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Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii , is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δ pigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δ pigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δ pigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions., Competing Interests: The authors declare no conflict of interest.

Unlike in infection and cancer, T cell exhaustion in autoimmune disease has not been clearly defined. Here we set out to understand inhibitory protein (PD-1, Tim3, CTLA4, Lag3) expression in CXCR5- and CXCR5+ CD8 and CD4 T cells in systemic lupus erythematosus. CXCR5+ CD8 and CD4 T cells express PD-1 and engage B cells in germinal center reactions, leading to autoantibody formation in autoimmunity. We hypothesized that CXCR5+ CD8 T cells develop an exhausted phenotype as SLE autoimmunity expands from initial to chronic, self-perpetuating disease due to chronic self-antigen exposure. Our results indicate that there is no exhaustion frequency differences between sexes, although disease kinetics vary by sex. CXCR5+ CD8 T cells express primarily IFNγ, known to promote autoimmune disease development, whereas CXCR5-CD8 T cells express TNFα and IFNγ as disease progresses from 2-6 months. Tim3 is the highest expressed inhibitory marker for all CD4 and CD8 T cell populations demonstrating potential for terminally exhausted populations. CTLA4 expression on CD4 T cells suggests potential tolerance induction in these cells. We identified exhaustion phenotypes within autoimmune disease that progress with increasing lupus erythematosus severity and possibly provide a feedback mechanism for immunological tolerance., Highlights: CXCR5- and CXCR5+ CD8 T cells expand with rate of disease in SLE mouse model.CXCR5+ CD8 T cells are low contributors to TNFα disease progression unlike CXCR5-CD8 T cells but may increase disease mechanisms through high IFNγ production.Inhibitory markers upregulate in frequency with the highest amounts seen in Tim3+ populations. Tim3+Lag3+ expression may be an indicator of terminal differentiation for all populations.Inhibitory marker expression frequency was unrelated to sex.

Introduction: Toxoplasma 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)., Methods: Therefore, 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., Results: Genetic 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 ., Discussion: Collectively, 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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kongsomboonvech, García-López, Njume, Rodriguez, Souza, Rosenberg and Jensen.)

Forward genetic approaches have been widely used in parasitology and have proven their power to reveal the complexities of host-parasite interactions in an unbiased fashion. Many aspects of the parasite's biology, including the identification of virulence factors, replication determinants, antibiotic resistance genes, and other factors required for parasitic life, have been discovered using such strategies. Forward genetic approaches have also been employed to understand host resistance mechanisms to parasitic infection. Here, we will introduce and review all forward genetic approaches that have been used to identify host factors involved with Apicomplexa infections, which include classical genetic screens and QTL mapping, GWAS, ENU mutagenesis, overexpression, RNAi and CRISPR-Cas9 library screens. Collectively, these screens have improved our understanding of host resistance mechanisms, immune regulation, vaccine and drug designs for Apicomplexa parasites. We will also discuss how recent advances in molecular genetics give present opportunities to further explore host-parasite relationships., Competing Interests: The authors declare that the review was conducted and written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sánchez-Arcila and Jensen.)

Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice ("bumble") did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection., Competing Interests: The authors have declared that no competing interests exist.

IL-2Rα, in part, comprises the high affinity receptor for IL-2, a cytokine important in immune proliferation, activation, and regulation. IL-2Rα deficient mice (IL-2Rα-KO) develop systemic autoimmune disease and die from severe anemia between 18 and 80 days of age. These mice develop kinetically distinct autoimmune progression, with approximately a quarter dying by 21 days of age and half dying after 30 days. This research aims to define immune parameters and cytokine signaling that distinguish cohorts of IL-2Rα-KO mice that develop early- versus late-stage autoimmune disease. To investigate these differences, we evaluated complete blood counts (CBC), antibody binding of RBCs, T cell numbers and activation, hematopoietic progenitor changes, and signaling kinetics, during autoimmune hemolytic anemia (AIHA) and bone marrow failure. We identified several alterations that, when combined, correlate to disease kinetics. Early onset disease correlates with anti-RBC antibodies, lower hematocrit, and reduced IL-7 signaling. CD8 regulatory T cells (Tregs) have enhanced apoptosis in early disease. Further, early and late end stage disease, while largely similar, had several differences suggesting distinct mechanisms drive autoimmune disease kinetics. Therefore, IL-2Rα-KO disease pathology rates, driven by T cell signaling, promote effector T cell activation and expansion and Treg dysfunction.

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., Competing Interests: The authors have declared that no competing interests exist.

Over the last century, the alarming surge in allergy and autoimmune disease has led to the hypothesis that decreasing exposure to microbes, which has accompanied industrialization and modern life in the Western world, has fundamentally altered the immune response. In its current iteration, the "hygiene hypothesis" suggests that reduced microbial exposures during early life restricts the production and differentiation of immune cells suited for immune regulation. Although it is now well-appreciated that the increase in hypersensitivity disorders represents a "perfect storm" of many contributing factors, we argue here that two important considerations have rarely been explored. First, the window of microbial exposure that impacts immune development is not limited to early childhood, but likely extends into the womb. Second, restricted microbial interactions by an expectant mother will bias the fetal immune system toward hypersensitivity. Here, we extend this discussion to hypothesize that the cell types sensing microbial exposures include fetal hematopoietic stem cells, which drive long-lasting changes to immunity., (Copyright © 2020 Apostol, Jensen and Beaudin.)

T cell exhaustion is a state of hyporesponsiveness that develops during many chronic infections and cancer. Neutralization of inhibitory receptors, or "checkpoint blockade," can reverse T cell exhaustion and lead to beneficial prognoses in experimental and clinical settings. Whether checkpoint blockade can resolve lethal acute infections is less understood but may be beneficial in vaccination protocols that fail to elicit sterilizing immunity. Since a fully protective vaccine for any human parasite has yet to be developed, we explored the efficacy of checkpoint inhibitors in a mouse model of Toxoplasma gondii reinfection. Mice chronically infected with an avirulent type III strain survive reinfection with the type I RH strain but not the MAS, GUY-DOS, and GT1 parasite strains. We report here that mouse susceptibility to secondary infection correlates with the initial parasite burden and that protection against the RH strain is dependent on CD8 but not CD4 T cells in this model. When given a lethal secondary infection, CD8 and CD4 T cells upregulate several coinhibitory receptors, including PD-1, TIM-3, 4-1bb, and CTLA-4. Moreover, the gamma interferon (IFN-γ) response of CD8 but not CD4 T cells is significantly reduced during secondary infection with virulent strains, suggesting that checkpoint blockade may reduce disease severity. However, single and combination therapies targeting TIM-3, CTLA-4, and/or PD-L1 failed to reverse susceptibility to secondary infection. These results suggest that additional host responses, which are refractory to checkpoint blockade, are likely required for immunity to this pathogen., (Copyright © 2018 American Society for Microbiology.)

For parasites that sequester themselves within a vacuole, new rules governing antigen presentation are coming into focus. Components of the host's autophagy machinery and the parasite's membranous nanotubular network within the parasitophorous vacuole play a major role in determining antigenicity of Toxoplasma proteins. As such, both parasite and vaccinologist may exploit these pathways to regulate the ever important CD8 T cell response to apicomplexan parasites., (Copyright © 2015. Published by Elsevier Ltd.)