Immune microniches shape intestinal Treg function

The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2 . Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in infammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identifed3,4 , less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5–7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and infammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria— but not embedded lymphoid aggregates—is the key microniche that supports efector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing infammation breaks down compartmentalization, leading to dominance of CD103+ SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor–ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies

Immune microniches shape intestinal Treg function.

Acknowledgements: The authors thank the Kennedy Institute of Rheumatology (KIR) Flow Cytometry Facility and the manager, J. Webber, for help with flow cytometry and FACS; the KIR Biomedical Services Unit, especially L. Barker for help with animal care and husbandry; the KIR microscopy facility and manager C. Lagerholm; I. Parisi, B. Stott and R. Cook for tissue processing and staining; A. Lee and M. Attar (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the generation and initial processing of sequencing data; S. van Dongen and P. V. Mazin and the Teichmann laboratory for discussion and support with scripts. We acknowledge the generous support of the Kennedy Trust for Rheumatology Research, IDRM and Carl Zeiss GMBH for the microscopy facilities (Zeiss 980) used in this research. We acknowledge the generous support of the Kennedy Trust for Rheumatology Research and a Wellcome Trust Multi-User Equipment Grant 202911/Z/16/Z for the microscope purchase (Zeiss 880 multiphoton) and facilities used in this research. Experimental design and summary diagrams were created with BioRender.com. Y.G. was funded by a Wellcome Trust Clinical Research Fellowship (CRTF), grant reference 201224/Z/16/Z. RB-C Grant 315307, Forskerprosjekt 2020, Researcher Project/International Mobility Grant from the Research Council of Norway and travel grant from the Per Brandtzæg’s Fund for Research in Mucosal Immunology. E.E.T. was supported by Wellcome Trust (095688/Z/11/Z and 212240/Z/18/Z, awarded to F.P.), Nuffield Department of Medicine, and MRC core grant reference MC_UU_00008. F.P. was supported by Wellcome Trust (095688/Z/11/Z and 212240/Z/18/Z). This research was funded in whole, or in part, by the Wellcome Trust 212240/Z/18/Z. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identified3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103+SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies

Induction of Metabolic Quiescence Defines the Transitional to Follicular B Cell Switch

Transitional B cells must actively undergo selection for self-tolerance before maturing into their resting follicular B cell successors. We found that metabolic quiescence was acquired at the follicular B cell stage in both humans and mice. In follicular B cells, the expression of genes involved in ribosome biogenesis, aerobic respiration, and mammalian target of rapamycin complex 1 (mTORC1) signaling was reduced when compared to that in transitional B cells. Functional metabolism studies, profiling of whole-cell metabolites, and analysis of cell surface proteins in human B cells suggested that this transition was also associated with increased extracellular adenosine salvage. Follicular B cells increased the abundance of the cell surface ectonucleotidase CD73, which coincided with adenosine 5′-monophosphate–activated protein kinase (AMPK) activation. Differentiation to the follicular B cell stage in vitro correlated with surface acquisition of CD73 on human transitional B cells and was augmented with the AMPK agonist, AICAR. Last, individuals with gain-of-function PIK3CD (PI3Kδ) mutations and increased pS6 activation exhibited a near absence of circulating follicular B cells. Together, our data suggest that mTORC1 attenuation may be necessary for human follicular B cell development. These data identify a distinct metabolic switch during human B cell development at the transitional to follicular stages, which is characterized by an induction of extracellular adenosine salvage, AMPK activation, and the acquisition of metabolic quiescence

Induction of Metabolic Quiescence Defines the Transitional to Follicular B Cell Switch

Transitional B cells must actively undergo selection for self-tolerance before maturing into their resting follicular B cell successors. We found that metabolic quiescence was acquired at the follicular B cell stage in both humans and mice. In follicular B cells, the expression of genes involved in ribosome biogenesis, aerobic respiration, and mammalian target of rapamycin complex 1 (mTORC1) signaling was reduced when compared to that in transitional B cells. Functional metabolism studies, profiling of whole-cell metabolites, and analysis of cell surface proteins in human B cells suggested that this transition was also associated with increased extracellular adenosine salvage. Follicular B cells increased the abundance of the cell surface ectonucleotidase CD73, which coincided with adenosine 5′-monophosphate–activated protein kinase (AMPK) activation. Differentiation to the follicular B cell stage in vitro correlated with surface acquisition of CD73 on human transitional B cells and was augmented with the AMPK agonist, AICAR. Last, individuals with gain-of-function PIK3CD (PI3Kδ) mutations and increased pS6 activation exhibited a near absence of circulating follicular B cells. Together, our data suggest that mTORC1 attenuation may be necessary for human follicular B cell development. These data identify a distinct metabolic switch during human B cell development at the transitional to follicular stages, which is characterized by an induction of extracellular adenosine salvage, AMPK activation, and the acquisition of metabolic quiescence

Extrafollicular IgD−CD27−CXCR5−CD11c− DN3 B cells infiltrate inflamed tissues in autoimmune fibrosis and in severe COVID-19

Summary: Although therapeutic B cell depletion dramatically resolves inflammation in many diseases in which antibodies appear not to play a central role, distinct extrafollicular pathogenic B cell subsets that accumulate in disease lesions have hitherto not been identified. The circulating immunoglobulin D (IgD)−CD27−CXCR5−CD11c+ DN2 B cell subset has been previously studied in some autoimmune diseases. A distinct IgD−CD27−CXCR5−CD11c− DN3 B cell subset accumulates in the blood both in IgG4-related disease, an autoimmune disease in which inflammation and fibrosis can be reversed by B cell depletion, and in severe COVID-19. These DN3 B cells prominently accumulate in the end organs of IgG4-related disease and in lung lesions in COVID-19, and double-negative B cells prominently cluster with CD4+ T cells in these lesions. Extrafollicular DN3 B cells may participate in tissue inflammation and fibrosis in autoimmune fibrotic diseases, as well as in COVID-19