Identification of a Master Regulator of Differentiation in Toxoplasma.

Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Alarmin S100A11 initiates a chemokine response to the human pathogen Toxoplasma gondii.

Toxoplasma gondii is a common protozoan parasite that infects up to one third of the world's population. Notably, very little is known about innate immune sensing mechanisms for this obligate intracellular parasite by human cells. Here, by applying an unbiased biochemical screening approach, we show that human monocytes recognized the presence of T. gondii infection by detecting the alarmin S100A11 protein, which is released from parasite-infected cells via caspase-1-dependent mechanisms. S100A11 induced a potent chemokine response to T. gondii by engaging its receptor RAGE, and regulated monocyte recruitment in vivo by inducing expression of the chemokine CCL2. Our experiments reveal a sensing system for T. gondii by human cells that is based on the detection of infection-mediated release of S100A11 and RAGE-dependent induction of CCL2, a crucial chemokine required for host resistance to the parasite.

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Rapid Enhancer Remodeling and Transcription Factor Repurposing Enable High Magnitude Gene Induction upon Acute Activation of NK Cells.

Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.

Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.

Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.

Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation.

The differentiation of helper T cells into effector subsets is critical to host protection. Transcription factors of the E-protein and Id families are important arbiters of T cell development, but their role in the differentiation of the TH1 and TFH subsets of helper T cells is not well understood. Here, TH1 cells showed more robust Id2 expression than that of TFH cells, and depletion of Id2 via RNA-mediated interference increased the frequency of TFH cells. Furthermore, TH1 differentiation was blocked by Id2 deficiency, which led to E-protein-dependent accumulation of effector cells with mixed characteristics during viral infection and severely impaired the generation of TH1 cells following infection with Toxoplasma gondii. The TFH cell-defining transcriptional repressor Bcl6 bound the Id2 locus, which provides a mechanism for the bimodal Id2 expression and reciprocal development of TH1 cells and TFH cells.

Both maternal IFNγ exposure and acute prenatal infection with Toxoplasma gondii activate fetal hematopoietic stem cells.

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but the fetal hematopoietic response to infection during pregnancy is not well-studied. Here, we investigated the fetal hematopoietic response to maternal infection with Toxoplasma gondii (T. gondii), an intracellular parasite that elicits Type II IFNγ-mediated maternal immunity. While it is known that maternal infection without direct pathogen transmission can affect fetal immune development, the effects of maternal IFNγ on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond to T. gondii infection with virulence-dependent changes in proliferation, self-renewal potential, and lineage output. Furthermore, maternal IFNγ crosses the fetal-maternal interface, where it is perceived by fetal HSCs. By comparing the effects of maternal IFNγ injection with maternal T. gondii infection, we reveal that the effects of IFNγ treatment mimic some aspects of the fetal HSC response to infection. Moreover, our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFNγ-induced inflammation. Altogether, our data disentangle the role of infection-induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors.

The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice.

As Toxoplasma gondii disseminates through its host, the parasite must sense and adapt to its environment and scavenge nutrients. Oxygen (O2) is one such environmental factor and cytoplasmic prolyl 4-hydroxylases (PHDs) are evolutionarily conserved O2 cellular sensing proteins that regulate responses to changes in O2 availability. Toxoplasma expresses 2 PHDs. One of them, TgPHYa hydroxylates SKP1, a subunit of the SCF-E3 ubiquitin ligase complex. In vitro, TgPHYa is important for growth at low O2 levels. However, studies have yet to examine the role that TgPHYa or any other pathogen-encoded PHD plays in virulence and disease. Using a type II ME49 Toxoplasma TgPHYa knockout, we report that TgPHYa is important for Toxoplasma virulence and brain cyst formation in mice. We further find that while TgPHYa mutant parasites can establish an infection in the gut, they are unable to efficiently disseminate to peripheral tissues because the mutant parasites are unable to survive within recruited immune cells. Since this phenotype was abrogated in IFNγ knockout mice, we studied how TgPHYa mediates survival in IFNγ-treated cells. We find that TgPHYa is not required for release of parasite-encoded effectors into host cells that neutralize anti-parasitic processes induced by IFNγ. In contrast, we find that TgPHYa is required for the parasite to scavenge tryptophan, which is an amino acid whose levels are decreased after IFNγ up-regulates the tryptophan-catabolizing enzyme, indoleamine dioxygenase (IDO). We further find, relative to wild-type mice, that IDO knockout mice display increased morbidity when infected with TgPHYa knockout parasites. Together, these data identify the first parasite mechanism for evading IFNγ-induced nutritional immunity and highlight a novel role that oxygen-sensing proteins play in pathogen growth and virulence.

Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection.

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.

Genome-wide and targeted CRISPR screens identify RNF213 as a mediator of interferon gamma-dependent pathogen restriction in human cells.

To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.

Deficiency in astrocyte CCL2 production reduces neuroimmune control of Toxoplasma gondii infection.

Toxoplasma gondii is an obligate intracellular parasite that infects one-third of the world's human population and establishes infection in the brain. Cerebral immune cell infiltration is critical for controlling the parasite, but little is known about the molecular cues guiding immune cells to the brain during infection. Activated astrocytes produce CCL2, a chemokine that mediates inflammatory monocyte recruitment to tissues by binding to the CCR2 receptor. We detected elevated CCL2 production in the brains of C57BL/6J mice by 15 days after T. gondii infection. Utilizing confocal microscopy and intracellular flow cytometry, we identified microglia and brain-infiltrating myeloid cells as the main producers of CCL2 during acute infection, and CCL2 was specifically produced in regions of parasite infection in the brain. In contrast, astrocytes became the dominant CCL2 producer during chronic T. gondii infection. To determine the role of astrocyte-derived CCL2 in mobilizing immune cells to the brain and controlling T. gondii infection, we generated GFAP-Cre x CCL2fl/fl mice, in which astrocytes are deficient in CCL2 production. We observed significantly decreased immune cell recruitment and increased parasite burden in the brain during chronic, but not acute, infection of mice deficient in astrocyte CCL2 production, without an effect on peripheral immune responses. To investigate potential mechanisms explaining the reduced control of T. gondii infection, we analyzed key antimicrobial and immune players in host defense against T. gondii and detected a reduction in iNOS+ myeloid cells, and T. gondii-specific CD4+ T cells in the knockout mice. These data uncover a critical role for astrocyte-derived CCL2 in immune cell recruitment and parasite control in the brain during chronic, but not acute, T. gondii infection.

The Toxoplasma gondii F-Box Protein L2 Functions as a Repressor of Stage Specific Gene Expression.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages require substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct perinucleolar sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNAseq data show that TgFBXL2 conditional depletion induces the expression of stage-specific genes including a large cohort of genes necessary for sexual commitment. Together, these data suggest that TgFBXL2 is a latent guardian of stage specific gene expression in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of development.

N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion.

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

The Transcription Factor T-bet Limits Amplification of Type I IFN Transcriptome and Circuitry in T Helper 1 Cells.

Host defense requires the specification of CD4+ helper T (Th) cells into distinct fates, including Th1 cells that preferentially produce interferon-γ (IFN-γ). IFN-γ, a member of a large family of anti-pathogenic and anti-tumor IFNs, induces T-bet, a lineage-defining transcription factor for Th1 cells, which in turn supports IFN-γ production in a feed-forward manner. Herein, we show that a cell-intrinsic role of T-bet influences how T cells perceive their secreted product in the environment. In the absence of T-bet, IFN-γ aberrantly induced a type I IFN transcriptomic program. T-bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, in addition to promoting Th1 effector commitment, T-bet acts as a repressor in differentiated Th1 cells to prevent abberant autocrine type I IFN and downstream signaling.

Role for Caspase-8 in the Release of IL-1ß and Active Caspase-1 from Viable Human Monocytes during Toxoplasma gondii Infection.

Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1ß. We previously showed that IL-1ß release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1ß release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1ß release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1ß release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1ß release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1ß, but resulted in the retention of mature IL-1ß within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1ß was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1ß release from viable cells. This study expands on the molecular pathways that promote IL-1ß in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1ß release during infection.

Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells.

Antigen (Ag) crosspresentation by dendritic cells (DCs) involves the presentation of internalized Ags on MHC class I molecules to initiate CD8+ T cell-mediated immunity in response to certain pathogens and tumor cells. Here, we identify the SNARE Sec22b as a specific regulator of Ag crosspresentation. Sec22b localizes to the ER-Golgi intermediate compartment (ERGIC) and pairs to the plasma membrane SNARE syntaxin 4, which is present in phagosomes (Phgs). Depletion of Sec22b inhibits the recruitment of ER-resident proteins to Phgs and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b-deficient DCs, crosspresentation is compromised after Ag phagocytosis or endocytosis and after invasion by T. gondii. Sec22b silencing inhibited Ag export to the cytosol and increased phagosomal degradation by accelerating lysosomal recruitment. Our findings provide insight into an intracellular traffic pathway required for crosspresentation and show that Sec22b-dependent recruitment of ER proteins to Phgs critically influences phagosomal functions in DCs.