Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4(+) and CD8(+) T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3(+) T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.

The study of the three protagonists in malaria-the Plasmodium parasite, the Anopheles mosquito, and the human host-is key to developing methods to control and eventually eliminate the disease. Genomic technologies, including the recent development of next-generation sequencing, enable interrogation of this triangle to an unprecedented level of scrutiny, and promise exciting progress toward real-time epidemiology studies and the study of evolutionary adaptation. We discuss the use of genomics by the International Centers of Excellence for Malaria Research, a network of field sites and laboratories in malaria-endemic countries that undertake cutting-edge research, training, and technology transfer in malarious countries of the world.

IFNγ signaling drives dendritic cells (DCs) to promote type I T cell (Th1) immunity. Here, we show that activation of DCs by IFNγ is equally crucial for the differentiation of a population of T-bet+ regulatory T (Treg) cells specialized to inhibit Th1 immune responses. Conditional deletion of IFNγ receptor in DCs but not in Treg cells resulted in a severe defect in this specific Treg cell subset, leading to exacerbated immune pathology during parasitic infections. Mechanistically, IFNγ-unresponsive DCs failed to produce sufficient amount of IL-27, a cytokine required for optimal T-bet induction in Treg cells. Thus, IFNγ signalling endows DCs with the ability to efficiently control a specific type of T cell immunity through promoting a corresponding Treg cell population.

Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.

Unlike most cells, protozoa in the phylum Apicomplexa divide by a distinctive process in which multiple daughters are assembled within the mother (schizogony or endodyogeny), using scaffolding known as the inner membrane complex (IMC). The IMC underlies the plasma membrane during interphase, but new daughters develop in the cytoplasm, as cytoskeletal filaments associate with flattened membrane cisternae (alveolae), which elongate rapidly to encapsulate subcellular organelles. Newly assembled daughters acquire their plasma membrane as they emerge from the mother, leaving behind vestiges of the maternal cell. Although the maternal plasma membrane remains intact throughout this process, the maternal IMC disappears - is it degraded, or recycled to form the daughter IMC? Exploiting fluorescently tagged IMC markers, we have used live-cell imaging, fluorescence recovery after photobleaching (FRAP) and mEos2 photoactivation to monitor the dynamics of IMC biogenesis and turnover during the replication of Toxoplasma gondii tachyzoites. These studies reveal that the formation of the T. gondii IMC involves two distinct steps - de novo assembly during daughter IMC elongation within the mother cell, followed by recycling of maternal IMC membranes after the emergence of daughters from the mother cell.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium's minimal information (MIxS) and NCBI's BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

The IL-27R, WSX-1, is required to limit IFN-γ production by effector CD4⁺ T cells in a number of different inflammatory conditions but the molecular basis of WSX-1-mediated regulation of Th1 responses in vivo during infection has not been investigated in detail. In this study we demonstrate that WSX-1 signalling suppresses the development of pathogenic, terminally differentiated (KLRG-1⁺) Th1 cells during malaria infection and establishes a restrictive threshold to constrain the emergent Th1 response. Importantly, we show that WSX-1 regulates cell-intrinsic responsiveness to IL-12 and IL-2, but the fate of the effector CD4⁺ T cell pool during malaria infection is controlled primarily through IL-12 dependent signals. Finally, we show that WSX-1 regulates Th1 cell terminal differentiation during malaria infection through IL-10 and Foxp3 independent mechanisms; the kinetics and magnitude of the Th1 response, and the degree of Th1 cell terminal differentiation, were comparable in WT, IL-10R1⁻/⁻ and IL-10⁻/⁻ mice and the numbers and phenotype of Foxp3⁺ cells were largely unaltered in WSX-1⁻/⁻ mice during infection. As expected, depletion of Foxp3⁺ cells did not enhance Th1 cell polarisation or terminal differentiation during malaria infection. Our results significantly expand our understanding of how IL-27 regulates Th1 responses in vivo during inflammatory conditions and establishes WSX-1 as a critical and non-redundant regulator of the emergent Th1 effector response during malaria infection.

Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism.

Disease progression in response to infection can be strongly influenced by both pathogen burden and infection-induced immunopathology. While current therapeutics focus on augmenting protective immune responses, identifying therapeutics that reduce infection-induced immunopathology are clearly warranted. Despite the apparent protective role for murine CD8⁺ T cells following infection with the intracellular parasite Leishmania, CD8⁺ T cells have been paradoxically linked to immunopathological responses in human cutaneous leishmaniasis. Transcriptome analysis of lesions from Leishmania braziliensis patients revealed that genes associated with the cytolytic pathway are highly expressed and CD8⁺ T cells from lesions exhibited a cytolytic phenotype. To determine if CD8⁺ T cells play a causal role in disease, we turned to a murine model. These studies revealed that disease progression and metastasis in L. braziliensis infected mice was independent of parasite burden and was instead directly associated with the presence of CD8⁺ T cells. In mice with severe pathology, we visualized CD8⁺ T cell degranulation and lysis of L. braziliensis infected cells. Finally, in contrast to wild-type CD8⁺ T cells, perforin-deficient cells failed to induce disease. Thus, we show for the first time that cytolytic CD8⁺ T cells mediate immunopathology and drive the development of metastatic lesions in cutaneous leishmaniasis.

In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8+ T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.

Interleukin-27 (IL-27) is a key immunosuppressive cytokine that counters T helper 17 (Th17) cell-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in T cells rapidly induced by IL-27. We found that IL-27 priming of naive T cells upregulated expression of programmed death ligand 1 (PD-L1) in a signal transducer and activator of transcription 1 (STAT1)-dependent manner. When cocultured with naive CD4(+) T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through a PD-1-PD-L1 interaction. In vivo, coadministration of naive TCR transgenic T cells (2D2 T cells) with IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Thus, these data identify a suppressive activity of IL-27, by which CD4(+) T cells can restrict differentiation of Th17 cells in trans.

Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown.

The OrthoMCL database (http://orthomcl.cbil.upenn.edu) houses ortholog group predictions for 55 species, including 16 bacterial and 4 archaeal genomes representing phylogenetically diverse lineages, and most currently available complete eukaryotic genomes: 24 unikonts (12 animals, 9 fungi, microsporidium, Dictyostelium, Entamoeba), 4 plants/algae and 7 apicomplexan parasites. OrthoMCL software was used to cluster proteins based on sequence similarity, using an all-against-all BLAST search of each species' proteome, followed by normalization of inter-species differences, and Markov clustering. A total of 511,797 proteins (81.6% of the total dataset) were clustered into 70,388 ortholog groups. The ortholog database may be queried based on protein or group accession numbers, keyword descriptions or BLAST similarity. Ortholog groups exhibiting specific phyletic patterns may also be identified, using either a graphical interface or a text-based Phyletic Pattern Expression grammar. Information for ortholog groups includes the phyletic profile, the list of member proteins and a multiple sequence alignment, a statistical summary and graphical view of similarities, and a graphical representation of domain architecture. OrthoMCL software, the entire FASTA dataset employed and clustering results are available for download. OrthoMCL-DB provides a centralized warehouse for orthology prediction among multiple species, and will be updated and expanded as additional genome sequence data become available.

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25-deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25(-/-) mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-gamma and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.

The non-photosynthetic plastid - or apicoplast - of Toxoplasma gondii and other apicomplexan parasites is an essential organelle and promising drug target. Most apicoplast proteins are encoded in the nucleus and targeted into the organelle through the apicoplast's four membranes courtesy of a bipartite N-terminal leader sequence comprising of an endomembrane signal peptide followed by a plastid transit peptide. Apicoplast transit peptides, like plant plastid transit peptides, have no primary consensus, are variable in length and may be distinguishable only by a relative depletion of negative charged residues and consequent enrichment in basic residues. In this study we examine the role of charged residues within an apicoplast transit peptide in T. gondii by point mutagenesis. We demonstrate that positive charged residues, combined with the absence of negatively charged amino acids, are essential for apicoplast transit peptide fidelity, as also observed in P. falciparum. Furthermore, we show that positive charge is more important at the transit peptide's N-terminus than its C-terminus, and that the nature of the positive residue and the exact position of the N-terminal positive charge are not important. These results suggest that a simple, rule-based prediction for T. gondii transit peptides, similar to that successfully implemented for P. falciparum should help to identify apicoplast proteins and facilitate the identification of drug targets in this important human pathogen.

Long-lived plasma cells (PCs) in the bone marrow (BM) are a critical source of antibodies after infection or vaccination, but questions remain about the factors that control PCs. We found that systemic infection alters the BM, greatly reducing PCs and regulatory T (Treg) cells, a population that contributes to immune privilege in the BM. The use of intravital imaging revealed that BM Treg cells display a distinct behavior characterized by sustained co-localization with PCs and CD11c-YFP+ cells. Gene expression profiling indicated that BM Treg cells express high levels of Treg effector molecules, and CTLA-4 deletion in these cells resulted in elevated PCs. Furthermore, preservation of Treg cells during systemic infection prevents PC loss, while Treg cell depletion in uninfected mice reduced PC populations. These studies suggest a role for Treg cells in PC biology and provide a potential target for the modulation of PCs during vaccine-induced humoral responses or autoimmunity.