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Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo.

Hammerschmidt SI, Ahrendt M, Bode U, Wahl B, Kremmer E, Förster R, Pabst O - J. Exp. Med. (2008)

Bottom Line: DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics.Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro.These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany.

ABSTRACT
T cells primed in the gut-draining mesenteric lymph nodes (mLN) are imprinted to express alpha4beta7-integrin and chemokine receptor CCR9, thereby enabling lymphocytes to migrate to the small intestine. In vitro activation by intestinal dendritic cells (DC) or addition of retinoic acid (RA) is sufficient to instruct expression of these gut-homing molecules. We report that in vivo stroma cells, but not DC, allow the mLN to induce the generation of gut tropism. Peripheral LN (pLN) transplanted into the gut mesenteries fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and prime T cells. DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics. Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro. These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

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Heterotopic chimeric Tx-pLN, but not orthotopic Tx-mLN, fail to generate gut-homing T cells in vivo. Cells isolated from OT-I or OT-II Ly5.1 mice were labeled with CFSE and adoptively transferred into mice that received Tx-pLN or Tx-mLN 8 wk before. 1 d later, a single dose of Ova was applied orally or injected s.c. (A) Representative results obtained for α4β7-integrin and CCR9 expression by OT-I T cells (DAPI−Vα2+Vβ5+CD8+) activated in the mLN, Tx-mLN, and Tx-pLN after oral antigen application and in pLN after s.c. injection of antigen. (B and C) Diagrams depict expression of α4β7-integrin and CCR9 as fold isotype control for OT-I T cells (DAPI−Vα2+Vβ5+CD8+; B) and OT-II T cells (DAPI−Ly5.1+Vβ5+CD4+; C). All experiments have been performed at least three times with two or more mice per group. Error bars represent SD.
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fig2: Heterotopic chimeric Tx-pLN, but not orthotopic Tx-mLN, fail to generate gut-homing T cells in vivo. Cells isolated from OT-I or OT-II Ly5.1 mice were labeled with CFSE and adoptively transferred into mice that received Tx-pLN or Tx-mLN 8 wk before. 1 d later, a single dose of Ova was applied orally or injected s.c. (A) Representative results obtained for α4β7-integrin and CCR9 expression by OT-I T cells (DAPI−Vα2+Vβ5+CD8+) activated in the mLN, Tx-mLN, and Tx-pLN after oral antigen application and in pLN after s.c. injection of antigen. (B and C) Diagrams depict expression of α4β7-integrin and CCR9 as fold isotype control for OT-I T cells (DAPI−Vα2+Vβ5+CD8+; B) and OT-II T cells (DAPI−Ly5.1+Vβ5+CD4+; C). All experiments have been performed at least three times with two or more mice per group. Error bars represent SD.

Mentions: To investigate the type of tissue tropism generated in such chimeric Tx-LN, TCR transgenic OT-I or OT-II T cells were fluorescently labeled with CFSE and adoptively transferred into nonmanipulated or transplanted recipients. The frequency of adoptively transferred cells homing into Tx-LN was comparable to endogenous mLN (unpublished data), indicating that Tx-LN were properly vascularized and equipped with the molecular machinery enabling efficient lymphocyte entry. On the subsequent day, mice were fed with a single dose of Ova, and a group of nontransplanted mice received a single s.c. injection of Ova. 3 d after antigen delivery, cells were isolated from gut-draining endogenous mLN or Tx-LN as well as endogenous skin-draining pLN and analyzed by flow cytometry. In line with previous reports, we observed that in nontransplanted WT mice, feeding of Ova induced T cell proliferation preferentially in the gut-draining mLN but not pLN (16). Expectedly, OT-I T cells proliferating in the endogenous mLN acquired a gut-homing phenotype, characterized by up-regulation of α4β7-integrin and CCR9 (Fig. 2, A and B). Expression of α4β7-integrin gradually increased with progressing cell divisions, whereas CCR9 was highly expressed already after one or two rounds of cell division (Fig. 2 B). Similarly, OT-II T cells up-regulated α4β7-integrin and CCR9 expression in the mLN after antigen feeding (Fig. 2 C), even though expression levels were lower compared with OT-I T cells. s.c. antigen delivery preferentially resulted in T cell proliferation in the draining inguinal LN, accompanied by no apparent expression of α4β7-integrin and CCR9 (Fig. 2, A and B).


Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo.

Hammerschmidt SI, Ahrendt M, Bode U, Wahl B, Kremmer E, Förster R, Pabst O - J. Exp. Med. (2008)

Heterotopic chimeric Tx-pLN, but not orthotopic Tx-mLN, fail to generate gut-homing T cells in vivo. Cells isolated from OT-I or OT-II Ly5.1 mice were labeled with CFSE and adoptively transferred into mice that received Tx-pLN or Tx-mLN 8 wk before. 1 d later, a single dose of Ova was applied orally or injected s.c. (A) Representative results obtained for α4β7-integrin and CCR9 expression by OT-I T cells (DAPI−Vα2+Vβ5+CD8+) activated in the mLN, Tx-mLN, and Tx-pLN after oral antigen application and in pLN after s.c. injection of antigen. (B and C) Diagrams depict expression of α4β7-integrin and CCR9 as fold isotype control for OT-I T cells (DAPI−Vα2+Vβ5+CD8+; B) and OT-II T cells (DAPI−Ly5.1+Vβ5+CD4+; C). All experiments have been performed at least three times with two or more mice per group. Error bars represent SD.
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Related In: Results  -  Collection

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fig2: Heterotopic chimeric Tx-pLN, but not orthotopic Tx-mLN, fail to generate gut-homing T cells in vivo. Cells isolated from OT-I or OT-II Ly5.1 mice were labeled with CFSE and adoptively transferred into mice that received Tx-pLN or Tx-mLN 8 wk before. 1 d later, a single dose of Ova was applied orally or injected s.c. (A) Representative results obtained for α4β7-integrin and CCR9 expression by OT-I T cells (DAPI−Vα2+Vβ5+CD8+) activated in the mLN, Tx-mLN, and Tx-pLN after oral antigen application and in pLN after s.c. injection of antigen. (B and C) Diagrams depict expression of α4β7-integrin and CCR9 as fold isotype control for OT-I T cells (DAPI−Vα2+Vβ5+CD8+; B) and OT-II T cells (DAPI−Ly5.1+Vβ5+CD4+; C). All experiments have been performed at least three times with two or more mice per group. Error bars represent SD.
Mentions: To investigate the type of tissue tropism generated in such chimeric Tx-LN, TCR transgenic OT-I or OT-II T cells were fluorescently labeled with CFSE and adoptively transferred into nonmanipulated or transplanted recipients. The frequency of adoptively transferred cells homing into Tx-LN was comparable to endogenous mLN (unpublished data), indicating that Tx-LN were properly vascularized and equipped with the molecular machinery enabling efficient lymphocyte entry. On the subsequent day, mice were fed with a single dose of Ova, and a group of nontransplanted mice received a single s.c. injection of Ova. 3 d after antigen delivery, cells were isolated from gut-draining endogenous mLN or Tx-LN as well as endogenous skin-draining pLN and analyzed by flow cytometry. In line with previous reports, we observed that in nontransplanted WT mice, feeding of Ova induced T cell proliferation preferentially in the gut-draining mLN but not pLN (16). Expectedly, OT-I T cells proliferating in the endogenous mLN acquired a gut-homing phenotype, characterized by up-regulation of α4β7-integrin and CCR9 (Fig. 2, A and B). Expression of α4β7-integrin gradually increased with progressing cell divisions, whereas CCR9 was highly expressed already after one or two rounds of cell division (Fig. 2 B). Similarly, OT-II T cells up-regulated α4β7-integrin and CCR9 expression in the mLN after antigen feeding (Fig. 2 C), even though expression levels were lower compared with OT-I T cells. s.c. antigen delivery preferentially resulted in T cell proliferation in the draining inguinal LN, accompanied by no apparent expression of α4β7-integrin and CCR9 (Fig. 2, A and B).

Bottom Line: DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics.Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro.These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany.

ABSTRACT
T cells primed in the gut-draining mesenteric lymph nodes (mLN) are imprinted to express alpha4beta7-integrin and chemokine receptor CCR9, thereby enabling lymphocytes to migrate to the small intestine. In vitro activation by intestinal dendritic cells (DC) or addition of retinoic acid (RA) is sufficient to instruct expression of these gut-homing molecules. We report that in vivo stroma cells, but not DC, allow the mLN to induce the generation of gut tropism. Peripheral LN (pLN) transplanted into the gut mesenteries fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and prime T cells. DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics. Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro. These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

Show MeSH