Accumulation of FDCs in reactive LNs does not rely on the recruitment or the proliferation of FDCs

One of the key functions of FDCs is the organization of GC structures devoted to the development and maturation of B cell responses (MacLennan, 1994; Allen et al., 2007; Wang et al., 2011; Victora and Nussenzweig, 2012). GCs contain enlarged FDC networks of unknown origin (Allen et al., 2004, 2007; Allen and Cyster, 2008). A previous report indicated that during carcinogenesis, at least 20% of carcinoma-associated fibroblasts originate from BM-derived mesenchymal stem cells (Quante et al., 2011). We thus sought to determine whether FDCs in inflamed follicles also arose from the recruitment of BM-derived stromal cells or derived from the proliferation of resident FDCs. We first addressed the contribution of blood-derived FDC precursors. To this aim, we took advantage of Wnt-1Cre Ubow mice in which cervical and auricular LNs display CFP⁺ and YFP⁺ FDCs at steady-state (Fig. 1 B). We injected an emulsion of OVA in CFA in the ears and rear footpads of these mice and analyzed the FDC networks of their auricular and popliteal LNs 3 wk later. As expected, confocal microscopy indicated that the FDC networks of inflamed auricular LNs contained CFP⁺ and YFP⁺ FDCs (Fig. 2 A, left). However, none of the inflamed popliteal LNs displayed CFP⁺ or YFP⁺ FDCs, ruling out the possibility that circulating neural crest–derived mesenchymal precursors migrated into reactive LNs and generated FDCs (Fig. 2 A, right). To exclude the implication of other circulating mesenchymal derived progenitors, we set up parabiotic pairs of mice consisting of CD21Cre Ubow^+/− µMT mice surgically attached to WT counterparts (Kitamura et al., 1991; Waskow, 2010; Fig. 2 B). In such parabionts, both mice share their blood circulation and thus a common probability to recruit a putative circulating FDC precursor. If such precursor arises from the CD21Cre Ubow^+/− µMT partner, it should generate CFP⁺ or YFP⁺ mature CD21/35⁺ FDCs upon its engraftment in the reactive LNs of the WT mouse. 3 mo after the surgery, the WT partners (all displaying full chimerism) were injected with CFA/OVA in their rear footpads. 3 wk later, their inflamed popliteal LNs were analyzed by confocal microscopy for the presence of CFP⁺ or YFP⁺ CD21/35⁺ FDCs. Analysis indicated that CFP⁺ or YFP⁺ CD21/35⁺ FDCs were absent in the inflamed follicles of the WT parabionts (Fig. 2 B) while all the follicles of CD21Cre Ubow^+/− µMT partners displayed YFP or CFP FDC networks as a result of WT B cell seeding. Altogether, our results rule out the possibility that reactive FDC networks incorporate putative blood-derived FDCs.

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Figure 2.

Reactive FDCs do not originate from circulating progenitors. (A) Wnt-1Cre Ubow^+/− mice were injected with an emulsion of CFA/OVA in their ears and rear footpads. 3 wk later, inflamed auricular and popliteal draining LNs were sectioned, stained for CD21/35 and IgD expression, and analyzed by confocal microscopy. Insets display high-magnification views of FDCs. Data are representative of 3 different experiments (2 mice per experiment, 4 analyzed LNs per mouse). (B) WT mice were joined surgically with naive CD21Cre Ubow^+/− µMt mice to create parabiotic mice. 3 mo later, when full chimerism was achieved, the WT partner was injected with CFA/OVA in its rear footpads. 3 wk later, popliteal LNs of both mice were sectioned, stained for CD21/35 and IgD expression, and the presence of colored FDCs in their LNs was analyzed by confocal microscopy. The development of mature FDC networks in the µMt mouse combined to the appearance of dTomato⁺ leukocytes in the WT partner indicates successful chimerism. Data are representative of 3 different experiments (2 mice per experiment). (C) CD21Cre Ubow^+/+ chimeras were injected with an emulsion of CFA/OVA in their ears and rear footpads. 3 wk later, inflamed draining LNs were sectioned, stained for CD21/35, and IgD expression and analyzed by confocal microscopy. (D) Quantification of CFP⁺⁺ CD21/35⁺ FDC clustering index in the follicles of CD21Cre Ubow^+/+ chimera. Horizontal bar = median. Data are representative of 4 different experiments (2 mice per experiment, 4 LNs analyzed per mouse). Each dot represents one follicle. In A and D, a two-tailed Student’s t test was used to determine significance. n.s = nonsignificant. Bars, 25 µm.

We next investigated the contribution of local FDC and VSC proliferation to the development of reactive follicles. To this aim, CD21Cre Ubow^+/− and ^+/+ chimeras were injected with CFA/OVA and their draining LNs were analyzed by confocal microscopy 3 wk later. As the LN FDCs of these mice did not assemble into monocolored clusters at steady-state (Fig. 1 E), any appearance of such clusters in inflamed LNs would reveal the proliferation of mature FDCs or VSCs. Confocal pictures and quantification indicated that FDCs were randomly distributed within reactive follicles (Fig. 2, C and D), suggesting that FDC and VSC proliferation does not constitute a major source of FDCs in reactive follicles.

FDC progenitors proliferate and differentiate in reactive follicles

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.

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Figure 3.

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.

As such columns and clusters were absent in the inflamed LNs of CD21Cre Ubow chimeric mice, we concluded that they should have been generated by FDC progenitors before their differentiation into mature CD21/35-expressing FDCs. We thus undertook the identification of these progenitors.

MRCs act as FDC progenitors during B cell follicle development and remodeling

The outer edge of follicles is populated by MRCs, a poorly defined stromal cell population thought to descend from lymphoid tissue organizer (LTo) cells (Katakai et al., 2008, 2012). MRCs express receptor activator of NF-κB ligand (RANK-L) and mucosal vascular addressin cell adhesion molecule 1 (MAdCam-1), and construct a reticulum of stromal cells between the SCS and the underlying FDC network. The unique location of MRCs combined with the arrangement of FDCs in clusters connected to the SCS in the primary follicles of Wnt-1Cre Ubow mice and in the GCs of immunized Ubow-CreERT2 mice raised the interesting possibility that MRCs could be the precursors of LN FDCs during ontogeny and inflammation (Figs. 1 B and 3 B).

To test this hypothesis, we first stained LN sections harvested from resting Wnt-1Cre Ubow^+/+ mice and inflamed Ubow-CreERT2 chimeras for RANK-L expression (Fig. 3, D and F; and Video 1). Confocal pictures indicated that in both types of mice, monocolored columns of stromal cells contained nonrandomly associated RANK-L^hi CD21/35⁻ MRCs juxtaposed to RANK-L⁻ CD21/35⁺ mature FDCs, suggesting that MRCs and FDCs derived from a common progenitor and/or that MRCs gave rise to the underlying FDCs (Fig. 3 E and Fig. S2).

To address the affiliation of MRCs and FDCs, we used another feature of the Ubow mouse. In the Ubow mouse, all cells express dTomato and upon Cre action, cells undergo a single recombination event that triggers YFP or CFP expression. Although Cre genetically switches off the production of dTomato in any Ubow-expressing cell, it does not act on the pool of cytoplasmic dTomato protein that should slowly decrease and eventually wash out (Fig. 4 A). In summary, any Ubow cell that expresses Cre should transiently coexpress dTomato and YFP (or CFP) before acquiring its final color (YFP or CFP). As a proof of principle, we treated Ubow^+/− CreERT2 mice with a single dose of tamoxifen and followed the colors of their blood lymphocytes over time (Fig. 4 B). At day 0, all lymphocytes exclusively expressed dTomato. 1 d later, dTomato⁺ lymphocytes started to express CFP or YFP. During the subsequent days, CFP and YFP expression progressively increased in dTomato-expressing cells. The first single, bright YFP⁺ (or CFP⁺) cells negative for dTomato expression appeared at day 6 while the remaining transitional cells gradually lost dTomato in an asynchronous manner, probably reflecting a gradual tamoxifen-induced recombination and intrinsic differences in fluorescent protein degradation (Fig. S3). Altogether, these results indicate that, upon Cre activation, any Ubow cell goes through a transitional step characterized by the coexpression of dTomato and CFP or YFP.

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Figure 4.

Cellular filiation in the Ubow mouse. (A) Schematic describing that when Ubow cells express Cre, they simultaneously lose dTomato and acquire CFP or YFP expression. Applied to a precursor/product relationship and in combination with the proper Cre reporter strain, this system unravels the transitional stage between progenitors and their differentiated cells. (B) Non-chimeric Ubow^+/−-CreERT2 mice were injected with a single dose of tamoxifen. Mice were bled every day and the relative proportion of CFP-, YFP-, and dTomato-expressing B220⁺ B cells were assessed by flow cytometry (same results were observed in CD3⁺ T cells). Data are representative of 3 different mice from 3 different experiments. (C) Schematic showing the expected phenotypes and colors of MRCs (RANK-L^hi CD21^neg) and immature (RANK-L^med CD21^med) and mature FDCs (RANK-L^neg CD21^hi) in CD21Cre Ubow mice according to the model in which MRCs give rise to FDCs.

Having validated the Ubow mouse as a powerful tool to track transitional cells, we used CD21Cre Ubow mice to determine the putative filiation of MRCs and FDCs during ontogeny and inflammation. If MRCs were FDC progenitors, they should proceed through sequential maturation steps. Upon differentiation, MRCs should evolve from their primordial phenotype (CD21/35⁻ RANK-L^hi dTomato^hi CFP/YFP^neg), then adopt a transitional stage (CD21/35⁺ RANK-L^med dTomato^med CFP/YFP^med), before acquiring the mature FDC status (CD21/35⁺ RANK-L^neg dTomato^neg CFP/YFP^hi; Fig. 4 C).

As LN MRCs reside below the SCS, we analyzed the outer follicle of inflamed LNs harvested from CD21Cre Ubow^+/+ chimeras. Confocal pictures indicated that CD21/35⁻ RANK-L^hi dTomato⁺ CFP^neg YFP^neg MRCs formed a cellular layer below the SCS of reactive follicles (Fig. 5 A). Interestingly, a discrete population of maturing CD21/35⁺ RANK-L^med dTomato^med CFP^med (or YFP^med) FDCs was immediately juxtaposed to this MRC layer and contiguous to the more central population of mature CD21/35⁺ RANK-L^neg dTomato^neg CFP^hi (or YFP^hi) FDCs. As LN FDCs cannot be efficiently isolated from nonirradiated LNs, we next quantified the colors and numbers of MRCs and transitional and mature FDCs on tissue sections. The great majority of CD21/35⁻ RANK-L⁺ MRCs expressed dTomato in the absence of YFP or CFP while almost all mature CD21/35⁺ RANK-L⁻ FDCs expressed YFP or CFP in absence of dTomato (Fig. 5 C). Interestingly, 77% of CD21/35⁺ RANK-L⁺ cells (172 out of 223 cells) coexpressed dTomato and YFP or CFP, confirming their transitional stage between MRCs and mature FDCs. In addition, these transitional cells represented 1.9% of all RANK-L⁺ cells (30 out of 1,514 cells) in control LNs while they represented 16.6% of all RANK-L⁺ cells (323 out of 1,935 cells) in inflamed LNs, suggesting that such transition was increased in reactive follicles. To rule out the contribution of an unknown follicular cell type to the generation of FDCs, we also analyzed the colors of all RANK-L⁻ CD21/35⁻ fluorescent cells present in the follicles of these mice. These data show that the majority of these cells expressed dTomato while very few of them coexpressed dTomato and YFP (or CFP), confirming that the transitional phenotype was almost exclusively observed in MRCs and FDCs. Further analysis indicated that dTomato⁺ RANK-L⁺ MRCs expressed CXCL13 but did not display the pre-FDC markers NG2 and Mfge-8 (Krautler et al., 2012; not depicted), supporting the recent evidence that splenic and LN FDC progenitors are of different origins (Castagnaro et al., 2013). These results indicate that MRCs give rise to FDCs during inflammation-induced remodeling of B cell follicles.

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Figure 5.

Tracking CD21⁻ FDC progenitors. (A) CD21Cre Ubow^+/+ chimeras were injected with an emulsion of CFA/OVA in their ears and rear footpads to trigger FDC remodeling in their draining LNs. 3 wk later, inflamed draining LNs were sectioned and stained for CD21/35 and RANK-L expression. (B) CD21Cre Ubow^+/− µMT mice were adoptively transferred with 6 × 10⁷ WT polyclonal B cells to trigger FDC development. 1 wk later, peripheral LNs were sectioned and stained for CD21/35 and RANK-L expression. In A and B, data are representative of 3 different experiments (3 mice per experiment, 4 LNs analyzed per mouse). Insets display high-magnification views of MRC and transitional and mature FDCs. (C) Confocal pictures acquired in all the experiments described in A and B were used to determine the colors and absolute numbers of MRCs, transitional and mature FDCs, and other cells present in B cell follicles. Bars, 25 µm.

We next analyzed the ability of MRCs to generate FDCs during follicle development. To this aim, we adoptively transferred WT B cells in nonirradiated CD21Cre Ubow^+/− µMt mice devoid of B cells and FDCs and determined the location, phenotype, and colors of MRCs and FDCs 1 wk later by confocal microscopy (Fig. 5, B and C). As expected, no FDCs and no transitional cells were observed in mice that were not injected with B cells (unpublished data) while LN B cell follicles of mice injected with B cells displayed MRCs, FDCs, and transitional cells. Transitional cells represented 20% of all RANK-L⁺ cells (362 out of 1,460 cells) and expressed dTomato together with YFP or CFP, demonstrating that these cells recently switched on the Cr2/CD21 promoter. Interestingly, 45% of CD21/35⁺ RANK-L⁻ FDCs (619 out of 1,370 cells) coexpressed dTomato and YFP (or CFP) 7 d after B cell transfer. Because at least 6 d was required to observe a full switch of colors in Ubow cells undergoing Cre-mediated recombination (Fig. 4 B), these transitional FDCs could represent newly differentiated cells that have not yet acquired their final color. To test this hypothesis, we analyzed the colors of FDCs in the LNs of CD21Cre Ubow^+/− µMt adoptively transferred with B cells 9 d before. As expected, the proportion of transitional CD21/35⁺ FDCs expressing both dTomato and YFP (or CFP) dropped to 12% (42 out of 350 CD21/35⁺ FDCs; not depicted) in these LNs, suggesting that these FDCs represent newly differentiated cells. Although we cannot rule out a contribution of other cell types to the generation of FDCs, our results strongly suggest that MRCs give rise to FDCs during B cell follicle development and inflammation-induced remodeling.

We thank Jean Livet for providing the brainbow 1.0L construct, Clément Ghigo and Jonathan Nowak for technical help, Emilie Narni-Mancinelli and Toby Lawrence for scientific comments and discussions, and the ImagImm photonic microscopy facility of the CIML.

This work was supported by grants from the Agence Nationale de la Recherche (ANR; ANR-RCS, ANR-STROMA, and ANR-10-INBS-04-01 France Bio Imaging), the Medical Research Council (MRC G0601156), and the Human Frontier Science Program (Young Investigator Grants RGY0077/2011 and RGP006/2009).

The authors have no conflicting financial interests.

Author contributions: M. Jarjour, I. Mondor, A. Jorquera, S. Wienert, and P. Narang performed the experiments. F. Klauschen, M. Jarjour, M.C. Coles, and M. Bajénoff designed the experiments and wrote the manuscript.