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Elucidating the functional anatomy of secondary lymphoid organs

Oliver Pabst, Heike Herbrand, Gunter Bernhardt and Reinhold Forster


Functional anatomy offers an attempt to exploit anatomical information as a platform from which to decipher mechanistic details of complex or multistep immunological processes. Immune function depends on structural organization, therefore this approach contributes to a comprehensive understanding of the immune system. Major advances in functional anatomy require progress in both experimental techniques and analytical equipment largely synonymous to renement of the anatomists favorite tool, the microscope. Here, we describe how currently available techniques co-operate to gain new insights into the biology of secondary lymphoid organs.
Addresses Institute of Immunology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany e-mail: foerster.reinhold@mh-hannover.de

surface epitopes of immune-competent cells have substantially contributed to our current understanding of the immune system. However, when evidence began to accumulate that the initiation of an immune response requires successive physical interactions of different types of immune cells [1,2,3], the importance of secondary lymphoid organs as providers of a suitable matrix for these processes was apparent. Thus, the task of functional anatomy, with respect to secondary lymphoid organs, aims to elucidate the impact of the complex 3D structures of lymphoid tissues and the dynamic spatial arrangement of cells within, for the initiation, propagation and termination of immune reactions. One of the key lessons learned from functional anatomy is that the structure of lymphoid organs determines the function of the cells within and that, conversely, an impaired function of cells frequently affects the structure of the entire organ [47]. Technique rarely wins a prize, yet its capacities set both the pace and the limitations in our efforts to understand complex immunological processes. Here, we give a brief overview of recent techniques and illustrate the tight association of structural and functional aspects in pivotal immunological questions: development and maintenance of lymphoid organs that enable the immune system to perform its routine functions. Recent technical advances now enable us to address the functional anatomy of even large and complex immunological compartments, such as the gut associated lymphoid tissue (GALT). The technical impetus on new insights in structure and function of GALT will therefore constitute a particular focus of this review.

Current Opinion in Immunology 2004, 16:394399 This review comes from a themed issue on Immunological techniques Edited by Peter Friedl Available online 15th June 2004 0952-7915/$ see front matter 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2004.05.014

Abbreviations GALT gut associated lymphoid tissue IL interleukin LT lymphotoxin PP Peyers patches

Introduction
Classical anatomy, the art of dissecting or articially separating the different parts of any organized body to discover their situation, structure and economy (Websters 1913 Dictionary), established the principal structure and organization of lymphoid organs. However, classical anatomy largely failed in elucidating the function of their major cellular players, the lymphocytes. Lymphocytes are small cells that carry a slight rim of cytoplasm without obvious subcellular structures that can be associated with functional properties. Even today, lymphocytes are frequently described as round cell inltrates in routine histopathology. To explore the function of lymphocytes, scientists began to isolate these cells from lymphoid organs and developed a plethora of novel techniques and tools during the past four decades. In particular, molecular in vitro approaches and the availability of monoclonal antibodies that identify dened
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Visualization of lymphoid organ structure


Most of our knowledge regarding the functional anatomy of lymphoid organs has not been derived from recent technical breakthroughs, such as two photon (2P) microscopy ([8] and see the article by Ulrich von Andrian in this issue) but rather beneted from many technical improvements modest on their own, but powerful when combined which allowed novel, exciting insights into the function of immune mechanisms. New uorochromes have been developed that show reduced bleaching and that cover the entire emission spectrum from ultraviolet to far red. Covalently linked to antibodies, these dyes now allow multiple color analyses with more than four different antibodies, as a routine application. Notably, the use of labeled antibodies not only allows detection of cell surface molecules but also provides easy access to techniques to visualize proliferating or apoptotic cells.
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Table 1 The pros and cons of different microscopic approaches. Microscopy technique Classical histology, using dyes such Hematoxilin and Eosin, and light microscopy Strength Good assessment of cell morphology and gross overview on tissue integrity Inexpensive Weakness No differentiation between subsets of lymphocyte and dendritic cells No information regarding positioning of defined cell populations to each other Fixation (shrinking) artefacts of cell morphology preventing subcellular analysis of surface receptors Usually limited to two markers (antibodies) per section Number of fluorochromes limited by excitation lines of lasers Time consuming Currently no automated image assembly

Immunohistochemistry

Confocal laser-scanningmicroscopy; multiphoton microscopy

Epifluorescence and digital imaging using fast and sensitive CCD cameras

No needs for sophisticated equipment Allows combination of imunohistology and classical histology High resolution imaging Allows the assessment of cell-cell interactions, 3D reconstruction of thick sections (up to 100 mm) Multicolor analysis Optical sectioning and 3D reconstruction Live cell imaging High resolution at up to 40x objectives Number of fluorochromes not limited by source of excitation Fast and sensitive Allows multicolor analysis combined with automated image assembly to analyze large coherent tissues

Section thickness should be <10 mm Co-localization experiments require intensive calculation Postprocessing by deconvolution frequently required

Of comparable impact in the renement of immunouorescent techniques has been the development of highly sensitive CCD camera systems that permit the detection of extremely weak uorescence signals as well as computer-based algorithms that facilitate the analysis and visualization of this complex information. The pros and cons of the different microscopic approaches to visualizing lymphoid tissue architecture are summarized in Table 1. Together with technical advances, a growing number of genetically manipulated animal models have been developed, enabling the exploration of the link between molecular and cellular aspects of lymphoid organ function. Besides conventional mouse mutants carrying deletions in one or more gene loci, conditional mutants have been established that allow a cell type or organ-specic deletion of genes. An alternative effect, the aberrant expression of a distinct gene, can be achieved in classical transgenic animal models. Although genetic manipulations are potent, their true dimensions are rarely fully recognized because the analysis will usually be restricted to a certain subset of tissues or a dened area within the organ of interest. This experimental problem has been successfully addressed by Marc Jenkins group [9], who applied a technique that enables the visualization of the seeding of an immune response throughout the whole body of the mouse (reviewed in [10]). Although this study primarily used well-established techniques, the consequent application of a rapid epiuorescent detection system in combination
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with the automated computer-based image assembly and analysis provided new insights into T-cell immunity. The authors were able to show that antigen-experienced T cells, located in non-lymphoid tissues, might play an essential role in protective immunity.

Lymphocyte migration patterns determine the anatomy of lymphoid organs


The key function of lymphoid organs is to provide a exible matrix that permits the establishment of separate areas, each distinguished by a characteristic microenvironment (e.g. B- and T-cell zones, see Figure 1a; v), thus enabling the efcient sequential interaction of different subsets of lymphoid and non-lymphoid cells. Any disturbance of the resulting architecture should therefore provoke impaired immune reactions. However there are only sporadic reports in the literature that support this hypothesis, although numerous animal models with disturbed organ architecture have been described. An especially well documented example are mice carrying a targeted deletion for the chemokine receptor CXCR5 (Figure 1). Animals that are decient for CXCR5 are devoid of inguinal lymph nodes and most Peyers patches (PP) of the intestine, with the preserved PP displaying disturbed organ architecture (Figure 1b; iv and v). Fluorescent dye-labeled cells, isolated from CXCR5decient animals and adoptively transferred into wild type recipients, migrated into the T-cell area but failed to enter the B-cell follicle, suggesting that impaired B-cell migration inside the lymphoid organ accounts for the disturbed architecture [6]. In continuation, it was shown that overexpression of CXCR5 prevents antigen-induced
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396 Immunological techniques

Figure 1

(i) Genomic organization (a)

(ii) Putative structure and topographie of gene product

(iii) Cells expressing gene product T T

(iv) Gross morphology of organ of interest

(v) Immunohistology of affected organ

T SS IIIII IVII VIV VI


CXCR5

T B T B FDC fected B B

B Organ 1 mm 200

PPP -

(b) T T B
NEO

B FDC B

T B B T 1 mm 200
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From genes to functional anatomy of lymphoid organs. This scheme illustrates how a single gene the chemokine receptor CXCR5 affects the structure of PP. (a) Upper panel: Schematic drawing of the CXCR5 locus (i), CXCR5 is a seven transmembrane-spanning receptor (ii) that is primarily expressed on B cells and a subpopulation of T helper cells termed follicular helper T cells (TFH). The chemokine CXCL13 [blue dots, (iii)] is the sole ligand for CXCR5 and is expressed by follicular dendritic cells (FDC). Mediated via CXCR5 signaling, B cells and those T cells that express CXCR5 are recruited to the B cell follicle, giving rise to the characteristic large and protruding PP [indicated by white arrow, (iv)], possessing the distinctive B cell follicles and inter-follicular T cell areas. (b) Lower panel: CXCR5 deficiency [replaced by a NEO-selectin cassette, (i)] interferes with the segregation of B and T cells [see (v), and compare to the corresponding upper panel], resulting in small PP [indicated by white arrow, (iv)], which lack T and B cell-rich areas. B cells are stained with anti-B220-FITC (green) and T cells with anti-Thy1.2-Cy5 (red). Furthermore, CXCR5-deficient mice have also severely reduced numbers of PP (not shown; see main body of the text). Parts of this figure have been originally published in [6].

migration of B cells from the follicle to the B cellT cell boundary in spleen [11]. Thus, CXCR5 is not only implicated in directed lymphocyte migration into lymphoid organs but also in their overall organization, due to its involvement in intra-organ trafc of distinct lymphoid subpopulations [12]. Unexpectedly, when CXCR5-decient animals were challenged according to standard immunization protocols, for example, with high doses of antigen in complete Freunds adjuvant, no obvious difference in the humoral immune response could be observed between knockout and wild-type animals [6]. Similarly, immunization with a T cell-dependent antigen that was precipitated with alum led to the formation of ectopic but functional germinal centers in the T cell zone of CXCR5 mutants [13]. However, when the same animals are subjected to an immunization regimen that has been adjusted to more natural conditions (low amount of antigens, weak adjuvant) the elicited B cell immune response strongly depends on an intact microenvironment; that is, the correct positioning
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of both B cells and follicular T helper (Th) cells within the B cell follicle (our unpublished data). These observations illustrate the exquisite importance of adequate experimental conditions in the analysis of complex scenarios such as immune responses. High doses of antigen might conceal existing defects in immune responses. Therefore, it seems worth re-evaluating these mice displaying disturbed lymphoid organs architecture but exerting apparently normal immune responses due to inappropriate confrontation with antigen. Aside from the chemokine system and adhesion molecules, the tumor necrosis factor/lymphotoxin (TNF/LT) family of cytokines are some of the most intensively studied players in secondary lymphoid organ development and organization [1416]. The role of LT in the formation of secondary lymphoid organs is illustrated by a set of conditionally targeted lymphotoxin-b (LTb)decient mice. Constitutive inactivation of LTb causes the complete absence of lymph nodes and PP, as well as profound defects in splenic architecture, affecting the
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structure of the marginal zone, the separation of B and T cell areas and the follicular dendritic cell network [4,17]. Interestingly, a panel of lymphotoxin-decient animals was generated by cell-type-specic inactivation of LTb, showing a graded disruption of splenic architecture, ranging from mild defects in T-cell-specic LTb knockouts to marked defects in B-cell-specic knockout animals and even more pronounced abnormalities in T- and B-cell-specic LTbdecient animals [18,19]. Concomitantly, the severity of the defects in splenic architecture that were observed in these animals correlates with the degree of impaired humoral immune responses to T-cell-dependent antigens [19]. Notably, the chemokine and the LT system cooperate with each other in the establishment of lymphoid organ architecture. CXCL13, the ligand for CXCR5, has been shown to be secreted by follicular dendritic cells, inducing the up-regulation of LTa1b2 that promotes follicular dendritic cell development, thereby establishing a positive feed-back loop that is essential for follicular development and homeostasis [20].

of this receptor by lymphoid tissue inducing cells. Finke et al. [24] demonstrated that transfer of wild-type inducer cells into newborn CXCR5 mutants was sufcient to restore the formation of PP. Recently, it has been demonstrated that CCR7 is also involved in lymphoid organogenesis, suggesting that additional chemokine receptors, or combinations thereof, might contribute to these processes [25,26].

Lymphoid structures in the murine intestine


In contrast to PP, other constituents of GALT are less well characterized. Anatomists have been analyzing the intestine for decades, yet the number of lymphoid structures discovered is still growing. Apart from the wellknown PP, in the small intestine, the existence of mature and immature isolated lymphoid follicles, cryptopatches, lymphocyte-lled villi and submucosal lymphoid aggregates has been reported [27,28,29,30]. Most of these structures have been described only recently and, in some cases, it is still unknown in which species each of these structures exist and of what signicance they are. The small size of these structures, consisting of only a few hundred cells, and their distribution in a large organ, impose the most obvious problems in their structural and functional characterization; thus, a proper analysis of these structures cannot rely on accidental cuts through the tissue but should comprise a systematic scan of large coherent areas of tissue, thereby allowing the evaluation of statistically signicant numbers of individual structures while retaining their localization within the organ and with respect to each other. To visualize small intestinal isolated lymphoid follicles, Lorenz and colleagues [30] applied the technique of fetal intestine whole-mount staining to adult intestines that were opened along the duct and analyzed from the luminal surface using lowpower stereo microscopy. Using this approach, the authors were able to inspect large parts of the intestine without examining hundreds of histological sections. However, in contrast to immunohistology, this technique cannot resolve structures at the cellular level and is restricted to the use of only a small set of antibodies. An alternative approach makes use of rapid epiuorescence microscopy in combination with an automated assembly of large composite images. Although still inferior to confocal microscopy, regarding resolution, this technique allows the identication of single cells (Table 1). A case study is given in Figure 2. Vertical sections (Figure 2a) and tangential sections (Figure 2b) through the crypt zone of murine small intestines were stained for nuclei (DAPI, blue), B cells (anti-B220, green) and T cells (anti-CD3, red). Using an automated image capture and assembly system, a composite picture was created from more than 500 individual images. In addition to the prominent PP, several small lymphoid aggregations were visible and these were subsequently analyzed with respect to their localization, size and cellular composition (our unpublished data). Therefore, this technique is well
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Lymphoid organ architecture and development are intimately linked


Most notably, in many cases the molecules that inuence lymphoid organ architecture also affect their development. The current model for this process suggests that, at the cellular level, lymphoid organ development initiates with the interaction of a hematopoetic inducer cell (initially described by Mebius and co-workers [21]) with a mesenchymal organizer cell [22,23]. At the molecular level, this crosstalk is based on a positive feedback loop that, again, involves members of the LT family and chemokines and, at least in some cases, interleukin (IL)-7 signaling. Most lymphoid organs develop during gestation, therefore an appropriate technique had to be developed to allow tracing of the distinct steps of this process. Developmental biologists have successfully developed the technique of whole-mount staining to visualize the spatial distribution of mRNA and proteins within entire embryos. Although the information suffers from limited resolution, this disadvantage is far more than compensated for by the successful visualization of precise spatial relationships between different structures within complex tissues. Consequently, the method of wholemount staining of fetal tissue was adapted to the needs of immunologists and was thereby able to reveal distinct steps in lymphoid organogenesis, as initially demonstrated for PP. In contrast to lymph nodes, PP do not develop at a predictable position but are randomly scattered along the intestine. Thus, only the examination of entire fetal intestines enabled the identication of early PP anlagen, composed of only a few hundred cells. The decisive role of CXCR5 in PP development, as suggested by the phenotype of the mouse mutant (see Figure 1b and previous section), could be pinned down to the expression
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398 Immunological techniques

Figure 2

(a)

(b)

static view and death of the investigated organ(ism), serial and high resolution tissue evaluation by immunouorescence will remain a powerful approach to decipher even dynamic events such as development and plasticity of immune organs. Therefore, much emphasis should be placed on further development of technical options, as well as on the renement of the experimental framework, such as improved animal models or experimental regimens. Apart from the genotype-to-phenotype approach, natural or experimentally induced disease models are of equal interest in this respect because they enable the study of environmental implications that are mediated by bioactive molecules or microorganisms.

Acknowledgements
This work was supported, in part, by Deutsche Forschungsgemeinschaft Grants, SFB621-A1, and Fo 334/1-1 to RF.

References and recommended reading


Papers of particular interest, published within the annual period of review, have been highlighted as:

1 mm
Current Opinion in Immunology

 of special interest  of outstanding interest Mempel TR, Henrickson SE, Von Andrian UH: T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 2004, 427:154-159. Following on from the work in [2] (in which the authors demonstrate that interactions between T cells and dendritic cells are short-lived and propose that multiple sequential interactions result in the accumulation of the signal intensity that eventually triggers immune reactions), this study conrmed multiple and sequential interactions of T cells and dendritic cells, during the initial phase of T-cell priming. 2. Gunzer M, Schafer A, Borgmann S, Grabbe S, Zanker KS, Brocker EB, Kampgen E, Friedl P: Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity 2000, 13:323-332. Friedl P, Gunzer M: Interaction of T cells with APCs: the serial encounter model. Trends Immunol 2001, 22:187-191. Alimzhanov MB, Kuprash DV, Kosco-Vilbois MH, Luz A, Turetskaya RL, Tarakhovsky A, Rajewsky K, Nedospasov SA, Pfeffer K: Abnormal development of secondary lymphoid tissues in lymphotoxin beta-decient mice. Proc Natl Acad Sci USA 1997, 94:9302-9307. Mackay F, Majeau GR, Lawton P, Hochman PS, Browning JL: Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice. Eur J Immunol 1997, 27:2033-2042. Forster R, Mattis AE, Kremmer E, Wolf E, Brem G, Lipp M: A putative chemokine receptor, BLR1, directs B cell migration to dened lymphoid organs and specic anatomic compartments of the spleen. Cell 1996, 87:1037-1047. Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, Lipp M: CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 1999, 99:23-33. Denk W, Strickler JH, Webb WW: Two-photon laser scanning uorescence microscopy. Science 1990, 248:73-76. Reinhardt RL, Khoruts A, Merica R, Zell T, Jenkins MK: Visualizing the generation of memory CD4 T cells in the whole body. Nature 2001, 410:101-105. 1. 

Immunofluorescence microscopy of large coherent areas of the small intestine. The small intestine of C57BL/6 mice was opened along the mesenterial axis and either used for (a) vertical or (b) tangential sections. Sections were stained for nuclei (DAPI, blue), B cells (anti-B220, green) and T cells (anti-CD3, red) and composite images were assembled. Arrowheads indicate PP that contain large B cell follicles (green) separated by the interfollicular T cell zones (red). Arrows point to small-sized lymphoid aggregations. The inset in (b) illustrates that this technique allows the analysis of these lymphoid aggregations at cellular resolution. White scale bars are equivalent to 1 mm.

3. 4.

suited to systematically addressing the composition, and possibly the function, of structures that we tentatively name solitary intestinal lymphoid tissue (SILT). In adoption, this approach may be used to characterize diseased tissue, as seen in animal models mimicking colitis [31]. To date, evaluation of these models has employed comparably crude parameters, such as a clinical score, colon length and thickness. In the past, a more detailed analysis of animal models mimicking colitis or other diseases was hampered by the problem of monitoring the immune reaction within complex organs, a limitation that now can be overcome through the techniques described here.

5.

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7.

Outlook
A technique such as 2P microscopy allows in-depth insights, up to several 100 mm, into a living lymphoid organ, while maintaining its integrity, thereby offering the opportunity to directly record cell movements and cellcell communication. However, operability is restricted by natural barriers, set by the optics and the living body itself. Although unlimited access inevitably means a
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from adjacent zones determines B-cell position. Nature 2002, 416:94-99. This paper provides in vivo evidence that the balance of responsiveness to the chemoattractants CXCL13 vs CCL19/CCL21, which are made in separate but adjacent zones, allows B-cell relocalization in response to antigen. 12. Ohl L, Bernhardt G, Pabst O, Forster R: Chemokines as organizers of primary and secondary lymphoid organs. Semin Immunol 2003, 15:249-255. 13. Voigt I, Camacho SA, de Boer BA, Lipp M, Forster R, Berek C: CXCR5-decient mice develop functional germinal centers in the splenic T cell zone. Eur J Immunol 2000, 30:560-567. 14. Tumanov AV, Kuprash DV, Nedospasov SA: The role of lymphotoxin in development and maintenance of secondary lymphoid tissues. Cytokine Growth Factor Rev 2003, 14:275-288. 15. Pfeffer K: Biological functions of tumor necrosis factor cytokines and their receptors. Cytokine Growth Factor Rev 2003, 14:185-191. 16. Spahn TW, Kucharzik T: Modulating the intestinal immune system: the role of lymphotoxin and GALT organs. Gut 2004, 53:456-465. 17. Koni PA, Sacca R, Lawton P, Browning JL, Ruddle NH, Flavell RA: Distinct roles in lymphoid organogenesis for lymphotoxins alpha and beta revealed in lymphotoxin beta-decient mice. Immunity 1997, 6:491-500. 18. Tumanov A, Kuprash D, Lagarkova M, Grivennikov S, Abe K,  Shakhov A, Drutskaya L, Stewart C, Chervonsky A, Nedospasov S: Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues. Immunity 2002, 17:239-250. In contrast to Peyers patches and lymph nodes, splenic architecture depends on LTb expressed on B cells. Furthermore, the correct lymphoid architectureisshown tobecrucialforaneffective humoral immuneresponse. 19. Tumanov AV, Grivennikov SI, Shakhov AN, Rybtsov SA, Koroleva EP, Takeda J, Nedospasov SA, Kuprash DV: Dissecting the role of lymphotoxin in lymphoid organs by conditional targeting. Immunol Rev 2003, 195:106-116. 20. Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, Browning JL, Lipp M, Cyster JG: A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000, 406:309-314. 21. Mebius RE, Rennert P, Weissman IL: Developing lymph nodes collect CD4RCD3-LTbetaR cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells. Immunity 1997, 7:493-504. 22. Yoshida H, Honda K, Shinkura R, Adachi S, Nishikawa S, Maki K, Ikuta K, Nishikawa SI: IL-7 receptor alphaR CD3(S) cells in the

embryonic intestine induces the organizing center of Peyers patches. Int Immunol 1999, 11:643-655. 23. Mebius RE: Organogenesis of lymphoid tissue. Nat Rev Immunol 2003, 3:292-303. 24. Finke D, Acha-Orbea H, Mattis A, Lipp M, Kraehenbuhl J:  CD4RCD3S cells induce Peyers patch development: role of alpha4beta1 integrin activation by CXCR5. Immunity 2002, 17:363-373. In this paper, evidence is presented that the organogenesis of Peyers patches can be restored in CXCR5-decient mice by injection of wildtype lymphoid tissue inducing cells; furthermore, CXCR5/CXCL13 is shown to activate a4b1 integrin, demonstrating the link between chemokine signaling and adhesion molecules that regulate the interaction of organizer and inducer cells during organ development. 25. Ohl L, Henning G, Krautwald S, Lipp M, Hardtke S, Bernhardt G, Pabst O, Forster R: Cooperating mechanisms of CXCR5 and CCR7 in development and organization of secondary lymphoid organs. J Exp Med 2003, 197:1199-1204. 26. Luther SA, Ansel KM, Cyster JG: Overlapping roles of CXCL13, interleukin 7 receptor alpha, and CCR7 ligands in lymph node development. J Exp Med 2003, 197:1191-1198. 27. Moghaddami M, Cummins A, Mayrhofer G: Lymphocyte-lled villi: comparison with other lymphoid aggregations in the mucosa of the human small intestine. Gastroenterology 1998, 115:1414-1425. 28. Hamada H, Hiroi T, Nishiyama Y, Takahashi H, Masunaga Y,  Hachimura S, Kaminogawa S, Takahashi-Iwanaga H, Iwanaga T, Kiyono H et al.: Identication of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 2002, 168:57-64. The authors describe the existence of isolated lymphoid follicles (ILF) along the anti-mesenteric wall in the murine small intestine. ILF are characterized by immunohistology and ow cytometry in wild type and various mutant mice. 29. Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, Ishikawa H: Identication of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kitR IL-7RR Thy1R lympho-hemopoietic progenitors develop. J Exp Med 1996, 184:1449-1459. 30. Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD: Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufcient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol 2003, 170:5475-5482. 31. Pizarro TT, Arseneau KO, Bamias G, Cominelli F: Mouse models for the study of Crohns disease. Trends Mol Med 2003, 9:218-222.

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Current Opinion in Immunology 2004, 16:394399