Interaction of Streptavidin-Based Peptide-MHC Oligomers (Tetramers) with Cell-Surface T Cell Receptors

he binding of oligomeric peptide–MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR–pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8[superscript pos] T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10–30% of cell-surface receptors), as is generally assumed in deriving affinity values (K[subscript D]), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k[subscript off] and k[subscript on]), but also on a multivalent association rate (μ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.National Institutes of Health (U.S.) (Grant P01 CA097296)National Institutes of Health (U.S.) (Grant R01 GM55767)National Institutes of Health (U.S.) (Grant PO1-AI071195)National Institutes of Health (U.S.). Pioneer Awar

Astrophysical Tests of Dark Matter with Maunakea Spectroscopic Explorer

We discuss how astrophysical observations with the Maunakea Spectroscopic Explorer (MSE), a high-multiplexity (about 4300 fibers), wide field-of-view (1.5 square degree), large telescope aperture (11.25 m) facility, can probe the particle nature of dark matter. MSE will conduct a suite of surveys that will provide critical input for determinations of the mass function, phase-space distribution, and internal density profiles of dark matter halos across all mass scales. N-body and hydrodynamical simulations of cold, warm, fuzzy and self-interacting dark matter suggest that non-trivial dynamics in the dark sector could have left an imprint on structure formation. Analysed within these frameworks, the extensive and unprecedented datasets produced by MSE will be used to search for deviations away from cold and collisionless dark matter model. MSE will provide an improved estimate of the local density of dark matter, critical for direct detection experiments, and will improve estimates of the J-factor for indirect searches through self-annihilation or decay into Standard Model particles. MSE will determine the impact of low mass substructures on the dynamics of Milky Way stellar streams in velocity space, and will allow for estimates of the density profiles of the dark matter halos of Milky Way dwarf galaxies using more than an order of magnitude more tracers. In the low redshift Universe, MSE will provide critical redshifts to pin down the luminosity functions of vast numbers of satellite systems, and MSE will be an essential component of future strong lensing measurements to constrain the halo mass function. Across nearly all mass scales, the improvements offered by MSE, in comparison to other facilities, are such that the relevant analyses are limited by systematics rather than statistics.Comment: 44 pages, 19 figures. To appear as a chapter for "The Detailed Science Case for the Maunakea Spectroscopic Explorer, 2019

The Role of T Cell Receptor: Peptide MHC Affinity in T Cell Activation

115 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.Engineering of TCRs requires stabilization of the receptor prior to engineering for higher affinity. Chapter 4 details the engineering of a high affinity TCR in its native state, without the need for stabilization, using a T cell display system. T cells bearing the newly isolated TCR bound pepMHC and were activated in the absence of co-receptor. This result demonstrates the feasibility of engineering full-length TCRs on the surface of T cells using a retroviral vector.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Single-Chain VαVβ T Cell Receptors Function Without Mispairing With Endogenous TCR Chains1

Transduction of exogenous T cell receptor (TCR) genes into patients’ activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These “mixed” heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vβ single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

<div><p>Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of <i>Staphylococcus aureus</i>. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of <i>Staphylococcus aureus</i> and <i>Streptococcus pyogenes</i>.</p></div

Multiplex assay in the presence of a mixture of toxins.

<p>Solutions containing a mixture of two or more toxins were tested in multiplex assays, at the indicated concentrations. Fluorescence emitted by each Vβ-immobilized bead due to toxin binding, was plotted on bar graphs shown. The error-bars represent standard deviations from two independent experiments. Similar data (but with higher background) were obtained in other experiments where unoccupied sites on biotin-Vβ immobilized-beads were not blocked with excess biotin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135986#pone.0135986.s005" target="_blank">S5 Fig</a>).</p

Multiplex assay with supernatants from cultures of various strains of <i>Staphylococcus aureus</i>.

<p>Supernatants (diluted 1:4) from cultures of 18 strains of <i>Staphylococcus aureus</i> obtained from the NARSA repository, were tested in a multiplex assay to determine their toxin expression profile. Fluorescence emitted by each Vβ-immobilized bead due to toxin binding, was plotted on bar graphs shown. Toxin(s) secreted by each strain was determined as an increase in fluorescence signal, compared to the background. The error-bars represent standard deviations from two independent experiments. Similar data (but with higher background) were obtained in other experiments where unoccupied sites on biotin-Vβ immobilized-beads were not blocked with excess biotin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135986#pone.0135986.s006" target="_blank">S6 Fig</a>).</p

Multiplex assay in the presence of one toxin.

<p>Solutions containing 50 ng/ml SEA, 10 ng/ml SEB, 10 ng/ml TSST-1, 50 ng/ml SpeA or 10 ng/ml SpeC were tested in multiplex assays. Fluorescence emitted by each Vβ-immobilized bead due to toxin binding, was plotted on bar graphs shown. In all cases, the toxins were detected by the beads immobilized with the high-affinity Vβ engineered for that toxin. The error-bars represent standard deviations from two independent experiments. Similar data (but with higher background) were obtained in other experiments where unoccupied sites on biotin-Vβ immobilized-beads were not blocked with excess biotin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135986#pone.0135986.s004" target="_blank">S4 Fig</a>).</p