Stromal cell-immune cell interactions.

Interaction between different types of hematopoietic cells is essential for proper functioning of the immune system. For instance, the cytokines produced by antigen-presenting dendritic cells will determine the type of T cell response that is induced. However, hematopoietic cells are also strongly influenced by the surrounding nonhematopoietic cells. The cells that form these microenvironments are collectively called stromal cells. Here, we focus on the stromal cells present within secondary lymphoid organs and discuss their importance for various aspects of the immune system.

Innate Lymphoid Cells: 10 Years On.

Innate lymphoid cells (ILCs) are lymphocytes that do not express the type of diversified antigen receptors expressed on T cells and B cells. ILCs are largely tissue-resident cells and are deeply integrated into the fabric of tissues. The discovery and investigation of ILCs over the past decade has changed our perception of immune regulation and how the immune system contributes to the maintenance of tissue homeostasis. We now know that cytokine-producing ILCs contribute to multiple immune pathways by, for example, sustaining appropriate immune responses to commensals and pathogens at mucosal barriers, potentiating adaptive immunity, and regulating tissue inflammation. Critically, the biology of ILCs also extends beyond classical immunology to metabolic homeostasis, tissue remodeling, and dialog with the nervous system. The last 10 years have also contributed to our greater understanding of the transcriptional networks that regulate lymphocyte commitment and delineation. This, in conjunction with the recent advances in our understanding of the influence of local tissue microenvironments on the plasticity and function of ILCs, has led to a re-evaluation of their existing categorization. In this review, we distill the advances in ILC biology over the past decade to refine the nomenclature of ILCs and highlight the importance of ILCs in tissue homeostasis, morphogenesis, metabolism, repair, and regeneration.

Fungi Take Control of Lymphocyte Recirculation.

Factors regulating leukocyte migration to neonatal lymph nodes are not sufficiently identified. In this issue of Immunity, Zhang et al. (2016) reveal that fungi drive emigration of gut DCs to lymph nodes, where these DCs instruct endothelial cell receptivity to leukocytes.

In-depth immune profiling of peripheral blood mononuclear cells in patients with pancreatic ductal adenocarcinoma reveals discriminative immune subpopulations.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time-of-flight to undertake an in-depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro-inflammatory cells, as CD86+ classical monocytes and memory T cells expressing CCR6+ and CXCR3+, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39+ regulatory T cells and CCR4+CCR6-CXCR3- memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.

Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling.

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

The role of CD56bright NK cells in neurodegenerative disorders.

Emerging evidence suggests a potential role for natural killer (NK) cells in neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise function of NK cells in these diseases remains ambiguous. The existence of two NK cell subsets, CD56bright and CD56dim NK cells, complicates the understanding of the contribution of NK cells in neurodegeneration as their functions within the context of neurodegenerative diseases may differ significantly. CD56bright NK cells are potent cytokine secretors and are considered more immunoregulatory and less terminally differentiated than their mostly cytotoxic CD56dim counterparts. Hence, this review focusses on NK cells, specifically on CD56bright NK cells, and their role in neurodegenerative diseases. Moreover, it explores the mechanisms underlying their ability to enter the central nervous system. By consolidating current knowledge, we aim to provide a comprehensive overview on the role of CD56bright NK cells in neurodegenerative diseases. Elucidating their impact on neurodegeneration may have implications for future therapeutic interventions, potentially ameliorating disease pathogenesis.

Unbiased method for spectral analysis of cells with great diversity of autofluorescence spectra.

Autofluorescence is an intrinsic feature of cells, caused by the natural emission of light by photo-excitatory molecular content, which can complicate analysis of flow cytometry data. Different cell types have different autofluorescence spectra and, even within one cell type, heterogeneity of autofluorescence spectra can be present, for example, as a consequence of activation status or metabolic changes. By using full spectrum flow cytometry, the emission spectrum of a fluorochrome is captured by a set of photo detectors across a range of wavelengths, creating an unique signature for that fluorochrome. This signature is then used to identify, or unmix, that fluorochrome's unique spectrum from a multicolor sample containing different fluorescent molecules. Importantly, this means that this technology can also be used to identify intrinsic autofluorescence signal of an unstained sample, which can be used for unmixing purposes and to separate the autofluorescence signal from the fluorophore signals. However, this only works if the sample has a singular, relatively homogeneous and bright autofluorescence spectrum. To analyze samples with heterogeneous autofluorescence spectral profiles, we setup an unbiased workflow to more quickly identify differing autofluorescence spectra present in a sample to include as "autofluorescence signatures" during the unmixing of the full stained samples. First, clusters of cells with similar autofluorescence spectra are identified by unbiased dimensional reduction and clustering of unstained cells. Then, unique autofluorescence clusters are determined and are used to improve the unmixing accuracy of the full stained sample. Independent of the intensity of the autofluorescence and immunophenotyping of cell subsets, this unbiased method allows for the identification of most of the distinct autofluorescence spectra present in a sample, leading to less confounding autofluorescence spillover and spread into extrinsic phenotyping markers. Furthermore, this method is equally useful for spectral analysis of different biological samples, including tissue cell suspensions, peripheral blood mononuclear cells, and in vitro cultures of (primary) cells.

Spatial immune composition of tumor microenvironment in patients with pancreatic cancer.

This study examined the composition of the immune microenvironment at different sites within resected pancreas specimens from patients with pancreatic ductal adenocarcinoma (PDAC). Therefore, single-cell suspensions were made from fresh tumor and non-tumorous tissue. Fourteen patients were included from whom twelve PDAC and five non-tumorous samples were obtained. These samples were analyzed with a nineteen marker panel on the Aurora spectral flow cytometer. Furthermore, slides from formalin-fixed paraffine PDACs of eight additional patients were stained with eight markers and analyzed by multispectral imaging. These corresponded to central tumor, periphery of the tumor, i.e., invasive front and resected lymph node and were divided into tumor and adjacent tissue. In the single-cell suspension, a decreased ratio between lymphoid and myeloid cells and between M1 and M2 macrophages was observed in the tumor tissue compared to non-tumorous tissue. Furthermore, an increase in CD169 + macrophages in patients undergoing neoadjuvant therapy was found. Using immunofluorescence, more macrophages compared to T cells were observed, as well as a lower ratio of CD8 to M2 macrophage, a higher ratio of CD4-CD8 T cells and a higher ratio of immune-suppressive cells to pro-inflammatory cells in the PDAC area compared to the adjacent non-tumorous tissue. Finally, there were more immune-suppressive cells in the central tumor area compared to the invasive front. In conclusion, we show a gradient in the immune-suppressive environment in PDAC from most suppressive in the central tumor to least suppressive in distant non-tumorous tissue.

Tertiary Lymphoid Structures: Diversity in Their Development, Composition, and Role.

Lymph node stromal cells coordinate the adaptive immune response in secondary lymphoid organs, providing both a structural matrix and soluble factors that regulate survival and migration of immune cells, ultimately promoting Ag encounter. In several inflamed tissues, resident fibroblasts can acquire lymphoid-stroma properties and drive the formation of ectopic aggregates of immune cells, named tertiary lymphoid structures (TLSs). Mature TLSs are functional sites for the development of adaptive responses and, consequently, when present, can have an impact in both autoimmunity and cancer conditions. In this review, we go over recent findings concerning both lymph node stromal cells and TLSs function and formation and further describe what is currently known about their role in disease, particularly their potential in tolerance.

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching.

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy.

Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation.

The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue-inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5-14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons.

CD62L Is a Functional and Phenotypic Marker for Circulating Innate Lymphoid Cell Precursors.

Innate lymphoid cells (ILCs) guard epithelial tissue integrity during homeostasis, but can be potent immune effector cells during inflammation. Precursors to all ILC subsets (ILC precursors [ILCP]) have been identified in human peripheral blood (PB). We found that during homeostasis, ILCP in PB of mouse and human expressed homing receptors for secondary lymphoid organs, mainly CD62L. These ILCP entered mouse lymph nodes in a CD62L-dependent way and relied on S1P receptors for their exit. Importantly, CD62L expression was absent on human ILCs expressing NKp44 in tonsils and PB of Crohn disease patients, and relatively fewer CD62L+ ILCP were present in PB of Crohn disease patients. These data are in agreement with selective expression of CD62L on nonactivated ILCP. As such, we conclude that CD62L not only serves as a functional marker of ILCP, but has potential to be used in the clinic as a diagnostic marker in inflammatory disorders.

Retinoic Acid and Immune Homeostasis: A Balancing Act.

In the immune system, the vitamin A metabolite retinoic acid (RA) is known for its role in inducing gut-homing molecules in T and B cells, inducing regulatory T cells (Tregs), and promoting tolerance. However, it was suggested that RA can have a broad spectrum of effector functions depending on the local microenvironment. Under specific conditions, RA can also promote an inflammatory environment. We discuss the dual role of RA in immune responses and how this might be regulated. Furthermore, we focus on the role of RA in autoimmune diseases and whether RA might be used as a therapeutic agent.

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity.

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. METHOD: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). RESULTS: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. CONCLUSION: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Innate lymphoid cells in secondary lymphoid organs.

The family of innate lymphoid cells (ILCs) has attracted attention in recent years as its members are important regulators of immunity, while they can also cause pathology. In both mouse and man, ILCs were initially discovered in developing lymph nodes as lymphoid tissue inducer (LTi) cells. These cells form the prototypic members of the ILC family and play a central role in the formation of secondary lymphoid organs (SLOs). In the absence of LTi cells, lymph nodes (LN) and Peyer's Patches (PP) fail to form in mice, although the splenic white pulp can develop normally. Besides LTi cells, the ILC family encompasses helper-like ILCs with functional distinctions as seen by T-helper cells, as well as cytotoxic natural killer (NK) cells. ILCs are still present in adult SLOs where they have been shown to play a role in lymphoid tissue regeneration. Furthermore, ILCs were implicated to interact with adaptive lymphocytes and influence the adaptive immune response. Here, we review the recent literature on the role of ILCs in secondary lymphoid tissue from the formation of SLOs to mature SLOs in adults, during homeostasis and pathology.

Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells To Induce Mucosal Tolerogenic Dendritic Cells.

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.

Lymphoid organs for peritoneal cavity immune response: milky spots.

Milky spots are located in the omentum of the peritoneal cavity and their classification as lymphoid organs has been debated. In this issue of Immunity, Rangel-Moreno et al. (2009) provide compelling data to consider them as unique secondary lymphoid organs.

Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.

Vitamin A Controls the Presence of RORγ+ Innate Lymphoid Cells and Lymphoid Tissue in the Small Intestine.

Changes in diet and microbiota have determining effects on the function of the mucosal immune system. For example, the active metabolite of vitamin A, retinoic acid (RA), has been described to maintain homeostasis in the intestine by its influence on both lymphocytes and myeloid cells. Additionally, innate lymphoid cells (ILCs), important producers of cytokines necessary for intestinal homeostasis, are also influenced by vitamin A in the small intestines. In this study, we show a reduction of both NCR(-) and NCR(+) ILC3 subsets in the small intestine of mice raised on a vitamin A-deficient diet. Additionally, the percentages of IL-22-producing ILCs were reduced in the absence of dietary vitamin A. Conversely, mice receiving additional RA had a specific increase in the NCR(-) ILC3 subset, which contains the lymphoid tissue inducer cells. The dependence of lymphoid tissue inducer cells on vitamin A was furthermore illustrated by impaired development of enteric lymphoid tissues in vitamin A-deficient mice. These effects were a direct consequence of ILC-intrinsic RA signaling, because retinoic acid-related orphan receptor γt-Cre × RARα-DN mice had reduced numbers of NCR(-) and NCR(+) ILC3 subsets within the small intestine. However, lymphoid tissue inducer cells were not affected in these mice nor was the formation of enteric lymphoid tissue, demonstrating that the onset of RA signaling might take place before retinoic acid-related orphan receptor γt is expressed on lymphoid tissue inducer cells. Taken together, our data show an important role for vitamin A in controlling innate lymphoid cells and, consequently, postnatal formed lymphoid tissues within the small intestines.