Biomedical subjects

Mark A Daniels

Publications and source records attributed to Mark A Daniels.

8 recordsLinked to original sources

PubMed2006

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling.

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

Animals↗

PubMed2006

The sialyltransferase ST3Gal-I is not required for regulation of CD8-class I MHC binding during T cell development.

The CD8 coreceptor plays a crucial role in thymocyte and T cell sensitivity by binding to class I MHC and recruiting downstream signaling molecules to the TCR. Previous studies reported considerable changes in TCR-independent CD8/class I MHC binding (i.e., CD8 noncognate interactions) during T cell development, changes that correlated with altered glycosylation of surface molecules. In particular, expression of the sialyltransferase ST3Gal-I has been proposed as a critical factor regulating the attenuation of CD8 avidity during the double-positive to CD8 single-positive progression. This hypothesis is strengthened by the fact that ST3Gal-I(-/-) animals show a profound disregulation of CD8 T cell homeostasis. In contrast to this model, however, we report in this study that ST3Gal-I deficiency had no detectable impact on CD8 noncognate binding to multimeric peptide/MHC class I ligands at any stage of thymocyte development. We also found that the susceptibility to CD8-induced cell death is not markedly influenced by ST3Gal-I deficiency. Thus, the profound effects of ST3Gal-I on CD8 T cell survival evidently do not involve a role for this enzyme in controlling CD8-class I binding.

Animals↗

PubMed2005

Loss of CD8 and TCR binding to Class I MHC ligands following T cell activation.

The capacity of T cells to bind peptide/MHC ligands changes with T cell development and differentiation. Here we study changes in peptide/MHC multimer binding following T cell activation. Surprisingly, T cell activation caused a marked reduction in specific peptide/MHC Class I multimer binding, which was distinct from transient TCR down-regulation, and was especially dramatic for engagement with low-affinity peptide/MHC ligands. Direct CD8-Class I interactions were also profoundly and rapidly impaired following T cell stimulation, even though surface CD8alpha and CD8beta levels were unchanged after activation, suggesting that decreased CD8 co-receptor binding contributes to this effect. Finally, we show that enzymatic desialylation restores much of the multimer binding on activated T cells, suggesting that altered glycosylation may inhibit TCR/CD8 binding to peptide/MHC ligands. These radical changes in activated T cells' ability to perceive peptide/MHC ligands may contribute to selective outgrowth of clones with high affinity for the stimulatory ligand.

Animals↗

PubMed2004

Cutting edge: LFA-1 integrin-dependent T cell adhesion is regulated by both ag specificity and sensitivity.

Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8(+) T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.

Animals↗

PubMed2004

Location of the epitope for an anti-CD8alpha antibody 53.6.7 which enhances CD8alpha-MHC class I interaction indicates antibody stabilization of a higher affinity CD8 conformation.

MHC class I tetramers are widely used, usually in combination with an antibody to CD8, to detect antigen specific T cells. Some anti-CD8alpha antibodies block the interaction of murine MHC class I tetramers with CD8 T cells, while others such as 53.6.7, enhance. To understand the molecular basis for this effect, we mapped the epitope for the enhancing antibody 53.6.7 and three other blocking antibodies using a panel of murine CD8alpha (Lyt-2) mutants expressed on COS-7 transfectants. Mutations in residues that contact MHC class I affected binding of the blocking antibodies. In contrast, antibody 53.6.7 was affected by a mutation in the residue T81A located on the D-E loop. In the cocrystal of CD8alphaalpha with MHC class I, two different complexes (A and B) were observed, indicating the existence of different CD8 conformations. The T81 residue does not make contact with MHC class I in either complex, however, neighboring residues in the D-E loop make very different contacts in the two different complexes. The most likely explanation for antibody enhancement of tetramer bindings is that binding of 53.6.7 to CD8alphabeta stabilizes a conformation with a higher affinity for interaction with MHC class I and suggests that the CD8 binding site is flexible.

Animals↗

PubMed2004

T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Amino Acid Sequence↗

PubMed2003

Thymocyte sensitivity and supramolecular activation cluster formation are developmentally regulated: a partial role for sialylation.

TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca(2+) flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.

Animals↗

PubMed2002

Sweet 'n' sour: the impact of differential glycosylation on T cell responses.

The fate and functional activity of T lymphocytes depend largely on the precise timing of gene expression and protein production. However, it is clear that post-translational modification of proteins affects their functional properties. Although modifications such as phosphorylation have been intensely studied by immunologists, less attention has been paid to the impact that changes in glycosylation have on protein function. However, there is considerable evidence that glycosylation plays a key role in immune regulation. We will focus here on examples in which differential glycosylation affects the development, survival or reactivity of T cells.

Animals↗