Lymph Node Genesis Is Induced by Signaling through the Lymphotoxin β Receptor

Immunity, Vol. 9, 71–79, July, 1998, Copyright 1998 by Cell Press

Lymph Node Genesis Is Induced by Signaling through the Lymphotoxin b Receptor Paul D. Rennert,* Danelle James, Fabienne Mackay, Jeffrey L. Browning, and Paula S. Hochman Department of Immunology and Inflammation Biogen, Incorporated Cambridge, Massachusetts 02142

Summary We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTb-R and TNF-R signaling. Lymph nodes developed in LTa2/2 mice treated in utero with agonist anti-LTb-R monoclonal antibody. Thus, LTb-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTab/LTb-R interaction were generated. Normal mice treated in utero with LTb-R-Ig and TNF-R55-Ig or antiTNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTa2/ 2 mice had disrupted cellular organization. Therefore, LTb-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes. Introduction Genetic deletion of the mouse lymphotoxin alpha (LTa) gene resulted in a striking phenotype characterized by the absence of lymph nodes (LN) and Peyer’s patches, providing evidence that LT ligands signal the genesis of peripheral lymphoid organs (De Togni et al., 1994; Banks et al., 1995). Since LN and Peyer’s patches were retained in TNF-R552/2 and TNF-R752/2 mice (Pfeffer et al., 1993; Rothe et al., 1993; Erickson et al., 1994), the signaling pathway was thought to be independent of homotrimeric LTa and TNF binding to the TNF receptors (TNF-R). It was therefore hypothesized that the membrane-bound heterotrimeric LTab protein signaled its unique receptor, LTb-R, to mediate LN genesis (Crowe et al., 1994; Erickson et al., 1994; Ware et al., 1995). Our previous studies showing that LN genesis was disrupted by in utero blockade of LTab using LTb-R-Ig fusion protein or anti-LTb MAb supported this hypothesis (Rennert et al., 1996). However, LTb-R-Ig treatment in utero did not block development of a subset of mucosal LN, including the mesenteric, cervical, lumbar, and sacral nodes, suggesting the existence of an LTab independent pathway of LN genesis (Rennert et al., 1996, 1997). This interpretation was supported by the description of the phenotype of LTb2/2 mice, which lacked most LN

* To whom correspondence should be addressed (e-mail: Paul_ [email protected]).

and also Peyer’s patches but retained mesenteric, cervical, and sacral LN (Alimzhanov et al., 1997; Koni et al., 1997). The maternal transfer and genetic deletion studies have not demonstrated that LTb-R is the receptor signaled by the LTab ligand to induce LN genesis. This is of particular importance since LT and TNF ligands bind multiple receptors, so alternative ligand/receptor interactions could explain the different LN phenotypes manifested in LTa2/ 2 and LTb2/2 mice. Human LTa homotrimers bind to TNF-R55 and TNF-R75 and with an apparent lower affinity to the HSV cellular receptor HVEM (Ware et al., 1995; Aggarwal and Natarajan, 1996; Mauri et al., 1998). Likewise, murine recombinant LTa binds mouse TNF-R55, although with 1,000- to 10,000fold less potent activity (Browning et al., 1997). The predominant human heteromeric LTab complex LTa1b2 binds LTb-R, as does the newly described TNF family ligand LIGHT, which itself is also a ligand for HVEM (Crowe et al., 1994; Browning et al., 1995; Mauri et al., 1998). The minor human form LTa2b1 binds to TNF-R55 and TNF-R75 and weakly to LTb-R (Browning et al., 1996; J. L. B., unpublished data). Mouse LTb-R is presumed to bind mouse LTab ligands similarly (Browning et al., 1997; Mackay et al., 1997b). Thus, there is the potential for multiple ligands to interact with LTb-R and for heteromeric receptor clustering by individual ligands, as recently described for the TNF receptors (Pinckard et al., 1997). It is not known whether other ligand/receptor interactions contribute to LN genesis. Complicating the analysis of pathways of LN genesis is the incomplete penetrance of the knockout phenotypes, as shown by the occasional presence of mesenteric and cervical LN in LTa2/2 mice and the occasional absence of any LN in one colony of LTb2/2 mice (De Togni et al., 1994; Banks et al., 1995; Alimzhanov et al., 1997). The in utero blockade experiments and the analyses of LTa2/ 2, LTb2/2, TNF2/2, and TNF-R552/ 2 mice also do not directly address whether LT and TNF ligand/ receptor interactions have additional roles in establishing and/or maintaining cellular organization in adult secondary lymphoid tissues. Data from LTa2/2, TNF-R552/2, TNF 2/ 2, and LTb2/2 mice have demonstrated that permanent interruption of LT and/or TNF signaling caused disruption in splenic marginal zone, lymphocyte, and follicular dendritic cell (FDC) organization and germinal center (GC) formation (reviewed in Liu and Banchereau, 1996; Matsumoto et al., 1997a; von Boehmer, 1997). Importantly, antagonism of LTab interactions using soluble LTb-R-Ig or anti-LTb MAb treatment in adult BALB/c mice caused disruptions in splenic T/B organization, marginal zone integrity, and FDC networks that were very similar to that seen in the LTa2/2 mice (Mackay et al., 1997a; Mackay and Browning, 1998). These studies showed that LTab is critical for the maintenance of cellular organization in the adult spleen. Similar conclusions can be drawn from studies showing that wild-type bone marrow transplantation restores normal cellular architecture to the spleen of LTa2/ 2 mice (Matsumoto et al., 1997b).

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It appears that lymphoid architecture is not controlled by one unique mechanism in all secondary lymphoid organs. In fact, the function of LT and TNF ligands in mediating cellular organization in LN is not well understood. Deletion or antagonism of the TNF/TNF-R55 pathway induced alterations in LN cellular populations that were similar to those observed in spleen, including a lack of organized FDC networks and disrupted B cell follicles (Fu et al., 1997; Pasparakis et al., 1997; Rennert et al., 1997). Despite this altered cellular organization, LN in TNF-R552/2 mice and in mice treated throughout life with a TNF-R55-Ig fusion protein supported GC formation (Pasparakis et al., 1997; Rennert et al., 1997). LN in mice expressing a TNF-R55-Ig transgene lacked B cell follicles and had displaced FDC networks (Ettinger et al., 1998). These studies have identified a role for the TNF/TNF-R55 pathway in maintaining B cell follicular structure and FDC networks in LN, as in spleen, but also suggested different requirements for GC formation in these two lymphoid compartments. Evidence that LT ligands function in establishing and maintaining LN cellular organization has come from analyses of the single mesenteric LN that occasionally develops in LTa2/2 mice and from LTb2/2 mice, which retain mesenteric, cervical, and sacral LN. In these mice, the organization/phenotype of LN B cell, high endothelial venule (HEV), and macrophage populations has been variably described as normal (Fu et al., 1987; Koni et al., 1997) or disrupted (Banks et al., 1995; Alimzhanov et al., 1997). Both LTa2/2 and LTb2/2 mice lack FDC networks, as do TNF2/2 and TNF-R552/2 mice (Fu et al., 1997; Pasparakis et al., 1997). B cell, HEV, and macrophage populations were unaffected in LN of adult mice treated with LTb-R-Ig fusion protein or in mice expressing an LTb-R-Ig transgene after birth (Ettinger et al., 1996; Mackay et al., 1997a), even though FDC networks were displaced or absent (Ettinger et al., 1998; Mackay and Browning, 1998). Thus, while LT ligands appear critical for LN genesis, it is not known to what extent LT is required for the establishment and maintenance of LN cellular organization in the adult. We have created a novel model to study LN genesis and cellular organization by utilizing an agonist antiLTb-R MAb in utero to reverse the genetic defect in LTa2/2 mice. Using these mice, which have LN but lack critical molecular interactions, we have examined the roles of the LTb-R and TNF-R pathways in LN genesis and in mediating LN cellular organization.

Results Activity of Agonistic Anti-LTb-R MAbs Previously, immobilized anti-human LTb-R MAbs were shown to induce the death of human adenocarcinoma WiDr cells in an interferon-g-dependent process (Browning et al., 1996). Therefore, hamster anti-murine LTb-R MAbs, which block soluble murine LTab-induced death of the murine fibroblastoid line WEHI-164 (Mackay et al., 1997b), were evaluated for agonist activity as well. MAb AC.H6, which effectively blocked LTab-induced cell death, also paradoxically potentiated TNF signaling of these same cells (Mackay et al., 1997b). This pattern

Figure 1. LTb-R-Specific MAb AF.H6 Has Antiproliferative Activity on Human WiDr Cells WiDr cells were grown for 4 days in the presence (circles) or absence (squares) of 50 U/ml interferon-g with a control hamster MAb (open) or AF.H6 (closed) at the indicated concentrations. Interferon-g alone has a slight antiproliferative effect and reduced the absorbance from 1.3 to 0.9.

was suggestive of mixed agonistic/antagonistic behavior, similar to that previously described for antibodies specific for TNF family receptors (Ledbetter et al., 1995; Browning et al., 1997). In such cases, an antibody can block ligand binding and thus receptor signaling but also effectively cross-link receptor in the absence of ligand and act as an agonist (Ledbetter et al., 1995). Alternative mechanisms may exist that involve MAb affinity and relative receptor density (Fadeel et al., 1997). The ability of anti-murine LTb-R MAbs AC.H6 and AF.H6 to act alone as agonists was tested on the murine WEHI 164 and human WiDr cell lines, as these hamster MAbs cross-reacted with human LTb-R (J. L. B., unpublished data). WiDr cells were grown for 4 days in the presence or absence of interferon-g with a control hamster MAb Ha4/8 or anti-LTb-R MAb AF.H6 and then analyzed for the extent of cell death. AF.H6 signaled WiDr cells to inhibit cell growth in an interferon-g-dependent manner, even without MAb immobilization (Figure 1). Neither soluble nor plastic-immobilized AC.H6 or AF.H6 induced WEHI 164 death, perhaps due to ineffective immobilization/orientation of the hamster MAb. Therefore, we evaluated MAb signaling of WEHI 164 cells by measuring murine LTb-R expression, as ligandinduced receptor down-regulation is commonly observed in the TNF-R family (Higuchi and Aggarwal, 1994). Using FACS analysis, murine LTab ligand binding was found to down-regulate LTb-R on L929 and WEHI 164 mouse cells (J. L. B., unpublished data). In experiments using MAbs, the mean fluorescence channel of intensity (MFCI) of LTb-R staining on WEHI 164 cells was 77 when untreated, 67 when incubated with control MAb Ha4/8, and 20 when incubated with anti-LTb-R MAb AF.H6 as assessed using biotinylated AC.H6, which binds a nonoverlapping epitope. Thus, MAb AF.H6 specifically induced LTb-R down-regulation. VCAM-1 expression was also increased on WEHI 164 cells, albeit weakly (2- to 3-fold), when stimulated by murine LTab ligand (J. L. B.,

LTb-R Mediates Lymph Node Genesis 73

Table 1. In Utero Treatment with Agonist Anti-LTb-R MAb Induces LN Formation in LTa2/2 Mice a MAb

Specificity

Initial Gestational Day Treated

Litters (#)

Progeny (#)

LN Identified

Ha/48 BB.F6 AF.H6

KLH LTb LTb-R

13 13 12–13

4 2 6

12 5 17

AF.H6

LTb-R

14–15

3

11

AF.H6 AF.H6

LTb-R LTb-R

16 17

1 1

4 3

None None Mandibular, cervical, brachial, axillary, cranial and caudal mesenteric, iliac, sacralb Mandibular, cervical, brachial, axillary, caudal mesenteric, iliacb Axillary None

a Pregnant LTa2/2 mice were injected i.v. with 250 mg MAb on the gestational day indicated and every 48 hr afterward. Pregnant mice were not treated after gd 17. Presence or absence of LN was identified in the progeny at 4 weeks of age. b Inguinal and popliteal LN were identified in two pups from one litter treated with AF.H6 beginning on gd 13 and in two pups from one litter treated with AF.H6 beginning on gd 14.

unpublished data), or MAbs AF.H6 and AC.H6. In experiments using MAbs, the MFCI of staining with rat antiVCAM-1 MAb 429 on WEHI 164 cells was 145 and 150 when unstimulated or incubated with Ha4/8, respectively, but was increased to 223 and 331 when incubated with LTb-R-specific MAbs AF.H6 and AC.H6, respectively. Collectively, these data indicate that AF.H6 and AC.H6 can directly signal through LTb-R in the absence of LTab ligand. LTb-R-Specific MAb Induced Embryonic Genesis of LN The generation of an agonist MAb specific for LTb-R allowed us to directly evaluate the role of this receptor in the genesis of LN. In these experiments, pregnant LTa2/2 mice were treated with agonist anti-LTb-R MAb AF.H6, control MAb Ha4/8, or anti-LTb MAb BB.F6 in utero beginning on gestational day (gd) 12 or 13 and every 48 hr thereafter until gd 17. These hamster MAbs were detected by ELISA in the plasma of the progeny of treated pregnant mothers immediately after birth, with AF.H6 typically present at a much lower level than BB.F6 or Ha4/8, consistent with the rapid clearance of AF.H6 from mouse circulation (Rennert et al., 1996; data not shown). Examination of 17 progeny from 6 litters born to anti-LTb-R-treated LTa2/2 mothers showed that mandibular, cervical, brachial, axillary, mesenteric, iliac, and sacral LN had been induced to develop (Table 1; Figure 2). Additional induction experiments in which treatment with anti-LTb-R MAb in utero was initiated on different gestational days suggested an order of development of LN. The mesenteric LN appeared to develop in two distinct phases, with cranially placed nodes developing prior to caudally placed nodes. Progeny born to mothers treated with anti-LTb-R beginning on gd 12 or 13 reproducibly developed as many as six mesenteric LN and sacral LN (Table 1), while those treated beginning on gd 14 or 15 had at most three mesenteric LN and no sacral LN. Peripheral LN development also depended on the gestational day that treatment commenced, since treatment beginning on gd 12–14 induced the formation of mandibular, brachial, axillary, and iliac LN, while treatment beginning on gd 15 failed to induce genesis of brachial and iliac LN in some litters. Treatment on gd 16 induced axillary LN genesis only, and treatment after

gd 16 resulted in no LN being formed (Table 1). In general, secondary lymphoid tissues known to develop late in gestation or at the time of birth (inguinal and popliteal LN, Peyer’s patches) did not form in the anti-LTb-Rtreated progeny. This suggests that as MAb AF.H6 cleared from circulation following the final treatment, signaling through LTb-R dropped below a threshold necessary to support LN genesis. This sequence of peripheral LN development was consistent with results from normal mice in which LN development was blocked with LTb-R-Ig fusion protein (Rennert et al., 1996). LN were not induced to form in progeny born to pregnant LTa2/2 mice treated with control MAbs (Table 1). These results formally show that signaling through LTb-R is sufficient to induce LN genesis and demonstrate that a phenotype caused by a genetic deletion can be permanently reversed by in utero signaling with a specific agonist MAb.

Figure 2. An Agonist LTb-R-Specific MAb Delivered In Utero Induces LN Genesis in LTa2/2 Mice Shown are brachial (A and B) and mesenteric (C and D) LN in adult LTa1/1 mice (A and C) and in 4-week-old LTa2/2 mice (B and D) that had been treated with 250 mg of anti-LTb-R MAb AF.H6 in utero on gestational days 13, 15, and 17.

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LN Development In Utero Was Blocked by Combined Dosing with LTb-R-Ig and Either TNF-R55-Ig or Anti-TNF MAb The induction of both peripheral and mucosal LN in LTa2/2 mice treated in utero with an agonist anti-LTb-R MAb was interesting, since in utero treatment with LTbR-Ig failed to block the development of mesenteric, cervical, sacral, and lumbar (ie., mucosal) LN in normal mice (Rennert et al., 1996, 1997). To determine which LT or TNF ligands and receptors function in LN genesis, we treated pregnant normal mice with LTb-R-Ig and TNFR55-Ig fusion proteins beginning on various gestational days and at 72 hr intervals thereafter. Surprisingly, although the development of mucosal LN could not be prevented by treating pregnant mice with as much as 400 mg of LTb-R-Ig or 400 mg of TNF-R55-Ig alone, the genesis was blocked using the combination of 100 mg of TNFR55-Ig and 100 mg of LTb-R-Ig. In these mice, the development of all LN, including mesenteric, cervical, sacral, and lumbar, was ablated (Figure 3; Table 2). The ability to block the genesis of mucosal LN depended on the gestational day on which treatment commenced. Treatment with 100 mg each of LTb-R-Ig and TNF-R55Ig on gd 11 was required to ablate all LN development, with greater numbers of mesenteric LN remaining in progeny born to mice treated progressively later in pregnancy (Table 2). The block in development of mesenteric LN appeared to have two distinct phases, with the cranial group of mesenteric LN developing prior to the caudal group (data not shown). Finally, the single most cranial mesenteric LN often developed even in mice treated in utero as early as gd 11. To demonstrate that the block in mucosal LN development was specific, pregnant mice were dosed beginning on gd 11 with 100 mg each of LTb-R-Ig and LFA-3-Ig. All of the progeny examined had mucosal LN (data not shown). To directly examine the potential role of TNF signaling in LN genesis, 100 mg of LTb-R-Ig fusion protein was administered to pregnant mice in combination with 250 mg of anti-TNF MAb TN3, beginning on day 11 of gestation. TN3 can be detected in the plasma of the progeny on the day of birth, indicating successful transfer of the MAb into the developing progeny (data not shown). Concommitant blockade of TNF and of LTab resulted in the ablation of all LN development, mimicking the results of dual TNF-R55-Ig/LTb-R-Ig treatment (Table 2). The ability to block LN genesis using TN3/LTb-R-Ig treatment was temporally regulated in the same manner as TNF-R55-Ig/LTb-R-Ig treatment (Table 2). Thus, TNF can deliver a signal for LN genesis during gestation.

LT and TNF Ligands Mediate LN Cellular Organization The in utero treatment strategy allowed us to investigate whether signaling by LT ligands during gestation is sufficient not only for LN genesis but also to establish the LN cellular organization thought to support immune responses in peripheral lymphoid organs of adult mice. To evaluate the role of LT ligands in establishing LN cellular organization, we examined the cellular populations and immune responsiveness of LN induced to develop in LTa2/ 2 mice. LTa2/2 mice were treated in utero

Figure 3. Development of All LN in Wild-Type Mice Can Be Ablated by Embryonic Treatment with Both LTb-R-Ig and TNF-R55-Ig Fusion Proteins Iliac and sacral LN are present in mice treated in utero with LFA-3Ig fusion protein (A); sacral LN remain in mice treated in utero with LTb-R-Ig fusion protein (B) but are absent in mice treated in utero with both LTb-R-Ig and TNF-R55-Ig fusion proteins (C). BALB/c mice were treated in utero with 100 mg of LFA-3-Ig, 100 mg of LTbR-Ig, or 100 mg each of LTb-R-Ig and TNF-R55-Ig fusion proteins on gd 11, 14, and 17.

with anti-LTb-R MAb AF.H6 beginning on gd 12 or 13 to induce the formation of LN. Since the half-life of AF.H6 in adult mouse circulation is approximately 1 day (J. L. B., unpublished data), and dosing occurs only until gd 17, AF.H6-induced LTb-R signaling is probably very transient. Four to six weeks after birth, we performed immunohistochemical analyses of the LN that had been induced to form in LTa2/2 mice. All LN that had developed in LTa2/ 2 mice were characterized by the localization of B cells in a thin zone that appeared coincident with the subcapsular sinus and segregated from the T cell area

LTb-R Mediates Lymph Node Genesis 75

Table 2. Development of All LN in Wild-Type Mice Can Be Ablated by Treatment In Utero with the Combination of LTb-R-Ig and TNF-R55-Ig or Anti-TNF MAb Reagent

Initial Gestational day Treated

mg Dosed

Litters (#)

Progeny (#)

LN Identifiedb

LFA-3-Ig TNF-R55-Ig TNF-R55-Ig LTb-R-Ig LTb-R-Ig TNF-R55-Ig/LTb-R-Ig TNF-R55-Ig/LTb-R-Ig TNF-R55-Ig/LTb-R-Ig TNF-R55-Ig/LTb-R-Ig

11 11 11 11 11 11 12–13 14 15

400 100 400 100 400 100 100 100 100

2 2 3 7 2 4 2 4 2

6 6 6 18 6 11 6 17 5

Anti-TNF/LTb-R-Ig Anti-TNF/LTb-R-Ig

11 12

250 MAb/100 protein 250 MAb/100 protein

2 2

8 7

6–8 mesenteric, sacral, 6–8 mesenteric, sacral, 6–8 mesenteric, sacral, 6–8 mesenteric, sacral, 6–8 mesenteric, sacral, 0–1 mesenteric 2–4 mesenteric, sacral 2–6 mesenteric, sacral, 6–8 mesenteric, sacral, brachial, axillary 0–1 mesenteric 2 mesenteric, sacral

ea ea ea ea

cervical, pLNb cervical, pLN cervical, pLN cervical cervical

cervical cervical,

a Pregnant BALB/c female mice were injected i.v. with the indicated dose of fusion proteins or MAb, followed by additional doses every 72 hr. Pregnant mice were not treated after gd 17. Presence or absence of LN was determined in the progeny at 4 weeks of age. b LN examined were peripheral LN (pLN) (mandibular, cervical, brachial, axillary, inguinal, popliteal, and iliac) and mucosal LN (cervical, mesenteric, and sacral).

(Figures 4A and 4B). In addition, sialoadhesin expression on macrophage populations within the subcapsular sinus was markedly down-regulated, as was PNAd expression on peripheral LN HEV and both PNAd and MAdCAM-1 expression on mesenteric LN HEV (Figures 4C–4F). FDC were absent, as shown by the loss of staining for FDCM1 (data not shown) and FDCM2 (Figures 4G and 4H). These results suggest that LTb-R signaling caused by treatment with anti-LTb-R MAb during gestation is not sufficient to establish the cellular organization within LN. Despite the disruption in B cell and FDC populations, mesenteric LN induced to develop in LTa2/2 mice formed GC (presumably in response to environmental antigens), as assessed by positive peanut agglutinin (PNA) staining (data not shown). To further examine the relative role of LTb-R and TNFR55 pathways in maintaining cellular organization in LN of adult mice, we treated 6- to 8-week-old BALB/c and C57Bl/6 mice every 72 hr with 100 mg of receptor-Ig fusion proteins (for 10 days) or 200 mg of MAbs (for 7 days). Peripheral lymphoid organs were harvested 24 hr after the final injection. Immunohistological examination of T/B cell organization in LN of normal adult BALB/c mice treated with TNF-R55-Ig fusion protein showed that B cells lose their typical follicular localization (Figure 5), a phenotype similar to that observed in the spleen (data not shown). Treatment with the anti-TNF-specific MAb TN3 induced a similar, although less marked, phenotype (Figure 5). Neither the LTa-specific MAb AF.B3 nor control MAb Ha4/8 disrupted B cell follicular localization (Figure 5), suggesting that TNF is the relevant ligand that engages TNF-R to maintain LN B cell follicles. Treatment of adult BALB/c mice with LTb-R-Ig or control LFA-3-Ig appeared to have no effect on T/B cellular organization, sialoadhesin expression, or HEV differentiation in LN (Figure 5; data not shown), although FDC staining was ablated after LTb-R-Ig treatment (Mackay and Browning, 1998). This contrasts with observations on the spleen from LTb-R-Ig-treated adult BALB/c mice, which had disrupted marginal zones, FDC, and B cell populations, as has been recently described (Mackay et al., 1997a; Mackay and Browning, 1998). Treatment

of C57Bl/6 mice with LTb-R-Ig, TNF-R55-Ig, and LFA3-Ig produced immunohistochemical results identical to those obtained using BALB/c mice. Taken together, these results suggest that postgestational LTab/LTb-R interaction is critical for the establishment of cellular organization in LN, including the formation of B cell follicles. However, once established, B cell follicle integrity in LN depends on signals through TNF-R rather than LTb-R. In contrast, FDC networks in LN require signals through both pathways to be maintained. Discussion Using a novel strategy of maternal transfer, we have shown that delivery of an agonist anti-LTb-R MAb to developing LTa2/ 2 mice induces the formation of LN. While manipulation of receptor signaling in vivo via injection of adult mice with agonist MAb is well established for TNF-R family members (Ogasawara et al., 1995; Tian et al., 1995; Shuford et al., 1997), this demonstrates that agonist signaling can permanently reverse a developmental defect caused by genetic deletion. Furthermore, our results show that the formation of all LN can be mediated via signaling through LTb-R and indicate that this pathway plays a dominant role in LN genesis. Since LTa2/2 mice lack essentially all LN (De Togni et al., 1994; Banks et al., 1995), this suggests that LN development is mediated by the interaction of LTab ligand with LTb-R. This result extends our previous studies in which LN development was blocked using LTb-R-Ig fusion protein (Rennert et al., 1996, 1997). These studies have produced a puzzling contradiction, as the results show that all LN can be induced to form via signaling through LTb-R, but not all LN formation is ablated by blocking LTab/LTb-R engagement during development. That these differences mimic those observed between the LTa2/ 2 and LTb 2/2 mice further complicates the synthesis of the available data. To untangle the ligand/receptor interactions that account for the genesis of mucosal LN (mesenteric, cervical, lumbar, and sacral) that are observed in mice treated with LTbR-Ig in utero and in LTb2/2 mice (Rennert et al., 1996,

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Figure 5. Antagonism of the TNF-R55 but Not the LTb-R Pathway in Normal Adult Mice Disrupts the Maintenance of Cellular Organization in LN

Figure 4. Embryonic Signaling of LTb-R Is Insufficient to Establish or Maintain the Cellular Organization within LN Induced to Form in LTa2/2 Mice Shown are immunohistochemical analyses of representative brachial LN from LTa1/1 mice (A, C, E, and G) and induced to form in LTa2/2 mice (B, D, F, and H). LN were dissected from 4-week-old mice and stained with antibodies to B220 ([A and B], green), CD4 ([A and B], red), PNAd (C and D), sialoadhesin (E and F), and FDCM2 (G and H).

1997; Alimzhanov et al., 1997; Koni et al., 1997), we employed a combination of antagonist receptor-Ig fusion proteins and blocking MAbs. Although previous studies using receptor blockade or knockout mice had indicated that TNF or LTa3 interactions with TNF-R are not necessary for LN genesis (Pfeffer et al., 1993; Rothe et al., 1993; Erickson et al., 1994; Pasparakis et al., 1996; Rennert et al., 1996), we have now shown that treatment of normal mice in utero with LTb-R-Ig and TNF-R55-Ig effectively blocked the development of all LN. Since results from LTb2/ 2 mice show that mucosal LN can form in the absence of LTb-containing ligands (Alimzhanov et al., 1997; Koni et al., 1997), and the available biochemical evidence indicates that LTa3 cannot bind LTb-R in either the human or murine systems (Crowe et al., 1994; Browning et al., 1995, 1997), the simplest interpretation of the available data is that in the absence of LTb-R signaling a TNF-R-dependent pathway mediates mucosal LN genesis. Specifically, LTab signals LTb-R to induce the formation of peripheral and mucosal LN, and, as shown by our results using anti-TNF MAb TN3 and LTb-R-Ig fusion protein in combination to block all LN

Normal adult mice were treated i.p. with purified fusion proteins for 10 days or with MAbs for 7 days starting at 6 weeks of age, and then LN were harvested for analysis of cellular organization in LN. Axillary LN from LFA-3-Ig (A), TNF-R55-Ig (B), LTb-R-Ig (C), MAb Ha4/8 (D), MAb TN3 (E), and MAb AF.B3 (F) treated normal adult mice were stained with antibodies to B220 (green) and CD4 (red).

genesis in utero, TNF/TNF-R engagement can provide a signal for the genesis of mucosal LN. The latter pathway is not distinguished in TNF-R2/2 or TNF2/ 2 mice, since LTb-R signaling is the dominant pathway for genesis of all LN, but is clearly revealed when the dominant pathway is blocked. The phenotype of the LTa2/2 mouse, which lacks all LN, suggests additional complexity. However, the accumulated data show that signaling by TNF can induce mucosal LN formation, although other variables may influence the efficacy of this pathway. This model provides an explanation for the incomplete penetrance of the knockout phenotypes of both the LTa2/ 2 and LTb2/ 2 mice (De Togni et al., 1994; Banks et al., 1995; Alimzhanov et al., 1997; Koni et al., 1997). The occasional mesenteric and cervical LN found in LTa2/ 2 mice may form due to TNF binding to TNF-R, although this interaction is clearly insufficient to induce the formation of all mucosal LN. It is unclear why this is so; however, an examination of TNF levels in LTa2/2 mice may provide some insight (Alexopoulou et al, 1998). Similarly, in one colony of LTb2/2 mice, only 75% of mice have mucosal LN (Alimzhanov et al., 1997), suggesting that TNF-R can signal to induce mucosal LN genesis but also showing that this interaction is insufficient to fully induce mucosal LN formation in the absence of LTb-containing ligands. It is not known if TNF expression is compromised in LTb2/2 mice.

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A preliminary report of the loss of all LN in the LTbR2/ 2 mouse (Alimzhanov et al., 1997; Fu¨tterer et al, 1998 [this issue of Immunity]) appears to be inconsistent with the model we have presented, unless the TNF-R pathway of LN genesis requires LTb-R/TNF-R heterocomplex formation or receptor clustering. Although TNFR55/TNF-R75 heterocomplexes have been observed (Pinckard et al., 1997), LTb-R-containing heterocomplexes have not been described. However, LTb-R activation by an agonist MAb can potentiate signaling by TNF, suggesting that cooperative signaling between the TNFR55 and LTb-R pathways can occur in both the human and murine systems (Browning et al., 1996, 1997). Other pathways for LN genesis have been hypothesized. For example, our results do not exclude the involvement of an LTa3/HVEM pathway in LN genesis (Mauri et al., 1998). It is also possible that a novel LTa ligand signals through LTb-R or an LTb-R signaling complex to induce the formation of mucosal LN, but in a manner that cannot be blocked by soluble LTb-R-Ig fusion protein. Finally, the recently described ligand LIGHT, which shares significant homology with LTb and binds specifically to LTb-R as a homotrimer (Mauri et al., 1998), could induce the genesis of the occasional single mesenteric LN found in LTa 2/2 mice. However, our results using TNF-R55-Ig or anti-TNF in combination with LTb-R-Ig to ablate all LN formation suggest that such pathways do not normally mediate LN genesis. Although the receptor agonist strategy showed that LTb-R signaling is the dominant pathway for LN genesis, our examination of peripheral and mucosal LN that developed in LTa2/2 mice shows unambiguously that gestational signaling through LTb-R is not sufficient for the establishment and maintenance of normal LN cellular organization. The near total loss of addressin and sialoadhesin expression on HEV and macrophages, respectively, and the dissolution of B cell follicles and FDC networks suggest that critical signals that would normally direct the establishment of fully developed microarchitecture in LN are absent in LTa2/2 mice. The developmental window during which LN cellular architecture is established may be very narrow, since B cell follicles are apparent 4 days after birth (Mebius et al., 1997). Expression of an LTb-R-Ig transgene beginning 3 days after birth did not appear to disrupt LN cellular organization, although this may have been due to insufficient protein expression (Ettinger et al., 1996). Likewise, our studies of LTb-R-Ig antagonism in adult BALB/c or C57Bl/6 mice indicate that, once established, cellular organization in LN does not require sustained LTb-R engagement, in contrast with the dramatically altered cellular organization in the spleen (Mackay et al., 1997). Strikingly, B cell follicles are maintained in LN of LTb-RIg-treated adult mice despite the loss of FDC networks (Mackay and Browning, 1998). Therefore, while the maintenance of splenic B cell follicular organization and FDC networks are mutually regulated and appear to require LTab/LTb-R engagement, LN B cell follicular organization remains functionally independent of LTb-R signaling and FDC networks. In contrast with our results using LTb-R-Ig, treatment of adult mice with TNF-R55-Ig caused effects on LN organization that closely resembled those seen in TNFR552/ 2 and TNF2/2 mice (Pasparakis et al., 1996, 1997;

Fu et al., 1997; Tkachuk et al., 1998). In these mice, the loss of B cell follicular organization was coincident with the loss of FDC networks. Furthermore, LN and splenic cellular organization was similarly affected, showing that these lymphoid organs have similar requirements for TNF-R signaling to maintain B cell follicular organization and FDC networks. As expected given the phenotype of the TNF2/2 mouse, B cell follicular organization was also disrupted by treatment with the anti-TNF MAb TN3, although to a lesser extent, possibly due to the shorter course of treatment (7 versus 10 days). These data demonstrate that the disruption in splenic and LN cellular organization observed in mice with blocked TNF/TNF-R engagement reflects the interruption of ongoing physiological processes rather than arrested tissue development as recently postulated (Ettinger et al., 1998). While the mechanisms that direct the establishment of cellular microarchitecture in secondary lymphoid organs are not understood, the recent identification of chemotactic agents expressed in LN and at high levels on HEV suggests possible signaling networks that could be disrupted in LTa2/ 2 and LTb2/2 mice (Forster et al., 1996; Adema et al., 1997; Yoshie et al., 1997; Gunn et al., 1998; Legler et al., 1998). It is intriguing to note that BLR-12/2 mice show a pattern of B cell follicular disruption that more closely resembles that of adult mice treated with LTb-R-Ig rather than TNF-R55-Ig; that is, the capacity of B cells to form follicles is normal in LN but not in spleen in these mice (Forster et al., 1996). It may be that the BLR-1 chemotactic pathway is dependent on the LTab/LTb-R pathway and not the TNF/TNFR55 pathway as has been speculated (Goodnow and Cyster, 1997). In summary, the novel strategy of maternal transfer has allowed us to show that signaling through LTb-R provides the dominant pathway for LN genesis and that signaling through TNF-R can provide a redundant pathway for mucosal LN genesis. Furthermore, LTb-R signaling is critical during neonatal life for the establishment of cellular architecture in LN and acts distinctly from TNF-R in maintaining lymphocyte and FDC populations in the spleen and LN of the secondary lymphoid system. Experimental Procedures Animals All animals were housed under conventional barrier protection and handled in accordance with institutional guidelines. LTa2/2 and LTa1/1 mice provided by Dr. David Chaplin and BALB/c and C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME) were bred in our animal facility. For some experiments, gd 14 timed pregnant BALB/c mice were purchased (Jackson). For experiments on adult animals, 6- to 8-week-old female BALB/c and C57Bl/6 mice (Jackson) were used. Monoclonal Antibodies, IgG1-Fusion Proteins, and Cytokines The generation of LT-specific MAbs and fusion proteins used in this study has been described previously (Browning et al., 1997; Mackay et al., 1997b). In brief, fusion proteins were comprised of the extracellular domain of murine LTb-R (LTb-R-Ig), murine TNF-R55 (TNFR55-Ig), or human LFA-3 (LFA-3-Ig) fused to the hinge, CH2, and CH3 domains of human IgG1. MAbs were raised in Armenian hamsters that had been immunized with purified murine LTab or proteinA-coupled recombinant LTb-R-Ig fusion protein. TN3, the blocking MAb specific for TNF (Sheehan et al., 1989), was generously provided by Dr. Robert Schreiber. The hybridoma secreting the control anti-KLH Armenian hamster Ig Ha4/8 was obtained from Dr. Donna

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Mendrick. Murine LTab (Mackay et al., 1997) and human IFNg were produced at Biogen.

Functional Characterization of Agonist Anti-LTb-R-Specific MAb Agonist activity of anti-LTb-R MAb AF.H6 was determined using cellular proliferation assays as described (Browning et al., 1996). In brief, serial dilutions of cytokines or MAbs were prepared in 50 ml of media in 96-well plates. WiDr cells (5000) in 50 ml of media were added with or without IFNg. After 4 days, 10 ml of 5 mg/ml MTT was added and allowed to incubate for 3 hr, at which point the formazin was dissolved by adding 100 ml of 10% SDS in 10 mM HCL. After a further 24 hr incubation at 378C, the OD was quantitated at 550 nm.

Acknowledgments We wish to thank the following individuals for creating, characterizing, and producing MAbs and receptor-Ig fusion proteins: Chris Benjamin, Irene Dougas-Sizing, Mohammad Zafari, Werner Meier, Konrad Miatkowski, Joe Amatucci, David Griffiths, Cathy Hession, Gerard Majeau, and Apinya Ngam-Ek. We thank David Chaplin for LTa2/2 mice, Robert Schreiber for MAb TN3, and Marie KoscoVilbois for MAbs FDC-M1 and FDC-M2.

Received May 4, 1998.

References LN Induction and Ablation Protocols To generate peripheral and mucosal LN in the progeny, pregnant female LTa2/2 mice were injected with 250 mg of agonist anti-LTb-R MAb AF.H6 via the tail vein beginning on gd 12 or 13, followed by additional injections i.v. every 48 hr. Other pregnant LTa2/2 mice were treated either with anti-LTb-R i.v. on different gestational days to examine the order in which LN could be induced to form or with control hamster MAbs HA/48 (anti-KLH) and BB.F6 (anti-LTb). For studies on the ablation of LN development, pregnant female BALB/c mice were injected i.v. multiple times with 250 mg of the TNF-specific blocking MAb TN3, or the LTa-specific blocking MAb AF.B3, or with 100 mg of purified murine LTb-R-Ig, murine TNF-R55-Ig, human LFA3-Ig, or with combinations thereof, beginning on gd 11, 12, 13, 14, or 15. MAbs were given at 48 hr intervals and fusion proteins at 72 hr intervals until gd 17. The LN induction and ablation protocols rely on the ability of yolk sac and placental Fc receptors to transport Ig fusion proteins from the circulatory system of the mother into the developing progeny, a method we refer to as maternal transfer. Such transport of Ig starts between gd 11 and 12 in the mouse (Morphis and Gitlin, 1970). In all experiments, we avoided daily dosing to alleviate stress on the pregnant mice. Representative progeny were examined for the presence of LN and Peyer’s patches at 4 weeks of age.

Treatment of Adult Mice with Receptor-IgG1 Fusion Proteins and MAbs For analysis of cellular organization in adult animals, 6-week-old BALB/c and C57Bl/6 mice were dosed i.p. every 72 hr with 100 mg of purified LTb-R-Ig, TNF-R55-Ig, or LFA-3-Ig fusion proteins for 10 days or 200 mg of MAbs AF.B3, TN3, or Ha/48 for 7 days. Mandibular, cervical, brachial, axillary, inguinal, mesenteric, iliac, and sacral LN and spleen were removed 72 hr after the final dose and frozen for immunohistochemistry as previously described (Rennert et al., 1996, 1997).

Immunohistochemistry Mouse spleen and LN were harvested, frozen, sectioned, and stained for B220, CD4, MAdCAM-1, the MECA79 antigens GlyCAM-1 and CD34 (peripheral node addressins [PNAd]), sialoadhesin, and peanut agglutinin (PNA) reactivity as previously described (Rennert et al., 1996, 1997). For staining of FDC, slides were incubated for 30 min with 0.3% H2O2/PBS to block endogenous peroxidase, then incubated for 1 hr with 10 mg/ml rat anti-mouse FDCM1 or rat antimouse-FDCM2 (generously provided by Dr. M. Vilbois-Kosco). After washing, the slides were incubated with 10 mg/ml horseradish peroxidase (HRP)-coupled goat anti-rat IgG (Jackson Immunoresearch, West Grove, PA), washed again, then fixed 1 min in methanol before initiating the enzymatic reaction. HRP activity was developed using the diaminobenzidine (DAB) tablet substrate kit (Sigma, St Louis, MO). The stained tissue sections were counterstained with Giemsa (Fluka, Buchs, Switzerland). All sections were viewed under 503 or 1003 optics and captured as digitized image files, as described previously (Rennert et al., 1996, 1997), using either single or dual filters. The images shown are representative of those obtained from a minimum of four animals per treatment group from a minimum of four independent experiments.

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