Background: How does a limited pool of <108 T cell receptors (TCRs) provide immunity to >1015 antigens?Results: A single TCR can respond to >one million different decamer peptides.Conclusion: This unprecedented level of receptor promiscuity explains how the naïve TCR repertoire achieves effective immunity.Significance: TCR degeneracy has enormous potential to be the root cause of autoimmune disease.

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of K D < ϳ80 M and ϳ35 M, respectively, to label human CTLs at 37°C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with K D values >200 M. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.In concert with the T cell receptor (TCR), 3 the coreceptors CD4 and CD8 participate in and enhance the process of antigen recognition by T cells through extracellular interactions with peptide-major histocompatibility complex (pMHC) molecules (1-3) and amplification of ensuing signal transduction events (4 -8). CD8 molecules are predominantly expressed as ␣␤ heterodimers on the surface of cytotoxic T lymphocytes (CTLs) (9), but are also found in ␣␣ homodimeric form on intraepithelial ␣␤ T lymphocytes, certain subsets of circulating activated CTLs, and the membranes of distinct cell lineages such as ␥␦ T cells, natural killer T cells, and dendritic cells (reviewed in Ref. 10). CD8␣␣ and CD8␣␤ bind directly to invariant domains of major histocompatibility complex class I (MHCI) molecules (11-13). Although CD8␣␣ and CD8␣␤ bind MHCI molecules with similar affinities (14), it is well established that CD8␣␣ is a much poorer coreceptor for CTLs than is CD8␣␤. Indeed, an emerging concept is that CD8␣␣ acts as an inhibitor of CTL activation (10). More generally, recent experimental evidence has lent credence to the hypothesis that efficient regulation of CTL activity is mediated by modifications of CD8 coreceptor functions in vivo. These modifications include switching to expression of the CD8␣␣ homodimer, post-translational changes of CD8␣␤ following activation (15, 16), and downregulation of CD8 expres...

SummaryThe development of fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers in conjunction with continuing advances in flow cytometry has transformed the study of antigen-specific T cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we bring together and discuss some of the 'tricks' that can be used to get the most out of pMHC multimers. These include: (1) simple procedures that can substantially enhance the staining intensity of cognate T cells with pMHC multimers; (2) the use of pMHC multimers to stain T cells with very-low-affinity Tcell receptor (TCR)/pMHC interactions, such as those that typically predominate in tumour-specific responses; and (3) the physical grading and clonotypic dissection of antigen-specific T cells based on the affinity of their cognate TCR using mutant pMHC multimers in conjunction with new approaches to the molecular analysis of TCR gene expression. We also examine how soluble pMHC can be used to examine T-cell activation, manipulate T-cell responses and study allogeneic and superantigen interactions with TCRs. Finally, we discuss the problems that arise with pMHC class II (pMHCII) multimers because of the low affinity of TCR/ pMHCII interactions and lack of 'coreceptor help'.

Flow cytometry with fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) tetramers has transformed the study of antigen-specific T-cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we demonstrate that the reversible protein kinase inhibitor (PKI) dasatinib improves the staining intensity of human (CD8+ and CD4+) and murine T-cells without concomitant increases in background staining. Dasatinib enhances the capture of cognate pMHC tetramers from solution and produces higher intensity staining at lower pMHC concentrations. Furthermore, dasatinib reduces pMHC tetramer-induced cell death and substantially lowers the T-cell receptor (TCR)/pMHC interaction affinity threshold required for cell staining. Accordingly, dasatinib permits the identification of T-cells with very low affinity TCR/pMHC interactions, such as those that typically predominate in tumour-specific responses and autoimmune conditions that are not amenable to detection by current technology.

New Findings r What is the central question of this study?In this study, we sought to survey the total transcript repertoire of human myometrium, comparing samples taken from patients at term who were not in labour with samples taken at term in spontaneous labour. These sequenced transcriptomes show the repertoire of possible proteins and their variants in myometrial smooth muscle, as well as transcriptional changes associated with term spontaneous labour. r What is the main finding and its importance?We describe, for the first time, the transcriptome of the human myometrial samples taken from patients prior to and after the onset of spontaneous labour. We document a significant number of novel transcripts of both protein-coding mRNA and microRNA. This information will be useful for the development of novel therapeutics and the formulation of new hypotheses to be tested by physiological experimentation.The transition of the human uterus from a quiescent to a contractile state takes place over a number of weeks. On such biological time scales, cellular phenotype is modified by changes in the transcriptome, which in turn is under the control of the underlying endocrine, paracrine, and biophysical processes resulting from the ongoing pregnancy. In this study, we characterize the transition of the human myometrial transcriptome at term from not in labour (NIL) to in labour (LAB) using high throughput RNA sequencing (RNA-seq). RNA was isolated from the myometrium of uterine biopsies from patients at term who were not in labour (n = 5) and at term in spontaneous labour (n = 5) without augmentation. A total of 143.6 million separate reads were sequenced, achieving, on average, ß13 times coverage of the expressed human transcriptome per sample. Principal component analysis indicated that the NIL and LAB transcriptomes could be distinguished as two distinct clusters. A comparison of the NIL and LAB groups, using three different statistical approaches (baySeq, edgeR, and DESeq), demonstrated an overlap of 764 differentially expressed genes. A comparison with currently available microarray data revealed only a partial overlap in differentially expressed genes. We conclude that the described RNA-seq data sets represent the first fully annotated catalogue of expressed mRNAs in human myometrium. When considered together, the full expression repertoire and the differentially

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Key Points• MHCI-restricted TCRs exhibit an explicit preference for a single MHCI-peptide length.• Effective CD8 ϩ T-cell immunity can only be achieved by length-matched Ag-specific T-cell clonotypes.␣␤-TCRs expressed at the CD8 ؉ T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8 ؉ T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8 ؉ T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8 ؉ T-cell clonotypes. (Blood. 2013;121(7):1112-1123)

We have observed a longitudinal nonlinear (second-harmonic) Kerr rotation up to 22° for an Fe(2 nm)/Cr(2 nm) multilayer on an S i0 2 substrate that shows a linear Kerr rotation o f only a few hundreths o f a degree. The latter value excludes any linearly induced mechanism and confirms the interface sensitivity o f this nonlinear Kerr technique. The enormous enhancement is shown to be in good agreement with recent predictions, based on spin dependent band structure calculations. The local optical field effects are shown to play an important role for the additional enhancements for such multilayers on a substrate. Besides their fundamental interest, these systems are also interesting for their potential in practical applications.One o f the most widely used techniques to study the magnetic properties o f magnetic thin films is the magneto optical Kerr effect (MOKE). In addition, MOKE is widely used for magnetic imaging. In this context, and also to study very thin films, large Kerr rotation angles are favorable.Recently, it has been pointed out by Pustogowa, Hiibner, and Bennemann that in nonlinear optical secondharmonic generation (SHG) from magnetic materials enormous enhancements of the Kerr angles can be expected [ In this paper we present experimental nonlinear Kerr data for a magnetic Fe/Cr multilayer in the longitudinal configuration. We show that extremely large nonlinear Kerr angles are found for this very thin magnetic layer, for which the linear magneto-optical Kerr rotation is (vanishingly) small. Experimentally is found to be largest for the ^-polarized input, with values between 5° and 2 2° for angles of incidence between 15° and 70°. For the p -input configuration we measure a rotation angle of 1° to 5° for incident angles between 15° and 7 0°. This is shown to be in good agreement with the results of band structure calculations on (bulk) Fe surfaces by Pustogowa, Hiibner, and Bennemann [9]. We will also show how the substrate, through local field effects, can influence the magnitude of the observed 4>k\The sample consisted of a thin Fe film (thickness 2 nm), covered with a 2 nm Cr film deposited by rf diode and dc magnetron sputtering, respectively. As a substrate we used a (100) silicon wafer, with a thermal oxide layer of about 525 nm. The substrate was on a rotating table, which moved with a velocity of 0 .9 6 and 3 .9 7 m /m in underneath the Fe and Cr targets, respectively. Both targets were equipped with screens for getting uniformity of the layer thickness better than 1%. The base pressure was 2 X 10~7 Torr and the Ar pressure was 5 mTorr. For the second harmonic experiments we used the 7 7 0 nm output of a mode-locked (8 0 MHz) Ti-sapphire laser. The pulse width was 7 0 fs and the input power was 100 mW focused on a spot diameter of 100 jmm. The experiments were done in the longitudinal configuration, i.e., the magnetization M was in the plane of the sample and in the optical plane of incidence (see inset, Fig. 1). Figure 1 shows the polarization dependence of the SH signal for an ^-polariz...