A computational approach to understanding the factors that shape the T cell receptor repertoire

The shapes of the naive and memory T cell receptor (TCR) repertoires are key determinants of whether an effective immune response will be mounted in the face of infection; a careful balance of diversity and specificity is necessary in keeping infection under control. The factors that shape the repertoires are many and varied, and a thorough understanding of these factors is important as we seek to characterise healthy immune responses and develop efficacious vaccines. The generation of a diverse range of TCRs is enabled by a random gene recombination process in the thymus, but this process does not produce different TCRs with equal probability. Previous studies of antigen-specific T cell responses have shown that a process of convergent recombination – whereby multiple recombination events converge to produce the same nucleotide sequence, and multiple nucleotide sequences converge to produce the same amino acid sequence – leads to variable TCR production frequencies. In this thesis, we have studied the role of these variable production frequencies in shaping the naive and memory TCRβ repertoires of CD8+ T cells. Millions of TCR sequences were obtained using traditional and high-throughput sequencing technologies, and analysed using novel computational methods. Higher frequency TCRβ clonotypes were more likely to be shared between individuals and present in both the naive and memory compartments. In turn, these higher-frequency clonotypes had the potential to be produced more efficiently during TCR gene recombination as demonstrated using computer simulations of a random recombination process. Furthermore, analysis of previously reported TCRα sequences from natural killer T cells and mucosal-associated invariant T cells in a variety of species showed that these ubiquitous receptors could also be efficiently made by the gene recombination process. This demonstrates that the effects of production efficiency are not limited to the TCRβ chain, nor are they limited to a particular species. These findings highlight the important role that variable production efficiencies play in shaping the naive TCR repertoire, resulting in a contoured clonotypic landscape in the thymus. This landscape provides the basis for memory and virus-specific immune responses, and thus has implications for vaccine design and disease control

Novel recombinant mycobacterium bovis BCG, ovine atadenovirus, and modified vaccinia virus ankara vaccines combine to induce robust human immunodeficiency virus-specific CD4 and CD8 T-cell responses in rhesus macaques

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA401 as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401 was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401 and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration

Stochastic expansions maintain the clonal stability of CD8+ T cell populations undergoing memory inflation driven by murine cytomegalovirus

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV

NKT and MAIT invariant TCRα sequences can be produced efficiently by VJ gene recombination

Semi-invariant T cell receptors (TCRs) found on natural killer T (NKT) and mucosal-associated invariant T (MAIT) cells are characterized by the use of invariant variable (V) and joining (J) gene combinations in the TCR α-chain, as well as ubiquitous canonical TCRα amino acid sequences that are dominant in many individuals and similar across species. That they are so prevalent indicates that they occupy an important niche within the immune system. However, these TCRs are produced by a largely random gene recombination process, which seems a risky approach for the immune system to acquire these innate-like cells. We surveyed studies reporting NKT and MAIT TCRα sequences for six and four different species, respectively. Although the germline nature of the canonical human and mouse NKT and mouse MAIT TCRα sequences and an overlap of nucleotides between the mouse MAIT-related Vα and Jα genes have been noted in previous studies, in this study we demonstrate that, for all reported species, the canonical TCRα amino acid sequences can be encoded by at least one germline-derived nucleotide sequence. Moreover, these nucleotide sequences can utilize an overlap between the Vα and Jα genes in their production, which enables them to be produced by a large variety of recombination mechanisms. We investigated the role of these TCRα features in the production of the canonical NKT and MAIT TCRα sequences. In computer simulations of a random recombination process involving the invariant NKT and MAIT TCRα gene combinations for each species, the canonical NKT and MAIT TCRα sequences were the first or second most generated of all sequences with the CDR3α length restrictions associated with NKT and MAIT cells. These results suggest that the immune machinery enables the canonical NKT and MAIT TCRα sequences to be produced with great efficiency through the process of convergent recombination, ensuring their prevalence across individuals and species

Extraction and characterization of the rhesus macaque T-cell receptor β-chain genes

Rhesus macaque models have been instrumental in the development and testing of vaccines before human studies and have provided fundamental insights into the determinants of immune efficacy in a variety of infectious diseases. However, the characterization of antigen-specific T-cell receptor (TCR) repertoires during adaptive immune responses in these models has earlier relied on human TCR gene assignments. Here, we extracted and characterized TCR β-chain (TRB) genes from the recently sequenced rhesus macaque genome that are homologous to the human TRB genes. Comparison of the rhesus macaque TRB genes with the human TRB genes showed an average best match similarity of 92.9%. Furthermore, we confirmed the usage of most rhesus macaque TRB genes by expressed TCRβ sequences within epitope-specific TCR repertoires. This primary description of the rhesus macaque TRB genes will provide a standardized nomenclature and enable better characterization of TCR usage in studies that use this species

Convergent recombination shapes the clonotypic landscape of the naïve T-cell repertoire

Adaptive T-cell immunity relies on the recruitment of antigen-specific clonotypes, each defined by the expression of a distinct T-cell receptor (TCR), from an array of naïve T-cell precursors. Despite the enormous clonotypic diversity that resides within the naïve T-cell pool, interindividual sharing of TCR sequences has been observed within mobilized T-cell responses specific for certain peptide–major histocompatibility complex (pMHC) antigens. The mechanisms that underlie this phenomenon have not been fully elucidated, however. A mechanism of convergent recombination has been proposed to account for the occurrence of shared, or “public,” TCRs in specific memory T-cell populations. According to this model, TCR sharing between individuals is directly related to TCR production frequency; this, in turn, is determined on a probabilistic basis by the relative generation efficiency of particular nucleotide and amino acid sequences during the recombination process. Here, we tested the key predictions of convergent recombination in a comprehensive evaluation of the naïve CD8+ TCRβ repertoire in mice. Within defined segments of the naïve CD8+ T-cell repertoire, TCRβ sequences with convergent features were (i) present at higher copy numbers within individual mice and (ii) shared between individual mice. Thus, the naïve CD8+ T-cell repertoire is not flat, but comprises a hierarchy of recurrence rates for individual clonotypes that is determined by relative production frequencies. These findings provide a framework for understanding the early mobilization of public CD8+ T-cell clonotypes, which can exert profound biological effects during acute infectious processes

Extraction and characterization of the rhesus macaque T‐cell receptor β‐chain genes

Rhesus macaque models have been instrumental in the development and testing of vaccines before human studies and have provided fundamental insights into the determinants of immune efficacy in a variety of infectious diseases. However, the characterization of antigen-specific T-cell receptor (TCR) repertoires during adaptive immune responses in these models has earlier relied on human TCR gene assignments. Here, we extracted and characterized TCR β-chain (TRB) genes from the recently sequenced rhesus macaque genome that are homologous to the human TRB genes. Comparison of the rhesus macaque TRB genes with the human TRB genes showed an average best match similarity of 92.9%. Furthermore, we confirmed the usage of most rhesus macaque TRB genes by expressed TCRβ sequences within epitope-specific TCR repertoires. This primary description of the rhesus macaque TRB genes will provide a standardized nomenclature and enable better characterization of TCR usage in studies that use this species

A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing

The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8+ T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals

Persistent survival of prevalent clonotypes within an immunodominant HIV gag-specific CD8+ T cell response

CD8+ T cells play a significant role in the control of HIV replication, yet the associated qualitative and quantitative factors that determine the outcome of infection remain obscure. In this study, we examined Ag-specific CD8+ TCR repertoires longitudinally in a cohort of HLA-B*2705+ long-term nonprogressors with chronic HIV-1 infection using a combination of molecular clonotype analysis and polychromatic flow cytometry. In each case, CD8+ T cell populations specific for the immunodominant p24 Gag epitope KRWIILGLNK (KK10; residues 263–272) and naturally occurring variants thereof, restricted by HLA-B*2705, were studied at multiple time points; in addition, comparative data were collected for CD8+ T cell populations specific for the CMV pp65 epitope NLVPMVATV (NV9; residues 495–503), restricted by HLA-A*0201. Dominant KK10-specific clonotypes persisted for several years and exhibited greater stability than their contemporaneous NV9-specific counterparts. Furthermore, these dominant KK10-specific clonotypes exhibited cross-reactivity with antigenic variants and expressed significantly higher levels of CD127 (IL-7Rα) and Bcl-2. Of note, we also found evidence that promiscuous TCR α-chain pairing associated with alterations in fine specificity for KK10 variants could contribute to TCR β-chain prevalence. Taken together, these data suggest that an antiapoptotic phenotype and the ability to cross-recognize variant epitopes contribute to clonotype longevity and selection within the peripheral memory T cell pool in the presence of persistent infection with a genetically unstable virus