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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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Lineage tracing of FDC progenitors. Ubow++-CreERT2 mice were irradiated and reconstituted with WT BM cells to generate chimeric mice with fluorescent stromal cells and nonfluorescent hematopoietic cells. Reconstituted chimeras were treated with tamoxifen, maintained under tamoxifen-free regimen during 1 wk, and injected with an emulsion of CFA/OVA in their ears and rear footpads (left side only). 3 wk later, inflamed popliteal and auricular LNs (B and C) and contralateral LNs (A) were sectioned, stained for CD21/35 (dashed line) and IgD (A and B) or RANK-L (D) expression, and analyzed by confocal microscopy. (C) Quantification of CFP++ YFP++ CFP+YFP+ CD21/35+ FDC clustering index. Horizontal bar = median. Data are representative of 3 different experiments (2 mice per experiment, 4 LNs analyzed per mouse). Each dot represents one follicle. (E) Comparison of MRC/FDC cluster formation as measured by the clustering index (see Materials and methods section and Fig. S2 for details) in original and simulated data. Each dot represents the cell clustering index of a cell cluster composed of MRCs and FDCs sharing a similar color, in original and Monte Carlo–simulated data. See also Video 1. (F) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ mice were stained for CD21/35 and RANK-L expression and analyzed by confocal microscopy. Insets in D and F represent magnified views of juxtaposed MRCs and FDCs sharing a similar color. Data are representative of 3 different experiments (2 mice per experiment, 2 LNs analyzed per mouse). In E and C, a two-tailed Student’s t test was used to determine significance. *, P < 0.08; **, P < 0.01. n.s. = nonsignificant. Bars, 25 µm.
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fig3: Lineage tracing of FDC progenitors. Ubow++-CreERT2 mice were irradiated and reconstituted with WT BM cells to generate chimeric mice with fluorescent stromal cells and nonfluorescent hematopoietic cells. Reconstituted chimeras were treated with tamoxifen, maintained under tamoxifen-free regimen during 1 wk, and injected with an emulsion of CFA/OVA in their ears and rear footpads (left side only). 3 wk later, inflamed popliteal and auricular LNs (B and C) and contralateral LNs (A) were sectioned, stained for CD21/35 (dashed line) and IgD (A and B) or RANK-L (D) expression, and analyzed by confocal microscopy. (C) Quantification of CFP++ YFP++ CFP+YFP+ CD21/35+ FDC clustering index. Horizontal bar = median. Data are representative of 3 different experiments (2 mice per experiment, 4 LNs analyzed per mouse). Each dot represents one follicle. (E) Comparison of MRC/FDC cluster formation as measured by the clustering index (see Materials and methods section and Fig. S2 for details) in original and simulated data. Each dot represents the cell clustering index of a cell cluster composed of MRCs and FDCs sharing a similar color, in original and Monte Carlo–simulated data. See also Video 1. (F) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ mice were stained for CD21/35 and RANK-L expression and analyzed by confocal microscopy. Insets in D and F represent magnified views of juxtaposed MRCs and FDCs sharing a similar color. Data are representative of 3 different experiments (2 mice per experiment, 2 LNs analyzed per mouse). In E and C, a two-tailed Student’s t test was used to determine significance. *, P < 0.08; **, P < 0.01. n.s. = nonsignificant. Bars, 25 µm.

Mentions: As FDCs were not recruited from the circulation and did not divide in reactive follicles, we reasoned that they could originate from the local proliferation of CD21/35-negative FDC progenitors. According to this scenario, these progenitors would divide and give rise to daughter cells that would randomly acquire CFP or YFP expression upon CD21 expression. This postmitotic acquisition of colors would thus explain the absence of monocolored FDC foci observed in CD21Cre Ubow inflamed LNs. To unravel such a CD21/35− progenitor, we developed a third fate mapping system in which all stromal cells could undergo combinatorial recombination of the Ubow construct. RAG-2°/° Ubow+/+ mice were crossed to tamoxifen Cre-inducible Ubiquitin-Cre-ERT2 mice (Hayashi and McMahon, 2002; hereafter referred to as Ubow-CreERT2), irradiated, and reconstituted with WT BM cells. Reconstituted chimeras were treated with tamoxifen for 3 consecutive days and then maintained under a tamoxifen-free regimen for 1 wk to eliminate any remaining tamoxifen activity (Fig. S3). Mice were then injected with an emulsion of CFA/OVA in their ears and rear footpads (left side only). Inflamed (left) and control (right) auricular and popliteal LNs were harvested 3 wk later and stained for CD21/35 expression (Fig. 3). We reasoned that if CD21/35− FDC progenitors proliferated during that period, they should have generated foci of colored FDCs around them and thus unraveled their own localization in the follicle. Confocal analysis revealed that very few sparse FDCs in control follicles underwent recombination (Fig. 3 A). On the contrary, inflamed follicles were populated by nonrandomly distributed monocolored columnar foci of FDCs often connected to the subcapsular sinus (SCS; Fig. 3, B and C), indicative of local cell proliferation.


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)

Lineage tracing of FDC progenitors. Ubow++-CreERT2 mice were irradiated and reconstituted with WT BM cells to generate chimeric mice with fluorescent stromal cells and nonfluorescent hematopoietic cells. Reconstituted chimeras were treated with tamoxifen, maintained under tamoxifen-free regimen during 1 wk, and injected with an emulsion of CFA/OVA in their ears and rear footpads (left side only). 3 wk later, inflamed popliteal and auricular LNs (B and C) and contralateral LNs (A) were sectioned, stained for CD21/35 (dashed line) and IgD (A and B) or RANK-L (D) expression, and analyzed by confocal microscopy. (C) Quantification of CFP++ YFP++ CFP+YFP+ CD21/35+ FDC clustering index. Horizontal bar = median. Data are representative of 3 different experiments (2 mice per experiment, 4 LNs analyzed per mouse). Each dot represents one follicle. (E) Comparison of MRC/FDC cluster formation as measured by the clustering index (see Materials and methods section and Fig. S2 for details) in original and simulated data. Each dot represents the cell clustering index of a cell cluster composed of MRCs and FDCs sharing a similar color, in original and Monte Carlo–simulated data. See also Video 1. (F) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ mice were stained for CD21/35 and RANK-L expression and analyzed by confocal microscopy. Insets in D and F represent magnified views of juxtaposed MRCs and FDCs sharing a similar color. Data are representative of 3 different experiments (2 mice per experiment, 2 LNs analyzed per mouse). In E and C, a two-tailed Student’s t test was used to determine significance. *, P < 0.08; **, P < 0.01. n.s. = nonsignificant. Bars, 25 µm.
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fig3: Lineage tracing of FDC progenitors. Ubow++-CreERT2 mice were irradiated and reconstituted with WT BM cells to generate chimeric mice with fluorescent stromal cells and nonfluorescent hematopoietic cells. Reconstituted chimeras were treated with tamoxifen, maintained under tamoxifen-free regimen during 1 wk, and injected with an emulsion of CFA/OVA in their ears and rear footpads (left side only). 3 wk later, inflamed popliteal and auricular LNs (B and C) and contralateral LNs (A) were sectioned, stained for CD21/35 (dashed line) and IgD (A and B) or RANK-L (D) expression, and analyzed by confocal microscopy. (C) Quantification of CFP++ YFP++ CFP+YFP+ CD21/35+ FDC clustering index. Horizontal bar = median. Data are representative of 3 different experiments (2 mice per experiment, 4 LNs analyzed per mouse). Each dot represents one follicle. (E) Comparison of MRC/FDC cluster formation as measured by the clustering index (see Materials and methods section and Fig. S2 for details) in original and simulated data. Each dot represents the cell clustering index of a cell cluster composed of MRCs and FDCs sharing a similar color, in original and Monte Carlo–simulated data. See also Video 1. (F) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ mice were stained for CD21/35 and RANK-L expression and analyzed by confocal microscopy. Insets in D and F represent magnified views of juxtaposed MRCs and FDCs sharing a similar color. Data are representative of 3 different experiments (2 mice per experiment, 2 LNs analyzed per mouse). In E and C, a two-tailed Student’s t test was used to determine significance. *, P < 0.08; **, P < 0.01. n.s. = nonsignificant. Bars, 25 µm.
Mentions: As FDCs were not recruited from the circulation and did not divide in reactive follicles, we reasoned that they could originate from the local proliferation of CD21/35-negative FDC progenitors. According to this scenario, these progenitors would divide and give rise to daughter cells that would randomly acquire CFP or YFP expression upon CD21 expression. This postmitotic acquisition of colors would thus explain the absence of monocolored FDC foci observed in CD21Cre Ubow inflamed LNs. To unravel such a CD21/35− progenitor, we developed a third fate mapping system in which all stromal cells could undergo combinatorial recombination of the Ubow construct. RAG-2°/° Ubow+/+ mice were crossed to tamoxifen Cre-inducible Ubiquitin-Cre-ERT2 mice (Hayashi and McMahon, 2002; hereafter referred to as Ubow-CreERT2), irradiated, and reconstituted with WT BM cells. Reconstituted chimeras were treated with tamoxifen for 3 consecutive days and then maintained under a tamoxifen-free regimen for 1 wk to eliminate any remaining tamoxifen activity (Fig. S3). Mice were then injected with an emulsion of CFA/OVA in their ears and rear footpads (left side only). Inflamed (left) and control (right) auricular and popliteal LNs were harvested 3 wk later and stained for CD21/35 expression (Fig. 3). We reasoned that if CD21/35− FDC progenitors proliferated during that period, they should have generated foci of colored FDCs around them and thus unraveled their own localization in the follicle. Confocal analysis revealed that very few sparse FDCs in control follicles underwent recombination (Fig. 3 A). On the contrary, inflamed follicles were populated by nonrandomly distributed monocolored columnar foci of FDCs often connected to the subcapsular sinus (SCS; Fig. 3, B and C), indicative of local cell proliferation.

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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Related in: MedlinePlus