Brian S. Sheridan

IL-17A–producing resident memory γδ T cells orchestrate the innate immune response to secondary oral Listeria monocytogenes infection

Significance Outbreaks of food-borne infections with Listeria monocytogenes can result in high mortality. Using a model of recombinant L. monocytogenes that models human infection in mice, we show that L. monocytogenes-specific memory γδ T cells in fact represent a resident memory (Trm) population in the mesenteric lymph node that secrete IL-17A and cluster with L. monocytogenes replication foci after secondary infection. Furthermore γδ Trms mediate the intranodal migration and redistribution of myeloid cells, which was necessary to contain the spread and growth of L. monocytogenes. Our findings demonstrate how γδ Trm cells orchestrate pathogen-induced innate immune responses. These observations provide the rationale for designing novel vaccination strategies to harness the ability of γδ Trm cells to provide protection against intestinal pathogens. Memory γδ T cells are important for the clearance of Listeria monocytogenes infection in the intestinal mucosa. However, the mechanisms by which memory γδ T cells provide protection against secondary oral infection are poorly understood. Here we used a recombinant strain of L. monocytogenes that efficiently invades the intestinal epithelium to show that Vγ4+ memory γδ T cells represent a resident memory (Trm) population in the mesenteric lymph nodes (MLNs). The γδ Trm exhibited a remarkably static pattern of migration that radically changed following secondary oral L. monocytogenes infection. The γδ Trms produced IL-17A early after rechallenge and formed organized clusters with myeloid cells surrounding L. monocytogenes replication foci only after a secondary oral infection. Antibody blocking studies showed that in addition to IL-17A, the chemokine receptor C-X-C chemokine receptor 3 (CXCR3) is also important to enable the local redistribution of γδ Trm cells and myeloid cells specifically near the sites of L. monocytogenes replication within the MLN to restrict bacterial growth and spread. Our findings support a role for γδ Trms in orchestrating protective immune responses against intestinal pathogens.

Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein. Opsonophagocytic efficacy was measured in vitro and in vivo by intravital microscopy in murine livers. In vivo protection was tested in murine models, including a novel model for dissemination in hvKp-colonized mice. Protective efficacy of monoclonal antibodies (MAbs) 4C5 (IgG1) and 19A10 (IgG3) was demonstrated both in murine sepsis and pulmonary infection. In hvKp-colonized mice, MAb treatment significantly decreased dissemination of hvKp from the gut to mesenteric lymph nodes and organs. Intravital microscopy confirmed efficient opsonophagocytosis and clearance of bacteria from the liver. In vitro studies demonstrate that MAbs work predominantly by promoting FcR-mediated phagocytosis but also indicate that MAbs enhance the release of neutrophil extracellular traps (NETs). In anticipation of increasing antibiotic resistance, we propose further development of these and other Klebsiella-specific MAbs for therapeutic use.

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection

Significance The control of a microbial infection by effector T cells is intrinsically linked to their migration. However, little is known about the mechanisms that control effector T-cell egress after infection. Sphingosine-1-phosphate receptor-1 (S1PR1) is a G-coupled protein receptor that plays an important role in naive T-cell egress from lymph nodes. However, less is known about its role in regulating effector T-cell trafficking during infection. Here, we used an inducible mouse model with temporally disrupted S1PR1 signaling exclusively in endogenous effector CD8 T cells to demonstrate that, after infection, even in the absence of retention signals such as CC chemokine receptor 7 (CCR7), intrinsic S1PR1 signaling is the overriding factor that regulates effector T-cell egress kinetics from the draining lymph node. Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1−/− effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1−/− T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1−/− effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.

Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection.

Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A ‘murinized’ Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the ‘gut-homing’ integrin α4β7 and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27− Ly6C− and CD69+ CD103− while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69−. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.

Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment

Bioactive sphingolipids are modulators of immune processes and their metabolism is often dysregulated in ulcerative colitis, a major category of inflammatory bowel disease (IBD). While multiple axes of sphingolipid metabolism have been investigated to delineate mechanisms regulating ulcerative colitis, the role of acid ceramidase (AC) in intestinal inflammation is yet to be characterized. Here we demonstrate that AC expression is elevated selectively in the inflammatory infiltrate in human and murine colitis. To probe for mechanistic insight into how AC up‐regulation can impact intestinal inflammation, we investigated the selective loss of AC expression in the myeloid population. Using a model of intestinal epithelial injury, we demonstrate that myeloid AC conditional knockout mice exhibit impairment of neutrophil recruitment to the colon mucosa as a result of defective cytokine and chemokine production. Furthermore, the loss of myeloid AC protects from tumor incidence in colitis‐associated cancer (CAC) and inhibits the expansion of neutrophils and granulocytic myeloid‐derived suppressor cells in the tumor microenvironment. Collectively, our results demonstrate a tissue‐specific role for AC in regulating neutrophilic inflammation and cytokine production. We demonstrate novel mechanisms of how granulocytes are recruited to the colon that may have therapeutic potential in intestinal inflammation, IBD, and CAC—Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., Obeid, L. M. Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment. FASEB J. 32, 2339–2353 (2018). www.fasebj.org

CCR2+ inflammatory monocytes are recruited to Yersinia pyogranulomas and dictate adaptive responses at the expense of innate immunity during oral infection

ABSTRACT Murine Ly6Chi inflammatory monocytes (IMs) require CCR2 to leave the bone marrow and enter mesenteric lymph nodes (MLNs) and other organs in response to Yersinia pseudotuberculosis infection. We are investigating how IMs, which can differentiate into CD11c+ dendritic cells (DCs), contribute to innate and adaptive immunity to Y. pseudotuberculosis. Previously, we obtained evidence that IMs are important for a dominant CD8+ T cell response to the epitope YopE69–77 and host survival using intravenous infections with attenuated Y. pseudotuberculosis. Here we challenged CCR2+/+ or CCR2−/− mice orally with wild-type Y. pseudotuberculosis to investigate how IMs contribute to immune responses during intestinal infection. Unexpectedly, CCR2−/− mice did not have reduced survival but retained body weight better and their MLNs cleared Y. pseudotuberculosis faster and with reduced lymphadenopathy compared to controls. Enhanced bacterial clearance in CCR2−/− mice correlated with reduced numbers of IMs in spleens and increased numbers of neutrophils in livers. In situ imaging of MLNs and spleens from CCR2-GFP mice showed that green fluorescent protein-positive (GFP+) IMs accumulated at the periphery of neutrophil-rich Yersinia-containing pyogranulomas. GFP+ IMs colocalized with CD11c+ cells and YopE69–77-specific CD8+ T cells in MLNs, suggesting that IM-derived DCs prime adaptive responses in Yersinia pyogranulomas. Consistently, CCR2−/− mice had reduced numbers of splenic DCs, YopE69–77-specific CD8+ T cells, CD4+ T cells, and B cells in organs and lower levels of serum antibodies to Y. pseudotuberculosis antigens. Our data suggest that IMs differentiate into DCs in MLN pyogranulomas and direct adaptive responses in T cells at the expense of innate immunity during oral Y. pseudotuberculosis infection.

Listeria Monocytogenes: A Model Pathogen Continues to Refine Our Knowledge of the CD8 T Cell Response

Listeria monocytogenes (Lm) infection induces robust CD8 T cell responses, which play a critical role in resolving Lm during primary infection and provide protective immunity to re-infections. Comprehensive studies have been conducted to delineate the CD8 T cell response after Lm infection. In this review, the generation of the CD8 T cell response to Lm infection will be discussed. The role of dendritic cell subsets in acquiring and presenting Lm antigens to CD8 T cells and the events that occur during T cell priming and activation will be addressed. CD8 T cell expansion, differentiation and contraction as well as the signals that regulate these processes during Lm infection will be explored. Finally, the formation of memory CD8 T cell subsets in the circulation and in the intestine will be analyzed. Recently, the study of CD8 T cell responses to Lm infection has begun to shift focus from the intravenous infection model to a natural oral infection model as the humanized mouse and murinized Lm have become readily available. Recent findings in the generation of CD8 T cell responses to oral infection using murinized Lm will be explored throughout the review. Finally, CD8 T cell-mediated protective immunity against Lm infection and the use of Lm as a vaccine vector for cancer immunotherapy will be highlighted. Overall, this review will provide detailed knowledge on the biology of CD8 T cell responses after Lm infection that may shed light on improving rational vaccine design.

Editorial: Vγ9Vδ2 T cells: triple costimulation goes the distance

Tcell populations are divided into distinct subsets based on their TCR use: ab T cells or gd T cells. Each T cell subset holds tremendous promise for adoptive immunotherapy as a result of its ability to eliminate pathogens and tumors [1]. Various avenues of basic biomedical research and ongoing clinical trials are currently exploring this potential. Whereas progress in understanding the cell biology of ab T cells has progressed rapidly, several factors have contributed to a slower emergence of biologic insight into gd T cells. This dichotomy may largely be explained by the difficulty of identifying gd T cell antigens compared with the relative ease of determining ab T cell antigen specificity. This difference has led to substantial tools to examine pathogenor tumor-specific ab T cells, while leaving gd T cell exploration to largely rely on phenotypic associations or TCR use, irrespective of specificity. In recent years, this has begun to change, with the spectrum of antigens recognized by human gd T cells beginning to emerge. Phosphorylated prenyl metabolites, lipids presented by CD1 family proteins, and cell stress-associated molecules, which may be up-regulated as a result of infection or malignant transformation, have been identified as important antigens recognized by human gd T cells. Importantly, for broad-based immunotherapeutic potential, gd T cells directly bind antigen, independent of HLA/peptide complexes and therefore, are potential universal T cell therapies against a wide array of tumor conditions as a result of their ability to recognize common cellular indicators of stress. Initial studies demonstrated a critical role of murine gd T cells in cancer immunosurveillance in chemically induced skin cancer, more than 1 decade ago [2]. More recently, a meta-analysis of expression signatures from 18,000 human tumors with overall survival data across 39 distinct malignancies explored cancer-wide prognostic signatures [3]. This groundbreaking analysis identified the presence of an intratumoral gd T cell signature as the most significant, favorable prognostic leukocyte population across a large collection of cancer types, providing a strong and renewed incentive to explore the therapeutic potential of gd T cells. Tumor-reactive gd T cells are multipotent effectors for tumor elimination. They can lyse tumor cells directly via perforin and granzymes or by engagement of death receptors, such as CD95. They are also a potent source of IFN-g, which may inhibit angiogenesis and enhance MHC class I expression by tumor cells. Lastly, extensive evidence has demonstrated a clear anti-tumor role for IL-17 production. However, recent data have also demonstrated that IL-17 may promote tumor progression under certain conditions, suggesting that any immunotherapy must fine tune the functional capacity and limit cellular plasticity of elicited immune responses based on tumor type [1]. In particular, Vg9Vd2 T cells have been a focus of substantial anti-tumor research, as they recognize a metabolic intermediate of the mevalonate pathway, isopentenyl pyrophosphate, which accumulates at high levels in cancer cells [4]. Emerging avenues of potential therapeutic modalities to use gd T cell immunotherapies have largely focused on 2 distinct approaches with the same goal, generating a large population of tumor-reactive Vg9Vd2 T cells. One approach uses in vivo methodology to expand gd T cells, whereas another approach uses large numbers of ex vivoexpanded and -activated gd T cells for direct infusion into cancer patients. To date, all clinical trials have targeted Vg9Vd2 T cell therapies, but progress with direct infusion of large numbers of gd T cells has been hindered by technical limitations in the efficient generation of large numbers of polyfunctional effector gd T cells ex vivo. In this issue of the Journal of Leukocyte Biology, Cho et al. [5] demonstrate that aAPCs, engineered to provide TCR ligation and costimulatory signals through CD80, 4-1BB, and CD83L in the presence of low levels of IL-2, induced long-lived and multifunctional Vg9Vd2 T cells from Vg9 T cells isolated from the peripheral blood of healthy human donors. Of note, some dual costimulation scenarios were able to induce efficient effector gd T cells with regard to cytokine production or