Lymphotoxin Signals CD4 T Cell Recruitment in the Absence Of

CD30 Is Required for CCL21 Expression and CD4 T Cell Recruitment in the Absence of Lymphotoxin Signals

This information is current as Vasileios Bekiaris, Fabrina Gaspal, Mi-Yeon Kim, David R. of September 27, 2021. Withers, Fiona M. McConnell, Graham Anderson and Peter J. L. Lane J Immunol 2009; 182:4771-4775; ; doi: 10.4049/jimmunol.0803481

http://www.jimmunol.org/content/182/8/4771 Downloaded from

References This article cites 30 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/182/8/4771.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD30 Is Required for CCL21 Expression and CD4 T Cell Recruitment in the Absence of Lymphotoxin Signals1

Vasileios Bekiaris,2* Fabrina Gaspal,† Mi-Yeon Kim,‡ David R. Withers,† Fiona M. McConnell,† Graham Anderson,† and Peter J. L. Lane3†

Lymphoid tissue inducer cells express a diverse array of tumor necrosis family ligands, including those that bind CD30 and the lymphotoxin ␤ receptor. Both of these signaling pathways have been linked with B/T segregation in the spleen. In this study, we have dissected a lymphotoxin-independent CD30-dependent signal for the induction of expression of the T zone chemokine, CCL21. Reduced expression of CCL21 due to CD30 deficiency was functionally significant: mice deficient in both lymphotoxin and CD30 (dKO) signals had significantly smaller accumulations of lymphocytes in their splenic white pulp areas, with no evidence of focal aggregation of T cells. Furthermore, recruitment of wild-type CD4 T cells was poor in dKO mice compared with both wild-type or lymphotoxin-deficient mice. Phylogeny suggests that CD30 signals predated those Downloaded from through the lymphotoxin ␤ receptor. We suggest that CD30 signals from lymphoid tissue inducer cells were a primitive mechanism to recruit and prime CD4 T cells. This would have been a stepping stone in the evolution of the highly organized lymphotoxin dependent B and T white pulp areas within which CD4-dependent memory Ab responses now develop. The Journal of Immunology, 2009, 182: 4771–4775.

he white pulp areas of the spleen are organized into B and tween T cells migrating on T zone stromal cells (11) and den- http://www.jimmunol.org/ T cell areas by the expression of homeostatic chemo- dritic cells (DCs)4 in the T zone. T kines within defined fixed stromal cells in B (CXCL13) The relationship between CCL21 and podoplanin implies that the and T (CCL19, CCL21) cell areas (1). Although required for the coexpression of the two molecules by T zone stromal cells is funda- formation of B follicles, B/T segregation is not dependent on mental to their function, and they require lymphotoxin ␤ receptor CXCL13, as B and T cells segregate normally in mice deficient (LT␤R) signals for the expression of both (12, 13). Recently, we in CXCR5 (2). Of the two CCR7 ligands, CCL21 is produced in identified a distinct role for another TNF receptor, CD30, in splenic greater abundance (100-fold greater protein levels than CCL19) B/T segregation (14). Despite normal levels of homeostatic chemo- (3). Furthermore, ectopic expression of CCL21 induces larger kines including CCL21, CD30-deficient (CD30Ϫ/Ϫ) mice demon- and more organized infiltrates than CCL19, so it is functionally strate impaired B/T segregation, with blurring of the normally crisp by guest on September 27, 2021 and quantitatively more important than CCL19. boundary between the B follicle and T zone. This defect maps to lack The main structural difference between CCL19 and CCL21 is of expression of podoplanin, supporting the view that podoplanin is that the latter has an extra 32 amino acid C terminus-containing functionally important for B/T segregation. basic amino acids (4, 5). Through this basic motif, it binds The work described above suggests that LT␤R and CD30-sig- negatively charged structures, including the highly glycosylated nals coordinate T zone stromal differentiation. Although lympho- mucin-type protein podoplanin (6) expressed on T zone stroma toxin signals can be provided by lymphocytes, lymphoid tissue (7). As a consequence of its immobilization, CCL21 is less inducer cells (LTi) can provide both LT␣ and CD30L (15). During chemotactic than soluble CCL19 (4, 5). The result of this im- splenic ontogeny, lymphotoxin-expressing LTi are the first cells to mobilization is that unlike CCL19, CCL21 does not desensitize associate with embryonic T zone stroma and activate the expres- its receptor (8). Furthermore, immobilization of CCL21 in vitro sion of VCAM-1 (16). At this stage of their development, LTi fail (simulating immobilization on podoplanin-expressing T zone to express CD30L (17), which appears around 1 wk of age, coin- stroma) stimulates integrin-independent T cell chemokinesis (9, cident with the up-regulation of podoplanin and B/T segregation 10), a process fundamental to the promotion of interactions be- (14). It was therefore possible that LTi CD30 signals were dependent on upstream lymphotoxin signals also provided by lymphocytes (14). Phylogeny, however, indicates otherwise: CD30 and its *La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; †Medical Re- ligand are present in mammalian and avian genomes, whereas the search Council Centre for Immune Regulation, Birmingham Medical School, Bir- ␤ mingham, United Kingdom; and ‡Department of Bioinformatics and Life Sciences, LT R and the lymphotoxin genes are exclusive to mammals (18). Soongsil University, Seoul, Korea So the effects of CD30 on chemokine expression are just as likely Received for publication October 16, 2008. Accepted for publication February to be independent of lymphotoxin. This suggested that CD30 13, 2009. might have effects on chemokine expression independent of lym- The costs of publication of this article were defrayed in part by the payment of page photoxin. To decide between the two scenarios, we generated mice charges. This article must therefore be hereby marked advertisement in accordance deficient in both CD30 and LT␣ and compared their phenotype to with 18 U.S.C. Section 1734 solely to indicate this fact. single deficient and wild-type (WT) mice. In this study, we 1 This work was supported by a Wellcome Programme Grant (to P.J.L.L. and G.A.). 2 Current address: La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037. 4 Abbreviations used in this paper: DC, dendritic cell; LT␤R, lymphotoxin ␤ receptor; 3 Address correspondence and reprint requests to Dr. P. J. L. Lane, Medical Research LTi, lymphoid tissue inducer cell; WT, wild type. Council Centre for Immune Regulation, Institute for Biomedical Research, Birming- ham Medical School, Birmingham, U.K. E-mail address [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803481 4772 CD30 INDUCES CCL21 EXPRESSION IN THE ABSENCE OF LYMPHOTOXIN

FIGURE 1. B/T segregation and white pulp size in WT and gene deﬁcient mice. A, WT, CD30Ϫ/Ϫ,LT␣Ϫ/Ϫ, and dKO mice were stained for IgM (green) and CD3 (red). B,LT␣Ϫ/Ϫ and dKO mice were stained for B220 (green) and CD3 (red). White pulp areas were demarcated by B220 expression as indicated by white lined areas. C, The graph shows the size of dKO (E) and LT␣Ϫ/Ϫ (F) white pulp areas; 12 dKO and 10 LT␣Ϫ/Ϫ mice were analyzed (each symbol represents a white pulp area). Each confocal micrograph (scale bar is 100 ␮m) was taken with a ϫ10 objective. Downloaded from

demonstrate that in the absence of lymphotoxin signals, CD30 http://www.jimmunol.org/ has a clear effect on CCL21 expression. Mice deﬁcient in both CD30 and lymphotoxin signals, have white pulp areas that are smaller and even less organized than those of lymphotoxin de- ﬁcient mice, and exhibit impaired recruitment of CD4 T cells.

Materials and Methods Mice All experiments were performed in accordance with U.K. laws and with the approval of the University of Birmingham ethics committee. Mice were by guest on September 27, 2021 bred in our animal facility and were of the C57BL/6 background. We generated CD30 and LT␣ double-deficient mice by crossing CD30Ϫ/Ϫ and LT␣Ϫ/Ϫ. Male or female mice were used when they were 6–10 wk old. Confocal microscopy and quantitative real-time PCR Tissue preparation, staining, and image acquisition and analysis were performed as previously described (14, 19). B cells were detected after staining with anti-mouse IgM conjugated to Rhodamine red (Jackson Immu- noResearch Laboratories) or anti-mouse B220 conjugated to Pacific blue (eBioscience). T cells were stained either with anti-mouse CD3e-FITC (eBioscience) or CD3-biotin (eBioscience) followed by streptavidin-Alexa Fluor-647 (Invitrogen). CD45.1ϩ transferred cells were detected in situ after staining with anti-mouse CD45.1-FITC (eBioscience). Sample preparation for and analysis of quantitative real-time PCR was as previously described (14, 19). Primer/probe sequences (ccl19, ccl21, cxcl13, ␤-actin) were published previously (14). Cell transfers and immunizations OT2 (20) spleen cells were adoptively transferred i.v. in WT, LT␣Ϫ/Ϫ,or dKO mice at a density of 5 ϫ 106 cells per mouse. One day later, mice were immunized i.p. with 200 ␮g OVA (Sigma-Aldrich), which was precipitated with a commercial alum gel, Alu-Gel-S (SERVA). Statistical analysis All statistical analyses were performed with the nonparametrical Mann- Whitney U test using StatView 5.0 ( p Ͻ 0.05 is considered significant).

Results FIGURE 2. T cell distribution between red and white pulp areas in Ϫ Ϫ Ϫ Ϫ Mice deficient in both CD30 and LT␣ have smaller splenic LT␣ / and dKO mice. Spleen sections from dKO (E) and LT␣ / (F) ϩ white pulp areas and impaired T cell retention relative to mice mice were stained and analyzed as in Fig. 1B. A, The percentage of CD3 ϩ ␮ 2 deficient in LT␣ alone pixels present in white and red pulp. B, CD3 pixels per m of total (white ϩ red pulp), white, or red pulp. C, CD3ϩ pixels present in total To test the hypothesis that CD30 and LT␣ might have independent (white ϩ red pulp), white, or red pulp. Seven dKO were compared with effects on B/T segregation, we made mice deficient in both. We five LT␣Ϫ/Ϫ mice. Each symbol represents one micrograph. The Journal of Immunology 4773 Downloaded from

FIGURE 4. Recruitment of primed CD4 T cells in WT and gene deﬁcient mice. CD45.1ϩ (red) OT2 cells were adoptively transferred into CD45.2ϩ WT (A), LT␣Ϫ/Ϫ (B)ordKO(C) recipient mice, which were immunized 24 h later and at day 5 postimmunization their spleens were stained for CD45.1 (red) and

B220 (green) and analyzed by confocal microscopy. D, Same micrograph as http://www.jimmunol.org/ in A; shows how each section was divided into white and red pulp for counting transferred cells. All spleen sections were also stained for CD4 and CD3 (data not shown) and transferred (CD45.1ϩCD4ϩCD3ϩB220Ϫ) cells were more accurately identiﬁed and counted in white and red pulp areas. Each confocal micrograph was taken with a ϫ10 objective (scale bar is 100 ␮m). Results are representative of ﬁve mice per strain.

tify whether there were specific differences between LT␣Ϫ/Ϫ and dKO white pulp areas, we evaluated the size of splenic by guest on September 27, 2021 white pulp areas (Fig. 1C). We found that the white pulp size of dKO (median ϭ 113000 ␮m2) was significantly reduced compared with LT␣Ϫ/Ϫ (median ϭ 175000 ␮m2) mice ( p Ͻ 0.0001) (Fig. 1C). The decreased white pulp areas observed in dKO mice were also ϩ ␮ 2 FIGURE 3. Expression of homeostatic chemokines in WT and gene de- correlated with decreased frequency of CD3 pixels/ m of total spleen (1.0 pixels/␮m2 dKO vs 1.22 pixels/␮m2 LT␣Ϫ/Ϫ; p ϭ ficient mice. Splenic mRNA expression of CXCL13 (A), CCL19 (B), CCL21 ϩ (C), relative to the house-keeping gene ␤-actin for dKO (E), LT␣Ϫ/Ϫ (F), 0.04) (Fig. 2B). When we calculated the percentage of CD3 pix- CD30Ϫ/Ϫ (Ⅺ), WT (᭜), and LT␤RϪ/Ϫ (Œ) mice; each symbol represents els in red and white pulp areas, we found that a higher percentage ϩ data from one mouse. A value of one means equal expression of target gene of CD3 pixels were located in the red pulp of dKO mice (Fig. and ␤-actin. 2A). However, this abnormal ratio was attributed solely to decreased CD3ϩ pixel densities in white pulp areas (1.7 pixels/␮m2 dKO vs 2.32 pixels/␮m2 LT␣Ϫ/Ϫ; p ϭ 0.0006) (Fig. 2B). Red pulp ϩ first compared the size of white pulp areas in WT, LT␣Ϫ/Ϫ, and CD3 frequency was not significantly different (0.64 pixels/␮m2 Ϫ Ϫ CD30Ϫ/Ϫ-deficient mice (Fig. 1A). WT mice as expected demon- dKO vs 0.69 pixels/␮m2 LT␣ / ; p ϭ 0.8). When the total white ϩ strate substantial white pulp areas with crisp segregation into B pulp was taken into account, the numbers of CD3 pixels/white Ϫ Ϫ and T cell areas. In contrast, CD30Ϫ/Ϫ mice present with impaired pulp was ϳ2-fold greater in LT␣ / vs dKO mice (median 4.1 ϫ B/T segregation and small T zones although B follicles and total 105 vs 2 ϫ 105 pixels/white pulp). However, the total numbers of ϩ white pulp areas are normal (14). In LT␣Ϫ/Ϫ mice, B follicles are CD3 pixels/red pulp was not significantly different ( p ϭ 0.6). In lost and the size of the white pulp areas is reduced. Although B/T contrast to CD3 cells, there were no significant differences in either ϩ segregation is disrupted, T cells are concentrated in the center of the total number of IgM pixels/␮m2 or in their distribution be- the white pulp areas whereas B cells are located peripherally. tween red and white pulp areas (data not shown). Furthermore, ϩ Spleens that lack both CD30 and LT␣ (dKO) have smaller white when we examined localization of CD11c DCs, there were no pulp areas (Fig. 1A), and there is little evidence that T cells are obvious differences in either total DC numbers, or their relative concentrated in the center of white pulp areas. distribution between red and white pulp areas (data not shown). To assess this defect quantitatively, we defined white pulp ␣ areas by staining for CD3 and B220, which demarcates the In the absence of LT , CD30 signals induce modest expression white pulp more clearly than IgM (Fig. 1B). No differences in of CCL21 white pulp areas were identified between WT and CD30Ϫ/Ϫ Our data suggested that there was a specific defect in localization mice using this method (data not shown and Ref. 14). To iden- of CD3ϩ T cells in white pulp areas in dKO vs LT␣Ϫ/Ϫ mice 4774 CD30 INDUCES CCL21 EXPRESSION IN THE ABSENCE OF LYMPHOTOXIN

ronment and CD4 T cell responses, we injected WT CD45.1ϩ OVA-specific OT2 CD4 cells (20) into CD45.2ϩ dKO, LT␣Ϫ/Ϫ, or WT hosts. All recipient mice were immunized with OVA and the numbers of OT2 cells in red and white pulp of the spleen were enumerated 5 days postimmunization. Transferred CD45.1ϩ CD4ϩ cells were easily detectable in the spleens of recipient immunized mice (Fig. 4). OT2 cells were identified by their coexpression of CD4, CD3, and CD45.1 in spleen sections. In WT mice, OT2 cells were clearly demarcated within the T zone areas, with few transgenic cells found outside this lo- cation (Fig. 4A). In LT␣Ϫ/Ϫ mice, OT2 cells were also concentrated in the T cell-rich central compartment of the white pulp areas (Fig. 4B). However, in dKO mice, OT2 cells were poorly localized to the small white pulp areas and could also be identified in the red pulp (Fig. 4C). The numbers of transferred OT2 cells in white and red pulp areas were quantified (an example of how the analysis was performed is shown in Fig. 4D). We found that the total number of

OT2 cells per section area (Fig. 5A), in both dKO (median ϭ 32 Downloaded from OT2 cells/section) and LT␣Ϫ/Ϫ hosts (median ϭ 41 OT2 cells/ section), was signiﬁcantly reduced compared with cells in WT hosts (median ϭ 81 OT2 cells/section) ( p Ͻ 0.0001). However, OT2 numbers in dKO recipient mice were also signiﬁcantly reduced compared with LT␣Ϫ/Ϫ mice ( p ϭ 0.04) (Fig. 5A). When

this was analyzed further, we found that numbers of OT2 cells/ http://www.jimmunol.org/ white pulp were significantly reduced in dKO mice (median ϭ 22 FIGURE 5. Distribution of recruited CD4 T cells in red and white pulp Ϫ Ϫ OT2 cells/white pulp) compared with LT␣ / (median ϭ 35 OT2 areas of WT and gene deficient mice. Spleen sections from immunized WT ϭ ϭ (᭜), LT␣Ϫ/Ϫ (F), and dKO (E) mice that received OT2 cells were analyzed cells/white pulp) ( p 0.002) and WT mice (median 75 OT2 Ͻ as described in Fig. 4. A, Numbers of OT2 cells in total (white ϩ red pulp), cells/white pulp) ( p 0.0001). In addition, a smaller percentage white, or red pulp of WT, LT␣Ϫ/Ϫ and dKO recipients. B, The percentage of of OT2 cells were contained in the white pulp of dKO compared Ϫ/Ϫ OT2 cells in white and red pulp of WT, LT␣Ϫ/Ϫ and dKO recipient mice. OT2 with either LT␣ or WT ( p Ͻ 0.0001) mice (Fig. 5B). cells were counted in white and red pulp and were expressed as a percentage relative to total (white ϩ red pulp) cells. Each symbol represents data from one Discussion micrograph. Results are representative of five mice per strain. Phylogeny suggests that the development of the capacity to make by guest on September 27, 2021 high affinity memory Ab responses was linked closely with the development of organized lymphoid structures (25), and we have reflected also in the decreased size of white pulp areas. Expression highlighted the central role of LTi in the development of both of homeostatic chemokines is central to B/T segregation. Although functions (15). LTi are found tightly associated with fixed B and T DCs produce CCL19, CCL21 is produced exclusively by stroma. zone stromal cells in secondary lymphoid organs (26, 27). They We have previously found no deficit in mRNA expression of any of constitutively express a diverse set of TNF ligands (TRANCE, Ϫ Ϫ these three chemokines in CD30 / mice (Fig. 3, A–C and Ref. TNF-␣ and lymphotoxins, ␣ and ␤, OX40, and CD30L). All of 14). In contrast, there is clear evidence that LT␣ drives mRNA these genes with the exception of OX40L have been linked with expression of all three chemokines (Fig. 3, A–C and Ref. 21). A the development and organization of secondary lymphoid structures Ϫ Ϫ comparison between dKO and LT␣ / mice revealed no differ- (14, 28, 29); in addition we have shown that OX40L and CD30L ence in expression of mRNA for CCL19 ( p ϭ 0.3) or CXCL13 together are critical for the generation and maintenance of CD4 ( p ϭ 0.6). In contrast, although median expression of CCL21 is memory (24, 26). Ϫ Ϫ 76-fold lower (ϳ1.3%) in LT␣ / vs WT mice, CCL21 levels are The crucial point about CD30 signals is that they alone are linked ϳ56-fold lower again in dKO vs WT mice, i.e., below the limits of with both functions: they contribute to organization in the presence of detection by quantitative PCR (Fig. 3C). This clearly shows that in lymphotoxin signals (14) but also to CD4 memory (23). Because a the absence of LT␣, CD30 signals induce modest expression comparison of mammalian genomes with those of lower vertebrates (ϳ1%) of CCL21, and that this correlates with the increased white suggested that CD30 signaling predated LT␤R signals, we speculated ϩ pulp areas and central localization of CD3 cells within these that a function for CD30 in organization might be revealed in LT␣Ϫ/Ϫ white pulp areas. Although LT␣ can interact with TNFR1, TNFR2, mice. To test this, we generated mice deficient in both CD30 and and HVEM, previous work has shown that its heterotrimer form, lymphotoxin (dKO), and compared their phenotype to LT␣Ϫ/Ϫ mice. LT␣1␤2, is the main switch for splenic CCL21 (12). LT␣1␤2 Our studies show that white pulp areas in dKO mice are significantly Ϫ Ϫ binds the LT␤R and accordingly we found that in LT␤R / mice smaller and less organized than in LT␣Ϫ/Ϫ mice and that this is cor- Ϫ Ϫ reduction of CCL21 matched that in LT␣ / mice (Fig. 3C). related with decreased expression of CCL21. Furthermore, we dem- onstrated a functional role for this decreased organization by showing Impaired CD4 T cell recruitment in mice double deficient for that recruitment and localization of WT CD4 T cells is impaired in ␣ ␣Ϫ/Ϫ CD30 and LT compared with LT mice dKO compared with LT␣Ϫ/Ϫ mice. Priming of CD4 T cells has been reported to be normal in LT␣Ϫ/Ϫ Although LT␤R and CD30 are both members of the TNF re- vs WT mice (22). In contrast, CD30 has a clear effect on CD4 T ceptor family, they are located on different chromosomes (in both cell survival independent of any effect on B/T segregation (23, 24). man and mouse), in different TNF receptor clusters. The TNF re- To distinguish between CD30 effects on forming the microenvi- ceptor cluster containing CD30 (TNFR2, CD30, 4–1BB, DR3, The Journal of Immunology 4775

HVEM, OX40, GITR) is principally linked with T cell memory 6. Kerjaschki, D., H. M. Regele, I. Moosberger, K. Nagy-Bojarski, B. Watschinger, (15, 30, 31) whereas the LT␤R cluster (TNFR1, LT␤R, and CD27) A. Soleiman, P. Birner, S. Krieger, A. Hovorka, G. Silberhumer, et al. 2004. Lymphatic neoangiogenesis in human kidney transplants is associated with im- is primarily associated with organization. The two gene clusters munologically active lymphocytic infiltrates. J. Am. Soc. Nephrol. 15: 603–612. are likely to be derived from tandem gene duplication (32), with 7. Farr, A. G., M. L. Berry, A. Kim, A. J. Nelson, M. P. Welch, and A. Aruffo. 1992. Characterization and cloning of a novel glycoprotein expressed by stromal cells subsequent separation and relocation to different chromosomes. in T-dependent areas of peripheral lymphoid tissues. J. Exp. Med. 176: 1477–1482. Evidence for this (besides the fact that both locations contain TNF 8. Forster, R., A. C. Davalos-Misslitz, and A. Rot. 2008. CCR7 and its ligands: receptor family members) is that both gene clusters encode struc- balancing immunity and tolerance. Nat. Rev. Immunol. 8: 362–371. 9. Woolf, E., I. Grigorova, A. Sagiv, V. Grabovsky, S. W. Feigelson, Z. Shulman, turally and functionally related serine proteases that catalytically T. Hartmann, M. Sixt, J. G. Cyster, and R. Alon. 2007. Lymph node chemokines activate complement component C4. On human chromosome 1, promote sustained T lymphocyte motility without triggering stable integrin ad- MASP2 (activates C4 via the mannan binding lectin pathway) is hesiveness in the absence of shear forces. Nat. Immunol. 8: 1076–1085. 10. Lammermann, T., B. L. Bader, S. J. Monkley, T. Worbs, R. Wedlich-Soldner, ϳ1MB away from human CD30. On human chromosome 12, the K. Hirsch, M. Keller, R. Forster, D. R. Critchley, R. Fassler, and M. Sixt. 2008. C1rs complex (activates C4 via the classical C1q pathway) is 0.7 Rapid leukocyte migration by integrin-independent flowing and squeezing. Na- MB from LT␤R. We think that the residual CD30-dependent in- ture 453: 51–55. 11. Bajenoff, M., J. G. Egen, L. Y. Koo, J. P. Laugier, F. Brau, N. Glaichenhaus, and duction of CCL21 expression in lymphotoxin deficient animals R. N. Germain. 2006. Stromal cell networks regulate lymphocyte entry, migra- could be the modern remainder of the CCL21 induction elicited by tion, and territoriality in lymph nodes. Immunity 25: 989–1001. 12. Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, M. D. Cooper, signals from the ancestral TNF receptor before its duplication. J. L. Browning, J. D. Sedgwick, and J. G. Cyster. 1999. Lymphotoxin ␣/␤ and tumor The main advantage conferred by gene duplication is the creation necrosis factor are required for stromal cell expression of homing chemokines in B of redundancy, which allows diversification of function among du- and T cell areas of the spleen. J. Exp. Med. 189: 403–412. 13. Ngo, V. N., R. J. Cornall, and J. G. Cyster. 2001. Splenic T zone development is plicated genes. Chromosomal translocation is equally important, as it B cell dependent. J. Exp. Med. 194: 1649–1660. Downloaded from creates opportunities for associations with unrelated genes to provide 14. Bekiaris, V., D. Withers, S. H. Glanville, F. M. McConnell, S. M. Parnell, new function. In this respect, the flanking genes of the LT␤R are M. Y. Kim, F. M. Gaspal, E. Jenkinson, C. Sweet, G. Anderson, and P. J. Lane. 2007. Role of CD30 in B/T segregation in the spleen. J. Immunol. 179: 7535–7543. instructive: CD4, which controls the development of CD4 T cells, is 15. Lane, P. J. L., M. C. Gaspal, and M.-Y. Kim. 2005. Two sides of a cellular coin: only 0.4mb from LT␤R, and the activation induced cytidine deami- CD4ϩCD3Ϫ cells orchestrate memory antibody responses and lymph node or- ganisation. Nat. Rev. Immunol. 5: 655–660. nase, required for somatic mutation and Ig class switching, 2.2 MB 16. Withers, D. R., M. Y. Kim, V. Bekiaris, S. W. Rossi, W. E. Jenkinson, F. Gaspal, away. Is this a chance association? We suspect not. Inactivation of any F. McConnell, J. H. Caamano, G. Anderson, and P. J. Lane. 2007. The role of http://www.jimmunol.org/ of these three genes results in failure to generate high affinity class lymphoid tissue inducer cells in splenic white pulp development. Eur. J. Immu- nol. 37: 3240–3245. switched Abs. Examination of the synteneic fish locus shows activa- 17. Kim, M.-Y., G. Anderson, I.-L. Martensson, L. Erlandsson, W. Arlt, A. White, tion induced cytidine deaminase and CD4 are linked with TNFR1 but and P. J. L. Lane. 2005. OX40-ligand and CD30-ligand are expressed on adult but ϩ Ϫ there is no true LT␤R homologue. This suggests to us that LT␤R- not neonatal CD4 CD3 inducer cells: evidence that IL7 signals regulate CD30- ligand but not OX40-ligand expression. J. Immunol. 174: 6686–6691. dependent organization (generation of B follicles) evolved in the con- 18. Kaiser, P., T. Y. Poh, L. Rothwell, S. Avery, S. Balu, U. S. Pathania, S. Hughes, text of genes also essential for the generation of high affinity Ab re- M. Goodchild, S. Morrell, M. Watson, et al. 2005. A genomic analysis of chicken sponses. We suspect that such linkage confers a minor advantage for cytokines and chemokines. J. Interferon Cytokine Res. 25: 467–484. 19. Bekiaris, V., O. Timoshenko, T. Z. Hou, K. Toellner, S. Shakib, F. Gaspal, the selection of new function, which when iterated over millions of F. M. McConnell, S. M. Parnell, D. Withers, C. D. Buckley, et al. 2008. Ly49Hϩ generations translates into a substantial advantage in performance. In NK cells migrate to and protect splenic white pulp stroma from murine cyto- by guest on September 27, 2021 megalovirus infection. J. Immunol. 180: 6768–6776. this regard, mammalian CD30 is also linked with the gene, podopla- 20. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR nin (1.8 MB distant), whose expression on splenic T zone stroma is expression in transgenic mice constructed using cDNA- based ␣- and ␤-chain CD30 dependent (14). genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76: 34–40. The data presented in this study therefore are consistent with the 21. Ansel, K. M., L. J. McHeyzer-Williams, V. N. Ngo, M. G. McHeyzer-Williams, proposal that CD30 and LT␤R are the descendants, by way of a gene and J. G. Cyster. 1999. In vivo-activated CD4 T cells upregulate CXC chemokine duplication event, of an ancestral TNF receptor which organized the receptor 5 and reprogram their response to lymphoid chemokines. J. Exp. Med. 190: 1123–1134. recruitment and survival of CD4 T cells through the induction of 22. Fu, Y. X., G. Huang, Y. Wang, and D. D. Chaplin. 2000. Lymphotoxin-␣-de- CCL21 expression. In this case, the coevolution of function for these pendent spleen microenvironment supports the generation of memory B cells and two related receptors would have fuelled the transition to the highly is required for their subsequent antigen-induced activation. J. Immunol. 164: 2508–2514. organized lymphotoxin and CD30 dependent B and T zone structures 23. Gaspal, F. M., M. Y. Kim, F. M. McConnell, C. Raykundalia, V. Bekiaris, and that today support the generation of high affinity CD4 dependent P. J. Lane. 2005. Mice deficient in OX40 and CD30 signals lack memory antibody responses because of deficient CD4 T cell memory. J. Immunol. 174: 3891–3896. memory Ab responses in mammalian immune systems. 24. Gaspal, F., V. Bekiaris, M. Kim, D. Withers, S. Bobat, I. MacLennan, G. Anderson, P. J. Lane, and A. F. Cunningham. 2008. Critical synergy of CD30 Disclosures and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella. The authors have no financial conflict of interest. J. Immunol. 180: 2824–2829. 25. Zapata, A., and C. T. Ameimiya. 2000. Phylogeny of lower vertebrates and their immunological structures. In Origin and Evolution of the Vertebrate Immune References System. G. W. Litman, ed. Springer, Berlin, pp. 67–110. 1. Cyster, J. G. 2003. Lymphoid organ development and cell migration. Immunol. 26. Kim, M. Y., F. M. Gaspal, H. E. Wiggett, F. M. McConnell, A. Gulbranson-Judge, ϩ Ϫ Rev. 195: 5–14. C. Raykundalia, L. S. Walker, M. D. Goodall, and P. J. Lane. 2003. CD4 CD3 2. Forster, R., A. E. Mattis, E. Kremmer, E. Wolf, G. Brem, and M. Lipp. 1996. A accessory cells costimulate primed CD4 T cells through OX40 and CD30 at sites putative chemokine receptor, blr1, directs B-cell migration to defined lymphoid where T cells collaborate with B cells. Immunity 18: 643–654. organs and specific anatomic compartments of the spleen. Cell 87: 1037–1047. 27. Kim, M. Y., F. M. McConnell, F. M. Gaspal, A. White, S. H. Glanville, 3. Luther, S. A., A. Bidgol, D. C. Hargreaves, A. Schmidt, Y. Xu, J. Paniyadi, V. Bekiaris, L. S. Walker, J. Caamano, E. Jenkinson, G. Anderson, and P. J. Lane. ϩ Ϫ M. Matloubian, and J. G. Cyster. 2002. Differing activities of homeostatic che- 2007. Function of CD4 CD3 cells in relation to B- and T-zone stroma in mokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruit- spleen. Blood 109: 1602–1610. ment and lymphoid neogenesis. J. Immunol. 169: 424–433. 28. Fu, Y. X., and D. D. Chaplin. 1999. Development and maturation of secondary 4. Hirose, J., H. Kawashima, M. Swope Willis, T. A. Springer, H. Hasegawa, lymphoid tissues. Annu. Rev. Immunol. 17: 399–433. O. Yoshie, and M. Miyasaka. 2002. Chondroitin sulfate B exerts its inhibitory 29. Mebius, R. E. 2003. Organogenesis of lymphoid tissues. Nat. Rev. Immunol. 3: effect on secondary lymphoid tissue chemokine (SLC) by binding to the C-ter- 292–303. minus of SLC. Biochim. Biophys. Acta 1571: 219–224. 30. Croft, M. 2003. Co-stimulatory members of the TNFR family: keys to effective 5. Ueno, T., K. Hara, M. S. Willis, M. A. Malin, U. E. Hopken, D. H. Gray, T-cell immunity? Nat. Rev. Immunol. 3: 609–620. K. Matsushima, M. Lipp, T. A. Springer, R. L. Boyd, et al. 2002. Role for CCR7 31. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cell re- ligands in the emigration of newly generated T lymphocytes from the neonatal sponses. Annu. Rev. Immunol. 23:23–68. thymus. Immunity 16: 205–218. 32. Ohno, S. 1970. Evolution by Gene Duplication. Springer-Verlag, New York.