Marie Malissen

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.

Mouse T cell antigen receptor: Structure and organization of constant and joining gene segments encoding the β polypeptide

The germ-line joining (J) gene segments and constant (C) genes encoding the beta chain of the mouse T cell antigen receptor have been isolated on a single cosmid clone. There are two constant genes, C beta 1 and C beta 2, each associated with a cluster of J beta gene segments. The nucleotide sequences of the C beta 2 gene and of the J beta 2 cluster gene segments have been determined. The coding sequence of the C beta 2 gene is very similar to the sequence of a cDNA clone encoded by the C beta 1 gene. The C beta 2 gene has four exons; exon-intron structure does not obviously correspond to the functional domains of the protein. The J beta 2 gene segment cluster contains six functional J gene segments. We have isolated specific probes for the C beta 1, C beta 2, J beta 1, and J beta 2 regions to examine DNA rearrangements in T lymphocytes. DNA rearrangements can occur in both J beta gene segment clusters, and both C beta genes appear functional.

The human t cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene

A cDNA clone YT35 , synthesized from poly(A)+ RNA of the human T cell tumor Molt 3, exhibits homology to the variable (V), joining (J), and constant (C) regions of immunoglobulin genes. We have isolated and sequenced the germ-line V and J gene segment counterparts to YT35 from a human cosmid library, and these failed to encode 14 nucleotides of the cDNA clone between the V and J regions. We postulate that these 14 nucleotides are encoded by a third gene segment analogous to the diversity (D) gene segments of immunoglobulin heavy chain genes. This T cell antigen receptor V gene appears to be assembled from three gene segments, V, D, and J, and accordingly most closely resembles immunoglobulin heavy chain V genes.

The T cell receptor β chain genes are located on chromosome 6 in mice and chromosome 7 in humans

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.

Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis

Neutropenia in mice and humans results in the generation of NK cells with an immature and hyporesponsive phenotype.

Direct evidence for chromosomal inversion during T-cell receptor beta-gene rearrangements.

A germline T-cell receptor variable region (Vβ) gene segment (Vβ14) has been mapped 10 kilobases to the 3′ side of the constant region (Cβ2) gene. The Vβ14 gene segment is in an inverted transcriptional polarity relative to the diversity-region (Dβ) and joining-region (Jβ) gene segments and the Cβ genes. Analyses of a T-cell clone (J6.19), which has productively rearranged the Vβ14 gene segment, indicate that the productive Vβ-Dβ-Jβ rearrangement and its reciprocal flank recombination product are linked and located at either border of a chromosomal inversion. These data demonstrate for the first time a linkage between mammalian V and C genes and verify that a functional T-cell receptor Vβ gene can be constructed through a chromosomal inversion.

Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment

The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated ζ chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.

The joining of germ-line Vα to Jα genes replaces the preexisting Vα-Jα complexes in a T cell receptor α,β positive T cell line

Abstract To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCRα and β genes in the progeny of the CD3 + , CD4−CD8− M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing Vα-Jα rearrangements by joining an upstream Vα gene to a downstream Jα segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCRβ genes are stable. These secondary recombinations are usually productive, and new forms of TCRα polypeptides are expressed in these cells in association with the original Cβ chain. Developmental control of this Vα-Jα replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.

Integrative biology of T cell activation

The activation of T cells mediated by the T cell antigen receptor (TCR) requires the interaction of dozens of proteins, and its malfunction has pathological consequences. Our major focus is on new developments in the systems-level understanding of the TCR signal-transduction network. To make sense of the formidable complexity of this network, we argue that fine-grained methods are needed to assess the relationships among a few components that interact on a nanometric scale, and those should be integrated with high-throughput -omic approaches that simultaneously capture large numbers of parameters. We illustrate the utility of this integrative approach with the transmembrane signaling protein Lat, which is a key signaling hub of the TCR signal-transduction network, as a connecting thread.

Mannosidase I inhibition rescues the human α-sarcoglycan R77C recurrent mutation

Limb girdle muscular dystrophy type 2D (LGMD2D, OMIM600119) is a genetic progressive myopathy that is caused by mutations in the human alpha-sarcoglycan gene (SGCA). Here, we have introduced in mice the most prevalent LGMD2D mutation, R77C. It should be noted that the natural murine residue at this position is a histidine. The model is, therefore, referred as Sgca(H77C/H77C). Unexpectedly, we observed an absence of LGMD2D-like phenotype at histological or physiological level. Using a heterologous cellular model of the sarcoglycan complex formation, we showed that the R77C allele encodes a protein that fails to be delivered to its proper cellular localization in the plasma membrane, and consequently to the disappearance of a positively charged residue. Subsequently, we transferred an AAV vector coding for the human R77C protein in the muscle of Sgca-null mice and were able to pharmacologically rescue the R77C protein from endoplasmic reticulum-retention using proteasome or mannosidase I inhibitors. This suggests a therapeutic approach for LGMD2D patients carrying mutations that impair alpha-sarcoglycan trafficking.