Anne-Marie K. Wegener

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down‐regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC‐mediated TCR down‐regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma‐negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC‐mediated down‐regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C‐terminal of S126 was required for TCR down‐regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane‐proximal di‐leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC‐mediated TCR down‐regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC‐mediated TCR down‐regulation in non‐mutated T cells, indicating that the TCR was down‐regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di‐leucine‐ or tyrosine‐based motifs is proposed.

Ceramide-induced TCR up-regulation.

The TCR is a constitutively recycling receptor meaning that a constant fraction of TCR from the plasma membrane is transported inside the cell at the same time as a constant fraction of TCR from the intracellular pool is transported to the plasma membrane. TCR recycling is affected by protein kinase C activity. Thus, an increase in protein kinase C activity affects TCR recycling kinetics leading to a new TCR equilibrium with a reduced level of TCR expressed at the T cell surface. Down-regulation of TCR expression compromises T cell activation. Conversely, TCR up-regulation is expected to increase T cell responsiveness. The purpose of this study was to identify and characterize potential pathways for TCR up-regulation. We found that ceramide affected TCR recycling dynamics and induced TCR up-regulation in a concentration- and time-dependent manner. Experiments applying phosphatase inhibitors indicated that ceramide-induced TCR up-regulation was most probably mediated by serine/threonine protein phosphatase 2A. Analyses of T cell variants demonstrated that TCR up-regulation was dependent on the presence of an intact CD3γ L-based motif and thus acted on TCR engaged in the recycling pathway. Finally, we showed that TCR up-regulation probably plays a physiological role by increasing T cell responsiveness. Thus, by affecting the TCR recycling kinetics, T cells have the potential both to up- and down-regulate TCR expression and thereby adjust T cell responsiveness.

T-cell receptor downregulation by ceramide-induced caspase activation and cleavage of the zeta chain.

Regulation of T‐cell receptor (TCR) cell surface expression levels is probably an important mechanism by which T‐cell responsiveness is controlled. Previously, two distinct pathways for TCR downregulation have been described. One is dependent on protein kinase C (PKC) and the leucine‐based receptor‐sorting motif (l‐based motif) of the CD3γ chain but independent of tyrosine kinases, whereas the other is dependent on the tyrosine kinase activation but independent of the PKC and the CD3γl‐based motif. In this study, we describe a new pathway for TCR downregulation distinct from both the PKC/CD3γl‐based motif‐dependent and the tyrosine kinase‐dependent pathways. This pathway is dependent on ceramide‐induced activation of caspases and correlate with caspase‐mediated cleavage of the ζ chain. Thus, a 10–15% downregulation of the TCR was induced following the treatment of the T cells with ceramide for 4 h. A close correlation between TCR downregulation, caspase activation, and cleavage of the ζ chain was found. Furthermore, the caspase inhibitors abolished the cleavage of the ζ chain and TCR downregulation in parallel with the inhibition of the caspase activity.

Cellular and molecular characteristics of transformed T cells from an antigen-specific T-cell line.

An anligen‐specific T‐cell line which transforms into T‐lymphoma cells in vitro but apparently not in vivo is described. Membrane markers, tumorigenicity and T‐cell receptor(TcR) Vα and Vβ‐ gene usage of the in vitro transformed T‐cell line were analysed to investigate whether the transformation event was poly‐, oligo‐, or monoclonal. The results indicate that the Tlymphoma has no chromosome abnormalities, contains no tumour‐inducing virus, can induee clone‐specific immunity, and is oligodonal wilh respect to TcR Vα and Vβ expression. The nature of the transformation event and clinical application of vaccination against Tlymphomas is diseussed. In addition, the expressed TcR Vα and Vβ repertoire of Con A T blasts was apparently not affected by the Igh‐I or the MHC haplotype, as investigated in lgh‐1 and MHC congeneic C57BI mice.

Biological transfer of the CBA Tcra locus into C57BL/6 mice.

We initiated breeding experiments in order to create Tcra locus-congenic mice. The CBA Tcra locus was backcrossed to C57BL/6 mice using a restriction fragment length polymorphism (RFLP) marker for Tcra-C : different fragments of genomic DNA cleaved with the BglI enzyme. Our results suggest that no recombination hotspots are present in the C57BL/6 or CBA Tcra locus and that the locus was less than 1 cM in size

Spatially conserved regulatory elements identified within human and mouse Cd247 gene using high-throughput sequencing data from the ENCODE project.

The Cd247 gene encodes for a transmembrane protein important for the expression and assembly of TCR/CD3 complex on the surface of T lymphocytes. Down-regulation of CD247 has functional consequences in systemic autoimmunity and has been shown to be associated with Type 1 Diabetes in NOD mouse. In this study, we have utilized the wealth of high-throughput sequencing data produced during the Encyclopedia of DNA Elements (ENCODE) project to identify spatially conserved regulatory elements within the Cd247 gene from human and mouse. We show the presence of two transcription factor binding sites, supported by histone marks and ChIP-seq data, that specifically have features of an enhancer and a promoter, respectively. We also identified a putative long non-coding RNA from the characteristically long first intron of the Cd247 gene. The long non-coding RNA annotation is supported by manual annotations from the GENCODE project in human and our expression quantification analysis performed in NOD and B6 mice using qRT-PCR. Furthermore, 17 of the 23 SNPs already known to be implicated with T1D were observed within the long non-coding RNA region in mouse. The spatially conserved regulatory elements identified in this study have the potential to enrich our understanding of the role of Cd247 gene in autoimmune diabetes.