The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.

Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127(+) ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

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.

Vitamin D downregulates the IL-23 receptor pathway in human mucosal group 3 innate lymphoid cells.

BACKGROUND: Vitamin D deficiency is a risk factor for inflammatory bowel disease (IBD). The IL-23-driven tissue-resident group 3 innate lymphoid cells (ILC3s) play essential roles in intestinal immunity, and targeting IL-23/12 is a promising approach in IBD therapy. OBJECTIVE: We set out to define the role of 1α,25-dihydroxy vitamin D3 (1,25D) in regulating functional responses of human mucosal ILC3s to IL-23 plus IL-1ß stimulation. METHODS: Transcriptomes of sorted tonsillar ILC3s were assessed by using microarray analysis. ILC3 cytokine production, proliferation, and differentiation were determined by means of flow cytometry, ELISA, and multiplex immunoassay. Intestinal cell suspensions and ILC3s sorted from gut biopsy specimens of patients with IBD were also analyzed along with plasma 25-hydroxy vitamin D3 (25D) detection. RESULTS: ILC3s stimulated with IL-23 plus IL-1ß upregulated the vitamin D receptor and responded to 1,25D with downregulation of the IL-23 receptor pathway. Consequently, 1,25D suppressed IL-22, IL-17F, and GM-CSF production from tonsillar and gut ILC3s. In parallel, 1,25D upregulated genes linked to the IL-1ß signaling pathway, as well as the IL-1ß-inducible cytokines IL-6, IL-8, and macrophage inflammatory protein 1α/ß. The 1,25D-triggered skewing in ILC3 function was not accompanied or caused by changes in viability, proliferation, or phenotype. Finally, we confirmed low 25D plasma levels in patients with IBD with active inflammation. CONCLUSION: In light of the beneficial targeting of IL-23/12 in patients with IBD, 1,25D appears as an interesting therapeutic agent that inhibits the IL-23 receptor pathway, providing a novel mechanism for how ILC3s could be manipulated to regulate intestinal inflammation.

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.

Tissue-infiltrating neutrophils represent the main source of IL-23 in the colon of patients with IBD.

OBJECTIVE: In IBD, interleukin-23 (IL-23) and its receptor (IL-23R) are implicated in disease initiation and progression. Novel insight into which cells produce IL-23 at the site of inflammation at an early stage of IBD will promote the development of new tools for diagnosis, treatment and patient monitoring. We examined the cellular source of IL-23 in colon tissue of untreated newly diagnosed paediatric patients with IBD. DESIGN: Colon tissues from IBD and non-IBD patients were analysed by quantitative real-time PCR (qPCR), immunofluorescence confocal microscopy and flow cytometry after appropriate sample preparation. Blood samples from IBD and non-IBD patients and healthy controls were analysed using flow cytometry and qPCR. RESULTS: We discovered that tissue-infiltrating neutrophils were the main source of IL-23 in the colon of paediatric patients with IBD, while IL-23(+) human leucocyte antigen-DR(+) or IL-23(+)CD14(+) cells were scarce or non-detectable, respectively. The colonic IL-23(+) neutrophils expressed C-X-C motif (CXC)R1 and CXCR2, receptors for the CXC ligand 8 (CXCL8) chemokine family, and a corresponding CXCR1(+)CXCR2(+)IL-23(+)subpopulation of neutrophils was also identified in the blood of both patients with IBD and healthy individuals. However, CXCL8-family chemokines were only elevated in colon tissue from patients with IBD. CONCLUSIONS: This study provides the first evidence of CXCR1(+)CXCR2(+)IL-23-producing neutrophils that infiltrate and accumulate in inflamed colon tissue of patients with IBD. Thus, this novel source of IL-23 may play a key role in disease progression and will be important to take into consideration in the development of future strategies to monitor, treat and prevent IBD.

Dysregulation of Group 3 Innate Lymphoid Cells in the Pathogenesis of Inflammatory Bowel Disease.

PURPOSE OF REVIEW: Here, we review recent literature indicating a role of innate lymphoid cells in human inflammatory bowel disease with a focus on the plastic population of ILC3. RECENT FINDINGS: Many studies suggest an involvement of ILC3 in human intestinal inflammation. ILC3 present the most abundant ILC subtype in the human intestine at steady state. In IBD, this composition is skewed towards ILCs showing an ILC1 phenotype and cytokine profile. This change is likely due to the microenvironment causing skewing of the functionally plastic ILC subsets. Interactions between ILCs and other cells are important to keep homeostasis and intestinal barrier integrity. The knowledge about the involvement of ILCs in IBD is rapidly increasing, and with the help of mouse models, new pathways and functions of ILCs are continuously unraveled. In the majority of human studies, a potential role for ILCs in Crohn's disease is found. However, less data is available for a possible role in ulcerative colitis. Results from mice are obtained from diverse model systems, and more research in this field is needed to clarify and integrate the current knowledge in order to improve treatment strategies for IBD patients.

Single-cell RNA-sequencing of virus-specific cellular immune responses in chronic hepatitis B patients.

Chronic hepatitis B (CHB) is a major global health challenge. CHB can be controlled by antivirals but a therapeutic cure is lacking. CHB is characterized by limited HBV-specific T cell reactivity and functionality and expression of inhibitory receptors. The mechanisms driving these T cell phenotypes are only partially understood. Here, we created a single-cell RNA-sequencing dataset of HBV immune responses in patients to contribute to a better understanding of the dysregulated immunity. Blood samples of a well-defined cohort of 21 CHB and 10 healthy controls, including a subset of 5 matched liver biopsies, were collected. scRNA-seq data of total immune cells (55,825) plus sorted HBV-specific (1,963), non-naive (32,773) and PD1+ T cells (96,631) was generated using the 10X Genomics platform (186,123 cells) or the full-length Smart-seq2 protocol (1,069 cells). The shared transcript count matrices of single-cells serve as a valuable resource describing transcriptional changes underlying dysfunctional HBV-related T cell responses in blood and liver tissue and offers the opportunity to identify targets or biomarkers for HBV-related immune exhaustion.

Human Th17- and IgG3-associated autoimmunity induced by a translocating gut pathobiont.

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

Proteomics of immune cells from liver tumors reveals immunotherapy targets.

Elucidating the mechanisms by which immune cells become dysfunctional in tumors is critical to developing next-generation immunotherapies. We profiled proteomes of cancer tissue as well as monocyte/macrophages, CD4+ and CD8+ T cells, and NK cells isolated from tumors, liver, and blood of 48 patients with hepatocellular carcinoma. We found that tumor macrophages induce the sphingosine-1-phospate-degrading enzyme SGPL1, which dampened their inflammatory phenotype and anti-tumor function in vivo. We further discovered that the signaling scaffold protein AFAP1L2, typically only found in activated NK cells, is also upregulated in chronically stimulated CD8+ T cells in tumors. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their anti-tumor activity synergistically with PD-L1 blockade in mouse models. Our data reveal new targets for immunotherapy and provide a resource on immune cell proteomes in liver cancer.

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.

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.

Distinct Alterations in the Composition of Mucosal Innate Lymphoid Cells in Newly Diagnosed and Established Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Innate lymphoid cells [ILC] have been suggested to play a role in inflammatory bowel disease [IBD]. Here, we investigated the ILC compartment in intestinal biopsies and blood from distinct patient groups with Crohn's disease [CD] and ulcerative colitis [UC], either newly diagnosed or with disease established for at least 1 year. This approach allowed us to simultaneously investigate temporal, disease-specific, and tissue-specific changes in ILC composition in IBD. METHODS: ILC subset frequencies, phenotype, and transcription factor profile in blood and intestinal biopsies were investigated by multi-parameter flow cytometry analysis. Endoscopic disease severity was judged using the ulcerative colitis endoscopic index of severity and the simple endoscopic score for Crohn's disease. RESULTS: The frequency of NKp44+ILC3 was decreased in inflamed tissue, both in patients with CD and those with UC, already at the time of diagnosis, and correlated with disease severity. Simultaneously, the frequency of ILC1 was increased in patients with CD, whereas the frequency of ILC2 was increased in patients with UC. However, in patients with established UC or CD, both ILC1 and ILC2 were increased. In contrast to the ILC composition in inflamed tissue, ILC in non-inflamed tissue or blood were unchanged compared with non-IBD controls. Finally, in patients undergoing treatment with an anti-α4ß7 antibody the frequencies of ILC in peripheral blood remained unchanged. CONCLUSIONS: We report both shared and distinct changes in ILC composition depending on diagnosis and disease duration. The alterations in ILC composition in IBD occur selectively at inflamed sites in the gut.

Flt3 ligand expands bona fide innate lymphoid cell precursors in vivo.

A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (ILC) lineage has been recently characterized. While specific requirements of transcription factors for CHILPs development has been partially described, their ability to sense cytokines and react to peripheral inflammation remains unaddressed. Here, we found that systemic increase in Flt3L levels correlated with the expansion of Lineage (Lin)negα4ß7+ precursors in the adult murine bone marrow. Expanded Linnegα4ß7+ precursors were bona fide CHILPs as seen by their ability to differentiate into all helper ILCs subsets but cNK in vivo. Interestingly, Flt3L-expanded CHILPs transferred into lymphopenic mice preferentially reconstituted the small intestine. While we did not observe changes in serum Flt3L during DSS-induced colitis in mice or plasma from inflammatory bowel disease (IBD) patients, elevated Flt3L levels were detected in acute malaria patients. Interestingly, while CHILP numbers were stable during the course of DSS-induced colitis, they expanded following increased serum Flt3L levels in malaria-infected mice, hence suggesting a role of the Flt3L-ILC axis in malaria. Collectively, our results indicate that Flt3L expands CHILPs in the bone marrow, which might be associated with specific inflammatory conditions.

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.