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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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Postmitotic LN MRC network is remodeled upon initial B cell colonization. (A) RAG-2°/° Ubow mice were irradiated and reconstituted with RAG-2°/° nonfluorescent BM cells to generate chimeric mice with dTomato-expressing stromal cells. Reconstituted chimeras were adoptively transferred or not with 6 × 107 CMFDA-labeled polyclonal WT B cells to trigger FDC development in recipient LNs. 1 d later, peripheral LNs were sectioned, stained for RANK-L expression, and analyzed by confocal microscopy. L displays the width of the MRC network based on RANK-L staining. (B) The number of RANK-L+ dTomato+ cellular bodies was manually counted on tissue sections and used to calculate the density of the dTomato+ RANK-L+ MRC network in the two types of LNs, either in its globality (left) or in the first 30 µm below the SCS (right). Data are representative of 3 different experiments (2 mice per experiment, 6 LNs analyzed per mouse). (C) Representative high-magnification views of the MRC network present in the 2 types of LNs. (D) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ RAG-2°/° mice were stained for RANK-L expression and analyzed by confocal microscopy. Note the presence of MRCs Foci (F) in the SCS of these LNs devoid of B cells. Insets on the right display high-magnification views of MRC clusters. (E) Quantification of CFP++ RANK-L+ MRC clustering index in the auricular/cervical LNs of Wnt-1Cre Ubow+/+ RAG-2°/° mice. A two-tailed Student’s t test was used to determine significance. *, P < 0.05; ***, P < 0.001. Bar = median. Data are representative of 5 individual mice (4 LNs analyzed per mouse) obtained in 2 independent experiments. Each dot represents one follicle. Bars, 25 µm.
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fig7: Postmitotic LN MRC network is remodeled upon initial B cell colonization. (A) RAG-2°/° Ubow mice were irradiated and reconstituted with RAG-2°/° nonfluorescent BM cells to generate chimeric mice with dTomato-expressing stromal cells. Reconstituted chimeras were adoptively transferred or not with 6 × 107 CMFDA-labeled polyclonal WT B cells to trigger FDC development in recipient LNs. 1 d later, peripheral LNs were sectioned, stained for RANK-L expression, and analyzed by confocal microscopy. L displays the width of the MRC network based on RANK-L staining. (B) The number of RANK-L+ dTomato+ cellular bodies was manually counted on tissue sections and used to calculate the density of the dTomato+ RANK-L+ MRC network in the two types of LNs, either in its globality (left) or in the first 30 µm below the SCS (right). Data are representative of 3 different experiments (2 mice per experiment, 6 LNs analyzed per mouse). (C) Representative high-magnification views of the MRC network present in the 2 types of LNs. (D) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ RAG-2°/° mice were stained for RANK-L expression and analyzed by confocal microscopy. Note the presence of MRCs Foci (F) in the SCS of these LNs devoid of B cells. Insets on the right display high-magnification views of MRC clusters. (E) Quantification of CFP++ RANK-L+ MRC clustering index in the auricular/cervical LNs of Wnt-1Cre Ubow+/+ RAG-2°/° mice. A two-tailed Student’s t test was used to determine significance. *, P < 0.05; ***, P < 0.001. Bar = median. Data are representative of 5 individual mice (4 LNs analyzed per mouse) obtained in 2 independent experiments. Each dot represents one follicle. Bars, 25 µm.

Mentions: Next, we assessed the contribution of MRC proliferation to the development of LN FDCs. The SLOs of RAG-2°/° mice lack FDC networks that can be reconstituted by an adoptive transfer of B cells (Fu et al., 1998). We adoptively transferred 6 × 107 WT B cells to RAG-2°/° mice, and 1 wk later, when many MRCs were maturing into FDCs (Fig. 5), mice were injected i.p with EdU. 1 d later, the percentage of EdU-labeled MRCs was analyzed by flow cytometry. Surprisingly, the percentage of proliferating (EdU+) MRCs in RAG-2°/° mice after B cell transfers was similar to that observed in mice that did not receive B cells (Fig. 6 E), suggesting that B cells did not trigger MRC proliferation but rather converted them into FDCs. To investigate this possibility, we determined the impact of B cells on the MRC network 24 h after their adoptive transfer, long before the appearance of the first mature FDCs (unpublished data). To this aim, we generated mice in which all stromal cells could be independently visualized in tissue section. RAG-2°/° Ubow mice were irradiated and reconstituted with nonfluorescent RAG-2°/° BM cells. 8 wk later, chimeric mice were adoptively transferred or not with CMFDA-labeled 6 × 107 B cells. 1 d later, LNs were sectioned, stained for RANK-L expression, and analyzed by confocal microscopy (Fig. 7 A). RANK-L+ dTomato+ MRCs formed a dense meshwork of short radioresistant stromal cells below the SCS of nontransferred mice (Fig. 7, A and C, left; Katakai et al., 2008). In contrast, upon B cell adoptive transfer, MRCs spread deeper in the follicle and harbored many cellular processes (Fig. 7, A and C, right), a morphological feature shared with mature FDCs (Fig. 5). Quantification of RANK-L+ dTomato+ cell bodies and mean width of the MRC territory indicated that B cells lead to the generation of a larger but less dense MRC network, even in the first 30-µm corridor abutting the SCS (Fig. 7 B). Accordingly, the total number of MRC cell bodies was similar in the MRC networks of control and B cell–injected mice, indicating that upon their arrival in the LN, B cells induce a topographic change rather than the proliferation of the preexisting MRC network.


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)

Postmitotic LN MRC network is remodeled upon initial B cell colonization. (A) RAG-2°/° Ubow mice were irradiated and reconstituted with RAG-2°/° nonfluorescent BM cells to generate chimeric mice with dTomato-expressing stromal cells. Reconstituted chimeras were adoptively transferred or not with 6 × 107 CMFDA-labeled polyclonal WT B cells to trigger FDC development in recipient LNs. 1 d later, peripheral LNs were sectioned, stained for RANK-L expression, and analyzed by confocal microscopy. L displays the width of the MRC network based on RANK-L staining. (B) The number of RANK-L+ dTomato+ cellular bodies was manually counted on tissue sections and used to calculate the density of the dTomato+ RANK-L+ MRC network in the two types of LNs, either in its globality (left) or in the first 30 µm below the SCS (right). Data are representative of 3 different experiments (2 mice per experiment, 6 LNs analyzed per mouse). (C) Representative high-magnification views of the MRC network present in the 2 types of LNs. (D) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ RAG-2°/° mice were stained for RANK-L expression and analyzed by confocal microscopy. Note the presence of MRCs Foci (F) in the SCS of these LNs devoid of B cells. Insets on the right display high-magnification views of MRC clusters. (E) Quantification of CFP++ RANK-L+ MRC clustering index in the auricular/cervical LNs of Wnt-1Cre Ubow+/+ RAG-2°/° mice. A two-tailed Student’s t test was used to determine significance. *, P < 0.05; ***, P < 0.001. Bar = median. Data are representative of 5 individual mice (4 LNs analyzed per mouse) obtained in 2 independent experiments. Each dot represents one follicle. Bars, 25 µm.
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fig7: Postmitotic LN MRC network is remodeled upon initial B cell colonization. (A) RAG-2°/° Ubow mice were irradiated and reconstituted with RAG-2°/° nonfluorescent BM cells to generate chimeric mice with dTomato-expressing stromal cells. Reconstituted chimeras were adoptively transferred or not with 6 × 107 CMFDA-labeled polyclonal WT B cells to trigger FDC development in recipient LNs. 1 d later, peripheral LNs were sectioned, stained for RANK-L expression, and analyzed by confocal microscopy. L displays the width of the MRC network based on RANK-L staining. (B) The number of RANK-L+ dTomato+ cellular bodies was manually counted on tissue sections and used to calculate the density of the dTomato+ RANK-L+ MRC network in the two types of LNs, either in its globality (left) or in the first 30 µm below the SCS (right). Data are representative of 3 different experiments (2 mice per experiment, 6 LNs analyzed per mouse). (C) Representative high-magnification views of the MRC network present in the 2 types of LNs. (D) Auricular/cervical LN sections from Wnt-1Cre Ubow+/+ RAG-2°/° mice were stained for RANK-L expression and analyzed by confocal microscopy. Note the presence of MRCs Foci (F) in the SCS of these LNs devoid of B cells. Insets on the right display high-magnification views of MRC clusters. (E) Quantification of CFP++ RANK-L+ MRC clustering index in the auricular/cervical LNs of Wnt-1Cre Ubow+/+ RAG-2°/° mice. A two-tailed Student’s t test was used to determine significance. *, P < 0.05; ***, P < 0.001. Bar = median. Data are representative of 5 individual mice (4 LNs analyzed per mouse) obtained in 2 independent experiments. Each dot represents one follicle. Bars, 25 µm.
Mentions: Next, we assessed the contribution of MRC proliferation to the development of LN FDCs. The SLOs of RAG-2°/° mice lack FDC networks that can be reconstituted by an adoptive transfer of B cells (Fu et al., 1998). We adoptively transferred 6 × 107 WT B cells to RAG-2°/° mice, and 1 wk later, when many MRCs were maturing into FDCs (Fig. 5), mice were injected i.p with EdU. 1 d later, the percentage of EdU-labeled MRCs was analyzed by flow cytometry. Surprisingly, the percentage of proliferating (EdU+) MRCs in RAG-2°/° mice after B cell transfers was similar to that observed in mice that did not receive B cells (Fig. 6 E), suggesting that B cells did not trigger MRC proliferation but rather converted them into FDCs. To investigate this possibility, we determined the impact of B cells on the MRC network 24 h after their adoptive transfer, long before the appearance of the first mature FDCs (unpublished data). To this aim, we generated mice in which all stromal cells could be independently visualized in tissue section. RAG-2°/° Ubow mice were irradiated and reconstituted with nonfluorescent RAG-2°/° BM cells. 8 wk later, chimeric mice were adoptively transferred or not with CMFDA-labeled 6 × 107 B cells. 1 d later, LNs were sectioned, stained for RANK-L expression, and analyzed by confocal microscopy (Fig. 7 A). RANK-L+ dTomato+ MRCs formed a dense meshwork of short radioresistant stromal cells below the SCS of nontransferred mice (Fig. 7, A and C, left; Katakai et al., 2008). In contrast, upon B cell adoptive transfer, MRCs spread deeper in the follicle and harbored many cellular processes (Fig. 7, A and C, right), a morphological feature shared with mature FDCs (Fig. 5). Quantification of RANK-L+ dTomato+ cell bodies and mean width of the MRC territory indicated that B cells lead to the generation of a larger but less dense MRC network, even in the first 30-µm corridor abutting the SCS (Fig. 7 B). Accordingly, the total number of MRC cell bodies was similar in the MRC networks of control and B cell–injected mice, indicating that upon their arrival in the LN, B cells induce a topographic change rather than the proliferation of the preexisting MRC network.

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