_:b555948645 . _:b555948633 . _:b555948534 . _:b555948646 . . _:b555948647 . _:b32449323 . . _:b555948648 . _:b555948649 . _:b555948529 . _:b555948650 . _:b555948651 . _:b32449332 . _:b555948456 . _:b555948652 . _:b555948653 . _:b555948528 . . _:b32449333 . . . _:b555948531 . . _:b555948551 . . _:b32449334 . _:b555948530 . _:b555948545 . . _:b32449335 . . . _:b555948541 . _:b555948569 . _:b555948620 . _:b32449328 . _:b555948540 . _:b32449329 . _:b555948531 . _:b555948543 . _:b32449342 "of conventional \u03B1\u03B2T cells, the finding that iNKT cells are dependent on CCR7 is compatible with their need to enter medullary thymic microenvironments during their development to gain access to trans-presentation of IL-15 by mTEC (>>9<<). Our findings also collectively highlight CCR7 as a common mechanism for the medullary relocation that occurs during the development of conventional \u03B1\u03B2T cells (10), \u03B3\u03B4T cells (15), and now CD1d-restricted iNKT cells." . . _:b32449330 . _:b32449314 . _:b555948645 . . _:b555948494 . _:b555948542 . _:b32449331 . _:b555948537 . _:b32449340 . . _:b555948536 . . _:b32449341 . _:b555948539 . _:b32449342 . . _:b555948527 . _:b555948538 . . _:b555948571 . _:b32449343 . . _:b555948524 . _:b32449336 . . _:b32449337 . _:b32449281 . . _:b32449338 . _:b32449280 . _:b32449270 "For example, although several studies have highlighted the important role played by mTEC in tolerance induction via the clonal deletion of autoreactive conventional \u03B1\u03B2T cells (3, >>4<<), the medulla also plays a key role in generating \u03B1\u03B2T cell subsets with key regulatory functions in the immune system (5)." . _:b32449339 . . . . . . . _:b555948589 . _:b555948610 . _:b555948619 . . _:b32449344 . _:b555948646 . _:b32449345 . _:b555948533 . _:b32449346 . _:b32449305 . _:b32449347 . _:b555948641 . _:b555948635 . _:b32449291 . . _:b32449305 "Calculation of cell frequency within a defined area was performed as previously described (>>34<<), where square fields of 100 \u00D7 100\u03BCm were arbitrarily set within either medullary or cortical regions, at least 100 \u03BCm from the CMJ." . . . . _:b555948628 . _:b32449294 . _:b555948561 . . . _:b555948458 . _:b555948521 . . _:b32449265 "introduction" . _:b555948543 . . _:b32449327 . _:b555948616 . . . . _:b555948455 . . _:b32449334 "In addition, given that access to the thymic medulla is important during the development of other \u03B1\u03B2T cell lineages (8, >>9<<), we compared chemokine receptor expression and requirements of Foxp3+ nT-Reg and CD1d-restricted iNKT cells with those of conventional \u03B1\u03B2T cells." . _:b555948604 . . _:b555948597 . _:b32449319 "As the impact of CCR7 deficiency on developing thymocytes is exacerbated in the presence of WT competitors (>>10<<), we performed similar experiments to assess the impact of CCR4 deficiency in a competitive setting." . _:b555948468 . . _:b555948634 . _:b32449346 "Such a scenario is perhaps consistent with the previously reported mislocalization of Foxp3+ cells in the thymus of Ccr7\u2212/\u2212 mice (>>16<<, 17). However, normal numbers of CD25+Foxp3\u2212 nT-Reg precursors in Ccr7\u2212/\u2212 mice (the generation of which is mTEC dependent) would suggest that developing nT-Reg still have sufficient access to medullary regions in the absence of CCR7." . _:b32449272 "Importantly, Foxp3+ nT-Reg development has been shown to be a multistage process (>>6<<, 7), with Foxp3\u2212CD25+ nT-Reg precursors in particular requiring interactions with mTEC for their generation (8)." . . _:b32449267 . . . . . . _:b555948467 . . _:b32449268 . . . _:b555948559 . . _:b555948652 . _:b32449339 . _:b555948497 . . _:b32449277 . _:b555948515 . . _:b555948478 . _:b32449287 "Although CCR7 deficiency does not totally eliminate SP thymocytes from thymic medullary regions (10, 12), pertussis toxin treatment has a more profound effect (>>18<<, 19), thereby implicating other chemokines receptors in cortex to medulla migration." . . _:b32449347 "Such a scenario is perhaps consistent with the previously reported mislocalization of Foxp3+ cells in the thymus of Ccr7\u2212/\u2212 mice (16, >>17<<). However, normal numbers of CD25+Foxp3\u2212 nT-Reg precursors in Ccr7\u2212/\u2212 mice (the generation of which is mTEC dependent) would suggest that developing nT-Reg still have sufficient access to medullary regions in the absence of CCR7. Although" . . _:b32449346 . _:b555948454 . _:b555948499 . _:b555948501 . _:b555948491 . . _:b555948606 . _:b555948487 . . . _:b555948609 . . _:b555948536 . . _:b32449283 "Indeed, Foxp3+ thymocytes have been reported to accumulate in the cortex of Ccr7\u2212/\u2212 mice (>>16<<, 17), although the impact of CCR7 deficiency on distinct nT-Reg progenitors and more mature Foxp3+ nT-Reg stages has not been fully addressed." . _:b32449310 "results" . _:b555948484 . _:b555948544 . . _:b32449288 . . _:b555948651 . _:b32449308 . . . . . _:b555948600 . _:b555948508 . _:b555948557 . _:b555948601 . _:b555948549 "2"^^ . _:b555948593 . . . _:b555948548 "2"^^ . _:b555948475 . . _:b32449309 "Quantitative PCR (qPCR) analysis of freshly sorted thymocyte populations was performed exactly as described (>>33<<). Primers used were as follows:" . _:b555948591 . _:b555948540 . _:b555948551 "2"^^ . . . . _:b32449289 . . _:b555948550 "2"^^ . . _:b555948578 . _:b555948545 "2"^^ . . _:b555948544 "2"^^ . _:b32449315 "Similar to conventional \u03B1\u03B2T cell development, Foxp3+ nT-Reg development in the thymus can be subdivided into distinct stages (6, >>7<<). As we recently showed that the generation of CD25+Foxp3\u2212 nT-Reg precursors is dependent on contact with mTEC (8), we next examined the expression of chemokine receptors at distinct stages in Foxp3+ nT-Reg development that might play a" . _:b555948547 "2"^^ . . _:b555948592 . . _:b555948546 "2"^^ . _:b32449328 "Thus, the reduction in iNKT cells seen in this study in the thymus of Ccr7\u2212/\u2212 mice fits well with the idea that CCR7 enables developing iNKT cells to enter the thymic medulla and gain access to mTEC, known providers of IL-15/IL-15R\u03B1 (>>9<<)." . _:b32449303 "Wild-type (WT) CD45.2+ C57BL/6, congenic CD45.1+ C57BL/6 (BoyJ), Rag2GFP (28), C57BL/6 Foxp3GFP reporter mice (29), Ccr4\u2212/\u2212 (30), Ccr7\u2212/\u2212 (31), Ccr4\u2212/\u2212xCcr7\u2212/\u2212, and Zap 70\u2212/\u2212 (>>32<<) were bred at the University of Birmingham in accordance with Home Office Regulations." . . _:b32449332 . _:b32449282 "Interestingly, a recent study also demonstrated a role for CCR7 in the medullary localization and thymic development of \u03B3\u03B4T cells (>>15<<), suggesting that it can influence multiple T cell lineages." . _:b555948557 "2"^^ . . _:b32449331 "Interestingly, although the medullary accumulation of SP thymocytes is reduced in Ccr7\u2212/\u2212 mice, small medullary areas are present that contain reduced numbers of SP4 and SP8 thymocytes (>>10<<), suggesting the involvement of additional chemokine receptors." . _:b555948556 "2"^^ . . _:b555948559 "2"^^ . _:b32449335 . _:b555948617 . _:b555948558 "2"^^ . _:b555948607 . _:b555948553 "2"^^ . _:b32449297 "Such findings argue against intrathymic redundancy of these chemokine receptors and demonstrate that Aire-mediated control of CCL17/CCL22 expression does not underlie the defective \u03B1\u03B2T cell development seen in adult Aire\u2212/\u2212 mice (>>27<<). Moreover, we reveal previously unreported roles for CCR7 in the development of T cell lineages that arise postnatally." . _:b555948518 . . _:b555948552 "2"^^ . _:b555948549 . _:b555948587 . . _:b555948483 . _:b555948555 "2"^^ . _:b555948548 . _:b555948548 . _:b555948554 "2"^^ . . _:b555948551 . _:b555948565 "2"^^ . _:b555948550 . _:b555948572 . _:b555948615 . _:b555948564 "2"^^ . . _:b555948545 . _:b32449265 _:b32449266 . _:b555948567 "2"^^ . . _:b32449265 _:b32449267 . _:b555948583 . _:b555948544 . _:b32449265 _:b32449268 . _:b32449265 _:b32449269 . _:b32449265 _:b32449270 . _:b32449279 "upregulation occurs during positive selection (10, 11) and plays a key role in tolerance induction, a finding compatible with its role in controlling thymocyte access to medullary microenvironments and its effects on TCR signaling (10, >>12<<). The role of CCR7 in cortex to medulla migration of SP thymocytes fits well with expression of both CCR7 ligands CCL19 and CCL21 in the medulla (13, 14)." . _:b555948576 . _:b555948566 "2"^^ . _:b32449265 _:b32449271 . _:b555948547 . . _:b32449265 _:b32449272 . _:b32449265 _:b32449273 . _:b32449265 _:b32449274 . _:b32449275 "we recently showed that mTEC play an important role during iNKT cell development in the thymus by providing IL-15 trans-presentation to expand newly generated iNKT cells following their CD1d-dependent positive selection in the cortex (>>9<<). Collectively, such observations indicate that a key shared step in the intrathymic development of multiple \u03B1\u03B2T cell lineages is the migration of thymocytes from the cortex to the medulla." . _:b555948561 "2"^^ . _:b555948553 . . _:b32449265 _:b32449275 . _:b555948546 . _:b32449265 _:b32449276 . _:b555948602 . _:b32449265 _:b32449277 . _:b32449265 _:b32449278 . _:b555948560 "2"^^ . _:b32449265 _:b32449279 . _:b555948557 . . _:b555948563 "2"^^ . . _:b555948556 . . . _:b32449344 "Our findings also collectively highlight CCR7 as a common mechanism for the medullary relocation that occurs during the development of conventional \u03B1\u03B2T cells (10), \u03B3\u03B4T cells (>>15<<), and now CD1d-restricted iNKT cells." . _:b555948562 "2"^^ . _:b555948636 . _:b555948546 . _:b555948559 . _:b555948612 . _:b555948573 "2"^^ . _:b555948558 . _:b555948560 . _:b555948550 . _:b555948572 "2"^^ . _:b555948553 . _:b555948575 "2"^^ . _:b32449345 "One possibility is that, as with \u03B1\u03B2T cells in the neonate (>>13<<), CCR7 plays a role in the emigration of Foxp3+ nT-Reg from the thymus and that increased numbers reflect their intrathymic accumulation." . _:b555948552 . _:b32449338 "Although CCR7 has been widely studied in the context of conventional \u03B1\u03B2T cell development (10, >>12<<, 13, 34, 41), less is known about its possible role in the intrathymic development and migration of other T cell subsets." . _:b555948522 . _:b32449277 "In particular, CCR7 upregulation occurs during positive selection (10, >>11<<) and plays a key role in tolerance induction, a finding compatible with its role in controlling thymocyte access to medullary microenvironments and its effects on TCR signaling (10, 12)." . _:b555948574 "2"^^ . . _:b555948555 . _:b32449312 . . _:b555948569 "2"^^ . _:b555948554 . _:b555948568 "2"^^ . _:b555948565 . _:b555948571 "2"^^ . _:b555948486 . _:b555948564 . _:b555948469 . _:b555948570 "2"^^ . . _:b555948567 . _:b32449330 . . _:b555948629 . _:b555948581 "2"^^ . _:b555948566 . . _:b555948461 . _:b32449331 . _:b555948577 . _:b555948580 "2"^^ . _:b555948561 . _:b555948632 . _:b32449274 "Importantly, Foxp3+ nT-Reg development has been shown to be a multistage process (6, 7), with Foxp3\u2212CD25+ nT-Reg precursors in particular requiring interactions with mTEC for their generation (>>8<<). In addition, we recently showed that mTEC play an important role during iNKT cell development in the thymus by providing IL-15 trans-presentation to expand newly generated iNKT cells following their CD1d-dependent positive selection in" . . _:b555948649 . _:b555948639 . . _:b555948583 "2"^^ . _:b555948560 . _:b555948446 "9"^^ . _:b555948582 "2"^^ . _:b555948563 . _:b32449312 "Of the molecules analyzed and consistent with in vitro thymocyte migration data and Ccr4 mRNA analysis (>>20<<), we found that CD4+8+CD69+ thymocytes undergoing thymic selection, but not preselection CD4+8+CD69- cells, expressed readily detectable cell surface expression of CCR4 (Fig." . _:b32449268 "In contrast, medullary areas contain more mature CD4+ and CD8+ single-positive (SP) cells that subsequently emigrate from the thymus to populate peripheral tissues (>>2<<). Importantly, the thymus medulla and medullary thymic epithelial cells (mTEC) in particular play important functional roles during the intrathymic development of multiple \u03B1\u03B2T cell subsets. For example, although several studies have" . _:b555948447 "7"^^ . . _:b555948577 "2"^^ . _:b555948562 . _:b555948445 "11"^^ . _:b555948538 . _:b32449343 . _:b32449271 . . _:b555948576 "2"^^ . _:b555948573 . _:b32449269 "For example, although several studies have highlighted the important role played by mTEC in tolerance induction via the clonal deletion of autoreactive conventional \u03B1\u03B2T cells (>>3<<, 4), the medulla also plays a key role in generating \u03B1\u03B2T cell subsets with key regulatory functions in the immune system (5)." . _:b555948579 "2"^^ . _:b555948445 . _:b555948572 . . . _:b555948578 "2"^^ . _:b555948575 . . _:b555948589 "2"^^ . _:b555948574 . _:b555948588 "2"^^ . _:b555948569 . . . _:b555948591 "2"^^ . _:b555948454 "6"^^ . _:b555948568 . _:b32449275 . _:b32449337 . _:b555948590 "2"^^ . _:b555948455 "6"^^ . _:b555948571 . _:b555948516 . _:b555948507 . . _:b555948585 "2"^^ . _:b555948570 . _:b555948452 "7"^^ . _:b32449308 "Soluble IL-15/IL-15R\u03B1 complexes were prepared as described previously (9, >>35<<). A total of 2.5 mg rIL-15 (PeproTech) was mixed with 15 mg IL-15R\u03B1 (R&D Systems) in 50 \u03BCl PBS and incubated for 30 min at 37\u00B0C and then made up to a total volume of 300 \u03BCl with PBS prior to i." . _:b32449319 . _:b555948584 "2"^^ . _:b555948453 "6"^^ . _:b555948581 . . _:b555948447 . _:b555948587 "2"^^ . _:b555948579 . _:b32449344 . _:b555948580 . _:b555948450 "7"^^ . . _:b555948586 "2"^^ . . _:b555948583 . _:b555948451 "7"^^ . _:b32449294 "Indeed, impaired CCR4-mediated thymocyte migration recently has been suggested (24) to help explain defects in the development of both conventional and Foxp3+ nT-Reg that are linked to the autoimmunity seen in Aire\u2212/\u2212 mice (>>22<<, 25). However, although CCR4 has been studied in the peripheral immune system, notably in the context of skin-homing of T cells (26), its role during the development of distinct \u03B1\u03B2T cell lineages in the adult thymus, either individually" . . . _:b32449336 . _:b555948597 "2"^^ . _:b555948582 . _:b32449327 "5D), correlating with the known effects of IL-15 on later stages of iNKT cell development (>>40<<). Thus, the reduction in iNKT cells seen in this study in the thymus of Ccr7\u2212/\u2212 mice fits well with the idea that CCR7 enables developing iNKT cells to enter the thymic medulla and gain access to mTEC, known providers of IL-15/IL-15R\u03B1 (9)." . _:b555948448 "7"^^ . . _:b555948596 "2"^^ . _:b555948577 . _:b555948490 . _:b32449313 "Moreover, analysis with additional markers including CD24, CD62L, Qa2, and notably levels of Rag2GFP (a direct indicator of thymocyte maturational status) (>>36<<) indicated the following developmental sequence within SP4 thymocytes:" . . . _:b555948449 "7"^^ . . _:b555948599 "2"^^ . _:b32449298 "materials and methods" . _:b555948576 . _:b32449278 "CCR7 upregulation occurs during positive selection (10, 11) and plays a key role in tolerance induction, a finding compatible with its role in controlling thymocyte access to medullary microenvironments and its effects on TCR signaling (>>10<<, 12). The role of CCR7 in cortex to medulla migration of SP thymocytes fits well with expression of both CCR7 ligands CCL19 and CCL21 in the medulla (13, 14)." . _:b555948461 "5"^^ . _:b555948598 "2"^^ . . . _:b555948642 . _:b555948579 . . _:b555948460 "5"^^ . _:b555948593 "2"^^ . _:b555948578 . . . _:b555948592 "2"^^ . _:b555948463 "5"^^ . _:b555948589 . _:b555948462 "5"^^ . _:b555948595 "2"^^ . _:b555948458 "6"^^ . _:b555948588 . _:b555948459 . . _:b555948594 "2"^^ . _:b555948591 . _:b32449307 "Soluble IL-15/IL-15R\u03B1 complexes were prepared as described previously (>>9<<, 35). A total of 2.5 mg rIL-15 (PeproTech) was mixed with 15 mg IL-15R\u03B1 (R&D Systems) in 50 \u03BCl PBS and incubated for 30 min at 37\u00B0C and then made up to a total volume of 300 \u03BCl with PBS prior to i." . _:b32449299 "Wild-type (WT) CD45.2+ C57BL/6, congenic CD45.1+ C57BL/6 (BoyJ), Rag2GFP (>>28<<), C57BL/6 Foxp3GFP reporter mice (29), Ccr4\u2212/\u2212 (30), Ccr7\u2212/\u2212 (31), Ccr4\u2212/\u2212xCcr7\u2212/\u2212, and Zap 70\u2212/\u2212 (32) were bred at the University of Birmingham in accordance with Home Office Regulations." . _:b555948653 . . _:b555948605 "2"^^ . _:b555948456 "6"^^ . _:b555948590 . _:b32449313 . _:b555948459 "5"^^ . _:b555948604 "2"^^ . . _:b555948457 "6"^^ . _:b555948585 . . . _:b32449320 . . _:b555948607 "2"^^ . _:b555948584 . _:b555948469 "4"^^ . _:b32449321 . . _:b555948606 "2"^^ . _:b555948587 . _:b555948468 "4"^^ . _:b555948630 . . . . _:b555948601 "2"^^ . _:b555948586 . _:b555948464 . _:b555948471 "4"^^ . . _:b555948600 "2"^^ . _:b555948597 . _:b555948585 . _:b555948470 "4"^^ . _:b32449315 . . _:b555948603 "2"^^ . _:b555948568 . _:b32449290 . _:b555948596 . . _:b32449292 "Moreover, Aire expression by MHC class IIhigh mTEC is known to influence intrathymic chemokine production (22, >>23<<), including the ligands for CCR4 (23)." . _:b555948465 "5"^^ . _:b555948602 "2"^^ . _:b555948549 . _:b555948599 . _:b555948445 . _:b32449265 . _:b32449322 . _:b555948613 "2"^^ . _:b555948464 "5"^^ . _:b555948446 . _:b555948447 . _:b555948598 . _:b555948448 . . _:b555948449 . _:b555948467 "4"^^ . _:b555948450 . _:b555948612 "2"^^ . . _:b555948451 . _:b555948593 . . _:b555948452 . _:b555948575 . _:b555948453 . _:b555948454 . _:b555948615 "2"^^ . _:b555948466 "5"^^ . _:b555948455 . _:b555948592 . _:b555948456 . _:b555948457 . . _:b555948477 "4"^^ . _:b555948458 . _:b555948614 "2"^^ . . _:b555948459 . _:b555948595 . _:b555948460 . _:b555948461 . . _:b555948476 "4"^^ . _:b555948462 . _:b555948609 "2"^^ . _:b555948463 . _:b32449311 . _:b32449285 "Although CCR7 deficiency does not totally eliminate SP thymocytes from thymic medullary regions (>>10<<, 12), pertussis toxin treatment has a more profound effect (18, 19), thereby implicating other chemokines receptors in cortex to medulla migration." . _:b555948594 . . _:b555948464 . _:b555948465 . _:b32449295 "Indeed, impaired CCR4-mediated thymocyte migration recently has been suggested (24) to help explain defects in the development of both conventional and Foxp3+ nT-Reg that are linked to the autoimmunity seen in Aire\u2212/\u2212 mice (22, >>25<<). However, although CCR4 has been studied in the peripheral immune system, notably in the context of skin-homing of T cells (26), its role during the development of distinct \u03B1\u03B2T cell lineages in the adult thymus, either individually or in" . _:b555948479 "4"^^ . _:b555948466 . _:b555948608 "2"^^ . _:b555948467 . _:b555948605 . _:b555948468 . _:b555948469 . _:b555948478 "4"^^ . _:b555948470 . _:b555948611 "2"^^ . _:b555948519 . _:b555948471 . _:b555948604 . _:b555948472 . _:b32449324 "Finally, given the requirements for medullary microenvironments during intrathymic iNKT cell development, we performed flow cytometric analysis of thymocytes using PBS57/CD1d tetramers (>>38<<), in combination with the iNKT cell maturation markers CD24, CD44 and NK1.1 (39)." . _:b555948595 . _:b555948473 . _:b32449293 "Moreover, Aire expression by MHC class IIhigh mTEC is known to influence intrathymic chemokine production (22, 23), including the ligands for CCR4 (>>23<<). Indeed, impaired CCR4-mediated thymocyte migration recently has been suggested (24) to help explain defects in the development of both conventional and Foxp3+ nT-Reg that are linked to the autoimmunity seen in Aire\u2212/\u2212 mice (22, 25)." . _:b555948473 "4"^^ . _:b555948474 . _:b555948610 "2"^^ . _:b555948475 . _:b555948607 . _:b555948476 . _:b555948477 . _:b32449306 . _:b32449302 "Wild-type (WT) CD45.2+ C57BL/6, congenic CD45.1+ C57BL/6 (BoyJ), Rag2GFP (28), C57BL/6 Foxp3GFP reporter mice (29), Ccr4\u2212/\u2212 (30), Ccr7\u2212/\u2212 (>>31<<), Ccr4\u2212/\u2212xCcr7\u2212/\u2212, and Zap 70\u2212/\u2212 (32) were bred at the University of Birmingham in accordance with Home Office Regulations." . _:b555948472 "4"^^ . _:b555948478 . _:b555948621 "2"^^ . _:b555948479 . _:b555948606 . _:b555948480 . _:b555948481 . _:b555948475 "4"^^ . _:b555948482 . _:b555948620 "2"^^ . _:b555948483 . _:b555948601 . _:b555948446 . _:b555948484 . _:b555948485 . _:b555948474 "4"^^ . _:b555948486 . _:b555948623 "2"^^ . _:b555948487 . _:b555948600 . . _:b555948488 . _:b555948594 . . _:b555948489 . . _:b555948485 "4"^^ . _:b555948490 . _:b555948622 "2"^^ . _:b555948613 . _:b555948491 . _:b555948603 . _:b555948492 . _:b555948493 . . _:b555948484 "4"^^ . _:b555948494 . _:b32449268 . _:b555948617 "2"^^ . _:b555948495 . _:b32449303 . _:b555948602 . _:b555948496 . _:b555948497 . _:b555948487 "4"^^ . _:b555948498 . _:b32449269 . _:b555948616 "2"^^ . _:b555948499 . _:b555948613 . _:b555948500 . _:b555948529 . . _:b555948486 "4"^^ . _:b555948501 . _:b555948502 . _:b32449316 . _:b32449270 . _:b555948619 "2"^^ . _:b555948503 . _:b555948612 . _:b555948504 . _:b555948505 . _:b32449285 . _:b555948481 "4"^^ . _:b555948506 . _:b32449271 . _:b555948618 "2"^^ . _:b555948507 . _:b555948615 . _:b555948508 . _:b555948509 . _:b555948582 . . _:b555948480 "4"^^ . _:b555948510 . _:b555948629 "2"^^ . _:b555948511 . _:b555948614 . _:b555948565 . _:b555948512 . _:b555948513 . _:b555948483 "4"^^ . _:b555948514 . _:b555948628 "2"^^ . _:b555948515 . _:b555948609 . _:b32449265 . _:b555948516 . _:b555948517 . _:b555948482 "4"^^ . . _:b555948518 . _:b32449266 . _:b555948631 "2"^^ . _:b555948519 . _:b555948608 . _:b555948520 . . _:b555948493 "3"^^ . _:b555948521 . _:b555948522 . _:b32449267 . _:b555948630 "2"^^ . _:b555948523 . _:b555948611 . _:b555948524 . _:b555948492 "3"^^ . _:b555948525 . _:b555948513 . _:b555948526 . _:b32449276 . _:b555948625 "2"^^ . . _:b32449329 "discussion" . _:b555948527 . _:b555948610 . _:b555948528 . _:b555948539 . _:b555948495 "3"^^ . _:b555948529 . _:b555948530 . _:b32449277 . _:b555948624 "2"^^ . . _:b555948531 . _:b32449343 "Our findings also collectively highlight CCR7 as a common mechanism for the medullary relocation that occurs during the development of conventional \u03B1\u03B2T cells (>>10<<), \u03B3\u03B4T cells (15), and now CD1d-restricted iNKT cells." . _:b555948621 . _:b555948532 . _:b555948494 "3"^^ . _:b555948533 . _:b555948534 . _:b32449278 . _:b555948627 "2"^^ . _:b555948535 . _:b555948620 . _:b555948536 . _:b32449339 "Although CCR7 has been widely studied in the context of conventional \u03B1\u03B2T cell development (10, 12, >>13<<, 34, 41), less is known about its possible role in the intrathymic development and migration of other T cell subsets." . _:b555948537 . _:b32449297 . . _:b555948489 "4"^^ . _:b555948538 . _:b32449279 . _:b555948626 "2"^^ . _:b555948514 . _:b555948465 . _:b555948539 . _:b555948623 . _:b555948540 . _:b555948541 . _:b555948488 "4"^^ . _:b555948542 . _:b32449337 "Although CCR7 has been widely studied in the context of conventional \u03B1\u03B2T cell development (>>10<<, 12, 13, 34, 41), less is known about its possible role in the intrathymic development and migration of other T cell subsets." . _:b32449272 . _:b555948637 "2"^^ . _:b555948471 . . _:b555948622 . _:b555948543 . . . _:b555948491 "3"^^ . _:b555948636 "2"^^ . _:b32449273 . _:b555948617 . _:b555948490 "3"^^ . _:b555948639 "2"^^ . _:b32449274 . _:b555948616 . _:b555948501 "3"^^ . _:b555948638 "2"^^ . _:b32449275 . _:b555948619 . _:b555948500 "3"^^ . _:b555948626 . . _:b32449341 . _:b555948633 "2"^^ . . _:b555948618 . _:b555948503 "3"^^ . . _:b32449333 . _:b555948632 "2"^^ . _:b555948629 . . _:b32449286 "Although CCR7 deficiency does not totally eliminate SP thymocytes from thymic medullary regions (10, >>12<<), pertussis toxin treatment has a more profound effect (18, 19), thereby implicating other chemokines receptors in cortex to medulla migration." . _:b555948502 "3"^^ . _:b555948462 . _:b555948635 "2"^^ . _:b555948453 . _:b555948628 . _:b555948497 "3"^^ . _:b555948570 . . _:b555948634 "2"^^ . _:b555948631 . _:b555948496 "3"^^ . _:b32449333 "In addition, given that access to the thymic medulla is important during the development of other \u03B1\u03B2T cell lineages (>>8<<, 9), we compared chemokine receptor expression and requirements of Foxp3+ nT-Reg and CD1d-restricted iNKT cells with those of conventional \u03B1\u03B2T cells." . . . _:b555948645 "2"^^ . _:b555948630 . _:b555948499 "3"^^ . _:b555948644 "2"^^ . . _:b555948625 . _:b555948498 "3"^^ . _:b32449329 _:b32449330 . _:b555948647 "2"^^ . _:b32449329 _:b32449331 . _:b555948624 . _:b32449329 _:b32449332 . _:b555948509 "3"^^ . _:b32449329 _:b32449333 . _:b32449329 _:b32449334 . _:b555948646 "2"^^ . _:b555948476 . . _:b32449329 _:b32449335 . _:b555948627 . _:b32449329 _:b32449336 . _:b555948508 "3"^^ . _:b32449329 _:b32449337 . . _:b32449329 _:b32449338 . _:b32449287 . _:b555948641 "2"^^ . . _:b32449329 _:b32449339 . _:b555948626 . _:b32449329 _:b32449340 . _:b555948588 . _:b555948511 "3"^^ . . _:b32449329 _:b32449341 . _:b32449329 _:b32449342 . _:b555948640 "2"^^ . _:b32449329 _:b32449343 . _:b555948637 . _:b555948510 "3"^^ . _:b32449289 "In line with this, positive selection is known to alter the in vitro responsiveness of thymocytes to several chemokines including CCL17 and CCL22 (>>20<<), representing ligands for CCR4 (21)." . _:b555948643 "2"^^ . _:b555948636 . _:b555948505 "3"^^ . _:b555948642 "2"^^ . _:b32449291 "Moreover, Aire expression by MHC class IIhigh mTEC is known to influence intrathymic chemokine production (>>22<<, 23), including the ligands for CCR4 (23)." . . _:b555948639 . _:b555948504 "3"^^ . _:b32449320 "T cell\u2013depleted mixed (50:50) bone marrow chimaeras between WT (CD45.1+):Ccr4\u2212/\u2212 (CD45.2+), or WT (CD45.1+):Ccr4\u2212/\u2212 (CD45.2+) partners were generated as previously described (>>10<<) and harvested after 5 wk (experimental design illustrated in Fig." . _:b32449273 . . _:b32449330 "With regard to the conventional \u03B1\u03B2T cell pool, CCR7 has been shown to play a prominent role in this process, with Ccr7\u2212/\u2212 mice displaying a reduced capacity for positively selected thymocytes to enter medullary areas of the thymus (>>10<<). Interestingly, although the medullary accumulation of SP thymocytes is reduced in Ccr7\u2212/\u2212 mice, small medullary areas are present that contain reduced numbers of SP4 and SP8 thymocytes (10), suggesting the involvement of additional" . _:b555948653 "2"^^ . _:b32449298 . _:b555948638 . _:b555948507 "3"^^ . _:b32449267 "In the thymus, the development of T cells bearing the \u03B1\u03B2TCR involves the progressive migration of immature thymocyte precursors through distinct intrathymic microenvironments formed from specialized thymic stromal cells (1, >>2<<). Although immature CD4\u22128\u2212 thymocytes are localized within subcapsular regions of the thymus, cortical regions house CD4+8+ thymocytes expressing low levels of \u03B1\u03B2TCR that represent precursors of conventional, natural Foxp3+ regulatory T" . _:b555948504 . _:b555948555 . _:b555948652 "2"^^ . _:b32449325 "for medullary microenvironments during intrathymic iNKT cell development, we performed flow cytometric analysis of thymocytes using PBS57/CD1d tetramers (38), in combination with the iNKT cell maturation markers CD24, CD44 and NK1.1 (>>39<<). In contrast to stages in both conventional and Foxp3+ regulatory T cell development, whereas intrathymic iNKT cell development involved expression of CCR7, CCR4 was notably absent, with the former being restricted to early developmental" . . _:b555948633 . _:b555948506 "3"^^ . _:b32449279 . _:b32449311 "TCR-mediated positive selection, we compared CD4+8+69+ thymocytes from WT mice, representing cells undergoing selection, with CD4+8+69\u2212 thymocytes from Zap70\u2212/\u2212 mice, which are blocked at a preselection stage of thymocyte development (>>32<<). Of the molecules analyzed and consistent with in vitro thymocyte migration data and Ccr4 mRNA analysis (20), we found that CD4+8+CD69+ thymocytes undergoing thymic selection, but not preselection CD4+8+CD69- cells, expressed readily" . _:b555948482 . _:b555948632 . _:b555948517 "3"^^ . _:b32449270 . _:b555948605 . . _:b555948495 . _:b32449310 . _:b555948635 . _:b555948477 . _:b555948516 "3"^^ . _:b555948512 . _:b555948649 "2"^^ . _:b555948634 . _:b32449336 "Such findings indicate that the defective Foxp3+ nT-Reg (22) and conventional \u03B1\u03B2T cell development (>>27<<) seen in Aire\u2212/\u2212 mice is not because of their reduced levels of the CCR4 ligands CCL17/CCL22." . _:b32449282 . _:b555948519 "3"^^ . _:b555948648 "2"^^ . _:b555948645 . _:b555948518 "3"^^ . _:b555948651 "2"^^ . _:b555948644 . _:b555948513 "3"^^ . . _:b555948650 "2"^^ . _:b555948449 . _:b32449272 . _:b555948647 . _:b555948512 "3"^^ . _:b32449329 . _:b555948537 . _:b555948646 . _:b555948515 "3"^^ . _:b32449286 . . _:b32449316 "As we recently showed that the generation of CD25+Foxp3\u2212 nT-Reg precursors is dependent on contact with mTEC (>>8<<), we next examined the expression of chemokine receptors at distinct stages in Foxp3+ nT-Reg development that might play a role in their migration to medullary microenvironments." . _:b555948481 . . _:b555948641 . _:b555948514 "3"^^ . . _:b555948640 . _:b555948525 "3"^^ . _:b555948599 . _:b32449325 . _:b555948643 . _:b555948524 "3"^^ . _:b555948642 . _:b555948527 "3"^^ . _:b32449321 "Although quantitation of SP thymocyte positioning by confocal analysis of frozen tissue sections confirmed a defect in medullary accumulation of Ccr7\u2212/\u2212 SP cells (>>10<<), no additive effect was observed in adult Ccr4\u2212/\u2212xCcr7\u2212/\u2212 DKO mice (Fig." . _:b555948653 . . _:b555948526 "3"^^ . _:b32449329 _:b32449344 . _:b32449329 _:b32449345 . _:b32449329 _:b32449346 . . . _:b32449347 . _:b555948652 . _:b32449329 _:b32449347 . _:b555948521 "3"^^ . _:b555948517 . _:b32449340 "Although CCR7 has been widely studied in the context of conventional \u03B1\u03B2T cell development (10, 12, 13, >>34<<, 41), less is known about its possible role in the intrathymic development and migration of other T cell subsets." . _:b555948520 "3"^^ . _:b555948637 . _:b555948603 . _:b555948523 "3"^^ . _:b32449292 . _:b555948649 . _:b555948522 "3"^^ . . _:b32449324 . . _:b32449293 . . _:b555948648 . _:b555948533 "2"^^ . . . . . _:b555948651 . _:b555948532 "2"^^ . _:b32449280 "The role of CCR7 in cortex to medulla migration of SP thymocytes fits well with expression of both CCR7 ligands CCL19 and CCL21 in the medulla (>>13<<, 14). Interestingly, a recent study also demonstrated a role for CCR7 in the medullary localization and thymic development of \u03B3\u03B4T cells (15), suggesting that it can influence multiple T cell lineages. Indeed, Foxp3+ thymocytes have been" . _:b32449288 "Although CCR7 deficiency does not totally eliminate SP thymocytes from thymic medullary regions (10, 12), pertussis toxin treatment has a more profound effect (18, >>19<<), thereby implicating other chemokines receptors in cortex to medulla migration." . _:b555948608 . _:b555948535 "2"^^ . _:b555948650 . . _:b32449273 "Importantly, Foxp3+ nT-Reg development has been shown to be a multistage process (6, >>7<<), with Foxp3\u2212CD25+ nT-Reg precursors in particular requiring interactions with mTEC for their generation (8)." . _:b555948496 . _:b32449326 . _:b555948534 "2"^^ . . _:b555948445 . . _:b32449310 _:b32449311 . _:b555948529 "3"^^ . . _:b555948528 "3"^^ . _:b555948447 . _:b32449301 . _:b555948460 . _:b555948531 "3"^^ . _:b555948446 . _:b555948556 . . _:b555948530 "3"^^ . _:b555948466 . . _:b32449323 . . _:b555948541 "2"^^ . . . _:b555948638 . _:b555948510 . _:b555948540 "2"^^ . _:b555948624 . _:b555948543 "2"^^ . . _:b555948542 "2"^^ . _:b555948453 . _:b555948463 . _:b555948537 "2"^^ . _:b555948622 . _:b555948511 . _:b555948452 . _:b555948536 "2"^^ . _:b555948455 . _:b555948539 "2"^^ . _:b32449310 _:b32449328 . _:b555948611 . _:b555948454 . _:b555948538 "2"^^ . _:b32449310 _:b32449324 . _:b555948598 . _:b32449298 _:b32449308 . _:b32449298 _:b32449309 . _:b32449310 _:b32449325 . _:b32449310 _:b32449326 . _:b555948449 . _:b32449310 _:b32449327 . _:b32449298 _:b32449304 . _:b555948473 . _:b32449310 _:b32449320 . _:b32449310 _:b32449321 . _:b32449298 _:b32449305 . _:b32449298 _:b32449306 . _:b32449309 . _:b555948448 . _:b32449310 _:b32449322 . _:b32449298 _:b32449307 . _:b32449310 _:b32449323 . _:b32449298 _:b32449300 . _:b32449310 _:b32449316 . . _:b32449298 _:b32449301 . _:b32449310 _:b32449317 . _:b32449310 _:b32449318 . _:b555948451 . _:b32449298 _:b32449302 . . _:b32449310 _:b32449319 . _:b32449298 _:b32449303 . _:b32449310 _:b32449312 . _:b32449310 _:b32449313 . . _:b32449310 _:b32449314 . _:b32449342 . _:b32449299 . _:b555948627 . _:b555948450 . . _:b32449298 _:b32449299 . _:b32449310 _:b32449315 . . _:b555948461 . . . _:b555948460 . _:b555948547 . _:b555948463 . _:b32449304 . _:b32449274 . _:b555948480 . _:b555948462 . _:b555948489 . _:b555948457 . _:b555948456 . _:b32449326 "development in the absence of CCR7 may, at least in part, be a consequence of diminished CCR7 dependent thymocyte migration into the thymic medulla, a microenvironment that provides IL15 trans-presentation during iNKT cell development (>>9<<). To test this, soluble IL-15/IL-15R\u03B1 complexes were injected into WT and Ccr7\u2212/\u2212 hosts, and iNKT cell development was examined after 4 d." . _:b555948459 . _:b32449304 "Purified populations of thymocytes and TEC were sorted from seven day fetal thymus organ cultures using a Mo-Flo XDP cell sorter, as described previously (>>33<<)." . _:b555948458 . _:b32449334 . . _:b555948625 . . _:b32449302 . _:b555948469 . _:b32449276 "In particular, CCR7 upregulation occurs during positive selection (>>10<<, 11) and plays a key role in tolerance induction, a finding compatible with its role in controlling thymocyte access to medullary microenvironments and its effects on TCR signaling (10, 12)." . _:b32449300 . _:b555948468 . _:b555948452 . . . _:b555948450 . . _:b555948648 . _:b555948647 . _:b555948471 . _:b555948470 . _:b555948552 . . . _:b555948465 . _:b32449281 "The role of CCR7 in cortex to medulla migration of SP thymocytes fits well with expression of both CCR7 ligands CCL19 and CCL21 in the medulla (13, >>14<<). Interestingly, a recent study also demonstrated a role for CCR7 in the medullary localization and thymic development of \u03B3\u03B4T cells (15), suggesting that it can influence multiple T cell lineages. Indeed, Foxp3+ thymocytes have been" . _:b555948464 . _:b32449328 . . . _:b555948467 . _:b555948466 . . _:b555948477 . . _:b32449306 "To investigate the role of CCR4 in T cell development in a competitive setting, mixed WT/WT and Ccr4\u2212/\u2212/WT chimaeras were established in a manner similar to that used to analyze the role of CCR7 (>>10<<). In brief, bone marrow cells were obtained from the femurs and tibias of Ccr4\u2212/\u2212 (CD45.2), C57BL/6 (CD45.2), or Boy J (CD45.1) mice." . _:b555948476 . _:b32449266 . _:b555948558 . _:b555948530 . _:b555948509 . _:b555948479 . _:b555948586 . . _:b32449271 "role played by mTEC in tolerance induction via the clonal deletion of autoreactive conventional \u03B1\u03B2T cells (3, 4), the medulla also plays a key role in generating \u03B1\u03B2T cell subsets with key regulatory functions in the immune system (>>5<<). Importantly, Foxp3+ nT-Reg development has been shown to be a multistage process (6, 7), with Foxp3\u2212CD25+ nT-Reg precursors in particular requiring interactions with mTEC for their generation (8)." . _:b555948474 . _:b555948492 . _:b555948478 . _:b555948623 . _:b32449301 "Wild-type (WT) CD45.2+ C57BL/6, congenic CD45.1+ C57BL/6 (BoyJ), Rag2GFP (28), C57BL/6 Foxp3GFP reporter mice (29), Ccr4\u2212/\u2212 (>>30<<), Ccr7\u2212/\u2212 (31), Ccr4\u2212/\u2212xCcr7\u2212/\u2212, and Zap 70\u2212/\u2212 (32) were bred at the University of Birmingham in accordance with Home Office Regulations." . _:b555948473 . _:b555948498 . _:b555948485 . . _:b555948528 . _:b555948472 . . _:b555948643 . _:b555948475 . . _:b555948474 . . _:b32449318 "mice at E18 of gestation, we found no overt differences between Ccr4\u2212/\u2212 and WT embryonic thymus lobes, in terms of total thymocyte numbers, cortical and medullary architecture and proportions of immature and mature thymocytes (Ref. >>37<<; data not shown). Thus, initial analysis suggests that absence of CCR4 during development does not have a major impact on fetal thymus populations." . _:b555948500 . _:b555948485 . _:b32449266 "In the thymus, the development of T cells bearing the \u03B1\u03B2TCR involves the progressive migration of immature thymocyte precursors through distinct intrathymic microenvironments formed from specialized thymic stromal cells (>>1<<, 2). Although immature CD4\u22128\u2212 thymocytes are localized within subcapsular regions of the thymus, cortical regions house CD4+8+ thymocytes expressing low levels of \u03B1\u03B2TCR that represent precursors of conventional, natural Foxp3+ regulatory" . . . _:b555948484 . . _:b555948451 . . _:b555948487 . _:b555948644 . _:b555948486 . _:b555948479 . _:b32449345 . _:b555948481 . . _:b32449284 . _:b555948480 . _:b32449285 . _:b32449296 "However, although CCR4 has been studied in the peripheral immune system, notably in the context of skin-homing of T cells (>>26<<), its role during the development of distinct \u03B1\u03B2T cell lineages in the adult thymus, either individually or in combination with CCR7, has not been studied." . . _:b555948483 . _:b32449286 . _:b32449338 . _:b555948493 . _:b555948482 . _:b555948554 . . _:b32449287 . _:b555948493 . _:b32449323 "Our previous studies have shown that the generation of both Foxp3+ nT-Reg and CD1d-restricted iNKT-cells depends on normal thymus medulla development and interactions with mTEC (8, >>9<<). Given the role of CCR7 during thymocyte migration, we next analyzed the intrathymic generation of these lineages in mice lacking CCR7, either individually or in combination with a lack of CCR4." . _:b32449269 . _:b32449284 "Indeed, Foxp3+ thymocytes have been reported to accumulate in the cortex of Ccr7\u2212/\u2212 mice (16, >>17<<), although the impact of CCR7 deficiency on distinct nT-Reg progenitors and more mature Foxp3+ nT-Reg stages has not been fully addressed." . . _:b32449280 . _:b555948492 . _:b32449318 . _:b555948506 . _:b32449281 . _:b555948573 . _:b555948495 . _:b32449317 . _:b32449282 . _:b555948494 . _:b555948520 . _:b32449284 . _:b555948503 . _:b32449283 . _:b555948489 . _:b32449307 . _:b555948567 . _:b555948505 . _:b32449278 . . _:b32449292 . . _:b555948488 . _:b555948584 . . _:b32449293 . _:b555948491 . _:b555948502 . _:b32449276 . _:b32449294 . . _:b555948490 . _:b555948614 . _:b555948562 . _:b32449295 . . _:b555948501 . . . _:b32449288 . _:b555948500 . _:b32449289 . . _:b555948503 . _:b555948535 . . _:b32449290 . _:b555948502 . _:b555948532 . _:b32449291 . _:b555948497 . _:b32449300 . . _:b555948496 . . _:b32449301 . . . _:b555948499 . . _:b32449302 . _:b555948523 . _:b555948498 . _:b32449303 . . . _:b555948509 . _:b32449296 . . . . _:b555948508 . . _:b32449297 . _:b555948544 . _:b555948590 . _:b555948545 . _:b555948511 . _:b555948546 . _:b555948547 . . _:b555948548 . _:b32449298 . _:b555948549 . _:b555948510 . _:b555948550 . _:b555948650 . _:b555948551 . _:b32449299 . _:b555948552 . _:b555948553 . _:b555948505 . . _:b555948554 . _:b555948555 . _:b32449308 . . _:b555948556 . _:b555948557 . _:b555948504 . _:b555948558 . _:b32449322 "Our previous studies have shown that the generation of both Foxp3+ nT-Reg and CD1d-restricted iNKT-cells depends on normal thymus medulla development and interactions with mTEC (>>8<<, 9). Given the role of CCR7 during thymocyte migration, we next analyzed the intrathymic generation of these lineages in mice lacking CCR7, either individually or in combination with a lack of CCR4." . . . _:b32449309 . _:b555948631 . _:b555948559 . _:b555948560 . _:b555948561 . _:b555948507 . _:b555948562 . _:b555948563 . _:b32449341 "Although CCR7 has been widely studied in the context of conventional \u03B1\u03B2T cell development (10, 12, 13, 34, >>41<<), less is known about its possible role in the intrathymic development and migration of other T cell subsets." . _:b555948621 . _:b32449310 . _:b555948564 . . _:b555948506 . _:b555948565 . _:b555948448 . _:b555948566 . _:b555948567 . _:b32449311 . _:b555948568 . _:b555948457 . _:b555948517 . _:b555948569 . _:b555948570 . _:b555948571 . _:b32449304 . _:b555948572 . . _:b32449314 "Similar to conventional \u03B1\u03B2T cell development, Foxp3+ nT-Reg development in the thymus can be subdivided into distinct stages (>>6<<, 7). As we recently showed that the generation of CD25+Foxp3\u2212 nT-Reg precursors is dependent on contact with mTEC (8), we next examined the expression of chemokine receptors at distinct stages in Foxp3+ nT-Reg development that might play" . _:b555948516 . _:b555948573 . _:b555948574 . _:b32449295 . _:b555948575 . _:b32449305 . _:b555948576 . _:b555948566 . _:b555948577 . _:b555948519 . _:b555948578 . "PMC0" . _:b32449306 . _:b555948579 . _:b555948580 . _:b555948581 . _:b555948518 . _:b555948582 . _:b555948618 . _:b555948596 . _:b555948583 . _:b32449307 . . _:b555948584 . _:b555948488 . _:b555948513 . _:b555948585 . _:b555948586 . _:b555948587 . _:b32449316 . _:b555948588 . _:b555948589 . _:b555948512 . _:b555948590 . _:b555948591 . _:b32449340 . _:b32449317 . _:b555948592 . _:b555948534 . _:b555948593 . _:b555948515 . _:b555948594 . _:b555948595 . _:b32449318 . _:b555948596 . _:b32449335 "Such findings indicate that the defective Foxp3+ nT-Reg (>>22<<) and conventional \u03B1\u03B2T cell development (27) seen in Aire\u2212/\u2212 mice is not because of their reduced levels of the CCR4 ligands CCL17/CCL22." . _:b32449265 _:b32449280 . _:b555948597 . _:b555948574 . _:b555948514 . _:b32449265 _:b32449281 . _:b555948598 . _:b555948526 . _:b32449265 _:b32449282 . . _:b555948599 . _:b32449319 . _:b32449265 _:b32449283 . _:b555948600 . _:b32449265 _:b32449284 . _:b555948601 . _:b555948525 . _:b32449265 _:b32449285 . _:b555948602 . _:b32449265 _:b32449286 . _:b555948603 . _:b32449312 . _:b32449265 _:b32449287 . _:b555948604 . _:b32449300 "Wild-type (WT) CD45.2+ C57BL/6, congenic CD45.1+ C57BL/6 (BoyJ), Rag2GFP (28), C57BL/6 Foxp3GFP reporter mice (>>29<<), Ccr4\u2212/\u2212 (30), Ccr7\u2212/\u2212 (31), Ccr4\u2212/\u2212xCcr7\u2212/\u2212, and Zap 70\u2212/\u2212 (32) were bred at the University of Birmingham in accordance with Home Office Regulations." . _:b32449265 _:b32449288 . _:b555948605 . _:b555948524 . _:b32449265 _:b32449289 . . _:b555948606 . _:b32449265 _:b32449290 . _:b555948607 . _:b32449313 . _:b32449290 "In line with this, positive selection is known to alter the in vitro responsiveness of thymocytes to several chemokines including CCL17 and CCL22 (20), representing ligands for CCR4 (>>21<<). Moreover, Aire expression by MHC class IIhigh mTEC is known to influence intrathymic chemokine production (22, 23), including the ligands for CCR4 (23). Indeed, impaired CCR4-mediated thymocyte migration recently has been suggested (24)" . _:b32449265 _:b32449291 . _:b555948608 . _:b32449265 _:b32449292 . _:b555948609 . _:b555948527 . _:b32449265 _:b32449293 . _:b555948610 . _:b32449265 _:b32449294 . _:b555948470 . _:b555948611 . _:b32449314 . _:b32449265 _:b32449295 . _:b555948612 . _:b32449265 _:b32449296 . . _:b555948613 . _:b555948526 . _:b32449265 _:b32449297 . _:b555948614 . _:b555948615 . _:b32449315 . _:b555948616 . . _:b555948472 . _:b555948617 . _:b555948521 . _:b555948618 . _:b555948619 . _:b32449324 . _:b555948620 . _:b555948621 . _:b555948520 . _:b555948622 . _:b555948525 . _:b555948623 . _:b32449325 . _:b555948624 . _:b555948625 . _:b555948523 . "10.4049%2Fjimmunol.1400993" . _:b555948626 . _:b555948627 . _:b32449326 . _:b555948628 . _:b555948640 . _:b555948629 . _:b32449283 . _:b555948522 . _:b555948630 . _:b555948631 . _:b32449327 . _:b555948632 . _:b555948541 . _:b555948633 . _:b555948533 . _:b555948634 . _:b555948635 . _:b32449320 . _:b32449317 "Unlike Ccr7\u2212/\u2212 mice, detailed analysis of intrathymic migration and T cell development in adult Ccr4\u2212/\u2212 mice (>>30<<) has not been performed." . _:b555948636 . _:b555948637 . _:b555948532 . _:b555948638 . _:b555948639 . _:b32449321 . _:b555948563 . _:b555948640 . _:b555948542 . _:b555948641 . _:b32449296 . _:b555948581 . _:b555948580 . _:b555948535 . _:b555948642 . _:b32449332 "With this in mind, and as positive selection is known to induce alterations in the responsiveness of developing thymocytes to multiple chemokines (>>20<<), we sought to identify additional chemokine receptors that may be involved in cortex-to-medulla migration." . _:b555948643 . _:b32449322 . _:b555948644 . _:b555948564 .