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Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells.

Jarjour M, Jorquera A, Mondor I, Wienert S, Narang P, Coles MC, Klauschen F, Bajénoff M - J. Exp. Med. (2014)

Bottom Line: In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat.Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ.We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation.

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Affiliation: Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille Université, UM2 Marseille, France Institut National de la Santé et de la Recherche Médicale (INSERM), UMR_S 1104 Marseille, France Centre National de la Recherche Scientifique (CNRS), UMR7280 Marseille, France Aix-Marseille Univ (AMU), F-13284 Marseille, France.

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MRCs proliferate during inflammation. (A) Flow cytometry gating strategies used to identify MRCs (gp38+ MAdCam-1+ CD45− CD31− CD21/35−) and FDCs (gp38+ CD45− CD31− CD21/35+) in LN cellular suspensions. WT mice (B) and CD21Cre Ubow++ chimeras (C) were subcutaneously injected or not with an emulsion of CFA/OVA. (B) 4, 9, or 20 d later, mice received a single i.p. injection of EdU to label proliferating cells. 1 d later, the percentages of EdU+ MRCs and EdU+ FDCs present in the peripheral inflamed LNs of WT mice were analyzed by flow cytometry. (C) The percentage of EdU+ MRCs (arrowheads) and EdU+ FDCs was determined at day 5 by confocal imaging in the inflamed LNs of CD21Cre Ubow++ chimeras. Insets display high-magnification views of EdU+ MRCs. Bars, 25 µm. Data are representative of 3 experiments (at least 4 mice pooled per group in B and 2 LNs analyzed per mouse in C). (D) WT mice were injected s.c. with CFA/OVA in ears and footpads, followed by BrdU injection (i.p) on day 4. This pulse of BrdU was followed by a chase period of 1 and 3 d. At the end of the chase period, the percentage of BrdU-labeled MRCs and FDCs was determined by flow cytometry. Data are representative of 3 different experiments (5 mice pooled per group). (E) RAG-2°/° mice were adoptively transferred or not with 6 × 107 WT polyclonal B cells. 1 wk later, mice received a single injection of EdU. The proportion of EdU+ cells among the MRCs and FDCs of peripheral LNs were analyzed one day later by flow cytometry. At this stage, no mature FDCs could be recovered from the LNs of both types of mice. Data are representative of 3 different experiments (5 mice pooled per group).
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fig6: MRCs proliferate during inflammation. (A) Flow cytometry gating strategies used to identify MRCs (gp38+ MAdCam-1+ CD45− CD31− CD21/35−) and FDCs (gp38+ CD45− CD31− CD21/35+) in LN cellular suspensions. WT mice (B) and CD21Cre Ubow++ chimeras (C) were subcutaneously injected or not with an emulsion of CFA/OVA. (B) 4, 9, or 20 d later, mice received a single i.p. injection of EdU to label proliferating cells. 1 d later, the percentages of EdU+ MRCs and EdU+ FDCs present in the peripheral inflamed LNs of WT mice were analyzed by flow cytometry. (C) The percentage of EdU+ MRCs (arrowheads) and EdU+ FDCs was determined at day 5 by confocal imaging in the inflamed LNs of CD21Cre Ubow++ chimeras. Insets display high-magnification views of EdU+ MRCs. Bars, 25 µm. Data are representative of 3 experiments (at least 4 mice pooled per group in B and 2 LNs analyzed per mouse in C). (D) WT mice were injected s.c. with CFA/OVA in ears and footpads, followed by BrdU injection (i.p) on day 4. This pulse of BrdU was followed by a chase period of 1 and 3 d. At the end of the chase period, the percentage of BrdU-labeled MRCs and FDCs was determined by flow cytometry. Data are representative of 3 different experiments (5 mice pooled per group). (E) RAG-2°/° mice were adoptively transferred or not with 6 × 107 WT polyclonal B cells. 1 wk later, mice received a single injection of EdU. The proportion of EdU+ cells among the MRCs and FDCs of peripheral LNs were analyzed one day later by flow cytometry. At this stage, no mature FDCs could be recovered from the LNs of both types of mice. Data are representative of 3 different experiments (5 mice pooled per group).

Mentions: WT mice were immunized s.c. with CFA/OVA to induce FDC network remodeling in their inflamed LNs. Animals were injected with EdU 4, 9, and 20 d after immunization, and 1 d later, LN cell suspensions were stained for EdU, CD21/35, CD45, CD31, gp38, and MAdCam-1 expression and analyzed by flow cytometry (Fig. 6), as previously described (Fletcher et al., 2011). The percentage of EdU+ MRCs (CD21/35− CD45− CD31− gp38+ MAdCam-1+) and EdU+ FDCs (CD21/35+ CD45− CD31− gp38+) was then determined for each time point (Fig. 6 A). Flow cytometry analysis revealed a burst of MRC proliferation at day 5 (13.4% of EdU+ MRCs vs. 1.9% in control LNs), followed by a progressive decrease of MRC proliferation over time (5.9% of EdU+ MRCs at day 10 and 5.2% at day 21; Fig. 6 B). As an alternative approach, we used confocal imaging to quantify the percentage of EdU+ MRCs and EdU+ FDCs in the LNs of CD21Cre Ubow chimeric mice subjected to the same experimental protocol. As depicted in Fig. 6 C, in situ quantification indicated that 9.7% (56 out of 574) MRCs and 1.5% (5 out of 329) FDCs incorporated EdU at day 5 after immunization. Altogether, our results demonstrate that MRCs actively proliferate in inflamed LNs.


Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells.

Jarjour M, Jorquera A, Mondor I, Wienert S, Narang P, Coles MC, Klauschen F, Bajénoff M - J. Exp. Med. (2014)

MRCs proliferate during inflammation. (A) Flow cytometry gating strategies used to identify MRCs (gp38+ MAdCam-1+ CD45− CD31− CD21/35−) and FDCs (gp38+ CD45− CD31− CD21/35+) in LN cellular suspensions. WT mice (B) and CD21Cre Ubow++ chimeras (C) were subcutaneously injected or not with an emulsion of CFA/OVA. (B) 4, 9, or 20 d later, mice received a single i.p. injection of EdU to label proliferating cells. 1 d later, the percentages of EdU+ MRCs and EdU+ FDCs present in the peripheral inflamed LNs of WT mice were analyzed by flow cytometry. (C) The percentage of EdU+ MRCs (arrowheads) and EdU+ FDCs was determined at day 5 by confocal imaging in the inflamed LNs of CD21Cre Ubow++ chimeras. Insets display high-magnification views of EdU+ MRCs. Bars, 25 µm. Data are representative of 3 experiments (at least 4 mice pooled per group in B and 2 LNs analyzed per mouse in C). (D) WT mice were injected s.c. with CFA/OVA in ears and footpads, followed by BrdU injection (i.p) on day 4. This pulse of BrdU was followed by a chase period of 1 and 3 d. At the end of the chase period, the percentage of BrdU-labeled MRCs and FDCs was determined by flow cytometry. Data are representative of 3 different experiments (5 mice pooled per group). (E) RAG-2°/° mice were adoptively transferred or not with 6 × 107 WT polyclonal B cells. 1 wk later, mice received a single injection of EdU. The proportion of EdU+ cells among the MRCs and FDCs of peripheral LNs were analyzed one day later by flow cytometry. At this stage, no mature FDCs could be recovered from the LNs of both types of mice. Data are representative of 3 different experiments (5 mice pooled per group).
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fig6: MRCs proliferate during inflammation. (A) Flow cytometry gating strategies used to identify MRCs (gp38+ MAdCam-1+ CD45− CD31− CD21/35−) and FDCs (gp38+ CD45− CD31− CD21/35+) in LN cellular suspensions. WT mice (B) and CD21Cre Ubow++ chimeras (C) were subcutaneously injected or not with an emulsion of CFA/OVA. (B) 4, 9, or 20 d later, mice received a single i.p. injection of EdU to label proliferating cells. 1 d later, the percentages of EdU+ MRCs and EdU+ FDCs present in the peripheral inflamed LNs of WT mice were analyzed by flow cytometry. (C) The percentage of EdU+ MRCs (arrowheads) and EdU+ FDCs was determined at day 5 by confocal imaging in the inflamed LNs of CD21Cre Ubow++ chimeras. Insets display high-magnification views of EdU+ MRCs. Bars, 25 µm. Data are representative of 3 experiments (at least 4 mice pooled per group in B and 2 LNs analyzed per mouse in C). (D) WT mice were injected s.c. with CFA/OVA in ears and footpads, followed by BrdU injection (i.p) on day 4. This pulse of BrdU was followed by a chase period of 1 and 3 d. At the end of the chase period, the percentage of BrdU-labeled MRCs and FDCs was determined by flow cytometry. Data are representative of 3 different experiments (5 mice pooled per group). (E) RAG-2°/° mice were adoptively transferred or not with 6 × 107 WT polyclonal B cells. 1 wk later, mice received a single injection of EdU. The proportion of EdU+ cells among the MRCs and FDCs of peripheral LNs were analyzed one day later by flow cytometry. At this stage, no mature FDCs could be recovered from the LNs of both types of mice. Data are representative of 3 different experiments (5 mice pooled per group).
Mentions: WT mice were immunized s.c. with CFA/OVA to induce FDC network remodeling in their inflamed LNs. Animals were injected with EdU 4, 9, and 20 d after immunization, and 1 d later, LN cell suspensions were stained for EdU, CD21/35, CD45, CD31, gp38, and MAdCam-1 expression and analyzed by flow cytometry (Fig. 6), as previously described (Fletcher et al., 2011). The percentage of EdU+ MRCs (CD21/35− CD45− CD31− gp38+ MAdCam-1+) and EdU+ FDCs (CD21/35+ CD45− CD31− gp38+) was then determined for each time point (Fig. 6 A). Flow cytometry analysis revealed a burst of MRC proliferation at day 5 (13.4% of EdU+ MRCs vs. 1.9% in control LNs), followed by a progressive decrease of MRC proliferation over time (5.9% of EdU+ MRCs at day 10 and 5.2% at day 21; Fig. 6 B). As an alternative approach, we used confocal imaging to quantify the percentage of EdU+ MRCs and EdU+ FDCs in the LNs of CD21Cre Ubow chimeric mice subjected to the same experimental protocol. As depicted in Fig. 6 C, in situ quantification indicated that 9.7% (56 out of 574) MRCs and 1.5% (5 out of 329) FDCs incorporated EdU at day 5 after immunization. Altogether, our results demonstrate that MRCs actively proliferate in inflamed LNs.

Bottom Line: In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat.Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ.We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille Université, UM2 Marseille, France Institut National de la Santé et de la Recherche Médicale (INSERM), UMR_S 1104 Marseille, France Centre National de la Recherche Scientifique (CNRS), UMR7280 Marseille, France Aix-Marseille Univ (AMU), F-13284 Marseille, France.

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