Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Generation of soluble T-cell receptor (TCR) Given the importance of understanding in explicit molecular terms the process by which T cells recognize pathogens and autoantigens, recent efforts have focused on production of recombinant soluble forms of the TCR heterodimer. Prokaryotic expression has yielded substantial quantities ofTCR protein, which, unfortunately, comprise a very low fraction of native or correctly refolded material (4-6). In eukaryotic expression systems, immunoglobulin chimeras with TCR a and 13 extracellular segments have been produced (7,8). In addition, glycosyl-phosphatidylinositol-linked chimeras (9, 10) released from the cell surface by phosphatidylinositol phospholipase C, as well as a and P3 chimeras fused to CD3; transmembrane and cytoplasmic segments using a thrombincleavable linker, have been generated (11). A major problem with each of these strategies is the inefficient pairing between a and P subunits. Monomers as well as homodimers, in addition to the desired heterodimers, are produced. This mispairing complicates protein purification and leads to a substantial reduction in useful protein yield.Here, we describe a method to facilitate TCR pairing through the use of synthetic peptide sequences that create a heterodimeric coiled coil (12). A TCR af3 heterodimer derived from the mouse N15 cytotoxic T lymphocyte (CTL) clone specific for a well-characterized vesicular stomatic virus octapeptide in the context of the Kb MHC class I molecule was chosen for the model (13,14). This approach offers a general method to express heterodimeric proteins. MATERIALS AND METHODSConstruction and Transfection of NlSaCand N1513Chhme-ras. The cDNAs encoding the N15 TCR were isolated by priming total RNA from the N15 CTL clone with oligonucleotides specific for the 3' untranslated region of constant region (C) a subunit and Ca2. cDNA was generated by reverse transcription and amplified by standard PCR technology. The full-length N15a and N15.3 cDNAs were subcloned into the pCRII vector (Invitrogen) for sequence analysis and further genetic manipulation. Two pairs of N15aC and N153?plasmids were engineered. The first pair, p2N15a; and p2N1513C, was created by replacing the variable (V) and C regions of 2B4a and 2B413 in the previously described constructs (11) with PCR-generated DNA fragments of Nl5a and N15P V and C regions. The second pair of plasmids was termed pcN15aC and pcN15I83. pcN15aC was generated by ligation of an EcoRI-HindIII fragment of the NlSa cDNA to a HindIII-EcoRI fragment encoding a segment of Ca fused to a linker sequence (SSADLVPRGSTTAPS) connecting Ca to the murine C-chain transmembrane region and cytoplasmic domain. The HindIII-EcoRI fragments were made using a combination of restriction site-containing oligonucleotides and PCR and subcloned into the EcoRI-digested pCRII vector. The N15,83 construct was generated in a similar fashion by ligation of a 5' EcoRJ-Nco I fragment from the N1513 cDNA with a Nco I-EcoRI fragment encoding a segment of Cp, the same linker and C do...

Norepinephrine (NE) can modulate dendritic cell (DC) activation in animal models, but the response of human DC to NE and other response modifiers is as yet not completely understood. Here we report the effect of NE on the cytokine response of a mixed population of human DC cells to extracellular stimuli. These cells were obtained by differentiating human cord blood CD34+ precursor cells. NE inhibited the lipopolysaccharide (LPS)-stimulated production of interleukin (IL)-23, IL-12 p40, tumor necrosis factor (TNF)-alpha and IL-6 whereas the expression of IL-10 was not significantly affected. Thus, human cord blood-derived DC respond to NE in a manner similar to mouse Langerhans cells (LC). Furthermore, forskolin also inhibited the LPS-induced levels of TNF-alpha, IL-12 p40, IL-23 p19 and IL-6, supporting the hypothesis that the effects of NE are mediated by cAMP. Data from experiments using inhibitors of adrenergic receptors suggest that NE acts through beta-adrenergic receptors. As IL-23 promotes the differentiation of CD4+ T cells required for T(H)1-mediated immunity, we suggest that NE decreases the differentiation of CD4+ T cells needed for T(H)1-mediated contact hypersensitivity and that NE is a candidate regulator of human DC functions in the skin.

On a daily basis, the skin is exposed to many environmental stressors and insults. Over a 24-h natural cycle, during the day, the skin is focused on protection; while at night, the skin is focused on repairing damage that occurred during daytime and getting ready for the next morning. Circadian rhythm provides the precise timing mechanism for engaging those different pathways necessary to keep a healthy skin through clock genes that are present in all skin cells. The strongest clue for determining cellular functions timing is through sensing light or absence of light (darkness). Here, we asked the question if blue light could be a direct entrainment signal to skin cells and also disrupt their circadian rhythm at night. Through a reporter assay for per1 transcription, we demonstrate that blue light at 410 nm decreases per1 transcription in keratinocytes, showing that epidermal skin cells can sense light directly and control their own clock gene expression. This triggers cells to "think" it is daytime even at nighttime. Elsewhere, we measured different skin cell damage because of blue light exposure (at different doses and times of exposure) vs. cells that were kept in full darkness. We show an increase in ROS production, DNA damage and inflammatory mediators. These deleterious effects can potentially increase overall skin damage over time and ultimately accelerates ageing.R esum e La peau est expos ee chaque jour a de nombreux facteurs de stress et traumatismes environnementaux. Pendant un cycle naturel de 24 heures, dans la journ ee, la peau est ax ee sur sa protection, tandis que la nuit, elle se concentre sur la r eparation des l esions survenues pendant la journ ee en pr eparation du lendemain matin. Le rythme circadien assure un m ecanisme de cadencement temporel pr ecis pour engager les diff erentes voies n ecessaires au maintien d'une peau saine a travers les g enes de l'horloge interne qui sont pr esents dans toutes les cellules cutan ees. La perception de la lumiere ou l'absence de lumi ere (obscurit e) est le plus fort indice pour d eterminer le cadencement des fonctions cellulaires. Nous nous sommes ici pos e la question de savoir si la lumi ere bleue serait un signal d'entraînement direct pour les cellules de la peau egalement capable de perturber leur rythme circadien la nuit.A travers un essai utilisant un g ene rapporteur pour la transcription de per1, nous d emontrons que la lumi ere bleue a 410 nm diminue la transcription de per1 dans les k eratinocytes, montrant que les cellules epidermiques peuvent d etecter directement la lumi ere et contrôler l'expression de leurs propres g enes horloges. Cela incite les cellules a « penser » que la journ ee a commenc e, même pendant la nuit. Par ailleurs, nous avons mesur e diff erentes l esions des cellules cutan ees suite a l'exposition a la lumi ere bleue ( a diff erentes doses et dur ees d'exposition) par rapport aux cellules qui etaient maintenues dans une obscurit e compl ete. Nous montrons une augmentation de la production d'esp eces r eactives de l'ox...

Affymetrix gene-expression analysis was performed on mRNAs from involved and noninvolved skin biopsies from three volunteers with Lentigo senilis. Of the 42,000 transcripts scanned, 17 were downregulated (<1.4 times below the control level) and 23 were upregulated (>1.9 times above the control level). A serine peptidase gene was downregulated in keeping with the suggestion that age spots are associated with impaired melanin degradation. Three genes involved in the keratinization and synthesis and the organization of fibers in the basement membrane were downregulated, two metalloproteinase genes were upregulated, as were six genes associated with the inflammatory response, in keeping with the postulate that the visible aspects of aged skin are causally linked with a microinflammatory response. The regulation of five genes associated with the Wnt family was altered. Antiapoptotic genes were downregulated, and six genes associated with transmembrane transport were upregulated.