William W. Agace

Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing

Gut-associated lymphoid tissue (GALT) dendritic cells (DCs) display a unique ability to generate CCR9+ α 4 β 7 + gut-tropic CD8+ effector T cells. We demonstrate efficient induction of CCR9 and α 4 β 7 on CD8+ T cells in mesenteric lymph nodes (MLNs) after oral but not intraperitoneal (i.p.) antigen administration indicating differential targeting of DCs via the oral route. In vitro, lamina propria (LP)–derived DCs were more potent than MLN or Peyers patch DCs in their ability to generate CCR9+ α 4 β 7 + CD8+ T cells. The integrin α chain CD103 (α E) was expressed on almost all LP DCs, a subset of MLN DCs, but on few splenic DCs. CD103+ MLN DCs were reduced in number in CCR7−/− mice and, although CD8+ T cells proliferated in the MLNs of CCR7−/− mice after i.p. but not oral antigen administration, they failed to express CCR9 and had reduced levels of α 4 β 7. Strikingly, although CD103+ and CD103− MLN DCs were equally potent at inducing CD8+ T cell proliferation and IFN-γ production, only CD103+ DCs were capable of generating gut-tropic CD8+ effector T cells in vitro. Collectively, these results demonstrate a unique function for LP-derived CD103+ MLN DCs in the generation of gut-tropic effector T cells.

Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions.

Chemokine receptor CX3CR1+ dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103+ DCs in the mucosa, which, through their ability to synthesize retinoic acid (RA), appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103+ DC, CX3CR1+ cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103+ DCs, but not CX3CR1-expressing cells, migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover, CX3CR1+ cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103+ DC. These findings indicate that selectively CD103+ DCs serve classical DC functions and initiate adaptive immune responses in local LNs, whereas CX3CR1+ populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens.

Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

A functionally distinct subset of CD103+ dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3+ T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and α4β7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103+ DCs. CD103+ SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103+ DCs do not derive from a CD103− SI-LP DC intermediate. The majority of CD103+ MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8+ and CD4+ T cells. In contrast, most CD103− MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103+ DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103+ MLN DCs isolated from SB Crohns patients. Thus, small intestinal CD103+ DCs represent a potential novel target for regulating human intestinal inflammatory responses.

Selective Generation of Gut Tropic T Cells in Gut-associated Lymphoid Tissue (GALT) Requirement for GALT Dendritic Cells and Adjuvant

In the current study, we address the underlying mechanism for the selective generation of gut-homing T cells in the gut-associated lymphoid tissues (GALT). We demonstrate that DCs in the GALT are unique in their capacity to establish T cell gut tropism but in vivo only confer this property to T cells in the presence of DC maturational stimuli, including toll-like receptor-dependent and -independent adjuvants. Thus, DCs from mesenteric LNs (MLNs), but not from spleen, supported expression of the chemokine receptor CCR9 and integrin α4β7 by activated CD8+ T cells. While DCs were also required for an efficient down-regulation of CD62L, this function was not restricted to MLN DCs. In an adoptive CD8+ T cell transfer model, antigen-specific T cells entering the small intestinal epithelium were homogeneously CCR9+α4β7 +CD62Llow, and this phenotype was only generated in GALT and in the presence of adjuvant. Consistent with the CCR9+ phenotype of the gut-homing T cells, CCR9 was found to play a critical role in the localization of T cells to the small intestinal epithelium. Together, these results demonstrate that GALT DCs and T cell expression of CCR9 play critical and integrated roles during T cell homing to the gut.

Essential role for CD103 in the T cell–mediated regulation of experimental colitis

The integrin CD103 is highly expressed at mucosal sites, but its role in mucosal immune regulation remains poorly understood. We have analyzed the functional role of CD103 in intestinal immune regulation using the T cell transfer model of colitis. Our results show no mandatory role for CD103 expression on T cells for either the development or CD4+CD25+ regulatory T (T reg) cell–mediated control of colitis. However, wild-type CD4+CD25+ T cells were unable to prevent colitis in immune-deficient recipients lacking CD103, demonstrating a nonredundant functional role for CD103 on host cells in T reg cell–mediated intestinal immune regulation. Non–T cell expression of CD103 is restricted primarily to CD11chighMHC class IIhigh dendritic cells (DCs). This DC population is present at a high frequency in the gut-associated lymphoid tissue and appears to mediate a distinct functional role. Thus, CD103+ DCs, but not their CD103− counterparts, promoted expression of the gut-homing receptor CCR9 on T cells. Conversely, CD103− DCs promoted the differentiation of IFN-γ–producing T cells. Collectively, these data suggest that CD103+ and CD103− DCs represent functionally distinct subsets and that CD103 expression on DCs influences the balance between effector and regulatory T cell activity in the intestine.

Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6C hi monocyte precursors

Macrophages (mφ) are essential for intestinal homeostasis and the pathology of inflammatory bowel disease (IBD), but it is unclear whether discrete mφ populations carry out these distinct functions or if resident mφ change during inflammation. We show here that most resident mφ in resting mouse colon express very high levels of CX3CR1, are avidly phagocytic and MHCIIhi, but are resistant to Toll-like receptor (TLR) stimulation, produce interleukin 10 constitutively, and express CD163 and CD206. A smaller population of CX3CR1int cells is present in resting colon and it expands during experimental colitis. Ly6ChiCCR2+ monocytes can give rise to all mφ subsets in both healthy and inflamed colon and we show that the CX3CR1int pool represents a continuum in which newly arrived, recently divided monocytes develop into resident CX3CR1hi mφ. This process is arrested during experimental colitis, resulting in the accumulation of TLR-responsive pro-inflammatory mφ. Phenotypic analysis of human intestinal mφ indicates that analogous processes occur in the normal and Crohns disease ileum. These studies show for the first time that resident and inflammatory mφ in the intestine represent alternative differentiation outcomes of the same precursor and targeting these events could offer routes for therapeutic intervention in IBD.

Regional specialization within the intestinal immune system

The intestine represents the largest compartment of the immune system. It is continually exposed to antigens and immunomodulatory agents from the diet and the commensal microbiota, and it is the port of entry for many clinically important pathogens. Intestinal immune processes are also increasingly implicated in controlling disease development elsewhere in the body. In this Review, we detail the anatomical and physiological distinctions that are observed in the small and large intestines, and we suggest how these may account for the diversity in the immune apparatus that is seen throughout the intestine. We describe how the distribution of innate, adaptive and innate-like immune cells varies in different segments of the intestine and discuss the environmental factors that may influence this. Finally, we consider the implications of regional immune specialization for inflammatory disease in the intestine.

CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa.

The recruitment of antigen-specific T lymphocytes to the intestinal mucosa is central to the development of an effective mucosal immune response, yet the mechanism by which this process occurs remains to be fully defined. Here we show that the CC chemokine receptor 9 (CCR9) is selectively and functionally expressed on murine alpha(E)beta(7)(+) naive CD8alphabeta(+) lymphocytes and a subset of recently activated CD69(+) CD8alphabeta(+) lymphocytes. Using a T cell receptor transgenic transfer model, we demonstrate that CCR9 expression is functionally maintained on CD8alphabeta(+) lymphocytes following activation in mesenteric lymph nodes but rapidly downregulated on CD8alphabeta(+) lymphocytes activated in peripheral lymph nodes. These recently activated CCR9(+) CD8alphabeta(+) lymphocytes selectively localized to the small-intestinal mucosa, and in vivo neutralization of the CCR9 ligand, CCL25, reduced the ability of these cells to populate the small-intestinal epithelium. Together these results demonstrate an important role for chemokines in the localization of T lymphocytes to the small-intestinal mucosa and suggest that targeting CCL25 and/or CCR9 may provide a means to selectively modulate small-intestinal immune responses.

Interleukin-8 and the neutrophil response to mucosal gram-negative infection.

Urinary tract infections activate a mucosal inflammatory response, which includes cytokine secretion and neutrophil influx. The mechanisms involved in the neutrophil influx have not been identified. Interleukin-8, a potent chemoattractant for neutrophils, is produced by urinary tract epithelial cell lines in vitro. This study analyzed the human IL-8 response to deliberate Escherichia coli infection of the urinary tract. Urine and serum samples were obtained before and after intravesical instillation of E. coli. Neutrophil numbers were determined on uncentrifuged urine, and IL-8 levels were measured by ELISA. A urinary IL-8 response was found in all patients after bacterial instillation, but no serum IL-8 was detected. There was a strong correlation between urinary IL-8 levels and urinary neutrophil numbers. The same E. coli strains used to colonize the patients stimulated IL-8 production in urinary tract epithelial cells. The level of IL-8 secreted by epithelial cell lines was influenced by the fimbrial properties of the E. coli. These results demonstrated that E. coli elicit a mucosal IL-8 response in humans, and suggested that IL-8 is involved in the onset of pyuria. Epithelial cells may be an important source of IL-8 during urinary tract infection.

Generation of gut-homing T cells and their localization to the small intestinal mucosa.

Summary:  The intestinal mucosa represents the largest body surface toward the external environment and harbors numerous T lymphocytes that take up resident within the intestinal epithelium or in the underlying lamina propria (LP). The intraepithelial lymphocytes include subsets of ‘unconventional’ T cells with unclear ontogeny and reactivity that localize to this site independently of antigen‐specific activation in secondary lymphoid organs. In contrast, the majority of the ‘conventional’ gut T cells are recruited into the intestinal mucosa subsequent to their activation in intestinal inductive sites, including Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs). T cells homing to the small intestine express a distinct pattern of homing molecules, allowing them to interact with and transmigrate across intestinal postcapillary endothelium. At least some of these homing molecules, including the integrin α4β7 and the chemokine receptor CCR9, are induced on T cells during their activation in PPs or MLNs. Mucosal dendritic cells (DCs) play a key role in this process, but not all intestinal DCs possess the ability to confer a gut‐homing capacity to T cells. Instead, functionally specialized CD103+ DCs derived from the small intestinal LP appear to selectively regulate T‐cell homing to the small intestine.