TNF Receptor-1 Is Required for the Formation of Splenic Compartments during Adult, but Not Embryonic Life

This information is current as Novica M. Milicevic, Karola Klaperski, Klaus Nohroudi, of October 1, 2021. Zivana Milicevic, Katja Bieber, Babett Baraniec, Maike Blessenohl, Kathrin Kalies, Carl F. Ware and Jürgen Westermann J Immunol published online 27 December 2010 http://www.jimmunol.org/content/early/2010/12/24/jimmun ol.1000740 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 27, 2010, doi:10.4049/jimmunol.1000740 The Journal of Immunology

TNF Receptor-1 Is Required for the Formation of Splenic Compartments during Adult, but Not Embryonic Life

Novica M. Milic´evic´,*,1 Karola Klaperski,†,1 Klaus Nohroudi,†,‡ Zˇ ivana Milic´evic´,* Katja Bieber,† Babett Baraniec,† Maike Blessenohl,† Kathrin Kalies,† Carl F. Ware,x and Ju¨rgen Westermann†

Lymphotoxin b-receptor (LTbR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTbR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTbR2/2, TNFR12/2 spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTbR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/

B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required Downloaded from both LTbR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases. The Journal of Immunology, 2011, 186: 000–000. http://www.jimmunol.org/ he morphological feature common to all secondary lym- of biological processes (8, 9), including the development of sec- phoid organs is their division into two main areas, the ondary lymphoid organs and the formation of T and B cell zones T T cell zone and the B cell zone [also called periarteriolar during embryonic life (ontogeny) (6, 10). It is found on a variety lymphatic sheath and follicle in the spleen, paracortex and follicle of cell types (including nonhematopoietic stroma cells, dendritic in lymph nodes, and interfollicular region and follicle in Peyer’s cells [DCs], macrophages) and has two membrane-bound ligands patches, respectively (1)]. By preferentially accumulating circu- (LTa1b2, LIGHT), which are preferentially expressed by hema- lating T lymphocytes in the T cell zone and circulating B lym- topoietic cells [e.g., lymphoid tissue inducer (LTi) cells and ac- phocytes in the B cell zone (2), secondary lymphoid organs fa- tivated T and B cells (6, 11, 12)]. During ontogeny, LTa1b2 by guest on October 1, 2021 cilitate the encounter of rare Ag-specific lymphocytes with APCs expressed by LTi cells binds to LTbR on stromal organizer cells, carrying their cognate Ag (3, 4), thereby making the activation which leads to the upregulation of adhesion molecules (e.g., of resting T and B cells possible. The subsequent interaction of ICAM-1, VCAM-1, mucosal addressin cell adhesion molecule-1) activated T and B cells occurs at the border between the T and and the secretion of (e.g., CCL19, CCL21, CXCL13) B cell zone (5). and so facilitates the segregation of T and B cells into discrete T 2 2 b-receptor (LTbR) and TNF receptor-1 (TNFR1) and B cell zones (6, 13). Thus, LTbR / mice lack lymph nodes are the main regulators for the development of secondary lym- and Peyer’s patches, and the spleen has no marginal zone and phoid organs (6, 7). LTbR is a molecule involved in a wide array shows poorly developed T and B cell zones, which in turn are unable to form germinal centers (6, 10, 14). TNFR1 is widely *Institute of Histology and Embryology, Faculty of Medicine, University of Beograd, expressed on all nucleated cells and especially on follicular DCs 11000 Beograd, Serbia; †Center for Structural and Cell Biology in Medicine, Institute (FDC) and macrophages (6). TNFR1 engages TNF (both soluble of Anatomy, University Lu¨beck, 23538 Lu¨beck, Germany; ‡Department I of Anat- a a omy, University of Cologne, 50931 Cologne, Germany; and xLaboratory of Molec- and membrane-bound) and LT 3 (soluble, LT ), which are mainly ular Immunology, Sandford|Burnham Medical Research Institute, La Jolla, CA 92037 produced by activated T and B cells, but also by FDCs, macro- 1N.M.M. and K.K. contributed equally to this work. phages, and mast cells (11). In TNFR1-deficient mice, lymph Received for publication March 9, 2010. Accepted for publication November 22, nodes are always and Peyer’s patches are usually present (15), and 2010. the splenic compartments including the marginal zone are rea- 2/2 This work was supported by the Ministry for Science and Technological Development sonably well developed (16); however, similar to LTbR mice, of Republic of Serbia (Grant 175005) and is a part of the Institutional Academic Co- germinal centers fail to form in response to Ag (6, 17). In contrast operation between Beograd and Lu¨beck (Project DEU/1033146), which is financially b supported by the Alexander von Humboldt-Foundation, Bonn, Germany. In addition, to embryonic life, the role of LT R and TNFR1 in the formation this work was supported by the research program Autoimmunity of the Medical Faculty of lymphoid tissues during adult life is not well defined (18, 19). in Lu¨beck and by the Deutsche Forschungsgemeinschaft (SFB 654; C4). This question is of great significance, because generation of new Address correspondence and reprint requests to Dr. Ju¨rgen Westermann, Center for lymphoid tissue with characteristically developed T and B cell Structural and Cell Biology in Medicine, Institute of Anatomy, University Lu¨beck, Ratzeburger Allee 160, D-23538 Lu¨beck, Germany. E-mail address: westermann@ zones (also called tertiary or ectopic lymphoid tissue) is observed anat.uni-luebeck.de in the course of many autoimmune and chronic inflammatory Abbreviations used in this article: DC, dendritic cell; FDC, follicular dendritic cell; diseases and has been implicated in their induction and mainte- LTbR, lymphotoxin b-receptor; LTi, lymphoid tissue inducer; TNFR1, TNF recep- nance (20–23). Elucidation of the molecular mechanisms involved tor-1; WT, wild-type. in the formation of lymphoid tissue during adult life could lead to Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 new targets for the treatment of these diseases.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000740 2 TNFR1 IS REQUIRED FOR THE FORMATION OF SPLENIC COMPARTMENTS

2 2 Because LTbR / mice lack lymph nodes and Peyer’s patches, mm) were cut, air-dried at room temperature for 2 h, and fixed in methanol- the role of LTbR and TNFR1 in the formation of T cell and B cell acetone (1:1 [v/v], 10 min at 220˚C) followed by fixation in 4% para- zones during ontogeny and adult life can only be compared using formaldehyde (45 min at 4˚C). Sections were incubated with the appro- b priate dilution of primary Ab for 1 h followed by secondary reagents with splenic tissue. To determine the role of LT R and TNFR1 in the an incubation time of 30 min each. Labeled cells were revealed either by compartmentalization of the spleen during ontogeny, we quanti- the Fast Blue or diaminobenzidine reaction. The sections were mounted tatively analyzed the size of splenic tissue in mice deficient in with water-soluble medium and coverslipped (3). respective signaling pathways. The role of LTbR and TNFR1 in The area of the different compartments was determined using an Axi- overt 200/HAL 100 microscope (Zeiss, Jena, Germany) and Palm@Robo the formation and function of lymphoid tissue generated during V2.2.2 software (P.A.L.M Microlaser Technologies, Bernried, Germany) adult life was studied by avascular implantation of splenic tissue and expressed as percentage of the total surface area. The area of the mixed (24, 25). After implantation into the greater omentum of the T/B cell zone was determined as follows. First, splenic sections were stained peritoneal cavity, the splenic tissue first becomes necrotic, because for either T cells or B cells, and the positively stained areas were determined the blood supply is lacking. Then, formation of splenic lymphoid (value 1 and value 2). On a consecutive section, T and B cell staining was performed together, and the resulting area was measured (value 3). By tissue starts de novo from surviving stromal cells and within 8 wk adding value 1 and 2, and then subtracting value 3, the size of the mixed T/B reproduces an equivalent to that of the adult organ (24, 25). By zone was obtained. These results were confirmed by simultaneously la- using splenic tissue from either LTbR2/2 or TNFR12/2 mice, it beling T and B cells with different fluorescent dyes (green and red, re- was possible to selectively investigate the molecular mechanisms spectively) and analyzing the overlapping area by confocal laser micro- scopy (3). operative in the formation of the lymphoid tissue during adult life. Our study shows that during ontogeny, only the LTbR pathway is Flow cytometry crucial for the development of the marginal zone and the com- Splenic tissues were weighed and single-cell suspensions prepared. After Downloaded from partments of the white pulp (T, B, and mixed T/B cell zones). In removal of RBCs, leukocyte numbers were determined, and the cells were contrast, during adult life, expression of TNFR1 is now also re- stained (28). Flow cytometry was performed on an FACSCalibur (BD Bio- quired. This should be considered when targeting the TNFR1 sciences, Heidelberg, Germany), and acquisition and analysis of the data were done using CellQuestPro (version 4.0.2; BD Biosciences). signaling pathway in patients suffering from diseases associated with the formation of ectopic lymphoid tissues. Laser microdissection and mRNA quantification This was done as described previously (29, 30). In brief, a pulsed UV laser Materials and Methods was used to dissect the lymphoid tissue compartments (Palm Microbeam, http://www.jimmunol.org/ Palm, Bernried, Germany). The dissected tissue was immediately dis- Mice solved in 350 ml guadinium-isothiocyanate–containing lysis buffer for iso- 2/2 b 2/2 lation of total RNA (RNeasy Kit; Qiagen, Hilden, Germany) and stored at C57BL/6NCrlBR, C57BL/6Ly5.1, TNFR-1 (26), and LT R (10) 2 mice were used. Wild-type (WT) and LTbR2/2 mice were obtained from 20˚C. After RNA isolation and cDNA synthesis, the cycle threshold Charles River Laboratories (Sulzfeld, Germany), whereas TNFR-12/2 mice value was determined for each (TaqMan System, ABI 7000; were provided by K. Pfeffer (Du¨sseldorf, Germany). Mice of both sexes Applied Biosystems, Darmstadt, Germany) and related to the expression of and 8–12-wk-old were used in experiments. The animals were housed the housekeeping . and bred under specific pathogen-free conditions in the Central Animal Stimulation of LTbR in vivo Facility of the University of Lu¨beck (Germany). Permission to perform 2 2 2 2 these animal experiments was issued by the Ministry of Nature and En- TNFR1 / splenic tissue was implanted into TNFR1 / mice (n =4) by guest on October 1, 2021 vironment (V 252-72241.122-1 [24-3/02]). receiving agonistic anti-LTbR Ab (rat IgG 4H8), which selectively induces signaling via the LTbR pathway (31). This group was compared with mice Implantation of splenic tissue treated with an appropriate control Ab (n = 4). The Abs were applied i.p. and treatment started 1 wk after the implantation. The mice received 200 Mice were anesthetized by i.p. injection of ketaminehydrochloride m m m (KetanestS; Parke-Davis, Berlin, Germany; 50 mg/kg body weight) and g for 2 wk and 100 g for a further 4 wk, resulting in a total of 800 g xylazinehydrochloride (Rompun; Bayer, Leverkusen, Germany; 5 mg/kg per mouse. Eight weeks after implantation, splenic tissue was removed and body weight) in 0.9% saline solution. The abdominal wall was opened analyzed by immunohistochemistry. To exclude a technical failure as cause for 1 cm, splenic arteries of the own spleen were coagulated electrically, and of the negative result, the experiment was repeated in the same way with the exception that the mice received 300 mg agonistic Ab (4H8) per week the spleen was removed (27). The spleen was cut into quarters, two of them m (total weight ∼40 mg) were implanted under the gastrosplenic ligament, for 6 wk (total: 1800 g). Again, no difference was seen between the and the abdominal wall was closed. In a first experiment, mice were an- treated and untreated group, although the injected Ab could be clearly alyzed 1, 3, 5, 8, and 16 wk postimplantation. Subsequently, the newly detected in the serum, and no host response against the rat Ab (4H8) was formed splenic tissue was removed 8 wk postimplantation. Although all initiated by the mouse host. mice used in the current study are bred on the same background, skin Statistical analysis transplantation was carried out in the WT to LTbR2/2 combination to exclude any rejection phenomena. Of 10 animals with transplanted skin, The statistical package SPSS 12.0G for Windows (SPSS, Chicago, IL) was none rejected the graft. To induce germinal centers in the spleen, mice used to calculate the means and SD and to determine significant differences were injected with 109 SRBC in 200 ml saline solution i.v., and 10 d later, (p , 0.05 in Mann–Whitney U test for two independent experimental the spleen was removed. groups). Abs Results The following cell populations were identified by immunohistochemistry: b red pulp macrophages (F4/80+), T cells (TCRb+), B cells (B220+), FDC During ontogeny, the LT R is instrumental for the segregation (FDC-M1+), germinal center (peanut agglutinin), and marginal metallo- of T and B cells within splenic tissue + philic macrophages (MOMA-1 ). With flow cytometry, the following mole- b cules were identified: CD4 (RM4-5), CD16/CD32 (2.4G2), CD21/CD35 To determine the role of TNFR1 and LT R in the compartmen- (7G6), CD23 (B3B4), CD25 (7D4), CD44 (IM-7), CD45.1 (A20), CD45RB talization of the spleen during ontogeny, a quantitative analysis of 2/2 2/2 (16A), B220 (RA3-6B2), ICAM-1/CD54 (3E2), L-selectin/CD62L WT, TNFR1 , and LTbR mouse spleens was performed. b (MEL-14), CD69 (H1.2F3), IA /MHC class II (25-9-17), IgD (11-26c.2a), Serial sections were stained by immunohistochemistry to outline b IgM (R6-60.2), LPAM-1 (DATK32), NK1.1 (PK136), TCR (H57-597), the red pulp, marginal zone, T cell zone (periarteriolar lymphatic and isotype control (R35-95). sheath), and the B cell zone (follicles) as compartments of the Immunohistochemistry white pulp (Fig. 1A,1B). Subsequently, the size of different com- One, 3, 5, 8, and 16 wk postsurgery, implanted splenic tissue was removed, partments was determined by morphometric analysis (Fig. 1C, snap-frozen in liquid nitrogen, and stored at 280˚C. Cryostat sections (12 1D). The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. Compartment size of WT spleen is similar to that of TNFR12/2 spleen but not to that of LTbR2/2 spleen. A, Serial cryostat sections from WT, TNFR12/2, and LTbR2/2 spleens were stained for macrophages (brown; F4/80+), B cells (blue; B220+), and T cells (brown; TCRb+) to outline the splenic compartments. Scale bar, 200 mm. B, Visualization of the mixed T/B cell zone by confocal laser microscopy. T cells (green) and B cells (red) were simultaneously labeled and the overlapping parts of T cell zone (dotted lines) and B cell zone (solid lines) determined. Scale bar, 50 mm. C and D, The size of the various compartments was determined quantitatively in spleens from intact WT, TNFR12/2, and LTbR2/2 mice. The bars represent the mean (6 SD) size of individual splenic compartments as percent of total spleen area (n = 6 animals per group, ages ranging between 8 and 12 wk). The Mann–Whitney U , test was used to indicate a significant difference between WT and knockout mice (*p 0.05). B, B cell zone; MZ, marginal zone; RP, red pulp; T, T cell by guest on October 1, 2021 zone; T/B, mixed T/B cell zone; WP, white pulp.

In the WT spleen, the red pulp comprised ∼50% of the splenic B cell zones during ontogeny, raising the question of whether the section, and the remaining 50% was occupied by the marginal same dominance occurs in lymphoid tissue formation in the adult. zone and the white pulp in equal parts (Fig. 1C). Within the white Comparable architecture of WT splenic tissue developed pulp, T and B cell zones were of comparable size, each zone during ontogeny and adult life comprising about one third of the white pulp (Fig. 1D). Inter- estingly, the remaining third was a mixed zone containing both T To study the formation of lymphoid tissue in adult animals, we and B cells (Fig. 1B,1D). Thus, even in normal WT spleens, examined splenic tissue formation following avascular implanta- segregation of T and B cells was incomplete, leaving a compart- tion of splenic tissue into the greater omentum within the peritoneal ment of considerable size that surrounded the T cell zone and in cavity (25). The optimal regeneration time was 8 wk post- which Tand B cells were tightly mixed. Surprisingly, in TNFR12/2 implantation (Fig. 2A–C). Although weight and leukocyte num- mice, the size of the different compartments was remarkably bers were reduced compared with the intact spleen (Table I), the similar to that of WT mice (Fig. 1A,1C,1D). Compared to the WT spleen, T and B cell zones were slightly reduced, whereas the mixed T/B cell zone was unchanged (Fig. 1D). In contrast, lack of Table I. Weight and cell number of intact spleen and newly formed the LTbR during ontogeny resulted in a significantly enlarged red splenic tissue pulp and a complete absence of the marginal zone (Fig. 1C). In addition, the size of the T cell zone was reduced, whereas that of the Weight Cell No. No. of 2 2 3 6 mixed T/B cell zone was enlarged (Fig. 1D). Both TNFR1 / and Splenic Tissue (mg) ( 10 /mg) Animals 2 2 LTbR / spleens were unable to form germinal centers upon Intact spleen stimulation with sheep RBCs (data not shown). Interestingly, leu- WT 91 6 16 1.12 6 0.37 11 TNFR2/2 65 6 10 0.98 6 0.19 7 kocyte numbers per milligram were in the same range in the three b 2/2 6 6 b LT R 113 18 0.89 0.31 8 groups, whereas splenic weight was higher in LT R-deficient mice Newly formed and lower in TNFR1-deficient mice compared with that of WT WT→WT 44 6 8 0.40 6 0.08 9 mice (Table I), which is in line with recently published data (31). TNFR12/2→TNFR12/2 37 6 8 0.20 6 0.08 5 2/2 2/2 These morphometric data demonstrate a prominent role of LTbR LTbR →LTbR 47 6 19 0.27 6 0.05 5 signaling, but not TNFR1, in regulating the segregation of T and Values are means 6 SD unless otherwise indicated. 4 TNFR1 IS REQUIRED FOR THE FORMATION OF SPLENIC COMPARTMENTS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Comparable cellular composition of splenic tissue de- veloped during ontogeny and adult life. A and B, Frequency of leukocyte subsets and surface molecule expression on T cells in normal (WT, de- veloped during ontogeny) and newly formed splenic tissue (WT→WT, developed in adult life) as determined by flow cytometry. The bars rep- resent the mean values (6 SD; n = 5–8 animals per group). C, Expression of various in the T cell zone (T) and B cell zone (B) of normal spleens (closed circle) and newly formed splenic tissue (open circle). In- FIGURE 2. Comparable architecture of splenic tissue developed during dicated is the mRNA expression determined by RT-PCR and related to the ontogeny and adult life. A, Eight weeks after implantation into the greater housekeeping gene (MLN 51). Each symbol represents mRNA expression omentum, the development of splenic tissue is complete (arrow). The in- of an individual animal. sert shows the newly formed splenic tissue in higher magnification (arrow). B, Cryostat sections of newly formed splenic tissue were immunostained newly formed splenic tissue contained all compartments of a nor- + + for T cells (brown; TCRb ) and B cells (blue; B220 ) 1, 3, and 5 wk mal spleen including the presence of FDC in the B cell zone (Fig. postimplantation of WT tissue into WT mice (WT→WT, developed during 2C, arrows). Then the size of the different compartments was m adult life). Scale bar, 500 m. C, Cryostat sections of WT spleen (WT, analyzed within the newly formed splenic tissue. Compared to developed during ontogeny) and newly formed splenic tissue (WT→WT, a normal spleen, the red pulp of the ectopically developed splenic developed during adult life) were triple-immunostained for T cells ( brown; TCRb+), B cells (blue; B220+), and marginal metallophilic mac- tissue was somewhat larger and the T cell zone smaller (Fig. 2D). rophages (MM; dark brown; MOMA-1+; upper row scale bar, 100 mm) and In addition, regenerated splenic tissue contained small areas of double-immunostained for T cells (light brown; TCRb+) and FDCs (blue; fibrotic tissues that are not seen in normal splenic tissue. However, FDC-M1+; lower row scale bar, 50 mm). All splenic compartments, in- in general, our data show that splenic tissue formed in the adult cluding FDCs in the B cell zone (arrow), reappeared in the splenic tissue mice was almost comparable to that formed during ontogeny. As newly formed during adult life. D, The size of the compartments of the normal spleen was analyzed and compared with splenic tissue newly formed during adult life 8 wk postimplantation. The bars represent the a significant difference between WT and transplanted mice (*p , 0.05). B, mean (6 SD) size of individual splenic zones as percent of total spleen B cell zone; MZ, marginal zone; RP, red pulp; T, T cell zone; T/B, mixed area (n = 6 animals per group). Mann–Whitney U test was used to indicate T/B cell zone; WP, white pulp. The Journal of Immunology 5 determined by flow cytometry, both tissues were similar regarding LTbR2/2 mice lack marginal zone B cells and are unable to form the frequency of T and B cells and their subsets (Fig. 3A) and their germinal centers upon stimulation (10, 14), implantation of WT surface molecule expression (Fig. 3B). Furthermore, isolation of tissue into LTbR2/2 mice was expected to show whether the T cell and B cell zones by laser microdissection and subsequent newly formed splenic tissue was able to compensate for these analysis of mRNA expression of various cytokines suggested no deficiencies. After implantation into LTbR2/2 mice, WT splenic gross differences between normal and newly formed splenic tissue compartments developed just as they did in WT recipients (data (Fig. 3C). not shown). Strikingly, marginal zone B cells that were present in normal spleens and absent in the spleens of LTbR2/2 mice were Comparable function of WT splenic tissue developed during clearly detectable in WT splenic tissue that formed in LTbR2/2 ontogeny and adult life mice (Fig. 4A). In addition, upon stimulation with sheep RBCs, To analyze the functional ability of newly formed splenic tissue, germinal centers developed in WT spleens, whereas in LTbR2/2 WT splenic tissue was implanted into LTbR2/2 mice. Because spleens, they did not (Fig. 4B). In contrast, germinal centers were Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Comparable function of splenic tissue developed during ontogeny and adult life. A, Marginal zone B cells were identified in cell suspensions of normal (WT) and LTbR2/2 spleens and of newly formed splenic tissue developed 8 wk postimplantation of WT tissue into adult LTbR2/2 recipients (WT→LTbR2/2). Flow cytometry was used to gate on B cells (B220+) and to determine among them the number of marginal zone B cells (CD21+, CD232, encircled area). Marginal zone B cells were present in normal spleens (WT), absent in LTbR2/2 spleens, and present in WT splenic tissue newly formed in adult LTbR2/2 mice. B, Normal (WT) and LTbR2/2 animals and LTbR2/2 animals 8 wk postimplantation of WT splenic (WT→LTbR2/2) were injected with 109 SRBCs into the tail vein. After 10 d, splenic tissue was analyzed for germinal center formation by immunohistochemistry (germinal center [GC], brown, peanut agglutinin+; scale bar, 100 mm). Germinal centers could be induced in normal spleens (WT) and in WT splenic tissue newly formed in adult LTbR2/2 mice but not in LTbR2/2 spleens. C, Eight weeks postimplantation of WT splenic tissue (CD45.1) into LTbR2/2 mice (CD45.2) .99% of the leukocytes within the newly formed splenic tissue (WT→LTbR2/2) were of host origin. Cell suspensions of splenic tissues were stained for the expression of CD45.1 and for T cells (TCRb) and analyzed by flow cytometry (percentage of CD45.1-positive cells in the upper and lower quadrant; left panel, 33.0 6 0.4/66.0 6 0.7; middle panel, 0.1 6 0.1/0.6 6 0.2; right panel, 0.2 6 0.1/0.7 6 0.1). Eight weeks postimplantation of WT splenic tissue into LTbR2/2 animals (WT→LTbR2/2), the regenerated spleen contained well-developed compartments. Statistical analysis showed that their sizes were comparable to that of regenerated splenic tissue of the WT→WT combination (Fig. 2C). All experiments depicted in this figure were repeated three to seven times with comparable results. 6 TNFR1 IS REQUIRED FOR THE FORMATION OF SPLENIC COMPARTMENTS readily identified within the newly formed splenic tissue that de- enlarged, whereas the white pulp was almost absent (Fig. 5, right veloped in LTbR2/2 mice after implantation of WT splenic tissue panel). The lack of the marginal zone from the newly formed (Fig. 4B). Together, these results demonstrate that the presence of TNFR12/2 splenic tissue (Fig. 5, right panel, right side) under- LTbR on stromal cells in the newly formed splenic tissue was able scores the importance of TNFR1 during adult life, because during to induce B cells of LTbR2/2 mice to become marginal zone ontogeny, the marginal zone is normally developed in the absence B cells and to provide an appropriate environment for T and of TNFR1 (Fig. 5, right panel, left side). Thus, in contrast to on- B cells of the LTbR2/2 host to generate germinal centers. To ex- togeny, in which only the LTbR is instrumental for the devel- clude the possibility that the cells present in the newly formed opment of T and B cell zones, during adult life, the TNFR1 is as splenic tissue were donor cells, which survived the necrotic phase important as the LTbR for splenic tissue formation. after splenic tissue implantation, a congenic animal system was used. CD45.1 WT splenic tissue was implanted into a CD45.2 In adult life, the TNFR1 functions independently of the LTbR LTbR2/2 host (Fig. 4C). Eight weeks later, the splenic tissue was in the formation of compartments within splenic tissue analyzed by flow cytometry and showed that .99.5% of the leu- kocytes within the newly formed tissue were of host origin (Fig. One major difference regarding the development of lymphoid tis- 4C). sue during ontogeny and adult life is that the number of LTi cells, which are the main source of ligands for the LTbR, decreases with b In adult life, both LT R and TNFR1 are involved in the age from ∼50% around birth to ,0.1% of leukocytes in adult life segregation of T and B cells within splenic tissue (7, 32, 33). Thus, there are two explanations for the fact that, in We showed that the avascular implantation of splenic tissue fully contrast to ontogeny, during adult life, the function of both the recapitulates the development of lymphoid tissue. Therefore, it can LTbR and TNFR1 pathways is necessary for the formation of Downloaded from be used as an appropriate model to examine the role of molecules lymphoid tissue. One possibility is that TNFR1 signaling indi- regulating the formation of lymphoid tissue in the adult animal. rectly increases the availability of LTbR ligands, and the other is Thus, splenic tissue from LTbR2/2 mice implanted into LTbR2/2 that TNFR1 signaling independently induces the formation of recipients attained a very low grade of structural organization (Fig. lymphoid tissue. 5, left panel). Semiquantitative evaluation showed that the newly To discriminate between the two possibilities, TNFR12/2 splenic 2/2 2/2 formed splenic tissue was comparable to that of the intact LTbR tissue was implanted into TNFR1 mice receiving an agonistic http://www.jimmunol.org/ spleen (Fig. 5, left panel). Very unexpectedly, however, splenic anti-LTbR Ab (rat IgG 4H8), which selectively induces signaling tissue from TNFR12/2 mice implanted into TNFR-12/2 recipients via the LTbR pathway (31). Eight weeks postimplantation, splenic failed to reproduce their original structure (Fig. 5, right panel). In tissue was removed and analyzed by immunohistochemistry. Sim- contrast to the intact TNFR12/2 spleen, in the newly formed tissue, ilar to the experiments described in Fig. 5, in the newly formed the marginal zone was completely absent, and T and B cell zones tissue, the marginal zone was completely absent, and T and B cell were only sparsely developed (Fig. 5, right panel). In addition, zones were only sparsely developed independently of whether semiquantitative evaluation revealed that the red pulp was grossly the animals received the agonistic Ab (4H8) or a control Ab (data by guest on October 1, 2021

FIGURE 5. In contrast to ontogeny, in adult life, the TNFR1 is necessary for the formation of T and B cell zones. In the left panel, cryostat sections of LTbR2/2 spleens and of splenic tissue formed 8 wk postimplantation of LTbR2/2 splenic tissue into adult LTbR2/2 recipient (LTbR2/2→LTbR2/2) are shown that were stained either for macrophages (brown; F4/80+; upper panels) or for T cells (light brown; TCRb+), B cells (blue; B220+), and marginal metallophilic macrophages (MM; no staining detectable; MOMA-1+; lower panels). Splenic tissue newly formed in adults (LTbR2/2→LTbR2/2) reproduced a structure comparable to that of LTbR2/2 spleens, which developed during ontogeny. In the right panel, cryostat sections of TNFR12/2 splenic tissues are shown. In this case, splenic tissue newly formed in adults (TNFR12/2→TNFR12/2) was not comparable to that of TNFR12/2 spleens, which developed during ontogeny. Scale bars, 100 mm. The size of the different organ compartments was semiquantitatively compared (+ + + +, larger than in WT; + + +, equal to WT; + +, smaller than in WT; +, much smaller than in WT; Ø, not present; n = 6 animals per group). B, B cell zone; MZ, marginal zone; RP, red pulp; T, T cell zone; T/B, mixed T/B cell zone; WP, white pulp. The Journal of Immunology 7

FIGURE 6. During adult life, LTa is upregulated in regenerated splenic tissue of WT mice but not of TNFR12/2 mice. A, Expression of TNF, LTa, CCL19, and CXCL13 in normal splenic tissue (developed during ontogeny) and regenerated splenic tissue (developed during adult life) of WT and 2/2

TNFR1 mice. Expression of CD3 (B) and CD19 (C) in normal splenic tissue (developed during ontogeny) and regenerated splenic tissue (developed Downloaded from during adult life) of WT and TNFR12/2 mice. Indicated is the mRNA expression determined by RT-PCR and related to the housekeeping gene (MLN 51). The bars represent the mean (6 SEM) of TNF and LTa expression (left ordinates in A), the ratio of CCL19 and CXCL13 expression (right ordinates in A), CD3 (B), and CD19 (C). Mann–Whitney U test was used to indicate a significant difference between normal and regenerated spleen (*p , 0.05) (n = 4–6 animals per group).

not shown). These results indicate that TNFR1 signaling is not and the TNFR1. This fact should be considered when targeting the http://www.jimmunol.org/ promoting splenic regeneration during adult life by acting via TNFR1 pathway during therapy of autoimmune and inflammatory LTbR signaling. diseases. To study which ligand might be involved in splenic regeneration, To study the role of LTbR and TNFR1 in the formation of TNF and LTa mRNA was determined. TNF mRNA expression in lymphoid tissue during adult life, we took advantage of the model normal spleens and splenic regenerates of WT mice were com- of avascular implantation of splenic tissue (25, 38). Despite an parable and similar to that in TNFR12/2 mice (Fig. 6A). However, initial necrosis due to the avascular implantation of the splenic LTa mRNA was significantly upregulated in splenic regenerates in fragments, stromal cells of donor origin survive (25, 39, 40), and WT mice (Fig. 6A). In contrast, LTa upregulation was not seen in our study demonstrates that the complex architecture of the spleen splenic regenerates in TNFR12/2 mice. The absence of LTa up- is rebuilt within 8 wk, and the different compartments are of al- by guest on October 1, 2021 regulation in splenic regenerates of TNFR12/2 mice correlated most normal size. Like the normal spleen, it contains a mixed T/ with a significantly increased ratio of the chemokines CCL19 and B cell zone (41, 42) that comprises about one third of the white CXCL13 (Fig. 6A). In addition, CD3 expression was reduced both pulp, completely encircles the T cell zone, and provides the ap- in WT and TNFR1-deficient regenerates (Fig. 6B), whereas CD19 propriate microenvironment for the interaction of T and B cells expression was increased only in WT regenerates (Fig. 6C). In- during a T cell-dependent B cell response (5). In addition, all terestingly, the expression of the ligands of LTbR, LTb and parameters investigated are comparable to that of the normal spleen LIGHT, were comparable in normal spleens and splenic regener- (lymphocytes subsets, adhesion molecule, cytokine expression, and ates in WT, TNFR12/2, and LTbR2/2 mice (Table II). Together, germinal center formation). Furthermore, when WT splenic tissue these results suggest that LTa probably expressed by B cells is implanted into LTbR2/2 mice, marginal zone B cells are found seems to play an important role during lymphoid tissue formation within the newly formed splenic tissue. The majority of them must in adults. be of host origin because only 0.7% of the B cells are of donor origin, whereas ∼3% of the B cells show the phenotype of marginal Discussion zone B cells. This demonstrates that the absence of marginal zone In the current study, we show by quantitative morphometry that B cells in LTbR2/2 mice (10) is due to the lack of LTbR expression during embryonic life, the LTbR is crucial for the formation of in the spleen and that the newly formed splenic tissue functions like splenic compartments (34–36), whereas the main function of the the normal spleen. The exact molecular pathway by which splenic TNFR1 is to complete the formation of lymphoid follicles from LTbR expression facilitates the maturation of marginal zone the already developed B cell zone (37). In contrast, during adult B cells is largely unknown. Finally, it has been shown that the life, the formation of lymphoid compartments requires both LTbR splenic regenerate is able to protect against various infections (38,

Table II. Expression of LTbR ligands in splenic tissue formed during ontogeny and adult life

WT TNFR12/2 LTbR2/2

Intact Spleen Transplanted Spleen Intact Spleen Transplanted Spleen Intact Spleen Transplanted Spleen (Ontogeny) (Adult Life) (Ontogeny) (Adult Life) (Ontogeny) (Adult Life) LTb 7.9 6 4.4 9.2 6 2.7 8.9 6 2.4 7.7 6 4.5 6.5 6 2.5 8.0 6 2.4 LIGHT 0.3 6 0.2 0.5 6 0.2 0.2 6 0.02 0.4 6 0.3 0.2 6 0.1 0.2 6 0.1 The mRNA expression determined by RT-PCR and related to the housekeeping gene (MLN 51) is indicated. Means 6 SD are given (n = 4–6). 8 TNFR1 IS REQUIRED FOR THE FORMATION OF SPLENIC COMPARTMENTS

43). Taken together, avascular implantation of splenic tissue is an Interestingly, established lymphoid tissue does not require excellent model to study lymphoid tissue formation in adult life not TNFR1 expression (52), indicating that not maintenance but the only in rats (24, 25) but also in mice (present study) with the great formation of new lymphoid tissue in adults necessitates TNFR1 advantage to use genetically manipulated animals. expression. Thus, the success of anti-TNF therapy in patients suf- Implantation of LTbR2/2 splenic tissue into LTbR2/2 hosts fering from autoimmune diseases such as rheumatoid arthritis results in splenic tissue indistinguishable from the intact spleen of (53–57) might be not only due to the neutralization of proin- LTbR2/2 mice (Fig. 5). This indicates that, as during ontogeny, flammatory molecules but also due to the selective targeting of also in adult life, the development of the marginal zone and the ectopic lymphoid tissue that initiates and maintains autoimmune compartments of the white pulp is LTbR dependent. Therefore, and inflammatory diseases (11, 58–60). the beneficial effect of inhibiting the LTbR pathway in many In summary, although the normal WT spleen and splenic tissue autoimmune diseases might be in part due to interference with the formed after avascular implantation of WT tissue in WT recipients development of ectopic lymphoid tissue (44). To our great sur- is very similar both structurally and functionally, the set of mol- prise, and in contrast to ontogeny, the implantation of TNFR12/2 ecules responsible for their development is different. During on- splenic tissue into TNFR12/2 hosts results in splenic tissue much togeny, the segregation and size of splenic compartments is main- less developed than that of the intact spleen of TNFR12/2 ani- ly LTbR dependent, whereas during adult life, both LTbR and mals (Fig. 5). What is the molecular basis for the additional re- TNFR1 are necessary. This might be one reason why secondary and quirement of the TNFR1 pathway for lymphoid tissue formation tertiary (ectopic) lymphoid tissues, despite all similarities, show during adult life? One explanation could be that during ontogeny, profound differences. For example, secondary lymphoid tissues are LTi cells constitutively express the ligands for the LTbR, thereby induced only at predetermined times and places, whereas ectopic facilitating the formation of lymphoid tissue (45). With increasing lymphoid tissues can be induced at any time and any place (61). Downloaded from age, however, their numbers in lymphoid organs decrease from With our approach, it is possible to identify molecular differences ∼50% of all CD45+ cells around birth to ,0.1% in adults (7, 32, between the formation of secondary and ectopic lymphoid tissues 33). This might be insufficient for the appropriate stimulation of and to use such differences to selectively treat autoimmune and the LTbR-expressing stromal organizer cells (32, 33). Therefore, inflammatory diseases. during adult life, ligands for the LTbR might be provided in a TNFR1-dependent fashion [e.g., TNFR1 ligation induces stroma Acknowledgments http://www.jimmunol.org/ cells to produce B cell activation factor of the TNF family (46), We thank M. Sixt (Mu¨nchen, Germany) and the members of our laboratory which in turn stimulates B cells to express LTbR ligands (47)]. for helpful comments, K. Pfeffer (Du¨sseldorf, Germany) for providing the However, injecting an agonistic anti-LTbR Ab (31) did not im- knockout mice, and C. Stamm, L. Gutjahr, M.-L. Leppin, and K. von prove the morphology of the newly formed tissue. In addition, Lingelsheim for technical assistance. although adult mice have only few LTi cells, they express different surface molecules than their neonatal counterparts, which could Disclosures compensate for their low numbers (48). Furthermore, adult lym- The authors have no financial conflicts of interest. phoid tissues contain activated B and T cells that also provide by guest on October 1, 2021 ligands for the LTbR (6). Taken together, the available data in- dicate that in adult life, the LTbR and TNFR1 pathway work in- References 1. Junt, T., E. Scandella, and B. Ludewig. 2008. Form follows function: lymphoid dependently of each other. tissue microarchitecture in antimicrobial immune defence. Nat. Rev. Immunol. 8: Thus, the current study demonstrates that during adult life, the 764–775. expression of TNFR1 is essential for the development of the mar- 2. Westermann, J., B. Engelhardt, and J. C. Hoffmann. 2001. Migration of T cells ginal zone and the segregation of T cell and B cell zones within in vivo: molecular mechanisms and clinical implications. Ann. Intern. Med. 135: 279–295. splenic tissue. This indicates that in contrast to embryonic life, 3. Westermann, J., U. Bode, A. Sahle, U. Speck, N. Karin, E. B. Bell, K. Kalies, during adult life, inflammation might be an important driving force and A. Gebert. 2005. Naive, effector, and memory T lymphocytes efficiently for the generation of lymphoid tissue. Recently, it was shown that scan dendritic cells in vivo: contact frequency in T cell zones of secondary lymphoid organs does not depend on LFA-1 expression and facilitates survival of inflammation (i.e., TNFR1 signaling) is necessary to induce adult effector T cells. J. Immunol. 174: 2517–2524. fibroblasts to become lymphoid stroma cells (49). In addition, the 4. Pape, K. A., D. M. Catron, A. A. Itano, and M. K. Jenkins. 2007. The humoral stimulation of adult fibroblasts in vitro with an agonistic LTbRAb immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles. Immunity 26: 491–502. alone did not induce stromal meshwork organization, whereas the 5. Garside, P., E. Ingulli, R. R. Merica, J. G. Johnson, R. J. Noelle, and additional presence of TNF led to the generation of a tridi- M. K. Jenkins. 1998. Visualization of specific B and T lymphocyte interactions mensional cellular meshwork (50). Furthermore, lymphoid tissue in the lymph node. Science 281: 96–99. 6. Ware, C. F. 2005. Network communications: , LIGHT, and TNF. formation during inflammation depends on the expression Annu. Rev. Immunol. 23: 787–819. of TNFR1 ligands (18), and the induction of lymph node-like 7. Scandella, E., B. Bolinger, E. Lattmann, S. Miller, S. Favre, D. R. Littman, structures in adults requires activated cells, whereas in neonate D. Finke, S. A. Luther, T. Junt, and B. Ludewig. 2008. Restoration of lymphoid organ integrity through the interaction of lymphoid tissue-inducer cells with animals they develop without prior activation (23). Lymphoid stroma of the T cell zone. Nat. Immunol. 9: 667–675. tissue formation in adult life seems to rely more on the TNFR1 8. Lo, J. C., Y. Wang, A. V. Tumanov, M. Bamji, Z. Yao, C. A. Reardon, G. S. Getz, ligand LTa and less on TNF because it is LTa, which is highly and Y. X. Fu. 2007. Lymphotoxin beta receptor-dependent control of lipid ho- expressed in regenerating lymphoid tissue (Fig. 6A). This obser- meostasis. Science 316: 285–288. 9. Ware, C. F. 2008. Targeting lymphocyte activation through the lymphotoxin and vation is in agreement with reports showing that formation of LIGHT pathways. Immunol. Rev. 223: 186–201. lymphoid tissue can be induced by overexpression of LTa (11, 10. Fu¨tterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, and K. Pfeffer. 1998. The 22). The present study indicates that the increased LTa expression lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9: 59–70. is probably caused by B cells that produce LTa and are found in 11. Gommerman, J. L., and J. L. Browning. 2003. Lymphotoxin/light, lymphoid WT splenic regenerates in increased numbers (Fig. 6C) (51). microenvironments and autoimmune disease. Nat. Rev. Immunol. 3: 642–655. Thus, the poor structure of splenic transplants in TNFR1-deficient 12. Summers-DeLuca, L. E., D. D. McCarthy, B. Cosovic, L. A. Ward, C. C. Lo, S. Scheu, K. Pfeffer, and J. L. Gommerman. 2007. Expression of lymphotoxin- mice might be due to their inability to recruit the appropriate alphabeta on antigen-specific T cells is required for DC function. J. Exp. Med. leukocyte subsets. 204: 1071–1081. The Journal of Immunology 9

13. Randall, T. D., D. M. Carragher, and J. Rangel-Moreno. 2008. Development of 37. Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Fo¨rster, J. D. Sedgwick, secondary lymphoid organs. Annu. Rev. Immunol. 26: 627–650. J. L. Browning, M. Lipp, and J. G. Cyster. 2000. A -driven positive 14. Rennert, P. D., J. L. Browning, R. Mebius, F. Mackay, and P. S. Hochman. 1996. feedback loop organizes lymphoid follicles. Nature 406: 309–314. Surface /beta complex is required for the development of 38. Leemans, R., W. Manson, J. A. Snijder, J. W. Smit, H. J. Klasen, T. H. The, and peripheral lymphoid organs. J. Exp. Med. 184: 1999–2006. W. Timens. 1999. Immune response capacity after human splenic autotrans- 15. Tumanov, A. V., S. I. Grivennikov, A. N. Shakhov, S. A. Rybtsov, E. P. Koroleva, plantation: restoration of response to individual pneumococcal vaccine subtypes. J. Takeda, S. A. Nedospasov, and D. V. Kuprash. 2003. Dissecting the role of Ann. Surg. 229: 279–285. lymphotoxin in lymphoid organs by conditional targeting. Immunol. Rev. 195: 39. Katakai, T., T. Hara, M. Sugai, H. Gonda, and A. Shimizu. 2003. Th1-biased 106-16–106-116. tertiary lymphoid tissue supported by CXC chemokine ligand 13-producing 16. Pasparakis, M., S. Kousteni, J. Peschon, and G. Kollias. 2000. Tumor necrosis stromal network in chronic lesions of autoimmune gastritis. J. Immunol. 171: factor and the p55TNF receptor are required for optimal development of the 4359–4368. marginal sinus and for migration of follicular dendritic cell precursors into 40. White, A., D. Carragher, S. Parnell, A. Msaki, N. Perkins, P. Lane, E. Jenkinson, splenic follicles. Cell. Immunol. 201: 33–41. G. Anderson, and J. H. Caaman˜o. 2007. Lymphotoxin a-dependent and -in- 17. Tkachuk, M., S. Bolliger, B. Ryffel, G. Pluschke, T. A. Banks, S. Herren, dependent signals regulate stromal organizer cell homeostasis during lymph R. H. Gisler, and M. H. Kosco-Vilbois. 1998. Crucial role of tumor necrosis node organogenesis. Blood 110: 1950–1959. factor receptor 1 expression on nonhematopoietic cells for B cell localization 41. Baje´noff, M., J. G. Egen, L. Y. Koo, J. P. Laugier, F. Brau, N. Glaichenhaus, and within the splenic white pulp. J. Exp. Med. 187: 469–477. R. N. Germain. 2006. Stromal cell networks regulate lymphocyte entry, migra- 18. Sacca, R., C. A. Cuff, W. Lesslauer, and N. H. Ruddle. 1998. Differential ac- tion, and territoriality in lymph nodes. Immunity 25: 989–1001. tivities of secreted lymphotoxin-alpha3 and membrane lymphotoxin-alpha1beta2 42. Cupedo, T., F. E. Lund, V. N. Ngo, T. D. Randall, W. Jansen, M. J. Greuter, R. de in lymphotoxin-induced inflammation: critical role of TNF receptor 1 signaling. Waal-Malefyt, G. Kraal, J. G. Cyster, and R. E. Mebius. 2004. Initiation of J. Immunol. 160: 485–491. cellular organization in lymph nodes is regulated by non-B cell-derived signals 19. Luther, S. A., A. Bidgol, D. C. Hargreaves, A. Schmidt, Y. Xu, J. Paniyadi, and is not dependent on CXC chemokine ligand 13. J. Immunol. 173: 4889– M. Matloubian, and J. G. Cyster. 2002. Differing activities of homeostatic 4896. chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell 43. Cooney, D. R., S. E. Swanson, J. C. Dearth, M. K. Dewanjee, and R. L. Telander. recruitment and lymphoid neogenesis. J. Immunol. 169: 424–433. 1979. Heterotopic splenic autotransplantation in prevention of overwhelming 20. Ludewig, B., B. Odermatt, S. Landmann, H. Hengartner, and R. M. Zinkernagel.

postsplenectomy infection. J. Pediatr. Surg. 14: 337–342. Downloaded from 1998. Dendritic cells induce autoimmune diabetes and maintain disease via de 44. Chiang, E. Y., G. A. Kolumam, X. Yu, M. Francesco, S. Ivelja, I. Peng, novo formation of local lymphoid tissue. J. Exp. Med. 188: 1493–1501. P. Gribling, J. Shu, W. P. Lee, C. J. Refino, et al. 2009. Targeted depletion of 21. Aloisi, F., and R. Pujol-Borrell. 2006. Lymphoid neogenesis in chronic in- lymphotoxin-alpha-expressing TH1 and TH17 cells inhibits autoimmune dis- flammatory diseases. Nat. Rev. Immunol. 6: 205–217. ease. Nat. Med. 15: 766–773. 22. Drayton, D. L., S. Liao, R. H. Mounzer, and N. H. Ruddle. 2006. Lymphoid 45. Mebius, R. E. 2003. Organogenesis of lymphoid tissues. Nat. Rev. Immunol. 3: organ development: from ontogeny to neogenesis. Nat. Immunol. 7: 344–353. 292–303. 23. Cupedo, T., W. Jansen, G. Kraal, and R. E. Mebius. 2004. Induction of secondary 46. Ohata, J., N. J. Zvaifler, M. Nishio, D. L. Boyle, S. L. Kalled, D. A. Carson, and and tertiary lymphoid structures in the skin. Immunity 21: 655–667. T. J. Kipps. 2005. Fibroblast-like synoviocytes of mesenchymal origin express

24. Westermann, J., R. Pabst, and E. Claassen. 1991. Histophysiology of auto- http://www.jimmunol.org/ functional B cell-activating factor of the TNF family in response to proin- transplanted splenic tissue. Res. Immunol. 142: 321–325. flammatory cytokines. J. Immunol. 174: 864–870. 25. Pabst, R., J. Westermann, and H. J. Rothko¨tter. 1991. Immunoarchitecture of 47. Schneider, P. 2005. The role of APRIL and BAFF in lymphocyte activation. regenerated splenic and lymph node transplants. Int. Rev. Cytol. 128: 215–260. Curr. Opin. Immunol. 17: 282–289. 26. Pasparakis, M., L. Alexopoulou, M. Grell, K. Pfizenmaier, H. Bluethmann, and 48. Kim, M. Y., S. Rossi, D. Withers, F. McConnell, K. M. Toellner, F. Gaspal, G. Kollias. 1997. Peyer’s patch organogenesis is intact yet formation of E. Jenkinson, G. Anderson, and P. J. Lane. 2008. Heterogeneity of lymphoid B lymphocyte follicles is defective in peripheral lymphoid organs of mice de- tissue inducer cell populations present in embryonic and adult mouse lymphoid ficient for and its 55-kDa receptor. Proc. Natl. Acad. Sci. USA 94: 6319–6323. tissues. Immunology 124: 166–174. 49. Peduto, L., S. Dulauroy, M. Lochner, G. F. Spath, M. A. Morales, A. Cumano, 27. Westermann, J., S. Michel, S. Lopez-Kostka, U. Bode, H. J. Rothko¨tter, ¨ M. Bette, E. Weihe, R. H. Straub, and R. Pabst. 1998. Regeneration of implanted and G. Eberl. 2009. Inflammation recapitulates the ontogeny of lymphoid stro- splenic tissue in the rat: re-innervation is host age-dependent and necessary for mal cells. J. Immunol. 182: 5789–5799. 50. Katakai, T., T. Hara, M. Sugai, H. Gonda, and A. Shimizu. 2004. Lymph node tissue development. J. Neuroimmunol. 88: 67–76. by guest on October 1, 2021 28. Milic´evic´, N. M., K. Nohroudi, Z. Milic´evic´, H. J. Hedrich, and J. Westermann. fibroblastic reticular cells construct the stromal reticulum via contact with 2005. T cells are required for the peripheral phase of B-cell maturation. Im- lymphocytes. J. Exp. Med. 200: 783–795. munology 116: 308–317. 51. Westermann, J., and R. Pabst. 1986. Autotransplantation of splenic fragments: 29. Kalies, K., M. Blessenohl, J. Nietsch, and J. Westermann. 2006. T cell zones of lymphocyte subsets in blood, lymph nodes and splenic tissue. Clin. Exp. lymphoid organs constitutively express Th1 cytokine mRNA: specific changes Immunol. 64: 188–194. during the early phase of an immune response. J. Immunol. 176: 741–749. 52. Mackay, F., G. R. Majeau, P. Lawton, P. S. Hochman, and J. L. Browning. 1997. 30. Kalies, K., P. Ko¨nig, Y. M. Zhang, M. Deierling, J. Barthelmann, C. Stamm, and Lymphotoxin but not tumor necrosis factor functions to maintain splenic ar- J. Westermann. 2008. Nonoverlapping expression of IL10, IL12p40, and IFN- chitecture and humoral responsiveness in adult mice. Eur. J. Immunol. 27: 2033– gamma mRNA in the marginal zone and T cell zone of the spleen after antigenic 2042. stimulation. J. Immunol. 180: 5457–5465. 53. Feldmann, M., and L. Steinman. 2005. Design of effective immunotherapy for 31. De Trez, C., K. Schneider, K. Potter, N. Droin, J. Fulton, P. S. Norris, S. W. Ha, human autoimmunity. Nature 435: 612–619. Y. X. Fu, T. Murphy, K. M. Murphy, et al. 2008. The inhibitory HVEM-BTLA 54. Haraoui, B. 2005. The anti-tumor necrosis factor agents are a major advance in pathway counter regulates lymphotoxin receptor signaling to achieve homeo- the treatment of rheumatoid arthritis. J. Rheumatol. Suppl. 72: 46–47. stasis of dendritic cells. J. Immunol. 180: 238–248. 55. Scott, D. L., and G. H. Kingsley. 2006. Tumor necrosis factor inhibitors for 32. Mebius, R. E., P. Rennert, and I. L. Weissman. 1997. Developing lymph nodes rheumatoid arthritis. N. Engl. J. Med. 355: 704–712. collect CD4+CD3- LTbeta+ cells that can differentiate to APC, NK cells, and 56. Kempen, J. H., E. Daniel, J. P. Dunn, C. S. Foster, S. Gangaputra, A. Hanish, follicular cells but not T or B cells. Immunity 7: 493–504. K. J. Helzlsouer, D. A. Jabs, R. O. Kac¸maz, G. A. Levy-Clarke, et al. 2009. 33. Kim, D., R. E. Mebius, J. D. MacMicking, S. Jung, T. Cupedo, Y. Castellanos, Overall and cancer related mortality among patients with ocular inflammation J. Rho, B. R. Wong, R. Josien, N. Kim, et al. 2000. Regulation of peripheral treated with immunosuppressive drugs: retrospective cohort study. BMJ 339: lymph node genesis by the tumor necrosis factor family member TRANCE. J. b2480. Exp. Med. 192: 1467–1478. 57. Chan, A. C., and P. J. Carter. 2010. Therapeutic antibodies for autoimmunity and 34. Gonzalez, M., F. Mackay, J. L. Browning, M. H. Kosco-Vilbois, and R. J. Noelle. inflammation. Nat. Rev. Immunol. 10: 301–316. 1998. The sequential role of lymphotoxin and B cells in the development of 58. Martin, F., and A. C. Chan. 2006. B cell immunobiology in disease: evolving splenic follicles. J. Exp. Med. 187: 997–1007. concepts from the clinic. Annu. Rev. Immunol. 24: 467–496. 35. Fu, Y. X., G. Huang, Y. Wang, and D. D. Chaplin. 1998. B lymphocytes induce 59. Carragher, D. M., J. Rangel-Moreno, and T. D. Randall. 2008. Ectopic lymphoid the formation of follicular dendritic cell clusters in a lymphotoxin alpha- tissues and local immunity. Semin. Immunol. 20: 26–42. dependent fashion. J. Exp. Med. 187: 1009–1018. 60. Can˜ete, J. D., R. Celis, C. Moll, E. Izquierdo, S. Marsal, R. Sanmartı´, A. Palacı´n, 36. Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, D. Lora, J. de la Cruz, and J. L. Pablos. 2009. Clinical significance of synovial M. D. Cooper, J. L. Browning, J. D. Sedgwick, and J. G. Cyster. 1999. Lym- lymphoid neogenesis and its reversal after anti-tumour necrosis factor alpha photoxin alpha/beta and tumor necrosis factor are required for stromal cell ex- therapy in rheumatoid arthritis. Ann. Rheum. Dis. 68: 751–756. pression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 61. Eberl, G. 2007. From induced to programmed lymphoid tissues: the long road to 189: 403–412. preempt pathogens. Trends Immunol. 28: 423–428.