Figure 1 BLC genomic locus and lack of BLC mRNA and protein in BLC-/- mice.   Full legend   High resolution image and legend (19k)

To test whether BLC is needed for organization of B cells in lymphoid follicles, sections of lymph nodes and spleen from BLC^-/- animals were analysed for B-cell distribution. In all these organs, B cells failed to organize in polarized follicular clusters, and instead appeared as a ring of cells at the perimeter of T-cell areas (Fig. 2a). The boundary between B-cell-rich areas and T zones was often poorly demarcated, with increased numbers of B cells and T cells in reciprocal areas ( Fig. 2a, b). In addition, staining spleen sections for immunoglobulin (Ig)M and IgD revealed a thickened ring of IgM^hi IgD^lo marginal zone B cells (Fig. 2a , top). The segregation of B cells between this area and the inner ring of B cells was also disrupted, with increased numbers of IgM^lo IgD^hi B cells located in the outer marginal zone (Fig. 2a, top).

Figure 2 Absence of primary follicles and FDCs in BLC-/- mice, and defective homing of CXCR5-/- B cells to lymph node follicles.   Full legend   High resolution image and legend (82k)

A key property of B-cell follicles is the presence of immune-complex-presenting follicular dendritic cells (FDCs)⁶. Staining for complement receptor-1 (CR1), which is highly expressed on FDCs and marginal zone B cells, revealed an absence of primary follicle FDCs in BLC-deficient spleen and lymph nodes (Fig. 2b). Previous studies in CXCR5-deficient mice established a requirement for this receptor in B-cell follicle formation in spleen and Peyer's patches, but did not indicate that the receptor has a role in lymph nodes¹. During our analysis of BLC expression in lymph nodes, however, we observed two types of B-cell-rich zones: polarized follicles that are BLC positive and contain FDCs but few T cells; and areas that lack staining for BLC or FDC markers, contain substantial numbers of T cells, and lack a polarized morphology (Fig. 2c, see also e, f). BLC staining was detected on a subset of CR1⁺ FDCs and on adjacent CR1^- follicular stromal cells ( Fig. 2d). Analysis of lymph nodes from CXCR5^-/- animals for these two types of B-cell areas revealed that they lack FDC-containing primary follicles (data not shown). In transfer experiments, CXCR5 ^+/+ B cells localized in FDC-containing follicles and FDC-deficient B-cell areas of wild-type lymph nodes (Fig. 2e). By contrast, CXCR5^-/- B cells failed to enter FDC-containing follicles (Fig. 2f). CXCR5^-/- B cells also did not migrate into follicles in the spleen and Peyer's patches, as previously observed¹; however, CXCR5^-/- B cells did localize in the FDC-deficient B-cell-rich areas in lymph nodes (Fig. 2f). B-cell homing to lymph node follicles therefore requires BLC and CXCR5, but neither molecule is necessary for migration to FDC-deficient B-cell-rich regions of secondary lymphoid tissues.

A lack of FDCs and organized follicles, as well as impaired development of several lymphoid organs, are phenotypes shared between BLC- and lymphotoxin (LT)-deficient mice⁴. Cell transfer experiments showed that B cells are an essential source of LT12 for the development of FDCs, but it was not clear whether LT12 was expressed by naive B cells^7-9. Although B cells express LT constitutively^10-12, surface expression of this subunit requires LT, and LT expression has only been reported after exposure to activating stimuli, such as CD40L or interleukion (IL)-4 (ref. 11 ), or treatment with endotoxin or phorbol esters¹³. Development of primary follicles is not associated with B-cell activation, however, as it occurs in germ-free mice¹⁴, Ig-transgenic mice¹⁵, T-cell-deficient mice¹⁶, CD40-deficient mice¹⁷ and IL4-deficient mice¹⁸. To investigate LT12 expression on naive B cells, we used a soluble form of the LT12 receptor, LTR-Ig, to stain freshly isolated cells (Fig. 3a–g ). Notably, a significant fraction of total B cells from wild-type spleen showed low but detectable LTR-Ig binding compared with LT- or LT- deficient spleen cells (Fig. 3c, d, g). An even larger fraction of lymph node B cells stained with LTR-Ig (Fig. 3e, f, g), whereas B cells in the blood (Fig. 3a, b, g ) and bone marrow (data not shown) were mostly negative. Similar levels of LT12 were observed on B cells in Ig-transgenic animals (Fig. 3g), showing that the expression was not induced by antigen. We then tested LT12 expression on B cells in BLC-deficient animals (Fig. 3h). LT12 expression on BLC ^-/- peripheral blood B cells was not different from wild-type controls, but expression on B cells from spleen and lymph nodes was significantly reduced (Fig. 3h), establishing that BLC is required for normal LT12 expression on naive B cells.

Figure 3 Membrane LT12 expression on B cells in peripheral lymphoid tissues and dependence on BLC.   Full legend   High resolution image and legend (79k)

To determine whether recirculating B cells upregulated LT12 expression as they moved into BLC-expressing lymphoid organs, we isolated cells from the blood of Ly5.1⁺ donor mice and transferred them into congenic Ly5.2⁺ recipients. At 0.5 and 6 h after transfer, recipient splenocytes were collected and stained with anti-Ly5.1 to detect the transferred cells, and with LTR-Ig (Fig. 3i). At the early time point, when most of the transferred B cells in the spleen are in the non-lymphoid area (data not shown), few of the cells expressed LT12 (Fig. 3i). Six hours after transfer, however, when many of the transferred cells are in BLC⁺ follicular areas (data not shown), LT12 expression was readily detectable (Fig. 3i). When B cells were taken from spleen and transferred intravenously to syngeneic recipients, LTR-Ig staining of cells in recipient blood and spleen soon after transfer had diminished to levels similar to endogenous blood B cells (Fig. 3i). The mechanism for this downregulation is not yet understood, but it suggests that as B cells leave a lymphoid organ and enter the blood they rapidly lose surface LT12 expression. In practical terms, this finding allowed us to use cells from spleen for further transfer experiments to test the mechanism of LT12 upregulation on cells entering lymphoid organs. Within 6 h of transfer, untreated spleen B cells expressed amounts of LT12 equal to endogenous B cells (Fig. 3i). By contrast, cells pretreated with pertussis toxin (PTX), an inhibitor of signalling by Gi-coupled receptors including all known chemokine receptors, failed to upregulate LT12 expression after transfer (Fig. 3i). Furthermore, minimal LT12 upregulation occurred when B cells from wild-type mice were transferred to BLC-deficient recipients (Fig. 3j, k). Notably, when B cells from BLC-deficient donors were transferred to wild-type recipients an exaggerated induction of LT12 occurred (Fig. 3l, m), perhaps because B cells that develop in BLC-deficient animals are hypersensitive to the chemokine. Together, these findings provide strong support for the hypothesis that recirculating B cells upregulate LT12 as they migrate to B-cell areas in response to BLC.

To test whether BLC directly induces LT12 expression, we incubated naive B cells in vitro with recombinant BLC (Fig. 4 ). BLC induced a dose-sensitive upregulation of LT12 on cultured B cells (Fig. 4a). The induction was inhibited by pre-incubation of the cells with PTX (Fig. 4b). Three other chemokines with chemotactic activity on naive B cells were also tested: the broadly expressed CXCR4 ligand, SDF1; and SLC and ELC, two related CC chemokines made in the T-cell area that are ligands for CCR7 (ref. 19). Each of these chemokines caused detectable increases in LT12 expression on B cells, although in all cases the maximal induction was lower than the induction by BLC (Fig. 4c; and data not shown). Although it is not yet clear whether these chemokines can induce LT12 expression on naive B cells in vivo, it seems possible that they contribute to the remaining expression in BLC ^-/- mice (Fig. 3h). BLC-mediated induction of LT12 was sensitive to actinomycin D, cycloheximide and wortmannin (Fig. 4d), providing evidence that BLC may signal through phosphatidylinositol-3-OH kinase to induce lymphotoxin transcription.

Figure 4 BLC induces LT12 upregulation on B cells in vitro.   Full legend   High resolution image and legend (73k)

Despite low expression of LT12 on naive B cells and the absence of primary follicle FDCs in BLC-deficient mice, germinal centres formed in lymph nodes and spleen after immunization with a T-cell-dependent antigen (Fig. 5a). These germinal centres were misplaced and usually smaller than those found in wild-type controls, yet they contained CR1⁺ FDC networks (Fig. 5a). Similar observations for spleen germinal centre formation have been made in CXCR5-deficient mice²⁰. Germinal centre FDCs, like those of primary follicles, require LT12 for their development and maintenance^{4, 21}. Staining with LTR-Ig revealed high LT12 expression on GL7 ⁺ germinal centre B cells in wild-type mice (Fig. 5b ). In contrast to our observations for naive B cells, LT12 expression on germinal centre B cells was not dependent on BLC ( Fig. 5b). We conclude that the requirement for BLC to induce LT12 is overridden in germinal centres, most likely by T-cell-derived signals. CD40 signalling is necessary for germinal centre formation, and might provide the required signal for inducing LT12. In agreement with this possibility, CD40-mediated induction of LT12 is independent of CXCR5 (Fig. 5c).

Figure 5 Misplaced FDC-containing germinal centres in BLC-/- mice and BLC-independent expression of LT12 on germinal centre B cells.   Full legend   High resolution image and legend (129k)

In summary, our findings indicate that BLC has dual roles in follicular compartmentalization of B cells: mediating B-cell attraction, and inducing increased LT12 expression on the recruited cells. LT12 then engages LTRs on non-haematopoietic stromal cells, promoting maturation of FDCs and leading to increased expression of BLC^{4, 5}. FDC maturation is regulated by signals in addition to LT12, including signals from tumour necrosis factor/tumour necrosis factor receptor-1 (ref. 4), and it remains to be determined whether these molecules function in the same or parallel pathways. In addition to showing the expression of LT12 on naive B cells, we have established the existence of a positive feedback loop between BLC and LT12. This feedback loop is likely to be critical in follicle development, causing the low amounts of BLC that are expressed independently of LT12 (ref. 5) to become upregulated as B cells are recruited. When the number of B cells entering an adult lymphoid organ increases, for example during an immune response, the feedback loop may help ensure that the follicular compartment can expand and accommodate the increased numbers of B cells. BLC-mediated B-cell recruitment and LT12 induction may also be involved in Peyer's patch organogenesis, because, in addition to requiring BLC and LT12, these structures depend strongly on B cells²². In support of this, ectopic expression of BLC induces B-cell-dependent and LT12-dependent lymphoid neogenesis²³.

A very early step in development of Peyer's patches and lymph nodes is the local accumulation of CD3^-IL7R⁺ cells that express LT12 (refs 24, 25,26). As some of these cells express CXCR5 (refs 25, 26), we propose that BLC functions in lymph node development by recruiting CXCR5⁺CD3^-IL7R⁺ cells and inducing them to upregulate LT12. Finally, the finding that germinal centre B cells express LT12 and that this expression is independent of BLC, together with the detection of CR1⁺ FDCs in germinal centres of BLC^-/- mice, reveals that the pathway controlling FDC development in germinal centres is distinct from the BLC-mediated feedback loop operating in primary follicles.

Methods
Generation of BLC^-/- mice A 10-kilobase (kb) EcoRI fragment containing the blc gene was isolated from a 129 mouse genomic DNA library (Genome Systems). A targeting vector was constructed in which nucleotides 18–116 of the second exon of blc were replaced with an in-frame stop codon followed by a Mengo virus internal ribosomal entry site, the gene encoding enhanced green fluorescent protein and, in reverse orientation, a loxP-flanked neomycin resistance gene (neo ^r). The linearized construct was electroporated into JM1 129 mouse ES cells (a gift from N. Killeen). G418 (200 µg ml ^-1) resistant colonies were screened by Southern hybridization of EcoRI-digested genomic DNA to a flanking 1.0-kb probe. Targeted clones were injected into C57BL/6 (B6) blastocysts. Chimaeric males were mated to B6 females, and germline transmission of the targeted allele was detected by Southern blot and by PCR using the primers: 5'-CGTCTATGTTCTTTGTCCAATGGG-3' (sense); 5'-CTTACACAACTTCAGTTTTGGGGC-3' (antisense; wild-type); and 5'-ACCTTGTATTCCTTTGTCGAGAGG-3' (antisense; mutant). Homozygous mutant mice were born at Mendelian ratios, were fertile and appeared healthy. Most mice used in this study were mixed 129 and B6 strain background carrying the original targeted allele, although the phenotype of these mice was indistinguishable from mice homozygous for a neo^r-deleted allele. Northern blot analysis was done as described5 using probes specific for blc exon 2 or EF-1. BLC protein was detected in heparin-sepharose precipitates of 1% NP40 spleen homogenates by western blotting with polyclonal goat anti-mouse BLC (R&D Systems). B6 wild-type mice and some of the LT ^-/- mice (on a mixed 129/B6 background²⁷) were from Jackson Labs. Other LT^-/- mice and the LT ^-/- mice were from a B6 colony^{5, 28}. Ig-transgenic mice were of the MD4 line¹⁵. To induce germinal centres, mice were injected intraperitoneally with 100 µg of TNP- or NP-chicken -globulin (Biosearch Technologies) diluted in 200 µl PBS and mixed with the Ribi Adjuvant System (Ribi Immunochem Research).