Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching’ can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases

Structural mechanism underpinning cross-reactivity of a CD8(+) T-cell clone that recognizes a peptide derived from human telomerase reverse transcriptase

T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8(+) T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection

Targeting of a Chlamydial Protease Impedes Intracellular Bacterial Growth

Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target

Genetic and Structural Basis for Selection of a Ubiquitous T Cell Receptor Deployed in Epstein-Barr Virus Infection

Despite the ∼1018 αβ T cell receptor (TCR) structures that can be randomly manufactured by the human thymus, some surface more frequently than others. The pinnacles of this distortion are public TCRs, which exhibit amino acid-identical structures across different individuals. Public TCRs are thought to result from both recombinatorial bias and antigen-driven selection, but the mechanisms that underlie inter-individual TCR sharing are still largely theoretical. To examine this phenomenon at the atomic level, we solved the co-complex structure of one of the most widespread and numerically frequent public TCRs in the human population. The archetypal AS01 public TCR recognizes an immunodominant BMLF1 peptide, derived from the ubiquitous Epstein-Barr virus, bound to HLA-A*0201. The AS01 TCR was observed to dock in a diagonal fashion, grasping the solvent exposed peptide crest with two sets of complementarity-determining region (CDR) loops, and was fastened to the peptide and HLA-A*0201 platform with residue sets found only within TCR genes biased in the public response. Computer simulations of a random V(D)J recombination process demonstrated that both TCRα and TCRβ amino acid sequences could be manufactured easily, thereby explaining the prevalence of this receptor across different individuals. Interestingly, the AS01 TCR was encoded largely by germline DNA, indicating that the TCR loci already comprise gene segments that specifically recognize this ancient pathogen. Such pattern recognition receptor-like traits within the αβ TCR system further blur the boundaries between the adaptive and innate immune systems

APLP1 is endoproteolytically cleaved by gamma-secretase without previous ectodomain shedding

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) and its homologs, the APP like proteins APLP1 and APLP2, is typically a two-step process, which is initiated by ectodomain-shedding of the substrates by alpha- or beta-secretases. Growing evidence, however, indicates that the cleavage process for APLP1 is different than for APP. Here, we describe that full- length APLP1, but not APP or APLP2, is uniquely cleaved by gamma-secretase without previous ectodomain shedding. The new fragment, termed sAPLP1gamma, was exclusively associated with APLP1, not APP, APLP2. We provide an exact molecular analysis showing that sAPLP1gamma was uniquely generated by gamma- secretase from full-length APLP1. Mass spectrometry analysis showed that the sAPLP1gamma fragment and the longest Abeta-like peptide share the C-terminus. This novel mechanism of gamma-secretase action is consistent with an ϵ-cut based upon the nature of the reaction in APP. We further demonstrate that the APLP1 transmembrane sequence is the critical determinant for gamma-shedding and release of full-length APLP1. Moreover, the APLP1 TMS is sufficient to convert larger type-I membrane proteins like APP into direct gamma-secretase substrates. Taken together, the direct cleavage of APLP1 is a novel feature of the gamma-secretase prompting a re-thinking of gamma-secretase activity modulation as a therapeutic strategy for Alzheimer disease

Anti-CD8 Antibodies Can Trigger CD8⁺ T Cell Effector Function in the Absence of TCR Engagement and Improve Peptide-MHCI Tetramer Staining

CD8⁺ T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide–MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8⁺ T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8⁺ T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8⁺ T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8⁺ T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8⁺ T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8⁺ T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8⁺ T cell signaling

„Poesie ist das schlechte Gewissen der Literatur“. Durs Grünbeins Frankfurter Poetikvorlesung

Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this ‘ground state’ immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8+ T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment

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