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.

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 ß-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.

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.

Convergent recombination shapes the clonotypic landscape of the naive 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(+) TCRß repertoire in mice. Within defined segments of the naïve CD8(+) T-cell repertoire, TCRß 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.

Long peptides induce polyfunctional T cells against conserved regions of HIV-1 with superior breadth to single-gene vaccines in macaques.

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.

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.HIVA(401) as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA(401) 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.HIVA(401) and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.

Extraction and characterization of the rhesus macaque T-cell receptor beta-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 beta-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 TCRbeta 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.

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.