CD4 ؉ T-cell entry to the intestinal mucosa is central to the generation of mucosal immunity as well as chronic intestinal inflammation, yet the mechanisms regulating this process remain poorly defined. Here we show that murine small intestinal CD4 ؉ lamina propria lymphocytes express a heterogeneous but restricted array of chemokine receptors including CCR5, CCR6, CCR9, CXCR3, and CXCR6. CD4 ؉ T-cell receptor transgenic OT-II cells activated in mesenteric lymph nodes acquired a distinct chemokine receptor profile, including expression of CCR6, CCR9, and CXCR3 that was only partially reproduced in vitro after priming with mesenteric lymph node dendritic cells. A subset of these effector CD4 ؉ T cells, expressing CD69 and ␣ 4 ␤ 7 , entered the intestinal lamina propria and the majority of these cells expressed CCR9. CCR9 ؊/؊ OT-II cells were disadvantaged in their ability to localize to the intestinal lamina propria; however, they were readily detected at this site and expressed ␣ 4 ␤ 7 , but little CCR2, CCR5, CCR6, CCR8, CCR10, CXCR3, or CXCR6. Thus, whereas CD4 ؉ T cells activated in gut-associated lymphoid tissue express a restricted chemokine receptor profile, including CCR9, targeting both CCR9-dependent and CCR9-independent entry mechanisms is likely to be important to maximally inhibit accumulation of these cells within the small intestinal mucosa. IntroductionThe intestinal lamina propria (LP) contains a large number of previously activated/memory CD4 ϩ T cells that play a central role in intestinal immunity and in the induction and maintenance of chronic intestinal inflammation. 1 T-cell entry into the intestinal mucosa is mediated by distinct sets of cell adhesion molecules expressed on the T cell and intestinal microvascular endothelial surface. Interaction between the gut-associated integrin ␣ 4 ␤ 7 , on the T-cell surface, with its ligand mucosal addresin cell adhesion molecule 1 (MAdCAM-1) on intestinal microvascular endothelium cells is important for T-cell entry into the LP. [2][3][4][5] In addition, antibody neutralization studies have suggested a role for P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) in effector CD4 ϩ T-cell entry to this site. 5 T-cell entry into nonlymphoid tissues is also regulated by chemokine/chemokine receptors and the chemokine receptor CCR9 is required for efficient effector CD8 ϩ T-cell localization to the small intestinal epithelium. 6,7 However, since CCR9 Ϫ/Ϫ mice have normal numbers of LP T cells, 8,9 the particular role of CCR9 or additional chemokine receptors in CD4 ϩ T-cell localization to the intestinal LP remains unclear.The ability of T cells to enter nonlymphoid effector tissues is acquired following T-cell priming in secondary lymphoid organs and is mediated, in part, through the de novo expression of chemokine receptors. 10,11 In vitro, the chemokine receptor profile induced following CD4 ϩ T-cell priming is highly dependent on the culture conditions and the nature of antigenpresenting cells. [12][13][14] In vivo, distinct subsets of B-helper ...

Migration of CD4+CD25+FOXP3+ regulatory T cells (Treg) is important for suppressing immune responses in different tissues. Previous studies show that the majority of Treg at birth express gut homing receptor α4β7 and that only few express CCR4, while the reverse pattern is found in adults. The age at which homing receptor switch occurs in vivo is not known. In this study, we show, in a prospective study of human infants from birth to 3 years of age, that homing receptor switch from α4β7 to CCR4 commences between 1 1/2 and 3 years of age and that Treg at that age also had started their switch to a memory phenotype. The majority of naive Treg express α4β7 in infants but not in adults, while the majority of memory Treg express CCR4 both infants and adults. The homing receptor expression on Treg corresponds to their actual migration properties, because Treg from cord blood migrate foremost toward the gut-associated chemokine CCL25. CD4+FOXP3+ T cell numbers increase rapidly in the circulation during the first days of life indicating conversion to suppressive Treg from CD25high Treg precursors. These findings suggest that the gut is the primary site of Treg stimulation to exogenous Ags during the first 18 mo of life and that homing receptor switch toward a more extra-intestinal phenotype occurs thereafter.

The CC chemokine receptor (CCR)9 is expressed on the majority of small intestinal, but few colonic, T cells, whereas its ligand CCL25 is constitutively expressed by small intestinal epithelial cells. As such, CCR9/CCL25 have been proposed to play a central role in regulating small intestinal but not colonic immune responses and thus to organize regionalized immunity within the intestinal mucosa. Here, we demonstrate that CCL25 is expressed at reduced levels by epithelial cells in the distal compared with proximal small intestine, which correlated with less efficient CCR9-dependent effector CD8␣␤ ؉ T cell entry into the ileal epithelium. In vitro-generated ␣4␤7 ؉ effector CD8␣␤ ؉ T cell entry into the lamina propria was less dependent on CCR9 than entry into the epithelium along the entire length of the small intestine and in particular in the ileum. CCR9-independent ␣4␤7 ؉ effector CD8␣␤ ؉ T cell entry was pertussis toxin-sensitive, suggesting a role for additional G␣ I-linked G protein-coupled receptors. Finally, in vivo-primed effector CD8␣␤ ؉ T cells displayed regionalized differences in their entry to the small intestinal epithelium with enhanced CCR9-independent entry to the ileum. These results highlight a hitherto underappreciated compartmentalization of immune responses within the small intestine and have direct implications for targeting strategies aimed at regulating T cell localization to the small intestinal mucosa.chemokine receptor ͉ intestinal mucosa

Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [ 3 H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1-0.25 mM Zn 2+ . The activity was abolished by site mutation of the predicted metalbinding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The V max for PAF hydrolysis was 374 µmol · h −1 · (mg of protein) −1 . The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer.

T cell-mediated immunity is a major component of antitumor immunity. In order to be efficient, effector T cells must leave the circulation and enter into the tumor tissue. Regulatory T cells (Treg) from gastric cancer patients, but not from healthy volunteers, potently inhibit migration of conventional T cells through activated endothelium. In this study, we compared T cells from colon cancer patients and healthy donors to determine the mechanisms used by Tregs from cancer patients to inhibit conventional T-cell migration. Our results showed that circulating Tregs from cancer patients expressed high levels of CD39, an ectoenzyme mediating hydrolysis of ATP to AMP, as a ratedetermining first step in the generation of immunosuppressive adenosine. Tumor-associated Tregs expressed even more CD39, and we therefore examined the importance of adenosine in Tregmediated inhibition of T-cell transendothelial migration in vitro. Exogenous adenosine significantly reduced migration of conventional T cells from healthy volunteers, and blocking either adenosine receptors or CD39 enzymatic activity during transmigration restored the ability of conventional T cells from cancer patients to migrate. Adenosine did not directly affect T cells or endothelial cells, but reduced the ability of monocytes to activate the endothelium. Taken together, our results indicate that Treg-derived adenosine acts on monocytes and contributes to reduced transendothelial migration of effector T cells into tumors. This effect of Tregs is specific for cancer patients, and our results indicate that Tregs may affect not only T-cell effector functions but also their migration into tumors.

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Intestinal-derived chemokines have a central role in orchestrating immune cell influx into the normal and inflamed intestine. Here, we identify the chemokine CCL6 as one of the most abundant chemokines constitutively expressed by both murine small intestinal and colonic epithelial cells. CCL6 protein localized to crypt epithelial cells, was detected in the gut lumen and reached high concentrations at the mucosal surface. Its expression was further enhanced in the small intestine following in vivo administration of LPS or after stimulation of the small intestinal epithelial cell line, mIC(c12), with IFNgamma, IL-4 or TNFalpha. Recombinant- and intestinal-derived CCL6 bound to a subset of the intestinal microflora and displayed antibacterial activity. Finally, the human homologs to CCL6, CCL14 and CCL15 were also constitutively expressed at high levels in human intestinal epithelium, were further enhanced in inflammatory bowel disease and displayed similar antibacterial activity. These findings identify a novel role for constitutively expressed, epithelial-derived chemokines as antimicrobial peptides in the intestinal mucosa.

<p>This supplementary figure shows CD73 expression by circulating (A) and colon (B) CD4+ T cells.</p>