T-cell receptors (TCRs) upon binding to peptide-MHC ligands transduce signals in T lymphocytes.Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (γδε) and ζ subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/ CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 ζ chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.

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.

CC chemokine receptor (CCR) 9, the receptor for the CC-chemokine CCL25/ thymus-expressed chemokine (TECK), is mainly expressed by thymocytes and by intraepithelial (IEL) and lamina propria lymphocytes of the small intestine. To study the biologic role of CCR9, a mouse strain was generated in which the CCR9 gene was deleted. In spite of the high level of CCR9 found in double-and singlepositive thymocytes and of the expression of its corresponding ligand on thymic stromal cells, CCR9 deletion had no major effect on intrathymic T-cell development. It was noted that there was only a one-day lag in the appearance of doublepositive cells during fetal ontogeny in CCR9 ؊/؊ thymi. When tested in chemotaxis assay, thymocytes isolated from CCR9 ؊/؊ mice failed to respond to TECK/ CCL25. Taken together, these results suggest that in thymocytes, CCR9 is the only physiologic receptor for TECK/CCL25, and that it is dispensable for proper T-cell development. Bone marrow pre-pro-B cells migrate in response to TECK/CCL25, but more mature B cells do not. Consistent with this observation, it was shown that there are fewer pre-pro-B cells in CCR9 ؊/؊ mice than in wild-type mice. However, this diminution does not appear to have a detectable effect on the generation of a normal complement of mature B cells. Finally, it was shown that in the small intestine of CCR9-deficient mice, the intraepithelial T-cell-to-epithelial cell ratio is decreased, an observation that can be accounted for by a marked diminution of the T-cell receptor ␥␦ ؉ compartment. IntroductionDuring their development in the thymus, T cells migrate from the outer capsule to the inner medulla, a process that allows their sequential interaction with different types of stromal cells. Following their exportation to the periphery, a sophisticated network of chemokines control their appropriate navigation to and from secondary lymphoid organs. [1][2][3] Likewise, the migration of developing T and B cells within their respective primary lymphoid organs is likely under the control of chemokines. 4,5 Consistent with the implication of G protein-coupled chemokine receptors in the intrathymic trafficking of T cells, the latter is inhibited by pertussis toxin. 6 Moreover, developing thymocytes express several chemokine receptors and their corresponding ligands produced by thymic stromal cells. 7 Recent studies, including our own, have described a novel thymus-expressed chemokine (TECK)/CCL25 8,9 and its receptor, CC chemokine receptor 9 (CCR9). 9-12 TECK/CCL25 is predominantly expressed by most thymic epithelial cortical cells, by a few thymic epithelial medullary cells, and by CD11b Ϫ thymic dendritic cells. 9,13 The level of CCR9 transcripts undergoes a 10-fold increase during the double-negative (DN) to double-positive (DP) transition. Among thymocytes, DP thymocytes have the highest CCR9 expression and this correlates with their ability to migrate in response to TECK/CCL25. CCR9 transcripts subsequently decrease in single-positive (SP) thymocytes. Therefore, TECK/ CCL25 may d...