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.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T cell receptor (TCR) contact residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both BCG vaccination and M. tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Adaptive T-cell immunity relies on the recruitment of antigenspecific 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.

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. Eur. J. Immunol. 2010. 40: 1973-1984 DOI 10.1002 Immunity to infection 1973 IntroductionDevelopment of an effective, accessible vaccine is the only realistic hope for halting the HIV-1/AIDS epidemic. Ideally, such a vaccine should induce broadly neutralizing antibodies and effective T cells at the same time; however, both of these goals face substantial and very different challenges [1]. A rational scientific strategy tackles and solves these roadblocks separately [2] before combining the two successful solutions into a single vaccine formulation.Here, we focus on the induction of protective T-cell responses. In order to act quickly and efficiently, protective T cells will be required to recognize all HIV-1 variants circulating in the target population as well as all escape mutants generated in individuals who did not repel the virus in the first place. We hypothesized that HIV-1 variation would be best tackled by focusing immune responses on domains that lie within functionally conserved regions of the virus [3]. Thus, we assembled immunogen HIVconsv as a single chimeric protein from the 14 most invariable segments of the HIV-1 proteome, each 27-128 amino acids in length, alternated individual regions among the four major clades (A, B, C, and D) to ensure equal representation and used clade consensus sequences to reflect variation within individual clades. As these regions are functionally conserved, T-cell escape mutations are expected to incur significant costs to viral fitness. Also, relative to non-protective responses induced during natural HIV-1 infection, HIVconsv refocuses T cells to subdominant epitopes, which may be important for protection [4][5][6]. Thus, the HIVconsv immunogen offers a simple and univ...

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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 Ankaravectored 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.

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.

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an antigen-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 antigen 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 antigen 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 antigen-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 antigen 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.