The Heterogeneity of Ly6Chi Monocytes Controls Their Differentiation into iNOS+ Macrophages or Monocyte-Derived Dendritic Cells.

Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c-MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3-CD11c-MHCII-PU.1lo monocytes differentiated into FcγRIII+PD-L2-CD209a-iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/- mice had reduced Flt3+CD11c-MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.

Chemoenzymatic Measurement of LacNAc in Single-Cell Multiomics Reveals It as a Cell-Surface Indicator of Glycolytic Activity of CD8+ T Cells.

Despite the rich information about the physiological state of a cell encoded in the dynamic changes of cell-surface glycans, chemical methods to capture specific glycan epitopes at the single-cell level are quite limited. Here, we report a chemoenzymatic method for the single-cell detection of N-acetyllactosamine (LacNAc) by labeling LacNAc with a specific DNA barcode. The chemoenzymatic labeling does not alter the transcriptional status of immune cells and is compatible with multiple scRNA-seq platforms. Integrated analysis of LacNAc and the transcriptome of T cells at the single-cell level reveals that the amount of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at basal levels in resting CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that LacNAc levels are positively correlated with the glycolytic activity of CD8+ T cells during differentiation. Taken together, our study demonstrates the feasibility of the chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with TCR sequence and cell-surface epitope information (i.e., scTCR and CITE-seq), and provides a new way to characterize the biological role of glycan in diverse physiological states.

Memory-T-cell-derived interferon-γ instructs potent innate cell activation for protective immunity.

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. Memory T cells controlled phagocyte, dendritic cell, and NK or NK T cell mobilization and induction of a strong program of differentiation, which included their expression of effector cytokines and microbicidal pathways, all of which were delayed in nonvaccinated hosts. Disruption of IFN-γ signaling in Ly6C+ monocytes, dendritic cells, and macrophages impaired these processes and the control of pathogen growth. These results reveal how memory T cells, through rapid secretion of IFN-γ, orchestrate extensive modifications of host innate immune responses that are essential for effective protection of vaccinated hosts.

A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

Inflammatory monocytes activate memory CD8(+) T and innate NK lymphocytes independent of cognate antigen during microbial pathogen invasion.

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. Here, we show that Ly6C(+)CCR2(+) inflammatory monocytes, a subset of monocytes, largely orchestrate memory CD8(+) T and NK lymphocytes activation by differentiating into interleukin-18 (IL-18)- and IL-15-producing cells in an inflammasome and type I interferon-IRF3-dependent manner. Memory CD8(+) T cells became potent effector cells by sensing inflammation 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. Thus, inflammatory monocyte-derived IL-18 and IL-15 are critical to initiate memory CD8(+) T and NK lymphocytes differentiation into antimicrobial effector cells.

Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Suppression of Th1 Priming by TLR2 Agonists during Cutaneous Immunization Is Mediated by Recruited CCR2+ Monocytes.

Effective subunit vaccines require the incorporation of adjuvants that stimulate cells of the innate immune system to generate protective adaptive immune responses. Pattern recognition receptor agonists are a growing class of potential adjuvants that can shape the character of the immune response to subunit vaccines by directing the polarization of CD4 T cell differentiation to various functional subsets. In the current study, we applied a high-throughput in vitro screen to assess murine CD4 T cell polarization by a panel of pattern recognition receptor agonists. This identified lipopeptides with TLR2 agonist activity as exceptional Th1-polarizing adjuvants. In vivo, we demonstrated that i.v. administration of TLR2 agonists with Ag in mice replicated the findings from in vitro screening by promoting strong Th1 polarization. In contrast, TLR2 agonists inhibited priming of Th1 responses when administered cutaneously in mice. This route-specific suppression was associated with infiltrating CCR2+ cells in the skin-draining lymph nodes and was not uniquely dependent on any of the well characterized subsets of dendritic cells known to reside in the skin. We further demonstrated that priming of CD4 T cells to generate Th1 effectors following immunization with the Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain, a lipoprotein-rich bacterium recognized by TLR2, was dependent on the immunization route, with significantly greater Th1 responses with i.v. compared with intradermal administration of BCG. A more complete understanding of route-dependent TLR2 responses may be critical for informed design of novel subunit vaccines and for improvement of BCG and other vaccines based on live-attenuated organisms.

Splenic Innate B1 B Cell Plasmablasts Produce Sustained Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-3 Cytokines during Murine Malaria Infections.

The physiopathology of malaria, one of the most deadly human parasitic diseases worldwide, is complex, as it is a systemic disease involving multiple parasitic stages and hosts and leads to the activation of numerous immune cells and release of inflammatory mediators. While some cytokines increased in the blood of patients infected with Plasmodium falciparum have been extensively studied, others, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), have not received much attention. GM-CSF and IL-3 belong to the ß common (ßc/CD131) chain family of cytokines, which exhibit pleiotropic functions, including the regulation of myeloid cell growth, differentiation, and activation. GM-CSF can be secreted by multiple cell types, whereas IL-3 is mostly restricted to T cells, yet innate response activator (IRA) B cells, a subset of innate B1 B cells, also produce significant amounts of these cytokines during bacterial sepsis via Toll-like receptor 4 (TLR4)/MyD88 sensing of lipopolysaccharides. Herein, using murine models of malaria, we report a sustained production of GM-CSF and IL-3 from IgM+ and IgM-/IgG+ CD138+ Blimp-1+ innate B1b B cell plasmablasts. IgM+ B1b B cells include IRA-like and non-IRA B cells and express higher levels of both cytokines than do their IgG+ counterparts. Interestingly, as infection progresses, the relative proportion of IgM+ B1 B cells decreases while that of IgG+ plasmablasts increases, correlating with potential isotype switching of GM-CSF- and IL-3-producing IgM+ B1 B cells. GM-CSF/IL-3+ B1 B cells originate in the spleen of infected mice and are partially dependent on type I and type II interferon signaling to produce both cytokines. These data reveal that GM-CSF and IL-3 are produced during malaria infections, initially from IgM+ and then from IgG+ B1b B cell plasmablasts, which may represent important emergency cellular sources of these cytokines. These results further highlight the phenotypic heterogeneity of innate B1 B cell subsets and of their possible fates in a relevant murine model of parasitic infection in vivo.

Memory CD8(+) T Cells: Innate-Like Sensors and Orchestrators of Protection.

Recent findings have revealed roles for systemic and mucosa-resident memory CD8(+) T cells in the orchestration of innate immune responses critical to host defense upon microbial infection. Here we integrate these findings into the current understanding of the molecular and cellular signals controlling memory CD8(+) T cell reactivation and the mechanisms by which these cells mediate effective protection in vivo. The picture that emerges presents memory CD8(+) T cells as early sensors of danger signals, mediating protective immunity both through licensing of cellular effectors of the innate immune system and via the canonical functions associated with memory T cells. We discuss implications for the development of T cell vaccines and therapies and highlight important areas in need of further investigation.

Monocyte-mediated defense against bacteria, fungi, and parasites.

Circulating blood monocytes are a heterogeneous leukocyte population that contributes critical antimicrobial and regulatory functions during systemic and tissue-specific infections. These include patrolling vascular tissue for evidence of microbial invasion, infiltrating peripheral tissues and directly killing microbial invaders, conditioning the inflammatory milieu at sites of microbial tissue invasion, and orchestrating the activation of innate and adaptive immune effector cells. The central focus of this review is the in vivo mechanisms by which monocytes and their derivative cells promote microbial clearance and immune regulation. We include an overview of murine models to examine monocyte functions during microbial challenges and review our understanding of the functional roles of monocytes and their derivative cells in host defense against bacteria, fungi, and parasites.

Memory CD8+ T Cells: Orchestrators and Key Players of Innate Immunity?

Over the past decades, the dichotomy between innate and adaptive immune responses has largely dominated our understanding of immunology. Upon primary encounter with microbial pathogens, differentiation of adaptive immune cells into functional effectors usually takes several days or even longer, making them contribute to host protection only late during primary infection. However, once generated, antigen-experienced T lymphocytes can persist in the organism and constitute a pool of memory cells that mediate fast and effective protection to a recall infection with the same microbial pathogen. Herein, we challenge this classical paradigm by highlighting the "innate nature" of memory CD8+ T cells. First, within the thymus or in the periphery, naïve CD8+ T cells may acquire phenotypic and functional characteristics of memory CD8+ T cells independently of challenge with foreign antigens. Second, both the "unconventional" and the "conventional" memory cells can rapidly express protective effector functions in response to sets of inflammatory cytokines and chemokines signals, independent of cognate antigen triggering. Third, memory CD8+ T cells can act by orchestrating the recruitment, activation, and licensing of innate cells, leading to broad antimicrobial states. Thus, collectively, memory CD8+ T cells may represent important actors of innate immune defenses.

STING-Licensed Macrophages Prime Type I IFN Production by Plasmacytoid Dendritic Cells in the Bone Marrow during Severe Plasmodium yoelii Malaria.

Malaria remains a global health burden causing significant morbidity, yet the mechanisms underlying disease outcomes and protection are poorly understood. Herein, we analyzed the peripheral blood of a unique cohort of Malawian children with severe malaria, and performed a comprehensive overview of blood leukocytes and inflammatory mediators present in these patients. We reveal robust immune cell activation, notably of CD14+ inflammatory monocytes, NK cells and plasmacytoid dendritic cells (pDCs) that is associated with very high inflammation. Using the Plasmodium yoelii 17X YM surrogate mouse model of lethal malaria, we report a comparable pattern of immune cell activation and inflammation and found that type I IFN represents a key checkpoint for disease outcomes. Compared to wild type mice, mice lacking the type I interferon (IFN) receptor exhibited a significant decrease in immune cell activation and inflammatory response, ultimately surviving the infection. We demonstrate that pDCs were the major producers of systemic type I IFN in the bone marrow and the blood of infected mice, via TLR7/MyD88-mediated recognition of Plasmodium parasites. This robust type I IFN production required priming of pDCs by CD169+ macrophages undergoing activation upon STING-mediated sensing of parasites in the bone marrow. pDCs and macrophages displayed prolonged interactions in this compartment in infected mice as visualized by intravital microscopy. Altogether our findings describe a novel mechanism of pDC activation in vivo and precise stepwise cell/cell interactions taking place during severe malaria that contribute to immune cell activation and inflammation, and subsequent disease outcomes.

A role for lipid bodies in the cross-presentation of phagocytosed antigens by MHC class I in dendritic cells.

Dendritic cells (DCs) have the striking ability to cross-present exogenous antigens in association with major histocompatibility complex (MHC) class I to CD8(+) T cells. However, the intracellular pathways underlying cross-presentation remain ill defined. Current models involve cytosolic proteolysis of antigens by the proteasome and peptide import into endoplasmic reticulum (ER) or phagosomal lumen by the transporters associated with antigen processing (TAP1 and TAP2). Here, we show that DCs expressed an ER-resident 47 kDa immune-related GTPase, Igtp (Irgm3). Igtp resides on ER and lipid body (LB) membranes where it binds the LB coat component ADFP. Inactivation of genes encoding for either Igtp or ADFP led to defects in LB formation in DCs and severely impaired cross-presentation of phagocytosed antigens to CD8(+) T cells but not antigen presentation to CD4(+) T cells. We thus define a new role for LB organelles in regulating cross-presentation of exogenous antigens to CD8(+) T lymphocytes in DCs.

The p60 and NamA autolysins from Listeria monocytogenes contribute to host colonization and induction of protective memory.

Inducing long-term protective memory CD8(+) T-cells is a desirable goal for vaccines against intracellular pathogens. However, the mechanisms of differentiation of CD8(+) T-cells into long-lived memory cells capable of mediating protection of immunized hosts remain incompletely understood. We have developed an experimental system using mice immunized with wild type (WT) or mutants of the intracellular bacterium Listeria monocytogenes (Lm) that either do or do not develop protective memory CD8(+) T-cells. We previously reported that mice immunized with Lm lacking functional SecA2, an auxiliary secretion system of gram-positive bacteria, did not differentiate functional memory CD8(+) T-cells that protected against a challenge infection with WT Lm. Herein we hypothesized that the p60 and NamA autolysins of Lm, which are major substrates of the SecA2 pathway, account for this phenotype. We generated Lm genetically deficient for genes encoding for the p60 and NamA proteins, ΔiapΔmurA Lm, and further characterized this mutant. Δp60ΔNamA Lm exhibited a strong filamentous phenotype, inefficiently colonized host tissues, and grew mostly outside cells. When Δp60ΔNamA Lm was made single unit, cell invasion was restored to WT levels during vaccination, yet induced memory T-cells still did not protect immunized hosts against recall infection. Recruitment of blood phagocytes and antigen-presenting cell activation was close to that of mice immunized with ΔActA Lm, which develop protective memory. However, key inflammatory factors involved in optimal T-cell programming such as IL-12 and type I IFN (IFN-I) were lacking, suggesting that cytokine signals may largely account for the observed phenotype. Thus, altogether, these results establish that p60 and NamA secreted by Lm promote primary host cell invasion, the inflammatory response and the differentiation of functional memory CD8(+) T-cells, by preventing Lm filamentation during growth and subsequent triggering of innate sensing mechanisms.

The T-Cell Inhibitory Molecule Butyrophilin-Like 2 Is Up-regulated in Mild Plasmodium falciparum Infection and Is Protective During Experimental Cerebral Malaria.

Plasmodium falciparum infection can result in severe disease that is associated with elevated inflammation and vital organ dysfunction; however, malaria-endemic residents gain protection from lethal outcomes and manifest only mild symptoms during infection. To characterize host responses associated with this more effective antimalarial response, we characterized whole-blood transcriptional profiles in Rwandan adults during a mild malaria episode and compared them with findings from a convalescence sample. We observed transcriptional up-regulation in many pathways, including type I interferon, interferon γ, complement activation, and nitric oxide during malaria infection, which provide benchmarks of mild disease physiology. Transcripts encoding negative regulators of T-cell activation, such as programmed death ligand 1 (PD-L1), programmed death 1 ligand 2 (PD-L2), and the butyrophilin family member butyrophilin-like 2 (BTNL2) were also increased. To support an important functional role for BTNL2 during malaria infection, we studied chimeric mice reconstituted with BTNL2(-/-) or wild-type hematopoietic cells that were inoculated with Plasmodium berghei ANKA, a murine model of cerebral malaria. We found that BTNL2(-/-) chimeric mice had a significant decrease in survival compared with wild-type counterparts. Collectively these data characterize the immune responses associated with mild malaria and uncover a novel role for BTNL2 in the host response to malaria.

Mechanisms of Memory T Cell Activation and Effective Immunity.

Effective immunization induces the development of populations of robust effector lymphocytes specific for the immunizing antigens. Amongst them are cytotoxic/CD8(+)T lymphocytes, which few will further differentiate into long-lived memory cells persisting in the host and exhibiting improved functional characteristics. The current model is that such memory cells can confer rapid host protection upon cognate antigen-mediated activation and direct killing of infected cells. In this chapter, we discuss work from our group and others that highlight the contribution of inflammatory cytokines to memory CD8(+) T cell activation and of cytolysis-independent mechanisms of host protection.

Inflammatory monocyte effector mechanisms.

Monocytes are blood-derived mononuclear phagocytic cells that traffic throughout the body and can provide rapid innate immune effector responses in response to microbial pathogen infections. Among blood monocytes, the most abundant subset in mice is represented by inflammatory Ly6C(+) CCR2(+) monocytes and is the functional equivalent of the CD14(+) monocytes in humans. Herein we focus on published evidence describing the exquisite functional plasticity of these cells, and we extend this overview to their multiples roles in vivo during host immune defenses against microbial pathogen infections, as antigen-presenting cells, inflammatory cells or Trojan horse cells.

Prenatal vitamin D deficiency exposure leads to long-term changes in immune cell proportions.

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We found that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Moreover, further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term offspring also confirm that maternal vitamin D levels in the second trimester significantly affect immune cell proportions in the offspring. These findings imply that the differentiation properties of hematopoiesis act as long-term memories of prenatal vitamin D deficiency exposure in later life.

Intratumoral CXCL12 Gradients Contextualize Tumor Cell Invasion, Migration and Immune Suppression in Breast Cancer.

Although the CXCL12/CXCR4 pathway has been prior investigated for its prometastatic and immuno- suppressive roles in the tumor microenvironment, evidence on the spatiotemporal regulation of these hallmarks has been lacking. Here, we demonstrate that CXCL12 forms a gradient specifically around cancer cell intravasation doorways, also known as Tumor Microenvironment of Metastasis (TMEM) doorways, thus facilitating the chemotactic translocation of prometastatic tumor cells expressing CXCR4 toward the perivascular TMEM doorways for subsequent entry into peripheral circulation. Fur- thermore, we demonstrate that the CXCL12-rich micro-environment around TMEM doorways may cre- ate immunosuppressive niches, whereby CD8 + T cells, despite being attracted to these regions, often exhibit reduced effector functions, limiting their efficacy. While the CXCL12/CXCR4 pathway can mini- mally influence the overall composition of immune cell populations, it biases the distribution of CD8 + T cells away from TMEM doorways, justifying its prior-established role as immunosuppressive factor for CD8 + T cells. Our research suggests that the complex interactions between CXCL12 and the various tumor and immune cell types contributes not only to the completion of the initial steps of the metastatic cascade, but also offers an immunological "sanctuary" to prometastatic tumor cells homed around TMEM doorways. Overall, our study enhances our current understanding on the mechanisms, via which CXCL12 orchestrates tumor cell behavior and immune dynamics, potentially guiding future thera- peutic strategies to combat breast cancer metastasis and improve anti-tumor immunity.