Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5

Abstract

Host resistance to Toxoplasma gondii relies on CD8 T cell IFN{gamma} 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 naive CD8 T cell IFN{gamma} responses to a vacuolar resident antigen of T. gondii, TGD057. Naive 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 IFN{gamma}. T57 IFN{gamma} responses to TGD057 were independent of the parasites 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 IFN{gamma} responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFN{gamma} 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 IFN{gamma} 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 IFN{gamma} responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFN{gamma} 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 IFN{gamma} 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, IFN{gamma}, 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 IFN{gamma} in response to infection. Importantly, CD8 T cell IFN{gamma} 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 IFN{gamma} responses to parasites.