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B Malissen

Publications and source records attributed to B Malissen.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1991

Wild type and tailless CD8 display similar interaction with microfilaments during capping.

We examined the influence of the intracytoplasmic region of CD8 alpha on capping and interaction with microfilaments. We used cell clones obtained by transfecting a CD4+ T-cell hybridoma with (a) T-cell receptor (TCR) alpha and beta chains from a cytolytic clone and (b) CD8 alpha genes that were either native or modified by extensive deletion of the intracytoplasmic region or replacement of the transmembrane and intracytoplasmic domains with those of a class I major histocompatibility complex gene (Letourneur et al. (1990). Proc. natn. Acad. Sci. U.S.A. 87, 2339-2343). Different cell surface structures were cross-linked with anti-T-cell receptor, anti-CD8 or anti-class I monoclonal antibodies and anti-immunoglobulin (Fab')2. Double labeling and quantitative image analysis were combined to monitor fluorescence anisotropy and correlation between different markers. Microfilaments displayed maximal polarization within two minutes. The correlation between these structures and surface markers was then maximal and started decreasing, whereas the redistribution of surface markers remained stable or continued. Furthermore, wild type and altered CD8 alpha exhibited similar ability to be capped and to induce co-capping of TCR and MHC (major histocompatibility complex) class I: the fraction of cell surface label redistributed into a localized cap ranged between 40% and 80%. Finally, cytochalasin D dramatically decreased CD8 capping in all tested clones. It is concluded that the transmembrane and/or intracellular domains of CD8 molecules are able to drive the extensive redistributions of membrane structures and cytoskeletal elements that are triggered by CD8 cross-linking.

Actin Cytoskeleton↗
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↗
PubMed1990

A signaling role for the cytoplasmic segment of the CD8 alpha chain detected under limiting stimulatory conditions.

To test for the functional importance of the cytoplasmic segment of the CD8 molecule, a mouse T-cell hybridoma expressing a T-cell receptor specific for the class I major histocompatibility complex product H-2Kb was transfected with a set of CD8 alpha-chain (Ly-2) and/or beta-chain (Ly-3) genes encoding polypeptides with carboxyl-terminal truncations or substitutions. When challenged with Kb-positive splenocytes, transfectants expressing Ly-2 homodimers that lacked cytoplasmic tails responded nearly as effectively as wild-type Ly-2 transfectants. However in marked contrast to the wild-type Ly-2 transfectants, tailless Ly-2 transfectants were greatly impaired in their ability to respond to Kb-transfected L cells. Coexpression of the Ly-3 gene did not restore this impaired response. The unique functional property of the Ly-2 alpha cytoplasmic segment was further supported by the analysis of a chimeric Ly-3 subunit in which the cytoplasmic segment was replaced by the one from the Ly-2 alpha subunit. When associated with a soluble Ly-2 subunit lacking a transmembrane segment, the chimeric Ly-3 was indeed sufficient to restore the response to Kb-transfected L cells. Since the lateral mobility of the tailless Ly-2 molecules on the cell surface was nearly identical to that of the wild-type Ly-2 molecules, their partially impaired function may indicate that they have lost their cis-acting signaling properties but retained their ability to bind class I products of the major histocompatibility complex.

Amino Acid Sequence↗
PubMed1989

Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and class I MHC-encoded proteins on T cells. Role of intracytoplasmic domains analyzed with hybrid CD8/class I molecules.

We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.

Amino Acid Sequence↗
PubMed1989

Visualizing interleukin 2 gene expression at the single cell level.

To analyze the expression of the interleukin (IL)2 gene at the single cell level, we have constructed a chimeric gene in which the regulatory sequences from the mouse IL2 gene were fused 5' proximal to the coding region of the Escherichia coli beta-galactosidase (lacZ) gene. Once stably introduced into a T cell hybridoma, the IL 2-lacZ reporter gene was shown to display a pattern of induction similar to the one observed for the resident IL 2 gene. Two points emerged from monitoring IL 2 expression for the resident IL2 gene. Two points emerged from monitoring IL 2 expression at the single cell level. First, upon activation the expression of the IL2-lacZ gene appears asynchronous. Second, at the peak of induction the percentage of beta-galactosidase+ cells never reached 100% and the level of beta-galactosidase reached among positive cells was highly heterogeneous within a cloned population of T cells. In combination with the possibility to sort viable lymphocytes according to lacZ expression, the IL2-lacZ reporter gene described herein should provide a way to isolate somatic cell mutants deficient in signal transduction by the T cell antigen receptor complex.

Animals↗
PubMed1989

Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.

We have isolated a variant of the DO-11.10.7 mouse T cell hybridoma which does not express functional T cell receptor alpha/beta chains. This variant, denoted 58 alpha-beta-, can be used as a recipient for T cell receptor alpha/beta gene transfer experiments to obtain cell lines which express only the products of the transfected alpha/beta genes at their surfaces. In the process of characterizing the defects affecting the 58 alpha-beta-T cell receptor genes, we have found that the parental BW5147 thymoma has undergone a previously unnoticed V alpha-J alpha rearrangement. This alpha rearrangement involves a V alpha pseudogene segment and accounts for the high level of alpha-mRNA transcripts present in the BW5147 alpha-beta- variant. Knowledge of the existence of this second, albeit nonfunctional, alpha-mRNA in BW5147 is of importance, since it could be, and actually already has been, mistakenly identified (due to partial nucleotide sequencing) in T hybrids as a functionally significant message donated by the normal T cell parent.

Animals↗
PubMed1989

Recognition of insulin on MHC-class-II-expressing L929 cells by antibody and T cells.

In the present experiments, we attempted to obtain evidence that T-cell receptors (Tcr) and immunoglobulins can react against the same antigen-peptide/MHC class II complexes on antigen-presenting cells (APC). Use was made of monoclonal APC, i.e. I-Ak alpha/beta gene-transfected L929 fibroblasts, monoclonal anti-insulin antibodies and selected insulin-specific T-cell lines. Evidence for similarities in antibody and T-cell recognition of insulin presented by I-Ak alpha/beta gene-transfected L-cell fibroblasts was not obtained after serious attempts. Furthermore, we found no evidence for synthesis of antibodies specific for insulin-peptide/MHC class II complexes.

Animals↗
PubMed1988

A T cell clone expresses two T cell receptor alpha genes but uses one alpha beta heterodimer for allorecognition and self MHC-restricted antigen recognition.

All of the T cell receptor alpha- and beta-chain rearrangements present in a dual reactive T cell clone were characterized. This clone exhibits allelic exclusion of its beta-chain genes in that only one of the two alleles is productively rearranged. Unexpectedly, it displays two productive V alpha-gene rearrangements, which are both transcribed into 1.5 kb mRNA. The contribution of each of the two productive alpha genes to the dual recognition was analyzed by gene transfer. To this end, each of the two alpha genes was separately transfected with the single productively rearranged beta gene. Transfer of only one of the two alpha beta combinations restored both allogeneic MHC recognition and self MHC-restricted antigen recognition. Thus, T cell dual recognition results from the cross-reactive recognition of an allo-MHC product by a single antigen-specific and MHC-restricted alpha beta T cell receptor. Furthermore, the presence of two productively rearranged alpha-chain genes in a T cell clone raises questions concerning the level at which allelic exclusion operates in T cells.

Animals↗
PubMed1988

Gene transfer of the Ly-3 chain gene of the mouse CD8 molecular complex: co-transfer with the Ly-2 polypeptide gene results in detectable cell surface expression of the Ly-3 antigenic determinants.

The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.

Amino Acid Sequence↗
PubMed1988

Clonal analysis of human T cell activation by the Mycoplasma arthritidis mitogen (MAS).

Mycoplasma arthritidis produces an as yet undefined soluble molecule (MAS) that has a potent mitogenic effect on T cells of several species. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by this mitogen. Reactivity to MAS is clonally expressed among T cell receptor (TcR) alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TcR alpha/beta chain-negative T lymphocyte clones expressing the CD3-associated TcR gamma chain. MAS is able to induce cytotoxicity and/or proliferation in these T cell clones. For triggering of these T cells, regardless of their phenotype of specificity, the presence of autologous, allogeneic or xenogeneic major histocompatibility complex (MHC) class II molecules on accessory cells or target cells is necessary. However, T cells do not immunologically recognize MAS on class II molecules, since a direct action of MAS on the T cells themselves can be demonstrated. Triggering of T cells by MAS can be blocked by monoclonal antibodies against CD2, CD3 and the TcR alpha/beta chain dimer. We discuss as a possible explanation that MAS is a functionally bivalent molecule cross-linking TcR and MHC class II molecules. Thus, the mechanism of T cell activation by MAS has striking similarities to the mechanisms by which Staphylococcal enterotoxins activate T cells. It is intriguing that a similar mitogenic principle has been developed by two evolutionary distinct pathogenic microorganisms.

Antigen-Antibody Reactions↗
PubMed1988

Structure-function analysis of Ia molecules: in-phase insertion mutagenesis of the amino-terminal domain of the E beta k polypeptide chain.

To identify which segments of the beta 1 domain of the E beta k polypeptide control T cell recognition of antigen, E beta genes were constructed with in-phase insertion mutations. Five independent mutants, with insertions mapping to positions 24, 50 and 93 of the E beta k polypeptide, were obtained. Cell lines expressing these mutated genes were analysed by microfluorometry using a panel of 20 anti-Ek monoclonal antibodies. None of the tested in-phase insertions has resulted in the loss of antibody binding sites. In striking contrast, mutations at position 93 and at a lesser level 50 were indicative of a crucial role of the corresponding regions in T-cell recognition, because they led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested. These data are discussed with regard to a model of the foreign antigen binding site of Ia molecules.

Amino Acid Sequence↗
PubMed1987

Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes.

The T cell receptor alpha and beta chain genes donated by an H-2 class I-specific, CD8-dependent cytotoxic T cell clone were transferred, alone or in combination with the Lyt-2 gene, into a class II-restricted, CD4+ T cell hybridoma. Two important points emerged. First, the alpha and beta T cell receptor genes endowed the recipient with the H-2 class I specificity of the donor only if the same cell had also been transfected with the Lyt-2 gene. Second, the functional Lyt-2 molecule was expressed on the transfected cells in the absence of the Lyt-3 polypeptide. These results demonstrate that, besides the T cell receptor, the Lyt-2 polypeptide is the only subset-specific molecule required to retarget a class II-reactive, CD4+ T cell line toward H-2 class I molecules.

Animals↗