These studies tested whether antigenic competition between T cells occurs. We generated CD8+ T cell responses in H-2b mice against the dominant ovalbumin epitope SIINFEKL (ova8) and subdominant epitope KRVVFDKL, using either vaccinia virus expressing ovalbumin (VV-ova) or peptide-pulsed dendritic cells. CD8+ T cell responses were visualized by major histocompatibility complex class I–peptide tetrameric molecules. Transfer of transgenic T cells with high affinity for ova8 (OT1 T cells) completely inhibited the response of host antigen-specific T cells to either antigen, demonstrating that T cells can directly compete with each other for response to antigen. OT1 cells also inhibited CD8+ T cell responses to an unrelated peptide, SIYRYGGL, providing it was presented on the same dendritic cells as ova8. These inhibitions were not due to a more rapid clearance of virus or antigen-presenting cells (APCs) by the OT1 cells. Rather, the inhibition was caused by competition for antigen and antigen-bearing cells, since it could be overcome by the injection of large numbers of antigen-pulsed dendritic cells. These results imply that common properties of T cell responses, such as epitope dominance and secondary response affinity maturation, are the result of competitive interactions between antigen-bearing APC and T cell subsets.

Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished. Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities. In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained. This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus. Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity. The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.

Multimeric peptide͞class II MHC staining reagents were synthesized and shown to bind with appropriate specificity to T cell hybridomas. A small, expanded population of T cells detected with one of these reagents in peptideimmunized C57BL͞10 mice persisted for several months. This population expanded further on secondary immunization. Equating the extent of binding of this reagent to T cell receptor affinity, we saw little correlation of immunizing peptide dose to T cell receptor affinity at the peak of the primary response. However, there was an inverse relation between peptide dose and the apparent receptor affinity of the T cells that were present several months after a primary response or after a secondary stimulation either in vivo or in vitro.Tracking the cellular dynamics of antigen-specific T cell responses in vivo has been difficult, not only because the responding precursors occur at low frequency, but also because the peptide͞MHC ligand for the T cell receptor (TCR) is cell bound and of low affinity. These latter problems have been overcome by methods for producing soluble MHC molecules bearing single peptides (1-4) and for producing fluorescent multimeric versions of these molecules (5-10). The cooperative binding achieved with these multivalent ligands produces an avidity high enough that antigen-specific T cells can be detected by flow cytometry. In examining the binding of these types of reagents to T cells with receptors of known affinity for monovalent peptide͞MHC, we observed a direct correlation between the extent of multimer binding and receptor affinity (10).The phenomenon of affinity maturation is well established in B cell responses. With multiple immunizations of T celldependent antigens, B cells bearing Ig receptors of steadily increasing affinity grow to dominate the B cell pool (11, 12). These cells appear to arise by selection of somatically mutated receptors as antigen becomes limiting (13)(14)(15). Recent studies have demonstrated that T cells having higher average affinity for peptide͞MHC are selected after multiple exposures to antigen (16,17). Whether this affinity maturation is primarily the result of the experience of repeated exposure to antigen or a shaping of the T cell repertoire during antigen waning (18-20) is unknown.We examined these questions for CD4 ϩ T cells responding to a peptide presented by the mouse class II MHC molecule, IA b . We tracked the frequency and relative affinities of peptide͞MHC-binding T cells during both primary and secondary immune responses. Our results show that with repeated or prolonged exposure to antigen, limiting doses of antigen select for T cells with higher-affinity receptors.

We show that the amino acid analogue betaine shares with small tetraalkylammonium ions [Melchior, W. B., Jr., & von Hippel, P. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 298-302] the ability to reduce or even eliminate the base pair composition dependence of DNA thermal melting transitions. The "isostabilizing" concentration of betaine (at which AT and GC base pairs are equally stable) is approximately 5.2 M. Betaine exerts its isostabilizing effect without appreciably altering the conformation of double-stranded DNA from the B form. The presence of > 5 M betaine also does not greatly change the behavior of DNA as a polyelectrolyte; this lack of effect on electrostatic interactions is expected because betaine exists as a zwitterion near neutral pH. Study of DNA melting transitions in high concentrations of betaine thus allows the experimental separation of compositional and polyelectrolyte effects on DNA melting. As a consequence, betaine solutions can also be used to investigate DNA-protein interactions under isostabilizing (or close to isostabilizing) conditions, which has not been possible using isostabilizing salts. This potential is illustrated by examining the highly salt concentration-dependent interaction of ribonuclease A with DNA in concentrated betaine solutions.

In a phase 2a trial of patients with moderate to severe Crohn's disease who had failed treatment with tumor necrosis factor antagonists, 8 and 24 weeks of treatment with MEDI2070 were associated with clinical improvement. ClinicalTrials.gov ID: NCT01714726.

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Background: Tezepelumab (AMG 157/MEDI9929), a first-in-class monoclonal antibody, targets thymic stromal lymphopoietin, a cytokine that is implicated in the pathogenesis of atopic dermatitis (AD).Objective: We sought to evaluate the efficacy and safety of tezepelumab in adults with moderate to severe AD. Methods:In this phase 2a study (NCT02525094), 113 patients were randomized 1:1 to subcutaneous tezepelumab 280 mg or placebo every 2 weeks, plus class 3 topical corticosteroids (TCS). The primary endpoint was the week 12 response rate for a $50% reduction in the Eczema Area and Severity Index (EASI50). Secondary endpoints including EASI75, Investigator's Global Assessment, SCORAD 50, SCORAD 75, pruritus numeric rating and 5-D itch scales, and exploratory endpoints (including EASI90) were assessed at weeks 12, and 16 (post hoc).Results: A numerically greater percentage of tezepelumab plus TCS-treated patients achieved EASI50 (64.7%) versus placebo plus TCS (48.2%; P = .091). Numerical improvements over placebo were demonstrated for week 12 secondary and exploratory endpoints, with further improvements at week 16. Treatment-emergent adverse events were similar between treatment groups.Limitations: Greater than expected response rates in placebo-treated patients were possibly attributable to TCS. Conclusion:Although not statistically significant, numerical improvements over placebo for all week 12 endpoints were demonstrated, with greater week 16 responses.

Complexes of Escherichia coli RNA polymerase with DNA containing the lambda PL promoter have been deposited on mica and imaged in air with a scanning force microscope. The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template. The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript. This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template.