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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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mLN- but not pLN-derived stroma cells express high levels of RALDH and support the induction of CCR9 on proliferating T cells. (A) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells and CD103+ as well as CD103− DC (CD11c+MHCII+) purified from pLN and mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR and is depicted as fold expression compared with GAPDH. Data depict the mean and SD of three to five independent experiments measured in duplicates. (B and C) CFSE-labeled OT-I cells were activated by anti-CD3/CD28 treatment in the presence of either pLN- or mLN-derived stroma cells with or without addition of retinol. Stroma cells were enriched by adherence and culture of digested LN cell suspensions over 10 d. Data shown represent the mean and SD of three independent experiments. (D) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells (open bars) and CD103+ DC (closed bars) of CCR7-deficient mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR. Bars depict the mean and SD of fold expression compared with GAPDH observed in two independent experiments. (E) CCR9 expression was analyzed after i.l. injection of antigen-loaded BM-DC into mLN afferent lymphatics of CCR7-deficient mice. In contrast to the situation in WT mice, BM-DC failed to support the induction of CCR9 on T cells in CCR7-deficient mice. Expression of α4β7-integrin was not significantly affected. Results depict data obtained in one out of two experiments performed with five mice. Error bars represent SD.
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fig5: mLN- but not pLN-derived stroma cells express high levels of RALDH and support the induction of CCR9 on proliferating T cells. (A) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells and CD103+ as well as CD103− DC (CD11c+MHCII+) purified from pLN and mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR and is depicted as fold expression compared with GAPDH. Data depict the mean and SD of three to five independent experiments measured in duplicates. (B and C) CFSE-labeled OT-I cells were activated by anti-CD3/CD28 treatment in the presence of either pLN- or mLN-derived stroma cells with or without addition of retinol. Stroma cells were enriched by adherence and culture of digested LN cell suspensions over 10 d. Data shown represent the mean and SD of three independent experiments. (D) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells (open bars) and CD103+ DC (closed bars) of CCR7-deficient mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR. Bars depict the mean and SD of fold expression compared with GAPDH observed in two independent experiments. (E) CCR9 expression was analyzed after i.l. injection of antigen-loaded BM-DC into mLN afferent lymphatics of CCR7-deficient mice. In contrast to the situation in WT mice, BM-DC failed to support the induction of CCR9 on T cells in CCR7-deficient mice. Expression of α4β7-integrin was not significantly affected. Results depict data obtained in one out of two experiments performed with five mice. Error bars represent SD.

Mentions: Because RA can mimic the effects of GALT DC in vitro and addition of RA antagonists counteracts the ability of GALT DC to generate gut-homing T cells, it is tempting to speculate that stroma cells might directly or indirectly influence RA levels in LN. To determine a potential contribution of stromal cells to RA production, we compared the expression of the RA producing enzymes RALDH1, 2, and 3 in purified stroma cells and CD103+ and CD103−DC isolated from mLN or pLN. Stroma cells were sorted as CD45−CD24−gp38+ cells to >95% purity and contained no detectable DC (unpublished data). As published previously, RALDH2 expression was substantially higher in CD103+ mLN DC compared with their CD103− counterparts (Fig. 5 A) (19). Surprisingly, CD103− pLN DC showed elevated expression levels of RALDH2. This indicates that RALDH2 expression by DC does not necessarily correlate with expression of CD103 or with their ability to instruct CCR9 expression on activated T cells. Interestingly, comparison of RALDH expression between pLN- and mLN-derived stroma cells revealed striking differences. Exclusively mLN-derived stroma cells showed robust expression of RALDH2, whereas expression of this enzyme was virtually absent from pLN stroma cells (Fig. 5 A). Moreover, expression of RALDH1 and 3 was substantially higher in mLN- compared with pLN-derived stroma cells (Fig. 5 A). In contrast, no differences were observed in expression of the Vitamin A–metabolizing alcohol dehydrogenases ADH1, ADH4, and ADH5 (unpublished data).


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)

mLN- but not pLN-derived stroma cells express high levels of RALDH and support the induction of CCR9 on proliferating T cells. (A) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells and CD103+ as well as CD103− DC (CD11c+MHCII+) purified from pLN and mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR and is depicted as fold expression compared with GAPDH. Data depict the mean and SD of three to five independent experiments measured in duplicates. (B and C) CFSE-labeled OT-I cells were activated by anti-CD3/CD28 treatment in the presence of either pLN- or mLN-derived stroma cells with or without addition of retinol. Stroma cells were enriched by adherence and culture of digested LN cell suspensions over 10 d. Data shown represent the mean and SD of three independent experiments. (D) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells (open bars) and CD103+ DC (closed bars) of CCR7-deficient mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR. Bars depict the mean and SD of fold expression compared with GAPDH observed in two independent experiments. (E) CCR9 expression was analyzed after i.l. injection of antigen-loaded BM-DC into mLN afferent lymphatics of CCR7-deficient mice. In contrast to the situation in WT mice, BM-DC failed to support the induction of CCR9 on T cells in CCR7-deficient mice. Expression of α4β7-integrin was not significantly affected. Results depict data obtained in one out of two experiments performed with five mice. Error bars represent SD.
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Related In: Results  -  Collection

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fig5: mLN- but not pLN-derived stroma cells express high levels of RALDH and support the induction of CCR9 on proliferating T cells. (A) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells and CD103+ as well as CD103− DC (CD11c+MHCII+) purified from pLN and mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR and is depicted as fold expression compared with GAPDH. Data depict the mean and SD of three to five independent experiments measured in duplicates. (B and C) CFSE-labeled OT-I cells were activated by anti-CD3/CD28 treatment in the presence of either pLN- or mLN-derived stroma cells with or without addition of retinol. Stroma cells were enriched by adherence and culture of digested LN cell suspensions over 10 d. Data shown represent the mean and SD of three independent experiments. (D) cDNA was prepared from sorted CD45−CD24−gp38+ stroma cells (open bars) and CD103+ DC (closed bars) of CCR7-deficient mLN. Expression of RALDH1, 2, and 3 was assessed by real-time PCR. Bars depict the mean and SD of fold expression compared with GAPDH observed in two independent experiments. (E) CCR9 expression was analyzed after i.l. injection of antigen-loaded BM-DC into mLN afferent lymphatics of CCR7-deficient mice. In contrast to the situation in WT mice, BM-DC failed to support the induction of CCR9 on T cells in CCR7-deficient mice. Expression of α4β7-integrin was not significantly affected. Results depict data obtained in one out of two experiments performed with five mice. Error bars represent SD.
Mentions: Because RA can mimic the effects of GALT DC in vitro and addition of RA antagonists counteracts the ability of GALT DC to generate gut-homing T cells, it is tempting to speculate that stroma cells might directly or indirectly influence RA levels in LN. To determine a potential contribution of stromal cells to RA production, we compared the expression of the RA producing enzymes RALDH1, 2, and 3 in purified stroma cells and CD103+ and CD103−DC isolated from mLN or pLN. Stroma cells were sorted as CD45−CD24−gp38+ cells to >95% purity and contained no detectable DC (unpublished data). As published previously, RALDH2 expression was substantially higher in CD103+ mLN DC compared with their CD103− counterparts (Fig. 5 A) (19). Surprisingly, CD103− pLN DC showed elevated expression levels of RALDH2. This indicates that RALDH2 expression by DC does not necessarily correlate with expression of CD103 or with their ability to instruct CCR9 expression on activated T cells. Interestingly, comparison of RALDH expression between pLN- and mLN-derived stroma cells revealed striking differences. Exclusively mLN-derived stroma cells showed robust expression of RALDH2, whereas expression of this enzyme was virtually absent from pLN stroma cells (Fig. 5 A). Moreover, expression of RALDH1 and 3 was substantially higher in mLN- compared with pLN-derived stroma cells (Fig. 5 A). In contrast, no differences were observed in expression of the Vitamin A–metabolizing alcohol dehydrogenases ADH1, ADH4, and ADH5 (unpublished data).

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