In vitro immunity: an overview of immunocompetent organ-on-chip models.

Impressive advances have been made to replicate human physiology in vitro over the last few years due to the growth of the organ-on-chip (OoC) field in both industrial and academic settings. OoCs are a type of microphysiological system (MPS) that imitates functional and dynamic aspects of native human organ biology on a microfluidic device. Organoids and organotypic models, ranging in their complexity from simple single-cell to complex multi-cell type constructs, are being incorporated into OoC microfluidic devices to better mimic human physiology. OoC technology has now progressed to the stage at which it has received official recognition by the Food and Drug Administration (FDA) for use as an alternative to standard procedures in drug development, such as animal studies and traditional in vitro assays. However, an area that is still lagging behind is the incorporation of the immune system, which is a critical element required to investigate human health and disease. In this review, we summarise the progress made to integrate human immunology into various OoC systems, specifically focusing on models related to organ barriers and lymphoid organs. These models utilise microfluidic devices that are either commercially available or custom-made. This review explores the difference between the use of innate and adaptive immune cells and their role for modelling organ-specific diseases in OoCs. Immunocompetent multi-OoC models are also highlighted and the extent to which they recapitulate systemic physiology is discussed. Together, the aim of this review is to describe the current state of immune-OoCs, the limitations and the future perspectives needed to improve the field.

Reconstructed Human Skin with Hypodermis Shows Essential Role of Adipose Tissue in Skin Metabolism.

BACKGROUND: Dysregulation of skin metabolism is associated with a plethora of diseases such as psoriasis and dermatitis. Until now, reconstructed human skin (RhS) models lack the metabolic potential of native human skin, thereby limiting their relevance to study human healthy and diseased skin. We aimed to determine whether incorporation of an adipocyte-containing hypodermis into RhS improves its metabolic potential and to identify major metabolic pathways up-regulated in adipose-RhS. METHODS: Primary human keratinocytes, fibroblasts and differentiated adipose-derived stromal cells were co-cultured in a collagen/fibrin scaffold to create an adipose-RhS. The model was extensively characterized structurally in two- and three-dimensions, by cytokine secretion and RNA-sequencing for metabolic enzyme expression. RESULTS: Adipose-RhS showed increased secretion of adipokines. Both RhS and adipose-RhS expressed 29 of 35 metabolic genes expressed in ex vivo native human skin. Addition of the adipose layer resulted in up-regulation of 286 genes in the dermal-adipose fraction of which 7 were involved in phase I (CYP19A1, CYP4F22, CYP3A5, ALDH3B2, EPHX3) and phase II (SULT2B1, GPX3) metabolism. Vitamin A, D and carotenoid metabolic pathways were enriched. Additionally, pro-inflammatory (IL-1ß, IL-18, IL-23, IL-33, IFN-α2, TNF-α) and anti-inflammatory cytokine (IL-10, IL-12p70) secretion was reduced in adipose-RhS. CONCLUSIONS: Adipose-RhS mimics healthy native human skin more closely than traditional RhS since it has a less inflamed phenotype and a higher metabolic activity, indicating the contribution of adipocytes to tissue homeostasis. Therefore it is better suited to study onset of skin diseases and the effect of xenobiotics.

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function.

BACKGROUND: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited. METHODS: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed. RESULTS: FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a- MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. CONCLUSION: This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.

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.

Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas.

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II.

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, Our data suggest that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.

Biodegradation of third-generation organic antifouling biocides and their hydrolysis products in marine model systems.

The environmental fate for some selected antifouling biocides, dichlofluanid, tolylfluanid, tralopyril, and medetomidine, is relatively poorly understood with nearly all data derived from the assessment reports. Water/sediment systems and biofilms were used to determine biodegradation of the antifouling biocides. Dichlofluanid and tolylfluanid are known to hydrolyze to form DMSA (N,N-dimethyl-N'-phenylsulfamide) and DMST (N,N-Dimethyl-N'-(4-methylphenyl)sulfamide), respectively. DMSA did not show biodegradation, but it was shown to transform abiotically into N,N-dimethylsulfamide (N,N-DMS). In contrast, the structurally similar DMST did show biodegradation with a half-life of 5.78 days. The resulting transformation product of the biodegradation of DMST is also N,N-DMS. N,N-DMS accounted for the majority of the mass balance after 27 days in the water/sediment systems. Moreover, the biofilm systems also degraded both DMSA and DMST to N,N-DMS. The hydrolysis product of tralopyril, called BCCPCA (3-bromo-5-(4-chlorophenyl)-4-cyano-1 H-pyrrole-3-carboxylic acid), was not metabolized in the experiments and remained persistent. For this compound, a new log Kow of 2.47 was determined since the previously reported Kow value seemed to overestimate sediment partitioning. Medetomidine was removed from the water/sediment system, though, not significantly more than the control. However, a transformation product (medetomidine-acid) was detected in the incubation but not in the control, pointing to limited biodegradation. These results show that tolylfluanid can be rapidly removed by biodegradation in the marine environment, while dichlofluanid, tralopyril, and medetomidine remained in the system for a longer period of time. The prolonged stability of these biocides could mean that there is potential for accumulation in the environment. This potential is also there for the DMSA (dichlofluanid) and DMST (tolylfluanid) derived transformation product N,N-DMS, which was recalcitrant.

A Multi-Organ-on-Chip Approach to Investigate How Oral Exposure to Metals Can Cause Systemic Toxicity Leading to Langerhans Cell Activation in Skin.

Investigating systemic toxicity in vitro is still a huge challenge. Here, a multi-organ-on-chip approach is presented as a typical case of topical exposure of oral mucosa to metals, which are known to activate the immune system and in turn may result in skin inflammation. Reconstructed human gingiva (RHG) and reconstructed human skin containing MUTZ-3-derived Langerhans cells (MUTZ-LC) in the epidermis (RHS-LC) were incorporated into a HUMIMIC Chip3plus, connected by dynamic flow and cultured for a total period of 72 h. Three independent experiments were performed each with an intra-experiment replicate in order to assess the donor and technical variations. After an initial culture period of 24 h to achieve stable dynamic culture conditions, nickel sulfate was applied topically to RHG for 24 h, and LC activation (maturation and migration) was determined in RHS-LC after an additional 24 h incubation time. A stable dynamic culture of RHG and RHS-LC was achieved as indicated by the assessment of glucose uptake, lactate production, and lactate dehydrogenase release into the microfluidics compartment. Nickel exposure resulted in no major histological changes within RHG or RHS-LC, or cytokine release into the microfluidics compartment, but did result in an increased activation of LC as observed by the increased mRNA levels of CD1a, CD207, HLA-DR, and CD86 in the dermal compartment (hydrogel of RHS-LC (PCR)). This is the first study to describe systemic toxicity and immune cell activation in a multi-organ setting and can provide a framework for studying other organoids in the future.

Abiotic fate of tolylfluanid and dichlofluanid in natural waters.

To prevent the growth of unwanted organisms on ship hulls, antifouling paints, containing biocides such as tolylfluanid (N-[dichlor(fluor)methyl]sulfanyl-N-(dimethylsulfamoyl)-4-methylaniline) and dichlofluanid (N-(dichlorfluormethylthio)-N',N'-dimethyl-N-phenylsulfamid), are applied. There are concerns over their occurrence and fate in the marine environment due to long-term immersion in water. In the present study, the hydrolysis and photolysis of these compounds were investigated. Results showed that tolylfluanid and dichlofluanid hydrolyzed completely to their respective hydrolysis products DMST (N,N-dimethyl-N'-p-tolylsulfamide) and DMSA (N,N-dimethyl-N'-phenylsulfamide) in coastal water within 24 h. Furthermore, the transformation of tolylfluanid and dichlofluanid under natural sunlight was determined in selected marine waters (coastal water and sea water) in comparison to deionized water. The experiments revealed that photodegradation rates of DMST and DMSA in coastal water were higher than in sea water or deionized water. The indirect phototransformation of the hydrolysis products with selected reactive species (triplet state organic matter, singlet oxygen, and hydroxyl radicals) showed that DMST and DMSA mainly display triplet reactivity. The measured half-lives of the hydrolysis products in natural waters were 2.7 and 23 days, with DMST being considerably faster transformed than DMSA. However, several direct and indirect photoproducts have been newly identified and measured. DMS (N,N-dimethylsulfamide), was identified as the major phototransformation product in natural waters. It is generated by indirect photodegradation processes and exhibits potential persistence in the environment.

A molecular map of murine lymph node blood vascular endothelium at single cell resolution.

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis.

The occurrence of modern organic antifouling biocides in Danish marinas.

Antifouling biocides are known to leach out of paints and into the aquatic environment. There is currently a data gap on the occurrence of the current antifouling biocides, as legislative changes caused a change in the antifouling market. Therefore, a comprehensive monitoring study was performed across 13 Danish marinas, both waters and sediments were analyzed, including a transect and a study with seasonal resolution. Three biocides, i.e., Medetomidine, Tralopyril, and DCOIT were not detected in any of the samples. More commonly found, in 11 of the 13 marinas, were the hydrolysis products of Dichlofluanid (DMSA) and Tolylfluanid (DMST). These biocides rapidly dropped in concentration and reached background levels around 200 m from the source. The antifouling biocide Irgarol 1051 was found in all sediment samples and half of all water samples. The concentrations of Irgarol were lower than previously monitored. The decrease can likely be attributed to legislative changes and its disapproval for use since 2016.

Phenotypical Characterization of Spleen Remodeling in Murine Experimental Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum or L. donovani infection. One of the main problems related to this disease is the emergence of severe clinical forms with a lethality of 5-20%, even while under specific treatment. In humans and other species susceptible to fatal VL, such as dogs and hamsters, the disruption of splenic white pulp (WP) is accompanied by disease progression. Control of VL progression is seen in BALB/c mice, as evidenced by a mild clinical presentation and controlled parasite replication in the liver and spleen. In this study, we investigated the features involved in the morphological remodeling of splenic compartments associated with the control of VL progression to death. Methods: We evaluated cohorts of BALB/c mice after 30, 60, and 90 days of infection by L. infantum. Spleen morphology, cell population subsets and cytokine production were studied in the spleen using flow- and histo-cytometry. Results: Intraperitoneal infection with 108 promastigotes of L. infantum led to progressive increases in spleen size at 60 and 90 days after infection. Splenomegaly was the only clinical sign of disease observed. At 30 days after infection, hyperplasia in the WP and decreased numbers of plasmacytoid dendritic cells were observed. The WP hyperplasia subsided at 60 days post-infection. However, the splenomegaly remained in association with increased numbers of macrophages, B and T lymphocytes and plasma cells. An increased number of lymphoid tissue inducer (LTi) cells was observed; these were distributed around the periarteriolar lymphoid sheath in control mice and scattered throughout the red pulp in the Leishmania-infected mice. After 90 days of infection, increased IL-6 and IFN-γ production was seen in the spleen, as well as higher frequencies of follicular and plasmacytoid dendritic cells. Conclusion: The data presented herein emphasizes the potential role of spleen remodeling in the control of severe forms of VL and highlights features potentially involved in this process.

Stromal cells and immune cells involved in formation of lymph nodes and their niches.

Secondary lymphoid organs are critical for efficient interaction between innate antigen presenting cells and adaptive lymphocytes in order to start adaptive immune responses. The efficiency by which these cellular subsets meet is highly increased by the orchestrating role of stromal cells within the secondary lymphoid organs. These cells provide cytokines, chemokines and cell surface receptors necessary for survival and guided migration. This increases the likelihood that antigen specific adaptive immune responses occur. Already from initial formation of secondary lymphoid organs, the interaction of immune cells with stromal cells is crucial and this interaction continues during immune activation. With the recent discovery of many stromal cell subsets new immune micro-niches with specific functions that are orchestrated by stromal cells will be discovered. Here, we will discuss how the development of lymph nodes as well as their specific niches is guided by the interaction of immune cells and stromal cells.

Lymph Node Stromal Cells Generate Antigen-Specific Regulatory T Cells and Control Autoreactive T and B Cell Responses.

Within lymph nodes (LNs), T follicular helper (TFH) cells help B cells to produce antibodies, which can either be protective or autoreactive. Here, we demonstrate that murine LN stromal cells (LNSCs) suppress the formation of autoreactive TFH cells in an antigen-specific manner, thereby significantly reducing germinal center B cell responses directed against the same self-antigen. Mechanistically, LNSCs express and present self-antigens in major histocompatibility complex (MHC) class II, leading to the conversion of naive CD4+ T cells into T regulatory (TREG) cells in an interleukin-2 (IL-2)-dependent manner. Upon blockade of TREG cells, using neutralizing IL-2 antibodies, autoreactive TFH cells are allowed to develop. We conclude that the continuous presentation of self-antigens by LNSCs is critical to generate antigen-specific TREG cells, thereby repressing the formation of TFH cells and germinal center B cell responses. Our findings uncover the ability of LNSCs to suppress the early activation of autoreactive immune cells and maintain peripheral tolerance.

Perivascular Adipose Tissue Controls Insulin-Stimulated Perfusion, Mitochondrial Protein Expression, and Glucose Uptake in Muscle Through Adipomuscular Arterioles.

Insulin-mediated microvascular recruitment (IMVR) regulates delivery of insulin and glucose to insulin-sensitive tissues. We have previously proposed that perivascular adipose tissue (PVAT) controls vascular function through outside-to-inside communication and through vessel-to-vessel, or "vasocrine," signaling. However, direct experimental evidence supporting a role of local PVAT in regulating IMVR and insulin sensitivity in vivo is lacking. Here, we studied muscles with and without PVAT in mice using combined contrast-enhanced ultrasonography and intravital microscopy to measure IMVR and gracilis artery diameter at baseline and during the hyperinsulinemic-euglycemic clamp. We show, using microsurgical removal of PVAT from the muscle microcirculation, that local PVAT depots regulate insulin-stimulated muscle perfusion and glucose uptake in vivo. We discovered direct microvascular connections between PVAT and the distal muscle microcirculation, or adipomuscular arterioles, the removal of which abolished IMVR. Local removal of intramuscular PVAT altered protein clusters in the connected muscle, including upregulation of a cluster featuring Hsp90ab1 and Hsp70 and downregulation of a cluster of mitochondrial protein components of complexes III, IV, and V. These data highlight the importance of PVAT in vascular and metabolic physiology and are likely relevant for obesity and diabetes.

Poor perfusion of the microvasculature in peritoneal metastases of ovarian cancer.

Most women with epithelial ovarian cancer (EOC) suffer from peritoneal carcinomatosis upon first clinical presentation. Extensive peritoneal carcinomatosis has a poor prognosis and its pathophysiology is not well understood. Although treatment with systemic intravenous chemotherapy is often initially successful, peritoneal recurrences occur regularly. We hypothesized that insufficient or poorly-perfused microvasculature may impair the therapeutic efficacy of systemic intravenous chemotherapy but may also limit expansive and invasive growth characteristic of peritoneal EOC metastases. In 23 patients with advanced EOC or suspicion thereof, we determined the angioarchitecture and perfusion of the microvasculature in peritoneum and in peritoneal metastases using incident dark field (IDF) imaging. Additionally, we performed immunohistochemical analysis and 3-dimensional (3D) whole tumor imaging using light sheet fluorescence microscopy of IDF-imaged tissue sites. In all metastases, microvasculature was present but the angioarchitecture was chaotic and the vessel density and perfusion of vessels was significantly lower than in unaffected peritoneum. Immunohistochemical analysis showed expression of vascular endothelial growth factor and hypoxia inducible factor 1α, and 3D imaging demonstrated vascular continuity between metastases and the vascular network of the peritoneum beneath the elastic lamina of the peritoneum. We conclude that perfusion of the microvasculature within metastases is limited, which may cause hypoxia, affect the behavior of EOC metastases on the peritoneum and limit the response of EOC metastases to systemic treatment.

The Role of Endothelial Cells and TNF-Receptor Superfamily Members in Lymphoid Organogenesis and Function During Health and Inflammation.

Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation.

Setmelanotide, a Novel, Selective Melanocortin Receptor-4 Agonist Exerts Anti-inflammatory Actions in Astrocytes and Promotes an Anti-inflammatory Macrophage Phenotype.

To date, available treatment strategies for multiple sclerosis (MS) are ineffective in preventing or reversing progressive neurologic deterioration, creating a high, and unmet medical need. One potential way to fight MS may be by limiting the detrimental effects of reactive astrocytes, a key pathological hallmark for disease progression. One class of compounds that may exert beneficial effects via astrocytes are melanocortin receptor (MCR) agonists. Among the MCR, MC4R is most abundantly expressed in the CNS and several rodent studies have described that MC4R is-besides neurons-expressed by astrocytes. Activation of MC4R in astrocytes has shown to have potent anti-inflammatory as well as neuroprotective effects in vitro, suggesting that this could be a potential target to ameliorate ongoing inflammation, and neurodegeneration in MS. In this study, we set out to investigate human MC4R expression and analyze its downstream effects. We identified MC4R mRNA and protein to be expressed on astrocytes and observed increased astrocytic MC4R expression in active MS lesions. Furthermore, we show that the novel, highly selective MC4R agonist setmelanotide ameliorates the reactive phenotype in astrocytes in vitro and markedly induced interleukin-6 and -11 production, possibly through enhanced cAMP response element-binding protein (CREB) phosphorylation. Notably, stimulation of human macrophages with medium from astrocytes that were exposed to setmelanotide, skewed macrophages toward an anti-inflammatory phenotype. Taken together, these findings suggest that targeting MC4R on astrocytes might be a novel therapeutic strategy to halt inflammation-associated neurodegeneration in MS.

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.