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Plasmacytoid dendritic cells (pDCs) are the most potent producer of type I interferon (IFN), but how pDC is primed in vivo is poorly defined. Using a mouse model of severe malaria, we have previously established that upon priming by CD169+ macrophages (MPs), pDC initiates type I IFN-I secretion in the bone marrow (BM) of infected mice via cell-intrinsic TLR7 sensing and cell-extrinsic STING sensing. Herein we show that CD169+ MP and TLR7 sensing are both required for pDC arrest during priming, suggesting CD169+ MP are the source of TLR7 ligands. We establish that TLR7 sensing in pDC and chemotaxis are both required for pDC arrest and functional communication with CD169+ MP in the BM. Lastly, we demonstrate that STING sensing in CD169+ MP control pDC initiation of type I IFN production while also regulating pDC clustering and retention/egress from the BM. Collectively, these results link pDC acquisition of type I IFN-secreting capacity with changes in their motility, homing and interactions with CD169+ MP during infection. Thus, targeting this cellular interaction may help modulate type I IFN to improve outcomes of microbial infections and autoimmune diseases.

Understanding how specific cancer mutations modulate the tumor microenvironment is essential for developing new precision cancer therapies. The histone methyltransferase MLL3 is a tumor suppressor that is frequently mutated together with p53 and PI3-kinase in breast cancer. Herein, using a mouse mammary-stem-cell-based tumor model and CRISPR gene editing, we recapitulated this genetic setting and revealed that Mll3 loss promotes tumorigenesis through enhancing tumor-intrinsic aggressiveness and establishing an immunosuppressive microenvironment. Specifically, HIF1α was upregulated in Mll3-mutant tumors and controlled proliferation and stem cell activity of the tumor cells. HIF1α also transcriptionally upregulated expression of the chemokine CCL2 by tumor cells. CCL2 further contributed to rapid CCR2-dependent accumulation of activated, proliferative KLRG1+Foxp3+ regulatory T (Treg) cells at very early stages of tumor initiation, promoting faster tumor onset. These results link MLL3 loss to HIF signaling, CCL2 and Treg cells, and the establishment of an early immune-suppressive microenvironment, a finding that may open new therapeutic opportunities for MLL3-mutant cancers.One Sentence SummaryMLL3 loss promotes tumorigenesis by enhancing recruitment of regulatory T cells.

Despite the rich information of a cell’s physiological state encoded in the dynamic changes of cell-surface glycans, methods of capturing glycosylation states at the single-cell level are quite limited. Here we report a chemoenzymatic single-cell N-acetyllactosamine (LacNAc) detection method via tagging the LacNAc with a specific DNA barcode. Compared to the lectin-based glycan detection, the chemoenzymatic labeling does not change the transcriptional status of immune cells and is more compatible with scRNA-seq. Integrated analysis of LacNAc and transcriptome of T cells at a single-cell level reveals that the quantity of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at the basal level in quiescent CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that the LacNAc level is positively correlated to the glycolytic activity of CD8+ T cells at all statues. Taken together, our study demonstrates the feasibility of chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with information of TCR sequence and cell-surface epitopes (i.e., scTCR and CITE-seq) and offers a new way to characterize the biological role of glycan in diversified physiological states.

Priming of naïve CD8+ T cells requires three signals: 1) cognate antigen, 2) co-stimulation, and 3) cytokines. These signals orchestrate CD8+ T cell differentiation. Particularly, strong T cell receptor (TCR) signals induce a more robust effector response, while weak TCR signals preferentially favor memory CD8+ T cells. However, memory cells induced upon varied TCR signaling strengths have been reported to be functionally similar. In this work, we show that, in contrast to this assumption, the strength of T cell receptor (TCR) signaling determines the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8+ T cells that are functionally different. We found that upon secondary antigen encounter, memory CD8+ T cells induced upon priming with lower TCR and IL-2 signals exhibited delayed cell cycle entry, impaired expansion and intracellular calcium accumulation compared to those primed with strong TCR and IL-2 signals. Furthermore, we also establish that the combination of both robust TCR and IL-2 signals, but not either one alone, are necessary to induce genome-wide changes in chromatin accessibility in regions targeting a wide range of biological processes consistent with their greater functional fitness. These notably included greater chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins. Taken together, these findings demonstrate that modulating TCR strength and IL-2 signaling at the time of CD8+ T cell priming, lead to the differentiation of functionally distinct pools of memory CD8+ T cells. Supported by T32GM7288

During Ag priming, naive CD4+ T cells differentiate into subsets with distinct patterns of cytokine expression that dictate to a major extent their functional roles in immune responses. We identified a subset of CD4+ T cells defined by secretion of IL-3 that was induced by Ag stimulation under conditions different from those associated with previously defined functional subsets. Using mouse models of bacterial and viral infections, we showed that IL-3–secreting CD4+ T cells were generated by infection at the skin and mucosa but not by infections introduced directly into the blood. Most IL-3–producing T cells coexpressed GM-CSF and other cytokines that define multifunctionality. Generation of IL-3–secreting T cells in vitro was dependent on IL-1 family cytokines and was inhibited by cytokines that induce canonical Th1 or Th2 cells. Our results identify IL-3–secreting CD4+ T cells as a potential functional subset that arises during priming of naive T cells in specific tissue locations.

Previous studies established that TCR signaling strength determines the size of CD8+ T cell expansion and memory pool. Here we hypothesized that TCR signaling strength further modulates the functional features of memory CD8+ T cells. We immunized mice transferred with naïve OT-I cells, which recognize the Ovalbumin (Ova)-derived epitope SIINFEKL, with Listeria monocytogenes expressing different variants, exposing OT-I cells to various TCR signaling strength. Transcriptomic analysis at early times after priming revealed that compared to OT-I cells primed with strong TCR signals, those primed with weak TCR signals upregulated genes involved in cell cycle (IL-2) and type I IFN response, suggesting that TCR signaling strength could determine how CD8+ T cells respond to cytokines at the time of priming. We postulated that disrupting cytokine signaling in T cells primed with weak TCR signals could alter memory T cell fate. As proposed, OT-I memory cells primed with weak TCR signals and lacking IL-2 or type I IFN cytokine signaling (Ifnar−/−, Il2ramut/mut) failed to re-expand upon secondary challenge compared to WT counterparts. Moreover, transcriptomic and epigenetic analyses of WT or Il2ramut/mut OT-I memory cells primed with strong versus weak TCR signals revealed that while gene expression remained similar, chromatin accessibility was significantly different. TCR and IL-2 signaling together regulate an epigenetic landscape reflecting a broad functional fitness of memory CD8+ T cells, whereas TCR signaling selectively imprinted an epigenetic landscape related to metabolism. These results suggest that epitope selection for effective CD8+ T cell vaccine design is pivotal to modulate the functional programming of memory T cells.

While type I interferon (IFN-I) is central to antiviral immunity, it can also promote pathology in bacterial and parasitic infections, and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) are able to produce 1,000× more IFN-I than any other cell upon sensing of nucleic acid ligands by TLR7 and TLR9. pDCs can obtain their ligands via direct cell-cell interactions, yet what regulates these interactions and whether pDCs need to receive additional signals to secrete IFN-I is unknown. Using a mouse model of severe malaria, we showed that pDCs initiate IFN-I production in the bone marrow (BM) of infected mice, and that this required CD169+ macrophages (MPs). IFN-I production by pDCs was also dependent on TLR7 and STING sensing in pDCs and CD169+ MPs, respectively. Through intravital microscopy (IVM), we further revealed that pDCs arrest and form clusters with CD169+MPs in the BM of malaria infected mice when peak IFN-I production occurs. Here, we hypothesize that pDC-MP interactions are required to license pDCs to produce IFN-I. We found that pDCs fail to arrest and form clusters with MPs in Tlr7−/− mice, and that blocking chemotaxis prevents pDC-MP interactions and subsequent IFN-I production, consistent with our hypothesis. Interestingly, while pDCs do not make IFN-I in STING-deficient mice, they still arrest and cluster around MPs. These clusters are much larger than in WT mice, suggesting that pDCs fail to receive another STING-dependent signal. Collectively, these results support a model in which pDC production of IFN-I is tightly regulated through a functional interaction with CD169+ MPs providing activating ligands and stimulatory signals. Targeting this interaction may represent a novel way to tune levels of IFN-I produced by pDCs.

Malaria remains a global burden with over 200 million infections and half a million deaths annually. While studies in human and surrogate mouse models reveal that B cells and CD4 T cells are essential, it is not known if and how sterilizing immunity against malaria may be achieved. Residents of endemic areas develop clinical immunity to Plasmodium falciparum over time yet protection is partial and requires repeated exposures. Herein we conducted a 2 year longitudinal study on patients with mild malaria in a hyperendemic region in Malawi. We categorized patients with a high number and short interval between reinfections (‘susceptible’) and with a low number and longer interval between reinfections (‘protected’). We hypothesized that clinical immunity correlates with the presence of specific immune cell subsets in protected versus susceptible patients. We conducted comprehensive cellular profiling on patient PBMCs, isolated during infection, by mass cytometry and a panel of 33 markers. We examined the frequency and activation of 40 immune cell subsets by unsupervised clustering analysis and discovered an expansion of CXCR3lo memory CD4T follicular helper cells (Tfh) in protected patients compared to susceptible patients. Importantly, with an in vitro antibody dependent opsonization assay we found increased phagocytosis of P. falciparum merozoites with plasma from protected versus susceptible patients, correlating CXCR3lo memory Tfh cell frequency with enhanced humoral responses. We speculate that CXCR3lo memory Tfh cells are critical for lasting protective responses against malaria in humans and may be essential for an efficacious future vaccine.

Malaria still remains a significant global health problem worldwide with approximately 200 million people infected, and half a million deaths every year. Most of these fatalities (80%) occur in children under the age of five, and the latest RTS, S, preventative vaccine trial only exhibited low protective efficacy (below 30%). The blood stage of Plasmodium infection accounts for the clinical symptoms of malaria, yet the immune mechanisms underlying disease outcomes are poorly understood. Here we have analyzed a cohort of severely infected Malawian children and revealed dramatic levels of inflammatory cytokines and extremely robust blood leukocyte activation, notably for CD14+CD16lo CCR2+ inflammatory monocytes, plasmacytoid DCs (pDCs), NK and T cells. We could recapitulate these observations using the Plasmodium yoelii (Py) 17X YM surrogate mouse model of lethal blood stage malaria. Using this experimental system, we demonstrate that type I interferon (IFN) signals as a key cytokine controlling lethal outcomes and immune cell activation. Plasmacytoid dendritic cells represented the main cellular source of type I IFN in the bone marrow and the blood of infected mice. Most interestingly, the activation of pDCs required CD169+ macrophages, and both cell types exhibited prolonged interactions in the bone marrow of infected mice by intravital microscopy. We will present additional data uncovering the molecular sensing pathways controlling these processes. Altogether our study establishes essential molecular pathways and cellular interactions that occur during this significant human parasitic infection, and suggest novel potential therapeutic targets involved in severe malaria.

Malaria is a severe and life-threatening disease that affects over 219 million people worldwide every year. In patients, a robust induction of the type I IFN pathway is observed, yet studies in mouse models have failed to determine whether type I IFN contributes to host protection or pathogenesis. We thus hypothesized that type I IFN is essential during blood stage malaria infection. Using Plasmodium yoelii (Py) YM which causes an acute lethal blood infection in C57BL/6 WT mice, we found that while WT mice rapidly succumbed, mice lacking the type I IFN receptor (IFNαβR-/-) mostly survived (>80%). Innate immune cells, particularly monocytes and NK cells, underwent early and robust activation and overwhelming production of inflammatory mediators in WT mice, whereas this was prevented in IFNαβR-/- mice. We are currently investigating precisely the cellular source of type I IFN and the mechanisms that regulate the activation of type I IFN-producing cells during this infection. Our results favor a model in which type I IFN promotes over-exuberant activation of innate monocytes and NK cells, leading to overwhelming inflammation and death of Py YM-infected mice. Overall, our work highlights a novel and unsuspected role for type I IFN in this significant human parasitic disease. We believe these results contribute to our understanding of the immunological cues leading to severe disease in malaria patients.

Cells of the innate immune system are essential for host defenses against primary microbial pathogen infections, yet their involvement in effective memory responses of vaccinated individuals has been poorly investigated. Here we show that memory T cells instruct innate cells to become potent effector cells in a systemic and a mucosal model of infection. Specifically, we found that memory T cells control phagocyte, dendritic cell and NK/NK T cell mobilization and induction of a strong program of differentiation which include their expression of effector cytokines and microbicidal pathways, all of which are delayed in non-vaccinated hosts. Disruption of IFN-gamma-signaling inside Ly6C+ monocytes, dendritic cells and macrophages impairs these processes and the control of pathogen growth. These results reveal how memory T cells, through rapid secretion of IFN-gamma, orchestrate extensive modifications of host innate immune responses that are essential for effective protection of vaccinated hosts.

Memory CD8+ T cells induced upon immunization exhibit improved functional features that contribute to protection of immunized hosts. Although both cognate antigen recognition and inflammation are important for memory CD8+ T cell reactivation, the relative contribution of these factors and the cell types providing these signals in vivo are poorly defined. In recent work, we have shown that Ly6C+CCR2+ inflammatory monocytes, a subset of monocytes, largely orchestrate memory CD8+ T and NK lymphocytes to become potent effector cells by sensing IL-18 and IL-15-derived inflammatory cytokines from monocytes independently of their cognate antigen. Like NK cells, they underwent rapid mobilization, upregulated intense and sustained effector functions during bacterial, viral and parasitic infections, and contributed to innate responses and protection in vivo. In our most recent experiments, we investigate further the molecular mechanisms of cross-talk between activated memory T cells and innate immune effector cells that allow for pathogen-specific protective immunity. Our results unravel a novel and unexpected level of antigen-dependent spatio-temporal tissue organization that is essential to achieve quick and efficient pathogen confinement and killing. This mechanism depends on memory T cell-derived IFN-γ and chemotactic cues, and on effector monocytes. We will present and discuss these data.