CD45RA+CD62L- ILCs in human tissues represent a quiescent local reservoir for the generation of differentiated ILCs.

Innate lymphoid cells (ILCs) are highly plastic and predominantly mucosal tissue-resident cells that contribute to both homeostasis and inflammation depending on the microenvironment. The discovery of naïve-like ILCs suggests an ILC differentiation process that is akin to naïve T cell differentiation. Delineating the mechanisms that underlie ILC differentiation in tissues is crucial for understanding ILC biology in health and disease. Here, we showed that tonsillar ILCs expressing CD45RA lacked proliferative activity, indicative of cellular quiescence. CD62L distinguished two subsets of CD45RA+ ILCs. CD45RA+CD62L+ ILCs (CD62L+ ILCs) resembled circulating naïve ILCs because they lacked the transcriptional, metabolic, epigenetic, and cytokine production signatures of differentiated ILCs. CD45RA+CD62L- ILCs (CD62L- ILCs) were epigenetically similar to CD62L+ ILCs but showed a transcriptional, metabolic, and cytokine production signature that was more akin to differentiated ILCs. CD62L+ and CD62L- ILCs contained uni- and multipotent precursors of ILC1s/NK cells and ILC3s. Differentiation of CD62L+ and CD62L- ILCs led to metabolic reprogramming including up-regulation of genes associated with glycolysis, which was needed for their effector functions after differentiation. CD62L- ILCs with preferential differentiation capacity toward IL-22-producing ILC3s accumulated in the inflamed mucosa of patients with inflammatory bowel disease. These data suggested distinct differentiation potential of CD62L+ and CD62L- ILCs between tissue microenvironments and identified that manipulation of these cells is a possible approach to restore tissue-immune homeostasis.

KLRG1 and NKp46 discriminate subpopulations of human CD117+CRTH2- ILCs biased toward ILC2 or ILC3.

Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117+ ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46+ ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1+ ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1+ ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1+ ILCs is consistent with the broad differentiation potential of these cells.

IL-1ß, IL-23, and TGF-ß drive plasticity of human ILC2s towards IL-17-producing ILCs in nasal inflammation.

Innate lymphoid cells (ILCs) are crucial for the immune surveillance at mucosal sites. ILCs coordinate early eradication of pathogens and contribute to tissue healing and remodeling, features that are dysfunctional in patients with cystic fibrosis (CF). The mechanisms by which ILCs contribute to CF-immunopathology are ill-defined. Here, we show that group 2 ILCs (ILC2s) transdifferentiated into IL-17-secreting cells in the presence of the epithelial-derived cytokines IL-1ß, IL-23 and TGF-ß. This conversion is abrogated by IL-4 or vitamin D3. IL-17 producing ILC2s induce IL-8 secretion by epithelial cells and their presence in nasal polyps of CF patients is associated with neutrophilia. Our data suggest that ILC2s undergo transdifferentiation in CF nasal polyps in response to local cytokines, which are induced by infectious agents.

Isolation of Human Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8+ T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc.

Innate Lymphoid Cells (ILCs): Cytokine Hubs Regulating Immunity and Tissue Homeostasis.

Innate lymphoid cells (ILCs) have emerged as an expanding family of effector cells particularly enriched in the mucosal barriers. ILCs are promptly activated by stress signals and multiple epithelial- and myeloid-cell-derived cytokines. In response, ILCs rapidly secrete effector cytokines, which allow them to survey and maintain the mucosal integrity. Uncontrolled action of ILCs might contribute to tissue damage, chronic inflammation, metabolic diseases, autoimmunity, and cancer. Here we discuss the recent advances in our understanding of the cytokine network that modulate ILC immune responses: stimulating cytokines, signature cytokines secreted by ILC subsets, autocrine cytokines, and cytokines that induce cell plasticity.

[Spontaneous regression of pancreatic arteriovenous malformation under the influence of severe acute pancreatitis: a case report].

A 49-year-old man was diagnosed with severe acute pancreatitis because of pancreatic arteriovenous malformation (AVM). The pancreatic AVM spontaneously regressed during conservative treatment for severe acute pancreatitis. Transarterial embolization of an aneurysm in an artery branch flowing into the pancreatic AVM was performed using metallic coils, following amelioration of severe acute pancreatitis. The complete elimination of the pancreatic AVM was confirmed 1 year after embolization, and the patient has had no recurrence of pancreatic AVM and pancreatitis for over 6 years. Most cases of pancreatic AVMs with acute pancreatitis require surgical resection. This is a rare case in which the pancreatic AVM spontaneously regressed under the influence of acute severe pancreatitis.

Human CD5+ Innate Lymphoid Cells Are Functionally Immature and Their Development from CD34+ Progenitor Cells Is Regulated by Id2.

Innate lymphoid cells (ILCs) have emerged as a key cell type involved in surveillance and maintenance of mucosal tissues. Mouse ILCs rely on the transcriptional regulator Inhibitor of DNA-binding protein 2 (Id2) for their development. Here, we show that Id2 also drives development of human ILC because forced expression of Id2 in human thymic progenitors blocked T cell commitment, upregulated CD161 and promyelocytic leukemia zinc finger (PLZF), and maintained CD127 expression, markers that are characteristic for human ILCs. Surprisingly CD5 was also expressed on these in vitro generated ILCs. This was not an in vitro artifact because CD5 was also found on ex vivo isolated ILCs from thymus and from umbilical cord blood. CD5 was also expressed on small proportions of ILC2 and ILC3. CD5+ ILCs were functionally immature, but could further differentiate into mature CD5- cytokine-secreting ILCs. Our data show that Id2 governs human ILC development from thymic progenitor cells toward immature CD5+ ILCs.

Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.

CD31, a Valuable Marker to Identify Early and Late Stages of T Cell Differentiation in the Human Thymus.

Although CD31 expression on human thymocytes has been reported, a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study, we provide a global picture of the evolution of CD31 expression from the CD34+ hematopoietic precursor to the CD45RA+ mature CD4+ and CD8+ single-positive (SP) T cells. Using nine-color flow cytometry, we show that CD31 is highly expressed on CD34+ progenitors and stays high until the early double-positive stage (CD3-CD4+CD8α+ß-). After ß-selection, CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3highCD4+CD8+ double-positive thymocytes. However, following positive selection, CD31 expression differs dramatically between CD4+ and CD8+ lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells, including a subset of CD45RA+CD31- mature CD4+ thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably, there is a substantial subset of semimature (CD45RA-) CD4 SP thymocytes that lack CD31 expression. Moreover, FOXP3+ and ICOS+ cells are overrepresented in this CD31- subpopulation. Despite this CD31-CD45RA- subpopulation, most egress-capable mature CD45RA+ CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: ß-selection, positive selection, and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases, our results suggest a significant role for CD31 during T cell development.

IL-1ß, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs.

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1ß (IL-1ß) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

MicroRNA-146a regulates survival and maturation of human plasmacytoid dendritic cells.

During microbial infections, plasmacytoid dendritic cells (pDCs) are a main source of type I interferons α/ß (IFN-α/-ß). Nucleic acids from microbes are sensed by Toll-like receptors 7/9 (TLR7/9), which are selectively expressed in pDCs. Activated pDCs also produce proinflammatory cytokines and upregulate costimulatory molecules. Together, this equips pDCs with the ability to prime T, B, and NK cells and conventional DCs, thereby initiating adaptive immune responses. To avoid deleterious effects to the host, tight regulation of pDC activation is required. Despite data linking aberrant activation of pDCs with autoimmune diseases, little is known about mechanisms controlling pDC activation. Here, we investigated the role of microRNA-146a (miR-146a) in TLR pathway regulation in human pDCs. MiR-146a expression was induced upon TLR7/9 signaling. Furthermore, ectopic miR-146a expression effectively impaired TLR-mediated signaling in pDCs as TLR-induced nuclear factor-κB activation was reduced. This consequently diminished the production of proinflammatory cytokines and reduced pDC survival. Moreover, miR-146a-expressing pDCs had decreased ability to induce CD4(+) T-cell proliferation likely due to reduced expression levels of major histocompatibility complex class II and costimulatory molecules. Our data unravel the crucial immunomodulatory role of miR-146a in pDCs and may add to our understanding of aberrant responses in autoimmune diseases.

IL-21-stimulated human plasmacytoid dendritic cells secrete granzyme B, which impairs their capacity to induce T-cell proliferation.

Plasmacytoid dendritic cells (pDCs) play a crucial role during innate immunity by secreting bulk amounts of type I interferons (IFNs) in response to Toll-like receptor (TLR)-mediated pathogen recognition. In addition, pDCs can also contribute to adaptive immunity by activation of antigen-specific T cells. Furthermore, it is well established that pDCs contribute to the pathogenesis of autoimmune diseases, including lupus. Interleukin-21 (IL-21) is a cytokine produced by activated CD4(+) T and natural killer T (NKT) cells and has a pleiotropic role in immunity by controlling myeloid DC-, NKT-, T-, and B-cell functions. It has remained elusive whether IL-21 affects pDCs. Here we investigate the role of IL-21 in human pDC activation and function and observe that IL-21 activates signal transducer and activator of transcription 3 in line with the finding that pDCs express the IL-21 receptor. Although IL-21 did not affect TLR-induced type I IFNs, IL-6, and TNF-α nor expression of major-histocompatibility-complex class II or costimulatory molecules, IL-21 markedly increased expression of the serine protease granzyme B (GrB). We demonstrate that GrB induction was, in part, responsible for IL-21-mediated downmodulation of CD4(+) T-cell proliferation induced by TLR preactivated pDCs. Collectively, our data provide evidence that pDCs are important cells to consider when investigating the role of IL-21 in immunity or pathogenesis.

IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells.

Interleukin (IL)-21-producing CD4(+)T cells are central to humoral immunity. Deciphering the signals that induce IL-21 production in CD4(+) T cells and those triggered by IL-21 in B cells are, therefore, of importance for understanding the generation of antibody (Ab) responses. Here, we show that IL-6 increased IL-21 production by human CD4(+) T cells, particularly in those that express the transcriptional regulator B cell lymphoma (BCL)6, which is required in mice for the development of C-X-C chemokine receptor type 5 (CXCR5(+)) IL-21-producing T follicular helper (T(FH)) cells. However, retroviral overexpression of BCL6 in total human CD4(+) T cells only transiently increased CXCR5, the canonical T(FH)-defining surface marker. We show here that IL-21 was required for the induction of Ab production by IL-6. In IL-21-treated B cells, signal transducer and activator of transcription (STAT)3 was required for optimal immunoglobulin production and upregulation of PR domain containing 1 (PRDM1(+)), the master plasma cell factor. These results, therefore, demonstrate the critical importance of STAT3 activation in B cells during IL-21-driven humoral immunity and suggest that BCL6 expression, although not sufficient, may serve as a platform for the acquisition of a T(FH)-like phenotype by human CD4(+) T cells.

The transcription factor Spi-B regulates human plasmacytoid dendritic cell survival through direct induction of the antiapoptotic gene BCL2-A1.

Plasmacytoid dendritic cells (pDCs) selectively express Toll-like receptor (TLR)-7 and TLR-9, which allow them to rapidly secrete massive amounts of type I interferons after sensing nucleic acids derived from viruses or bacteria. It is not completely understood how development and function of pDCs are controlled at the transcriptional level. One of the main factors driving pDC development is the ETS factor Spi-B, but little is known about its target genes. Here we demonstrate that Spi-B is crucial for the differentiation of hematopoietic progenitor cells into pDCs by controlling survival of pDCs and its progenitors. In search for Spi-B target genes, we identified the antiapoptotic gene Bcl2-A1 as a specific and direct target gene, thereby consolidating the critical role of Spi-B in cell survival.

Functional CD47/signal regulatory protein alpha (SIRP(alpha)) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo.

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2(-/-)IL-2Rγc(-/-) mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4(+) or CD8(+) single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo.

Synergy between IL-15 and Id2 promotes the expansion of human NK progenitor cells, which can be counteracted by the E protein HEB required to drive T cell development.

The cytokine IL-15 and the inhibitor of DNA binding (Id)2, which negatively regulates the activity of basic helix-loop-helix transcription factors, have been shown to play key roles in NK cell development. Consistent with this, exogenous IL-15 added to human thymic progenitor cells stimulated their development into NK cells at the expense of T cells both in fetal thymic organ culture and in coculture with stromal cells expressing the Notch ligand Delta-like 1. Overexpression of Id2 in thymic progenitor cells stimulated NK cell development and blocked T cell development. This, in part, is attributed to inhibition of the transcriptional activity of the E protein HEB, which we show in this study is the only E protein that enhanced T cell development. Notably, Id2 increased a pool of lineage CD1a-CD5+ progenitor cells that in synergy with IL-15 furthered expansion and differentiation into NK cells. Taken together, our findings point to a dualistic function of Id2 in controlling T/NK cell lineage decisions; T cell development is impaired by Id2, most likely by sequestering HEB, whereas NK cell development is promoted by increasing a pool of CD1a-CD5+ NK cell progenitors, which together with IL-15 differentiate into mature NK cells.

Thymic stromal lymphopoietin induces early human B-cell proliferation and differentiation.

Thymic stromal lymphopoietin (TSLP) is a cytokine that binds the IL-7-receptor-alpha chain and a unique TSLP receptor (TSLPR) chain. The role of TSLP in human B-cell development has not been elucidated. We show that TSLPR transcripts are expressed most prominently in CD34(+) cells from fetal liver and BM. In general, cell surface expression of TSLPR was low, except on a subset of multilineage-commited progenitor cells. TSLP induced the tyrosine-phosphorylation of STAT5 and the proliferation of multilineage-commited progenitor cells, pro-B cells and pre-B cells. Compared with IL-7, the levels of proliferation after stimulation of the B-cell progenitors with TSLP were lower. Expression of the BCR on the cell surface of fetal cells was inversely correlated to TSLP or IL-7 responsiveness. Pre-B cells from fetal BM, but not fetal liver, were refractory to TSLP or IL-7 stimulation. When employing an in vitro B-cell differentiation culture system starting from CD34(+)CD38(-) multipotent HSC, IL-7 induced a short wave of precursor cell expansion but did not result in long-term survival of mature B cells. TSLP was capable of increasing the proportion and the absolute numbers of more mature human B cells. Overall, we provide evidence that TSLP supports human B-cell differentiation from fetal hematopoietic progenitors.

Isolation and in vitro generation of gene-manipulated human plasmacytoid and conventional dendritic cells.

Our understanding of human lymphocyte development has increased significantly over the past 20 years. In particular, our insight into human T- and B-cell development has improved (1, 2). Nonetheless, there are many gaps in our understanding, particularly regarding the early stages of development of hematopoietic progenitor cells (HPCs) into downstream lineage-biased and lineage-restricted precursors and the molecular mechanisms underlying these activities. The same holds true for our knowledge of human dendritic cell (DC) development. While the amount of data on the different subsets of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) rapidly increases in mice (3, 4), the developmental stages of different DC subsets in humans remain poorly defined (2). The relatively easy access to patient material and therefore human precursor cells that can be isolated from these tissues combined with the availability of in vitro and in vivo differentiation assays allows studies in the field of human hematopoietic development, including that of DCs. In addition, the opportunities to manipulate gene expression, by stable overexpression of a gene of interest or RNA interference-mediated knockdown, generate valuable information about the mechanisms underlying lineage commitment and differentiation.

In vivo modulation of gene expression by lentiviral transduction in "human immune system" Rag2-/- gamma c -/- mice.

Over the last two decades, several humanized mouse models have been used to experimentally analyze the function and development of the human immune system. Recent advances have lead to the establishment of new murine-human chimeric models with improved characteristics, both in terms of human engraftment efficiency and in situ multilineage human hematopoietic development. We describe here the use of newborn BALB/c Rag2(-/-)gamma(c) (-/-) mice as recipients of human hematopoietic progenitor cells to produce "human immune system" (HIS) (BALB-Rag/gamma) mice, using human fetal liver progenitors. The two major subsets of the human dendritic cell lineage, namely, BDCA2(+)CD11c(-) plasmacytoid dendritic cells and BDCA2(-)CD11c(+) conventional dendritic cells, can be found in HIS (BALB-Rag/gamma) mice. In order to manipulate the expression of genes of interest, the human hematopoietic progenitor cells can be genetically engineered ex vivo by lentiviral transduction before performing xenograft transplantation. Using this mouse model, the human immune system can be assessed for both fundamental and pre-clinical purposes.