Analysis of human lung mast cells by single cell RNA sequencing.

Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MCT and MCTC, where MCT express the mast cell protease tryptase and MCTC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MCT subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG. However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak.

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.

Identification of resident memory CD8+ T cells with functional specificity for SARS-CoV-2 in unexposed oropharyngeal lymphoid tissue.

Cross-reactive CD4+ T cells that recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more commonly detected in the peripheral blood of unexposed individuals compared with SARS-CoV-2­reactive CD8+ T cells. However, large numbers of memory CD8+ T cells reside in tissues, feasibly harboring localized SARS-CoV-2­specific immune responses. To test this idea, we performed a comprehensive functional and phenotypic analysis of virus-specific T cells in tonsils, a major lymphoid tissue site in the upper respiratory tract, and matched peripheral blood samples obtained from children and adults before the emergence of COVID-19 (coronavirus disease 2019). We found that SARS-CoV-2­specific memory CD4+ T cells could be found at similar frequencies in the tonsils and peripheral blood in unexposed individuals, whereas functional SARS-CoV-2­specific memory CD8+ T cells were almost only detectable in the tonsils. Tonsillar SARS-CoV-2­specific memory CD8+ T cells displayed a follicular homing and tissue-resident memory phenotype, similar to tonsillar Epstein-Barr virus­specific memory CD8+ T cells, but were functionally less potent than other virus-specific memory CD8+ T cell responses. The presence of preexisting tissue-resident memory CD8+ T cells in unexposed individuals could potentially enable rapid sentinel immune responses against SARS-CoV-2.

Tissue-specific transcriptional imprinting and heterogeneity in human innate lymphoid cells revealed by full-length single-cell RNA-sequencing.

The impact of the microenvironment on innate lymphoid cell (ILC)-mediated immunity in humans remains largely unknown. Here we used full-length Smart-seq2 single-cell RNA-sequencing to unravel tissue-specific transcriptional profiles and heterogeneity of CD127+ ILCs across four human tissues. Correlation analysis identified gene modules characterizing the migratory properties of tonsil and blood ILCs, and signatures of tissue-residency, activation and modified metabolism in colon and lung ILCs. Trajectory analysis revealed potential differentiation pathways from circulating and tissue-resident naïve ILCs to a spectrum of mature ILC subsets. In the lung we identified both CRTH2+ and CRTH2- ILC2 with lung-specific signatures, which could be recapitulated by alarmin-exposure of circulating ILC2. Finally, we describe unique TCR-V(D)J-rearrangement patterns of blood ILC1-like cells, revealing a subset of potentially immature ILCs with TCR-δ rearrangement. Our study provides a useful resource for in-depth understanding of ILC-mediated immunity in humans, with implications for disease.

Crohn's Disease Is Associated With Activation of Circulating Innate Lymphoid Cells.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with disturbed mucosal innate lymphoid cell (ILC) composition, which is correlated to the degree of intestinal inflammation. However, it remains unclear whether circulating ILCs are dysregulated in patients with IBD. METHODS: Blood samples from 53 patients with Crohn's disease (CD), 43 patients with ulcerative colitis (UC), and 45 healthy control subjects (HC) were analyzed by flow cytometry for markers of ILC subsets (ILC1, ILC2, and ILC precursors [ILCp]) and selected IBD-relevant proteins, as predicted by previous genome-wide association studies. A dimensionality reduction approach to analyzing the data was used to characterize circulating ILCs. RESULTS: The frequency of ILCp expressing the ILC3 activation markers NKp44 and CD56 was increased in CD versus HC and UC (NKp44) or in CD versus HC (CD56), whereas the CD45RA+ ILCp were reduced in CD versus UC. Furthermore, the activation marker HLA-DR was increased on ILC1 and ILC2 in CD versus HC. Interestingly, the IBD-related protein SLAMF1 was upregulated on ILC2 from both CD and UC samples as compared with HC samples. In active CD, SLAMF1+ ILC2 frequency was negatively correlated with disease severity (Harvey-Bradshaw index). The characterization of SLAMF1+ ILC2 revealed a higher expression of the ILC2 markers CRTH2, CD161, and GATA3 as compared with SLAMF1- ILC2. CONCLUSIONS: In line with the systemic nature of CD inflammation, our findings point toward the activation of ILCs in the blood of patients with CD. Furthermore, in active CD, circulating SLAMF1+ ILC2 are increased in patients with less active disease, introducing SLAMF1+ ILC2 as interesting therapeutic targets deserving further exploration.

Cytokines regulate the antigen-presenting characteristics of human circulating and tissue-resident intestinal ILCs.

ILCs and T helper cells have been shown to exert bi-directional regulation in mice. However, how crosstalk between ILCs and CD4+ T cells influences immune function in humans is unknown. Here we show that human intestinal ILCs co-localize with T cells in healthy and colorectal cancer tissue and display elevated HLA-DR expression in tumor and tumor-adjacent areas. Although mostly lacking co-stimulatory molecules ex vivo, intestinal and peripheral blood (PB) ILCs acquire antigen-presenting characteristics triggered by inflammasome-associated cytokines IL-1ß and IL-18. IL-1ß drives the expression of HLA-DR and co-stimulatory molecules on PB ILCs in an NF-κB-dependent manner, priming them as efficient inducers of cytomegalovirus-specific memory CD4+ T-cell responses. This effect is strongly inhibited by the anti-inflammatory cytokine TGF-ß. Our results suggest that circulating and tissue-resident ILCs have the intrinsic capacity to respond to the immediate cytokine milieu and regulate local CD4+ T-cell responses, with potential implications for anti-tumor immunity and inflammation.

Suppression of Aiolos and Ikaros expression by lenalidomide reduces human ILC3-ILC1/NK cell transdifferentiation.

The Ikaros family of transcription factors (TFs) are important regulators of lymphocyte function. However, their roles in human innate lymphoid cell (ILC) function remain unclear. Here, we found that Ikaros (IKZF1) is expressed by all ILC subsets, including NK cells, in blood, tonsil, and gut, while Helios (IKZF2) is preferentially expressed by ILC3 in tonsil and gut. Aiolos (IKZF3) followed the expression pattern of T-bet and Eomes, being predominantly expressed by ILC1 and NK cells. Differentiation of IFN-γ-producing ILC1 and NK cells from ILC3 by IL-1ß plus IL-12-stimulation was associated with upregulation of T-bet and Aiolos. Selective degradation of Aiolos and Ikaros by lenalidomide suppressed ILC1 and NK cell differentiation and expression of ILC1 and NK cell-related transcripts (LEF1, PRF1, GRZB, CD244, NCR3, and IRF8). In line with reduced ILC1/NK cell differentiation, we observed an increase in the expression of the ILC3-related TF Helios, as well as ILC3 transcripts (TNFSF13B, IL22, NRP1, and RORC) and in the frequency of IL-22 producing ILC3 in cultures with IL-1ß and IL-23. These data suggest that suppression of Aiolos and Ikaros expression inhibits ILC1 and NK cell differentiation while ILC3 function is maintained. Hence, our results open up for new possibilities in targeting Ikaros family TFs for modulation of type 1/3 immunity in inflammation and cancer.

Cytokine-induced endogenous production of prostaglandin D2 is essential for human group 2 innate lymphoid cell activation.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and maintenance of type 2 immune responses. The prostaglandin (PG) D2-chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) receptor axis potently induces cytokine production and ILC2 migration. OBJECTIVE: We set out to examine PG production in human ILC2s and the implications of such endogenous production on ILC2 function. METHODS: The effects of the COX-1/2 inhibitor flurbiprofen, the hematopoietic prostaglandin D2 synthase (HPGDS) inhibitor KMN698, and the CRTH2 antagonist CAY10471 on human ILC2s were determined by assessing receptor and transcription factor expression, cytokine production, and gene expression with flow cytometry, ELISA, and quantitative RT-PCR, respectively. Concentrations of lipid mediators were measured by using liquid chromatography-tandem mass spectrometry and ELISA. RESULTS: We show that ILC2s constitutively express HPGDS and upregulate COX-2 upon IL-2, IL-25, and IL-33 plus thymic stromal lymphopoietin stimulation. Consequently, PGD2 and its metabolites can be detected in ILC2 supernatants. We reveal that endogenously produced PGD2 is essential in cytokine-induced ILC2 activation because blocking of the COX-1/2 or HPGDS enzymes or the CRTH2 receptor abolishes ILC2 responses. CONCLUSION: PGD2 produced by ILC2s is, in a paracrine/autocrine manner, essential in cytokine-induced ILC2 activation. Hence we provide the detailed mechanism behind how CRTH2 antagonists represent promising therapeutic tools for allergic diseases by controlling ILC2 function.

An Optimized Protocol for the Isolation and Functional Analysis of Human Lung Mast Cells.

Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Prostaglandin E2 suppresses human group 2 innate lymphoid cell function.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are involved in the initial phase of type 2 inflammation and can amplify allergic immune responses by orchestrating other type 2 immune cells. Prostaglandin (PG) E2 is a bioactive lipid that plays protective roles in the lung, particularly during allergic inflammation. OBJECTIVE: We set out to investigate how PGE2 regulates human ILC2 function. METHODS: The effects of PGE2 on human ILC2 proliferation and intracellular cytokine and transcription factor expression were assessed by means of flow cytometry. Cytokine production was measured by using ELISA, and real-time quantitative PCR was performed to detect PGE2 receptor expression. RESULTS: PGE2 inhibited GATA-3 expression, as well as production of the type 2 cytokines IL-5 and IL-13, from human tonsillar and blood ILC2s in response to stimulation with a combination of IL-25, IL-33, thymic stromal lymphopoietin, and IL-2. Furthermore, PGE2 downregulated the expression of IL-2 receptor α (CD25). In line with this observation, PGE2 decreased ILC2 proliferation. These effects were mediated by the combined action of E-type prostanoid receptor (EP) 2 and EP4 receptors, which were specifically expressed on ILC2s. CONCLUSION: Our findings reveal that PGE2 limits ILC2 activation and propose that selective EP2 and EP4 receptor agonists might serve as a promising therapeutic approach in treating allergic diseases by suppressing ILC2 function.

ILC-poiesis: Making Tissue ILCs from Blood.

The development of human innate lymphoid cells (ILCs) remains poorly characterized. In a recent issue of Cell, Lim et al. show that human peripheral-blood CD117+ ILCs harbor ILC precursors (ILCPs) derived from hematopoietic stem cells. Peripheral-blood ILCPs can generate all ILC subsets in vivo and in vitro.

Functional Analyses of the Crohn's Disease Risk Gene LACC1.

BACKGROUND: Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression. METHODS: We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function. RESULTS: FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems. CONCLUSION: FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

Downregulation of MicroRNA-21 in Colonic CD3+ T Cells in UC Remission.

BACKGROUND: In active inflammatory bowel disease (IBD), microRNA expression profiling consistently features disease-specific signatures, and microRNA-21 (miR-21) has been shown to be upregulated in the inflamed colon of patients with active ulcerative colitis (UC). However, the cellular sources of miR-21 expression in IBD tissues have not yet been identified. We sought to determine the expression levels of miR-21 and one of its downstream target genes, programmed cell death 4 (PDCD4), in CD3 T cells isolated from the colonic mucosa of patients with active IBD, inactive IBD, and non-IBD controls. METHODS: Colonic biopsies were treated with collagenase V. CD3 T cells were isolated using MACS CD3 positive selection. Total RNA was converted to cDNA. Real-time PCR reactions were performed with PCR primers for miR-21, SNORD95, PDCD4, and GAPDH. RESULTS: The expression of miR-21 was statistically significantly downregulated in CD3 T cells from patients with UC in remission as compared to active disease (P = 0.0193). miR-21 negatively regulates PDCD4 expression. As predicted, the mRNA level of PCDC4 in CD3 T cells was upregulated in UC and Crohn's disease in remission as compared to active disease (UC active versus UC remission: P = 0.0008, Crohn's disease active versus Crohn's disease remission: P = 0.0215) and in patients with UC in remission as compared to healthy controls (P = 0.0226). CONCLUSIONS: Although miR-21 expression is downregulated, PDCD4 is upregulated in CD3 T cells during the remission phase of UC. Our results indicate that miR-21 and related pathways in colonic T cells may play a role in limiting pathogenic T-cell responses and may constitute future target candidates to induce remission in UC.