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Escherichia coli heat-labile enterotoxin (Etx) is an oral adjuvant in mice. We show that this is also true for rats. To understand this adjuvant activity we examined lymph dendritic cells (DC) migrating from the intestine to mesenteric lymph nodes (MLN) in animals fed Etx. These DC can prime antigen-specific antibody responses. We show that in rats the small intestine contains 7-24 million DC and 8 × 10of these migrate to MLN each day. Surprisingly, Etx does not stimulate increased migration of lymph DC. However, oral Etx affects the activation, antigen transport and localization of migratory DC. Specifically, expression of CD25 increases on the CD172a subset of lymph DC. Oral Etx also increases the number of CD172a lymph DC containing co-administered ovalbumin. CD172a lymph DC treated with Etx in vitro, or purified from the lymph of animals fed Etx, stimulate stronger proliferative responses from primed T cells. Etx also directs more of the CD172a lymph DC into the central region of the MLN T cell areas. This change in DC localization is associated with an increase in the expression of CCR7. These data help advance our understanding of the role of DC in initiating mucosal immune responses in vivo. [ABSTRACT FROM AUTHOR]

The murine serous cavities contain a rare and enigmatic population of short-lived F4/80 lo MHCII + macrophages but what regulates their development, survival, and fate is unclear. Here, we show that mature F4/80 lo MHCII + peritoneal macrophages arise after birth, but that this occurs largely independently of colonization by microbiota. Rather, microbiota specifically regulate development of a subpopulation of CD11c + cells that express the immunoregulatory cytokine RELM-α, are reliant on the transcription factor EGR2, and develop independently of the growth factor CSF1. Furthermore, we demonstrate that intrinsic expression of RELM-α, a signature marker shared by CD11c + and CD11c - F4/80 lo MHCII + cavity macrophages, regulates survival and differentiation of these cells in the peritoneal cavity in a sex-specific manner. Thus, we identify a previously unappreciated diversity in serous cavity F4/80 lo MHCII + macrophages that is regulated by microbiota, and describe a novel sex and site-specific function for RELM-α in regulating macrophage endurance that reveals the unique survival challenge presented to monocyte-derived macrophages by the female peritoneal environment., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the T H 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C + MHCII + monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The gut microbiota's function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium , rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development., Competing Interests: All authors declare no conflicts of interest., (© 2021 The Author(s).)

The use of helminth infections as tools to understand the type 2 immune response is a well-established technique and important to many areas of immunological research. The phenotype and function of immune cell populations at the site of infection is a key determinant of pathogen clearance. However, infections with helminths such as the murine nematode Heligomosmoides polygryrus cause increased mucus production and thickening of the intestinal wall, which can result in extensive cell death when isolating and analysing cells from the lamina propria (LP). Populations of larger immune cells such as macrophages and dendritic cells are often trapped within mucus or dying tissues. Here we describe an optimised protocol for isolating LP leukocytes from the small intestine of H.polygyrus -infected mice, and we demonstrate phenotypic and functional identification of myeloid and CD4 + T cell subsets using cytokine staining and flow cytometry. Our protocol may provide a useful experimental method for the immunological analysis of the affected tissue site during helminth infections., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103 + /CD141 + DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8 + (but not CD4 + ) T cells in vitro and in vivo . Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8 + T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The identification of conventional dendritic cells (cDCs) in the intestinal mucosa has been hampered by the difficulties associated with isolating cells from the intestine and by the fact that overlapping markers have made it complicated to discriminate them accurately from other intestinal mononuclear phagocytes such as macrophages (MFs). Here we detail the protocols we have developed to isolate live leukocytes from both murine and human small and large intestines and describe reliable strategies which can be used to identify bona fide cDCs in such preparations.

Mononuclear phagocytes (MPs) in the murine intestine, comprising dendritic cells (DCs) and macrophages (Mϕs), perform disparate yet complementary immunological functions. Functional analyses of these distinct MP subsets have been complicated by the substantial overlap in their surface phenotypes. Here, we review recent findings that have enabled more accurate definition of these MP subsets. We discuss these recent advances in the context of the current understanding of the functions of DCs and Mϕs in the maintenance of intestinal homeostasis, and how their functions may alter when homeostasis is disrupted., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Dendritic cells (DCs) play a critical key role in the initiation of immune responses to pathogens. Paradoxically, they also prevent potentially damaging immune responses being directed against the multitude of harmless antigens, to which the body is exposed daily. These roles are particularly important in the intestine, where only a single layer of epithelial cells provides a barrier against billions of commensal microorganisms, pathogens, and food antigens, over a huge surface area. In the intestine, therefore, DCs are required to perform their dual roles very efficiently to protect the body from the dual threats of invading pathogens and unwanted inflammatory reactions. In this review, we first describe the biology of DCs and their interactions with other cells types, paying particular attention to intestinal DCs. We, then, examine the ways in which this biology may become misdirected, resulting in inflammatory bowel disease. Finally, we discuss how DCs potentiate immune responses against viral, bacterial, parasitic infections, and their importance in the pathogenesis of prion diseases. We, therefore, provide an overview of the complex cellular interactions that affect intestinal DCs and control the balance between immunity and tolerance.

Dendritic cells (DCs) migrating via lymph are the primary influence regulating naive T cell differentiation, be it active immunity or tolerance. How DCs achieve this regulation in vivo is poorly understood. Intestinal DCs are in direct contact with harmless or pathogenic luminal contents, but may also be influenced by signals from epithelial cells, macrophages, or other resident or immigrant cells. To understand the role of TLR7 and TLR8 in regulating intestinal DC function, we fed a TLR7/8 ligand (resiquimod (R-848)) to rats and mice and examined DC in pseudoafferent lymph (rat) and mesenteric lymph nodes (MLNs). Oral R-848 induced a 20- to 30-fold increase in DC output from the intestine within 10 h due to a virtually total release of lamina propria DCs. This resulted in an accumulation of DCs in the MLNs that in mice was completely TNF-alpha dependent. Surprisingly, intestinal lymph DCs (iL-DCs) released by R-848 did not up-regulate CD86, but did up-regulate CD25. In contrast, MLN-DCs from R-848-stimulated rats and mice expressed high levels of CD86. This DC activation in MLNs was dependent on type 1 IFNs. The major source of these rapidly released cytokines is plasmacytoid DCs (pDCs) and not classical DCs, because depletion of pDCs significantly reduces the R-848-stimulated increase in serum cytokine levels as well as the accumulation and activation of DCs in MLNs. These experiments show that TLR-mediated regulation of iL-DC functions in vivo is complex and does not depend only on direct iL-DC stimulation, but can be regulated by pDCs.

Dendritic cells (DCs) are crucial in immune induction. Not only do they collect antigens in peripheral tissues, and transport and process them for presentation to lymphocytes in draining lymph nodes, but they also regulate the immune response by modulating T-cell differentiation. Intestinal and hepatic DCs migrating in lymph can be collected from rats under near-physiological conditions. Initially, the mesenteric or celiac lymph nodes are removed from young rats (30 min). The afferent and efferent lymph vessels subsequently heal, permitting DCs to enter the thoracic duct. After at least 6 wk, the duct is cannulated (40 min). Lymph can be collected for up to 48 h. DCs can subsequently be identified, enriched and sorted to high degrees of purity. This two-stage technique generates large numbers of immunologically relevant DCs under near-physiological conditions. Lymph collection requires 2-3 h per animal over 6 wk.

Recent evidence demonstrates that dendritic cells (DCs) can insert dendrites between the epithelial cells that form the barrier protecting the body from the gut contents. Although first observed almost a decade ago, this is a controversial area of DC biology and the physiological importance of this phenomenon is only now being clarified. A recent study by Niess and colleagues shows that this behaviour enables efficient sampling of both invasive and non-invasive bacteria and might enhance the ability of an organism to resist infections by a pathogenic strain of Salmonella.

Melanomas are promising targets for immunotherapy, as they express a number of tissue-specific antigens against which immune responses can be elicited. We have previously described transgenic mice in which malignant cutaneous melanomas are produced. The 1042 melanoma cell line, derived from a primary melanoma in one of these mice, was used here to generate tumours by subcutaneous inoculation in syngeneic animals. All mice injected with 1 x 10(6) cells of the 1042 cell line developed a tumour. CD4+ T cells, CD8+ T cells and macrophages infiltrated the tumours. Treatment with dendritic cells pulsed with peptides from melanogenic proteins slowed tumour growth and resulted in increased numbers of infiltrating lymphocytes and macrophages, expansion of CD4+ T cells specific for 1042 cell antigens, and increased levels of 1042-specific immunoglobulin G1 (IgG1) and IgM in serum. The frequency of cytotoxic T lymphocytes (CTLs) specific for the MART-1 melanocytic antigen did not increase after dendritic cell treatment. Indeed, the presence of CD8+ T cells was apparently not required for the anti-tumour effects: slowing of tumour growth was not abrogated in animals depleted of CD8+ T cells using antibodies, or in syngeneic CD8-/- animals. In contrast, treatment with dendritic cells + peptides was ineffective after depletion of CD4+ T cells and in syngeneic CD4-/- mice. This experimental system therefore provides an opportunity to investigate CD4-dependent anti-tumour effector mechanisms, and for studies designed to activate the quiescent CTLs which infiltrate melanomas.

Murine dendritic cells (DCs) are widely used for experimental vaccinations in mouse models. A high-yield method for freezing and thawing batches of these cells, if compatible with retention of cell immunophenotype, would reduce the time required for repeated preparations from DC precursors in bone marrow (BM), as well as variability among lots. Following depletion of specific lineages, murine bone marrow cells from C57BL/6 inbred-strain mice were grown in medium containing 10% fetal calf serum (FCS) and granulocyte/macrophage colony-stimulating factor (GM-CSF); after 6 days, large numbers of immature DCs were obtained. The immature cells were frozen in complete medium with GM-CSF and 10% DMSO, at a cell density of 5x10(6) DCs/ml. After thawing, 80% of DCs survived; they were induced to mature by addition of lipopolysaccharide (LPS). In comparison with fresh DCs, the thawed DCs had similar morphology, purity, and expression of class I (H-2D(b) and H-2K(b)) and class II major histocompatibility complex (MHC) proteins, as well as CD11b, CD11c, CD40, CD80, and CD86 molecules. Freeze-thawing did not affect trafficking to T cell areas of spleen, nor reduce the capacity to stimulate an alloresponse. Frozen-thawed cells were also proficient at uptake, processing, and presentation of native or denatured ovalbumin (OVA) protein to a peptide-specific T cell hybridoma, and were able to induce T cell responses in vivo after being loaded with denatured OVA protein. The ability to freeze and thaw DCs, and to obtain high yields without altering their essential properties, will facilitate future immunotherapy experiments in laboratory mouse models.

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in alpha-beta hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR., (Copyright 2002 Wiley-Liss, Inc.)