithelial cells, cells of neuroectodermal origin etc. Thymic induction of unresponsiveness: Transgenic mice were prepared with the a, beta TCR genes from a CD8-dependent and Kb-specific CTL clone. In homozygous H-2b mice clonotype+ cells were found in the thymus but none or few in the periphery, suggesting that deletion occurs in the thymus although lack of migration to the periphery has not been ruled out. In H-2 heterozygous mice (TCR.H-2kxb) deletion was incomplete in the thymus and clonotype+ cells accumulated substantially in the periphery. Most of them had downregulated their CD8 molecules. The clonotype+ cells in both the thymus and periphery were unresponsive to the Kb antigen in vitro, suggesting the induction of anergy in the thymus. The inefficient negative selection in H-hkxb heterozygous mice is probably due to the lower Kb expression in comparison to H-2b homozygous mice. We then further reduced the amount of Kb expression in the thymus by constructing Kb transgenic mice using the 0.8 Kb fragment of the keratin IV promoter. In the thymus expression was only observed on a subset of medullary epithelial cells. When these mice were crossed with the TCR transgenic mice than no deletion was observed in the thymus. However, the clonotype+ CD8+ thymocytes could not respond to Kb in vitro, indicating that they had been rendered anergic in the thymus. These data show that unresponsiveness can be induced in the thymus, that the extent of clonal deletion can vary greatly owing to a change in antigen expression by a factor of two as in H-2 heterozygous versus homozygous mice, and that expression of Kb on a few medullary thymic epithelial cells is sufficient to induce anergy. Peripheral induction of unresponsiveness: To study the consequence of tissue-specific tolerogen expression we have generated transgenic mice expressing Kb exclusively in cells of neuroectodermal origin (GFAP.Kb mice) in the liver (alumin.Kb mice), or in certain epithelial cells outside the thymus (2.4 keratin IV.Kb mice). Consistent with the absence of Kb expression in the thymus there was no deletion of clonotype+ CD8+ cells in the thymus, and the thymocytes were fully functional.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

A novel type of aberrant T cell receptor alpha-chain gene rearrangement. Implications for allelic exclusion and the V-J recombination process.

In the process of analyzing the contribution of nonproductive alpha- and beta-chain gene rearrangements to the allelic exclusion of TCR gene expression, we have found a novel type of aberrant alpha-gene rearrangement. In one alpha-allele of the mouse KB5-C20 T cell clone, a J alpha gene segment has been abutted precisely to a sequence that does not display any homology to known V and D gene segment. The appended sequence originates from within the V alpha locus and is located, in the germ-line, 1 kb upstream of a member of the V alpha 2-gene segment subfamily. No recombination signal sequences have been found contiguous to the recombination point. These observations indicate that in normal T lymphocytes, TCR alpha-genes may be affected by aberrant rearrangements similar to those that predominate in human T cell tumors containing chromosome 14 inversion or translocation. Furthermore, compilation of published data and cloning and sequencing of three additional alpha-alleles has allowed us to examine the status of alpha-loci in nine mouse T cell clones expressing functional alpha beta-heterodimers. Interestingly, in contrast to the situation observed at the beta-locus, only 1 of 18 analyzed alpha-alleles has retained a germ-line unrearranged configuration. In addition, in each T cell clone, alpha-rearrangements on homologous chromosomes were unevenly distributed over the J alpha region and shown to generally involve neighboring J alpha gene segments.

Alleles↗
PubMed1990

Genomic organization of the mouse T cell receptor V alpha family.

Based on the analysis of V alpha gene segment deletions in a panel of T lymphomas, we have constructed a map of the mouse T cell receptor alpha/delta region and assigned the relative position of 72 distinct V gene segments. Three major observations have emerged from such studies. First, members of a given V alpha subfamily are not organized in discrete units along the chromosome but largely interspersed with members of other V alpha subfamilies. Second, analysis of the deletion map suggests the existence of repetitive patterns (V alpha clusters) in the chromosomal distribution of the V alpha gene segments. Third, the present-day organization of the V alpha/delta region may be readily explained by a series of sequential duplications involving three ancestral V alpha clusters. Direct evidence for the existence of these unique structural features has been gained by cloning approximately 370 kb of DNA and positioning 26 distinct V alpha gene segments belonging to six different subfamilies. Finally, the relationships existing between the V alpha/delta gene segment organization and usage are discussed in terms of position-dependent models.

Animals↗