Human Tfh and Tfr cells: identification and assessment of their migration potential.

The ability of follicular T cells to migrate into B-cell follicles is central for them to participate in germinal center responses. The chemokine receptor CXCR5 is expressed by both Tfh and Tfr cells and is the defining marker for follicular T cells. In addition, Tfh and Tfr cells express additional chemokine receptors to enable them to interact with B cells and other cell types. CXCR5(+) Tfh and Tfr cells are divided into CCR7(+) perifollicular cells and CCR7(-) follicular cells. Most of the CXCR5(+) CCR7(-) Tfh cells reside in germinal centers and are called GC T cells. The methods to identify human Tfh and Tfr cell subsets based on chemokine receptors and other antigens and assess their migration potential are provided in this article.

Short-chain fatty acids activate GPR41 and GPR43 on intestinal epithelial cells to promote inflammatory responses in mice.

BACKGROUND & AIMS: Short-chain fatty acids (SCFAs), the most abundant microbial metabolites in the intestine, activate cells via G-protein-coupled receptors (GPRs), such as GPR41 and GPR43. We studied regulation of the immune response by SCFAs and their receptors in the intestines of mice. METHODS: Inflammatory responses were induced in GPR41(-/-), GPR43(-/-), and C57BL6 (control) mice by administration of ethanol; 2, 4, 6-trinitrobenzene sulfonic-acid (TNBS); or infection with Citrobacter rodentium. We examined the effects of C rodentium infection on control mice fed SCFAs and/or given injections of antibodies that delay the immune response. We also studied the kinetics of cytokine and chemokine production, leukocyte recruitment, intestinal permeability, and T-cell responses. Primary colon epithelial cells were isolated from GPR41(-/-), GPR43(-/-), and control mice; signaling pathways regulated by SCFAs were identified using immunohistochemical, enzyme-linked immunosorbent assay, and flow cytometry analyses. RESULTS: GPR41(-/-) and GPR43(-/-) mice had reduced inflammatory responses after administration of ethanol or TNBS compared with control mice, and had a slower immune response against C rodentium infection, clearing the bacteria more slowly. SCFAs activated intestinal epithelial cells to produce chemokines and cytokines in culture and mice after administration of ethanol, TNBS, or C rodentium. These processes required GPR41 and GPR43 and were required to recruit leukocytes and activate effector T cells in the intestine. GPR41 and GPR43 activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways in epithelial cells to induce production of chemokines and cytokines during immune responses. CONCLUSIONS: SCFAs activate GPR41 and GPR43 on intestinal epithelial cells, leading to mitogen-activated protein kinase signaling and rapid production of chemokines and cytokines. These pathways mediate protective immunity and tissue inflammation in mice.

Retinoic acid determines the precise tissue tropism of inflammatory Th17 cells in the intestine.

Th17 cells are major effector T cells in the intestine, but the regulation of their tissue tropism within the gut is poorly understood. We investigated the roles of vitamin A and retinoic acid in generation of inflammatory Th17 cells with distinct tissue tropisms within the intestine. We found that Th17 cells with distinct tissue tropisms and pathogenic activities are generated depending on the available concentration of retinoic acid (RA). In contrast to the widespread perception that RA would suppress the generation of Th17 cells, we provide evidence that RA is actually required for generation of Th17 cells with specific tissue tropisms within the gut. Th17 cells induced at suboptimal serum concentrations of RA migrated and induced moderate inflammation mainly in the large intestine, whereas the Th17 cells induced with optimal levels of exogenous RA (approximately 10 nM) migrated to the small intestine and induced more severe inflammation. The Th17 cells, induced in the presence or absence of RA, differentially expressed the trafficking receptors CCR9 and alpha4beta7. CCR9 is required for Th17 cell migration to the small intestine, whereas alpha4beta7 is required for the migration of Th17 cells throughout the whole intestine. Our results identified RA as a major signal that regulates the generation of gut Th17 cells with distinct capacities in migration and inflammatory activities. The results indicate also that specific gut tropism of Th17 cells is determined by the combination of trafficking receptors regulated by the RA signal.

High and low vitamin A therapies induce distinct FoxP3+ T-cell subsets and effectively control intestinal inflammation.

BACKGROUND & AIMS: Retinoic acid plays a positive role in induction of FoxP3(+) regulatory T cells. Because retinoic acid is produced as a metabolite of vitamin A in the intestine and FoxP3(+) T cells regulate intestinal inflammation, we investigated the impact of vitamin A status on the regulatory T cells and inflammation in the intestine. METHODS: The SAMP1/YP model is a mouse model of Crohn's disease. We made vitamin A-deficient, vitamin A-excessive, and normal SAMP1/YP mice and assessed the intestinal inflammation. We also investigated the phenotype and function of FoxP3(+) T cells induced in different levels of vitamin A availability in regulation of intestinal inflammation in a T-cell-induced inflammation model in SCID mice. RESULTS: The limited and excessive vitamin A conditions induced distinct FoxP3(+) T-cell subsets in vivo, and both ameliorated the intestinal inflammation in SAMP1/YP mice. The limited vitamin A condition greatly induced unusual CD103(+)CCR7(+) FoxP3(+) cells, while the high vitamin A condition induced CCR9(+)alpha4beta7(+) FoxP3(+) T cells in the intestine. Both FoxP3(+) T-cell populations, when transferred into mice with ongoing intestinal inflammation, were highly effective in reversing the inflammation. Blockade or lack of occupancy of RARalpha is a mechanism to induce highly suppressive CD103(+)CCR7(+) FoxP3(+) cells in both the thymus and periphery in limited vitamin A availability. CONCLUSIONS: Our results identify novel pathways of inducing highly suppressive FoxP3(+) regulatory T cells that can effectively control intestinal inflammation. The results have significant ramifications in treating inflammatory bowel diseases.

Vitamin A metabolites induce gut-homing FoxP3+ regulatory T cells.

In this study, we report a novel biological function of vitamin A metabolites in conversion of naive FoxP3- CD4+ T cells into a unique FoxP3+ regulatory T cell subset (termed "retinoid-induced FoxP3+ T cells") in both human and mouse T cells. We found that the major vitamin A metabolite all-trans-retinoic acid induces histone acetylation at the FoxP3 gene promoter and expression of the FoxP3 protein in CD4+ T cells. The induction of retinoid-induced FoxP3+ T cells is mediated by the nuclear retinoic acid receptor alpha and involves T cell activation driven by mucosal dendritic cells and costimulation through CD28. Retinoic acid can promote TGF-beta1-dependent generation of FoxP3+ regulatory T cells but decrease the TGF-beta1- and IL-6-dependent generation of inflammatory Th17 cells in mouse T cells. Retinoid-induced FoxP3+ T cells can efficiently suppress target cells and, thus, have a regulatory function typical for FoxP3+ T cells. A unique cellular feature of these regulatory T cells is their high expression of gut-homing receptors that are important for migration to the mucosal tissues particularly the small intestine. Taken together, these results identify retinoids as positive regulatory factors for generation of gut-homing FoxP3+ T cells.

Identification of a chemokine network that recruits FoxP3(+) regulatory T cells into chronically inflamed intestine.

BACKGROUND AND AIMS: It has been unclear which chemokine network is involved in migration of T-cell subsets to chronically inflamed lesions of the intestine. SAMP1/YP mice develop a spontaneous chronic transmural intestinal lesion specifically in the ileum. Using these mice, we investigated the gut chemokine network involved in specific migration of T-cell subsets to the inflamed lesion of the intestine. METHODS: We performed expression analyses of chemokines and their receptors, chemokine receptor blocking studies, and migration studies in vitro and in vivo to identify the gut chemokine network induced in intestinal inflammation and to determine its role in migration of conventional and FoxP3(+) suppressor T cells to the inflamed intestine. RESULTS: The expression of homeostatic chemokines was largely unchanged in the inflamed lesion of SAMP1/YP mice compared with control mice. However, an additional chemokine axis (CCL5-CCR5) was up-regulated in the inflamed intestine of SAMP1/YP mice compared with control mice. Activated T cells of SAMP1/YP mice compared with control mice were hyperresponsive to CCL5 in chemotaxis. CCR5(+) T cells preferentially migrated to the inflamed lesion, which can be blocked by a CCR5 antagonist. Importantly, the FoxP3(+) regulatory T cells of the inflamed lesion of SAMP1/YP mice highly expressed CCR5. CCR5 blockade suppressed the migration of FoxP3(+) T cells into the inflamed intestine and significantly exacerbated the intestinal inflammation. CONCLUSIONS: The CCL5-CCR5 chemokine axis is involved in preferential recruitment of FoxP3(+) regulatory T cells, which prevents further exacerbation of chronic inflammation in the intestine.

FoxP3+ T cells undergo conventional first switch to lymphoid tissue homing receptors in thymus but accelerated second switch to nonlymphoid tissue homing receptors in secondary lymphoid tissues.

Forkhead box P3 (FoxP3)-positive T cells are a specialized T cell subset for immune regulation and tolerance. We investigated the trafficking receptor switches of FoxP3(+) T cells in thymus and secondary lymphoid tissues and the functional consequences of these switches in migration. We found that FoxP3(+) T cells undergo two discrete developmental switches in trafficking receptors to migrate from primary to secondary and then to nonlymphoid tissues in a manner similar to conventional CD4(+) T cells as well as unique to the FoxP3(+) cell lineage. In the thymus, precursors of FoxP3(+) cells undergo the first trafficking receptor switch (CCR8/CCR9-->CXCR4-->CCR7), generating mostly homogeneous CD62L(+)CCR7(+)CXCR4(low)FoxP3(+) T cells. CXCR4 expression is regained in FoxP3(+) thymic emigrants in the periphery. Consistent with this switch, recent FoxP3(+) thymic emigrants migrate exclusively to secondary lymphoid tissues but poorly to nonlymphoid tissues. The FoxP3(+) thymic emigrants undergo the second switch in trafficking receptors for migration to nonlymphoid tissues upon Ag priming. This second switch involves down-regulation of CCR7 and CXCR4 but up-regulation of a number of memory/effector type homing receptors, resulting in generation of heterogeneous FoxP3(+) T cell subsets expressing various combinations of trafficking receptors including CCR2, CCR4, CCR6, CCR8, and CCR9. A notable difference between the FoxP3(+) and FoxP3(-) T cell populations is that FoxP3(+) T cells undergo the second homing receptor switch at a highly accelerated rate compared with FoxP3(-) T cells, generating FoxP3(+) T cells with unconventionally efficient migratory capacity to major nonlymphoid tissues.

Human CD57+ germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination.

BACKGROUND: The function of CD57+ CD4+ T cells, constituting a major subset of germinal center T (GC-Th) cells in human lymphoid tissues, has been unclear. There have been contradictory reports regarding the B cell helping function of CD57+ GC-Th cells in production of immunoglobulin (Ig). Furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ GC-Th cells in inducing B cell Ig synthesis. RESULTS: We demonstrated that CD57+ GC-Th cells are highly efficient in helping B cell production of all four subsets of Ig (IgM, IgG, IgA and IgE) compared to other T-helper cells located in germinal centers or interfollicular areas. CD57+ GC-Th cells were particularly more efficient than other T cells in helping GC-B cells but not naive B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and class switch recombination in developing B cells. IgG1-3 and IgA1 were the major Ig isotypes induced by CD57+ GC-Th cells. CD40L, but not IL-4, IL-10 and IFN-gamma, was critical in CD57+ GC-Th cell-driven B cell production of Ig. However, IL-10, when added exogenously, significantly enhanced the helper activity of CD57+ GC-Th cells, while TGF-beta1 completely and IFN-gamma partially suppressed the CD57+ GC-Th cell-driven Ig production. CONCLUSIONS: CD57+CD4+ T cells in the germinal centers of human lymphoid tissues are the major T helper cell subset for GC-B cells in Ig synthesis. Their helper activity is consistent with their capacity to induce AID and class switch recombination, and can be regulated by CD40L, IL-4, IL-10 and TGF-beta.