T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer

TCR‐induced alteration of primary MHC peptide anchor residue

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells

Mutation of recombinant complement component C9 reveals the significance of the N-terminal region for polymerization

Complement component C9 binds to C5b-8 sites on target cells and polymerizes to form the membrane attack complex (MAC). The aim of the work reported here was to discover which region within C9 was responsible for protecting the globular protein against self-polymerization. Computer prediction modelling highlighted the domain at the N-terminus of C9, which was then investigated by site-directed mutagenesis. The mutated proteins were expressed using insect cells infected with baculovirus. Removal of 16, 20 or 23 amino acids at the N-terminus of C9 resulted in inactivation due to self-polymerization. In contrast, removal of 4, 8 or 12 amino acids resulted in a C9 that did not polymerize spontaneously, had two to threefold enhanced lytic activity on erythrocytes, and had increased binding to C5b-8 sites on rat neutrophils. These results suggest that the domain within the first 16 amino acids at the N-terminus of C9 is crucial in preventing the self-polymerization of the globular protein. We have also found that C9 contains a motif (27WSEWS31) common to a family of cytokine receptors that is similar to a tryptophan-rich motif (WEWWR) of the membrane pore formers, thiol-activated cytolysins. Mutation of this motif in C9 resulted in polymerized protein, consistent with this site keeping the N-terminus in a protected conformation and preventing premature self-polymerization

Peptide length determines the outcome of TCR/peptide-MHCI engagement

αβ-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

Peptide length determines the outcome of TCR/peptide-MHCI engagement

αβ-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