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Originally published online as doi:10.1189/jlb.0105047 on November 10, 2005

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(Journal of Leukocyte Biology. 2005;78:1106-1117.)
© 2005 by Society for Leukocyte Biology

Fas receptor signaling is requisite for B cell differentiation

Valérie Pasqualetto*, Florence Vasseur*, Flora Zavala{dagger}, Elke Schneider{ddagger} and Sophie Ezine*,1

* INSERM U.591, Institut Necker, Paris, France; and
{dagger} 1NSERM U580 and
{ddagger} CNRS UMR 8147, Hôpital Necker, Paris, France

1Correspondence: INSERM U.591, Institut Necker, 156 rue de Vaugirard, 75730 Paris, Cedex 15, France. E-mail: ezine{at}necker.fr


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ABSTRACT
 
The Fas/Fas ligand (FasL) pathway has been largely implicated in the homeostasis of mature cells. However, it is still unclear whether it plays a role at the progenitor level. To address this issue, we created chimeric mice by transferring C57BL/6 bone marrow (BM) cells of the lpr (Fas–FasL+) or gld (Fas+FasL–) genotype into Rag-2–/– hosts of the same genetic background. In this model, the consequences of a deficient Fas/FasL pathway on lymphoid differentiation could be evaluated without endogenous competition. Analysis of the chimerism revealed a differential sensitivity of hematopoietic lineages to the lack of Fas receptor signaling. While donor-derived myelo-monocytic cells were similarly distributed in all chimeric mice, mature B cells were deleted in the BM and the spleen of lpr chimera, leading to the absence of the marginal zone (MZ) as detected by immunohistology. In contrast, B cell hematopoiesis was complete in gld chimera but MZ macrophages undetectable. These defects suggest a direct and determinant dual role of FasL regulation in negative selection of B cells and in maintenance of the MZ.

Key Words: FasL • gld • lpr • marginal zone


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INTRODUCTION
 
In normal mice, Fas (CD95/APO-1) and Fas ligand (FasL) genes encode 45 and 40 kDa membrane proteins, respectively, which belong to the tumor necrosis factor (TNF)/nerve growth factor receptor family [1 , 2 ]. In mice homozygous for the lpr mutation the defective expression of Fas is caused by the insertion of a retroviral transposon into the second intron of the gene, thus preventing its normal transcription. In gld mice, a point mutation at the protein level generates a nonfunctional form of FasL on the cell surface. Defective interactions between Fas and FasL give rise to a similar progressive disease in lpr and gld mice, characterized by lymphadenopathy, splenomegaly, hyper-gammaglobulinemia, and autoimmune disease [3 4 5 ].

Fas and FasL play important roles in lymphocyte homeostasis as evidenced, in vivo, by the characteristic lymphadenopathy in deficient mice [3 ]. This pathway has been implicated in the control of T cell expansion during the immune response [6 ] and in the cytolytic activity of activated T cells [7 ]. Its immunoregulatory functions are exerted mainly through induction of activation-induced cell death of T cells [8 ], B cells [9 ], and macrophages [10 ]. FasL expression on T cells participates also in the deletion of activated B cells expressing Fas [11 ].

However, dysregulation of the Fas/FasL system can also affect earlier events in murine hematopoietic stem cell development. Indeed, we have shown that increased extramedullary hematopoiesis occurs in fetal and adult lpr and gld mice [12 ]. It results mainly in the expansion of the myelo-monocytic compartment in the spleen, and the bone marrow (BM) stem cell content is equivalent to that of age-matched controls [12 ].

Studies on hematopoietic repopulation by lpr BM cells have been hampered so far by the lack of an experimental model ensuring their stable engraftment without a typical "wasting syndrome" [13 ]. We have circumvented this difficulty by analyzing the reconstitution of the hematopoietic compartment in Rag-2–/– hosts grafted with lpr BM cells [14 , 15 ]. We studied the kinetics of lymphoid development, as well as the restoration of the splenic compartments using immunohistology. We provide evidence, at one month posttransplantation, of progressive loss of mature B cells and transitional T1 cells, followed by disorganization of the splenic micro-architecture and elimination of the marginal zone (MZ). The origin of these defects is an arrest of differentiation at the pro-B cell stage in the BM of lpr chimera. Our data imply that abnormal regulation of Fas/ FasL is directly involved in negative selection of B cell progenitors in these chimera.


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MATERIALS AND METHODS
 
Mice
C57BL/6 (B6; Ly5.2, Thy1.2), C57BL/6-lpr/lpr (lpr; Ly5.2, Thy1.2), C57BL/6-gld/gld (gld; Ly5.2, Thy1.2), C57BL/6-Rag-2–/– (Ly5.1, Thy1.2; referred to as Rag-2–/–), and C57BL/6-Rag2–/–-lpr/lpr (Ly5.1,Thy1.2; referred to as Rag2–/–-lpr/lpr) mice were purchased from CDTA-CNRS (Orléans, France) and maintained in our animal facility. Rag-2–/– and B6 mice were used between 6 and 12 weeks of age, while lpr and gld mice were between 2 to 4 months old. Rag-2–/–-lpr/lpr were generated in our facility. To study germinal center formation, chimeric mice were injected intraperitoneally at the indicated times, with 2 x 108 sterile sheep red blood cells (SRBC) and spleens were removed 8-10 days later for histological analysis.

Depletion of T and B cells
BM cells were depleted of mature T and B cells by sequential incubation at 4°C for 30 min with anti-CD3 (KT3) [16 ] and anti-B220 (RA3-6B2) rat anti-mouse mAb, respectively, followed by treatment with anti-rat magnetic beads (Dynal, Biosys, France).

BM transfer
Rag-2–/– mice were subjected to 600 rad irradiation 3 to 4 h before i.v. reconstitution with 1 x 106 T and B cell-depleted BM cells from sex-matched donors. B6, lpr and gld chimera were thus generated in Rag-2–/– recipients grafted with the respective donors.

Cytokine assay
TNF-{alpha} was measured with a commercial ELISA from R & D Systems (Minneapolis, MN), according to the manufacturer’s instructions.

Immunohistology
Spleens were embedded in OCT-compound, frozen on dry ice, and stored at –20°C. Cryostat sections of 5-µm thickness were fixed in acetone for 10 min and air-dried. After washing with PBS, the sections were pre-incubated with avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA) and stained with rat antibodies specific for T cells (clone KT3) [16 ], B cells (clone RA3-6B2), follicular dendritic cells (FDC; clones FDC M1 and FDC M2, a gift from M. Kosco-Vilbois, Geneva, Switzerland) [17 ], marginal-zone macrophages (clone ER-TR9) [18 ] (BMA), marginal metallophilic macrophages (clone MOMA-1) [19 ] (Serotec), MadCAM-1 vascular addressin (clone MECA 367, BD Biosciences, San Jose, CA) [20 ], reticular fibroblasts (clone ER-TR7) [21 ], and red pulp macrophages (clone F4/80) [22 ]. These antibodies were detected with biotin-conjugated mouse anti-rat IgG Ab (Jackson Immunoresearch Lab, West Grove, PA) or with biotin-conjugated mouse anti-rat IgM Ab (BD Biosciences), followed by incubation with avidin-biotin peroxydase-complexes (Vectastain ABC Kit, Vector Laboratories). The histochemical color development was achieved by the Vectastain DAB (3,3'-diaminobenzidine) substrate kit (Vector Laboratories). Dendritic cells were revealed by anti-CD11c (clone N418) followed by incubation with biotin-conjugate mouse anti-hamster IgG Ab (BD Biosciences). Finally, the sections were counterstained with hematoxylin. Germinal centers were stained with the plant lectin peanut agglutinin (PNA) conjugated with horseradish peroxidase (Vector Laboratories).

Flow cytometric analysis
Single-cell suspensions prepared from thymus, spleen, lymph nodes, and BM were subjected to fluorescence staining with appropriate Abs. To block nonspecific Fc receptor binding, cells were preincubated for 10 min at room temperature with supernatant from the 2.4G2 hybridoma cell line, which produces antibodies against CD16 and CD32. Abs labeled with FITC, PE, and cychrome were used, as well as streptavidin-Tricolor (TC) or -allophycocyanine (APC) as second-step reagents. Staining was performed with the following mAbs: anti-TCR{alpha}β (clone H57-597), anti-IgM (clone AF6-78), anti-IgD (clone 217-170), anti-CD19 (clone 1D3, BD Biosciences), anti-CD21 (clone 7G6), anti-CD23 (clone B3B4), anti-Fas (clone Jo2, BD Biosciences), anti-FasL (clone MFL3, BD Biosciences), anti-macrophage (clone M1-70), and anti-granulocytes (clone RA3-8C5). Anti-CXCR5 (BLR1) [23 ] was a gift from R. Forster (Berlin, Germany). Flow cytometry analysis was performed with a FacScan or a Facscalibur (BD Biosciences). A minimum of 20,000 events was acquired and analyzed with CellQuest Software (BD Biosciences).

Bromodeoxyuridine (BrdUrd) incorporation
BrdUrd (Sigma Chemical Co., St. Louis, MO) was injected (1 mg/mouse) i.p. twice, at 30-min intervals. To detect and characterize DNA-synthesizing cells, spleens were removed 30 min after the second injection. Surface-stained CD4 (-PE) and CD8 (-Cychrome, BD Biosciences) cells were fixed and permeabilized in PBS containing 1% paraformaldehyde plus 0.01% Tween-20 for 24–48 h at 4°C in the dark. After washing in PBS and then in DNase buffer 1x, cells were incubated for 30 min at 37°C in the same buffer containing 50 U of DNase I (Pharmacia, Uppsala, Sweden). After another washing in PBS, they were incubated at room temperature in PBS containing 0.5% Tween-20 and FITC-conjugated anti-BrdUrd mAb (BD Biosciences).

Deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay
Flow cytometry was used for the detection of cells undergoing apoptosis. Surface-stained CD4, CD8, or B220 cells were fixed and permeabilized in PBS containing 4% paraformaldehyde and 0.5% Tween-20 for 5 min at 37°C. Cells were then washed and incubated with TUNEL mix containing 5 Units terminal transferase (TdT) for 30 min at 37°C in the dark. TdT was omitted from the mix to be used with the negative controls.

Statistics
All results were expressed as mean ± SEM, and significance was evaluated according to Student’s t-test.


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RESULTS
 
Generation of the splenic architecture in Rag-2–/– mice having received lpr BM
It is not clear yet at what stage of lymphoid development the Fas/FasL pathway comes into play. To address the question of the in vivo requirement of FasL signaling during myeloid and lymphoid lineage development from the donor type, a previously developed chimera model was used [14 , 15 ]. Sublethally irradiated Rag-2–/– hosts having received T and B cell-depleted BM cells from B6 (Fas+FasL+), lpr (Fas–FasL+), or gld (Fas+FasL–) donor mice were analyzed for up to 7 months.

Immuno-histological analyses were performed in spleen sections, using specific markers to define the micro-architecture as well as the major populations colonizing and developing within the recipient’s spleen after transplantation of lpr BM: the development of lymphoid and myeloid Fas– populations was studied within a wild type environment.

In untreated Rag-2–/– mice, due to the lack of mature T and B cells, the white pulp is not fully mature. Only small rare clusters (5–10 cells) of B220+ cells representing the pro-B cell compartment were found scattered over the sections (not shown).

After transplantation of lpr BM, CD3+ T cells were observed as early as 20 days post-graft when B220+ lpr B cells segregated into distinct compartments (not shown). At 1 month, B6 (Fig. 1A a ), lpr (Fig. 1Ae) and gld (not shown) chimera had apparently normal B cell follicles. FDC, identified by FDC M1 [17 ] and FDC M2 markers (data not shown), were also present. T cells were concentrated in the white pulp, around the central arterioles forming the periarteriolar lymphatic sheaths (not shown).


Figure 1
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  		Figure 1. Generation of marginal zone components in 1-month-old B6 and lpr chimera (A), compared with gld chimera (B). Spleen tissue from the indicated chimera was sectioned and stained to detect B cells (anti-B220; Aa and e), MZ macrophages (ERTR-9; Ab and f; Ba), marginal metallophilic macrophages (MOMA-1; Ac and g; Bb) and anti-mucosal addressin cell adhesion molecule (MadCam-1; Ad and h; Bc). Incomplete MZ in lpr (Af and g, arrows) and gld (Ba and b, arrows) chimera are depicted; macrophages arise in the same area as pointed by the arrows. More than six chimera per group were analyzed (original magnification: x100).

The marginal zone (MZ) is an important element surrounding the T/B regions and separating the white pulp from the red pulp in the spleen. It is composed of several populations: marginal zone macrophages (ER-TR9+) [18 ] and marginal zone B cells (MZB) in the outer skirt; marginal metallophilic macrophages (MOMA-1+) [19 ] in the inner marginal sinus; and MadCAM-1+ [20 ] endothelial cells, which line the marginal sinus. In untreated Rag2–/– hosts, only MadCAM-1+ expression is detected around the central arterioles (not shown).

Following BM transfer, all components of the MZ appeared. In B6, lpr and gld chimera MadCAM-1+ sinus lining cells clearly developed (Fig. 1A d and h ; Fig. 1B c ). Around the white pulp, ER-TR9+ cells formed a nearly complete ring in B6 chimera (Fig. 1A b) or an association of few cells, in proper location in lpr (Fig. 1A f) and gld (Fig. 1B a) chimera. MOMA-1+ macrophages formed only incomplete lining (Fig. 1A c and g ; Fig. 1B b ). In all chimera, the red pulp components F4/80+ macrophages and CD11c+ DC were present (Fig. 2 ).


Figure 2
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  		Figure 2. Intact red pulp in lpr chimera 1 month post-graft. Presence of tissue macrophages (F4/80) and DC (N418) in B6 chimera (a, b) and lpr chimera (c, d); more than six mice per group were analyzed (original magnification: x100).

Thus, one month post-graft, following B cell follicle formation, MadCAM-1+ cells and ER-TR9+ cells are the first to be installed and MOMA-1+ cells are still in formation in B6 chimera. In contrast, in spite of a normal T and B cell compartment segregation, in lpr chimera ERTR-9+ cells were scarce, as was the case for gld chimera. We noted that in lpr and gld chimera ERTR-9+ cells and MOMA-1+ cells appeared in the same location, suggesting that they are probably induced by the same signals. Thus, MZ components are not generated with the same kinetics in lpr and gld chimera compared with their B6 counterpart, even though all unmanipulated BM donor mice (B6, lpr, and gld) have a complete MZ (not shown).

Disruption of the white pulp in 2-month-old lpr chimera
Subsequently, important modifications regarding splenic architecture of lpr chimera occurred. At 2 months post-transfer, alterations in lymphocyte positioning became obvious. The clear demarcation between T and B cell zones was lost, and lymphoid cells were scattered all over the tissue, although aggregates of B220+ cells were still found in lpr chimera (Fig. 3e ). Reticular fibroblasts that express ER-TR7 are localized in the red pulp and surrounded the T cell areas in the white pulp. These fibroblasts are absent in B cell follicules [21 ]. The progressive loss of the white pulp organization was confirmed by invasion of ER-TR7+ fibroblasts in the spleen in lpr chimera (not shown).


Figure 3
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  		Figure 3. Disruption of B cell follicles in 2-month-old lpr chimera compared with B6 chimera. Spleen tissue from the indicated chimera was sectioned and stained to detect T cells (anti-CD3; a, d), B cells (anti-B220; b, e), and FDC networks (FDC M1; c, f). More than 8 mice per group were analyzed (original magnification: x100).

FDC networks were not detected in 2-month-old lpr chimera (Fig. 3f) but remained associated with B220+ cells follicles in gld (not shown) and B6 chimera controls (Fig. 3c) until 7 months. Furthermore, immunization with sheep red blood cells (SRBC) did not lead to GC formations in lpr chimera, as evaluated by peanut agglutinin (PNA) staining (data not shown). To find out whether the disorganization in the splenic lymphoid compartment 2 months after transplantation was accompanied by modifications of the MZ, we analyzed its components using the appropriate histological markers.

Figure 4A and 4B shows the representation of MZ populations in 2-month-old B6, lpr, and gld chimera. Neither ER-TR9+ nor MOMA-1+ macrophages were detected in recipients transplanted with lpr BM (Fig. 4A f and g , respectively) or gld BM (Fig. 4B b and c , respectively) as compared with controls (Fig. 4A b and c , respectively). On endothelial cells lining the marginal sinus, MadCAM-1 expression is no longer detected in lpr chimera (Fig. 4A h) but still abundant in gld chimera (Fig. 4B d)


Figure 4
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  		Figure 4. Immunological analysis of spleen marginal zone in lpr chimera compared with B6 chimera (A) and gld chimera (B). Sections are from 2-month-old mice stained with anti-mouse B220 (B cells; Aa and e; Ba); ERTR-9 (MZ macrophages; Ab and f; Bb), MOMA-1 (marginal metallophilic macrophages; Ac and g; Bc) and anti-MadCAM-1 (MECA 367, mucosal addressin cell adhesion molecule-1; Ad and h; Bd). Complete loss of the MZ in lpr chimera and partial loss in gld chimera (only MadCAM-1+ cells detected). These pictures are representative of a minimum of four animals per group.

Thus, progressive loss of mature B cells in the spleen could have led to the disruption of the white pulp and complete elimination of the MZ in lpr chimera. In contrast, the partial depletion of the MZ in gld chimera (ERTR9+ and MOMA-1+ cells absent), in spite of the presence of intact B cells follicles, suggests a dual role of the Fas/FasL pathway in the generation and the maintenance of these components.

Cellular distribution in secondary lymphoid organs in lpr BM-> Rag-2–/– chimera
As revealed by histology analysis, the B cell compartment was greatly disturbed in the spleen of lpr chimera. Then, we determine absolute cell numbers by morphology using viable dye (Trypan blue) in suspensions from the spleen, lymph nodes, thymus, and BM of chimera. The peak of spleen repopulation by donor cells occurred 1 month after BM transfer (Fig. 5A ), when the cell number in lpr chimera was not different from the B6 counterpart (66±20x106 cells in lpr chimera, n=9) vs. 46 ± 19 x 106 cells in B6 chimera (n=6). However, by 2 months, this organ had developed an atrophy in lpr chimera (27±5x106 cells, n=5), while controls remained stable (46±18x106 cells in B6 chimera, n=7). Thereafter, splenic regression in lpr chimera came to a standstill (6±1x106 cells, n=5), while full repopulation persisted in B6 chimera (87±14x106 cells, n=4) up to 150 days. This dramatic decrease in cell numbers was also observed in the LN of lpr chimera, dropping from 5 ± 3 x 106 cells (n=9) at one month to 1 ± 0.6 x 106 cells (n=7) at 2 months, when lymph nodes were no longer detectable. In B6 chimera, LN repopulation increased from 18 ± 8 x 106 cells after one month (n=9) to 22 ± 5 x 106 cells after 2 months (n=9) and remained stable henceforth. Normal development of spleens [43±9x106 cells after 1 month (n=4) and 51 ± 3 x 106 cells (n=5) after 2 months] and lymph nodes [31±17x106 cells (n=3) after one month and 60 ± 8 x 106 cells (n=3) after 2 months] was observed in gld chimera. Thus, while peripheral repopulation took place and was maintained in B6 and gld chimera, lpr chimerism led to progressive atrophy, first in LN and later on in the spleen.


Figure 5
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  		Figure 5. (A) Spleen and lymph node (mesenteric, axillary, inguinal) cellularities in B6 and lpr chimera. Analysis of absolute numbers in single cell suspensions from 8–10 mice per time point was performed in eight independent experiments in spleen and lymph nodes of B6 BM-> Rag-2–/– hosts (bold line) and lpr BM-> Rag-2–/– hosts (thin line) at different times after the graft. (B) Analysis of Fas expression in the thymus, the spleen, and the BM of lpr BM-> B6Ly5.1 hosts, 5 weeks post-graft. Surface expression of anti-Fas antibody (bold-lined histogram) are represented with the isotype control (thin-lined histogram). Three independent experiments were analyzed, and representative results are shown.

Absence of Fas expression by donor cells in lpr chimera
The loss of lymphocytes in the peripheral lymphoid organs of lpr chimera prompted us to evaluate the modulation of Fas expression. Indeed, transplanted lpr BM cells and their progeny could have express elevated levels of Fas protein. Up-regulation of Fas receptor that can induce an apoptotic signal could be responsible for the lymphopenia detected. Initially, the lymphopenia was observed in sublethally irradiated Rag2–/– host mice grafted with T and B cell-depleted lpr BM. Using wild-type B6 host grafted with lpr BM in identical conditions, chimera developed similar lymphopenia around 3 months. This finding suggests that the host phenotype was not involved in these processes. Figure 5B shows in such chimera (lpr BM-> B6 Ly5.1), Fas expression, detected by flow cytometry in the thymus, spleen, and BM of a 5-week-old chimera mouse. This model allowed us to examine the presence of Fas receptor presence among lymphoid T cells (CD4+ and CD8+) of host and donor type, as well as in B cells (B220+) and macrophages (Mac1+); therefore host cells represent an internal control for Fas expression among hematopoietic cells. Data show that Fas is expressed on most host T cells but absent in lpr donor type T cells generated. In the BM, host type Mac1+ cells are the main population expressing Fas, compared with endogenous B220+ cells. These analyses support the findings that lpr progeny do not express Fas receptor after transplantation into B6 mice (Fig. 5B) and Rag2–/– mice (not shown).

Moreover, neither difference in FasL expression by PCR analysis on ex vivo isolated cells nor by intracellular staining on anti-CD3 + PMA stimulated cells was observed in B6 and lpr chimera. Thus, in lpr chimera the lymphopenia observed is not linked to Fas induced cell death among host and donor lymphoid cells.

To examine thoroughly the undergoing apoptosis, we analyzed by flow cytometry and the TUNEL method the spleen and the BM of lpr BM-> Rag2–/– chimera. In the spleen, up to 2 months the ratio of TUNEL-positive (apoptotic) cells among T and B donor cells compared with donor B6 is constant and around 2 (not shown). However, in the BM, a higher difference was detected among B220+ cells in lpr chimera compared with B6 chimera: as early as 20 days post-graft, TUNEL-positive cells are sixfold more elevated in lpr B220+ cells compared with B6 B220+ cells (Fig. 6A ). Therefore, the BM represents the site where apoptosis started and that might hamper correct B cell production.


Figure 6
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  		Figure 6. (A) Detection of the cells undergoing apoptosis by TUNEL assay in the BM. Analysis of dUTP-labeled cells percentages in B220 positive population in B6 and lpr chimera mice at 20 days post-graft. These pictures are representative of three animals. (B) Analysis of T and B cell percentages in the spleen evaluated in TCR-{alpha}β (T cells {square}) and B220 (B cells {blacksquare}) positive populations in B6, lpr, and gld chimera at 1 and 2 months post-graft. Error bars do not exceed 10% of the values. The T/B ratios are, respectively, at 1 and 2 months post-graft, for B6 chimera 0.33 and 0.47; for gld chimera 0.61 and 0.59; for lpr chimera 1.44 and 1.38. This ratio in lpr chimera increases with time (2.45 at 4 months and 5.33 at 5 months). Results represent data from 8 independent experiments.

Progressive loss of transitional T1 and mature B cells in the spleen of lpr chimera
The decreased cellularity in lpr chimera could originate from deficient regeneration of both B and T cell subsets in the spleen or from B cells alone. Evaluation of these two compartments revealed a lower incidence of B cells as early as 1 month post-transplantation and represented no more than 12% of the spleen 5 months later (data not shown), as compared with B6 chimera. In 2 months post-graft, the number of B cells declined progressively relative to B6 controls (9.75±3.4x106, n=6, in lpr chimera vs. 29.5±7.6x106, n=5, in controls). Analysis of T cell and B cell percentages in lymphoid compartment revealed a T/B ratio in lpr chimera, which reached 1.3 vs. 0.3 in B6 controls and continued to increase over time (T/B=5.3 in 5-month-old lpr chimera). In gld chimera the T/B ratios were not modified, as compared with controls (Fig. 6B) .

Regarding the B cell lineage, four subsets can be distinguished. Transitional B cells of Type 1 (T1, IgMhiIgDlo), which are BM immigrants and give rise to transitional Type 2 (T2, IgMhiIgDhi). Mature B cells can be generated from T1 and/or T2 cells. The MZB population (IgMhi CD21+CD23–) is probably derived from mature B cells. Type 2 and mature B cells are located in the follicles, while T1 and MZB are in the MZ [24 ].

At one month post-graft, the T1 subset was diminished in lpr vs. gld chimera; however, the T2 B cell subset was present (Fig. 7A ). From 6 weeks, histology studies and phenotypic analysis indicated a gradual reduction of mature B cells and MZB in lpr chimera (not shown).


Figure 7
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  		Figure 7. (A) Splenic transitional T1 (lower right), T2 (upper right), and mature (upper left) B cell subpopulations were evaluated in the B cell (detected by anti-CD19 antibody) subset. Data show a typical experiment in a 1-month-old set of chimera. (B) The CXCR5 chemokine receptor (anti-BLR1 antibody) was analyzed in the B cell subset in B6 (thin line –––), gld (dotted line ······), and lpr (bold line ––), 1 month post-BM transfer into Rag-2–/– hosts. A representative experiment is shown. Background level is represented by gating on T cells (– – –).

Since both recirculation and maturation of B cells were deffective in lpr chimera, it could be argued that their homing functions were defective. However, the chemokine receptor CXCR5 detected by anti-BLR1 antibody [23 ] was maintained on B cells in lpr chimera (Fig. 7B) . Hence, reduction of the immature T1 B cell subset might be the consequence of a dysregulation at the progenitor level.

Pro-B cell progenitor accumulation in lpr chimera
Apoptosis data analyzed by TUNEL assay suggested the BM as crucial site directly involved in lymphopenia. Early depletion of immature T1 B cells in the spleen of lpr chimera might result from the absence of appropriate progenitors among donor lpr BM and/or from an intrinsic B cell defect in the chimera.

We thus analyzed the BM of the lpr chimera. Figure 8A represents the absolute numbers of hematopoietic populations among donor type cells in the BM. All lineages were present with a major contribution of the myeloid compartment (Mac1+), which was similarly distributed among B6 and lpr chimera. Regarding lymphoid progenitors, members of the B cell lineage in the B220+ compartment were present in both B6 and lpr chimera. However, as early as 2 months post-graft, the B220+IgM+ mature B cells subset started to decrease in the BM of lpr chimera (Fig. 8B ) as well as the B220+IgM– immature population (2.4±1.6x106, n=5, in lpr chimera vs. 15.0±5.8x106, n=6 in B6 chimera). Conversely, the B220lowCD43low donor type pro-B population was increased in lpr chimera (Fig. 8C) , pointing to this early developmental stage as an essential checkpoint. Thus, an arrest of differentiation hampered the generation of mature B cells.


Figure 8
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  		Figure 8. (A) Analysis of the medullary compartments of the B6 ({square}) and lpr ({blacksquare}) chimera. BM cells were analyzed for B cells (B220), granulocytes (GR1), and macrophages (Mac1) at different time after BM graft. (B) Analysis of mature B cell subsets: IgM+ populations were evaluated among B220+ cells. (C) Evaluation of pro-B (B220low CD43low) and pre-B cells (B220low CD43neg) among B220+ BM cells from chimera. Each bar represents 2–6 mice.

However, we checked that lpr BM contains appropriate B cell progenitors. Thus, when grafted to sublethally irradiated Rag2–/–-lpr/lpr hosts, T- and B cell depleted lpr BM cells efficiently and permanently repopulate all hematopoietic lineages and no lymphopenia appeared; the absolute cell numbers in the spleen were 134 ± 14 x 106 (n=3) at one month and 99 ± 14 x 106 (n=5) at 4 months post-graft. Large B cell follicles were maintained in the spleen (not shown). Thus, lpr BM is competent per se: in a Fas receptor deficient environment, B cell hematopoiesis of lpr origin is complete.

Transient proliferation of CD8+ T cells in lpr chimera
T cells were enriched in spleens of lpr chimera as a consequence of the reduction of B cell populations. One month after transplantation absolute T cell counts were higher in lpr chimera (38.5±12.1x106 T cells, n=6) than in the B6 control counterpart (7.4±3.8x106 T cells, n=5).

We determined the absolute number of CD4+ and CD8+ T cells in lpr chimera to evaluate their relative contribution to this increase. As early as 1 month post-graft, the CD4/CD8 ratio was inverted in the spleen of lpr chimera (CD4/CD8=0.7), as compared with their B6 counterpart (CD4/CD8=1.7), proving the preferential expansion of CD8+ T cells (Fig. 9A ). As shown in Table 1 , percentages as well as absolute numbers of CD8+ T cells were 3–4 times higher in lpr than in B6 chimera, indicating a bias toward CD8+ T cells in the early phase of peripheral reconstitution. This early prevalence of CD8+ T cells in the spleen is the result of their preferential proliferation over CD4+ T cells, as measured by BrdU incorporation (Fig. 9B) . Both CD4+ and CD8+ were CD44–CD62L+, signifying their naive stage of development (Fig. 9C) .


Figure 9
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  		Figure 9. Phenotypic and functional characterization of T cells. (A) Analysis of CD4+/CD8+ T cell subsets in the spleen of 1-month-old chimera mice. A representative experiment is shown. (B) BrdU incorporation in splenic T cells of 1-month-old chimera; BrdU incorporation in a typical experiment performed in B6 (bold line ––) and lpr (dotted line ·····) chimera, showing, respectively, the percentages of total (B6: 6.5% and lpr: 29.8%), CD4+ (B6: 1.4% and lpr: 3.3%) and CD8+ (B6: 3.5% and lpr: 8.6%) T cells. (C) A typical experiment in 3-month-old chimera examining the status of T cells in the spleen.


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  		Table 1. T Cell Subset Development in the Spleen of B6 and lpr Chimera

Presence of TNF-{alpha} in the serum of lpr and gld chimera
To find out whether the striking cellular modifications were a direct consequence of FasL production by lpr cells or required other cytokines produced in response to FasL, we analyzed serum levels of pro-inflammatory factors, such as TNF-{alpha}.

Results summarized in Fig. 10 show that TNF-{alpha} was present in the serum of all three chimera, although mice transplanted with lpr and gld BM produced more TNF-{alpha} than their B6 counterpart. We found no major differences in the amount of soluble receptors (sTNF-R-p55 and sTNF-R-p75) between B6 and lpr chimera in the course of this study (data not shown). These data suggest that TNF-{alpha} cannot be held responsible for the defects observed in lpr chimera since it was also present in the serum of mice having received gld BM, where no abnormalities (cellular and histological) occurred. Membrane-bound TNF-{alpha} was not detected on effector cells (not shown), which makes its implication in the destruction of the splenic micro-architecture highly improbable. Whether a control system ensuring the protection of the graft exists in gld but not in lpr chimera remains an open question.


Figure 10
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  		Figure 10. TNF-{alpha} assay in the serum of chimera mice by ELISA. The data were obtained in 1- and 2-month-old chimera. Each bar is representative of one individual mouse.


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DISCUSSION
 
In this study, we took advantage of a chimera model established in Rag-2–/– mice transplanted with lpr BM to investigate whether Fas/FasL (CD95/CD95L) interactions are required during hematopoietic differentiation in vivo. In this system grafted Fas-deficient (lpr) hematopoietic cells develop but mature B cells are deleted and the spleen microarchitecture disrupted with loss of distinct T/B compartments and of the MZ (Table 2 ). In contrast, BM from gld donor (FasL deficient) reconstitute permanently the B cell lineage of Rag-2–/– hosts. Consequently, Fas expression on B progenitors protect them from negative selection in a wild-type environment. However, B cell hematopoiesis is completed when lpr BM cells are grafted in a Fas-deficient host (Rag-2–/–-lpr/lpr). Thus, development of B cells expressing a Fas deficiency is under the control of the non-lymphoid environment.


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  		Table 2. Summary of Histological Observations of the Splenic Microarchitecture in Various Chimera and Their Controls

The chimera model we developed (lpr BM-> Rag-2–/– hosts) enabled us to examine the gradual reconstitution of the host’s lymphoid organs by donor cells lacking an operative Fas pathway. Full reconstitution of the B cell compartment could not be achieved in the spleen of 1-month-old lpr chimera. First, this could be due to an intrinsic defect in lpr BM donor cells. However, lpr medullary cells from unmanipulated mice differentiate correctly in IgM+ cells as described previously [25 ], indicating that they are competent. Second, B cell differentiation might be defective in lpr chimera; indeed, we showed that T1 immature transitional B cells are reduced in the spleen and pro-B cells accumulate in the BM. Thus, B cell hematopoiesis is severely disturbed in lpr chimera. In contrast, in gld chimera a permanent B cell hematopoiesis takes place. Therefore, Fas expression on B progenitors is sufficient to maintain survival and a full B cell differentiation. Several mechanisms have been suggested to document on the escape from the Fas deletional pathway [26 ]. We concentrated on evaluating the permissive environment that allowed Fas deficient hematopoietic cells to generate B cells. The contact with a Fas deficient non-lymphoid environment (Rag-2–/–-lpr-lpr hosts) can sustain B lymphopoiesis of lpr progenitor cells (Table 2) . Thus, absence of Fas/FasL signaling can lead to complete B cell maturation. In contrast, a Fas/ FasL dysregulation generates two statuses: in gld chimera, with inefficient donor FasL, signaling with engagement of non-lymphoid (host) FasL and in Fas expressing (donor) gld cells give rise to mature B cells. However, in lpr chimera, engagement of FasL expressing lymphoid lpr cells on Fas positive host stroma cells might lead to destruction of environmental niches involved in B cell generation. Stroma cells are crucial participants of the B cell differentiation interacting by interleukins they produce to promote pro-B cell differentiation [27 , 28 ]. Thus, according to this hypothesis, our data support the concept that FasL produced by lymphoid cells induces more damages than when produced by non-lymphoid cells.

Together with a significant diminution of B cells in the spleen of lpr chimera, the architecture is progressively dismantled (Table 2) . B cell positioning, rather than homing, is affected, since no clear demarcation between red and white pulps is detected 2 months after grafting with loss of the MZ. Similar alterations were observed in lpr BM-> sublethaly irradiated B6 hosts (data not shown), thus excluding, their host-type origin. Our data are consistent with recently published work on the role of B cells in the development and maintenance of the MZ [29 ]. However, MZ macrophages (ERTR-9 and MOMA-1) were also absent in 2-month-old gld chimera, where B cells are present in the spleen. Similarly, B cell reconstitution is normal in lpr BM-> Rag-2–/–-lpr-lpr hosts, but no MZ macrophages are detected (not shown). Thus, the requirements for the generation of the MZ are different than those for B cells.

In addition to the B cell defect, we observed a complete disappearance of LN in lpr chimera after 2 months, concomitantly with the loss of the MZ in the spleen. According to some studies in mutant mice [30 , 31 ] these elements share a common organization, which might explain their similar dependency on correct Fas/FasL signaling.

The increase in mature CD8+ T cells in the spleen of lpr relative to B6 chimera is one of the earliest events following Fas–FasL+ hematopoietic cell grafting. BrdU incorporation revealed that CD8+ donor-derived lpr T cells proliferate 2–3 times more than their B6 counterpart in B6 chimera. Fink’s group has provided ample evidence that FasL co-stimulates the in vivo proliferation of CD8+ T cells [32 , 33 ]. Thymic differentiation was normal in lpr chimera revealing that T cell progenitors as well as thymic stroma cells were not impaired in these mice.

Mutations affecting TNF family members and their receptors also result in disruption of the splenic follicular structure [16 , 34 35 36 37 38 39 40 41 ]. Our model presents the advantage that cellular and histological events resulting from deficient Fas/FasL signaling on hematopoietic cells occur gradually and can be examined one after the other. This approach allowed us to demonstrate that the cross-talk between lymphoid progenitors and non-lymphoid cells is dependent on the expression of Fas and FasL, respectively, to ensure complete B cell differentiation and probably related signals to generate the MZ. In conclusion, our data support a complementary role of the TNF receptor family members in the positioning of B cells and a participation of FasL in their negative selection.


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ACKNOWLEDGEMENTS
 
This work was supported by the "Association de la recherche sur le cancer" (ARC) and the "Institut de la Santé et de la Recherche Médicale" (INSERM). We thank Daniel Corcos, Martine Papiernik, and Alf Eaton for critically reading the manuscript, Georg Kraal and Reina Mebius for encouragements in the initial parts of this work, R. Foerster, M. Kosko-Vilbois, G. Kraal, and P. Leenen for generously providing antibodies, Renée Communal for secretarial assistance, Eric Le Gall for artwork, and Sandrine Leaument for mouse breeding.

Received January 25, 2005; revised April 22, 2005; accepted July 21, 2005.


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