Mature Follicular Dendritic Cell Networks Depend on Expression of Lymphotoxin beta  Receptor by Radioresistant Stromal Cells and of Lymphotoxin beta  and Tumor Necrosis Factor by B Cells

By Robert Endres,* Marat B. Alimzhanov,Dagger Thomas Plitz,* Agnes Fütterer,* Marie H. Kosco-Vilbois,§ Sergei A. Nedospasov,parallel Klaus Rajewsky,Dagger and Klaus Pfeffer*

From the * Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, D-81675 Munich, Germany; the Dagger  Institute for Genetics, University of Cologne, D-50931 Cologne, Germany; § Serono Pharmaceutical Research Institute, Plan-les-Ouates, CH-1228, Switzerland; and the parallel  Engelhardt Institute of Molecular Biology and Belozersky Institute of Physico-Chemical Biology, 117984 Moscow, Russia

    Abstract
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The formation of germinal centers (GCs) represents a crucial step in the humoral immune response. Recent studies using gene-targeted mice have revealed that the cytokines tumor necrosis factor (TNF), lymphotoxin (LT) alpha , and LTbeta , as well as their receptors TNF receptor p55 (TNFRp55) and LTbeta R play essential roles in the development of GCs. To establish in which cell types expression of LTbeta R, LTbeta , and TNF is required for GC formation, LTbeta R-/-, LTbeta -/-, TNF-/-, B cell-deficient (BCR-/-), and wild-type mice were used to generate reciprocal or mixed bone marrow (BM) chimeric mice. GCs, herein defined as peanut agglutinin-binding (PNA+) clusters of centroblasts/centrocytes in association with follicular dendritic cell (FDC) networks, were not detectable in LTbeta R-/- hosts after transfer of wild-type BM. In contrast, the GC reaction was restored in LTbeta -/- hosts reconstituted with either wild-type or LTbeta R-/- BM. In BCR-/- recipients reconstituted with compound LTbeta -/-/BCR-/- or TNF-/-/BCR-/- BM grafts, PNA+ cell clusters formed in splenic follicles, but associated FDC networks were strongly reduced or absent. Thus, development of splenic FDC networks depends on expression of LTbeta and TNF by B lymphocytes and LTbeta R by radioresistant stromal cells.

Key words: lymphotoxintumor necrosis factorbone marrow transferfollicular dendritic cellgerminal center
    Introduction
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the course of a humoral immune response, germinal centers (GCs)1 represent the microenvironment where antigen-specific B cells efficiently undergo clonal expansion, diversification of their Ig genes, selection for clones bearing B cell receptors (BCRs) of high affinity to the antigen, and differentiation into memory or plasma cells (for reviews, see references 1). To support the GC reaction, B cells, T cells, and follicular dendritic cells (FDCs), the three major cellular constituents of GCs, have to interact in an intricate way. In brief, FDCs trap and retain native antigen complexed with antibody and complement, and also provide contact-dependent antigen-nonspecific costimulatory signals that facilitate chemotaxis and proliferation of B cells (4, 5). It is believed that the trapped antigen can be best endocytosed by B cells with increased affinity of their BCRs to the antigen (4, 5). These B cells then present the processed antigen to CD4+ T cells, which on their part, together with FDCs, support further proliferation and differentiation of the selected B cells towards preplasma cells or memory B cells (4, 5).

Over the past few years, several members of the TNF/ lymphotoxin (LT) receptor and ligand families have been shown to play essential roles in the induction of GCs. Prominent members mediating this function are the TNFRp55, the LTbeta R, and their ligands TNF, LTalpha , and LTbeta . Both LTalpha and TNF form homotrimers (6, 7) which signal via TNFRp55 and TNFRp75 (8, 9). LTalpha 3 is only secreted by activated lymphocytes and NK cells (7), whereas TNF3 exists both in a membrane-bound and soluble form (10) and can be produced by many different cell types, including macrophages, T, and B cells (11). LTalpha can also associate with LTbeta , a membrane-bound type II protein (14, 15). The LTalpha 1beta 2 heterotrimer engages the LTbeta R, which is expressed on macrophages and in lymphoid and visceral tissues, but not on T or B lymphocytes (16). Mice deficient in TNFRp55, LTbeta R, TNF, or LTalpha all lack GCs, as defined by the absence of both peanut agglutinin-binding (PNA+) clusters of centroblasts/centrocytes and associated FDC networks in B cell areas (19). LTbeta -/- mice differ in their phenotype in that some small PNA+ clusters are detectable in the B cell areas, but associated FDC networks are absent or strongly reduced (22). Expression of LTalpha and TNFRp55 by B cells and radioresistant stromal cells, respectively, was shown to be required for GC formation (25). However, no data concerning the cell lineages required to express LTbeta R, LTbeta , and TNF for GC development are available.

This study demonstrates that engagement of the LTbeta R on radioresistant stromal cells is mandatory for creating an intact splenic microarchitecture, allowing T-B cell segregation and establishment of GCs. Moreover, for full development of splenic FDC networks, B lymphocytes are required to produce both LTbeta and TNF.

    Materials and Methods
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mice.

LTbeta - and LTbeta R-deficient mice were generated as described previously (21, 22). BCR-deficient mice (30) and TCR beta /delta -deficient mice (31) were purchased from The Jackson Laboratory. Ly5.1+ C57BL/6 mice were supplied by Dr. H.R. Rodewald (Basel Institute for Immunology, Basel, Switzerland). TNF-deficient mice (19) were provided by Dr. G. Kollias (Hellenic Pasteur Institute, Athens, Greece). Mice were maintained and bred in a conventional mouse facility in isolated cages according to German guidelines for animal care. 8-12-wk-old mice were taken for experiments.

Bone Marrow Transfer.

Bone marrow (BM) cells were harvested by flushing femurs and tibias of donor mice with cold RPMI 1640 medium (Seromed) supplemented with 10% heat-inactivated FCS (Seromed), 2 mM L-glutamine (Seromed), 50 µM 2-ME (GIBCO BRL), 50 µg/ml streptomycin (Seromed), and 100 U/ml penicillin. Cells were washed and depleted of mature T cells using magnetic beads coated with anti-Thy1.2 mAb (Dynal) according to the manufacturer's protocol. After the depletion, cells were counted, washed, and resuspended in PBS. Recipient mice were irradiated with 9.5 Gy using a 137Cs irradiator (Buchler) and injected intravenously with 2 × 106 BM cells in 0.2 ml PBS. For mixed BM transfer, 1.5 × 106 cells of each genotype were injected in a total volume of 0.2 ml PBS.

Immunization.

6-8 wk after the BM transfer, the mice received an intraperitoneal injection of 5 µg of alum-precipitated (4-hydroxy-3-nitrophenyl-acetyl)-chicken gamma globulin (molar ratio 19:1; NP19-CG) in 0.2 ml PBS.

Evaluation of Chimerism.

10 d after immunization, mixed BM chimeric mice were killed, and BM, spleens, lymph nodes, and sera were collected. One half of each spleen was frozen for immunohistology, and the other half and all lymph nodes were used to purify B lymphocytes. Single cell suspensions were prepared using nylon cell strainers (Becton Dickinson). Cells were washed and incubated in Tris-buffered ammonium chloride solution to lyse erythrocytes. Cells were then washed in RPMI 1640 medium and in PBS containing 0.5% BSA. At this stage, chimerism of some mice was determined by flow cytometry on a FACScan®, using mAbs directed against the Ly5.1 (CD45.1) and Ly5.2 (CD45.2) isoforms conjugated to FITC or PE (PharMingen). B cells were purified by magnetic cell sorting using mouse CD45R (B220) microbeads, MACS® VS+ separation columns and a MACS® magnet (Miltenyi Biotec) according to the instructions provided by the manufacturer. The selected fractions were additionally depleted of remaining T cells by magnetic cell sorting using anti-Thy1.2-coated Dynabeads (Dynal). BM cells were depleted of T cells and of IgM+/IgG+ cells using a cocktail of Dynabeads coated with anti-Thy1.2 or anti-IgM/IgG antibodies (Dynal). The purity of cell populations was confirmed by flow cytometry. The percentage of LTbeta -/- cells in the purified populations was determined by Southern blot hybridization of BamHI-digested genomic DNA using an SphI-PstI fragment of the murine LTbeta promoter (nucleotides 2977-3411, sequence data available from EMBL/GenBank/DDBJ under accession no. U06950) as a probe. Similarly, the percentage of TNF-/- cells was determined by Southern blot hybridization of EcoRI- digested genomic DNA using a PCR-generated fragment of exon 4 of the TNF gene (PCR primers 5'-AGGTCACTGTCCCAGCATCT and 5'-GTCAGCCGATTTGCTATCTCA) as a probe. Quantifications of chimerism were performed by densitometry using a PhosphorImager (Molecular Dynamics).

Immunohistochemistry.

Tissue samples were embedded in tissue-freezing medium (Leica) and snap-frozen in 2-methylbutane (Merck) prechilled by liquid nitrogen. Cryostat sections (7 µm) were fixed for 8 min in acetone (Merck), dried, and preincubated for 30 min with PBS containing 5% (vol/vol) goat serum, 1% (wt/vol) BSA, and 0.15% (vol/vol) hydrogen peroxide (Sigma). Blocking of endogenous biotin was performed using an avidin-biotin blocking kit (Vector) according to the manufacturer's protocol. For double labeling, sections were incubated for 30 min with (a) biotinylated PNA diluted 1:500 (Vector) and rat anti- mouse CR1 (CD35) diluted 1:100 (clone 8C12; PharMingen); (b) biotinylated PNA diluted 1:500 (Vector) and rat anti-mouse B220 diluted 1:100 (clone RA3-6B2; PharMingen); and (c) biotinylated mouse anti-mouse IgD diluted 1:50 (clone 1.3-5), rat anti-mouse CD4 diluted 1:100 (GK1.5; PharMingen), and rat anti-mouse CD8 used as a 1:2 diluted hybridoma supernatant (clone 53.6-72; American Type Culture Collection). Rat IgG2a and IgG2b (PharMingen) were used as isotype controls. Single labeling was performed with FDC-M1 mAb (clone 4C11). After washing, alkaline phosphatase (AP)-conjugated streptavidin (Sigma) and/or horseradish peroxidase-coupled mouse anti-rat IgG (Dianova) were added. After 30 min incubation and washing, color development for bound AP and horseradish peroxidase was consecutively performed with an AP reaction kit (Vector) according to the manufacturer's instructions and with 3-aminoethyl-carbazole (Sigma) as described (32). In addition, fluorescent microscopy was used for analysis of sections labeled with PNA and FDC-M2 mAb (clone 209) as described previously (25).

Measurement of Antigen-specific IgG.

10 d after immunization, NP-specific IgG antibodies were detected using sandwich ELISAs with NP12-BSA- or NP5-BSA-conjugated ELISA plates (10 µg/ml in carbonate buffer [pH 9.5]). Murine NP-specific IgG antibodies were detected with an AP-conjugated goat anti- mouse IgG antiserum (Dianova). The substrate used was p-nitrophenyl phosphate (Sigma). For calculation of arbitrary binding units of NP-specific IgG antibodies, the standard NP-reactive mAb, N1G9 (33), was included on each ELISA plate.

    Results
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
GC Formation and Intact Splenic Architecture Require Expression of LTbeta R on Radioresistant Cells.

To address which cell types have to express LTbeta or LTbeta R to initiate and maintain GC reactions, a reciprocal BM transplantation approach was used. BM from C57BL/6 wild-type donors was transferred into myeloablatively irradiated LTbeta R-/- recipients (B6 right-arrow LTbeta R-/-) and vice versa (LTbeta R-/- right-arrow B6). In the same manner, LTbeta R-/- right-arrow LTbeta -/-, B6 right-arrow LTbeta -/-, and LTbeta -/- right-arrow B6 chimeric mice were generated. As a control, BM from C57BL/6 wild-type donors was taken to repopulate irradiated C57BL/6 wild-type recipients (B6 right-arrow B6). After 8 wk, mice were immunized intraperitoneally with 5 µg NP19-CG adsorbed to alum, and 10 d later spleens and sera were taken for analysis. For detection of GCs, a double labeling of spleen sections was performed using the plant lectin PNA and the mAb 8C12. PNA binds to centroblasts/centrocytes, whereas the mAb 8C12 is directed against the murine complement receptor CR1, which is highly expressed on FDCs and at lower levels by B lymphocytes (34). As shown in Fig. 1, E-H, in the spleens of B6 right-arrow LTbeta R-/- animals only a few, small PNA+ cell aggregates were observed, and FDC networks were completely absent. A similar phenotype was found in immunized LTbeta R-/- mice (21). An aliquot of the wild-type BM used to reconstitute the LTbeta R-/- recipients was also transferred into LTbeta -/- recipients. In these mice, the wild-type BM-derived cells were capable of restoring GC formation, proving the intrinsic competence of the donor BM cells (data not shown). Morphologically intact GCs, i.e., PNA+ cell clusters in association with FDC networks, were also detectable in spleens from LTbeta R-/- right-arrow LTbeta -/- (Fig. 1, I and J), LTbeta R-/- right-arrow B6 (data not shown), and B6 right-arrow B6 (Fig. 1, A and B) chimeras. In contrast, wild-type mice reconstituted with LTbeta -/- BM had fewer and smaller PNA+ cell clusters with almost undetectable FDC networks (Fig. 1, M and N). Presence or absence of FDC networks in association with PNA+ cell clusters was confirmed by double labeling with PNA and FDC-M2 mAbs or by single labeling with FDC-M1 mAbs (data not shown).


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Fig. 1.   Splenic GC development in reciprocal BM chimeric mice. Irradiated recipients (n = 3-5 per group) were reconstituted with BM from donors as indicated. After 8 wk, chimeras were immunized intraperitoneally with 5 µg NP19-CG adsorbed to alum. 10 d later, chimeras were killed and splenic cryosections were labeled with anti-CR1 (brown) and PNA (blue); anti-B220 (brown) and PNA (blue); or anti-CD4 (brown), anti-CD8 (brown), and anti-IgD (blue).

The anatomical localization of GCs was determined by labeling splenic sections with either PNA and anti-B220 mAbs or anti-IgD, anti-CD4, and anti-CD8 mAbs. Downregulation of IgD on most GC B cells and relatively low numbers of T cells scattered in GCs compared with compact T cell areas in periarteriolar lymphoid sheaths served as criteria for the identification of GCs. In LTbeta R-/- right-arrow LTbeta -/- (Fig. 1, K and L), LTbeta R-/- right-arrow B6 (data not shown), and B6 right-arrow B6 (Fig. 1, C and D) chimeric mice, all GCs were correctly localized in the B cell areas. Conversely, in spleens of LTbeta R-/- mice reconstituted with wild-type BM, PNA+ cell aggregates were found around central arterioles (Fig. 1, G and H). T and B cells segregated normally in LTbeta R-/- right-arrow LTbeta -/- (Fig. 1 L), LTbeta R-/- right-arrow B6 (data not shown), and B6 right-arrow B6 (Fig. 1 D) chimeric mice, forming distinct periarteriolar lymphoid sheaths and B cell follicles. In contrast, in B6 right-arrow LTbeta R-/- mice, T and B cells were mixed despite the presence of hematopoietically derived LTbeta R+/+ donor cells (Fig. 1 H). Taken together, the failure of LTbeta R+/+ BM-derived cells to restore GCs and an intact splenic architecture in LTbeta R-/- recipients provides evidence that LTbeta R on radioresistant stromal cells is required for these functions. However, LTbeta R-/- BM-derived cells were capable of establishing GCs in LTbeta -/- recipients. This shows that for GC development in adult mice the presence of LTbeta R on radiosensitive BM-derived cells is dispensable, whereas the presence of LTbeta on these cells is necessary. The latter conclusion is further supported by the finding that transfer of LTbeta -/- BM into wild-type C57BL/6 recipients severely impaired GC formation.

LTbeta and TNF from B Cells Are Required for Formation of Mature FDC Networks.

Expression of LTbeta and TNF by hematopoietic cell lineages is required for GC formation (results above, and reference 35). Yet it is unclear whether a single hematopoietic lineage is necessary and, perhaps, sufficient for the production of LTbeta and/or TNF, or whether different cellular sources can redundantly provide these ligands in GC reactions. In particular for TNF, the latter appears to be possible, since a great variety of cell types (e.g., macrophages, granulocytes, T cells, B cells, dendritic cells) can produce both soluble and membrane-bound TNF. Similarly, LTbeta can be synthesized by three distinct cell types, namely T, B, and NK cells (15, 16, 36). Thus, to address the question of which cell type is required to express LTbeta and TNF for GC establishment, compound BM chimeric mice were made. BM cells from BCR-deficient donors were mixed in a 1:1 ratio with BM cells from TNF-/- or LTbeta -/- donors and transferred into myeloablatively irradiated BCR-/- recipients (LTbeta -/- + BCR-/- right-arrow BCR-/-; TNF-/- + BCR-/- right-arrow BCR-/-). Control groups were established by reconstituting myeloablatively irradiated BCR-/- recipients with mixed BM from Ly5.1+ C57BL/6 wild-type donors and LTbeta -/- or TNF-/- donors (LTbeta -/- + B6 right-arrow BCR-/-; TNF-/- + B6 right-arrow BCR-/-). Since BCR-/- BM cannot give rise to mature B cells, peripheral B cells in the LTbeta -/- + BCR-/- right-arrow BCR-/- and TNF-/- + BCR-/- right-arrow BCR-/- chimeras were genetically deficient in LTbeta and TNF, respectively. All other radiosensitive BM-derived cell populations consisted of a mixture of wild-type and gene-targeted cells. In control groups (LTbeta -/- + B6 right-arrow BCR-/-; TNF-/- + B6 right-arrow BCR-/-), all BM-derived cell populations---including B cells---were composed of wild-type and genetically deficient cells. After 6-7 wk, mice were immunized intraperitoneally with 5 µg of alum-precipitated NP19-CG, and 10 d later BM, spleen, lymph nodes, and serum were taken for analysis. BM chimerism and B cell chimerism were determined by Southern blotting and/or flow cytometric analysis of the CD45 isoforms Ly5.1 and Ly5.2 (see Materials and Methods, and Table I). BM chimerism was found to be comparable between experimental and control groups (Table I). Immunohistochemical analysis of splenic sections from LTbeta -/- + BCR-/- right-arrow BCR-/- chimeras revealed the presence of few PNA+ cell clusters. Associated FDC networks were absent or significantly reduced in size (Fig. 2, E and F). Most of the PNA+ cell clusters were localized within B cell areas (Fig. 2, E-H). Albeit segregated from the T cell areas, the B cell areas did not represent well-defined B cell follicles. Spleens of LTbeta -/- + B6 right-arrow BCR-/- control mice showed numerous PNA+ cell clusters in association with large FDC networks, most of them correctly localized within B cell follicles (Fig. 2, A-D). With regard to FDC network formation, the absence of TNF production by B cells in TNF-/- + BCR-/- right-arrow BCR-/- chimeras resulted in a phenotype similar to the one found in LTbeta -/- + BCR-/- right-arrow BCR-/- chimeras: few PNA+ cell clusters contained considerably underdeveloped FDC networks, and most clusters lacked immunohistochemically detectable networks altogether (Fig. 2, M and N). However, in these mice, approximately two thirds of the PNA+ cell clusters were located around central arterioles in T cell areas (Fig. 2, O and P) and only one third was found in B cell areas (not shown). B cells segregated from T cells and some B cell follicles were observed (not shown). In TNF-/- + B6 right-arrow BCR-/- control chimeras, most PNA+ cell clusters associated with FDC networks were readily detectable in distinct B cell follicles (Fig. 2, I-L). All results regarding splenic FDC network formation in compound BM chimeras were confirmed by double labeling with PNA and FDC-M2 mAbs (data not shown). Taken together, the results demonstrate that expression of both LTbeta and TNF by B cells is required for the development of mature splenic FDC networks, but not for the formation of PNA+ cell clusters.

                              
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Table I
Degree of Chimerism in Compound BM Chimeras


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Fig. 2.   Splenic GC development in compound BM chimeric mice. Irradiated recipients (n = 4 per group) were reconstituted with a 1:1 mixture of BM cells originating from the two donors indicated. After 6-7 wk, the mixed BM chimeras were immunized intraperitoneally with 5 µg NP19-CG adsorbed to alum. 10 d later, chimeras were killed and splenic cryosections were labeled with anti-CR1 (brown) and PNA (blue); anti-B220 (brown) and PNA (blue); or anti-CD4 (brown), anti-CD8 (brown), and anti-IgD (blue).

Specific Primary IgG Responses.

To investigate whether the observed abnormal GC reactions correlated with impaired specific IgG responses, NP-specific IgG titers were quantified in the sera of animals 10 d after immunization. Densely or sparsely haptenated BSA (NP12-BSA or NP5-BSA) was used for coating of ELISA plates, allowing detection of both low and high affinity NP-specific IgG. B6 right-arrow B6 and LTbeta R-/- right-arrow B6 chimeras responded to immunization with a comparable production of specific IgG, whereas B6 right-arrow LTbeta R-/- animals did not mount a significant primary IgG response (Fig. 3, A and B). Moreover, significant defects were not observed in B6 right-arrow LTbeta -/-, LTbeta -/- right-arrow B6, or LTbeta R-/- right-arrow LTbeta -/- chimeras (data not shown). In B6 right-arrow LTbeta R-/- chimeras, the impaired primary IgG response correlated with multiple defects in the organization of peripheral lymphoid tissues such as lack of lymph nodes and Peyer's patches, disruption of T-B cell segregation, and aberrant PNA+ cell clusters without FDC networks in the spleen (macroscopic examination, and Fig. 1, E-H). In contrast, LTbeta -/- + BCR-/- right-arrow BCR-/- and TNF-/- + BCR-/- right-arrow BCR-/- mice contained lymph nodes, PNA+ cell clusters, and distinct T and B cell areas, yet differed from their control groups regarding splenic FDC network formation (Fig. 2). Here, differences in NP-specific IgG titers between experimental and control groups were not statistically significant (Fig. 3, C and D), implying that a lack or strong reduction of splenic FDC networks does not necessarily lead to impaired specific primary IgG responses.


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Fig. 3.   NP-specific IgG titers in the sera of reciprocal (A and B) and mixed BM chimeras (C and D). Chimeric mice were made and immunized as described in the legends to Figs. 1 and 2 (n = 3-5 per group). Sera were taken before and 10 d after immunization. The amounts of anti-NP antibodies were determined as described in Materials and Methods. Note that two different batches of NP19-CG adsorbed to alum were used for immunization, precluding a direct comparison of values from A and B with those from C and D. bullet , All values were below the detection limit.

    Discussion
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mice deficient for the TNFRp55 or the LTbeta R lack FDC networks and correctly localized PNA+ cell clusters, demonstrating the requirement of signals from these receptors for GC formation (20, 21). Development of PNA+ cell clusters and FDC networks in TNFRp55-/- mice is not rescued by transplantation of wild-type BM (25, 26), whereas TNFRp55-/- BM is as efficient as wild-type BM in reconstituting these structures in LTalpha -/- mice (26). These data indicate that for establishment of GCs, TNFRp55 is required on radioresistant stromal cells and not on radiosensitive BM-derived cells (25, 26). In the present study, LTbeta R-/- right-arrow LTbeta -/-, LTbeta R-/- right-arrow B6, and B6 right-arrow LTbeta R-/- BM chimeric mice were used for the analysis of GC development, and evidence is provided that for formation of FDC networks expression of LTbeta R, like TNFRp55, is required on radioresistant cells, but not on BM-derived radiosensitive cells. Since FDCs are known to withstand high doses of irradiation (37), it is likely that putative FDC precursors are radioresistant cells that depend on signals from the TNFRp55 (25, 26) and the LTbeta R (this study) for differentiation to mature FDCs (Fig. 4). Alternatively, it is conceivable that radioresistant stromal cells different from FDC precursors exist that provide molecules needed for FDC maturation and depend on signals from the TNFRp55 and/or the LTbeta R in order to fulfill this function.


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Fig. 4.   Model of the molecular interactions essential for the establishment of splenic FDC networks associated with PNA+ cell clusters. B cells provide the ligands LTalpha (references 28, 29), TNF, and LTbeta required for an effective engagement of the TNFRp55 (references 25, 26) and the LTbeta R on specific radioresistant cells, which most likely represent FDC precursors. In this cell population, both signaling pathways have to be functional for mature splenic FDC networks to form. Note that the present data do not elucidate whether signaling from the TNFRp55 and the LTbeta R has to occur simultaneously or consecutively in the putative FDC precursors. TNFRI, TNFRp55.

Comparable to LTbeta R-/- mice, mice with a disrupted LTalpha gene are devoid of marginal zones, proper T-B cell segregation, correctly localized PNA+ cell clusters, and FDC networks (20, 38, 39). Recently, experiments by Fu et al. (28) and Gonzalez et al. (29) led to the identification of the cellular source of LTalpha needed for the establishment of FDC networks and correctly localized PNA+ cell clusters in the spleen. The first group used BM from LTalpha -/- mice mixed together with BM from TCR-/- or BCR-/- mice to reconstitute LTalpha -/- mice. In chimeric mice, all T or B lymphocytes were deficient in LTalpha , whereas the other cells of hematopoietic origin consisted of a mixture of LTalpha -/- and LTalpha +/+ genotypes. The formation of PNA+ cell clusters and FDC networks was precluded only in BM chimeras in which B cells were deficient for LTalpha , indicating that LTalpha -producing B cells are essential for the establishment of GCs (28). Gonzalez et al. (29) adoptively transferred purified lymphocytes into SCID mice and also showed the dependence of the FDC network on LTalpha -producing B lymphocytes. Since LTalpha together with LTbeta can form ligands specific for LTbeta R (16), it was suggested that LTalpha 1beta 2 heterotrimers on B cells are required for FDC development. In line with this hypothesis, wild-type B cells transferred into SCID mice that were simultaneously treated with LTbeta R-Fc fusion protein did not induce FDC networks (29). In our study, a mixture of BM cells from BCR-/- and LTbeta -/- mice were transferred into irradiated BCR-/- recipients. In the absence of LTbeta -producing B cells, but not of LTbeta -producing T cells (Endres, R., M.B. Alimzhanov, and K. Pfeffer, unpublished results), FDC networks were strongly reduced or absent. However, PNA+ cell clusters in B cell areas were observed. These results are in contrast to the findings of Fu and colleagues, who did not detect any correctly localized PNA+ cell clusters and FDC-specific labeling in BCR-/- + LTalpha -/- right-arrow LTalpha -/- compound BM chimeras (28). This discrepancy may be explained by different experimental conditions, i.e., Fu et al. reconstituted LTalpha -/- mice, which show a severely disorganized splenic architecture (20, 38), whereas here B cell-deficient mice are reconstituted, which apart from missing B cells and FDC networks appear to have a conserved splenic architecture (Endres, R., M.B. Alimzhanov, and K. Pfeffer, unpublished results). Alternatively, it is conceivable that LTbeta -/- B cells still produce LTalpha homotrimers which engage the TNFRp55 and thereby induce GC formation, although much less efficiently than wild-type B cells. However, since signaling via the LTbeta R appears indispensable for FDC development (this study, and reference 21), in order to explain the appearance of few underdeveloped FDC clusters in LTbeta -/- + BCR-/- right-arrow BCR-/- chimeras and in LTbeta -/- mice (this study, and references 22, 24), one has to assume that ligands other than LTalpha 1beta 2 can engage the LTbeta R on radioresistant cells at least to some extent. A recently identified member of the TNF ligand family, LIGHT, may serve this function, since it was shown to bind to the LTbeta R (40).

Besides LTalpha 3 homotrimers, soluble and membrane-bound forms of TNF3 signal via the TNFRp55. TNF-/- and TNFRp55-/- mice have comparable phenotypes in that they lack B cell follicles, FDC networks, and correctly localized PNA+ cell clusters (19, 20). Since TNF is known to be produced by many cell types (11), we asked whether B cell-derived TNF is required for the induction of splenic FDC networks by generating compound TNF-/- + BCR-/- right-arrow BCR-/- BM chimeras. Surprisingly, these chimeras did not contain mature splenic FDC networks, showing a phenotype similar to the one observed in chimeras devoid of LTbeta -producing B cells. Thus, TNF is yet another mediator in B cell-pre-FDC interactions which lead to FDC network development. In contrast to TNF-/- mice (19), TNF-/- + BCR-/- right-arrow BCR-/- chimeras have few underdeveloped FDC networks associated with PNA+ cell clusters. This indicates that, in the absence of B cell- derived TNF, TNF from cells other than B cells can provide signals for FDC network development albeit to a limited extent. It is noteworthy that Alexopoulou et al. observed a reduced production of TNF in LTalpha -/- mice after LPS treatment (24). The authors suggested that in the LTalpha -/- mouse strain (38), TNF gene expression is altered by the neor cassette used to inactivate the LTalpha gene (24). The mutation introduced in the LTbeta gene (22) presumably does not interfere with the expression of the neighboring TNF and LTalpha genes. Therefore, differences in GC formation between chimeras devoid of LTalpha -producing B cells versus LTbeta -producing B cells could result from different amounts of TNF produced by LTalpha -/- and LTbeta -/- B cells, respectively. LTalpha -/- B cells, but not LTbeta -/- B cells, might fail to provide local TNF concentrations high enough to allow formation of few underdeveloped GCs.

One simple model of splenic FDC network maturation, which accommodates data from several groups (25, 26, 28, 29, and this study), is depicted in Fig. 4. Radioresistant stromal cells have to receive at least two different signals, one via the LTbeta R (this study) and the other via the TNFRp55 (25, 26), in order to give rise to a local FDC network. Most likely, these radioresistant cells represent hitherto unidentified FDC precursors, either residing at the site of B cell accumulation or attracted to this location by the B cells. Alternatively, the radioresistant cells are not FDC precursors, but serve as inducible sources of unknown factors required for differentiation of FDC precursors to mature local FDC networks. The cytokines required for FDC network development, TNF3, LTalpha 3 and LTalpha 1beta 2, are provided by B lymphocytes (28, 29, and this study). To date, the temporal and spatial interrelations of TNFRp55 and LTbeta R transduced signals remain unclear. It is possible that these two signals act during distinct stages of FDC differentiation.

    Footnotes

Address correspondence to Klaus Pfeffer, Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 9, D-81675 Munich, Germany. Phone: 49-89-4140-4132; Fax: 49-89-4140-4183; E-mail: klaus.pfeffer{at}lrz.tu-muenchen.de

Received for publication 8 September 1998 and in revised form 13 October 1998.

   The continuous and generous support of H. Wagner is greatly appreciated. The authors thank E. Schaller, U. Huffstadt, S. Weiss, and K. Mink for expert technical assistance. The authors are also grateful to H.R. Rodewald and G. Kollias for providing Ly5.1+ C57BL/6 and TNF-/- mice, respectively, and thank H. Neubauer, T. Novobrantseva, S. Scheu, and H. Häcker for critical reading of the manuscript and for scientific advice.
   R. Endres and M.B. Alimzhanov contributed equally to this work.

This study was supported by the Deutsche Forschungsgemeinschaft (grant Pf 259/2-4) and the Sonderforschungsbereich 391 and 243, Klinische Forschergruppe Postoperative Immunparalyse und Sepsis (grant Si208/5-1). M.B. Alimzhanov was supported by a fellowship from the European Molecular Biology Organization. S.A. Nedospasov is an international research scholar of the Howard Hughes Medical Institute.

Abbreviations used in this paper AP, alkaline phosphatase; FDC, follicular dendritic cell; GC, germinal center; LT, lymphotoxin; BCR, B cell receptor; PNA, peanut agglutinin.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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