The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure of this balance leads to ...conditions such as inflammatory bowel diseases, food allergies and invasive gastrointestinal infections
. Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (T
) cells and FOXP3
regulatory T (pT
) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively
. The gut-draining lymph nodes (gLNs) are key sites for orchestrating adaptive immunity to luminal perturbations
. However, it is unclear how they simultaneously support tolerogenic and inflammatory reactions. Here we show that gLNs are immunologically specific to the functional gut segment that they drain. Stromal and dendritic cell gene signatures and polarization of T cells against the same luminal antigen differ between gLNs, with the proximal small intestine-draining gLNs preferentially giving rise to tolerogenic responses and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted the targeting of distal gLNs for vaccination and the maintenance of duodenal pT
cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, with effects on both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage antigen targeting to specific gut segments for therapeutic immune modulation.
Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content
, regulating both physiological intestinal functions such as nutrient absorption and motility
..., and brain-wired feeding behaviour
. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology
. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.
Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria and epithelium, ensuring appropriate balance between tolerance and resistance. By combining ...mouse genetics with single-cell and chromatin analyses, we uncovered the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (T
) CD4
T cells from mLN, lamina propria and intestinal epithelium segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4 programming and acquisition of an intraepithelial profile. T
cell fate mapping coupled with RNA sequencing and assay for transposase-accessible chromatin followed by sequencing revealed that the T
cell program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4-lineage-defining transcription factor ThPOK results in premature acquisition of an intraepithelial lymphocyte profile by mLN T
cells, partially recapitulating epithelium imprinting. Thus, coordinated replacement of the circulating lymphocyte program with site-specific transcriptional and chromatin changes is necessary for tissue imprinting.
The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. ...However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4+CD8αα+ IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4+ T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4+CD8αα+ IEL differentiation. However, TCR ablation on differentiated CD4+CD8αα+ IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4+CD8αα+ IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4+ T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.
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•Intraepithelial CD4+CD8αα+ IEL differentiation is accompanied by clonal restriction•TCR and local antigen presentation are required for CD4+CD8αα+ IEL differentiation•TCR engagement is dispensable for CD4+CD8αα+ IEL maintenance
Immune cell adaptation to the harsh intestinal environment is key for healthy gut maintenance. Bilate et al. showed that a subpopulation of intraepithelial lymphocytes is clonally expanded and depends on antigen receptor signaling for their differentiation. Once differentiated, these cells can thrive without antigen, highlighting the ability of these cells to maintain function under different contexts.
Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in ...detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3
T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (T
) cells. Ectopic expression of Foxp3 in T
cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by T
cells as a potential regulator of GC lifetimes.
The commensal microbiota has a high impact on health and disease by modulating the development and homeostasis of host immune system. Immune cells are involved in virtually every aspect of the wound ...repair process; however, the impact of commensal microbiota on skin wound healing is largely unknown. In this study, we evaluated the influence of commensal microbiota on tissue repair of excisional skin wounds by using germ-free (GF) Swiss mice. We observed that macroscopic wound closure rate is accelerated in the absence of commensal microbiota. Accordantly, histologically assessed wound epithelization was accelerated in GF in comparison with conventional (CV) Swiss mice. The wounds of GF mice presented a significant decrease in neutrophil accumulation and an increase in mast cell and macrophage infiltration into wounds. Interestingly, alternatively activated healing macrophage-related genes were highly expressed in the wound tissue of GF mice. Moreover, levels of the anti-inflammatory cytokine IL-10, the angiogenic growth factor VEGF and angiogenesis were higher in the wound tissue of those mice. Conversely, scarring and levels of the profibrogenic factor TGF-β1 were greatly reduced in GF mice wounded skin when compared with CV mice. Of note, conventionalization of GF mice with CV microbiota restored wound closure rate, neutrophil and macrophage accumulation, cytokine production, and scarring to the same extent as CV mice. Overall, our findings suggest that, in the absence of any contact with microbiota, skin wound healing is accelerated and scarless, partially because of reduced accumulation of neutrophils, increased accumulation of alternatively activated healing macrophages, and better angiogenesis at wound sites.
Germinal centres, the structures in which B cells evolve to produce antibodies with high affinity for various antigens, usually form transiently in lymphoid organs in response to infection or ...immunization. In lymphoid organs associated with the gut, however, germinal centres are chronically present. These gut-associated germinal centres can support targeted antibody responses to gut infections and immunization
. But whether B cell selection and antibody affinity maturation take place in the face of the chronic and diverse antigenic stimulation characteristic of these structures under steady state is less clear
. Here, by combining multicolour 'Brainbow' cell-fate mapping and sequencing of immunoglobulin genes from single cells, we find that 5-10% of gut-associated germinal centres from specific-pathogen-free (SPF) mice contain highly dominant 'winner' B cell clones at steady state, despite rapid turnover of germinal-centre B cells. Monoclonal antibodies derived from these clones show increased binding, compared with their unmutated precursors, to commensal bacteria, consistent with antigen-driven selection. The frequency of highly selected gut-associated germinal centres is markedly higher in germ-free than in SPF mice, and winner B cells in germ-free germinal centres are enriched in 'public' clonotypes found in multiple individuals, indicating strong selection of B cell antigen receptors even in the absence of microbiota. Colonization of germ-free mice with a defined microbial consortium (Oligo-MM
) does not eliminate germ-free-associated clonotypes, yet does induce a concomitant commensal-specific B cell response with the hallmarks of antigen-driven selection. Thus, positive selection of B cells can take place in steady-state gut-associated germinal centres, at a rate that is tunable over a wide range by the presence and composition of the microbiota.
During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This ...anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by "inertia." We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.
The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4
T lymphocytes differentiate into functional subtypes with ...regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4
T cells that can differentiate into CD4IELs remains unknown. We identified β-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, β-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.
Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and ...long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.
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•Influenza infection induces long-lived germinal centers (LLGCs)•LLGCs are composed of “founder” and naive “invader” cells•Invaders are not flu specific but compete with founders for affinity maturation•Late founder clones export highly mutated memory cells that can be recalled
B cells continue to hone their affinity for antigen in lymph-node germinal centers long after an initial respiratory virus infection, but as time goes on, new B cells that are less specific come to dominate the population. This work suggests that there are limits to how much a germinal-center reaction can hone the B cell and antibody repertoire over time.
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