The molecular and cellular events that initiate the formation of T and B cell areas in developing lymph nodes are poorly understood. In this study we show that formation of the lymphoid architecture in murine neonatal lymph nodes evolves through a series of distinct stages. The initial segregation of T and B cells is regulated in a CXCL13-independent manner, characterized by the localization of B cells in a ring-like pattern in the outer cortex on day 4. However, during this CXCL13-independent phase of lymph node modeling, CXCL13 is expressed and regulated in a lymphotoxin-α1β2 (LTα1β2)-dependent manner. Surprisingly, neonatal B cells are unable to respond to this chemokine and also lack surface LTα1β2 expression. At this time, CD45+CD4+CD3 cells are the predominant LTα1β2-expressing cells and are also capable of responding to CXCL13. From day 4 on, architectural changes become CXCL13 dependent, and B cells become fully CXCL13 responsive, express LTα1β2, and cluster in anatomically distinct follicles. Because the initial induction of CXCL13 is dependent on LTα1β2, a role for CD45+CD4+CD3 cells in inducing chemokine expression in the developing lymph nodes is proposed and, as such, a role in initiation of the shaping of the microenvironment.

One of the hallmarks of the adaptive immune system is the presence of secondary lymphoid organs, which provide the appropriate environment for activation and expansion of naive lymphocytes in response to signals delivered by APCs. Lymph nodes (LNs),3 Peyer’s patches (PPs), and spleen contain anatomically distinct, B cell-rich microenvironments in which an environment permissive for affinity maturation and the generation of immunological memory is created. As such, the correct formation of lymphoid follicles is important for the ability to mount adaptive immune responses (1, 2, 3, 4, 5). The generation of gene-targeted mice provided insights into the cellular and molecular pathways governing the establishment of B cell follicles, implicating several members of the TNF superfamily as pivotal regulators of this process (6, 7, 8, 9, 10, 11, 12, 13). Mice with deficient TNF receptor I (TNF-RI) or lymphotoxin-β receptor (LTβ-R) signaling lack follicle formation in the spleen, whereas impaired LTβ-R signaling in addition leads to a block in the development of LNs and PPs (8, 9, 12, 14, 15, 16, 17, 18). The cellular source of LTα1β2 and TNF-α was determined in a series of splenocyte and bone marrow transfer studies (6, 19, 20). B cells have been shown to deliver the signals necessary for both the induction of follicular dendritic cells (FDCs) as well as the maintenance of lymphoid follicles in adult animals. In spleen, engagement of the LTβ-R by B cells results in increased production of the chemokine CXCL13 by stromal cells. Binding of CXCL13 to its receptor, CXCR5, reciprocally induces signaling events, leading to the additional up-regulation of surface LTα1β2 on B cells and subsequent clustering of these cells, effectively establishing a positive feedback loop important for the integrity of B cell follicles. This is illustrated by the fact that in the absence of CXCL13, follicles in both spleen and LNs fail to form (21). The role of B cells in this process was further dissected by preventing B cells from expressing LTα1β2, by selectively ablating the LTβ gene in B cells. In the spleen, this resulted in the absence of FDCs and disruption of the B cell follicles. However, LNs were much less affected, and follicles and FDCs could still be detected (22). Therefore, although B cell-derived signals appear important in the formation and maintenance of lymphoid follicles in the spleen, the requirements for follicle formation in LNs are less clear.

In this study we analyzed the cellular and molecular mechanisms governing the initial segregation of T and B cells in neonatal LNs. The initial migration of B cells to the outer cortex of the LNs occurs in a CXCL13-independent manner, because these processes are indistinguishable in C56BL/6 and CXCL13−/− mice. Non-B cells regulate the induction of CXCL13, which depends on LTα1β2 expression. We propose a role for LTα1β2+CD45+CD4+CD3 cells in inducing CXCL13 and possibly additional chemokines responsible for the earliest B cell migration and, therefore, in the molding of the LN architecture.

C57BL/6 mice were purchased from Harlan (Horst, The Netherlands) and were kept under routine laboratory conditions. LTα−/− and SCID mice were bred and kept at the in-house animal facilities of the Vrije Universiteit Medical Center. CXCL13−/− mice were described previously (21) and were bred and kept at the in-house animal facilities of Trudeau Institute.

For flow cytometry and immunohistology, the following Abs were used: 6B2 (anti-B220), GK-1.5 (anti-CD4), 2.4G2 (anti-CD16/CD32; all were affinity-purified from hybridoma cell culture supernatants with protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) and labeled in our laboratory if needed), FDC-M2 (anti-FDC; AMS Biotechnology, Oxfordshire U.K.), anti-IgM F(ab)2 (Cappel, West Chester, PA), anti-IgD-PE (DakoCytomation, Glostrup, Denmark), H139.52.1 (anti κ-L chain-FITC; Beckman Coulter, Fullerton, CA), 1D3 (anti-CD19; BD Pharmingen), and 2G8 (anti-CXCR5; BD Pharmingen). Biotin-labeled anti-CXCL13 and anti-CCL21 were purchased from R&D Systems Europe (Oxon, U.K.). For surface LTα1β2 detection, cells were pretreated with 2.4G2 (anti-CD16/32) supplemented with 5% normal mouse serum for 30 min and subsequently incubated with an LTβR-human IgG fusion protein for 60 min (15). Anti-human-PE (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as second-step conjugate. Splenocytes from adult LTα−/− mice were used as negative controls.

Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences). 7-Aminoactinomycin D (Molecular Probes, Eugene, OR) was used to exclude dead cells.

Chemotactic activity was determined using a Transwell system with 5-μm pores (Costar, Corning, NY). Recombinant murine CXCL13 (1 μg/ml; R&D Systems Europe) was used in the lower compartment, whereas 2.5 × 105 to 1 × 106 cells were placed in the upper compartment. Cells were allowed to migrate for 3 h at 37°C, then migrated cells were analyzed by flow cytometry.

Six-micron cryosections were fixed in dehydrated acetone for 5 min and air-dried for an additional 10 min. Sections were incubated with primary Ab for 1 h at room temperature, followed by a 30-min incubation with a Fluor-Alexa-labeled conjugate (Molecular Probes) when needed. Sections were embedded in Fluorstab (ICN Biomedicals, Aurora, OH) and analyzed on an Eclipse E800 microscope (Nikon Europe, Haarlem, The Netherlands).

For visualization of chemokines, sections were incubated overnight at 4°C with chemokine-specific Abs. The Alexa-Fluor 594 tyramide amplification system was subsequently used to enhance the signal according to the manufacturer’s protocol (Molecular Probes).

RNA was extracted on the day of birth from whole mesenteric lymph nodes (C57BL/6) or from mesenteric lymph node rudiments (LTα−/−) using TRIzol (Invitrogen Life Technologies, Gaithersburg, MD) and reverse transcribed with oligo(dT)12–18 (Invitrogen Life Technologies) and random hexamer primers (Invitrogen Life Technologies) using standard protocols. cDNA was analyzed for the expression of CXCL13 and LTβ-R by TaqMan PCR assay using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA).

In situ hybridization was performed on 6-μm cryostat sections using a digoxigenin-labeled CXCL13 probe as described previously (23).

To gain insight into the cellular and molecular processes underlying the initial shaping of the LN microdomains, immunohistochemical analysis of inguinal, axillary, brachial, and mesenteric LNs was performed on the day of birth and 4 and 7 days after birth. Three distinct phases could be distinguished in the segregation of T and B lymphocytes and the formation of B cell follicles. On the day of birth (day 0) the developing LNs showed no apparent organization, with B cells scattered throughout the LN, and T cells being virtually absent (stage I; Fig. 1,A). Starting on approximately day 2, the first wave of αβTCR+ thymic emigrants colonized the LNs, and B cells started to localize to the outer cortex (24, 25). On day 4, the first phase of B cell migration was completed, and B cells were organized in a ring-like structure in the outer cortex of the LNs, the paracortex being occupied by numerous T lymphocytes. However, no follicles were detectable at this time (stage II; Fig. 1,B). Between days 4 and 7, B cells localized in cortical clusters, and the first clearly distinguishable B cell follicles became evident on day 7 (stage III, Fig. 1, C and D). The generation of these follicles was accompanied by the emergence of FDCs, as detected by the FDC-M2 Ab (Fig. 1 D) as well as positive staining for complement receptor 1 (data not shown). Because FDCs are dependent on TNF-α and LTα1β2 for their development (6, 19), this suggests that insufficient levels of these signals are available before day 7.

FIGURE 1.

Formation of B cell follicles during the first week after birth. Peripheral LNs (PLNs) and MLNs were dissected on the day of birth, day 4, and day 7. A–C, B cells were detected with anti-B220 Abs (green), and T cells with anti-CD3ε (red). A, On the day of birth, virtually no T cells are present. B cells appear scattered throughout the organ, with no apparent organization. B, On day 4, B cells have migrated to the outer cortex. T cells now occupy the paracortical region. B cells appear as a ring-like structure in which no follicles can be distinguished. C, From day 7 on, organized B cell follicles become apparent; D, now containing FDCs (FDC-M2; red). At least 10 animals were analyzed per time point; representative PLNs are shown.

FIGURE 1.

Formation of B cell follicles during the first week after birth. Peripheral LNs (PLNs) and MLNs were dissected on the day of birth, day 4, and day 7. A–C, B cells were detected with anti-B220 Abs (green), and T cells with anti-CD3ε (red). A, On the day of birth, virtually no T cells are present. B cells appear scattered throughout the organ, with no apparent organization. B, On day 4, B cells have migrated to the outer cortex. T cells now occupy the paracortical region. B cells appear as a ring-like structure in which no follicles can be distinguished. C, From day 7 on, organized B cell follicles become apparent; D, now containing FDCs (FDC-M2; red). At least 10 animals were analyzed per time point; representative PLNs are shown.

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Development and maintenance of splenic B cell follicles depends on local production of CXCL13, regulated largely by LTα1β2-expressing B cells (6, 19, 20, 21, 22). To determine the role of CXCL13 in the organization of developing LNs, expression of CXCL13 mRNA in newborn LNs was analyzed by RT-PCR (Fig. 2,A). Mesenteric LNs (MLNs) were dissected on the day of birth, day 2, and day 8, and total RNA was extracted. By means of this method, CXCL13 mRNA was already detected within the developing LNs on the day of birth. To address whether this early expression of CXCL13 was dependent on LTα1β2 signaling, as described for the adult spleen, CXCL13 expression was compared between C57BL/6 MLNs isolated at birth and rudimentary MLNs (rMLNs) from LTα−/− mice (Fig. 2 B). To control for possible differences in stromal cell populations between these two mouse strains, CXCL13 levels were normalized to the expression of the LTβ-R, expressed on stromal cells. Quantitative analysis of LTα−/− and C57BL/6 mice revealed a 5-fold decrease in CXCL13 message in the absence of LTα1β2 signaling in total LNs as well as in sorted CD45 stromal cells. These data indicate that in the developing LNs also CXCL13 is regulated in an LTα1β2-dependent manner.

FIGURE 2.

Lymphotoxin-dependent CXCL13 expression in developing lymph nodes. A, Newborn MLNs were analyzed by RT-PCR on the day of birth and days 2 and 8 after birth. Already at birth, CXCL13 mRNA was readily detectable by RT-PCR. B, Newborn MLNs were isolated from C57BL/6 mice, and rudimentary MLNs from LTα−/− mice; mRNA was isolated from the total organ or from sorted CD45 stromal cells. CXCL13 message was normalized to LTβ-R message using TaqMan analysis. In the absence of a LTα1β2 signal, an average 5-fold reduction in CXCL13 message was observed, both in total LN as well as in sorted CD45 stromal cells. C. Popliteal LNs (PLNs) were dissected on the day of birth and on days 2, 4, and 7 after birth, and CXCL13 mRNA was visualized by in situ hybridization. At birth, no CXCL13 message could be detected; the first signal became apparent on day 2. CXCL13 was expressed in the outer cortex, although not in a follicular pattern. On day 4, CXCL13 is organized in a follicle-like expression pattern, which was even more obvious on day 10.

FIGURE 2.

Lymphotoxin-dependent CXCL13 expression in developing lymph nodes. A, Newborn MLNs were analyzed by RT-PCR on the day of birth and days 2 and 8 after birth. Already at birth, CXCL13 mRNA was readily detectable by RT-PCR. B, Newborn MLNs were isolated from C57BL/6 mice, and rudimentary MLNs from LTα−/− mice; mRNA was isolated from the total organ or from sorted CD45 stromal cells. CXCL13 message was normalized to LTβ-R message using TaqMan analysis. In the absence of a LTα1β2 signal, an average 5-fold reduction in CXCL13 message was observed, both in total LN as well as in sorted CD45 stromal cells. C. Popliteal LNs (PLNs) were dissected on the day of birth and on days 2, 4, and 7 after birth, and CXCL13 mRNA was visualized by in situ hybridization. At birth, no CXCL13 message could be detected; the first signal became apparent on day 2. CXCL13 was expressed in the outer cortex, although not in a follicular pattern. On day 4, CXCL13 is organized in a follicle-like expression pattern, which was even more obvious on day 10.

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Finally, the pattern of CXCL13 expression within developing LNs was monitored by in situ hybridization. Using this method, LNs isolated on the day of birth showed no detectable expression of CXCL13 (Fig. 2,C), and the first CXCL13 message was detected on days 1–2 after birth (Fig. 2,D, black arrows). At this time, expression was restricted to the outer cortex, but no clear follicular expression pattern could be distinguished. From day 4 on, CXCL13 expression emerged into a follicular constellation, concordant with the initiation of B cell clusters in the outer cortex (Figs. 2,E and 1,B). The process of B cell clustering was completed on day 7, when B cells had organized into tightly packed follicles, then strong CXCL13 expression could be detected within the follicles (Fig. 1, C and D, and Fig. 2 F). These data show that the LTα1β2-dependent follicular expression of CXCL13 is accompanied by localization of B cells in these putative follicular areas in the outer cortex.

The fact that in adult CXCL13−/− mice, B cell follicles were disrupted indicated the necessity of CXCL13 for follicle maintenance and/or development (21). Therefore, we set out to determine during which stage of development follicle formation in CXCL13-deficient mice is halted, and MLNs dissected from neonatal CXCL13−/− mice were analyzed by immunofluorescence. In contrast to the defective LN architecture reported in MLNs of adult mice, neonatal CXCL13−/− MLNs appeared completely normal until 4 days after birth (stage II; Fig. 3,A). As can easily be appreciated from Fig. 3,A, the initial migration of B cells to the outer cortex seen during stage II (Fig. 1,B) was completed in CXCL13−/− mice by day 4. This indicates that the first phase of B cell migration to the outer cortex was regulated in a CXCL13-independent manner. The subsequent progression to stage III, clustering of B cells in anatomically distinct follicles between days 4 and 7, did not occur in CXCL13−/− mice (Fig. 3 A) and was therefore dependent on CXCL13.

FIGURE 3.

CXCL13-independent initiation of architectural changes in developing lymph nodes. A, MLNs isolated from day 4 and adult CXCL13−/− mice were analyzed by immunofluorescence. B cells were detected with anti-B220 Abs (green), and T cells with anti-CD3ε (red). Development of the lymphoid architecture appeared normal until day 4. B, To determine the CXCL13 responsiveness of neonatal B cells, day 0, day 4, and adult B cells from MLNs were allowed to migrate in a Transwell assay in response to 1 μg/ml CXCL13 for 3 h. The average specific migration of adult B cells was set at 100%, and neonatal values are shown as the relative migration. On the day of birth B cells are virtually unresponsive, whereas on day 4 migratory capacity is comparable to that of adult cells, albeit with a higher degree of variety than seen in adult cells. Shown are averages from three independent experiments. C, B cells from newborn and adult MLNs were stained for CXCR5. Newborn B cells express lower levels of CXCR5 than adult B cells.

FIGURE 3.

CXCL13-independent initiation of architectural changes in developing lymph nodes. A, MLNs isolated from day 4 and adult CXCL13−/− mice were analyzed by immunofluorescence. B cells were detected with anti-B220 Abs (green), and T cells with anti-CD3ε (red). Development of the lymphoid architecture appeared normal until day 4. B, To determine the CXCL13 responsiveness of neonatal B cells, day 0, day 4, and adult B cells from MLNs were allowed to migrate in a Transwell assay in response to 1 μg/ml CXCL13 for 3 h. The average specific migration of adult B cells was set at 100%, and neonatal values are shown as the relative migration. On the day of birth B cells are virtually unresponsive, whereas on day 4 migratory capacity is comparable to that of adult cells, albeit with a higher degree of variety than seen in adult cells. Shown are averages from three independent experiments. C, B cells from newborn and adult MLNs were stained for CXCR5. Newborn B cells express lower levels of CXCR5 than adult B cells.

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The findings that B cells only start organizing in a CXCL13-dependent manner after day 4 prompted us to determine the capacity of neonatal B cells to migrate in response to CXCL13. B cells were isolated from MLNs on the day of birth, on day 4, and from adult mice and tested for their CXCL13 responsiveness in an in vitro Transwell assay (Costar, Corning, NY) (Fig. 3,B). Shown are specific migrations relative to migration of B cells from adult MLNs. Surprisingly, on the day of birth, B cells were virtually CXCL13 unresponsive (Fig. 3 B). The ability to respond to CXCL13 was acquired during the first days of life, reaching levels comparable to those in adults on day 4. However, at this time the B cell pool was still highly heterogeneous with regard to CXCL13-induced migration. To find an explanation for this unexpected unresponsiveness of newborn B cells, neonatal and adult MLNs were dissected, and expression levels of CXCR5, the receptor for CXCL13 (26), were compared between newborn and adult B cells. In agreement with their unresponsiveness to CXCL13 in Transwell assays, neonatal MLN-derived B cells showed lower expression of CXCR5 compared with adult B cells, explaining the inability of these cells to respond to CXCL13. The acquisition of normal levels of expression and full CXCL13 responsiveness represents an additional, previously unappreciated, developmental step in B cell maturation.

Because neonatal LN-derived B cells still have to acquire a distinct feature of mature B cells (CXCL13 responsiveness), an additional phenotypic analysis was performed to assess the degree of maturity of the B cell pool in the neonatal LNs. As shown in Fig. 4,A, phenotypically mature IgM+IgD+ B cells were found on the day of birth in MLNs (Fig. 4,A). These data were confirmed by the fact that ∼40% of CD19+ cells at birth expressed the κ-L chain (Fig. 4 B), indicative of progression beyond the pre-B cell stage. Therefore, a large part of the neonatal MLN B cells appeared phenotypically mature despite their low expression of CXCR5.

FIGURE 4.

Newborn LNs contain phenotypically mature, but LTα1β2-negative, B cells. On the day of birth, B cells from MLNs were analyzed for the expression of surface Igs. A, Gated on CD19+ cells, ∼33% of the B cells in newborn LNs express both IgM and IgD; B, roughly the same percentage of CD19+ B cells expresses the κ-L chain, indicative of maturation beyond the pre-B cell stage. C and D, Surface expression of LTα1β2 was evaluated by staining with a soluble LTβ-R-IgG fusion protein. Splenic B cells from LTα−/− mice were used as a negative control, and B cells from either day 0 MLNs or day 4 MLNs were compared with B cells from adult C57BL/6 MLNs. At birth, no surface LTα1β2 could be detected on B cells. Expression was instigated on approximately day 2 (data not shown) and reached levels comparable to adult values on day 4. The percentage LTα1β2-expressing B cells of the total B cells is shown.

FIGURE 4.

Newborn LNs contain phenotypically mature, but LTα1β2-negative, B cells. On the day of birth, B cells from MLNs were analyzed for the expression of surface Igs. A, Gated on CD19+ cells, ∼33% of the B cells in newborn LNs express both IgM and IgD; B, roughly the same percentage of CD19+ B cells expresses the κ-L chain, indicative of maturation beyond the pre-B cell stage. C and D, Surface expression of LTα1β2 was evaluated by staining with a soluble LTβ-R-IgG fusion protein. Splenic B cells from LTα−/− mice were used as a negative control, and B cells from either day 0 MLNs or day 4 MLNs were compared with B cells from adult C57BL/6 MLNs. At birth, no surface LTα1β2 could be detected on B cells. Expression was instigated on approximately day 2 (data not shown) and reached levels comparable to adult values on day 4. The percentage LTα1β2-expressing B cells of the total B cells is shown.

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In the spleen, induction of surface LTα1β2 on B cells is the second feature attributed to CXCR5 signaling. To test whether the newborn CXCR5low B cells from MLNs had initiated expression of LTα1β2, MLNs from mice on the day of birth and those from adult mice were analyzed for the expression of LTα1β2 (Fig. 4,C). Spleen cells from LTα−/− mice were used as negative controls. In conjunction with their hampered migratory response to CXCL13, day 0 MLN B cells were also surface LTα1β2 negative. The expression of LTα1β2 became apparent from day 2 (data not shown) and reached adult levels on day 4 (Fig. 4 C). This shows that although newborn B cells appear phenotypically mature, their low levels of CXCR5 lead to CXCL13 unresponsiveness as well as lack of surface LTα1β2.

CXCL13 expression in newborn LNs was regulated in an LTα1β2-dependent manner (Fig. 2,B), whereas B cells were LTα1β2 negative at this time (Fig. 4,C). As a consequence, during LN ontogeny, CXCL13 is likely to be induced independently of B cell-derived signals. To further address this issue, protein expression of CXCL13 as well as the other homeostatic chemokine, CCL21, was analyzed in LNs of mice devoid of B cells. Peripheral LNs (inguinal, axillary, and brachial) from SCID mice, which lack mature T and B cells, and control C57BL/6 mice were dissected on days 3 and 10 as were those from adult animals (6–8 wk old), and CXCL13 as well as CCL21 were determined by immunofluorescence (Fig. 5). Visualization of chemokine protein by this method is not as sensitive as detection of mRNA by in situ hybridization, and in C57BL/6 mice, CXCL13 protein is detected no earlier than day 10 on, whereas CCL21 can be visualized from days 1–2 on (Fig. 5). Remarkably, similar to wild-type mice, lymphocyte-deficient SCID mice showed the first detectable CXCL13 protein expression on day 10 (Fig. 5,B). In LNs from adult SCID mice, CXCL13 was expressed in a ring-like pattern in the outer cortex of the lymph nodes (Fig. 5,C). Notably, in the absence of lymphocytes, CXCL13 expression failed to become organized in a follicular pattern. This indicates that induction of CXCL13 occurred independently of B cells, yet follicular organization was still B cell dependent. The kinetics of expression of CCL21 in LNs devoid of B cells was similar to those in C57BL/6 mice (Fig. 5); it was already abundantly present on day 3. Induction of these chemokines in LNs therefore occurs independently of B cells.

FIGURE 5.

Production of homeostatic chemokines in SCID mice. LNs from SCID and C57BL/6 mice were analyzed for the expression of CXCL13 and CCL21 in combination with CD4 on days 3 and 7 after birth and in adult animals. Chemokines were stained in red, and CD4 in green. A, Three days after birth, LNs from SCID mice showed no expression of CXCL13, but showed a strong production of CCL21. This was similar to the chemokine expression patterns seen in C57BL/6 LNs using this method. B, At 10 days after birth, SCID lymph nodes showed the first CXCL13 expression in addition to the already present CCL21, again in agreement with C57BL/6 LNs. C, At 37 days after birth, CXCL13 was abundantly expressed in the cortical region of the SCID lymph nodes, colocalizing with the CD4+ cells. CCL21 expression was comparable to that at earlier time points. In C57BL/6 mice, CXCL13 was localized in the follicular areas, with CCL21 being expressed in the paracortex.

FIGURE 5.

Production of homeostatic chemokines in SCID mice. LNs from SCID and C57BL/6 mice were analyzed for the expression of CXCL13 and CCL21 in combination with CD4 on days 3 and 7 after birth and in adult animals. Chemokines were stained in red, and CD4 in green. A, Three days after birth, LNs from SCID mice showed no expression of CXCL13, but showed a strong production of CCL21. This was similar to the chemokine expression patterns seen in C57BL/6 LNs using this method. B, At 10 days after birth, SCID lymph nodes showed the first CXCL13 expression in addition to the already present CCL21, again in agreement with C57BL/6 LNs. C, At 37 days after birth, CXCL13 was abundantly expressed in the cortical region of the SCID lymph nodes, colocalizing with the CD4+ cells. CCL21 expression was comparable to that at earlier time points. In C57BL/6 mice, CXCL13 was localized in the follicular areas, with CCL21 being expressed in the paracortex.

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Because induction of CXCL13 was regulated in an LTα1β2-dependent, B cell-independent manner, newborn LNs were studied to identify an alternative LTα1β2-expressing cell population (Fig. 6,A). Staining of MLNs on the day of birth for B220 and surface LTα1β2 led to the identification of a single discrete LTα1β2+ population, lacking expression of B220. Analysis of this population showed that the predominant cell type in the newborn LN that expresses LTα1β2 is CD4+ and CD3. These CD45+CD4+CD3 cells have previously been shown to be the inducer cells of PPs and nasal-associated lymphoid tissue organogenesis, and are the putative inducers of LN formation during embryonic development (27, 28, 29, 30, 31). At birth, CD45+CD4+CD3 cells make up ∼40–50% of all hemopoietic cells, and upon adoptive transfer, these cells home to the B cell follicles (32, 33). These features make LTα1β2+CD45+CD4+CD3 cells likely candidates for inducing LTα1β2-dependent chemokines in the developing LN. SCID mice contain a normal population of LTα1β2+CD45+CD4+CD3 cells (32), and in Fig. 5, A and B, the predominant localization of LTα1β2+CD45+CD4+CD3 cells at the outer cortex of the SCID lymph nodes could clearly be seen due to the absence of CD4+TCR+ lymphocytes. This cortical distribution was apparent around day 2 after birth in both SCID and C57BL/6 mice (32) and coincided with the migration of B cells to this area during stage II of follicle formation. Furthermore, similar to the migration of B lymphocytes, this migration of CD45+CD4+CD3 cells occurred independently of CXCL13, because a predominant cortical localization of these cells was seen in CXCL13−/− mice (Fig. 6,B). However, in contrast to neonatal B cells, CD45+CD4+CD3 cells showed a very potent migratory response to CXCL13 in vitro (Fig. 6 C) (34). Therefore, LTα1β2+CD45+CD4+CD3 cells display many of the features attributed to B cells in the adult spleen for induction of the LTα1β2-CXCL13 feedback loop, indicating that LTα1β2+CD45+CD4+CD3 cells may very well be responsible for the induction of homeostatic chemokines during postnatal development and initiation of the lymphoid microdomains.

FIGURE 6.

Properties of CD45+CD4+CD3 cells in newborn LNs. Newborn MLNs were dissected and analyzed for surface LTα1β2 expression using a LTβ-R-IgG fusion protein. A, The only cells expressing LTα1β2 at birth are CD45+CD4+CD3 cells. B, LTα1β2+CD45+CD4+CD3 cells localize to the outer cortex in a CXCL13-independent manner, as shown in MLN from day 4 CXCL13−/− mice. T cells appear red (anti CD3ε) or yellow (anti-CD3ε together with anti-CD4), and CD45+CD4+CD3 cells appear green (anti-CD4). C, In contrast to newborn B cells, CD45+CD4+CD3 cells show a vigorous response to CXCL13 in an in vitro Transwell assay. Shown are specific migrations within one experiment. A representative example of three independent experiments is shown.

FIGURE 6.

Properties of CD45+CD4+CD3 cells in newborn LNs. Newborn MLNs were dissected and analyzed for surface LTα1β2 expression using a LTβ-R-IgG fusion protein. A, The only cells expressing LTα1β2 at birth are CD45+CD4+CD3 cells. B, LTα1β2+CD45+CD4+CD3 cells localize to the outer cortex in a CXCL13-independent manner, as shown in MLN from day 4 CXCL13−/− mice. T cells appear red (anti CD3ε) or yellow (anti-CD3ε together with anti-CD4), and CD45+CD4+CD3 cells appear green (anti-CD4). C, In contrast to newborn B cells, CD45+CD4+CD3 cells show a vigorous response to CXCL13 in an in vitro Transwell assay. Shown are specific migrations within one experiment. A representative example of three independent experiments is shown.

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The shaping of the cellular architecture within developing LNs requires an intricate interplay among several cell types and soluble factors. Postnatal segregation of lymphocytes and arrangement into functionally and anatomically distinct microdomains is a multistep process, in which several distinct stages can be discerned (Figs. 1 and 7). During the first stage (stage I), from day of birth until day 2, LNs are still devoid of αβTCR+ T cells, and the main hemopoietic cell types present are B cells, LTα1β2+CD45+CD4+CD3 cells, and γδT cells (25). During this time, no cellular organization is apparent. The transition from stage I to stage II is marked by an influx of the first αβTCR+ T cells. In conjunction with the influx of T cells, which accumulate in the paracortex of the LN, B cells and CD45+CD4+CD3 cells start to migrate toward the outer cortex. In this study CXCL13 expression was up-regulated, in an LTα1β2-dependent manner, most likely induced by LTα1β2+CD45+CD4+CD3 cells. During stage II, the pattern of CXCL13 mRNA expression indicates the creation of a microenvironment permissive for follicle formation, culminating on day 4 in the organized follicular expression of this chemokine. Stage III is initiated when B cells become responsive to CXCL13, and CXCL13-dependent migration can occur, leading to the clustering of B cells in the outer cortex and the expression of surface LTα1β2. The latter enables B cells to contribute to the sustained production of CXCL13 in a similar way as described for the spleen (21). The LN architecture is completed on day 7, when FDCs become apparent, and B cells are clustered in anatomically distinct, tightly packed follicles (Fig. 7).

FIGURE 7.

Model for the initiation of lymphoid segregation and follicle formation in LNs. During the segregation of T and B cells and the formation of lymphoid follicles, three distinct stages can be perceived. At birth, no αβTCR+ cells are present, and B cells and CD45+CD4+CD3 cells make up the main hemopoietic cell types (stage I). During stage I, CXCL13 is expressed in the outer cortex (left panel). Organization is initiated on approximately day 2, when the first T cells enter the LN. B cells and CD45+CD4+CD3 cells now start to localize to the outer cortex, forming a ring-like pattern on day 4 (stage II). This migration is regulated in a CXCL13-independent manner, whereas expression of CXCL13 is organized in an follicular constellation on day 4. From day 4 on, B cells become CXCL13-responsive and start to cluster in follicular structures. At 7 days after birth, the first anatomically distinct B cell follicles can be detected as well as the first FDCs (stage III). The migrational events during stage III are CXCL13-dependent, and abundant CXCL13 expression can be found within the follicles.

FIGURE 7.

Model for the initiation of lymphoid segregation and follicle formation in LNs. During the segregation of T and B cells and the formation of lymphoid follicles, three distinct stages can be perceived. At birth, no αβTCR+ cells are present, and B cells and CD45+CD4+CD3 cells make up the main hemopoietic cell types (stage I). During stage I, CXCL13 is expressed in the outer cortex (left panel). Organization is initiated on approximately day 2, when the first T cells enter the LN. B cells and CD45+CD4+CD3 cells now start to localize to the outer cortex, forming a ring-like pattern on day 4 (stage II). This migration is regulated in a CXCL13-independent manner, whereas expression of CXCL13 is organized in an follicular constellation on day 4. From day 4 on, B cells become CXCL13-responsive and start to cluster in follicular structures. At 7 days after birth, the first anatomically distinct B cell follicles can be detected as well as the first FDCs (stage III). The migrational events during stage III are CXCL13-dependent, and abundant CXCL13 expression can be found within the follicles.

Close modal

The observed discrepancy between CXCL13 mRNA and protein levels indicates that low levels of CXCL13 protein are likely to be present before day 10. However, because development of the lymphoid architecture in C57BL/6 mice and that in CXCL13−/− mice are completely identical until day 4, this low level of expression is obviously not essential for the early phases of T/B segregation. Furthermore, differences in the sensitivity of the CCL21 and CXCL13 Abs might exist, which could hamper determination of the exact onset of protein expression.

Recently, the importance of B cells in maintenance of the architecture in spleen and LNs of adult mice was questioned (22). Tumanov et al. (22) showed that mice with a B cell-specific LTβ deletion display normal chemokine levels, FDC, and follicular structures in the LN. Furthermore, it was shown that T cell-derived LTα1β2 is sufficient to maintain the lymphoid architecture. This indicates that during adult life the lymphoid architecture is preserved by a combination of T and B cell-derived signals. However, it has to be noted that all these observations were made 8–10 days after immunization with sheep RBC. Because LTα1β2 levels on T cells are dependent on activation state, it is conceivable that high levels of LTα1β2 on activated T cells leads to the generation of a normal lymphoid architecture through reorganization of the already present CXCL13 expression into a follicular pattern.

During the first days after birth (stages I and II), the neonatal B cell pool needs to undergo additional maturation steps before being able to contribute to the LTα1β2-CXCL13 feedback loop. Complete CXCL13 responsiveness is not reached until approximately day 4, when B cells show CXCL13-dependent migration and express LTα1β2. The CXCL13-LTα1β2 positive feedback loop, which was shown to be instrumental in generating tightly packed B cell follicles and maintaining these structures in spleens of adult mice, is therefore in all probability in LNs initiated by non-B cell-derived LTα1β2. The only cells in neonatal LNs that express LTα1β2 are CD45+CD4+CD3 cells; therefore, these cells are the most likely candidate to induce the expression of homeostatic chemokines.

Before the CXCL13-mediated migration leading to follicle formation in stage III, the initiation of T/B cell segregation as well as migration of CD45+CD4+CD3 cells during stage II are regulated independently of CXCL13. This initial segregation of T and B cells is therefore mediated by alternative chemokines. These chemokines are not dependent on T cells for their induction, because in athymic nude mice, B cell migration to the outer cortex occurs via the same kinetics as in C57BL/6 mice (data not shown). Moreover, induction is most likely LTα1β2-independent, because in solitary MLNs, which occasionally develop in LTα-deficient animals, normal T/B segregation was found (35). It is conceivable that CD45+CD4+CD3 cells are also essential for inducing these unidentified chemokines. To establish the involvement of LTα1β2+CD45+CD4+CD3 cells in this process, we have extensively tried to eliminate these cells in vivo using depleting Abs; however, this proved infeasible, probably due to a combination of low complement levels in neonates and the high degree of apoptosis resistance of CD45+CD4+CD3 cells (our unpublished observations). Identification of the signals and chemokines that mediate this CXCL13-independent migration provides novel challenges for future research.

This study provides new insights into the complex interactions underlying the generation of distinct microdomains in which T and B lymphocytes are allowed to efficiently sample Ags presented by APCs. This is a phenomenon seen in secondary lymphoid organs as well as in ectopic tertiary lymphoid structures commonly found in several chronic inflammatory conditions (17, 36, 37, 38, 39, 40, 41, 42). There is accumulating evidence for a high degree of homology between the generation of inflammatory lymphoid structures and the normal development of the lymphoid architecture (36, 43, 44). This indicates that understanding the factors that shape the developing LNs will probably lead to new insights into the formation of ectopic lymphoid structures and will eventually open new therapeutic avenues leading to the targeted disruption or prevention of formation of these structures during chronic inflammation.

We thank Dr. Paul D. Rennert (Biogen, Cambridge, MA) for providing LTβ-R-IgG and for critical reading of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Netherlands Organization for Scientific Research (Grant 901-06-340; to T.C.), the Royal Netherlands Academy of Arts and Sciences (to R.E.M.), National Institutes of Health Grants AI50844 (to F.E.L.) and HL69409 (to T.D.R), and the Trudeau Institute (to F.E.L. and T.D.R.). DNAX is supported by the Schering Plough.

3

Abbreviations used in this paper: LN, lymph node; FDC, follicular dendritic cell; LTα1β2, lymphotoxin-α1β2; LTβ-R, lymphotoxin-β receptor; MLN, mesenteric LN; PP, Peyer’s patch; TNF-RI, TNF receptor I.

1
MacLennan, I. C..
1994
. Germinal centers.
Annu. Rev. Immunol.
12
:
117
.
2
Kelsoe, G..
1996
. Life and death in germinal centers (redux).
Immunity
4
:
107
.
3
Kelsoe, G., B. Zheng.
1993
. Sites of B-cell activation in vivo.
Curr. Opin. Immunol.
5
:
418
.
4
Tarlinton, D..
1998
. Germinal centers: form and function.
Curr. Opin. Immunol.
10
:
245
.
5
Tarlinton, D. M., K. G. Smith.
2000
. Dissecting affinity maturation: a model explaining selection of antibody-forming cells and memory B cells in the germinal centre.
Immunol. Today
21
:
436
.
6
Endres, R., M. B. Alimzhanov, T. Plitz, A. Fütterer, M. H. Kosco-Vilbois, S. A. Nedospasov, K. Rajewsky, K. Pfeffer.
1999
. Mature follicular dendritic cell networks depend on expression of lymphotoxin β receptor by radioresistant stromal cells and of lymphotoxin β and tumor necrosis factor by B cells.
J. Exp. Med.
189
:
159
.
7
Ettinger, R., J. L. Browning, S. A. Michie, W. van Ewijk, H. O. McDevitt.
1996
. Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble lymphotoxin-β receptor-IgG1 fusion protein.
Proc. Natl. Acad. Sci. USA
93
:
13102
.
8
Banks, T. A., B. T. Rouse, M. K. Kerley, P. J. Blair, V. L. Godfrey, N. A. Kuklin, D. M. Bouley, J. Thomas, S. Kanangat, M. L. Mucenski.
1995
. Lymphotoxin-α-deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness.
J. Immunol.
155
:
1685
.
9
Alimzhanov, M. B., D. V. Kuprash, M. H. Kosco-Vilbois, A. Luz, R. L. Turetskaya, A. Tarakhovsky, K. Rajewsky, S. A. Nedospasov, K. Pfeffer.
1997
. Abnormal development of secondary lymphoid tissues in lymphotoxin β-deficient mice.
Proc. Natl. Acad. Sci. USA
94
:
9302
.
10
Futterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, K. Pfeffer.
1998
. The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.
Immunity
9
:
59
.
11
Körner, H., M. Cook, D. S. Riminton, F. A. Lemckert, R. M. Hoek, B. Ledermann, F. Köntgen, B. Fazekas de St. Groth, J. D. Sedgwick.
1997
. Distinct roles for lymphotoxin-α and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue.
Eur. J. Immunol.
27
:
2600
.
12
Togni, P. d., J. Goellner, N. H. Ruddle, P. R. Streeter, A. Fick, S. Mariathasan, S. C. Smith, R. Carlson, L. P. Shornick, J. Strauss-Schoenberger, et al
1994
. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.
Science
264
:
703
.
13
Pasparakis, M., L. Alexopoulou, M. Grell, K. Pfizenmaier, H. Bluethmann, G. Kollias.
1997
. Peyer’s patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor.
Proc. Natl. Acad. Sci. USA
94
:
6319
.
14
Ettinger, R., R. Mebius, J. L. Browning, S. A. Michie, S. van Tuijl, G. Kraal, W. van Ewijk, H. O. McDevitt.
1998
. Effects of tumor necrosis factor and lymphotoxin on peripheral lymphoid tissue development.
Int. Immunol.
10
:
727
.
15
Rennert, P. D., J. L. Browning, R. Mebius, F. Mackay, P. S. Hochman.
1996
. Surface lymphotoxin α/β complex is required for the development of peripheral lymphoid organs.
J. Exp. Med.
184
:
1999
.
16
Rennert, P. D., D. James, F. Mackay, J. L. Browning, P. S. Hochman.
1998
. Lymph node genesis is induced by signaling through the lymphotoxin β receptor.
Immunity
9
:
71
.
17
Fu, Y. X., D. D. Chaplin.
1999
. Development and maturation of secondary lymphoid tissues.
Annu. Rev. Immunol.
17
:
399
.
18
Miyawaki, S., Y. Nakamura, H. Suzuka, M. Koba, R. Yasumizu, S. Ikehara, Y. Shibata.
1994
. A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice.
Eur. J. Immunol.
24
:
429
.
19
Fu, Y. X., G. Huang, Y. Wang, D. D. Chaplin.
1998
. B lymphocytes induce the formation of follicular dendritic cell clusters in a lymphotoxin α-dependent fashion.
J. Exp. Med.
187
:
1009
.
20
Gonzalez, M., F. Mackay, J. L. Browning, M. H. Kosco-Vilbois, R. J. Noelle.
1998
. The sequential role of lymphotoxin and B cells in the development of splenic follicles.
J. Exp. Med.
187
:
997
.
21
Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Förster, J. Sedgwick, J. L. Browning, M. Lipp, J. G. Cyster.
2000
. A chemokine-driven positive feedback loop organizes lymphoid follicles.
Nature
406
:
309
.
22
Tumanov, A., D. Kuprash, M. Lagarkova, S. Grivennikov, K. Abe, A. Shakhov, L. Drutskaya, C. Stewart, A. Chervonsky, S. Nedospasov.
2002
. Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues.
Immunity
17
:
239
.
23
Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, M. D. Cooper, J. L. Browning, J. D. Sedgwick, J. G. Cyster.
1999
. Lymphotoxin α/β and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen.
J. Exp. Med.
189
:
403
.
24
Friedberg, S. H., I. L. Weissman.
1974
. Lymphoid tissue architecture. II. Ontogeny of peripheral T and B cells in mice: evidence against Peyer’s patches as the site of generation of B cells.
J. Immunol.
113
:
1477
.
25
Mebius, R. E., P. R. Streeter, S. Michie, E. C. Butcher, I. L. Weissman.
1996
. A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3 cells to colonize lymph nodes.
Proc. Natl. Acad. Sci. USA
93
:
11019
.
26
Forster, R., A. E. Mattis, E. Kremmer, E. Wolf, G. Brem, M. Lipp.
1996
. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen.
Cell
87
:
1037
.
27
Fukuyama, S., T. Hiroi, Y. Yokota, P. D. Rennert, M. Yanagita, N. Kinoshita, S. Terawaki, T. Shikina, M. Yamamoto, Y. Kurono, et al
2002
. Initiation of NALT organogenesis is independent of the IL-7R, LTβR, and NIK signaling pathways but requires the Id2 gene and CD3CD4+CD45+ cells.
Immunity
17
:
31
.
28
Finke, D., H. Acha-Orbea, A. Mattis, M. Lipp, J. Kraehenbuhl.
2002
. CD4+CD3 cells induce Peyer’s patch development: role of α4β1 integrin activation by CXCR5.
Immunity
17
:
363
.
29
Eberl, G., S. Marmon, M. J. Sunshine, P. D. Rennert, Y. Choi, D. R. Littman.
2004
. An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells.
Nat. Immunol.
5
:
64
.
30
Cupedo, T., G. Kraal, R. E. Mebius.
2002
. The role of CD45+CD4+CD3 cells in lymphoid organ development.
Immunol. Rev.
189
:
41
.
31
Mebius, R. E..
2003
. Organogenesis of lymphoid tissues.
Nat. Rev. Immunol.
3
:
292
.
32
Mebius, R. E., P. Rennert, I. L. Weissman.
1997
. Developing lymph nodes collect CD4+CD3LTb+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells.
Immunity
7
:
493
.
33
Kim, D., R. E. Mebius, J. D. MacMicking, S. Jung, T. Cupedo, Y. Castellanos, J. Rho, B. R. Wong, R. Josien, N. Kim, et al
2000
. Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE.
J. Exp. Med.
192
:
1467
.
34
Honda, K., H. Nakano, H. Yoshida, S. Nishikawa, P. Rennert, K. Ikuta, M. Tamechika, K. Yamaguchi, T. Fukumoto, T. Chiba, et al
2001
. Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer’s patch organogenesis.
J. Exp. Med.
193
:
621
.
35
Fu, Y.-X., G. Huang, M. Matsumoto, H. Molina, D. D. Chaplin.
1997
. Independent signals regulate development of primary and secondary follicle structure in spleen and mesenteric lymph node.
Proc. Natl. Acad. Sci. USA
94
:
5739
.
36
Ruddle, N. H..
1999
. Lymphoid neo-organogenesis: lymphotoxin’s role in inflammation and development.
Immunol. Res.
19
:
119
.
37
Salomonsson, S., P. Larsson, P. Tengner, E. Mellquist, P. Hjelmstrom, M. Wahren-Herlenius.
2002
. Expression of the B cell-attracting chemokine CXCL13 in the target organ and autoantibody production in ectopic lymphoid tissue in the chronic inflammatory disease Sjogren’s syndrome.
Scand. J. Immunol.
55
:
336
.
38
Weyand, C. M., P. J. Kurtin, J. J. Goronzy.
2001
. Ectopic lymphoid organogenesis: a fast track for autoimmunity.
Am. J. Pathol.
159
:
787
.
39
Shi, K., K. Hayashida, M. Kaneko, J. Hashimoto, T. Tomita, P. E. Lipsky, H. Yoshikawa, T. Ochi.
2001
. Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients.
J. Immunol.
166
:
650
.
40
Hjelmstrom, P..
2001
. Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines.
J. Leukocyte Biol.
69
:
331
.
41
Amft, N., S. J. Curnow, D. Scheel-Toellner, A. Devadas, J. Oates, J. Crocker, J. Hamburger, J. Ainsworth, J. Mathews, M. Salmon, et al
2001
. Ectopic expression of the B cell-attracting chemokine BCA-1 (CXCL13) on endothelial cells and within lymphoid follicles contributes to the establishment of germinal center-like structures in Sjogren’s syndrome.
Arthritis Rheum.
44
:
2633
.
42
Hjelmstrom, P., J. Fjell, T. Nakagawa, R. Sacca, C. A. Cuff, N. H. Ruddle.
2000
. Lymphoid tissue homing chemokines are expressed in chronic inflammation.
Am. J. Pathol.
156
:
1133
.
43
Nishikawa, S. I., H. Hashi, K. Honda, S. Fraser, H. Yoshida.
2000
. Inflammation, a prototype for organogenesis of the lymphopoietic/hematopoietic system.
Curr. Opin. Immunol.
12
:
342
.
44
Cupedo, T., R. E. Mebius.
2003
. Role of chemokines in the development of secondary and tertiary lymphoid tissues.
Semin. Immunol.
15
:
243
.