The Journal of Immunology

Akt1 and Akt2, isoforms of the serine threonine kinase Akt, are essential for T cell development. However, their role in peripheral T cell differentiation remains undefined. Using mice with germline deletions of either Akt1 or Akt2, we found that both isoforms are important for Th17 differentiation, although Akt2 loss had a greater impact than loss of Akt1. In contrast to defective IL-17 production, Akt2-/- T cells exhibited enhanced IL-4 production in vitro under Th2 polarizing conditions. In vivo, Akt2-/- mice displayed significantly diminished IL-17A and GM-CSF production following immunization with myelin oligodendrocyte glycoprotein (MOG). This dampened response was associated with further alterations in Th cell differentiation including decreased IFN{gamma} production but preserved IL-4 production, and preferential expansion of regulatory T cells compared to non-regulatory CD4 T cells. Taken together, we identify Akt2 as an important signaling molecule in regulating peripheral CD4 T cell responses.

Cytomegalovirus (CMV) infection alters natural killer (NK) cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the Fc receptor {gamma} chain (Gene: FCER1G, FcR{gamma}), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (Fc{gamma}RIIIA) signaling apparatus, which includes FcR{gamma}, CD3{zeta}, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcR{gamma} chain caused a modest increase in TNF production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcR{gamma} or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3{zeta} chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.

Intracellular bacteria produce antigens, which serve as potent activators of {gamma}{delta} T cells. Phosphoantigens are presented via a complex of Butyrophilins (BTN) to signal infection to human V{gamma}9+V{delta}2+ T cells. Here, we established an in vitro system allowing for studies of V{gamma}9+V{delta}2+ T cell activity in coculture with epithelial cells infected with the intracellular bacterial pathogen Listeria monocytogenes. We report that the V{gamma}9+V{delta}2+ T cells efficiently purge such cultures from infected cells. This effector function requires the expression of members of the BTN3A family on epithelial cells. Specifically, the BTN3A1 and BTN3A3 are redundant in their ability to present antigen to V{gamma}9+V{delta}2+ T cells. Since BTN3A1 is the only BTN3A associated with phosphoantigen presentation our study suggests that BTN3A3 may present different classes of antigens to mediate V{gamma}9+V{delta}2+ T cell effector function against L. monocytogenes-infected epithelia.

Characterization of the functional effects of cDC2s in vivo requires model systems in which cDC2s are depleted. Previous literature has reported a loss of cDC2s in mice lacking the transcription factor IRF21,2. We sought to further characterize the cDC2 defect in these animals. Here, we find that the requirement for IRF2 in cDC2 development and survival is cell-extrinsic and correlated to the development of dermatitis in the Irf2-/- model system. We also find that Flt3L-mediated in vitro development of cDC1s and cDC2s, but not pDCs, is abrogated in Irf2-/- bone marrow, as well as in wild-type bone marrow cultured with IFN. Loss of interferon (IFN) signaling in Irf2-/- mice restored cDC2 development in vivo and cDC1 and cDC2 development in vitro. We therefore conclude that IRF2 is required for cDC2 development in a cell-extrinsic manner dependent on IFN signaling.

Mucosal-associated invariant T (MAIT) cells are donor unrestricted T cells capable of both antigen-specific adaptive responses and cytokine driven innate-like functions. Although human MAIT cells uniformly express ROR{gamma}t and IL23R, they generally produce IFN-{gamma}, and only a small fraction produces IL-17. Recent studies show that combined TCR and cytokine stimulation can elicit functional heterogeneity in blood-derived MAIT cells. Here, we investigate the role of IL-23/IL-23R signaling in mediating the function and transcriptional profiles of lung MAIT cell clones. We demonstrate that BAL-derived lung MAIT cell clones exhibit distinct cytokine profiles and variable IL23R expression. Short-term IL-23 stimulation triggers clone-specific transcriptional programs and IL23R-dependent upregulation of type 17-associated genes. Prolonged conditioning of lung MAIT cell clones with TCR (5-OP-RU) and cytokine (IL-23) stimulation induces stable IL-17A production along with unique transcriptional changes. TCR + IL-23 conditioning alone upregulates clone-specific and shared cytoskeletal/structural gene programs, whereas subsequent PMA/Ionomycin stimulation further induces IL-12 family signaling and metabolic genes. Together, these findings demonstrate that IL23R expression and TCR signaling are required for IL-17A production, highlighting that these conditions may be met in tissue environments where MR1-specific antigens and proinflammatory cytokines coexist.

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

Notch ligands are present during the interactions between T cells and dendritic cells (DC) and induce a myriad of effects that facilitate the activation of T cells, including the induction of T cell regulation, survival, and cytokine production. Although the ligands Delta-like 4 and Delta-like 1 are expressed as a function of DC activation, the notch ligand Jagged-1 is constitutively expressed on DC. We sought to determine the role of Jagged-1 in the interactions between CD4+ T cells and DC. We observed that Jagged-1 regulates Foxp3 expression, and Cd11cCre+Jaggedff mice have an altered expression of Foxp3 in effector cells that arise as a result of infection with the mycobacterium Bacille Calmette-Guerin. The observed changes in Foxp3 expression were correlated with an increase in cytokine production from cultures of antigen-stimulated draining lymph nodes.

Ets1 is a lymphoid-enriched transcription factor that regulates B and T cell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop spontaneous autoimmune disease with high levels of autoantibodies. Naive CD4+ T cells isolated from Ets1 KO mice differentiate more readily to Th17 cells that secrete IL-17, a cytokine implicated in autoimmune disease pathogenesis. To determine if increased IL-17 production contributes to the development of autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL-17 receptor A subunit (IL17RA KO) to generate double knock out (DKO) mice. We found that the absence of IL17RA signaling did not prevent or ameliorate the autoimmune phenotype of Ets1 KO mice, but rather that DKO animals exhibited worse symptoms with striking increases in activated B cells and secreted autoantibodies. This was correlated with a prominent increase in the numbers of T follicular helper (Tfh) cells. In addition to the autoimmune phenotype, DKO mice also showed signs of immunodeficiency and developed spontaneous skin lesions colonized by Staphylococcus xylosus. When DKO mice were experimentally infected with S. aureus they were unable to clear the bacteria, suggesting a general immunodeficiency to Staphylococcal species. {gamma}{delta} T cells are important for control of skin Staphylococcal infections. We found that mice lacking Ets1 have a complete deficiency of the {gamma}{delta} T cell subset dendritic epidermal T cells (DETC), which are involved in skin wound healing responses. To determine if loss of DETC might promote susceptibility to Staph infection, we depleted DETC from IL17RA KO mice and found that the combined loss of DETC and IL-17 signaling leads to a failure to clear the infection. Our studies suggest that defects in wound healing, such as that caused by loss of DETC, can cooperate with impaired IL-17 responses to lead to increased susceptibility to skin Staph infections.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by suppressing excessive activation of effector T cells. Although several mechanisms of Treg-mediated suppression have been described, the molecular signals that contribute to this regulation remain incompletely understood. WNT signaling, best known for its roles in development and tissue homeostasis, has recently emerged as an important regulator of immune function, but its contribution to Treg-mediated immune suppression is largely unknown. Here, we show that Tregs preferentially express multiple canonical WNT ligands, including WNT2B, WNT3, WNT7B, and WNT10B, compared with conventional CD4+ T cells. These WNT proteins were detected intracellularly in Tregs, and WNT2B and WNT3 were actively secreted into culture supernatants. Conventional CD4+ T cells expressed Frizzled receptors capable of sensing these ligands. Pharmacological inhibition of canonical WNT signaling using the antagonist mDKK-1 enhanced CD4+ T cell activation and proliferation and increased pro-inflammatory cytokine expression, while anti-inflammatory IL-10 remained unchanged. Together, these findings identify Tregs as a source of canonical WNT ligands and suggest that Treg-derived WNT signaling contributes to the suppression of effector CD4+ T cell responses. This work reveals a previously underappreciated pathway through which Tregs regulate immune activity and identifies WNT signaling as a potential target for modulating inflammatory immune responses.

Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have four sialidases, but their biological functions are unclear. In this report, we show that incubation of human neutrophils with the human sialidase NEU3, but not NEU1, NEU2 or NEU4, inducess human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed neutrophil markers CD66, CD11B, and CD18 to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

Neutrophils are crucial to innate immune responses to microbes. The crosslinking of opsonized pathogens by Fc gamma receptors (Fc{gamma}Rs) on neutrophil surfaces mediates multiple antimicrobial functions, including phagocytosis and the production of reactive oxygen species (ROS). Fc{gamma}RIIIb (CD16b) is the most abundant receptor on human neutrophils. It is a GPI-anchored receptor that lacks an intracellular domain. The exact mechanisms by which Fc{gamma}RIIIb transduces signals remain unclear. A rare Fc{gamma}RIIIb-deficient phenotype has been reported in apparently healthy subjects, which is intriguing given the abundance of this receptor on neutrophil surfaces and its crucial role in neutrophil activation by immune complexes. Here, we identified 2 healthy brothers lacking Fc{gamma}RIIIb on neutrophils and characterized their neutrophil activation through Fc{gamma}R crosslinking by immune complexes. Sequencing of the FCGR3B gene revealed mutations in exon 2 resulting in translation loss. In the absence of stimulation, Fc{gamma}RIIIbnull neutrophils showed unaltered levels of Fc{gamma}RIIa, TLR-2, TLR-4 and TLR-6, but significantly higher Fc{gamma}RIIIa and Fc{gamma}RIa. Upon challenge with E. coli immune complexes, increased surface expression of Fc{gamma}RIa, TLR-4, and M integrin (CD11b) was observed exclusively in Fc{gamma}RIIIbnull neutrophils. Antibacterial functions stimulated by immune complexes were significantly lower in Fc{gamma}RIIIbnull neutrophils, including phagocytic capacity and ROS production compared to Fc{gamma}RIIIb-expressing neutrophils. Overall, the absence of Fc{gamma}RIIIb on human neutrophils correlated with impaired antimicrobial functions following stimulation through Fc{gamma}Rs. This study provides new insights into the functional relevance of Fc{gamma}RIIIb and emphasizes the importance of this receptor in neutrophil responses to bacteria.

Although the Src family kinase (SFK) Lyn is known to be involved in induction and maintenance of peripheral B cell tolerance, the molecular basis of its action in this context remains unclear. This question has been approached using conventional as well as B cell-targeted knockouts of Lyn, with varied conclusions likely confused by collateral loss of Lyn functions in B cell and myeloid cell development and activation. Here we utilized a system in which Lyn gene deletion is tamoxifen inducible and B cell restricted. This system allows acute elimination of Lyn in B cells without off-target effects. This genetic tool was employed in conjunction with immunoglobulin transgenic mice in which peripheral B cells are autoreactive. DNA reactive Ars/A1 B cells require continuous inhibitory signaling, mediated by the inositol phosphatase SHIP-1 and the tyrosine phosphatase SHP-1, to maintain an unresponsive (anergic) state. Here we show that Ars/A1 B cells require Lyn to establish and maintain B cell unresponsiveness. Lyn primarily functions by restricting PI3K-dependent signaling pathways. This Lyn-dependent mechanism complements the impact of reduced mIgM BCR expression to restrict BCR signaling in Ars/A1 B cells. Our findings suggest that a subset of autoreactive B cells requires Lyn to become anergic and that the autoimmunity associated with dysregulated Lyn function may, in part, be due to an inability of these autoreactive B cells to become tolerized.

CD8 T cell memory differentiation endows T cells with an ability to rapidly induce effector functions upon pathogen re-encounter. While it is well established that substantial epigenetic remodeling occurs during the effector stage of the immune response, the signaling events that imprint CD8 T cells with these stable epigenetic programs are not well-defined. To gain insight into the signaling determinants of effector-associated epigenetic programming among CD8 T cells, we explored the role of IL-12 in the imprinting of IFNg expression during human CD8 T cell priming. We observed that TCR-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promotor. However, TCR stimulation in the presence of the inflammatory cytokine, IL-12, resulted in significant and stable demethylation of the IFNg locus that was commensurate with an increase in IFNg expression. We further show that IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent passive demethylation in an ex vivo human CAR T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector epigenetic programming in CD8 T cells during the primary immune response and serve as proof of concept that signal 3 cytokines can be used to guide the induction of epigenetically regulated traits among T cells used for adoptive immunotherapies.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I interferon and TGF{beta}, that underlie this dysregulation. However, the role of cell-cell interactions in mediating changes in NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell co-culture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro co-cultures in which NK cells from healthy donors were incubated with monocytes from COVID-19+ or healthy donors. Co-culture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from transwell co-cultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines as well as TGF{beta}. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19. BACKGROUNDNatural killer (NK) cells are innate lymphocytes that are critical antiviral effectors. Because of their role in controlling acute viral infections, multiple studies have evaluated the role of NK cells in SARS-CoV-2 infection. Such studies revealed that NK cell phenotype and function are significantly altered by severe COVID-19; the peripheral NK cells of severe COVID-19 patients are highly activated and proliferative(1-5), with increased expression of cytotoxic molecules, Ki-67, and several surface markers of activation(3, 5-8). However, these NK cells also have dysfunctional cytotoxic responses to both tumor target cells(1, 2, 9, 10) and SARS-CoV-2-infected target cells(9, 10). Given that peripheral NK cells are thought to migrate to the lung during COVID-19(11-13), these results suggest that the NK cells of severe COVID-19 patients may be incapable of mounting a successful antiviral response to SARS-CoV-2 infection. Although the unique phenotype and dysfunctionality of NK cells in severe COVID-19 has been well-characterized, the processes underlying these phenomena have not. Only one study has conducted in vitro mechanistic experiments to identify a possible cause of NK cell dysfunction: Witkowski et al. identified serum-derived TGF{beta} as a suppressor of NK cell functionality in severe COVID-19 patients(9). However, this study did not identify the source of serum TGF{beta}. Additionally, given the high degree of complexity within the immune system, there are likely other causes of NK cell dysfunction in COVID-19 that have thus far remain unexplored. One such mechanism may be the myriad of interactions between NK cells and other peripheral immune cells. NK cells are known to interact with CD4 and CD8 T cells, dendritic cells, neutrophils, and macrophages/monocytes(14), which can prime NK cell cytotoxicity or induce tolerance. Previous work by our lab suggested the potential for NK cell-monocyte crosstalk in severe COVID-19 through the expression of ligands for NK cell activating receptors on the monocytes of these patients(3). Crosstalk between NK cells and monocytes plays a role in regulating the NK cell response to other infections, including HIV-1(15, 16), mouse(17) and human cytomegalovirus(18), and malaria(19) through mechanisms including secretion of NK cell-regulating cytokines by monocytes. In this study, we used a combination of computational and in vitro methods to dissect the interactions between NK cells and monocytes in severe COVID-19. We utilized primary NK cells and monocytes from a large cohort of COVID-19 patients to demonstrate that co-culture of healthy NK cells with monocytes from severe COVID-19 donors can partially recapitulate the activated phenotype observed in the NK cells from COVID-19 patients. We then interrogated the mechanisms by which this activation occurs by performing NK cell-monocyte co-cultures in a transwell setting and using O-link to analyze the cytokines present in this system. Collectively, our work identifies monocytes as a driver of NK cell activation in severe COVID-19 and reveals interactions between NK cells and monocytes that may underlie this process.

Selenoproteins are involved in immune cell metabolism, yet the roles of these proteins in T cell development and function remain largely unknown. The Trsp gene encodes the selenocysteine tRNA (tRNASec) required for translation of all selenoproteins. In this study, we found that Trsp was required for thymopoiesis, with the majority of tRNASec-deficient T cells not progressing beyond double negative 3 stage, with egressed thymocytes undergoing peripheral homeostatic expansion. Trsp-deficient CD4+ T cells exhibited impairments in TCR and IL-2 signaling and did not cause inflammation in experimental models. On the other hand, Trsp-deficient regulatory T (Treg) cells exhibited defects in suppressive function ex vivo and Treg-specific Trsp deletion using Trspfl/flFoxp3YFP-Cre (Trsp!{iota}Treg) mice caused fatal autoimmunity similar to FOXP3-deficient mice. Reducing oxidative stress via 2-HOBA administration prolonged survival in these Trsp!{iota}Treg mice. These findings indicate that tRNASec is required for T cell homeostasis and may be therapeutic targets in inflammation. One sentence summaryTrsp, a gene required for translation of all selenoproteins, is essential for all T cell development and function, especially regulatory T cells.

Following acute viral infection, naive CD4+ T cells differentiate into T follicular helper (Tfh) and T helper 1 (Th1) cells that generate long-lived memory cells. However, it is unclear how memory Tfh and Th1 cells maintain their lineage commitment. We demonstrate that Tfh and Th1 lineages acquire distinct Dnmt3a-dependent de novo DNA methylation programs that are preserved into memory. Dnmt3a deletion impairs lineage commitment and functionality of memory Th1 and Tfh cells, resulting in aberrant Runx1 upregulation that represses germinal center Tfh cell differentiation. In contrast, transient pharmacological DNA methyltransferase inhibition during priming impairs repression of Tfh-associated genes while properly silencing Runx1, and results in enhanced Tfh cell functionality in primary and secondary responses to viral infections. Together, these findings demonstrate that Dnmt3a-mediated epigenetic programing is required to enforce T helper lineage commitment and preserve Tfh and Th1-specific functions during the recall response to infection, and reveal novel strategies to improve long-lived adaptive immunity against infectious diseases. SUMMARYThis article demonstrates that Dnmt3a-dependent epigenetic programing regulates functionality and plasticity of Th1 and Tfh memory cells. Furthermore, early pharmacological inhibition of such programing enhances GC Tfh cell differentiation, suggesting novel strategies for modulating the immune response to viral infections.

Mechanisms that restrict class switch recombination (CSR) to IgE limit the subsequent production of IgE antibodies and therefore the development of allergic disease. Mice with impaired B cell receptor (BCR) signaling have significantly increased IgE responses, consistent with a role for BCR signaling in IgE regulation. While prior work focused on BCR signaling in IgE-expressing cells to explain these findings, it has been reported that BCR signaling can reduce CSR. Therefore, we investigated the possibility that IgE CSR might be particularly sensitive to inhibition by BCR signaling in unswitched B cells. We found that immunization of mice with high-affinity antigen resulted in reduced representation of IgE-expressing cells among germinal center B cells and plasma cells relative to a low-affinity antigen. Mechanistic experiments with cultured mouse B cells demonstrated that BCR ligands selectively inhibited IgE CSR in a dose-, affinity-, and avidity-dependent manner. Signaling via Syk was required for the inhibition of IgE CSR following BCR stimulation, whereas inhibition of the PI3K subunit p110{delta} increased IgE CSR independently of BCR ligation. The inhibition of IgE CSR by BCR ligands synergized with IL-21 or TGF{beta}1. BCR ligation also inhibited CSR to IgE in human tonsillar B cells, and this inhibition was also synergistic with IL-21. These findings establish that IgE CSR is uniquely susceptible to inhibition by BCR signaling in mouse and human B cells, with important implications for the regulation and pathogenesis of allergic disease.

The immune regulatory gene PTPN22 is expressed in all immune cells and encodes Lyp in humans and the ortholog PEP in mice. The PTPN22 alternative allele, 1858C>T, is expressed in 5-15% of the North American population and is strongly associated with the development of autoimmune disease while simultaneously capable of providing protection during virus infection and cancer. In murine models, significant progress has been made in elucidating the molecular mechanisms that PEP and its pro-autoimmune variant (PEP-R619W) modulate T cell function, yet their influence on non-T cell pathways, such as antigen presenting cell cytokine production, remains less defined. Previously, it was reported that PEP promotes type I interferon (IFN-I) production in dendritic cells (DCs) and macrophages following TLR4 stimulus. Here, we show that contrary to previous results, both PEP-WT and the PEP-R619W variant do not promote IFN-I production in DCs and macrophages following exposure to LPS, 3p-hpRNA, or coronavirus MHV A59. We attribute the prior findings to mouse strain-specific differences and conclude that factors independent of PEP may be regulating IFN-I production in these studies. We further show that PEP and its R619W variant distinctly modulate the production of TNF, IL-12 and IL-2 in DCs following LPS stimulus. Taken together, our results challenge the current understanding of the role of PEP during inflammation while providing new insight into how the PEP-R619W variant may alter myeloid cell function during disease.

Macrophages, as key sentinel cells of the innate immune system, can retain memory of prior stimulus exposure. Interferon gamma (IFN{gamma}) plays a central role in maintaining trained immunity in vivo and can induce potent memory in macrophages. Such memory is associated with the formation of de novo enhancers that alter gene expression responses to subsequent stimuli. However, how such enhancers are maintained after cytokine exposure remains unclear. We report that durable IFN{gamma}-induced enhancers can last for days after cytokine washout, yet the underlying persistence mechanism is not cell-intrinsic. IFN{gamma}-treated macrophages continue to exhibit JAK/STAT signaling days after cytokine removal. Blocking IFN{gamma} signaling with a JAK inhibitor or anti-IFN{gamma} neutralizing antibodies after cytokine removal is sufficient to reverse IFN{gamma}-induced enhancers and erase the potentiated state of the treated macrophages. Our findings suggest that epigenetic changes in macrophages do not inherently encode innate immune memory or a "potentiated" macrophage state, but in fact are themselves dependent on ongoing cytokine signaling. These findings suggest new possibilities for pharmacologic interventions to reverse aberrantly trained immune states associated with pathology.

Placental immune responses are highly regulated to strike a balance between protection and tolerance. For relatively mild infections, protection encompasses both the mother and fetus; however, during worsening conditions, protection becomes exclusively reserved for the mother. Previously, we and others have shown that the host factor Perforin-2 plays a central role in protecting mice and cells against infection. Here, we analyzed Perforin-2 activity in the mouse placenta to determine whether Perforin-2 plays a similarly protective role. We show that Perforin-2 is critical for inhibiting Listeria monocytogenes colonization of the placenta and fetus and that this protection is due to both maternal and fetal-encoded Perforin-2. Perforin-2 mRNA is readily detectable in individual immune cells of the decidua and these levels are further enhanced specifically in decidual macrophages during high-dose infections that result in fetal expulsion. Unexpectedly, inductive Perforin-2 expression in decidual macrophages did not occur during milder infections in which fetal viability remained intact. This pattern of expression significantly differed from that observed in splenic macrophages in which inductive Perforin-2 expression was observed in both high and mild infection conditions. In the placenta, inductive Perforin-2 expression in decidual macrophages was co-incident with their polarization from a M2 to M1 phenotype that normally occurs in the placenta during high-burden infections. Our results suggest that Perforin-2 is part of a host response that is protective either for both the mother and fetus in milder infections or exclusively for the mother during high-dose infections.