Innate lymphoid cell differentiation and functions in intestinal homeostasis and disease

The gastrointestinal (GI) tract, which includes the small and the large intestine, is considered the largest immunological organ that plays a pivotal role in food digestion, nutrient absorption and fuel generation. Inflammatory Bowel Disease (IBD), which is broadly divided into Crohn’s disease (CD) and Ulcerative Colitis (UC), are multifactorial chronic intestinal inflammatory conditions that affect both pediatric and adult patients. Patients with IBD have a significantly increased risk of developing colorectal cancer (CRC). The immune system is undoubtably a major factor in disease pathogenesis and understanding what goes awry to cause disease is of great importance. This thesis focuses on a particular immune cell type called innate lymphoid cells (ILCs), and follows our journey to understand their role in the intestine of pediatric and adult patients with IBD as well as in CRC. Since the discovery of ILCs is fairly recent, a lot remains unknown regarding their phenotype and function at steady and disease state, particularly in humans. In this thesis we performed immunophenotypic, functional, transcriptional as well as epigenetic assays to understand many aspects of their biology, differentiation and interactions with their adaptive counterparts, T cells. In Paper I, we described the presence of CD45RA+ ILCs with naïve features in the tonsil that are transcriptionally, epigenetically and functionally distinct from the differentiated ILC subsets. We demonstrated that (CD45RA+)CD62L− ILCs were accumulated in the inflamed gut of adult patients with IBD and compared to their tonsil counterpart, these cells showed preferential differentiation towards IL22-producing ILC3s. In Paper II, we determined the landscape of innate and adaptive lymphocytes in pediatric IBD (pIBD) through single-cell RNA sequencing. First, we demonstrated that ILCs are altered in pIBD, and in line with Paper I, we show that increased frequency of CD62L− ILCs is also a feature in pediatric IBD patients. Also, we were able to uncover shared and unique transcriptional signatures between ILCs and T cells and identify with a neighbor-based computational method the most and least inflamed cells in our dataset. In Paper III, we explored the antigen-presenting properties of circulating and tissue-resident intestinal ILCs. Specifically, we were able to demonstrate that human circulating HLADR+ ILCs were able to internalize, process and present antigen to memory CD4+ T cells, and that this process is regulated by IL-1β through NF-κΒ signaling while it is suppressed by TGF-β. Additionally, via confocal microscopy we found that intestinal HLADR+ ILCs were located in close proximity to T cells, raising the possibility that the antigen presentation by ILCs could occur in vivo. Overall, the research work included in this thesis contributes to advancing our understanding of ILCs in intestinal homeostasis and disease. The characterization of novel ILC states and functions in complex intestinal diseases such as IBD and CRC could pave the way for unraveling mechanisms that drive these diseases and potentially facilitate the development of more effective therapies for those patients

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

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 SARS-CoV-2 are more commonly detected in the peripheral blood of unexposed individuals compared to SARS-CoV-2-reactive CD8+ T cells. However, large numbers of memory CD8+ T cells reside in tissues, feasibly harboring localized SARS-CoV-2-specific 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. We found that SARS-CoV-2-specific memory CD4+ T cells could be found at similar frequencies in the tonsils and peripheral blood in unexposed individuals, whereas functional SARS-CoV-2-specific memory CD8+ T cells were almost only detectable in the tonsils. Tonsillar SARS-CoV-2-specific memory CD8+ T cells displayed a follicular homing and tissue-resident memory phenotype, similar to tonsillar Epstein-Barr virus-specific memory CD8+ T cells, but were functionally less potent than other virus-specific memory CD8+ T cell responses. The presence of pre-existing tissue-resident memory CD8+ T cells in unexposed individuals could potentially enable rapid sentinel immune responses against SARS-CoV-2

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-1 beta and IL-18. IL-1 beta drives the expression of HLA-DR and co-stimulatory molecules on PB ILCs in an NF-kappa B-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-beta. 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. Murine ILCs can modulate T cell responses in MHCII-dependent manner. Here the authors show that human ILCs process and present antigens and induce T-cell responses upon exposure to IL-1-family cytokines; along with the article by Lehmann et al, this work elucidates how cytokines set context specificity of ILC-T cell crosstalk by regulating ILC antigen presentation.Funding Agencies|Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation; Swedish Research CouncilSwedish Research Council; Centre for Innovative Medicine; Jonasson center at the Royal Institute of Technology, Sweden; board of research at the Karolinska InstituteKarolinska Institutet; research committee at the Karolinska hospital; German Research Foundation (Deutsche Forschungsgemeinschaft)German Research Foundation (DFG) [RA 2986/1-1]; Swedish Cancer Foundation [130396, 160664, 170082]; Swedish Research CouncilSwedish Research Council [521-2013-2791]; Swedish Society for Medical Research [4-140/2014]; Swedish Foundation for Strategic ResearchSwedish Foundation for Strategic Research [FFL15-0120]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [4-1198/2016]; EMBO long-term fellowshipEuropean Molecular Biology Organization (EMBO) [ALTF 786-2013]; Karolinska InstitutetKarolinska Institutet; ERC-2013-ADG [341038]</p

Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice

The intestinal microbiota influences immune maturation during childhood, and is implicated in early-life allergy development. However, to directly study intestinal microbes and gut immune responses in infants is difficult. To investigate how different types of early-life gut microbiota affect immune development, we collected fecal samples from children with different allergic heredity (AH) and inoculated germ-free mice. Immune responses and microbiota composition were evaluated in the offspring of these mice. Microbial composition in the small intestine, the cecum and the colon were determined by 16S rRNA sequencing. The intestinal microbiota differed markedly between the groups of mice, but only exposure to microbiota associated with AH and known future allergy in children resulted in a T helper 17 (Th17)-signature, both systemically and in the gut mucosa in the mouse offspring. These Th17 responses could be signs of a particular microbiota and a shift in immune development, ultimately resulting in an increased risk of allergy

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

Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF

Abstract Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142− /low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142− /low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation