Ana I. Costa

Naive CD8+ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics

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.

A Comparative Study of HLA Binding Affinity and Ligand Diversity: Implications for Generating Immunodominant CD8+ T Cell Responses

Conventional CD8+ T cell responses against intracellular infectious agents are initiated upon recognition of pathogen-derived peptides presented at the cell surface of infected cells in the context of MHC class I molecules. Among the major MHC class I loci, HLA-B is the swiftest evolving and the most polymorphic locus. Additionally, responses restricted by HLA-B molecules tend to be dominant, and most associations with susceptibility or protection against infectious diseases have been assigned to HLA-B alleles. To assess whether the differences in responses mediated via two major HLA class I loci, HLA-B and HLA-A, may already begin at the Ag presentation level, we have analyzed the diversity and binding affinity of their peptide repertoire by making use of curated pathogen-derived epitope data retrieved from the Immune Epitope Database and Analysis Resource, as well as in silico predicted epitopes. In contrast to our expectations, HLA-B alleles were found to have a less diverse peptide repertoire, which points toward a more restricted binding motif, and the respective average peptide binding affinity was shown to be lower than that of HLA-A-restricted epitopes. This unexpected observation gives rise to new hypotheses concerning the mechanisms underlying immunodominance of CD8+ T cell responses.

HLA class I allele promiscuity revisited

The peptide repertoire presented on human leukocyte antigen (HLA) class I molecules is largely determined by the structure of the peptide binding groove. It is expected that the molecules having similar grooves (i.e., belonging to the same supertype) might present similar/overlapping peptides. However, the extent of promiscuity among HLA class I ligands remains controversial: while in many studies T cell responses are detected against epitopes presented by alternative molecules across HLA class I supertypes and loci, peptide elution studies report minute overlaps between the peptide repertoires of even related HLA molecules. To get more insight into the promiscuous peptide binding by HLA molecules, we analyzed the HLA peptide binding data from the large epitope repository, Immune Epitope Database (IEDB), and further performed in silico analysis to estimate the promiscuity at the population level. Both analyses suggest that an unexpectedly large fraction of HLA ligands (>50%) bind two or more HLA molecules, often across supertype or even loci. These results suggest that different HLA class I molecules can nevertheless present largely overlapping peptide sets, and that “functional” HLA polymorphism on individual and population level is probably much lower than previously anticipated.

Comprehensive Analysis of the Naturally Processed Peptide Repertoire : Differences between HLA-A and B in the Immunopeptidome

The cytotoxic T cell (CTL) response is determined by the peptide repertoire presented by the HLA class I molecules of an individual. We performed an in-depth analysis of the peptide repertoire presented by a broad panel of common HLA class I molecules on four B lymphoblastoid cell-lines (BLCL). Peptide elution and mass spectrometry analysis were utilised to investigate the number and abundance of self-peptides. Altogether, 7897 unique self-peptides, derived of 4344 proteins, were eluted. After viral infection, the number of unique self-peptides eluted significantly decreased compared to uninfected cells, paralleled by a decrease in the number of source proteins. In the overall dataset, the total number of unique self-peptides eluted from HLA-B molecules was larger than from HLA-A molecules, and they were derived from a larger number of source proteins. These results in B cells suggest that HLA-B molecules possibly present a more diverse repertoire compared to their HLA-A counterparts, which may contribute to their immunodominance. This study provides a unique data set giving new insights into the complex system of antigen presentation for a broad panel of HLA molecules, many of which were never studied this extensively before.

CD8+ TCR Repertoire Formation Is Guided Primarily by the Peptide Component of the Antigenic Complex

CD8+ T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8+ T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide–MHC I complex on the molecular frontline of CD8+ T cell–mediated immune surveillance.

Hla-b molecules target more conserved regions of the Hiv-1 proteome

Background: HLA-B alleles of HIV-infected individuals have been shown to have a major impact on their rate of progression toward AIDS, and the T-cell responses they restrict are immunodominant. Objective: We sought to identify whether the association of HLA-B alleles with rate of progression toward AIDS is due to targeting of more restricted and thus more conserved regions of the HIV-1 proteome. Methods: Each residue of the HIV-1 consensus subtype B sequence was coded according to the presence/absence of an epitope, using the compiled epitope data available in the HIV-LANL immunology database. The Shannon entropy for each HXB2 position was calculated using pre-aligned HIV-1 clade B sequences as a measure of its degree of conservation. We then compared the entropy of empty versus epitope-containing positions and HLA-B-restricted versus HLA-A-restricted positions. Results: Positions containing CD8+ epitopes were significantly more conserved than corresponding empty positions. Moreover, residues targeted by HLA-B alleles in the HIV-1 proteome were significantly more conserved than the ones targeted by HLA-A alleles. Analysing a recent dataset, we found that B epitope regions contain significantly more escape mutations and reversions, which might be the reason why we find them to be more conserved. Conclusion: Our results suggest that epitopes in HIV-1 targeted by HLA-B alleles lie in more constrained regions of its proteins, in which mutations might have a higher fitness cost and tend to revert. Consequently, HLA-B-restricted cytotoxic T-lymphocyte (CTL) responses may persist longer. This may be one of the factors contributing to the immunodominance and impact of HLA-B-restricted CTL responses on disease progression.

In vitro expansion of antigen-specific CD8+ T cells distorts the T-cell repertoire

Short-term in vitro expansion of antigen-specific T cells is an appreciated assay for the analysis of small memory T-cell populations. However, how well short-term expanded T cells represent the direct ex vivo situation remains to be elucidated. In this study we compared the clonality of Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific CD8(+) T cells directly ex vivo and after in vitro stimulation with antigen. Our data show that the antigen-specific T cell repertoire significantly alters after in vitro culture. Clear shifts in clonotype hierarchy were observed, with the most dominant ex vivo clonotype decreasing after stimulation at the expense of several previously subdominant clonotypes. Notably, these alterations were more pronounced in polyclonal T-cell populations compared to mono- or oligoclonal repertoires. Furthermore, TCR diversity significantly increased after culture with antigen. These results suggest that the T-cell repertoire is highly subjective to variation after in vitro stimulation with antigen. Hence, although short-term expansion of T cells provides a simple and efficient tool to examine antigen-specific immune responses, caution is required if T-cell populations are expanded prior to detailed, clonotypic analyses or other repertoire-based investigations.

Complex T-Cell Receptor Repertoire Dynamics Underlie the CD8+ T-Cell Response to HIV-1

ABSTRACT Although CD8+ T cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naive individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8+ T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, as evidenced by discordant clonotype-specific transitions directed against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8+ T-cell clonotypes orchestrated at the TCR-antigen interface. IMPORTANCE We describe a relation between viral epitope mutation, antigen-specific T-cell expansion, and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provides insights into the process of coadaptation between the human immune system and a rapidly evolving lentivirus.

Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation.

Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8+ cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8+ CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC–MS/MS. Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV.

The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein.

Misfolded proteins from the endoplasmic reticulum (ER) are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD) pathway to downregulate human leukocyte antigen (HLA) class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129) as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo–Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING) domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins.