Defining rules of CD8(+) T cell expansion against pre-erythrocytic Plasmodium antigens in sporozoite-immunized mice.

BACKGROUND: Whole Plasmodium sporozoites serve as both experimental tools and potentially as deployable vaccines in the fight against malaria infection. Live sporozoites infect hepatocytes and induce a diverse repertoire of CD8(+) T cell responses, some of which are capable of killing Plasmodium-infected hepatocytes. Previous studies in Plasmodium yoelii-immunized BALB/c mice showed that some CD8(+) T cell responses expanded with repeated parasite exposure, whereas other responses did not. RESULTS: Here, similar outcomes were observed using known Plasmodium berghei epitopes in C57BL/6 mice. With the exception of the response to PbTRAP, IFNγ-producing T cell responses to most studied antigens, such as PbGAP50, failed to re-expand in mice immunized with two doses of irradiated P. berghei sporozoites. In an effort to boost secondary CD8(+) T cell responses, heterologous cross-species immunizations were performed. Alignment of P. yoelii 17XNL and P. berghei ANKA proteins revealed that >60 % of the amino acids in syntenic orthologous proteins are continuously homologous in fragments ≥8-amino acids long, suggesting that cross-species immunization could potentially trigger responses to a large number of common Class I epitopes. Heterologous immunization resulted in a larger liver burden than homologous immunization. Amongst seven tested antigen-specific responses, only CSP- and TRAP-specific CD8(+) T cell responses were expanded by secondary homologous sporozoite immunization and only those to the L3 ribosomal protein and S20 could be re-expanded by heterologous immunization. In general, heterologous late-arresting, genetically attenuated sporozoites were better at secondarily expanding L3-specific responses than were irradiated sporozoites. GAP50 and several other antigens shared between P. berghei and P. yoelii induced a large number of IFNγ-positive T cells during primary immunization, yet these responses could not be re-expanded by either homologous or heterologous secondary immunization. CONCLUSIONS: These studies highlight how responses to different sporozoite antigens can markedly differ in recall following repeated sporozoite vaccinations. Cross-species immunization broadens the secondary response to sporozoites and may represent a novel strategy for candidate antigen discovery.

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations.

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins.

Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses.

In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T(H)1, T(H)2 or T(H)17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T(reg)). T(reg) cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T(H)1 and T(H)2 cytokine production. Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets. Here we show that in mouse T(reg) cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T(H)2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T(reg) cells with the ability to suppress T(H)2 responses. Indeed, ablation of a conditional Irf4 allele in T(reg) cells resulted in selective dysregulation of T(H)2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T(reg) cells. Our results indicate that T(reg) cells use components of the transcriptional machinery, promoting a particular type of effector CD4(+) T cell differentiation, to efficiently restrain the corresponding type of the immune response.

Decoding cell lineage from acquired mutations using arbitrary deep sequencing.

Because mutations are inevitable, the genome of each cell in a multicellular organism becomes unique and therefore encodes a record of its ancestry. Here we coupled arbitrary single primer PCR with next-generation DNA sequencing to catalog mutations and deconvolve the phylogeny of cultured mouse cells. This study helps pave the way toward construction of retrospective cell-fate maps based on mutations accumulating in genomes of somatic cells.

Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells.

Transcription factor Foxp3 (forkhead box P3), restricted in its expression to a specialized regulatory CD4+ T-cell subset (T(R)) with a dedicated suppressor function, controls T(R) lineage development. In humans and mice, Foxp3 deficiency results in a paucity of T(R) cells and a fatal breach in immunological tolerance, causing highly aggressive multi-organ autoimmune pathology. Here, through genome-wide analysis combining chromatin immunoprecipitation with mouse genome tiling array profiling, we identify Foxp3 binding regions for approximately 700 genes and for an intergenically encoded microRNA. We find that a large number of Foxp3-bound genes are up- or downregulated in Foxp3+ T cells, suggesting that Foxp3 acts as both a transcriptional activator and repressor. Foxp3-mediated regulation unique to the thymus affects, among others, genes encoding nuclear factors that control gene expression and chromatin remodelling. In contrast, Foxp3 target genes shared by the thymic and peripheral T(R) cells encode primarily plasma membrane proteins, as well as cell signalling proteins. Together, our studies suggest that distinct transcriptional sub-programmes implemented by Foxp3 establish T(R) lineage during differentiation and its proliferative and functional competence in the periphery.

Identification of novel HLA class II target epitopes for generation of donor-specific T regulatory cells.

Therapies capable of generating host T regulatory cells (T(R)) responsive to donor-specific HLA-class II minor histocompatibility antigens have the potential to promote tolerance of a transplanted organ. Our group has developed a novel approach for the identification of potentially therapeutic T(R) target antigens. We perform parallel non-synonymous SNP genotyping of HLA-identical subject pairs to identify peptide variations expressed by only one of the two subjects. Variant peptide pairs are then evaluated for binding a shared HLA-class II allele. Minor peptides predicted to bind HLA-class II with greater affinity than the common variant peptide are tested for HLA class II binding and in vitro induction of suppressive CD4+ T cells. Using this approach we have identified multiple pairs of variant peptides capable of differential binding and induction of suppressive CD4+ T cells. These data demonstrate the feasibility of identifying potentially therapeutic HLA class II minor antigens for generation of donor-specific T(R).

Phylogenetic analysis of developmental and postnatal mouse cell lineages.

Fate maps depict how cells relate together through past lineage relationships, and are useful tools for studying developmental and somatic processes. However, with existing technologies, it has not been possible to generate detailed fate maps of complex organisms such as the mouse. We and others have therefore proposed a novel approach, "phylogenetic fate mapping," where patterns of somatic mutation carried by the individual cells of an animal are used to retrospectively deduce lineage relationships through phylogenetic inference. Here, we have cataloged genomic polymorphisms at 324 mutation-prone polyguanine tracts for nearly 300 cells isolated from a single mouse, and have explored the cells' lineage relationships both phylogenetically and through a network-based approach. We present a model of mouse embryogenesis, where an early period of substantial cell mixing is followed by more coherent growth of clones later. We find that cells from certain tissues have greater numbers of close relatives in other specific tissues than expected from chance, suggesting that those populations arise from a similar pool of ancestral lineages. Finally, we have investigated the dynamics of cell turnover (the frequency of cell loss and replacement) in postnatal tissues. This work offers a longitudinal study of developmental lineages, from conception to adulthood, and provides insight into basic questions of mouse embryology as well as the somatic processes that occur after birth.

Tanglegrams: A Reduction Tool for Mathematical Phylogenetics.

Many discrete mathematics problems in phylogenetics are defined in terms of the relative labeling of pairs of leaf-labeled trees. These relative labelings are naturally formalized as tanglegrams, which have previously been an object of study in coevolutionary analysis. Although there has been considerable work on planar drawings of tanglegrams, they have not been fully explored as combinatorial objects until recently. In this paper, we describe how many discrete mathematical questions on trees "factor" through a problem on tanglegrams, and how understanding that factoring can simplify analysis. Depending on the problem, it may be useful to consider a unordered version of tanglegrams, and/or their unrooted counterparts. For all of these definitions, we show how the isomorphism types of tanglegrams can be understood in terms of double cosets of the symmetric group, and we investigate their automorphisms. Understanding tanglegrams better will isolate the distinct problems on leaf-labeled pairs of trees and reveal natural symmetries of spaces associated with such problems.

Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens.

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner.

Distinct classes of protective immunity are guided by activation of STAT transcription factor family members in response to environmental cues. CD4+ regulatory T cells (T(regs)) suppress excessive immune responses, and their deficiency results in a lethal, multi-organ autoimmune syndrome characterized by T helper 1 (TH1) and T helper 2 (TH2) CD4+ T cell-dominated lesions. Here we show that pathogenic TH17 responses in mice are also restrained by T(regs). This suppression was lost upon T(reg)-specific ablation of Stat3, a transcription factor critical for TH17 differentiation, and resulted in the development of a fatal intestinal inflammation. These findings suggest that T(regs) adapt to their environment by engaging distinct effector response-specific suppression modalities upon activation of STAT proteins that direct the corresponding class of the immune response.

Ancient haplotypes of the HLA Class II region.

Allelic variation in codons that specify amino acids that line the peptide-binding pockets of HLA's Class II antigen-presenting proteins is superimposed on strikingly few deeply diverged haplotypes. These haplotypes appear to have been evolving almost independently for tens of millions of years. By complete resequencing of 20 haplotypes across the approximately 100-kbp region that spans the HLA-DQA1, -DQB1, and -DRB1 genes, we provide a detailed view of the way in which the genome structure at this locus has been shaped by the interplay of selection, gene-gene interaction, and recombination.

Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa.

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.

Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa.

The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.

Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.