Regulation of T cell repertoires by commensal microbiota.

The gut microbiota plays an important role in regulating the host immune systems. It is well established that various commensal microbial species can induce the differentiation of CD4+ T helper subsets such as Foxp3+ regulatory T (Treg) cells and Th17 cells in antigen-dependent manner. The ability of certain microbial species to induce either Treg cells or Th17 cells is often linked to the altered susceptibility to certain immune disorders that are provoked by aberrant T cell response against self-antigens. These findings raise an important question as to how gut microbiota can regulate T cell repertoire and the activation of autoreactive T cells. This review will highlight microbiota-dependent regulation of thymic T cell development, maintenance of T cell repertoire in the secondary lymphoid tissues and the intestine, and microbiota-mediated modulation of autoreactive and tumor neoantigen-specific T cells in autoimmune diseases and tumors, respectively.

Redefining the Foreign Antigen and Self-Driven Memory CD4+ T-Cell Compartments via Transcriptomic, Phenotypic, and Functional Analyses.

Under steady-state conditions, conventional CD4+ T lymphocytes are classically divided into naïve (CD44lo CD62Lhi) and memory (CD44hi CD62Llo) cell compartments. While the latter population is presumed to comprise a mixture of distinct subpopulations of explicit foreign antigen (Ag)-specific "authentic" memory and foreign Ag-independent memory-phenotype (MP) cells, phenotypic markers differentially expressed in these two cell types have yet to be identified. Moreover, while MP cells themselves have been previously described as heterogeneous, it is unknown whether they consist of distinct subsets defined by marker expression. In this study, we demonstrate using combined single-cell RNA sequencing and flow cytometric approaches that self-driven MP CD4+ T lymphocytes are divided into CD127hi Sca1lo, CD127hi Sca1hi, CD127lo Sca1hi, and CD127lo Sca1lo subpopulations that are Bcl2lo, while foreign Ag-specific memory cells are CD127hi Sca1hi Bcl2hi. We further show that among the four MP subsets, CD127hi Sca1hi lymphocytes represent the most mature and cell division-experienced subpopulation derived from peripheral naïve precursors. Finally, we provide evidence arguing that this MP subpopulation exerts the highest responsiveness to Th1-differentiating cytokines and can induce colitis. Together, our findings define MP CD4+ T lymphocytes as a unique, self-driven population consisting of distinct subsets that differ from conventional foreign Ag-specific memory cells in marker expression and establish functional relevance for the mature subset of CD127hi Sca1hi MP cells.

A blend of broadly-reactive and pathogen-selected Vγ4 Vδ1 T cell receptors confer broad bacterial reactivity of resident memory γδ T cells.

Although murine γδ T cells are largely considered innate immune cells, they have recently been reported to form long-lived memory populations. Much remains unknown about the biology and specificity of memory γδ T cells. Here, we interrogated intestinal memory Vγ4 Vδ1 T cells generated after foodborne Listeria monocytogenes (Lm) infection to uncover an unanticipated complexity in the specificity of these cells. Deep TCR sequencing revealed that a subset of non-canonical Vδ1 clones are selected by Lm infection, consistent with antigen-specific clonal expansion. Ex vivo stimulations and in vivo heterologous challenge infections with diverse pathogenic bacteria revealed that Lm-elicited memory Vγ4 Vδ1 T cells are broadly reactive. The Vγ4 Vδ1 T cell recall response to Lm, Salmonella enterica serovar Typhimurium (STm) and Citrobacter rodentium was largely mediated by the γδTCR as internalizing the γδTCR prevented T cell expansion. Both broadly-reactive canonical and pathogen-selected non-canonical Vδ1 clones contributed to memory responses to Lm and STm. Interestingly, some non-canonical γδ T cell clones selected by Lm infection also responded after STm infection, suggesting some level of cross-reactivity. These findings underscore the promiscuous nature of memory γδ T cells and suggest that pathogen-elicited memory γδ T cells are potential targets for broad-spectrum anti-infective vaccines.

Microbiota-derived lactate promotes hematopoiesis and erythropoiesis by inducing stem cell factor production from leptin receptor+ niche cells.

Although functional interplay between intestinal microbiota and distant sites beyond the gut has been identified, the influence of microbiota-derived metabolites on hematopoietic stem cells (HSCs) remains unclear. This study investigated the role of microbiota-derived lactate in hematopoiesis using mice deficient in G-protein-coupled receptor (Gpr) 81 (Gpr81-/-), an established lactate receptor. We detected significant depletion of total HSCs in the bone marrow (BM) of Gpr81-/- mice compared with heterogenic (Gpr81+/-) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for the proliferation of HSCs, decreased significantly in leptin receptor-expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of the BM from Gpr81-/- mice compared with Gpr81+/- mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment also required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated SCF secretion from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggest that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Structural specificities of cell surface ß-glucan polysaccharides determine commensal yeast mediated immuno-modulatory activities.

Yeast is an integral part of mammalian microbiome, and like commensal bacteria, has the potential of being harnessed to influence immunity in clinical settings. However, functional specificities of yeast-derived immunoregulatory molecules remain elusive. Here we find that while under steady state, ß-1,3-glucan-containing polysaccharides potentiate pro-inflammatory properties, a relatively less abundant class of cell surface polysaccharides, dubbed mannan/ß-1,6-glucan-containing polysaccharides (MGCP), is capable of exerting potent anti-inflammatory effects to the immune system. MGCP, in contrast to previously identified microbial cell surface polysaccharides, through a Dectin1-Cox2 signaling axis in dendritic cells, facilitates regulatory T (Treg) cell induction from naïve T cells. Furthermore, through a TLR2-dependent mechanism, it restrains Th1 differentiation of effector T cells by suppressing IFN-γ expression. As a result, administration of MGCP display robust suppressive capacity towards experimental inflammatory disease models of colitis and experimental autoimmune encephalomyelitis (EAE) in mice, thereby highlighting its potential therapeutic utility against clinically relevant autoimmune diseases.

Listeria monocytogenes Establishes Commensalism in Germ-Free Mice Through the Reversible Downregulation of Virulence Gene Expression.

The intestine harbors a complex community of bacterial species collectively known as commensal microbiota. Specific species of resident bacteria, as known as pathobiont, have pathogenic potential and can induce apparent damage to the host and intestinal inflammation in a certain condition. However, the host immune factors that permit its commensalism under steady state conditions are not clearly understood. Here, we studied the gut fitness of Listeria monocytogenes by using germ-free (GF) mice orally infected with this food-borne pathogen. L. monocytogenes persistently exists in the gut of GF mice without inducing chronic immunopathology. L. monocytogenes at the late phase of infection is not capable of infiltrating through the intestinal barrier. L. monocytogenes established the commensalism through the reversible down regulation of virulence gene expression. CD8+ T cells were found to be sufficient for the commensalism of L. monocytogenes. CD8+ T cells responding to L. monocytogenes contributed to the down-regulation of virulence gene expression. Our data provide important insights into the host-microbe interaction and have implications for developing therapeutics against immune disorders induced by intestinal pathogens or pathobionts.

Complement C5a promotes antigen cross-presentation by Peyer's patch monocyte-derived dendritic cells and drives a protective CD8+ T cell response.

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.

Mucin degrader Akkermansia muciniphila accelerates intestinal stem cell-mediated epithelial development.

Mucin-degrading bacteria are densely populated in the intestinal epithelium; however, their interaction with intestinal stem cells (ISCs) and their progeny have not been elucidated. To determine whether mucin-degrading bacteria play a role in gut homeostasis, mice were treated with Akkermansia muciniphila, a specialized species that degrades mucin. Administration of A. muciniphila for 4 weeks accelerated the proliferation of Lgr5+ ISCs and promoted the differentiation of Paneth cells and goblet cells in the small intestine (SI). We found similar effects of A. muciniphila in the colon. The levels of acetic and propionic acids were higher in the cecal contents of A. muciniphila-treated mice than in PBS-treated mice. SI organoids treated with cecal contents obtained from A. muciniphila-treated mice were larger and could be diminished by treatment with G protein-coupled receptor (Gpr) 41/43 antagonists. Pre-treatment of mice with A. muciniphila reduced gut damage caused by radiation and methotrexate. Further, a novel isotype of the A. muciniphila strain was isolated from heathy human feces that showed enhanced function in intestinal epithelial regeneration. These findings suggest that mucin-degrading bacteria (e.g., A. muciniphila) may play a crucial role in promoting ISC-mediated epithelial development and contribute to intestinal homeostasis maintenance.

Author Correction: Requirements for the differentiation of innate T-bethigh memory-phenotype CD4+ T lymphocytes under steady state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dietary Glucose Consumption Promotes RALDH Activity in Small Intestinal CD103+CD11b+ Dendritic Cells.

Retinal dehydrogenase (RALDH) enzymatic activities catalyze the conversion of vitamin A to its metabolite Retinoic acid (RA) in intestinal dendritic cells (DCs) and promote immunological tolerance. However, precise understanding of the exogenous factors that act as initial trigger of RALDH activity in these cells is still evolving. By using germ-free (GF) mice raised on an antigen free (AF) elemental diet, we find that certain components in diet are critically required to establish optimal RALDH expression and activity, most prominently in small intestinal CD103+CD11b+ DCs (siLP-DCs) right from the beginning of their lives. Surprisingly, systematic screens using modified diets devoid of individual dietary components indicate that proteins, starch and minerals are dispensable for this activity. On the other hand, in depth comparison between subtle differences in dietary composition among different dietary regimes reveal that adequate glucose concentration in diet is a critical determinant for establishing RALDH activity specifically in siLP-DCs. Consequently, pre-treatment of siLP-DCs, and not mesenteric lymph node derived MLNDCs with glucose, results in significant enhancement in the in vitro generation of induced Regulatory T (iTreg) cells. Our findings reveal previously underappreciated role of dietary glucose concentration in establishing regulatory properties in intestinal DCs, thereby extending a potential therapeutic module against intestinal inflammation.

Requirements for the differentiation of innate T-bethigh memory-phenotype CD4+ T lymphocytes under steady state.

CD4+ T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells exert innate-like effector function during host defense, but whether MP CD4+ T cells are functionally heterogeneous and, if so, what signals specify the differentiation of MP cell subpopulations under homeostatic conditions is still unclear. Here we characterize MP lymphocytes as consisting of T-bethigh, T-betlow, and T-bet- subsets, with innate, Th1-like effector activity exclusively associated with T-bethigh cells. We further show that the latter population depends on IL-12 produced by CD8α+ type 1 dendritic cells (DC1) for its differentiation. Finally, our data demonstrate that this tonic IL-12 production requires TLR-MyD88 signaling independent of foreign agonists, and is further enhanced by CD40-CD40L interactions between DC1 and CD4+ T lymphocytes. We propose that optimal differentiation of T-bethigh MP lymphocytes at homeostasis is driven by self-recognition signals at both the DC and Tcell levels.

Dietary Antigens Induce Germinal Center Responses in Peyer's Patches and Antigen-Specific IgA Production.

The primary induction sites for intestinal IgA are the gut-associated lymphoid tissues (GALT), such as Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). The commensal microbiota is known to contribute to IgA production in the gut; however, the role of dietary antigens in IgA production is poorly understood. To understand the effect of dietary antigens on IgA production, post-weaning mice were maintained on an elemental diet without any large immunogenic molecules. We found that dietary antigens contribute to IgA production in PPs through induction of follicular helper T cells and germinal center B cells. The role of dietary antigens in the PP responses was further confirmed by adding bovine serum albumin (BSA) into the elemental diet. Although dietary antigens are important for PP responses, they have fewer effects than the microbiota on the development and maturation of ILFs. Furthermore, we demonstrated that dietary antigens are essential for a normal antigen-specific IgA response to Salmonella typhi serovar Typhimurium infection. These results provide new insights into the role of dietary antigens in the regulation of mucosal immune responses.

Bone marrow CX3CR1+ mononuclear cells relay a systemic microbiota signal to control hematopoietic progenitors in mice.

The microbiota regulate hematopoiesis in the bone marrow (BM); however, the detailed mechanisms remain largely unknown. In this study, we explored how microbiota-derived molecules (MDMs) were transferred to the BM and sensed by the local immune cells to control hematopoiesis under steady-state conditions. We reveal that MDMs, including bacterial DNA (bDNA), reach the BM via systemic blood circulation and are captured by CX3CR1+ mononuclear cells (MNCs). CX3CR1+ MNCs sense MDMs via endolysosomal Toll-like receptors (TLRs) to produce inflammatory cytokines, which control the basal expansion of hematopoietic progenitors, but not hematopoietic stem cells, and their differentiation potential toward myeloid lineages. CX3CR1+ MNCs colocate with hematopoietic progenitors at the perivascular region, and the depletion of CX3CR1+ MNCs impedes bDNA influx into the BM. Moreover, the abrogation of TLR pathways in CX3CR1+ MNCs abolished the microbiota effect on hematopoiesis. These studies demonstrate that systemic MDMs control BM hematopoiesis by producing CX3CR1+ MNC-mediated cytokines in the steady-state.

Food antigens drive spontaneous IgE elevation in the absence of commensal microbiota.

Immunoglobulin E (IgE), a key mediator in allergic diseases, is spontaneously elevated in mice with disrupted commensal microbiota such as germ-free (GF) and antibiotics-treated mice. However, the underlying mechanisms for aberrant IgE elevation are still unclear. Here, we demonstrate that food antigens drive spontaneous IgE elevation in GF and antibiotics-treated mice by generating T helper 2 (TH2)-skewed T follicular helper (TFH) cells in gut-associated lymphoid tissues (GALTs). In these mice, depriving contact with food antigens results in defective IgE elevation as well as impaired generation of TFH cells and IgE-producing cells in GALT. Food antigen-driven TFH cells in GF mice are mostly generated in early life, especially during the weaning period. We also reveal that food antigen-driven TFH cells in GF mice are actively depleted by colonization with commensal microbiota. Thus, our findings provide a possible explanation for why the perturbation of commensal microbiota in early life increases the occurrence of allergic diseases.

Interleukin-2/antibody complex expanding Foxp3＋ regulatory T cells exacerbates Th2-mediated allergic airway inflammation.

Foxp3＋ regulatory CD4＋ T (Treg) cells play an essential role in preventing overt immune responses against self and innocuous foreign antigens. Selective expansion of endogenous Treg cells in response to the administration of interleukin (IL)-2/antibody complex, such as the IL-2/JES6-1 complex (IL-2C) in mice, is considered an attractive therapeutic approach to various immune disorders. Here, we investigated the therapeutic potential of IL-2C in allergic airway inflammation models. IL-2C treatment ameliorated Th17-mediated airway inflammation; however, unexpectedly, IL-2C treatment exacerbated Th2-mediated allergic airway inflammation by inducing the selective expansion of Th2 cells and type-2 innate lymphoid cells. We also found that IL-2 signaling is required for the expansion of Th2 cells in lymphoproliferative disease caused by Treg cell depletion. Our data suggest that IL-2C is selectively applicable to the treatment of allergic airway diseases depending on the characteristics of airway inflammation. [BMB Reports 2019; 52(4): 283-288].

Gut microbiota regulates lacteal integrity by inducing VEGF-C in intestinal villus macrophages.

A lacteal is a blunt-ended, long, tube-like lymphatic vessel located in the center of each intestinal villus that provides a unique route for drainage of absorbed lipids from the small intestine. However, key regulators for maintaining lacteal integrity are poorly understood. Here, we explore whether and how the gut microbiota regulates lacteal integrity. Germ depletion by antibiotic treatment triggers lacteal regression during adulthood and delays lacteal maturation during the postnatal period. In accordance with compromised lipid absorption, the button-like junction between lymphatic endothelial cells, which is ultrastructurally open to permit free entry of dietary lipids into lacteals, is significantly reduced in lacteals of germ-depleted mice. Lacteal defects are also found in germ-free mice, but conventionalization of germ-free mice leads to normalization of lacteals. Mechanistically, VEGF-C secreted from villus macrophages upon MyD88-dependent recognition of microbes and their products is a main factor in lacteal integrity. Collectively, we conclude that the gut microbiota is a crucial regulator for lacteal integrity by endowing its unique microenvironment and regulating villus macrophages in small intestine.

Unregulated antigen-presenting cell activation by T cells breaks self tolerance.

T cells proliferate vigorously following acute depletion of CD4+ Foxp3+ T regulatory cells [natural Tregs (nTregs)] and also when naive T cells are transferred to syngeneic, nTreg-deficient Rag1-/- hosts. Here, using mice raised in an antigen-free (AF) environment, we show that proliferation in these two situations is directed to self ligands rather than food or commensal antigens. In both situations, the absence of nTregs elevates B7 expression on host dendritic cells (DCs) and enables a small subset of naive CD4 T cells with high self affinity to respond overtly to host DCs: bidirectional T/DC interaction ensues, leading to progressive DC activation and reciprocal strong proliferation of T cells accompanied by peripheral Treg (pTreg) formation. Likewise, high-affinity CD4 T cells proliferate vigorously and form pTregs when cultured with autologous DCs in vitro in the absence of nTregs: this anti-self response is MHCII/peptide dependent and elicited by the raised level of B7 on cultured DCs. The data support a model in which self tolerance is imposed via modulation of CD28 signaling and explains the pathological effects of superagonistic CD28 antibodies.

Microbiota-Derived Lactate Accelerates Intestinal Stem-Cell-Mediated Epithelial Development.

Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/ß-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-protein-coupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner.

Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3+ regulatory T cells.

Dysregulation of intestinal microflora is linked to inflammatory disorders associated with compromised immunosuppressive functions of Foxp3+ T regulatory (Treg) cells. Although mucosa-associated commensal microbiota has been implicated in Treg generation, molecular identities of the "effector" components controlling this process remain largely unknown. Here, we have defined Bifidobacterium bifidum as a potent inducer of Foxp3+ Treg cells with diverse T cell receptor specificity to dietary antigens, commensal bacteria, and B. bifidum itself. Cell surface ß-glucan/galactan (CSGG) polysaccharides of B. bifidum were identified as key components responsible for Treg induction. CSGG efficiently recapitulated the activity of whole bacteria and acted via regulatory dendritic cells through a partially Toll-like receptor 2-mediated mechanism. Treg cells induced by B. bifidum or purified CSGG display stable and robust suppressive capacity toward experimental colitis. By identifying CSGG as a functional component of Treg-inducing bacteria, our studies highlight the immunomodulatory potential of CSGG and CSGG-producing microbes.

Spontaneous Proliferation of CD4+ T Cells in RAG-Deficient Hosts Promotes Antigen-Independent but IL-2-Dependent Strong Proliferative Response of Naïve CD8+ T Cells.

The fast and intense proliferative responses have been well documented for naïve T cells adoptively transferred into chronic lymphopenic hosts. This response known as spontaneous proliferation (SP), unlike antigen-independent lymphopenia-induced proliferation (LIP), is driven in a manner dependent on antigens derived from commensal microbiota. However, the precise nature of the SP response and its impact on homeostasis and function for T cells rapidly responding under this lymphopenic condition are still unclear. Here we demonstrate that, when naïve T cells were adoptively transferred into specific pathogen-free (SPF) but not germ-free (GF) RAG-/- hosts, the SP response of these cells substantially affects the intensity and tempo of the responding T cells undergoing LIP. Therefore, the resulting response of these cells in SPF RAG-/- hosts was faster and stronger than the typical LIP response observed in irradiated B6 hosts. Although the intensity and tempo of such augmented LIP in SPF RAG-/- hosts were analogous to those of antigen-dependent SP, the former was independent of antigenic stimulation but most importantly, dependent on IL-2. Similar observations were also apparent in other acute lymphopenic settings where antigen-dependent T cell activation can strongly occur and induce sufficient levels of IL-2 production. Consequently, the resulting T cells undergoing IL-2-driven strong proliferative responses showed the ability to differentiate into functional effector and memory cells that can control infectious pathogens. These findings therefore reveal previously unappreciated role of IL-2 in driving the intense form of T cell proliferative responses in chronic lymphopenic hosts.