Lymphotoxin Signal Promotes Thymic Organogenesis by Eliciting RANK Expression in the Embryonic Thymic Stroma

This information is current as Yasuhiro Mouri, Masashi Yano, Miho Shinzawa, Yusuke of September 26, 2021. Shimo, Fumiko Hirota, Yumiko Nishikawa, Takuro Nii, Hiroshi Kiyonari, Takaya Abe, Hisanori Uehara, Keisuke Izumi, Koji Tamada, Lieping Chen, Josef M. Penninger, Jun-ichiro Inoue, Taishin Akiyama and Mitsuru Matsumoto

J Immunol 2011; 186:5047-5057; Prepublished online 25 Downloaded from March 2011; doi: 10.4049/jimmunol.1003533 http://www.jimmunol.org/content/186/9/5047 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/03/25/jimmunol.100353 Material 3.DC1 References This article cites 54 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/186/9/5047.full#ref-list-1

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Lymphotoxin Signal Promotes Thymic Organogenesis by Eliciting RANK Expression in the Embryonic Thymic Stroma

Yasuhiro Mouri,* Masashi Yano,* Miho Shinzawa,† Yusuke Shimo,† Fumiko Hirota,* Yumiko Nishikawa,* Takuro Nii,* Hiroshi Kiyonari,‡ Takaya Abe,‡ Hisanori Uehara,x Keisuke Izumi,x Koji Tamada,{ Lieping Chen,‖ Josef M. Penninger,# Jun-ichiro Inoue,† Taishin Akiyama,†,** and Mitsuru Matsumoto*

It has recently become clear that signals mediated by members of the TNFR superfamily, including lymphotoxin-b receptor (LTbR), receptor activator for NF-kB (RANK), and CD40, play essential roles in organizing the integrity of medullary thymic epithelial cells (mTECs) required for the establishment of self-tolerance. However, details of the mechanism responsible for the

unique and cooperative action of individual and multiple TNFR superfamily members during mTEC differentiation still remain Downloaded from enigmatic. In this study, we show that the LTbR signal upregulates expression of RANK in the thymic stroma, thereby promoting accessibility to the RANK ligand necessary for mTEC differentiation. Cooperation between the LTbR and RANK signals for optimal mTEC differentiation was underscored by the exaggerated defect of thymic organogenesis observed in mice doubly deﬁcient for these signals. In contrast, we observed little cooperation between the LTbR and CD40 signals. Thus, the LTbR signal exhibits a novel and unique function in promoting RANK activity for mTEC organization, indicating a link between thymic organogenesis

mediated by multiple cytokine signals and the control of autoimmunity. The Journal of Immunology, 2011, 186: 5047–5057. http://www.jimmunol.org/

he thymus provides a microenvironment in which T cells Aire deficiency (5–8). The significance of TRA gene expression in gain the ability to discriminate between self and nonself the thymic stroma for the establishment of central tolerance has T (1, 2). Developing thymocytes recognizing self-Ags in been further supported by the fact that mice deficient in several the thymic stroma either develop into immunoregulatory T cells signal-transducing molecules and NF-kB components downstream or are deleted by apoptosis, depending on the strength and/or na- of TNFR superfamily (TNFRsf) members have a similar or, in ture of the reactivity with self-Ags (3). Medullary thymic epithelial many cases, more profound reduction of TRA gene expression (9– cells (mTECs) seem to play pivotal roles in this cross talk with 15), and it has been shown that embryonic thymi taken from these thymocytes by expressing a set of self-Ags (4). This scenario has mice induce autoimmune disease phenotypes when grafted into by guest on September 26, 2021 been supported by gene expression studies showing that mTECs recipient mice. In this regard, it is important to emphasize that, in are a specialized cell type in which promiscuous expression of contrast to Aire-deficient mice, the reduction of TRA gene ex- a broad range of tissue-restricted Ag (TRA) genes is an autono- pression in these mice is strongly associated with a defect in the mous property (1). Remarkably, mice deficient for autoimmune mTEC differentiation program; the thymi show easily discernable regulator (Aire), a mouse homolog of the gene responsible for the structural abnormalities such as a small medulla, a paucity of development of autoimmune polyendocrinopathy-candidiasis-ecto- mTECs including Aire-expressing cells, and loss of architectural dermal dystrophy in humans, show reduced expression of many, integrity of mTECs, as assessed by morphological observation though not all, TRA genes from mTECs, which has been impli- and flow cytometric analysis. Subsequently, one of the upstream cated in the development of autoimmune pathogenesis caused by TNFRsf members responsible for thymic organogenesis was iden-

*Division of Molecular Immunology, Institute for Enzyme Research, The University Education, Culture, Sports, Science and Technology of Japan (to M.M., T.A., and J.I.), of Tokushima, Tokushima 770-8503, Japan; †Division of Cellular and Molecular and by the Takeda Science Foundation (to M.M.). Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Address correspondence and reprint requests to Prof. Mitsuru Matsumoto or Dr. ‡Laboratory for Animal Resources and Genetic Engineering, Center for Developmen- x Taishin Akiyama, Division of Molecular Immunology, Institute for Enzyme Re- tal Biology, RIKEN Kobe, Kobe 650-0047, Japan; Department of Molecular and search, The University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Environmental Pathology, Institute of Health Biosciences, The University of Tokush- { Japan (M.M.) or Division of Cellular and Molecular Biology, Institute of Medical ima Graduate School, Tokushima 770-8503, Japan; Marlene and Stewart Greene- ‖ Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, baum Cancer Center, University of Maryland, Baltimore, MD 21201; Depart- Japan (T.A.). E-mail addresses: [email protected] (M.M.) and taishin@ ment of Oncology and Institute for Cell Engineering, The Johns Hopkins University ims.u-tokyo.ac.jp (T.A.) School of Medicine, Baltimore, MD 21205; #Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria; and **Precursory Re- The online version of this article contains supplemental material. search for Embryonic Science and Technology, Japan Science and Technology Abbreviations used in this article: Aire, autoimmune regulator; 2-DG, 29-deoxygua- Agency, Saitama 332-0012, Japan nosine; DKO, double knockout; E, embryonic day; EpCAM, epithelial cell adhesion Received for publication October 25, 2010. Accepted for publication February 18, molecule; FTOC, fetal thymus organ culture; IKKa,IkB kinase a; K5, keratin 5; KI, 2011. knockin mice; KO, knockout; LTbR, lymphotoxin-b receptor; MHC II, MHC class II; mLT, membrane-bound form of lymphotoxin; mTEC, medullary thymic epithelial This work was supported in part by Grants-in-Aid for Scientific Research from the cell; NIK, NF-kB–inducing kinase; RANK, receptor activator for NF-kB; RANKL, Japan Society for the Promotion of Science and from the Ministry of Education, RANK ligand; Sap1, salivary protein 1; sLT, secreted form of lymphotoxin; TNFRsf, Culture, Sports, Science and Technology of Japan (to M.M.), by Grants-in-Aid for TNFR superfamily; TRA, tissue-restricted Ag; TRAF, TNFR-associated factor; Scientific Research from the Precursory Research for Embryonic Science and Tech- UEA-1, Ulex europaeus agglutinin 1. nology program of the Japan Science and Technology Agency (to T.A.), by a grant from the Japanese Society for the Promotion of Science (to T.A.), by a Cooperative Ó Research grant from the Institute for Enzyme Research, The University of Tokushima Copyright 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 (to M.M. and T.A.), by grants for Priority Area Research from the Ministry of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003533 5048 LYMPHOTOXIN ELICITS THYMIC STROMAL RANK EXPRESSION tified as receptor activator for NF-kB (RANK) (16). These studies Thus, cytokine signals mediated by multiple TNFRsf members ex- clearly suggested a link between cytokine-mediated thymic or- hibit unique cooperation to achieve the mTEC organization re- ganogenesis and the establishment of central tolerance (1, 17). quired for establishment of self-tolerance in the thymus. Besides the individual role of each TNFRsf signal in the production and maintenance of mTECs, combined actions of multiple Materials and Methods TNFRsf members, RANK and CD40, have recently been dem- Mice onstrated (18) (see below). Ltbr-KO (accession number CDB0531K at the Center for Developmental Among the TNFRsf members studied so far, RANK seems to Biology, RIKEN Kobe) were generated by gene targeting. Briefly, the have the strongest impact on the organization mTECs, at least on targeting vector was constructed by replacing a 39 portion of Ltbr exon 1 the basis of examination of the individual thymic phenotypes of together with the whole of exon 2–5 with a LacZ and neomycin resistance gene (LacZ-neor) (Supplemental Fig. 1A). The targeting vector was in- mice deficient for each of the TNFRsf members (19). It has been troduced into TT2 embryonic stem cells, and the homologous recombinant suggested that RANK is required for the initial differentiation clones were first identified by PCR and confirmed by Southern blot anal- phase of mTECs during embryogenesis and that lymphoid tissue ysis (37). After the targeted embryonic stem cells had been injected into inducer cells with a CD4+CD32 cell signature are the main source morula-stage embryos, the resulting chimeric male mice were mated with of the RANK ligand (RANKL) in this period (16). In the postnatal C57BL/6 females (CLEA Japan) to establish germline transmission (Sup- plemental Fig. 1B–D). Lta-KO (38), Light-KO (39), Rankl-KO (40), and phase, the CD40 signal also becomes indispensable (20), and co- Aire/GFP-knockin mice (KI) (41) were generated as described pre- operation between RANK and CD40 signals is required for the op- viously. aly mice (33, 34) and BALB/cnu/nu mice were purchased from timal organization and homeostasis of mTECs (18). Furthermore, CLEA Japan, and Cd40-KO (42) were from The Jackson Laboratory. The mature single-positive thymocytes play essential roles in pro- mice were maintained under pathogen-free conditions, and the protocols Downloaded from used in this study were in accordance with the Guidelines for Animal viding the ligands for RANK and CD40 in postnatal mice (21, 22). Experimentation of Tokushima University School of Medicine and con- Compared with the essential roles of the RANK and CD40 ducted with the approval of the RIKEN Kobe Animal Experiment Com- signals in thymic organogenesis, the functional significance of the mittee. b b lymphotoxin- receptor (LT R) signal for thymic organogenesis Immunohistochemistry is still poorly defined (23). This is rather unexpected when consi- Immunohistochemical analysis of the thymus with rat ER-TR5 mAb dering the dominant role of the LTbR signal over the RANK http://www.jimmunol.org/ (43), rat anti-epithelial cell adhesion molecule (EpCAM) mAb (BD Bio- signal in secondary lymphoid organogenesis; mice deficient in sciences), and rabbit polyclonal anti-keratin 5 (K5) Ab (Covance) was LTbR (Ltbr-knockout [KO]) lack both lymph nodes and Peyer’s performed as described previously (41). Rat anti-Aire mAb (clone RF33-1; patches and show a disorganized splenic structure, whereas mice IgG1) recognizing the COOH-terminal portion of mouse Aire was pro- deficient in RANK (Rank-KO) lack only lymph nodes, but possess duced in our laboratory. UEA-1 was from Vector Laboratories. For the Peyer’s patches and have an apparently normal splenic structure detection of autoantibodies, serum from untreated mice or thymic chimeras was incubated with various organs obtained from Rag2-deficient mice. (24–28). Although thymic alterations such as a reduction in the Alexa 488-conjugated anti-mouse IgG Ab (Invitrogen) was used for the + number of Ulex europaeus agglutinin 1 (UEA-1) mature mTECs detection. and defective three-dimensional organization of mTECs have been

TEC preparation and flow cytometric analysis by guest on September 26, 2021 noticed in postnatal Ltbr-KO (29), production of Aire-expressing mTECs remains unchanged in the absence of the LTbR signal (9, Preparation of TECs and flow cytometric analysis with an FACSCalibur 16, 29–32). This relatively mild phenotype in Ltbr-KO is more (BD Biosciences) were performed as described previously (18, 41). The mAbs used were anti-CD4, anti-CD8, anti-CD45, anti–TER-119, anti- remarkable when one considers the severe thymic disorganization EpCAM, anti-CD80, anti-B220, anti–I-A/I-E, and anti-Ly51, all pur- + characterized by lack of Aire mTECs in a natural strain of mice chased from eBioscience except for anti-EpCAM mAb. with NF-kB–inducing kinase (NIK) mutation [aly mice (33, 34)] Thymus grafting and FTOC (9, 29) or in mice deficient in IkB kinase a (IKKa), downstream of NIK (10, 11). In marked contrast to the different phenotypes Thymus grafting was performed as described previously (9). Briefly, thymic of thymic organogenesis, Ltbr-KO and aly mice (and Ikka-KO) lobes were isolated from embryos at 14.5 d postcoitus and then cultured for 4 d on top of Nuclepore filters (Whatman) placed on RPMI 1640 medium exhibit common abnormalities of secondary lymphoid organo- (Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), 2 genesis (35), suggesting that NIK-IKKa constitutes a component mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 downstream of LTbR that is essential for secondary lymphoid or- mM 2-ME, hereafter referred to as R10, and supplemented with 1.35 mM a 29-deoxyguanosine (2-DG) (Sigma-Aldrich). Five thymic lobes were ganogenesis, whereas for thymic organogenesis, NIK-IKK ad- nu/nu b grafted under the renal capsule of BALB/c mice. After 6–8 wk, re- ditionally acts downstream of other receptor(s) beyond LT R. In constitution of peripheral T cells was determined by flow cytometric this context, it would be important to look for any cooperative analysis, and then thymic chimeras were used for analyses. For the as- action of LTbR together with other TNFRsf members, especially sessment of mTEC differentiation using FTOC, 2-DG–treated fetal thymic RANK and CD40. Accordingly, an exact picture of the role of stroma was cultured in R10 supplemented with rRANKL (1 mg/ml; Wako), m b m LTbR in organization of mTEC integrity is still lacking (23, 30– CD40L (5 g/ml; R&D Systems), agonistic anti-LT R mAb AC.H6 [2 g/ ml (44)], and their combinations, as described previously (18). mTEC 32, 36). differentiation and induction of gene expression from thymic stroma were We have approached these issues by generating both Ltbr/Cd40 assessed by flow cytometric analysis and real-time PCR, respectively. double-deficient mice (Ltbr/Cd40-double KO [DKO]) and Rank Real-time PCR and semiquantitative RT-PCR ligand/Ltbr double-deficient mice (Rankl/Ltbr-DKO), and using fetal thymus organ culture (FTOC) to further explore the mech- RNA was extracted from thymic stroma with RNeasy Mini Kits (Qiagen) anisms underlying the cooperation by TNFRsf members. The and made into cDNA with SuperScript III RT Kits (Invitrogen) in accor- b dance with the manufacturer’s instructions. Real-time PCR (41) and results suggested that the LT R signal regulates the development semiquantitative RT-PCR (9) were performed as described previously. The of mTECs cooperatively with the RANK signal, but not with the primers and the probes for the real-time PCR are as follows. Rank primers: CD40 signal. Through this newly identified cooperation between 59-TCCTGGGCTTCTTCTCAGAT-39 and 59-CACATCTGATTCCGTTGT- LTbR and RANK, the LTbR signal was found to upregulate the CC-39; Rank probe: 59-FAM-TGGACCAACTGCACCCTCCTTG-39; salivary protein 1 (Sap1)primers:59-ACTCCTTGTGTTGCTTGGTGTTT-39 and expression of RANK in immature mTECs (most likely presum- 59-TCGACTGAATCAGAGGAATCAACT-39; Sap1 probe: 59-FAM-TTCA- ptive mTEC progenitors), thereby promoting subsequent differ- CCAGCAGAATCAGCAGTTCCAGAA-39; Hprt primers: 59-TGAAGAGC- entiation of the mTECs after induction by the RANK signal. TACTGTAATGATCAGTCAAC-39 and 59-AGCAAGCTTGCAACCTTAA- The Journal of Immunology 5049

CCA-39;andHprt probe: 59-FAM-TGCTTTCCCTGGTTAAGCAGTACAG- abundance of mTECs expressing Aire among UEA-1+ mTECs CCC-39. was not affected (Supplemental Fig. 4B).

Statistical analysis Lack of obvious cooperation between LTbR and CD40 signals for thymic organization and self-tolerance All results are expressed as mean 6 SEM. Statistical analysis was performed using Student’s two-tailed unpaired t test for comparisons between The role of the LTbR signal in thymic organogenesis might be , two groups. Differences were considered significant at p 0.05. better understood if its cooperative action with other signals could be clarified. Because the defect of thymic organization in aly mice is much more profound than that in Ltbr-KO (10, 29), it is rea- Results sonable to speculate that NIK is additionally acting downstream b Roles of LT R and its ligands in thymic organogenesis of other TNFRs beyond LTbR in this process. One obvious can- To gain an insight into the precise roles of the LTbR signal in didate for such interaction is CD40, because it activates the non- thymic organogenesis, we first performed immunohistochemical canonical NIK–RelB activation pathway (46), and the additional analysis using mice deficient in LTbR and mice deficient in two lack of a CD40 signal in mice deficient in RANK accelerates the known ligands of LTbR (i.e., the membrane-bound form of lym- defect of thymic organogenesis (18). We therefore examined the photoxin [mLT] and LIGHT) (27) (Supplemental Fig. 2A). Im- cooperation between the LTbR and CD40 signals in thymic or- munohistochemical analyses demonstrated that wild-type mouse ganogenesis by establishing mice deficient in both LTbR and thymus contained medullary areas that varied in size, as recog- CD40. The thymi of Cd40-KO had well-developed medullas with nized by reactivity with the ER-TR5 mAb (43), some of the larger well demarcated shapes, similar to those of wild-type mice, and Downloaded from areas probably containing a few medullary islets (45) (Fig. 1A). immunohistochemistry demonstrated no major structural altera- Areas binding with UEA-1 showed considerable overlap with tions (Fig. 1C, Supplemental Figs. 2B, 4), although flow cyto- ER-TR5+ medullary areas. In contrast, the individual ER-TR5+ metric analysis revealed a reduced percentage of MHC class II medullary areas in Lta-KO, which lack both mLT (LTa1b2) and (MHC II)+UEA-1+ mTECs (18). Mice doubly deficient in LTbR the secreted form of LT (LTa3; sLT), were smaller in size and less and CD40 (Ltbr/Cd40-DKO) showed no additional defect of

frequently connected with each other, as also demonstrated by medullary organization beyond that evident in single Ltbr-KO http://www.jimmunol.org/ UEA-1 staining. At higher magnification, disruption of the three- (compare Fig. 1A and 1C, Supplemental Fig. 2B). Flow cyto- dimensional organization of mTECs, as assessed by UEA-1 stain- metric analysis also supported this view, except that there was ing, was also evident in Lta-KO (Supplemental Fig. 2B). Of note, a small reduction of mTECs expressing MHC IIlow (mTEClow), it has been reported that Ltb-KO showed no significant redu- but not MHC IIhigh (mTEChigh) [(18), Fig. 2A], in Ltbr/Cd40-DKO ction in the total mass of mTECs, although changes in the UEA-1+ compared with single Ltbr-KO (Supplemental Fig. 3B). This was cell distribution pattern have been pointed out (29) (Fig. 1B). in marked contrast to that resulting from cooperation between the Light-KO showed no major alteration of medullary organization, RANK and CD40 signals (18), indicating the existence of unique as assessed using ER-TR5 mAb and UEA-1 staining, at either low functional cooperation among the TNFRsf members. The apparent or high magnification (Fig. 1A, Supplemental Fig. 2B). Impor- lack of cooperation between LTbR and CD40 was also supported by guest on September 26, 2021 tantly, however, Lta/Light-DKO showed even smaller medullas by the fact that production of Aire+ mTECs was not impaired in than those in Lta-KO at lower magnification (Fig. 1A); the size of either Ltbr-KO or Ltbr/Cd40-DKO (Supplemental Fig. 4A); al- each UEA-1+ area was further reduced in comparison with that in though the total numbers of Aire+ mTECs per medulla were again Lta-KO. It was also noteworthy that, in Lta/Light-DKO, each reduced in both Ltbr-KO and Ltbr/Cd40-DKO due to the smaller UEA-1+ area was significantly smaller than the corresponding ER- size of each medulla, the relative abundance of mTECs expressing TR5+ area, giving the UEA-1+ areas an appearance resembling Aire among UEA-1+ mTECs was not affected in these strains small nodules, a feature that was not evident in Lta-KO (Fig. 1A). (Supplemental Fig. 4B). At higher magnification, the Lta/Light-DKO thymus also exhibi- The autoimmune pathologies in untreated mice described above ted a more disorganized three-dimensional organization of UEA- were examined histologically together with autoantibody pro- 1+ mTECs than was the case in Lta-KO (Supplemental Fig. 2B). duction at between 16 and 20 wk of age. Lta-KO, Lta/Light-DKO, The exaggerated thymic disorganization due to additional lack of and Ltbr-KO showed lymphoid cell aggregates in the liver and LIGHT in Lta-KO is in marked contrast to that seen in Ltb-KO lung (Supplemental Fig. 5A,5B), consisting of B220+, CD4+, and (lacking only mLT and not sLT), in which the introduction of CD8+ lymphocytes (data not shown). Interestingly, introduction of LIGHT deficiency results in no additional deterioration of thymic CD40 deficiency into Ltbr-KO (i.e., Ltbr/Cd40-DKO) abolished organogenesis (29) (Fig. 1B) (see Discussion). The more profound these pathological changes (Supplemental Fig. 5A). Overall, pro- defect of mTEC organization in Lta/Light-DKO in comparison duction of IgG-class autoantibodies was not obvious in untreated with Lta-KO (or Light-KO) suggests that LTa and LIGHT have mice, including Ltbr/Cd40-DKO (Supplemental Fig. 5C) [partly some redundancy of action in thymic organogenesis. Ltbr-KO due to the defect of class switching in these strains (24)], and thus showed poorly connected small medullas with small nodule-like the autoimmune pathogenesis seen in Ltbr-KO was not further UEA-1+ clusters similar to those seen in Lta/Light-DKO (Fig. 1A, accelerated by additional lack of the CD40 signal. Supplemental Fig. 2B). Similarity in impaired development of The lack of an obvious combined effect of the LTbR and CD40 mTECs in Lta/Light-DKO and Ltbr-KO was also supported by the signals for establishing central tolerance was also examined in flow cytometric analysis (Supplemental Fig. 3A), suggesting that thymus graft experiments. Lymphoid cell infiltration was observed the major ligands for LTbR in thymic organogenesis might be in the stomach, salivary glands, and pancreas of nude mice grafted mLT and LIGHT. with embryonic thymi from Ltbr-KO (Supplemental Fig. 6A,6B), Despite the disrupted organization of mTECs in Lta-KO, Lta/ and sera from these recipient mice contained IgG-class autoanti- Light-DKO, and Ltbr-KO, production of Aire+ mTECs was re- bodies against gastric mucosa (Supplemental Fig. 6C,6D), contained in these animals (Supplemental Fig. 4A); although the total sistent with a previous report (30). Despite the retention of thy- numbers of Aire+ mTECs per medulla were apparently reduced mic organization in Cd40-KO, as described above, nude mice due to the smaller size of each medulla in these strains, the relative grafted with embryonic thymi from Cd40-KO showed patho- 5050 LYMPHOTOXIN ELICITS THYMIC STROMAL RANK EXPRESSION

FIGURE 1. Thymic organization of mice deficient in TNF/TNFRsf members. Organization of the thymic medulla was assessed by immunohistochemistry using ER-TR5 mAb (in red) and UEA-1 binding (in green) (A, C) from mice deficient in LTbR and its two known ligands (A) or from mice deficient in CD40 and both LTbR and CD40 (C). B, Summary of the defect in mTEC integrity from each of the KO strains we pro- pose. The biological significance of the binding between sLT and HVEM is not clear (27) and is parenthesized. Note that Lta/Light-DKO show a more profound defect of mTEC organization than Lta-KO Downloaded from and that Lta/Light-DKO and Ltbr-KO show similar poorly connected small medullas with small nodule- like UEA-1+ areas (A). Also, note the phenotypic difference in thymic organogenesis between Lta/Light- DKO and Ltb/Light-DKO (and most likely already between Lta-KO and Ltb-KO). Furthermore, no obvious difference was found between Ltbr-KO and http://www.jimmunol.org/ Ltbr/Cd40-DKO (compare A and C). A and C, Scale bars, 1 mm. One representative experiment from a total of more than three repeats is shown. aConclusions from the current study. bResults summarized from the previous report (29) for comparison. by guest on September 26, 2021

logical changes in the stomach, salivary gland, and pancreas (Sup- were reduced in Ltbr-KO, whereas in Rankl-KO reduction of plemental Fig. 6A) together with autoantibody production against mTEChigh was more prominent than that of mTEClow. Rankl/Ltbr- these organs (Supplemental Fig. 6C). Importantly, beyond the lack DKO showed markedly more severe reduction of mTECs than of LTbR or CD40 signal alone, no augmentation of autoimmune mice with each deficiency alone. Rankl/Cd40-DKO showed the pathological changes together with autoantibody production was most severe reduction of mTECs for both mTEChigh and mTEClow discernable in nude mice grafted with embryonic thymi from among the strains tested. Percentages of cortical thymic epithelial Ltbr/Cd40-DKO (Supplemental Fig. 6A,6C). Taken together, cells remained intact in all of the strains (Fig. 2B). these results suggest that there is no obvious cooperation between Immunohistochemical staining of the thymic medulla using the LTbR and CD40 signals in the thymic organization required anti-K5 Ab, UEA-1 binding, and anti-EpCAM/anti-Aire mAbs for establishment of self-tolerance, in marked contrast to the co- gave results consistent with those obtained by flow cytometry (Fig. operation between the RANK and CD40 signals we reported 2C); disruption of UEA-1 binding together with the defective previously (18). production of Aire+ mTECs was more obvious in Rankl/Ltbr- DKO than in Ltbr-KO or Rankl-KO, and Rankl/Cd40-DKO Cooperation between LTbR and RANK signals for thymic again showed the most severe defect. Thus, the role of the organization LTbR signal in the organization of thymic organogenesis was We next addressed whether the LTbR and RANK signals exert any underscored by its cooperative action with RANK. cooperative action on thymic organogenesis by establishing mice b deficient in both signals (i.e., mice deficient for both LTbR and LT R elicits RANK expression in mTECs of fetal thymus RANKL [Rankl/Ltbr-DKO]) and compared them with those de- Having established that the LTbR signal cooperates with the ficient in LTbR (Ltbr-KO) or RANKL (Rankl-KO) alone. Flow RANK signal, but not with the CD40 signal, to ensure the correct cytometric analysis demonstrated that both Ltbr-KO and Rankl- development of mTECs, we investigated the mechanism of this KO had reduced percentages of UEA-1+ mTECs to a similar ex- phenomenon using FTOC. We first prepared 2-DG–treated em- tent, although there was a phenotypic difference between the two bryonic thymic stroma from Aire/GFP-KI (41) at embryonic day strains (Fig. 2A,2B); mTECs for both mTEChigh and mTEClow (E) 14.5 and stimulated them with rRANKL, rCD40L, ago- The Journal of Immunology 5051

FIGURE 2. Differential effects of multiple combinations of TNFRsf signal loss on mTEC differentiation. A, mTEC differentiation in the thymus of adult mice deficient in TNFRsf signals was assessed by flow-cytometric analysis using anti-MHC II (I-A/ I-E) mAb and UEA-1 binding. Thymic cell sus- pensions were stained with UEA-1, anti-MHC II, anti-CD45, and TER-119 mAbs and analyzed by flow cytometry. A subset of MHC IIhigh in UEA-1+ cells (gated for CD452TER-1192 cells) is labeled as high, and a subset of MHC IIlow in UEA-1+ cells is labeled as low in the figure (rightmost panel). Percentages of the cells in the indicated areas are included. B, Summary of the ratios of mTECs

(subsets of UEA-1+MHC IIhigh and UEA-1+MHC Downloaded from IIlow) and cortical thymic epithelial cells (subsets of UEA-12MHC II+) in thymic stromal cells (gated for CD452TER-1192 cells) of the mice shown in A. Note that concomitant loss of RANKL and LTbR exerted an additive effect of each deﬁciency on mTEC differentiation, although it was less profound than that due to concomitant loss of RANKL and http://www.jimmunol.org/ CD40. C, Organization of the thymic medulla was assessed by immunohistochemistry using anti-K5 mAb (top panel), UEA-1 binding (middle panel), and anti-EpCAM (in red)/anti-Aire (in green) mAbs (bottom panel). Scale bar, 200 mm. One representative experiment from a total of more than three repeats is shown. *p , 0.05, **p , 0.01, Student t test. n = 3 for each genotype. by guest on September 26, 2021

nistic anti-LTbR mAb (44), and their multiple combinations. in vivo developmental program of mTECs induced by the signals Stimulation with rRANKL clearly induced the development of of individual members of the TNFRsf or their combinations. mTECs expressing the UEA-1 ligand (Fig. 3A), Aire (as assessed Because the LTbR signal enhanced the differentiation of mature by GFP expression) (Fig. 3B), and the Sap1 gene (Fig. 3C), an mTECs induced by RANK in FTOC, despite the fact that the Aire-dependent TRA gene (5). In contrast, stimulation with LTbR signal alone did not induce such effects (Fig. 3), we hy- rCD40L or agonistic anti-LTbR mAb had only a modest or no pothesized that the LTbR signal might facilitate more efficient effect on these activities, respectively, which appeared to accord reception of the RANK signal by mTECs. We therefore examined well with the phenotypes of mice that were deficient in each the effect of LTbR on the induction of RANK expression in FTOC TNFR signal; the thymic phenotypes of Rankl-KO were more and found that RANK expression in the thymic stroma detected by obvious than those of Cd40-KO or Ltbr-KO (Figs. 1, 2). We then real-time PCR was enhanced at day 4 after treatment with ago- investigated the combined effect of these stimuli on the induction nistic anti-LTbR mAb (Fig. 4A). In contrast, addition of rCD40L of mTECs together with TRA gene expression in FTOC. Addition to FTOC induced no such effect. Interestingly, stimulation with of rCD40L to rRANKL stimulation exerted no additional effect on rRANKL also upregulated the expression of RANK, suggesting mTEC development (Fig. 3A,3B) and Sap1 gene expression (Fig. a possible self-amplification mechanism that might explain, at 3C) beyond that induced by rRANKL alone. In contrast, addition least in part, why RANK has the strongest impact among TNFRsf of agonistic anti-LTbR mAb to rRANKL stimulation clearly members known so far on the induction of mTEC differentiation augmented mTEC development and Sap1 gene expression. This (19). Currently available mAbs directed against RANK did not seems remarkable when considering that LTbR stimulation alone allow us to evaluate the expression of RANK in the thymic stroma did not have any obvious effect on mTEC development and Sap1 using flow cytometry (Y. Mouri, T. Akiyama, and M. Matsumoto, gene expression. When agonistic anti-LTbR mAb and rCD40L unpublished observations). were added together in FTOC, we observed no obvious effect, The expression of RANK in the thymic stroma induced by LTbR perhaps being consistent with the phenotypes of mice doubly was not confined to the experimental setting of FTOC. RANK deficient in LTbR and CD40. Thus, the cytokine-directed process expression in the embryonic thymi of Ltbr-KO was indeed re- of mTEC differentiation assessed using FTOC well reflects the duced in comparison with that in control heterozygous littermates 5052 LYMPHOTOXIN ELICITS THYMIC STROMAL RANK EXPRESSION

FIGURE 3. Effects of combinations of TNFRsf stimuli on mTEC differentiation in FTOC. A, 2-DG– treated embryonic thymic stromas from Aire/GFP-KI mice (41) at E14.5 were cultured in media supplemented with rRANKL, rCD40L, agonistic anti-LTbR mAb (aLTbR), and their multiple combinations for 4 d. Single cells were mechanically released from the Downloaded from thymic stroma and subjected to ﬂow cytometric analysis using UEA-1 and anti-MHC II mAb after gating for CD452TER-1192 cells. B, Aire-expressing cells were monitored as GFP+MHC IIhigh mTECs. Percen- tages of the cells in the indicated areas are included. One representative experiment from a total of six http://www.jimmunol.org/ repeats is shown. C, Total RNAs were extracted from embryonic thymic stroma 4 d after stimulation, and expression of the Sap1 gene was assessed by real-time PCR. Results are expressed as the mean 6 SEM for triplicate wells for each sample. Numbers are the relative gene expression level in comparison with that of the Hprt gene. One representative experiment from a total of three repeats is shown. by guest on September 26, 2021

(Fig. 4B), indicating a requirement for the LTbR signal to ensure 3A). In contrast, rRANKL stimulation upregulated RANK tran- appropriate RANK expression at the embryonic stage in vivo. scripts more slowly, and this upregulation continued for 72 h (Fig. The observed upregulation of RANK induced by the LTbR 4C, upper panel). Concomitant induction of Sap1 gene expression signal can be explained in terms of expansion of the stromal was observed at 72 h only after rRANKL stimulation (Fig. 4C, mTEC population expressing RANK. Alternatively, the LTbR lower panel). These results are consistent with the idea that the signal may enhance the per-cell expression of RANK in pre- LTbR signal induces RANK expression in pre-existing mTECs on existing mTECs. To investigate these possibilities, we performed a per-cell basis, rather by expanding the population of RANK- a kinetic study of RANK expression together with an assessment expressing mTECs. In contrast, upregulation of RANK induced by of mTEC development using FTOC. We harvested RNAs from rRANKL appeared to be associated with expansion of the RANK- total embryonic thymi cultured with agonistic anti-LTbR mAb or expressing mTEC population (Fig. 4D, bottom panel) accompa- rRANKL at different time points. We observed upregulation of nied by TRA gene expression in induced mature mTECs. RANK transcripts as early as 6 h after stimulation with agonistic b anti-LTbR mAb, and RANK expression reached a plateau at 24 h, The LT R signal conditions mTECs to receive the RANK remaining at the same level thereafter until at least 72 h of ob- signal for differentiation servation (Fig. 4C, upper panel). mTEC development assessed by As we had clariﬁed that the development of mature mTECs trig- ﬂow cytometry in the same kinetic study showed no obvious effect gered by the RANK signal is enhanced by the LTbR signal through after stimulation with agonistic anti-LTbR mAb during the course upregulation of RANK in mTECs, we hypothesized that LTbR of observation (Fig. 4D, middle panel) as analyzed on day 4 (Fig. helps to condition mTECs for receiving the RANK signal to fa- The Journal of Immunology 5053

FIGURE 4. LTbR signal elicits RANK expression in the embryonic thymic stroma. A, Embryonic thymi were stimulated with agonistic anti-LTbR mAb, rRANKL, or rCD40L for 4 d, andexpressionoftheRank gene was assessed by real-time PCR, as described in the legend for Fig. 3C. One representative experiment from a total of four repeats is shown. B, Rank gene expression from embryonic thymi at E14.5 was assessed by real-time PCR. RNAs were harvested from total thymi of five heterozygous (control; clear circles) and homozygous LTbR-deficient mice (solid circles) for each. One circle corresponds to one mouse analyzed. C, Kinetics of Rank and Sap1 gene expression after stimulation with agonistic anti-LTbR mAb (gray columns) or rRANKL (black columns) at indicated time points. White columns, medium alone. Rank gene expression from thymic stroma induced by the LTbR signal was rapid and preceded that induced by the RANK signal. Relative expres- Downloaded from sion was calculated by determining the values of Rank (upper panel)andSap1 gene (lower panel) expression assessed by real-time PCR at each time point, using that in the absence of stimuli as 1. One representative experiment from a total of three repeats is shown. D, mTEC development http://www.jimmunol.org/ assessed by flow cytometric analysis in the same kinetic study as that demonstrated in C.Cells were analyzed for UEA-1 binding and MHC II expression after gating for CD452TER-1192 EpCAM+ cells. **p , 0.01. cilitate further differentiation; the LTbR signal might act on im- it plays an important role for the promotion of thymic organo- mature mTECs prior to their differentiation mediated by the genesis by optimizing the RANK signal through induction of

RANK signal. We therefore examined whether upregulation of RANK expression in the thymic stroma. In this context, it is by guest on September 26, 2021 RANK by the LTbR signal is prerequisite per se for the combined effect of LTbR and RANK by sequential treatment of embryonic thymi with agonistic anti-LTbR mAb and rRANKL. FTOC with agonistic anti-LTbR mAb for the ﬁrst 3.5 d followed by switching to rRANKL stimulation for an additional 3 d resulted in the development of more mature mTECs in comparison with those obtained by FTOC using a reverse order of treatment (i.e., FTOC with rRANKL for the ﬁrst 3.5 d, followed by switching to agonistic anti-LTbR mAb for an additional 3 d) (Fig. 5). Treatment with agonistic anti-LTbR mAb and rRANKL throughout the culture period (i.e., for 6.5 d) exerted a minimal and a strong effect, respectively. These results support the idea that the LTbR signal makes the embryonic thymic stroma more receptive to the RANK signal for mTEC differentiation through upregulation of RANK expression.

The LTbR signal regulates the initial development of mTECs in b the fetus FIGURE 5. LT R signal conditions mTECs to receive the RANK signal for differentiation. FTOCs were supplemented with rRANKL or with ag- Adult Ltbr-KO showed alteration of the thymic medullary archi- onistic anti-LTbR mAb for the first 3.5 d, followed by switching to ago- tecture (Fig. 1). However, based on our results demonstrating the nistic anti-LTbR mAb or rRANKL for an additional 3 d. FTOC ability of the LTbR signal to promote accessibility to the RANK supplemented with agonistic anti-LTbR mAb followed by rRANKL signal in FTOC, and the fact that the RANK signal promotes showed more mature mTEC development compared with that from FTOC b mTEC differentiation at the embryonic stage (16), we assumed treated with rRANKL, followed by agonistic anti-LT R mAb; the latter that the process of mTEC development at the embryonic stage culture yielded results similar to those for FTOC supplemented with rRANKL for 3.5 d followed by no supplementation for an additional 3 might already be affected in the absence of LTbR. We therefore d(upper left panel). Treatment with agonistic anti-LTbR mAb and directly examined the development of mTECs from Ltbr-KO at rRANKL throughout the culture period (i.e., for 6.5 d) exerted a minimal + E14.5 and found that this strain had lower numbers of UEA-1 and strong effect, respectively. Cells were analyzed for UEA-1 binding and mTECs expressing MHC II than their control littermates, as MHC II expression after gating for CD452TER-1192EpCAM+ cells. assessed by flow cytometry (Fig. 6A,6B). Thus, although the Percentages of cells in the indicated regions are included. One represen- LTbR signal is not absolutely necessary for mTEC development, tative experiment from a total of five repeats is shown. 5054 LYMPHOTOXIN ELICITS THYMIC STROMAL RANK EXPRESSION

Discussion In the current study, we investigated the roles of the LTbR signal in thymic organogenesis, focusing especially on its functional cooperation with two other TNFRsf members, RANK and CD40. Our results demonstrated that the LTbR signal orchestrates thymic organogenesis at the embryonic stage through cooperation with the RANK signal, but not with the CD40 signal. The LTbR signal upregulated RANK expression in stromal cells, allowing immature mTECs, possibly including mTEC progenitors, to become more receptive to the ligand for RANK, thus promoting their differentiation. This supportive role of the LTbR signal in RANK activity FIGURE 6. LTbR signal controls the initial phase of mTEC de- may at least partly explain why the thymic phenotype of Ltbr-KO velopment at the embryonic stage. A, mTEC development from Ltbr-KO is relatively mild in comparison with that of mice deficient in the was assessed at E14.5. Representative FACS profiles assessed with UEA-1 RANK signal, although the LTbR signal in mTECs is no doubt binding and MHC II expression after gating for CD452TER-1192 indispensable for preventing autoimmunity. EpCAM+ cells from heterozygous (control; left panel) and homozygous One of the most intriguing observations in this study was the LTbR-deficient mice (right panel). B, Percentages of the cells with UEA- different effects of various combinations of deficiencies of three + + 2 2 + 1 MHC II cells (gated for CD45 TER-119 EpCAM cells) for each TNFRsf members (LTbR, RANK, and CD40) on mTEC devel- individual mouse were plotted. One dot corresponds to one mouse ana- opment, which were not easily predictable from the phenotypes Downloaded from , lyzed. **p 0.01. of each TNFR deficiency alone. The order of the gross severity of important to emphasize that lack of the LTbR signal at the em- the defective thymic organization based on medullary size bryonic stage results in smaller ER-TR5+ medullas with disruption and the composition and/or structural organization of stromal cells of the three-dimensional organization of UEA-1+ mTECs at the assessed using immunohistochemistry and flow cytometric anal- adult stage (Fig. 1), which is associated with a propensity for ysis of mTECs was: Rank-KO (most severe; assuming that the development of autoimmunity. Thus, the LTbR signal controls the Rankl-KO we analyzed had an equivalent phenotype to Rank- http://www.jimmunol.org/ initial phase of mTEC development at the embryonic stage, thus KO), Ltbr-KO (intermediate), and Cd40-KO (mildest). Never- playing an important role in the control of autoimmunity in later theless, Rankl/Cd40-DKO showed a more severe thymic pheno- life. type than that of Rankl/Ltbr-DKO. Furthermore, Ltbr/Cd40-DKO Genes encoding both receptors (i.e., LTbR and RANK) and their showed no additional defect, if any, beyond that seen in Ltbr-KO. corresponding ligands (i.e., LTa/LTb and RANKL) were ex- This differential effect of various combinations of TNFRsf defi- pressed in embryonic thymi (Supplemental Fig. 7). Although ciencies might be due to the functional characteristics of each CD40 was expressed in embryonic thymi, CD40L expression was TNFR signal in terms of the developmental stage of action (i.e., fetus versus postnatal), the responding cell type(s) (e.g., pre-

low, suggesting that the CD40L–CD40 axis may not be involved by guest on September 26, 2021 in the initial phase of thymic organogenesis at the embryonic cursor, immature or mature mTECs), the mode of action (i.e., stage. Instead, the CD40L–CD40 axis may become relevant after difference in the target genes controlled by the transcriptional the postnatal stage (18, 20, 21). activities), and their combinations. We speculate that the actions of the RANK and CD40 signals have significant redundancy at Differential requirement of NIK and TNFR-associated factor 6 critical point(s) for mTEC differentiation, because the defect of for upregulation of RANK expression induced by the LTbR and thymic organization in Rankl/Cd40-DKO was much more pro- RANK signals found than that in Rankl-KO, despite the apparently normal thy- Given that upregulation of RANK by the LTbR signal is pre- mic architecture of Cd40-KO. Consistent with this view, the requisite for the normal developmental process of mTECs, we combined effect of stimuli exerted by rRANKL and rCD40L on investigated the signaling pathways involved in this process. We mTEC differentiation in FTOC was rather weak; addition of first examined whether upregulation of RANK induced by the rCD40L did not induce any further effect other than that of LTbR or RANK signal requires normal NIK activity. In contrast to rRANKL. In contrast, the actions of the RANK and LTbR signals FTOC using material from control aly/+ mice, FTOC using ma- seem to be distinct, and the simultaneous stimuli exerted by terial from NIK mutant aly mice showed neither mTEC de- rRANKL and agonistic anti-LTbR mAb showed an additive ef- velopment (Fig. 7A) nor upregulation of RANK expression (Fig. fect. Indeed, we demonstrated that the LTbR signal contributes to 7B) after any of the tested stimuli, indicating that upregulation of mTEC differentiation in a stepwise manner with the RANK sig- RANK expression by the LTbR or RANK signal is totally NIK nal; the LTbR signal elicits RANK expression, thereby permitting dependent. RANKL to efficiently control the differentiation program of im- We then investigated the requirement for TNFR-associated fac- mature mTECs thereafter. In contrast, crossing Cd40-KO onto tor (TRAF) 6 in this process. FTOC using material from Traf6- Ltbr-KO resulted in no obvious effect beyond the dominant phe- KO demonstrated no mTEC development after rRANKL stimu- notype attributable to LTbR deficiency, and simultaneous stimulation (18). Similarly, RANK expression induced by rRANKL was lation with agonistic anti-LTbR mAb and rCD40L exerted no greatly diminished (Fig. 7C). In contrast, upregulation of RANK effect in FTOC, suggesting that there is little functional associa- induced by the LTbR signal was indistinguishable between con- tion between the LTbR and CD40 signals in thymic organogen- trol mice and Traf6-KO, in agreement with the minimal involve- esis. These results illustrated many aspects of the unique and ment of TRAF6 downstream of LTbR assessed with embryonic cooperative action of multiple TNFRsf members in the cytokine- fibroblasts (12). These results suggest that NIK is indispensable mediated mTEC differentiation program. for the upregulation of RANK induced by both the LTbR and Even after alteration of the thymic structure in postnatal Ltbr- RANK signals, whereas TRAF6 is required for the activity in- KO had been reported (29), the precise roles of LTbR in thymic duced by RANK, but not by LTbR. organogenesis remained enigmatic and were investigated only The Journal of Immunology 5055 Downloaded from

FIGURE 7. Differential requirement of NIK and TRAF6 for upregulation of RANK expression. A, mTEC development from aly/+ (control; upper panels) and NIK-mutant aly/aly mouse thymi (lower panels) was assessed after stimulation with agonistic anti-LTbR mAb, rRANKL, or their combination for 4 d. aly mouse thymi did not show any mTEC development, as assessed by UEA-1 binding and MHC II expression. Cells were analyzed after gating for http://www.jimmunol.org/ CD452TER-1192EpCAM+ cells. One representative experiment from a total of two repeats is shown. B, Rank gene expression assessed by real-time PCR from the samples shown in A. Black columns, aly/+ mice; white columns, aly mice. C, TRAF6 is required for upregulation of RANK induced by the RANK signal but not by the LTbR signal. Black columns, control mice; white columns, Traf6-KO. Numbers are gene expression levels relative to that of the Gapdh gene. One representative experiment from a total of three repeats is shown. recently. Expression of CCL19/EBL-1 ligand chemokine was re- onic stage results in a defect of mTEC organization in the post- duced in mTECs from Lta-KO, Ltb-KO, or Ltbr-KO (31, 36). natal period; the size of each medulla was smaller, and they were Conversely, in vivo agonistic stimulation of LTbR in adult Lta-KO poorly connected to each other in the thymus of adult Ltbr-KO. has been demonstrated to upregulate expression of the CCL19 Given that many aspects of thymic organization are receptive to by guest on September 26, 2021 gene from mTEClow (31). Interestingly, in vivo agonistic stimu- thymocyte cross talk even in the postnatal period (47), the de- lation of LTbR in Lta-KO also increased the UEA-1 binding to fective thymic organization in adult Ltbr-KO suggests that the mTEClow together with an increase in the expression of some TRA requirement for the LTbR signal to ensure proper organization of genes such as Crp. Among the genes upregulated by in vivo ag- the thymic epithelium may not be confined to the embryonic stage. onistic stimulation of LTbR in adult mice (31) or genes differ- Instead, the continued presence of the LTbR signal provided by entially expressed in mTECs between postnatal Ltbr-KO and thymocytes as a cross talk may also play a role in homeostasis of control mice (30, 32), the Rank gene, which we identified in the the thymic microenvironment during the postnatal period. Indeed, current study as an LTbR-responsive gene relevant to thymic or- a recent study has suggested that continued mTEC development to ganogenesis in the fetus, was not detected in these previous the stage of expression of involucrin, a marker of terminally dif- studies. One obvious reason for this could be that the effect of the ferentiated epithelium (48), maps to activation of the LTbR signal LTbR signal in thymic organogenesis is dependent on the de- pathway by mature thymocytes in adult mice (49). velopmental stage (i.e., fetal versus postnatal) as suggested by Although both the LTbR and RANK signals augmented the LTbR-mediated secondary lymphoid organogenesis; lymph nodes expression of RANK in the thymic stroma, there was a kinetic and developed in Lta-KO when agonistic LTbR stimulation was ini- functional difference between the two. RANK expression induced tiated in utero before day 17 of gestation, whereas no such effect by the LTbR signal was rapid, and this alone was not associated was observed when the same treatment was applied beyond this with mTEC differentiation. In contrast, RANK expression induced developmental window (44). We speculate that analysis of gene by the RANK signal itself was more gradual and associated with expression in mTECs at the embryonic stage might identify Rank mTEC differentiation together with the expression of TRA genes as a gene that is differentially expressed between Ltbr-KO and such as Sap1. We speculate that this kinetic difference between the control mice. Thus, factors influencing thymic organogenesis need two signals through LTbR with RANK enables effective mTEC to be studied in a developmental stage-dependent manner, a pre- development in a physiological setting, as we demonstrated by the cedent of which is the main source of RANKL; in the initial efficient mTEC differentiation after sequential stimulation with differentiation phase of embryos, Aire+ mTECs develop through LTbR, followed by RANK stimulation in FTOC. Thus, the LTbR interaction with lymphoid tissue inducer cells expressing the signal may play a more important role in the initial phase of RANKL, whereas in the postnatal stage, Aire+ mTECs are mTEC development, whereas the RANK signal ensures the dif- maintained by the RANKL (together with the CD40L) provided ferentiation of mTECs together with their maintenance in the later by mature CD4 single-positive T cells to ensure their turnover stages of embryo development. and/or survival (16, 18, 20–22). In the search for additional receptor(s) beyond LTbR that could We have suggested that a defect in the upregulation of RANK in fill the gap between LTbR deficiency and NIK mutation or IKKa immature mTECs through loss of the LTbR signal at the embry- deficiency for thymic phenotypes, we hypothesized that such 5056 LYMPHOTOXIN ELICITS THYMIC STROMAL RANK EXPRESSION putative receptor(s) should primarily activate the non-canonical The fact that LTa deficiency (lacking both mLT and sLT) and NF-kB activation pathway, as is the case for LTbR. This as- LTb deficiency (lacking mLT but not sLT) do not have the same sumption was based on the fact that, for secondary lymphoid or- consequences for thymic organogenesis is reminiscent of the fact ganogenesis, Ltbr-KO show essentially the same phenotype as that Ltb-KO showed less profound lymph node genesis pheno- mice deficient in the NIK–IKKa signal pathway (35) and that the types (i.e., the presence of mesenteric lymph nodes) in contrast to NIK–IKKa signal pathway is a unique component of the non- the case of Lta-KO (51, 52). The phenotypic difference in thymic canonical NF-kB activation pathway (46). We therefore expec- organogenesis between Lta/Light-DKO and Ltb/Light-DKO (and ted that crossing Ltbr-KO with other mouse strains deficient in most likely already between Lta-KO and Ltb-KO) (29 and this TNFRsf members that can activate the non-canonical NF-kB study) may suggest a role of sLT in thymic organogenesis (also pathway, such as RANK and CD40, would produce thymic phe- for lymph node genesis). If this is the case, then interaction be- notypes similar to those of NIK mutant aly mice, but it was not the tween sLT (i.e., LTa3) and TNFR (TNFRsf1a/1b) and/or HVEM case. Although it is still possible that there are other combinations (TNFRsf14) might play some role in thymic organogenesis. Al- of deficiency for non-canonical NF-kB activating receptor(s) be- ternatively, there may be a weak in vivo interaction between sLT yond LTbR plus RANK or LTbR plus CD40, which we tested in and LTbR (53), although an in vitro study did not demonstrate the current study, the fact that the role of NIK in NF-kB activation such binding (54). In either case, it would be important to em- is a component of not only the non-canonical but also the canoni- phasize that deficiency of LTa or LTbR results in a different cal NF-kB activation pathway, at least downstream of some phenotype of thymic organogenesis and that LIGHT deficiency TNFRsf member such as CD27 (50), merits attention. Although additional to LTa deficiency fills this gap. This is remarkable the exact roles of the canonical NF-kB activation pathway for when considering that a deficiency of LTa or LTbR produces very Downloaded from thymic organogenesis have not yet been fully defined, it is pos- similar defects in secondary lymphoid organogenesis. Thus, it is sible that the combined defect in the canonical and non-canonical now evident that there is a critical requirement for LIGHT (but NF-kB activation pathways in NIK mutant aly mice might account only in combination with mLT) through LTbR in thymic organ- for the more severe thymic phenotype in comparison with Ltbr- ogenesis. KO, where the defect is essentially confined to the non-canonical

NF-kB activation pathway (46). Thus, it is possible that mTEC Acknowledgments http://www.jimmunol.org/ differentiation requires intact NF-kB activities mediated by both We thank Drs. W. van Ewijik and P.D. Rennert for mAbs ER-TR5 and AC. the canonical and non-canonical NF-kB activation pathways. In H6, respectively. this scenario, redundancy and/or cross talk between the canonical and non-canonical NF-kB activation pathways for thymic organ- Disclosures ogenesis is likely, although the functional segregation of the two The authors have no financial conflicts of interest. pathways seems to be more blurred than previously thought. In contrast to the complexity of NIK-mediated thymic organogenesis References with respect to the upstream receptor(s), TRAF6-dependent thy- 1. Kyewski, B., and L. Klein. 2006. A central role for central tolerance. Annu. Rev. by guest on September 26, 2021 mic organogenesis seems to be better defined; Rankl/Cd40-DKO Immunol. 24: 571–606. showed a severe defect of thymic organogenesis similar to that in 2. Hogquist, K. A., T. A. Baldwin, and S. C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5: 772–782. Traf6-KO, suggesting that the upstream receptor(s) responsible for + 3. Sakaguchi, S. 2004. Naturally arising CD4 regulatory t cells for immunologic TRAF6-dependent thymic organogenesis is very likely RANK and self-tolerance and negative control of immune responses. Annu. Rev. Immunol. CD40 (18). However, the precise mechanisms underlying the 22: 531–562. 4. Anderson, G., P. J. Lane, and E. J. Jenkinson. 2007. Generating intrathymic cooperation between the RANK and CD40 signals require further microenvironments to establish T-cell tolerance. Nat. Rev. Immunol. 7: 954–963. study. 5. Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. P. Berzins, S. J. Turley, Finally, an integrated and detailed phenotypic analysis of mice H. von Boehmer, R. Bronson, A. Dierich, C. Benoist, and D. Mathis. 2002. b Projection of an immunological self shadow within the thymus by the aire deficient in the LT R signal would help to clarify many aspects of protein. Science 298: 1395–1401. LT biology. Ltbr-KO showed clear reduction of UEA-1+ mTECs 6. Mathis, D., and C. Benoist. 2009. Aire. Annu. Rev. Immunol. 27: 287–312. associated with loss of the characteristic three-dimensional orga- 7. Peterson, P., T. Org, and A. Rebane. 2008. Transcriptional regulation by AIRE: molecular mechanisms of central tolerance. Nat. Rev. Immunol. 8: 948–957. nization and also a reduction of absolute numbers (29 and in this 8. Gardner, J. M., A. L. Fletcher, M. S. Anderson, and S. J. Turley. 2009. AIRE in study). In contrast, Ltb-KO (not Lta-KO) showed no significant the thymus and beyond. Curr. Opin. Immunol. 21: 582–589. 9. Kajiura, F., S. Sun, T. Nomura, K. Izumi, T. 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