Nicolas Blanchard

T cells are critical to control Toxoplasma gondii (T. gondii) parasite infection. Yet, our understanding of T. gondii-specific CD8+ T cell responses in humans remains scarce. Here, we studied 56 HLA-A2+ healthy blood donors, including 40 latently infected subjects. In addition to routine T. gondii serodiagnosis, we quantified IgG specific for the bradyzoite-restricted Brain Cyst Load-associated Antigen (BCLA) and we enumerated blood-circulating parasite-specific CD8+ T cells by IFN-γ ELISPOT using a panel of 29 T. gondii peptides, previously reported to be HLA-A2 ligands. We identified a set of 16 epitopes that stimulates detectable CD8+ T cell responses in 50% routine T. gondii seropositive subjects, yet none was a universal immunodominant T. gondii epitope. Among individuals with either positive BCLA-specific serology and/or detectable T. gondii-specific T cell response, T. gondii-specific ELISPOT responses were inversely correlated with BCLA-specific IgG titers, suggesting that stronger CD8+ T cell responses may contribute to reduce chronic parasite burden or that certain long-standing infections may result in waning of antibody levels but persistence of CD8+ T cell memory. Furthermore, 2 out of 56 subjects displayed null T. gondii-specific humoral responses across 4 serological assays, but had measurable CD8+ T cell responses to 2 T. gondii peptides. This study sheds light on the complexity of T. gondii-specific immune responses in humans and rationalizes the cellular and serological toolkits for immune monitoring of latent T. gondii infection in humans.

Chronic inflammation, resulting from infections, is characterized by increased levels of cytokines including interleukin-1 (IL-1), but little is known about how IL-1 contributes to cognitive impairment, potentially via epigenetic processes. Here we demonstrate that mice chronically infected with the parasite Toxoplasma gondii exhibit impaired spatial memory, which is dependent on neuronal IL-1 signaling and mimicked by chronic exposure to IL-1β. Both T. gondii infection and chronic IL-1β drive H2A.X-dependent DNA double-strand break signaling in hippocampal neurons and invalidating neuronal H2A.X-dependent signaling blocks memory impairments caused by either exposure. Our results highlight the instrumental role of cytokine-induced double-strand-break-dependent signaling in spatial memory defects, which may be relevant to multiple brain diseases.

Preventing T cell migration to the central nervous system (CNS) has remarkable therapeutic effects in relapsing-remitting multiple sclerosis (RRMS) but is poorly effective against the progressive form (PMS). Disability progression in PMS likely results from an interplay between smoldering local inflammation and neurodegeneration. The mechanisms sustaining the chronicity of PMS are poorly understood. Here, we investigated the potential role of tissue-resident memory CD4 ⁺ T cells (CD4 ⁺ Trm cells) in sustaining chronic CNS autoimmunity. We showed that CD4 ⁺ Trm cells were present in the CNS of mice with chronic experimental autoimmune encephalomyelitis (EAE) and in brain tissues from persons with PMS. Using flow cytometry and immunohistofluorescence analysis, we revealed the presence of bona fide CD4 ⁺ Trm cells expressing characteristic Trm cell surface markers, including CD69, CXCR6, P2RX7, and CD49a, in the CNS of mice with EAE and in the brains of persons with PMS. These T cells also expressed the transcription factor Hobit in mice with chronic EAE. Single-cell transcriptomic analysis uncovered the transcriptional heterogeneity and inflammatory potential of CD4 ⁺ Trm cells, and, accordingly, these cells localized within CNS inflammatory lesions of mice with EAE and persons with PMS. Last, either genetic or pharmacological depletion of CD4 ⁺ Trm cells combined with antibody-mediated depletion of the recirculating CD4 ⁺ T cell compartment alleviated neurological signs during the chronic phase of EAE. Our results indicate that CD4 ⁺ Trm cells contribute to maintain a chronic inflammatory state in the CNS and suggest that therapeutic strategies for PMS should consider targeting the CNS-resident T cell compartment.

By eliciting immune activation in the digestive tract, intestinal pathogens may perturb gut homeostasis. Some gastrointestinal infections can indeed increase the risk of developing post-infectious irritable bowel syndrome (PI-IBS). Intriguingly, the prevalent foodborne parasite Toxoplasma gondii has not been linked to the development of PI-IBS and the impact of this infection on colon homeostasis remains ill-defined. We show in a mouse model that latent T. gondii decreases visceral nociceptive responses in an opioid signaling-dependent manner. Despite the accumulation of Th1 and cytotoxic T cells in the colon of latently infected mice, the selective invalidation of enkephalin gene in T cells ruled out the involvement of T cell-derived enkephalins in hypoalgesia. These findings provide clues about how this widespread infection durably shapes the gut immune landscape and modifies intestinal physiological parameters. They suggest that in contrast to other gut microbes, T. gondii infection could be negatively associated with abdominal pain.

By eliciting immune activation in the digestive tract, intestinal pathogens may perturb gut homeostasis. Some gastrointestinal infections can indeed increase the risk of developing post-infectious irritable bowel syndrome (PI-IBS). Intriguingly, the prevalent foodborne parasite Toxoplasma gondii has not been linked to the development of PI-IBS and the impact of this infection on colon homeostasis remains ill-defined. We show in a mouse model that latent T. gondii decreases visceral nociceptive responses in an opioid signaling-dependent manner. Despite the accumulation of Th1 and cytotoxic T cells in the colon of latently infected mice, the selective invalidation of enkephalin gene in T cells ruled out the involvement of T cell-derived enkephalins in hypoalgesia. These findings provide clues about how this widespread infection durably shapes the gut immune landscape and modifies intestinal physiological parameters. They suggest that in contrast to other gut microbes, T. gondii infection could be negatively associated with abdominal pain. Teaser: Latent toxoplasmosis decreases gut nociception in the mouse, suggesting a negative association with abdominal pain in humans

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr), but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell–mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a, and CD103 are sequentially acquired by brain parasite–specific CD8+ T cells throughout infection and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell–dependent manner and is associated with effective parasite control during chronic stage. Conditional invalidation of Transporter associated with Antigen Processing (TAP)-mediated major histocompatibility complex (MHC) class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulates the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell–mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections.

Chronic T. gondii infection induces brain-resident CD8+ T cells (bTr) but their protective functions and differentiation cues remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are sufficient to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection, and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner, and is associated with effective parasite control during chronic stage. Conditional invalidation of TAP-mediated MHC class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulate the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses upon T. gondii latency vs. encephalitis revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are functionally heterogeneous and autonomously ensure parasite control during T. gondii latent infection. Their differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections.

Chronic inflammation characterized by increased cytokine levels, such as interleukin-1 (IL-1), accompanies many neurological diseases but little is known about IL-1 contribution to cognitive impairment and its interplay with epigenetic processes, including the DNA double-strand break (DSB) response. Here, we demonstrate that H2A.X-dependent DSB signaling in hippocampal neurons drives cognitive deficits upon chronically elevated IL-1. Mice persistently and latently infected with Toxoplasma gondii display impaired spatial memory consolidation along with elevated IL-1β in the hippocampus. We find that neuronal IL-1 signaling in excitatory neurons is required for the spatial memory deficits caused by T. gondii infection and by chronic systemic infusion of IL-1β. In both cases, the deficit in spatial memory was prevented by the abrogation of neuronal H2A.X-dependent signaling. Our results highlight the instrumental role of cytokine-induced DSB-dependent signaling in spatial memory defects. This novel pathological mechanism in inflammation control of neuronal function may extend to several neurological diseases.

Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, specific therapeutic strategies targeting this developmental stage need to be found. T. gondii contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage.

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8⁺ T cells are found in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8⁺ T cell entry into the CNS. Here, we show that brain endothelial cells process and cross-present Ag, leading to effector CD8⁺ T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8⁺ T cell crawling and diapedesis resulting in brain endothelial cell apoptosis and BBB breakdown. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages but still reduced motility of Ag-specific CD8⁺ T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8⁺ T cell entry into the CNS and triggers CD8⁺ T cell-mediated focal BBB breakdown.