Introduction

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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 LTR, and their ligands TNF, LT, and LT. Both LT and TNF form homotrimers (6, 7) which signal via TNFRp55 and TNFRp75 (8, 9). LT₃ 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). LT can also associate with LT, a membrane-bound type II protein (14, 15). The LT₁₂ heterotrimer engages the LTR, which is expressed on macrophages and in lymphoid and visceral tissues, but not on T or B lymphocytes (16). Mice deficient in TNFRp55, LTR, TNF, or LT 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). LT^/ 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 LT 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 LTR, LT, and TNF for GC development are available.

This study demonstrates that engagement of the LTR 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 LT and TNF.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Mice.

Bone Marrow Transfer.

Immunization.

Evaluation of Chimerism.

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Immunohistochemistry.

Measurement of Antigen-specific IgG.

	Results

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GC Formation and Intact Splenic Architecture Require Expression of LTR on Radioresistant Cells.

To address which cell types have to express LT or LTR 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 LTR^/ recipients (B6  LTR^/) and vice versa (LTR^/  B6). In the same manner, LTR^/  LT^/, B6  LT^/, and LT^/  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  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  LTR^/ animals only a few, small PNA⁺ cell aggregates were observed, and FDC networks were completely absent. A similar phenotype was found in immunized LTR^/ mice (21). An aliquot of the wild-type BM used to reconstitute the LTR^/ recipients was also transferred into LT^/ 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 LTR^/  LT^/ (Fig. 1, I and J), LTR^/  B6 (data not shown), and B6  B6 (Fig. 1, A and B) chimeras. In contrast, wild-type mice reconstituted with LT^/ 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).

View larger version (129K): [in this window] [in a new window]   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 LTR^/  LT^/ (Fig. 1, K and L), LTR^/  B6 (data not shown), and B6  B6 (Fig. 1, C and D) chimeric mice, all GCs were correctly localized in the B cell areas. Conversely, in spleens of LTR^/ 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 LTR^/  LT^/ (Fig. 1 L), LTR^/  B6 (data not shown), and B6  B6 (Fig. 1 D) chimeric mice, forming distinct periarteriolar lymphoid sheaths and B cell follicles. In contrast, in B6  LTR^/ mice, T and B cells were mixed despite the presence of hematopoietically derived LTR^+/+ donor cells (Fig. 1 H). Taken together, the failure of LTR^+/+ BM-derived cells to restore GCs and an intact splenic architecture in LTR^/ recipients provides evidence that LTR on radioresistant stromal cells is required for these functions. However, LTR^/ BM-derived cells were capable of establishing GCs in LT^/ recipients. This shows that for GC development in adult mice the presence of LTR on radiosensitive BM-derived cells is dispensable, whereas the presence of LT on these cells is necessary. The latter conclusion is further supported by the finding that transfer of LT^/ BM into wild-type C57BL/6 recipients severely impaired GC formation.

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

Expression of LT 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 LT 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, LT 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 LT 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 LT^/ donors and transferred into myeloablatively irradiated BCR^/ recipients (LT^/ + BCR^/  BCR^/; TNF^/ + BCR^/  BCR^/). Control groups were established by reconstituting myeloablatively irradiated BCR^/ recipients with mixed BM from Ly5.1⁺ C57BL/6 wild-type donors and LT^/ or TNF^/ donors (LT^/ + B6  BCR^/; TNF^/ + B6  BCR^/). Since BCR^/ BM cannot give rise to mature B cells, peripheral B cells in the LT^/ + BCR^/  BCR^/ and TNF^/ + BCR^/  BCR^/ chimeras were genetically deficient in LT and TNF, respectively. All other radiosensitive BM-derived cell populations consisted of a mixture of wild-type and gene-targeted cells. In control groups (LT^/ + B6  BCR^/; TNF^/ + B6  BCR^/), all BM-derived cell populationsincluding B cellswere 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 LT^/ + BCR^/  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 LT^/ + B6  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^/  BCR^/ chimeras resulted in a phenotype similar to the one found in LT^/ + BCR^/  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  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 LT and TNF by B cells is required for the development of mature splenic FDC networks, but not for the formation of PNA⁺ cell clusters.

View larger version (130K): [in this window] [in a new window]   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).

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  B6 and LTR^/  B6 chimeras responded to immunization with a comparable production of specific IgG, whereas B6  LTR^/ animals did not mount a significant primary IgG response (Fig. 3, A and B). Moreover, significant defects were not observed in B6  LT^/, LT^/  B6, or LTR^/  LT^/ chimeras (data not shown). In B6  LTR^/ 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, LT^/ + BCR^/  BCR^/ and TNF^/ + BCR^/  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.

View larger version (35K): [in this window] [in a new window]   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. , All values were below the detection limit.

	Discussion

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Mice deficient for the TNFRp55 or the LTR 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 LT^/ 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, LTR^/  LT^/, LTR^/  B6, and B6  LTR^/ BM chimeric mice were used for the analysis of GC development, and evidence is provided that for formation of FDC networks expression of LTR, 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 LTR (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 LTR in order to fulfill this function.

View larger version (57K): [in this window] [in a new window]   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 LT (references 28, 29), TNF, and LT required for an effective engagement of the TNFRp55 (references 25, 26) and the LTR 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 LTR has to occur simultaneously or consecutively in the putative FDC precursors. TNFRI, TNFRp55.

Comparable to LTR^/ mice, mice with a disrupted LT 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 LT needed for the establishment of FDC networks and correctly localized PNA⁺ cell clusters in the spleen. The first group used BM from LT^/ mice mixed together with BM from TCR^/ or BCR^/ mice to reconstitute LT^/ mice. In chimeric mice, all T or B lymphocytes were deficient in LT, whereas the other cells of hematopoietic origin consisted of a mixture of LT^/ and LT^+/+ genotypes. The formation of PNA⁺ cell clusters and FDC networks was precluded only in BM chimeras in which B cells were deficient for LT, indicating that LT-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 LT-producing B lymphocytes. Since LT together with LT can form ligands specific for LTR (16), it was suggested that LT₁₂ 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 LTR-Fc fusion protein did not induce FDC networks (29). In our study, a mixture of BM cells from BCR^/ and LT^/ mice were transferred into irradiated BCR^/ recipients. In the absence of LT-producing B cells, but not of LT-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^/ + LT^/  LT^/ compound BM chimeras (28). This discrepancy may be explained by different experimental conditions, i.e., Fu et al. reconstituted LT^/ 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 LT^/ B cells still produce LT homotrimers which engage the TNFRp55 and thereby induce GC formation, although much less efficiently than wild-type B cells. However, since signaling via the LTR appears indispensable for FDC development (this study, and reference 21), in order to explain the appearance of few underdeveloped FDC clusters in LT^/ + BCR^/  BCR^/ chimeras and in LT^/ mice (this study, and references 22, 24), one has to assume that ligands other than LT₁₂ can engage the LTR 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 LTR (40).

Besides LT₃ 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^/  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 LT-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^/  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 LT^/ mice after LPS treatment (24). The authors suggested that in the LT^/ mouse strain (38), TNF gene expression is altered by the neo^r cassette used to inactivate the LT gene (24). The mutation introduced in the LT gene (22) presumably does not interfere with the expression of the neighboring TNF and LT genes. Therefore, differences in GC formation between chimeras devoid of LT-producing B cells versus LT-producing B cells could result from different amounts of TNF produced by LT^/ and LT^/ B cells, respectively. LT^/ B cells, but not LT^/ 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 LTR (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, LT₃ and LT₁₂, are provided by B lymphocytes (28, 29, and this study). To date, the temporal and spatial interrelations of TNFRp55 and LTR transduced signals remain unclear. It is possible that these two signals act during distinct stages of FDC differentiation.