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autoimmune or autoaggressive disease. Trapani, J.A. (2009). Immunity 30, this issue, Podack, E.R., and Tschopp, J. (1982). Proc. Natl. The new climax of research on Perforin, 684–695. Acad. Sci. USA 79, 574–578. as reported here, may soon be followed Blumenthal, R., Millard, P.J., Henkart, M.P., Rey- Slade, D.J., Lovelace, L.L., Chruszcz, M., Minor, by the development of novel therapeutics nolds, C.W., and Henkart, P.A. (1984). Proc. Natl. W., Lebioda, L., and Sodetz, J.M. (2008). J. Mol. Acad. Sci. USA 81, 5551–5555. Biol. 379, 331–342. targeted to enhance or diminish Perforin cytotoxicity, to enhance our fitness and Hadders, M.A., Beringer, D.X., and Gros, P. (2007). Spielman, J., Lee, R.K., and Podack, E.R. (1998). Science 317, 1552–1554. J. Immunol. 161, 7063–7070. survival. Kagi, D., Ledermann, B., Burki, K., Seiler, P., Oder- Stepp, S.E., Dufourcq-Lagelouse, R., Le Deist, F., REFERENCES matt, B., Olsen, K.J., Podack, E.R., Zinkernagel, Bhawan, S., Certain, S., Mathew, P.A., Henter, R.M., and Hengartner, H. (1994). Nature 369, J.I., Bennett, M., Fischer, A., de Saint Basile, G., 31–37. and Kumar, V. (1999). Science 286, 1957–1959. Baran, K., Dunstone, M., Jenny, D., Ciccone, A., Brownie, K.A., Clarke, C.J.P., Lukoyanova, N., Podack, E.R., and Dennert, G. (1983). Nature 302, Young, J.D., Cohn, Z.A., and Podack, E.R. (1986). Saibil, H., Whisstock, J.C., Voskoboinik, I., and 442–445. Science 233, 184–190.

Lymphoid Organs for Peritoneal Cavity Immune Response: Milky Spots

Reina E. Mebius1,* 1Department of Molecular Cell Biology and Immunology, VUMC, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands *Correspondence: [email protected] DOI 10.1016/j.immuni.2009.04.005

Milky spots are located in the omentum of the peritoneal cavity and their classification as lymphoid organs has been debated. In this issue of Immunity, Rangel-Moreno et al. (2009) provide compelling data to consider them as unique secondary lymphoid organs.

The omentum is formed by a double layer et al., 2002). As a consequence, Cxcl13À/À marrow. Antigens injected into the perito- of mesothelial cells that connects the mice, as well as mice deficient for the neal cavity of SLP mice were shown to stomach, pancreas, , and colon (Wil- CXCL13 receptor, CXCR5, have strongly collect in the milky spots, resulting in the liams and White, 1986). Embedded within reduced titers of natural antibodies (Ansel generation of antigen-specific antibodies. the omentum are opaque structures, which et al., 2002; Hopken et al., 2004). Therefore, Furthermore, res- are clusters of leukocytes, called milky milky spots are an important source of ponses, supporting isotype switching, spots. Milky spots are mainly composed natural antibodies. somatic hypermutation, and some affinity of and B1 cells, resembling Because milky spots mainly consist maturation, as well as proliferation of T cells the cellular composition found in the perito- of macrophages and B1 cells, and are in response to intraperitoneally injected neal cavity. B1 cells form a unique subset of described to lack dendritic cells as well as antigens, could be observed in the milky B cells that can be distinguished from follicular dendritic cells, controversy exists spots. Not only are these milky spots occu- conventional B (B2) cells by expression of as to whether to mark these milky spots as pied by locally activated , but distinct cell-surface markers and antigen secondary lymphoid organs. In this issue of also lymphocytes that encounter antigens receptors that can bind common bacterial Immunity, Rangel-Moreno et al. (2009) elsewhere were shown to recirculate epitopes, as well as by their recognized addressed the immunological potential of through the milky spots. These experi- potential to produce natural antibodies milky spots in the absence of lymph nodes, ments clearly identified the milky spots that provide a first protection to bacterial spleen, and Peyer’s patches. Here, they as part of the general surveillance route infections. B1 cells are localized in distinct used splenectomized lymphotoxin-alpha of antigen-experienced lymphocytes in anatomical locations, such as the perito- (LTa)-deficient mice (LtaÀ/À), which as search for their antigen. neal and pleural cavities, but are also a result of their deficiency already lacked What does it take to enter and leave the present in the spleen. For their localization lymph nodes and Peyer’s patches. These milky spots? Milky spots contain high to these body cavities, but not the spleen, animals, devoid of secondary lymphoid endothelial venules (HEVs) that express the chemokine CXCL13 is of great impor- organs, were reconstituted with wild-type the peripheral addressin tance because in Cxcl13À/À mice, B1 cells (SLP mice) and compared (PNAd) as well as the mucosal addressin as well as milky spots are absent from the to irradiated C57BL/6 mice that were simi- (MAdCAM-1), permitting entry of lympho- pleural and peritoneal cavities (Ansel larly reconstituted with wild-type bone cytes from the bloodstream into the milky

670 Immunity 30, May 22, 2009 ª2009 Elsevier Inc. Immunity Previews

peritoneal cavity and back to the milky (Browning et al., 2005). It is tempting to spots. speculate that HEVs are in fact essential The overall architecture of the milky for the maintenance of milky spots. spots is unique because the B cell clusters Absolutely required for milky spots do not appear to contain centrally posi- formation is the chemokine CXCL13, and tioned networks of follicular dendritic cells in analogy with the development of lymph (FDCs). FDCs in conventional lymphoid nodes and Peyer’s patches, the cells that organs are located in the center of B cell make CXCL13 could be denoted as ‘‘orga- follicles and express CD35 and CD21, as nizer cells.’’ Rangel-Moreno et al. (2009) well as FDCM1 and FDCM2, while they show that CXCL13 expression colocalizes produce CXCL13. In milky spots, how- in CD11b+ cells,presumably macrophages, ever, only some expression of CD35 could and stromal cells in an atypical cup-like be detected in the center of the B cell structure around the B cell areas (Figure 1). clusters, whereas expression of CXCL13 In line with this is the reported expression of was very uncharacteristically located on CXCL13 by peritoneal macrophages as the outside of these clusters. Expression well as radio-resistant cells in the omentum of CXCL13 colocalized with FDCM1+ and (Ansel et al., 2002). Therefore, these CD11b+ cells. The lack of conventional CXCL13-producing stromal cells and FDCnetworks may accountforthe reduced macrophages can be considered as the ability to support affinity maturation in mice milky-spot-organizing cells. The macro- that only contain milky spots (Rangel-Mor- phages in the omentum certainly fulfill the eno et al., 2009). requirement of organizer cells because in Figure 1. Schematic Overview of Milky Spot Not only are milky spots different from parabiosed mice (mice with joined blood Function, Architecture, and Formation lymph nodes and Peyer’s patches in their circulation), only 10% of these cells were Milky spots are formed by clustered B cells, with , CD11c+ cells, and some cellular composition and morphology, but shown to be replaced by cells from the T cells within the B cell areas. CXCL13-producing also their formation seems to follow distinct partner over a period of 8 weeks (Ansel + + cells, which colocalize with CD11b and FDCM1 cues. In man, the formation of milky spots et al., 2002) and are thus sessile cells that cells, are positioned in a cup-like structure around the B cell clusters. In these milky spots, B and starts at 20 weeks of gestation, when clus- could organize a structure. activation can take place, and germinal centers, ters of monocytes and macrophages can The inducing signal for CXCL13 expres- supporting isotype switching, somatic hypermuta- be observed, and true milky spots are sion is, unlike in LN and PP development, tion, and some affinity maturation, can also be apparent at week 35. This development is not delivered by LTi cells, B cells, LTa, found. clearly much later than that of human lymph LTb,orTNFa (Ansel et al., 2002; Rangel- nodes, which can be dissected out starting Moreno et al., 2009). One can envision that spots (Berberich et al., 2008; Rangel-Mor- at week 13 of gestation (Cupedo et al., activation of macrophages and perhaps eno et al., 2009). In fact, milky spots were 2009). For the formation of lymph nodes even stromal cells, i.e., by bacterial proposed to be the port of entry for B1 and Peyer’s patches, it is now well estab- encounter or particulate antigens, may cells that migrate from the bloodstream lished that lymphoid tissue inducer cells lead to the induction of CXCL13 within the toward the peritoneal cavity (Ansel et al., (LTi cells), expressing LTab,trigger milky spots. Consistent with this model is 2002). For the entry of B2 cells into the lymphoid tissue stromal organizer cells the observation that germ-free BALB/c milky spots, only a4b7-MAdCAM-1 inter- that express the lymphotoxin-b receptor mice have strikingly reduced titers of natural action was shown to be essential, (LTbR). This interaction, which leads to the antibodies to phosphorylcholine, a cell- whereas L-selectin was not needed (Ber- production of chemokines, expression of surface component of several bacteria berich et al., 2008). However, the entry adhesion molecules, and synthesis of cyto- (Gearhart et al., 1975). This is similar to from the bloodstream into the milky spots kines, is required for the formation of LNs Cxcl13À/À mice, which lack milky spots. must have additional requirements, given and PPs (Vondenhoff et al., 2009). For milky Perhaps also CXCL13 expression in the that the cellular composition of the milky spot formation, Rangel-Moreno et al. omentum is strongly reduced in germ-free spots is very different from lymph nodes (2009) show that LTi cells are dispensable mice, consequently leading to reduction or and Peyer’s patches, which express the and thus that milky spots follow a distinct absence of milky spots. Indeed, germ-free same addressins on HEVs. In addition, developmental program. Although milky rats were reported to have strongly reduced Rangel-Moreno et al. (2009) showed that spots are strongly reduced in absence of numbers of milky spots (Beelen et al., 1980). a flow from the peritoneal cavity to the LTa,LTa is not crucial to induce essential The localization of the CD11b+ cells in the milky spots allows entry of antigens as chemokines because CXCL13 was nor- omentum seems to be independent of well as lymphocytes into the milky spots. mally expressed in the omentum of LtaÀ/À CXCL13 because these cells were present For lymphocytes, this process requires mice. The function of LTa is more likely to in distinct areas of the omentum in receptors that are sensitive to pertussis be related to regulating the function of Cxcl13À/À mice and it is therefore likely toxin treatment. Altogether, it seems that HEVs, which are absent in milky spots of that additional signals that determine the cells enter the milky spots from the blood- LtaÀ/À mice. The importance of LTbR position of CD11b+ cells are involved in stream, whereas they may thereafter signaling for HEV function and mainte- milky spot formation. It remains to be seen move freely from the milky spots to the nance has been reported for lymph nodes whether their positioning is determined

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during a certain time window in develop- system, such as Treg cells, Th17 cells, Cupedo, T., Crellin, N.K., Papazian, N., Rombouts, ment. Future studies will also have to and IgA-producing B cells, are also found, E.J., Weijer, K., Grogan, J.L., Fibbe, W.E., Corne- lissen, J.J., and Spits, H. (2009). Nat. Immunol. resolve whether indeed bacterial compo- and perhaps generated, in milky spots. 10, 66–74. nents and perhaps other stimuli evoke the Future studies are needed to address these expression of CXCL13 in the peritoneal questions. Gearhart, P.J., Sigal, N.H., and Klinman, N.R. cavity. (1975). J. Exp. Med. 141, 56–71. Now that we know that activation of T Hopken, U.E., Achtman, A.H., Kruger, K., and Lipp, lymphocytes can take place in the milky REFERENCES M. (2004). J. Leukoc. Biol. 76, 709–718. spots it remains to be seen whether these cells will subsequently leave the milky Ansel, K.M., Harris, R.B., and Cyster, J.G. (2002). Rangel-Moreno, J., Moyron-Quiroz, J.E., Carra- spots and if so, to which part of the body Immunity 16, 67–76. gher, D.M., Kusser, K., Hartson, L., Moquin, A., and Randall, T.D. (2009). Immunity 30, this issue, they preferentially migrate. Analysis of Beelen, R.H., Fluitsma, D.M., and Hoefsmit, E.C. 731–743. homing receptors expressed on lympho- (1980). J. Reticuloendothel. Soc. 28, 585–599. cytes activated in milky spots should give Berberich, S., Dahne, S., Schippers, A., Peters, T., Vondenhoff, M.F., Greuter, M., Goverse, G., Ele- a first indication. Furthermore, the associa- Muller, W., Kremmer, E., Forster, R., and Pabst, O. waut, D., Dewint, P., Ware, C.F., Hoorweg, K., tion of the omentum with the gastrointes- (2008). J. Immunol. 180, 2196–2203. Kraal, G., and Mebius, R.E. (2009). J. Immunol. 182, 5439–5445. tinal tract makes one wonder whether Browning, J.L., Allaire, N., Ngam-Ek, A., Notidis, subsets that are characteristi- E., Hunt, J., Perrin, S., and Fava, R.A. (2005). Williams, R., and White, H. (1986). Curr. Probl. cally associated with the mucosal immune Immunity 23, 539–550. Surg. 23, 789–865.

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