Parasite fate and involvement of infected cells in the induction of CD4+ and CD8+ T cell responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses

A Mendelian Disease Of Autoimmunity Reveals Gimap5 As A Novel Member Of The Ragulator Complex

The incidence of autoimmune diseases, many of which lack effective treatments, is rapidly increasing in the developed world. Mendelian diseases allow the study of autoimmunity in humans, enabling new insights into the underlying pathology. In this study, I have identified a patient cohort suffering from a novel recessive Mendelian disease of immune dysregulation characterized by severe lymphopenia, splenomegaly, thrombocytopenia and liver failure. Whole exome sequencing revealed mutations in GIMAP5, a small GTPase primarily expressed in T, NK and endothelial cells. The missense mutations in these patients destabilize the protein in vitro and lead to a near complete loss of protein in patient cells. Animal models lacking GIMAP5 develop a disease remarkably similar to that observed in the human patients; however, the molecular role of this gene in the immune system remains obscure. To address this, I defined the interactome of GIMAP5 via immunoprecipitation and high-throughput mass spectrometry. This revealed a robust interaction with all seven members of the Ragulator complex which I went on to confirm via endogenous co-immunoprecipitation and proximity ligation assays. This complex has recently been described as a key regulator of mTORC1, Erk signaling and lysosome positioning. In order to study the functional relevance of this interaction I utilized an in vitro CRISPR mediated approach to knockout Gimap5 in murine primary T cells. I observed a very rapid and robust induction of apoptosis accompanied by significant increases in ceramide levels following the loss of GIMAP5 which is consistent with the lymphopenic phenotype. Future studies will relate the increased ceramide to the Ragulator complex and induction of apoptosis in GIMAP5-deficient T cells and leverage these findings to develop novel treatments for GIMAP5 deficiency and other autoimmune diseases

Genome-wide mouse mutagenesis reveals CD45-mediated T cell function as critical in protective immunity to HSV-1.

International audienceHerpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc(L3X)), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc(L3X) mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc(L3X) mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc(L3X) mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4⁺ and CD8⁺ T cells and could be attributed to function of CD4⁺ T helper 1 (Th1) cells in CD8⁺ T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development

Differences in pH sensitivity of two fluorescent markers can be used to distinguish parasites that have been phagocytosed from those that actively invade host cells.

<p>Fluorescence intensity of mCherry-expressing <i>cpsII</i> parasites labeled with CellTrace Violet and incubated overnight at varying pH in buffer solutions consisting of citric acid and disodium phosphate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004047#ppat.1004047-McIlvaine1" target="_blank">[93]</a> was measured by flow cytometry (a). Violet and mCherry fluorescence of immortalized murine bone marrow-derived macrophages exposed to Violet-labeled, mCherry-expressing <i>cpsII</i> parasites pre-treated with DMSO (top) or the irreversible inhibitor of invasion 4-p-bpb (bottom) 1 hour and 18 hours following exposure to parasites, measured by flow cytometry (b). Images of mCherry^+veViolet^+ve and mCherry^+veViolet^−ve bone marrow-derived macrophages 18 hours following exposure to Violet-labeled, mCherry-expressing <i>cpsII</i> parasites pre-treated with 4-p-bpb or DMSO (c). Violet and mCherry fluorescence of cells isolated from the PECS of mice 18 hours post-administration of 10⁶ DMSO-treated or 4-p-bpb-treated parasites (d). Cytospin analysis was performed on Violet^+ve cells isolated by FACS sorting, obtained from the PECS of a mouse 18 hours after vaccination with Violet-labeled <i>cpsII</i> parasites (e). Images of mCherry^+veViolet^+ve and mCherry^+veViolet^−ve cells isolated from the PECS of mice 18 hours post-administration of 10⁶ DMSO-treated or 4-p-bpb-treated Violet-labeled, mCherry-expressing <i>cpsII</i> parasites (f).</p

The fate of heat-killed and live <i>cpsII</i> parasites in host cells.

<p>C57BL/6 bone marrow derived macrophages were infected with <i>cpsII</i> parasites and examined using immunofluorescence assays (a). Bone marrow-derived macrophages activated with IFN-γ (100 U/ml) and LPS (0.1 ng/ml) were infected with freshly lysed or heat-killed parasites for 3 hours. Intracellular parasites were stained for host Irgb6 or LAMP1 recruitment in green. Parasites were stained with a mouse monoclonal antibody to GRA1 to identify the parasitophorous vacuole or rabbit polyclonal sera against GRA7 in red. IFN-γ and LPS activated bone marrow-derived macrophages were infected with freshly lysed <i>cpsII</i> parasites and fixed at 3, 24, 48 and 120 hours post-infection (b). Parasite vacuoles were identified with rabbit polyclonal sera to GRA7 (red) and host LAMP1 was identified with a rat monoclonal antibody. Scale bar = 10 µm. Electron micrograph images of infected macrophages treated with IFN-γ (50 units/ml) and LPS (10 ng/ml) or untreated at 2 hours post-infection (c). Parasites persist in intact vacuoles and do not display blebbing or disruption of the parasitophorous vacuole. Some <i>cpsII</i> parasites were found to exhibit non-productive cell division in IFN-γ and LPS- treated or untreated macrophages when examined 24 hours post-infection (d). Scale bars = 1.5 µm.</p

Composition of total cell populations, mCherry^+veViolet^−ve cell populations, and mCherry^+veViolet^+ve populations from the PECS of naïve and vaccinated mice.

<p>Mice were vaccinated with 10⁶ Violet-labeled, mCherry-expressing <i>cpsII</i> parasites intraperitoneally and sacrificed 18 hours post-vaccination. Cell type composition of total peritoneal cell populations in naïve and vaccinated mice, and the cell type composition of mCherry^+veViolet^−ve cells and mCherry^+veViolet^+ve cells in vaccinated mice were examined. Representative flow plots demonstrating infected cells and cells that have phagocytosed <i>T. gondii</i> for each major cell type present in the PECS are shown (a). The composition of the PECS in naïve mice and vaccinated mice, and the composition of infected cells (mCherry^+veViolet^+ve) and cells that have phagocytosed <i>T. gondii</i> (mCherry^+veViolet^−ve) are depicted (b). Percentages of macrophages and dendritic cells in the total peritoneal cell population in vaccinated mice are compared to the percentages of infected cells that are macrophages and dendritic cells (c). T/B/NK cells are identified by expression of CD3, CD19, or NK1.1. Dendritic cells were identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^HI,MHCII^HI. Monocytes and neutrophils were defined as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^LOW-INT,Gr-1^+ve. Macrophages were identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^LOW-INT,Gr-1^−ve,CD11b^INTorHI. *p<0.05; ***p<0.0005. AVG±STDEV. A paired, two-tailed student's t test was used to analyze the data in (c). Results shown are from one representative experiment. Similar results were obtained over the course of seven separate experiments.</p

Active invasion is required for adaptive immune responses to <i>T. gondii</i>.

<p><i>cpsII</i>-OVA parasites were heat-killed, treated with the invasion inhibitor 4-p-bpb or left untreated and administered to mice intraperitoneally. Tetramer-specific and total CD4⁺ (a) and CD8⁺ (b) T cell responses were measured from cells isolated from the spleen and lymph nodes (pooled) 10 days post-vaccination. Flow plots are gated on Foxp3^−ve CD4⁺ T cells (a, top) or CD4⁺ T cells (a, bottom) and the population examined at the bottom of A was enriched for tetramer^+ve cells. Flow plots in B are gated on CD8⁺ T cells. *p<0.05; **p<0.005. AVG±SE.</p

Dendritic cells are required for optimal CD4⁺ and CD8⁺ T cell responses.

<p>CD11c-DTR mice were administered diphtheria toxin 1 day prior to <i>cpsII</i>-OVA vaccination. At the time of vaccination, some mice were sacrificed to determine the efficiency of depletion. Percentages and numbers of dendritic cells from the spleen are shown. FACS plots are gated on CD3⁻,CD19⁻,NK1.1⁻ cells (a). Eight days following vaccination, mice were sacrificed and total and tetramer-specific CD4⁺ and CD8⁺ T cell responses were analyzed. Total CD4⁺ T cell responses from the spleens are shown (b, top). Tetramer-specific CD4⁺ T cell responses from pooled lymph nodes and splenocytes were determined in a separate experiment (b, bottom). Flow plots are gated on CD4⁺ T cells (b), and the population examined was magnetically enriched for the tetramer^+ve population (b, bottom). Total and OVA-specific CD8⁺ T cell responses from the PECS are depicted (c), and flow plots are gated on CD8+ T cells. Significant differences in tetramer and total CD8⁺ T cell responses between WT and CD11c-DTR mice were also apparent in the spleen. *p<0.05; **p<0.005. AVG±SE.</p

Activation status of mCherry^+veViolet^−ve and mCherry^+veViolet^+ve macrophages and dendritic cells.

<p>Mice were administered parasites as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004047#ppat-1004047-g003" target="_blank">Figure 3</a>. At 18 hours post-vaccination, expression of the antigen presentation molecules MHCI (a) and MHCII (b) and expression of the costimulatory molecules CD86 (c) and CD40 (d) on CD11b^HI macrophages and dendritic cells was determined by flow cytometry. Macrophages are identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^−ve,Gr-1^−ve,CD11b^HI cells. Dendritic cells are identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^HI,MCHII^HI. Confidence intervals were determined using the Bonferroni correction method. *p<0.017; **p<0.0017; ***p<0.00017. AVG±SE. Paired, two-tailed student's t tests were used to compare expression levels of molecules on populations within <i>cpsII</i>-vaccinated mice.</p