Search

Sarcoidosis is a systemic inflammatory disease mainly affecting the lungs. The hallmark of sarcoidosis are granulomas that are surrounded by activated T cells, likely targeting the disease-inducing antigen. IFNγ-producing Th1 and Th17.1 T cells are elevated in sarcoidosis and associate with disease progression. Monocytes and dendritic cells (DCs) are antigen-presenting cells (APCs) and required for T cell activation. Several subsets of monocytes and DCs with different functions were identified in sarcoidosis. However, to what extent different monocyte and DC subsets can support activation and skewing of T cells in sarcoidosis is still unclear. In this study, we performed a transcriptional and functional side-by-side comparison of sorted monocytes and DCs from matched blood and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients. Transcriptomic analysis of all subsets showed upregulation of genes related to T cell activation and antigen presentation in DCs compared with monocytes. Allogeneic T cell proliferation was higher after coculture with monocytes and DCs from blood compared with BAL and DCs induced more T cell proliferation compared with monocytes. After coculture, proliferating T cells showed high expression of the transcription factor Tbet and IFNγ production. We also identified Tbet and RORγt coexpressing T cells that mainly produced IFNγ. Our data show that DCs rather than monocytes from sarcoidosis patients have the ability to activate and polarize T cells towards Th1 and Th17.1 cells. This study provides a useful in vitro tool to better understand the contribution of monocytes and DCs to T cell activation and immunopathology in sarcoidosis.

Löfgren’s syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3–restricted manner. Using ELISPOT analysis, a greater number of IFN-γ– and IL-2–secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

Background: Immunohistochemical analysis of granule-associated proteases has revealed that human lung mast cells constitute a heterogeneous population of cells, with distinct subpopulations identified. However, a systematic and comprehensive analysis of cell-surface markers to study human lung mast cell heterogeneity has yet to be performed. Methods: Human lung mast cells were obtained from lung lobectomies, and the expression of 332 cell-surface markers was analyzed using flow cytometry and the LEGENDScreen kit. Markers that exhibited high variance were selected for additional analyses to reveal whether they were correlated and whether discrete mast cell subpopulations were discernable. Results: We identified the expression of 102 surface markers on human lung mast cells. Several markers showed high continuous variation in expression within the mast cell population. Six of these markers were correlated: SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR. The expression of these markers was also correlated with the size and granularity of mast cells. However, no marker produced an expression profile consistent with a bi- or multimodal distribution. Conclusions: LEGENDScreen analysis identified more than 100 cell-surface markers on mast cells, including 23 that, to the best of our knowledge, have not been previously described on human mast cells. Several of the newly described markers are known to be involved in sensing the microenvironment, and their identification can shed new light on mast cell functions. The exhaustive expression profiling of the 332 surface markers failed to detect distinct mast cell subpopulations. Instead, we demonstrate the continuous nature of human lung mast cell heterogeneity.

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.

Background Group 2 innate lymphoid cells (ILC2s) play critical roles in induction and exacerbation of allergic airway inflammation. Thus clarification of the mechanisms that underlie regulation of ILC2 activation has received significant attention. Although innate lymphoid cells are divided into 3 major subsets that mirror helper effector T-cell subsets, counterpart subsets of regulatory T cells have not been well characterized. Objective We sought to determine the factors that induce regulatory innate lymphoid cells (ILCregs). Methods IL-10+ ILCregs induced from ILC2s by using retinoic acid (RA) were analyzed with RNA-sequencing and flow cytometry. ILCregs were evaluated in human nasal tissue from healthy subjects and patients with chronic rhinosinusitis with nasal polyps and lung tissue from house dust mite– or saline-treated mice. Results RA induced IL-10 secretion by human ILC2s but not type 2 cytokines. IL-10+ ILCregs, which were converted from ILC2s by means of RA stimulation, expressed a regulatory T cell–like signature with expression of IL-10, cytotoxic T lymphocyte–associated protein 4, and CD25, with downregulated effector type 2–related markers, such as chemoattractant receptor–homologous molecule on TH2 cells and ST2, and suppressed activation of CD4+ T cells and ILC2s. ILCregs were rarely detected in human nasal tissue from healthy subjects or lung tissue from saline-treated mice, but numbers were increased in nasal tissue from patients with chronic rhinosinusitis with nasal polyps and in lung tissue from house dust mite–treated mice. Enzymes for RA synthesis were upregulated in airway epithelial cells during type 2 inflammation in vivo and by IL-13 in vitro. Conclusion We have identified a unique immune regulatory and anti-inflammatory pathway by which RA converts ILC2s to ILCregs. Interactions between airway epithelial cells and ILC2s play an important roles in the generation of ILCregs.

Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, Fc + RI, 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. Funding Agencies|Swedish Research Council-Medicine and Health; Heart-Lung Foundation; Ollie and Elof Ericssons foundation; Ellen; Walter and Lennart Hesselmans foundation; European Cooperation in Science and Technology COST Action, Developmental Origins of Chronic Lung Disease [BM1201]; ChAMP (Centre for Allergy Research Highlights Asthma Markers of Phenotype) consortium - Swedish Foundation for Strategic Research; Karolinska Institutet; AstraZeneca & Science for Life Laboratory Joint Research Collaboration; Vardal Foundation

Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and maintenance of type 2 immune responses. The prostaglandin (PG) DWe set out to examine PG production in human ILC2s and the implications of such endogenous production on ILC2 function.The effects of the COX-1/2 inhibitor flurbiprofen, the hematopoietic prostaglandin DWe show that ILC2s constitutively express HPGDS and upregulate COX-2 upon IL-2, IL-25, and IL-33 plus thymic stromal lymphopoietin stimulation. Consequently, PGDPGD

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 he ...

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., Graphical abstract