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OX40 Ligand and CD30 Ligand Are Expressed on Adult but Not Neonatal CD4 + CD3− Inducer Cells: Evidence That IL-7 Signals Regulate CD30 Ligand but Not OX40 This information is current as Ligand Expression of September 26, 2021. Mi-Yeon Kim, Graham Anderson, Andrea White, Eric Jenkinson, Wiebke Arlt, Inga-Lill Martensson, Lena Erlandsson and Peter J. L. Lane

J Immunol 2005; 174:6686-6691; ; Downloaded from doi: 10.4049/jimmunol.174.11.6686 http://www.jimmunol.org/content/174/11/6686 http://www.jimmunol.org/ References This article cites 28 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/174/11/6686.full#ref-list-1

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

OX40 Ligand and CD30 Ligand Are Expressed on Adult but Not Neonatal CD4؉CD3؊ Inducer Cells: Evidence That IL-7 Signals Regulate CD30 Ligand but Not OX40 Ligand Expression1

Mi-Yeon Kim,* Graham Anderson,* Andrea White,* Eric Jenkinson,* Wiebke Arlt,† Inga- Lill Martensson,‡ Lena Erlandsson,‡ and Peter J. L. Lane2*

In this report, we have examined the expression of the T cell survival signals, OX40 ligand (OX40L) and CD30 ligand (CD30L) on CD4؉CD3؊CD11c؊B220؊IL-7R␣؉ inducer cells from birth to adulthood in mice. We found that adult but not neonatal

inducer cells expressed high levels of OX40L and CD30L, whereas their expression of TNF-related activation-induced cytokine Downloaded from (TRANCE) and receptor activator of NF-␬B (RANK) was comparable. The failure of neonatal inducer cells to express the ligands that rescue T cells helps to explain why exposure to Ag in neonatal life induces tolerance rather than immunity. The expression of OX40L and CD30L on inducer cells increased gradually in the ﬁrst few weeks of life achieving essentially normal levels around the time mice were weaned. We found that IL-7 signaling through the common cytokine receptor ␥-chain was critical for the optimal expression of both TNF-related activation-induced cytokine and CD30L but not OX40L. Furthermore, glucocorticoids,

which potently suppress T effector function, did not inﬂuence the expression of OX40L and CD30L in the presence of IL-7. The http://www.jimmunol.org/ Journal of Immunology, 2005, 174: 6686–6691.

he capacity of animals to mount high affinity long-lived ficient in ROR␥ (5) lack lymph nodes and Peyer’s patches; mice Ab responses is associated with the development of seg- deficient in TRANCE (6) and RANK (7) signals lack most lymph regated B and T cell areas in secondary lymphoid tissue nodes; mice deficient in IL-7 (8) and ␥ chain signals (9) lack T c and also the development of lymph nodes. Recent work has iden- Peyer’s patch anlagen and some lymph nodes; and CXCR5-defi- ϩ Ϫ Ϫ Ϫ ϩ tified a key role for a CD4 CD3 CD11c B220 IL-7R␣ cell cient mice have impaired Peyer’s patch formation and are deficient (inducer cell) in this process (1). These cells are found in neonatal in some lymph nodes (10). lymph nodes before they are colonized by lymphocytes (2). They In adult mice, we identified the adult equivalent of inducer cells, by guest on September 26, 2021 3 are one of only a few cell types that express lymphotoxin (LT) and reported that they were located in B follicles and at the inter- ␣ ␤ ␣ ␤ ␤ 1 2 (LT 1 2) (2), which is the ligand for the LT receptor that face between the B and T cell areas (11). These cells had a very plays a critical role in organizing secondary lymphoid tissues (3, similar phenotype to those described in the neonate (11), but in 4). Mice deficient in this signaling pathway lack lymph nodes, and addition to the expression of LT␣1␤2 and TRANCE, these cells the spleen is not organized into B and T cell areas. Evidence that were also found to express high levels of the TNF ligands for ␤ LT receptor signals from inducer cells initiate a sequence of OX40 and CD30. We found evidence for direct interactions be- events that leads to the development of lymph nodes comes from tween primed CD4 T cells and these cells during the course of mice deficient in these cells. Inducer cells express IL-7 receptor ␣ immune responses, and showed that the survival of primed T cells chain (IL-7R␣), common cytokine receptor ␥-chain (␥ chain, c was partially dependent on OX40 signals from these cells. Because CD132), ROR␥ (retinoic acid receptor-related orphan nuclear hor- OX40 and CD30 are genetically linked in human (chromosome 1) mone receptor), CXCR5, TNF-related activation-induced cytokine and mouse (chromosome 4) (12) and share common signaling (TRANCE), and receptor activator of NF-␬B (RANK). Mice de- pathways, it seemed likely that there were redundant signaling pathways (13). Recently, we have generated and examined the *Medical Research Council Centre for Immune Regulation, †Division of Medical phenotype of mice deficient in both OX40 and CD30 and shown ‡ Sciences, Birmingham Medical School, Birmingham, United Kingdom; and Labo- that they lack T cell memory for Ab production, and this failure ratory of Lymphocyte Signaling and Development, The Babraham Institute, Cam- bridge, United Kingdom results from deficient OX40 and CD30 signals delivered by in- Received for publication December 8, 2004. Accepted for publication March ducer cells (14). 15, 2005. In this study, we have investigated the expression of OX40 li- The costs of publication of this article were defrayed in part by the payment of page gand (OX40L) and CD30 ligand (CD30L) during the development charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of inducer cells from the neonatal period to adulthood. We found that inducer cells isolated from neonatal mice did not express 1 This study was supported by a Wellcome Programme Grant (to P.J.L.L.). OX40L and CD30L, whereas their expression of TRANCE and 2 Address correspondence and reprint requests to Dr. Peter J. L. Lane, Medical Re- search Council Centre for Immune Regulation, Birmingham Medical School, Vincent RANK was similar to adult mice. This suggested that there were Drive, Birmingham B15 2TT, UK. E-mail address: [email protected] specific signals that induced expression of OX40L and CD30L on 3 ␥ Abbreviations used in this paper: LT, lymphotoxin; c chain, common cytokine inducer cells. We found that mice deficient in either IL-7 signals or receptor ␥-chain; TRANCE, TNF-related activation-induced cytokine; RANK, recep- ␥ tor activator of NF-␬B; OX40L, OX40 ligand; CD30L, CD30 ligand; Tg, transgenic; c chain had substantially reduced CD30L expression, whereas FDC, follicular dendritic cell. OX40L expression was comparable. Furthermore, addition of IL-7

FIGURE 1. Expression of TRANCE, RANK, OX40L, and CD30L on inducer cells isolated from the spleens of varying ages. Staining of adult RAG-deﬁcient Downloaded from cells was used to compare levels of expression at each of the time points indicated. Closed histograms are control staining with biotinylated rat Abs. A, Inducer cells from RAG-deﬁcient mice. Results are representative of six separate experiments. B, Inducer cells from neonatal and adult wild-type mice. Results are representative of two separate experiments. http://www.jimmunol.org/ by guest on September 26, 2021

to neonatal inducer cells specifically up-regulated CD30L expres- Taconic Farms. Spleens from IL-7-deficient mice were provided by I.-L. sion. The IL-7-induced CD30L expression was also observed on Martensson (Babraham Institute, Cambridge, UK), and spleens from RAG2Ϫ/Ϫ mice deficient in ␥ chain and IL-7R␣-deficient mice were pro- memory phenotype CD4 and CD8 T cells as well as naive CD8 T c vided by R. Zamoyska (National Institute for Medical Research, cells, suggesting that there is a common IL-7-dependent pathway London, UK). for CD30L expression. We failed to identify specific signals that up-regulated OX40L expression on neonatal inducer cells. The ex- Preparation of inducer cells pression of OX40L and CD30L was not dependent on MHC class Cell suspensions for isolation of inducer cells were made from the spleens I or II signals or T cells. Furthermore, we show that although as follows. Spleens were cut into small fragments and then cultured with glucocorticoids down-regulated CD30L and TRANCE expression Collagenase D (1 mg/ml) (Roche Diagnostics) for 45 min at 37°C. Di- gested fragments were crushed between gauze. After depletion of RBC on inducer cells, OX40L expression was only modestly affected. with Gey’s solution, the cell suspensions were pooled and resuspended in However, in the presence of IL-7, CD30L and TRANCE expres- MACS buffer containing 10% mouse serum per the manufacturer’s instruc- sion was restored on glucocorticoid-treated cells. tions. CD11cϩ cells were depleted with MACS anti-mouse CD11c mi- crobeads (Miltenyi Biotec) using autoMACS (Miltenyi Biotec). CD4ϩ cells from CD11cϪ cells were enriched with MACS anti-mouse CD4 mi- Materials and Methods crobeads and positively selected. The resulting populations were cultured Mice overnight. CD4ϩCD3ϪB220Ϫ cells from CD4-enriched population were Normal, Rag1 deficient (Rag1Ϫ/Ϫ), and OX40 and CD30 double deficient stained with appropriate Abs and analyzed by flow cytometry. Ϫ/Ϫ Ϫ/Ϫ (OX40 and CD30 ) C57BL/6 mice were bred and maintained in our In vitro culture with IL-7 or steroid hormones animal facility. Rag1Ϫ/Ϫ mice were used as a source of inducer cells (Figs. 1, 4A, and 6). CD3⑀ transgenic (Tg) 26 mice that lack CD3ϩ T cells (15) T cells from normal mouse spleen and inducer cells from RAG-deficient were obtained from The Jackson Laboratory, and MHC class II-deficient mouse spleen were cultured with 100 ng/ml IL-7 (PeproTech) and/or with and MHC class I and II double deficient mice (16) were obtained from 100 nM endogenous human glucocorticoid cortisol (ϭ hydrocortisone) 6688 DIFFERENTIAL EXPRESSION OF CD30L AND OX40L ON INDUCER CELLS

FIGURE 2. Expression of IL- ␣ ␥ 7R , c chain, TRANCE, RANK, OX40L, and CD30L on inducer cells isolated from IL-7-deficient, IL-7R␣- Ϫ/Ϫ ␥ deficient, and RAG c chain-deficient mice compared with control RAGϪ/Ϫ mice. Results are representative of two separate experiments. To compare levels of expression, inducer cells were isolated from the various knockout and control mice contemporaneously. Closed histograms are control staining with biotinylated rat Abs. Downloaded from

(Sigma-Aldrich) and 100 nM synthetic glucocorticoid dexamethasone isolated at the same time from control adult RAG-deﬁcient mice (Sigma-Aldrich). were used to compare levels of expression. It is technically difﬁcult

Abs for staining to do this because primed T cells associate with inducer cells. http://www.jimmunol.org/ Nevertheless, the same pattern of expression of TRANCE, RANK, Anti-CD4 PE, anti-B220 FITC, anti-B220 allophycocyanin, anti-CD3 OX40L, and CD30L was observed on inducer cells isolated from FITC, anti-CD8 allophycocyanin, anti-CD44 FITC, and IL-7R␣ PE and ␥ wild-type neonatal and adult mice. biotinylated mAbs against CD4, OX40L, CD30L, and c chain were purchased from BD Pharmingen. Biotinylated mAbs against TRANCE and ␥ RANK were obtained from R&D Systems. As the second step staining IL-7 and c chain signaling regulates CD30L but not OX40L reagents, streptavidin cychrome was purchased from BD Pharmingen. expression on inducer cells Because inducer cells express IL-7R␣ and ␥ chain, we first tested Results c ϩ Ϫ whether signaling through these pathways regulated CD30L or Differential expression of OX40L and CD30L on CD4 CD3 OX40L expression. To compare the expression directly, inducer by guest on September 26, 2021 inducer cells from neonatal to adulthood cells were isolated from control and deficient mice and stained Adult inducer cells are unusual in that they show constitutive ex- with the same concentrations of Abs on the same day. Analysis of pression of high levels of the TNF ligands for OX40 and CD30 IL-7- and IL-7R␣-deficient mice showed that OX40L expression that are expressed on activated T cells (11). However, early in was essentially unaffected and this was also true of RAGϪ/Ϫ mice neonatal life these cells play a key role in the development and with concomitant ␥c chain deficiency (Fig. 2). In contrast, the ex- organization of secondary lymphoid tissue (1). At this stage of pression of TRANCE but not its receptor, RANK, was impaired in ␣ ␥ development, exposure to foreign Ags is more likely to tolerize IL-7-, IL-7R -, and c chain-deficient mice, and there was also than immunize the murine immune system. We were interested to substantially reduced expression of CD30L. This suggested that know at what stage in ontogeny the expression of OX40L and IL-7 signals up-regulated TRANCE and CD30L expression. We CD30L appears. Inducer cells were isolated from day 4 neonatal also found that the numbers of inducer cells isolated from mice ␥ spleens from RAG-deficient mice to exclude contaminating lym- deficient in IL-7 and c chain signals was greatly reduced (Fig. 3). phocytes. These cells, like their adult equivalents, expressed To test directly whether IL-7 induced CD30L expression, in- TRANCE and RANK at comparable levels (Fig. 1A) and also ex- ducer cells from neonatal and adult mice were cultured in vitro ␣ ␥ pressed IL-7R and c chain (data not shown). However, at this stage of development, the expression of OX40L and CD30L was lacking (Fig. 1A). This was also observed in CD3⑀Tg26 mice that fail to generate any CD3ϩ T cells but have normal B cell numbers (data not shown). Some inducer cells from 9-day-old mice expressed OX40L (there was a bimodal distribution of OX40Lϩ and OX40LϪ cells). This increased from 13.7% on day 4 to 35.5% on day 9, 71.1% on day 14, and 94.6% in adult 6-wk-old mice. In contrast, the expression of CD30L did not reveal a bimodal distribution. Rather there was a gradual increase in intensity of CD30L expression with time from 8.4% at day 4 to 96.7% at 6 wk of age. For each of these time points, control cells from adult RAG-deficient mice were isolated at the same time to compare levels of expression of TNF family members. To confirm that the results we obtained in RAG-deficient mice FIGURE 3. Numbers of inducer cells isolated from the spleens of gene- were also true in wild-type mice, we isolated inducer cells from deficient mice. Results are representative of at least two separate experi- day 1 neonatal and adult spleens of wild-type mice (Fig. 1B). Cells ments. Bars show SD between experiments. The Journal of Immunology 6689

FIGURE 4. Modiﬁcation of TRANCE, OX40L, and CD30L on inducer cells and memory CD44high CD4 and CD8 T cells by IL-7. A, Adult and neonatal (1-wk old) inducer cells from RAG-deﬁcient mice were cultured in vitro for 6 days without (top panel) and with (bottom panel) IL-7 (100 ng/ml). B, Splenocytes from normal C57BL/6 mice were cultured for 1 day without (top panel) and with (bottom panel) IL-7 (100 ng/ml). His- tograms show expression on CD44high CD4 and CD44high CD8 T cells. Results are representative of two separate experiments. Closed histograms are control staining with biotinylated rat Abs. Downloaded from

with 1–100 ng/ml IL-7 (data shown of 100 ng/ml IL-7) for 6 days Effects of glucocorticoids on TNF expression on inducer cells http://www.jimmunol.org/ (Fig. 4A). Compared with freshly isolated or untreated cells, IL-7 Because glucocorticoids are commonly used to suppress immune clearly induced CD30L expression on neonatal inducer cells and responses, we investigated whether they affected expression of enhanced CD30L expression by adult inducer cells (Fig. 4A). IL-7 TNF ligands on adult inducer cells. After 2 days of incubation with also clearly up-regulated TRANCE expression but OX40L expres- the endogenous glucocorticoid cortisol or the synthetic glucocor- sion was unaffected. This effect of IL-7 was not attributable to ticoid dexamethasone, inducer cells down-regulated the expression selective survival of inducer cells expressing high levels of of both CD30L and TRANCE, although the expression of OX40L TRANCE and CD30L, because the viability and cell number of was only modestly attenuated (Fig. 6A). This was associated with inducer cells from IL-7 treated and control cultures were similar impaired survival of inducer cells after 6 days of culture with dexa- by guest on September 26, 2021 (data not shown). methasone (Fig. 6B). The effects of dexamethasone did not depend The induction of CD30L by IL-7 was not restricted to inducer cells. Both CD44high populations of CD4 and CD8 T cells up- regulated CD30L expression in response to IL-7 (Fig. 4B), and this was also true for CD44low CD8 naive T cells (data not shown). Whereas IL-7 augmented CD30L expression, TRANCE or OX40L expression was not induced on T cells by IL-7. IL-7 signals up-regulated CD30L and TRANCE expression, and this was also true for IL-2 (data not shown). This suggests that this ␥ signal was mediated by c chain. In contrast, other cytokines that ␥ signal through c chain (17), IL-4, IL-9, and IL-15, had little effect on the expression of OX40L, CD30L, or TRANCE (data not shown).

MHC class I and II expression does not affect OX40L and CD30L expression Because inducer cells express CD4, we tested whether interaction with MHC class I and class II molecules were key to regulating expression of OX40L and CD30L. This was not the case although OX40L and CD30L expression was slightly lower in mice deficient in MHC class I and II molecules (Fig. 5). Also CD3ϩ T cells did not influence inducer cell development as mice lacking T cells, but normal numbers of B cells (15) showed the highest levels of expression of OX40L and CD30L (Fig. 5), and the numbers of inducer cells isolated per spleen was consistently augmented com- Ϫ Ϫ FIGURE 5. Expression of TRANCE, RANK, OX40L, and CD30L on pared with RAG / mice (Fig. 3). There is some evidence for inducer cells from MHC deficient, CD3⑀tg26 (T cell deficient), and OX40 reverse signaling through TNF ligands (18, 19) but there was no and CD30 double deficient (OX40Ϫ/Ϫ & CD30Ϫ/Ϫ) mice compared with evidence that this was important for inducer cells. Mice deficient control RAGϪ/Ϫ mice. Results are representative of at least two separate in OX40 and CD30 had normal numbers (data not shown), and experiments. To compare levels of expression, inducer cells were isolated their expression of TRANCE, RANK, OX40L, and CD30L was from the various knockout and control mice contemporaneously. Closed also normal (Fig. 5). histograms are control staining with biotinylated rat Abs. 6690 DIFFERENTIAL EXPRESSION OF CD30L AND OX40L ON INDUCER CELLS

OX40L and CD30L on inducer cells, by 2 wk of age both molecules were expressed, and almost normal levels were achieved by 3 wk, the time mice were weaned. These data suggest that there must be distinct signaling pathway/s that regulate expression of these molecules on inducer cells after birth. We demonstrated that IL-7 signals to inducer cells up-regulate TRANCE and CD30L expression. On adult inducer cells, CD30L expression was augmented, and on neonatal inducer cells, CD30L expression was induced by IL-7. Furthermore, mice deﬁcient in ␣ ␥ IL-7, IL-7R , and c chain had reduced CD30L expression. In contrast to CD30L, OX40L expression was not dependent on IL-7 ␥ or c chain, and we could identify no other cytokine signals (IL-3, IL-4, IL-9, IL-10, IL-12, or IL-15) that would induce its expression on neonatal inducer cells (data not shown). IL-7 mediated up-regulation of CD30L expression was not ex- clusive to inducer cells because memory CD4 and CD8 T cells also up-regulated CD30L expression in the presence of IL-7, suggesting that there is a common signaling pathway in T cells and in-

ducer cells. We have observed previously that IL-4, which also Downloaded from ␥ signals via c chain-like IL-7, does not affect the expression of either OX40L or CD30L on inducer cells (21). Its effects on CD4 T cells are also different: IL-4 down-regulated OX40L and CD30L expression on primed CD4 T cells, whereas IL-7 induced CD30L but not OX40L expression. This indicates that although the cyto-

␥ http://www.jimmunol.org/ kines, IL-4 and IL-7, both share signaling pathways through c ␥ chain, there must be distinct signaling pathways that are c chain independent, particularly for IL-4. Because inducer cells express CD4, we tested whether MHC class I or II molecules might be required for their development and the expression of TNF ligands. No differences in number or phenotype were observed. Similarly the expression of these molecules was normal in mice deficient for OX40 and CD30 expression suggesting that reverse signaling through OX40L and CD30L was unimportant for inducer cell development. by guest on September 26, 2021 Finally we investigated whether glucocorticoids, which are potent suppressors of T cell-mediated effector responses, modified FIGURE 6. Effects of glucocorticoids on inducer cell expression and the expression of TNF ligands on inducer cells. After 2 days of survival. Control (ethanol solvent), cortisol (100 nM), DEX (100 nM dexa- culture with cortisol or the more potent synthetic glucocorticoid, methasone), DEX ϩ IL-7 (100 nM dexamethasone and 100 ng/ml IL-7), or dexamethasone, inducer cells down-regulated both CD30L and IL-7 (100 ng/ml) alone. Results are representative of two separate exper- TRANCE expression compared with control. After 6 days of cul- ␣ iments. A, The expression of IL-7R , TRANCE, OX40L, and CD30L on ture, there were significantly fewer inducer cells in the dexametha- ء day 2. Closed histograms are control staining with biotinylated rat Abs. , sone-treated cultures, presumably due to down-regulation of Receptor down-modulation by IL-7. B, The survival on day 6. Bars show TRANCE-dependent survival signals for inducer cells, but OX40L SD between experiments. expression was spared. The decreased survival in the presence of glucocorticoids was reversed if inducer cells were cultured in the presence of IL-7. The failure of glucocorticoids to attenuate the on down-regulation of IL-7R␣ because levels were unaffected by expression of the T cell survival signal, OX40L explain why glu- treatment (Fig. 6A). Furthermore, the effects of dexamethasone on cocorticoids, which are effective at suppressing effector T cell re- the expression of CD30L and TRANCE and their survival were sponses (22, 23), fail to eliminate CD4 T cell memory (24). prevented by addition of IL-7. In this study, we show that OX40L and CD30L expression on adult inducer cells, which we have linked with CD4 T cell survival Discussion and memory for Ab responses, is not expressed in neonatal life on Here we report that inducer cells isolated from neonatal mice lack inducer cells. We provide evidence that the expression of ␥ expression of the T cell survival molecules, OX40L and CD30L, TRANCE and CD30L on inducer cells is regulated by IL-7, c although expression of TRANCE and RANK was normal. We chain, and glucocorticoid signals. In contrast, although OX40L have found that mice deficient in both OX40 and CD30 show was not expressed on neonatal inducer cells, regulation of its ex- grossly impaired CD4 T cell memory responses because they fail pression was not affected by the above signals. We suggest that the to receive survival signals from inducer cells (14). The data pro- deficient expression of OX40L and CD30L on neonatal inducer vided here on neonatal inducer cells suggest that T cells primed in cells could be a contributory mechanism for neonatal tolerance the neonate would be rendered effectively deficient in OX40 and induction. CD30 survival signals from inducer cells, so they would not be- We do not know whether inducer cells isolated from adults that come memory cells. This observation may help explain the phe- express OX40L and CD30L are the direct descendants of neonatal nomenon of neonatal tolerance first described 50 years ago by inducer cells, although labeling studies with thymidine analogues Medawar et al. (20). Although neonatal mice did not express suggest that they turnover slowly (25). We have provided evidence The Journal of Immunology 6691 that CD30L expression can be induced by IL-7, but we do not development in mice lacking expression of the common cytokine receptor gamma know which cell provides this signal in secondary lymphoid tissue. chain. Immunity 2: 223–228. 10. Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Forster, J. D. Sedgwick, An intriguing possibility is that it comes from follicular dendritic J. L. Browning, M. Lipp, and J. G. Cyster. 2000. A chemokine-driven positive cells (FDCs) (26) in B follicles close to where inducer cells are feedback loop organizes lymphoid follicles. Nature 406: 309–314. found. FDCs depend on B cell LT␣1␤2 for their survival (27), and 11. Kim, M. Y., F. M. Gaspal, H. E. Wiggett, F. M. McConnell, A. Gulbranson-Judge, C. Raykundalia, L. S. Walker, M. D. Goodall, and we have found that inducer cells isolated from neonatal mice with P. J. Lane. 2003. CD4ϩCD3Ϫ accessory cells costimulate primed CD4 T cells B cells (CD3⑀Tg26 mice) did have higher levels of CD30L (data through OX40 and CD30 at sites where T cells collaborate with B cells. Immunity not shown) than when isolated from RAG-deficient mice. How- 18: 643–654. 12. Locksley, R. M., N. Killeen, and M. J. Lenardo. 2001. The TNF and TNF re- ever, this cannot be the sole explanation, because adult inducer ceptor superfamilies: integrating mammalian biology. Cell 104: 487–501. cells isolated from RAG-deficient mice that lack FDCs have high 13. Chung, J. Y., Y. C. Park, H. Ye, and H. Wu. 2002. All TRAFs are not created levels of CD30L. In any case, we have not identified the signals equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. J. Cell Sci. 115: 679–688. that regulate OX40L expression. 14. Gaspal, F. M. C., M. Kim, F. M. McConnell, C. Raykundalia, V. Bekiaris, and Inducer cells have been reported in the blood (28), and we found P. J. L. Lane. 2005. Mice deficient in OX40 and CD30 signals lack memory that in the adult they express L-selectin (11), so precursors could antibody responses because of deficient CD4 T cell memory. J. Immunol. 174: 3891–3896. potentially migrate from the blood into secondary lymphoid or- 15. Hollander, G. A., B. Wang, A. Nichogiannopoulou, P. P. Platenburg, gans. It could be that the neonatal population that we isolate is W. van Ewijk, S. J. Burakoff, J. C. Gutierrez-Ramos, and C. Terhorst. 1995. simply replaced by an adult population that expresses OX40L and Developmental control point in induction of thymic cortex regulated by a sub- population of prothymocytes. Nature 373: 350–353. CD30L. Nevertheless, understanding the molecular mechanism 16. Crump, A. L., M. J. Grusby, L. H. Glimcher, and H. Cantor. 1993. Thymocyte that regulates this transition may provide new approaches to mod- development in major histocompatibility complex-deficient mice: evidence for Downloaded from ifying pathogenic T cell responses that are refractory to conven- stochastic commitment to the CD4 and CD8 lineages. Proc. Natl. Acad. Sci. USA 90: 10739–10743. tional immune suppression including glucocorticoid therapy. 17. Kovanen, P. E., and W. J. Leonard. 2004. Cytokines and immunodeficiency dis- eases: critical roles of the ␥-dependent cytokines interleukins 2, 4, 7, 9, 15, and Acknowledgments 21, and their signaling pathways. Immunol. Rev. 202: 67–83. 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