Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T . gondii , TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T . gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T . gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.
Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the ‘nutrient sensitized’ Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged “bubble vacuoles” with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.
Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.AUTHOR SUMMARYParasites are excellent “students” of our immune system as they can deflect, antagonize and confuse the immune response making it difficult to vaccinate against these pathogens. In this report, we analyzed how a widespread parasite of mammals, Toxoplasma gondii, manipulates an immune cell needed for immunity to many intracellular pathogens, the CD8 T cell. Host pathways that govern CD8 T cell production of the immune protective cytokine, IFNγ, were also explored. We hypothesized the secreted Toxoplasma virulence factor, ROP5, work to inhibit the MHC 1 antigen presentation pathway therefore making it difficult for CD8 T cells to see T. gondii antigens sequestered inside a parasitophorous vacuole. However, manipulation through T. gondii ROP5 does not fully explain how CD8 T cells commit to making IFNγ in response to infection. Importantly, CD8 T cell IFNγ responses to T. gondii require the pathogen sensor NLRP3 to be expressed in the infected cell. Other proteins associated with NLRP3 activation, including members of the conventional inflammasome activation cascade pathway, are only partially involved. Our results identify a novel pathway by which NLRP3 regulates T cell function and underscore the need for inflammasome-activating adjuvants in vaccines aimed at inducing CD8 T cell IFNγ responses to parasites.
IntroductionToxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic “Regulator Of CD8 T cell Response” (ROCTR).MethodsTherefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages.ResultsGenetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii.DiscussionCollectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.
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