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The Journal of Immunology, 2009, 182, 2610 -2619
Copyright © 2009 by The American Association of Immunologists, Inc.
doi:10.4049/jimmunol.0801141
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Chemokine Receptor CXCR5 Supports Solitary Intestinal Lymphoid Tissue Formation, B Cell Homing, and Induction of Intestinal IgA Responses

Sarvari Velaga*, Heike Herbrand*, Michaela Friedrichsen*, Tian Jiong{dagger}, Martina Dorsch{ddagger}, Matthias W. Hoffmann{dagger}, Reinhold Förster* and Oliver Pabst1,*

* Institute of Immunology, {dagger} Department of Visceral and Transplantation Surgery, and {ddagger} Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Solitary intestinal lymphoid tissue (SILT) comprises a spectrum of phenotypically diverse lymphoid aggregates interspersed throughout the small intestinal mucosa. Manifestations of SILT range from tiny lymphoid aggregates almost void of mature lymphocytes to large structures dominated by B cells. Large SILT phenotypically resemble a single Peyer’s patch follicle, suggesting that SILT might contribute to intestinal humoral immune responses. In this study, we track the fate of individual SILT in vivo over time and analyze SILT formation and function in chemokine receptor CXCR5-deficient mice. We show that, in analogy to Peyer’s patches, formation of SILT is invariantly determined during ontogeny and depends on CXCR5. Young CXCR5-deficient mice completely lack SILT, suggesting that CXCR5 is essential for SILT formation during regular postnatal development. However, microbiota and other external stimuli can induce the formation of aberrant SILT distinguished by impaired development of B cell follicles in CXCR5-deficient mice. Small intestinal transplantation and bone marrow transplantation reveal that defect follicle formation is due to impaired B cell homing. Moreover, oral immunization with cholera toxin or infection with noninvasive Salmonella fail to induce efficient humoral immune responses in CXCR5-deficient mice. Bone marrow transplantation of CXCR5-deficient recipients with wild-type bone marrow rescued B cell follicle formation in SILT but failed to restore full humoral immune responses. These results reveal an essential role of CXCR5 in Peyer’s patch and SILT development and function and indicate that SILT do not fully compensate for the lack of Peyer’s patches in T cell-dependent humoral immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the small intestines of mice and humans, Peyer’s patches (PP)2 provide an anatomical platform supporting the induction of immune responses. PP are uniquely adapted to the particular requirements in the intestine by combining Ag uptake mechanisms via M cells and a microenvironment facilitating the interaction of APCs and mature lymphocytes in one organ (1). Consistently, PP are regarded to chiefly contribute to the induction of intestinal IgA responses. Besides PP, numerous lymphoid structures that have been assigned different names and functions are present in the gut. Small-sized lymphoid aggregates located in the intestinal crypts have been named cryptopatches (CP) (2). CP contain almost exclusively cells that have not yet been allocated to any hematopoietic cell lineage and express the stem cell factor receptor c-Kit. These cells have been thought to represent T cell precursors and CP were thus regarded as sites of extrathymic T cell development (2, 3). Phenotypically different lymphoid aggregates are the isolated lymphoid follicles (ILF) (4). ILF contain a dome region harboring dendritic cells, a prominent B cell follicle, and sporadically germinal centers, thereby resembling a single dome of a PP. Moreover, the epithelium of ILF is equipped with M cells that allow the uptake of material from the intestinal lumen into the ILF (for recent reviews, see Refs. 5 and 6).

We have recently provided evidence that CP and ILF do not represent separate types of lymphoid structures, but form a dynamic continuum of interconvertible structures, with CP representing precursors of ILF (7). Consistently, numerous lymphoid aggregates in the intestine combine phenotypical traits of both, CP and ILF, indicative for transitional structures in the CP/ILF continuum (7, 8). We have thus suggested to refer to these interconvertible structures as solitary intestinal lymphoid tissue (SILT). The spectrum of SILT observed in germfree mice is dominated by CP-like structures, whereas colonization of germfree mice induces a shift in the spectrum of SILT to include ILF (9). Transition of CP into ILF is induced by the nucleotide-binding oligomerization domain-containing innate receptor (NOD1) expressed in epithelial cells when recognizing Gram-negative bacteria (10). Importantly, bacterial colonization does not affect the overall number of SILT, indicating that intestinal microbiota stimulate the progression of CP to ILF but no de novo formation of SILT (9).

The development of PP has been intensively studied and much of the current knowledge on lymphoid organogenesis can be compiled in a model that initially has been put forward by Nishikawa et al. (11). In essence, the interaction of hematopoietic lymphoid tissue inducer cells (LTIC) with mesenchymal organizer cells is thought to drive the progressive production of chemokines, which in turn mediate the recruitment of cells into the forming organ anlage (12). Heterotrimeric lymphotoxin {alpha}1β2 expressed by LTIC interacts with the lymphotoxin-β receptor present on the organizer cells. This signal triggers the organizer cells to secrete the chemokine CXCL13 that signals back to the LTIC via the cognate receptor CXCR5, thereby augmenting the expression of lymphotoxin (for recent reviews, see Refs. 13 and 14). Interference with this feedback loop abolishes PP development as evidenced by lack of PP in mice deficient for lymphotoxin, lymphotoxin-β receptor, CXCL13, or CXCR5. In addition to LTIC, CXCR5 is expressed by B cells and regulates B cell homing and follicle formation in lymphoid organs (15, 16). In particular, B cell homing into PP depends on CXCR5, since in PP a unique type of high endothelial venules localized in the B cell follicles displays luminal CXCL13 and facilitates the direct entry of B cells (17).

The development of SILT shares important features with PP development. Like PP, SILT are absent in the intestines of lymphotoxin-{alpha}, lymphotoxin-β and lymphotoxin-β receptor-deficient mice (4, 18). Moreover, SILT, as well as PP, fail to form in mice lacking the orphan nuclear receptor ROR{gamma}t, which is required for the development and maintenance of LTIC (19).

The chemokine receptor CCR6 is expressed on B cells in ILF. Transition of CP to ILF, which is mainly characterized by the influx of B lymphocytes, is triggered through expression of CCR6 ligands by epithelial cells (10) and blocked in CCR6-deficient mice. In contrast, the overall number of CP and accumulation of dendritic cells are unaffected in these mutants (20). Lymphocyte influx into PP is also reduced in CCR6-deficient mice, resulting in smaller PP (21). Another chemokine receptor strongly influencing the phenotype of SILT is CCR7. Absence of CCR7 signaling results in heavily enlarged SILT that are, however, unchanged in frequency and cellular composition when compared with wild-type mice. This phenotype is conferred by bone marrow-derived cells and is independent of the presence of intestinal bacteria, suggesting that hypertrophy of SILT in CCR7-deficient mice does not depend on external stimulation (9).

In this study, we show that just like PP formation, regular initiation of SILT development depends on CXCR5. However, pathways independent of CXCR5 can be triggered in CXCR5-deficient mice through microbiota and other stimuli resulting in the formation of SILT lacking B cell follicles. Impaired follicle formation results from defect homing of CXCR5-deficient B cells. SILT in CXCR5-deficient mice fail to support robust IgA responses upon Salmonella infection or oral immunization with cholera toxin. Bone marrow reconstitution of CXCR5-deficient recipients with wild-type bone marrow cells rescues B cell deficiency in SILT but fails to restore the induction of IgA, indicating that SILT do not fully compensate for the lack of PP in CXCR5-deficient mice in T cell-dependent humoral immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and bone marrow chimera

BALB/c, C57BL/6, C57BL/6-Ptprca (designated here as Ly5.1 mice), C57BL/6-Tg(ACTbEGFP)1Osb/J (designated here EGFP mice), B6.Cg-IghaThy1aGpi1a/J (designated here as Igha mice), B6.Igh-6tm1cgn (designated here as µMT-H2B), and B6.Cg-Blr1tm1Lipp/J backcrossed for at least 15 generations to C57BL/6 mice (designated here as CXCR5-deficient mice) were bred at the central animal facility of the Hannover Medical School under specific pathogen-free (SPF) conditions. C57BL/6 mice were additionally purchased from Charles River. CXCR5-deficient germfree mice were generated and bred at the central animal facility at the Hannover Medical School and analyzed at the age of 8, 10, or 20 wk. For generation of bone marrow chimeras, recipients were lethally irradiated with a single dose of 10 Gy and transplanted with 107 congenic bone marrow cells purified by discontinuous Lympholite-M gradient centrifugation. Chimeras were analyzed 8 wk after transplantation. Chimerism was above 90% for all chimeras used in the immunization experiments. All animal experiments have been performed in accordance with institutional guidelines and have been approved by the local committees.

Oral immunizations and Salmonella infections

For all infection experiments, an attenuated, noninvasive streptomycin-resistant derivative of the Salmonella enterica serovar typhimurium strain SL1344, which is referred to as SL1344 aroA ssrB (22, 23), was used. Bacteria were grown in Luria-Bertani broth until an OD of 1.35 was reached, washed twice with PBS, and resuspended at a density of 108 bacteria/100 µl of PBS. Mice continuously received streptomycin (5 mg/ml) via the drinking water starting 1 day before infection and were inoculated orally with 100 µl of bacterial suspension with a feeding needle. The number of inoculated bacteria was confirmed by serial plating of appropriate dilutions onto Luria-Bertani agar plates and overnight incubation. Intestinal colonization with Salmonella was confirmed by weekly plating of feces. Ig levels in the serum and intestinal washes were determined 4 wk after infection. For oral immunization, mice received 20 µg of cholera toxin (Sigma-Aldrich) in 100 µl of PBS by gavage and were analyzed 10 days after immunization. Intestinal washes were collected by flushing 10 cm of the small intestine with 400 µl of PBS containing 0.1 mg/ml trypsin inhibitor, 50 mM EDTA, and 0.1% BSA.

ELISA

Ninety-six-well Nunc microprep plates were coated with 1 µg/ml cholera toxin or Salmonella LPS (Sigma-Aldrich) and incubated at 4°C overnight. Plates were washed once with PBS/0.05% Tween 20 and blocked with PBS/2% BSA for 1 h at 37°C. Samples were diluted in PBS/0.5% BSA/0.1% Tween 20 and incubated for 1 h at 37°C. Plates were thoroughly washed with PBS/0.05% Tween 20 and incubated with alkaline phosphatase-conjugated Abs appropriately diluted in PBS/0.5% BSA/0.1% Tween 20 for 1 h at 37°C. Plates were washed with PBS/0.05% Tween 20 before adding 3,3',5,5'-tetramethylbenzidine dihydrochloride (Sigma-Aldrich). Absorbances were measured at 450 nm.

Intestinal surgery

Mouse vascularized small bowel transplantation was performed as previously described (24), with some modifications: C57BL/6 Ly5.1 mice were used as donors and congenic CXCR5-deficient Ly5.2 mice as recipients. Briefly, under the combined anesthesia of ketamine and rompun, the donor jejunum and proximal part of the ileum was isolated with attached superior mesenteric artery and portal vein. After luminal irrigation and vascular perfusion, the graft was stored at 4°C in Ringer’s solution until implantation. The graft portal vein and superior mesenteric artery were anastomosed to the recipient’s inferior vena cava and abdominal aorta, respectively, in an end-to-side fashion. The proximal end of the graft was exteriorized as a stoma. The graft secretion was drained into host alimentary tract by an end-to-side anastomosis between graft ileal end and host jejunum. Mice were analyzed 8 wk after transplantation. To track the position of SILT over time, C57BL/6 mice were lethally irradiated and reconstituted with GFP-expressing bone marrow cells (see before). Eight weeks after reconstitution, the small intestine was exposed under anesthesia by laparotomy and the pattern of SILT was documented using a fluorescent stereo microscope (MZ16FA; Leica). Subsequently, the intestine was reintroduced and the abdomen closed in a single-layer discontinuous suture. Three weeks later, the mice were sacrificed, intestinal fragments were aligned based on the position of individual PP, and the location of SILT was reevaluated.

Antibodies

The following Abs and conjugates were used in this study: goat anti-mouse IgA-peroxidase (Zymed Laboratories), goat anti-mouse IgM-peroxidase (Nordic Immunology), rat anti-mouse IgG-peroxidase (Jackson ImmunoResearch Laboratories), anti-CD4 (clone RM4-5), anti CD19-allophycocyanin-Cy7 (clone 1D3), anti-CD21/35-FITC (clone 7G6), anti-CD45.2-PerCP-Cy5 (clone 104), anti-TER-119-biotin, anti-CD11c-biotin (clone HL3), anti-CD117-PE (clone 2B8), anti-Ly5.2-FITC (all BD Pharmingen), anti-CD19-PE (Southern Biotechnology Associates), anti-CD11b-biotin (clone M1/70.15; Caltag Laboratories), anti-CD117-allophycocyanin (clone ACK2; eBioscience), anti-Ly5.1-biotin (clone A20; Chemicon International), anti-IgD, anti-GR-1, anti-CD4, anti-B220, anti-CD3, and anti-CXCR5 were provided by E. Kremmer (GSF National Research Center for Environment and Health, Munich, Germany). Anti-B220, anti-CD3, and anti-CD4 were coupled to Cy3 (Jackson ImmunoResearch Laboratories), FITC (Sigma-Aldrich), Cy5 (GE Healthcare), or Pacific Orange (Invitrogen) according to the manufacturers’ instructions. Anti-CXCR5 was detected using mouse anti-rat-Cy5 (Jackson ImmunoResearch Laboratories). Anti-human EBNA1 mAb (E1-BS 1H4) was used as isotype control. Biotinylated Abs were recognized by streptavidin coupled to PerCP (BD Biosciences) or to Cy3 (Jackson ImmunoResearch Laboratories) or Alexa Fluor 488 (Invitrogen).

Cell preparation and flow cytometry

Adult mice were sacrificed by CO2 inhalation and cervical dislocation. The small intestines were flushed with PBS and opened along the mesenteric border. SILT were cut out with a stab knife (5.0-mm blade; Fine Science Tools) using a stereo microscope. To obtain single-cell suspensions, SILT were placed in PBS containing 3% FCS and passed through a nylon mesh. Lineage-positive cells were excluded by incubation with a mixture of biotinylated Abs directed against CD3, B220, CD11c, CD11b, TER-119, and GR-1 that were detected by streptavidin conjugated to PerCP. Dead cells were excluded by 4',6-diamidino-2-phenylindole (DAPI) staining. Data were acquired using an LSR II flow cytometer (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences) or FlowJo (Tree Star).

Immunofluorescence microscopy

Immunohistochemistry was performed as described previously (7). In brief, isolated small intestines were flushed with cold PBS, opened along the mesenteric site, and 2-cm-long pieces were flattened on filter paper. Tissue was frozen in OCT on dry ice. Eight-micrometer cryosections were prepared, air dried, and fixed for 10 min in ice-cold acetone. Sections were rehydrated with TBST (0.1 M Tris (pH 7.5), 0.15 M NaCl, and 0.1% Tween 20) and blocked with TBST containing 5% rat or mouse serum depending on the source of Ab. Sections were incubated with different sets of Abs diluted in TBST supplemented with 2.5% rat or mouse serum. Sections were washed with TBST containing 1% FCS and nuclei were stained with DAPI and covered with MOWIOL. Fluorescence images were made using an IX81 microscope and analySIS D software (Olympus) and composite images were taken using a Zeiss Axiovert 200M microscope and analyzed with Axiovision software.

Statistical analysis

Cholera toxin and Salmonella-specific Ig levels were normalized to the levels observed in wild-type mice. Statistical analysis was performed using GraphPad Prism software. To test the significance of data, we used an unpaired Student’s t test. Data are expressed as means ± SD. Statistical differences of the mean values are indicated as follows: *, p < 0.05 and **, p < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
SILT constitute stable lymphoid aggregates in vivo

Analyzing the frequency of SILT present under various experimental conditions, we observed that the total number of SILT per small intestine is invariant (7, 9). This suggests that SILT, once established during postnatal development, might not newly form or disappear. However, definitive proof of this concept requires the tracking of individual SILT in the same animal in vivo over time. To this aim, we established a new method to visualize SILT. Adult wild-type C57BL/6 mice were lethally irradiated and reconstituted with bone marrow cells constitutively expressing the GFP in all cell types. In these chimeras, all hematopoietic cells constitutively expressed the GFP (GFP-chimera). In GFP-chimeras, PP and SILT were readily visible as brightly fluorescent aggregates from the luminal as well as the serosal side (Fig. 1A). High magnification revealed that from the luminal side SILT appeared as fluorescent, dome- shaped aggregates with reduced height compared with surrounding villi (Fig. 1A, left). Cryosections stained with anti-B220 and anti-CD11c Abs confirmed that fluorescent aggregates observed in GFP-chimeras contained characteristic cell types observed in SILT. Furthermore, SILT in GFP-chimeras encompassed the full spectrum of SILT typically observed in unmanipulated wild-type mice (data not shown).Counting the entire number of SILT detectable by this method, we observed a density of 41 structures per cm2 of the intestinal wall, yielding a total of 1100 ± 200 SILT/small intestine (n = 5, C57BL/6 wild-type recipients). This number reasonably matches the frequency of 58 SILT/cm2, corresponding to ~1500 SILT/small intestine that was obtained by the analysis of horizontal cryosections (7) and fits with the original estimate of 1500 CP/small intestine (2). These observations demonstrate that in GFP-chimeras SILT can be identified as aggregates of fluorescent cells and that GFP-chimeras provide a convenient tool to determine the frequency and positioning of SILT in vivo.


Figure 1
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  		FIGURE 1. Tracking SILT in vivo over time using GFP-chimeras. Lethally irradiated adult C57BL/6 mice were reconstituted with bone marrow cells expressing the GFP in all cells (GFP-chimera). A, left image, SILT can easily be identified from the luminal side as fluorescent dome-shaped structures with reduced height compared with surrounding villi. More dispersed fluorescent cells can be observed within the villi. A, right image, From the serosal side PP and SILT of GFP-chimeras appear as brightly fluorescent aggregates. Some SILT are marked by white arrows and PP appear as large multinodular structures. B, Stability of SILT pattern over time. The upper micrograph shows the distribution of SILT in the small intestine of a GFP-chimera exposed under anesthesia by laparotomy (day 0). Three weeks after surgery mice were sacrificed and the distribution of SILT in the same gut segment was reanalyzed (day 21). Dotted white lines facilitate identification of SILT pattern; white arrows indicate SILT that was spotted only at the second but not the first inspection of the small intestine. C, Summarized results for six jejunal gut segments derived from six individual mice analyzed at intervals of 3 wk. Sections depicted in B refer to mouse 2 in the table.

 
We next analyzed the positioning of SILT in the intestines of GFP-chimeras exposed under anesthesia by laparotomy. Subsequently, the intestines were reintroduced into the body cavity and the body wall was closed by suturing. Three weeks after surgery, the mice were sacrificed and the positioning of SILT was analyzed a second time. A rough alignment of intestinal fragments was achieved based on the positioning of PP. Interestingly, we invariably observed an identical pattern of SILT at the two time points (Fig. 1, B and C). Thus, even though we cannot rule out that the pattern of SILT might change after longer time periods, these observations provide the first direct in vivo evidence for the stability of SILT.

Expression of the chemokine receptor CXCR5 in SILT cells

Development of PP during gestation as well as homing of naive B cells into mature PP depend on the chemokine receptor CXCR5 (15, 17). To delineate a potential role for CXCR5 in SILT development and homeostasis, we analyzed the expression of CXCR5 in SILT cells. SILT were microdissected from the intestines of BALB/c mice and analyzed for the expression of c-Kit, CD4, and CXCR5 on lineage– (lin–: CD3–B220–CD11b–CD11c–Ter-119–) and CD19+ cells by flow cytometry. This analysis revealed the presence of a CD4+ and a CD4– subpopulation among the c-Kit+lin– SILT cells (Fig. 2A). Consistently, we could identify a subset of CD3–c-Kit+ cells in SILT that costained with anti-CD4 Abs by immunofluorescent microscopy (Fig. 2E). Expectedly, CD19+ B cells in SILT expressed CXCR5 at similar levels as PP B cells (Fig. 2C and data not shown). In contrast, lin–c-Kit+ isolated from SILT showed a dual expression pattern with lin–c-Kit+CD4+ cells expressing low albeit detectable levels of CXCR5, whereas lin–c-Kit+CD4– cells do not express CXCR5 (Fig. 2, B and D). This indicates that expression of CD4 might be suited to discriminate functionally different subtypes of lin–c-Kit+ cells in SILT. However, at present it is unclear whether coexpression of CD4 and CXCR5 on a fraction of lin–c-Kit+ cells in SILT distinguishes a functionally unique subset of cells or represents a temporal variation in the phenotype or status of these cells. Indeed, we observed that both CD4+ and CD4–lin–c-Kit+ cells isolated from microdissected SILT expressed high levels of ROR{gamma}t as judged by quantitative real-time PCR (data not shown). ROR{gamma}t has also been reported to be expressed in lin–c-Kit+ lamina propria cells (25). In any case, our data reveal that at least two distinct populations of cells present in SILT, i.e., CD19+ B cells and lin–c-Kit+CD4+ cells, express CXCR5, suggesting that CXCR5 might fulfill several tasks in SILT formation and function.


Figure 2
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  		FIGURE 2. Expression of CXCR5 in SILT cells. A–D, Expression of CXCR5 was analyzed by flow cytometry. Single-cell suspensions were prepared from SILT microdissected from the small intestines of BALB/c mice and stained for CXCR5, c-Kit, CD4, and a lineage mixture containing Abs directed against CD3, B220, CD11c, CD11b, TER-119, and GR-1. A, CD4 expression defines two subpopulations of c-Kit+lin– SILT cells. B–D, Histograms illustrate weak expression of CXCR5 on lin–c-Kit+CD4+ cells (B), profound levels of CXCR5 on CD19+ cells (C), and no detectable CXCR5 expression on lin–cKit+CD4– cells (D). Light gray lines indicate isotype control stainings. E, Location of c-Kit+CD4+CD3– cells in SILT. Horizontal sections from the small intestine of a C57BL/6 mouse were stained for c-Kit (blue), CD4 (green), and CD3 (red). c-Kit+CD4+CD3– cells (marked by white arrows) constitute a subpopulation of all c-Kit+ cells present in SILT.

 
CXCR5 is essential for regular SILT formation during postnatal development

Formation of PP during fetal development depends on CXCR5 (15). Penetrance of this phenotype is subject to genetic background variations (26). However, we never observed any macroscopically discernible PP in the colony of CXCR5-deficient mice backcrossed to the C57BL/6 background for at least 15 generations that was used throughout this study (data not shown). Impaired PP development can be rescued by adoptive transfer of wild-type LTIC isolated from fetal mesenteric lymph nodes into newborn CXCR5-deficient mice, indicating that CXCR5-sufficient LTIC can induce lymphoid organ development (27). We thus speculated that CXCR5 might serve an inductive function during SILT development. Yet, when analyzing cryosections of small intestines of adult 8- to 10-wk-old CXCR5-deficient mice, lymphoid aggregates were readily detectable, indicating that SILT can develop independent of CXCR5 (Figs. 3 and 4C). Moreover, the density of structures, i.e., the number of structures per area, closely matched the density of SILT observed in wild-type mice (data not shown). In contrast, at 4 wk of age, SILT were readily detectable in the small intestines of wild-type but not CXCR5-deficient mice (Fig. 3). Thus, CXCR5 appears to be indispensable for the regular developmental program generating SILT during early postnatal development. However, CXCR5-independent mechanisms can lead to the generation of lymphoid aggregations in the intestines of 8-wk-old CXCR5-deficient mice.


Figure 3
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  		FIGURE 3. CXCR5 is important for timely SILT formation. SILT of CXCR5-deficient mice at different ages were analyzed in comparison to wild-type mice. SILT were found to be absent in CXCR5-deficient mice at 4 wk of age, when they could readily be detected in wild-type mice. The number of SILT in 8-wk-old CXCR5-deficient mice kept under SPF conditions matched that of adult wild-type mice, whereas there were few, if any, SILT present in 8-wk-old germfree CXCR5-deficient mice. Micrographs depicted are composite images derived from cryosections from the small intestines of wild-type, SPF CXCR5-deficient, and germfree CXCR5-deficient mice that were stained with DAPI (blue) and CD11c (red). SILT structures are encircled with dashed white lines and scale bars represent 100 µm.

 

Figure 4
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  		FIGURE 4. CXCR5-deficient mice possess enlarged SILT devoid of B cell follicles. SILT of wild-type and CXCR5-deficient mice were classified regarding size and B cell content. Structures that in horizontal sections occupy areas of <5,000 µm2, 5,000–10,000 µm2, 10,000–15,000 µm2, 15,000–20,000 µm2, 20,000–50,000 µm2, and >50,000 µm2 were assigned classes I–VI, respectively. Additionally, structures were classified according to their B cell content: class A structures contain <5% of B cells, class B 5–20%, class C 20–50%, and class D structures have a prominent B cell follicle in their center that constitutes >50% of the SILT’s cellular mass. A, Images of representative class A–D structures derived from horizontal sections through the crypt zone of C57BL/6 small intestines stained for B220 (red) and c-Kit (blue). B, Graphical presentation of the SILT spectrum of C57BL/6 wild-type mice (267 SILT analyzed pooled from four mice) kept under SPF conditions. The diagram illustrates that an increase in size generally comes along with a higher content in B cells. C, The spectrum of SILT in SPF-kept mice deficient for the chemokine receptor CXCR5 clearly differed from that of wild-type mice: The diagram reveals a shift toward larger structures containing a lower amount of B cells (159 SILT analyzed pooled from four mice). D, B cell numbers were even more reduced in 20-wk-old germfree CXCR5-deficient mice as evidenced by a further increase in the proportion of class A structures. In contrast, the hyperplasia of structures was found to be independent of intestinal colonization as germfree and SPF- kept CXCR5-deficient mice had a similarly enlarged size spectrum of SILT compared with wild-type mice (64 SILT analyzed pooled from three mice).

 
Initiation of SILT formation does not depend on microbial stimulation as revealed by the presence of normal numbers of SILT in germfree mice (9). However, microbial stimulation drives the recruitment of B cells into SILT, yielding the progression of small-sized CP-like SILT into B cell follicle-containing ILF. We thus speculated that the CXCR5-independent formation of SILT in adult mice might be driven by microbiota. Indeed, we observed that the small intestine of adult 8-wk-old germfree CXCR5-deficient mice harbored very few SILT compared with mice housed under SPF conditions (Fig. 3). This suggests that microbiota can promote SILT development in the absence of CXCR5. However, stimulation by live microbiota is not a prerequisite for SILT formation in CXCR5-deficient germfree mice, because we observed an increase in the number of lymphoid aggregates in 20-wk-old compared with 8-wk-old germfree CXCR5-deficient mice (Fig. 4D and data not shown).

The spectrum of SILT can be illustrated based on size and B cell content

We have previously suggested to display the spectrum of SILT present in wild-type mice by using a size-based classification system (7). In this study, we report a refined model that includes two independent parameters to display the spectrum of SILT. This model is particularly suited when SILT cannot be appropriately characterized by a single parameter, e.g., in numerous gene-deficient mouse mutants. As previously described, we classified SILT according to size: SILT covering <5,000 µm2, 5,000–10,000 µm2, 10,000–15,000 µm2, 15,000–20,000 µm2, 20,000–50,000 µm2 and >50,000 µm2 of section area at the center of the structure were assigned to size classes I–VI, respectively (9). Additionally, the same structures were scored according to their B cell content: SILT containing <5% of B cells were designated class A, structures containing up to 20% of B cells but no clear B cell follicle were designated class B, SILT with up to 50% of B cells and a discernible B cell core were designated class C, and SILT displaying a prominent B cell follicle constituting >50% of the overall cell number were designated class D (Fig. 4A). For technical reasons, B cells in SILT were routinely identified by using anti-B220 stainings. However, B220-expressing cells in SILT could also be stained using anti-IgD Abs, indicating that the vast majority of all B220 staining cells in SILT are indeed naive B cells (data not shown and Ref. 7). Classifying the spectrum of SILT in C57BL/6 wild-type mice as described above yields a two-dimensional histogram (Fig. 4B), illustrating that in wild-type mice an increase in size coincides with a higher B cell content (Fig. 4B) as previously reported (7).

SILT in CXCR5-deficient intestines fail to accumulate B cells

SILT in adult 8-wk-old CXCR5-deficient mice encompassed a size spectrum of structures that was shifted in favor of large-sized SILT, i.e., SILT classified to size classes IV, V, and VI, when compared with wild-type mice (cf Fig. 4, B and C). The cellular composition of SILT in CXCR5-deficient mice displayed striking differences compared with wild-type controls. In wild-type mice, the majority of SILT contains a substantial number of B cells and are thus classified as classes C and D (Fig. 4B). In contrast, SILT in CXCR5-deficient mice were almost completely devoid of B cells as evidenced by the prevalence of class A and B structures (Fig. 4C). The size spectrum of SILT in 20-wk-old germfree CXCR5-deficient mice resembled that of CXCR5-deficient mice kept under SPF conditions (Fig. 4D). However, germfree CXCR5-deficient mice showed a further decreased content of B cells as evidenced by an increased proportion of class A structures compared with CXCR5 mutants kept under SPF conditions (cf Fig. 4, C and D). This suggests that microbial stimulation contributes to the accumulation of B cells in SILT of CXCR5-deficient mice. In CXCR5-deficient mice, the impact of intestinal microflora on the composition of SILT is not as obvious as in wild-type mice (9), as a pronounced shift toward B cell-depleted structures is already evident under SPF conditions.

Reduction of class C and D structures in CXCR5-deficient mice might be attributable to the incapability of CXCR5-defcient B cells to enter the structures. In a previous study, we could show that following small bowel transplantation B cells rapidly exchange between host and graft tissue and immunofluorescence microscopy revealed that the vast majority of such B cells localized in SILT (7). Thus, to evaluate the role of CXCR5 in the homing of B cells to SILT, we performed small bowel transplantations. Small intestinal fragments isolated from Ly5.1+ wild-type donors were grafted into congenic Ly5.2+ CXCR5-deficient recipients. The spectrum of SILT present in the wild-type graft 60 days after transplantation closely resembled those observed in the CXCR5-deficient host intestine or in untreated CXCR5-deficient mice (cf Figs. 5, A and B, to 4C). Immunofluorescent microscopy revealed that the few scattered B cells present in the SILT of the grafted intestine were of host origin (B220+Ly5.2+Ly5.1–; Fig. 5C), whereas the majority of c-Kit+ cells were of donor origin (cKit+Ly5.2–Ly5.1+; Fig. 5C). Thus, following intestinal transplantation, SILT B cells egress from the grafted tissue and cannot be replaced by host CXCR5-deficient B cells, even though a CXCR5-sufficient population of c-Kit-expressing cells is retained in the graft. We therefore suggest that the shift in the SILT spectrum in the graft tissue directly reflects the impaired homing of host CXCR5-deficient B cells into SILT.


Figure 5
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  		FIGURE 5. Small bowel transplantations revealed that B cell scarcity in CXCR5-deficient SILT is caused by impaired homing of CXCR-deficient B cells. Small bowel transplantations were performed by grafting small intestinal fragments isolated from Ly5.1+ wild-type donors into congenic Ly5.2+ CXCR5-deficient recipients. A and B, Eight weeks after transplantation, the spectrum of SILT in the host and graft intestine was determined as described for Fig. 4. SILT present in the grafted intestine showed a phenotype comparable to SILT observed in the host intestine and in unmanipulated CXCR5-deficient mice (47 SILT (grafted intestine) and 44 SILT (host intestine) analyzed pooled from four mice; cf with Fig. 4C). C, Immunofluorescent microscopy revealed that the few scattered B220+ (blue) cells present in SILT of the grafted intestine were of host (Ly5.2, green) origin, indicating that B cells in SILT rapidly exchange between the host and the graft intestine (7 ). In contrast, c-Kit+ (blue) cells in the graft have not been exchanged by host-derived cells as evidenced by expression of the graft marker Ly5.1 (red) by the majority of c-Kit+ (blue) cells in the graft.

 
To confirm this hypothesis by an independent experimental approach, we transplanted irradiated wild-type Ly5.1+ recipients with equal mixtures of bone marrow derived from CXCR5-deficient Ly5.2+ mice and B cell-deficient Ly5.1+ mice. In these chimeras, all B cells will lack CXCR5, whereas any other cell types will contain a substantial number of CXCR5-sufficient wild-type cells. Consistent with our observations obtained by small bowel transplantations, SILT in such mixed bone marrow chimeras were almost completely void of B cells (Fig. 6, A and B). In yet another approach, we reconstituted CXCR5-deficient recipients with equal mixtures of wild-type Ly5.1+ and CXCR5-deficient Ly5.2+ bone marrow cells. In the resulting chimeras, CXCR5-sufficient and CXCR5-deficient hematopoietic cells can be distinguished using congenic markers, allowing us to directly compare the ability of wild-type and CXCR5-deficient cells to settle into SILT. In such chimeras, we observed the formation of B cell follicles and furthermore confirmed that the vast majority of B cells in SILT originated from CXCR5-sufficient Ly5.1 B cells (purple cells in Fig. 6C that express Ly5.1 (blue) and B220 (red)). This indicates that lack of CXCR5 expression on B cells is sufficient to cause the scarcity of B cells observed in SILT of CXCR5-deficient mice and suggests that CXCR5 expression on B cells is required for B cell homing into SILT.


Figure 6
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  		FIGURE 6. Chemokine receptor CXCR5 is important for the homing of B cells into SILT. Mixed bone marrow chimeras were generated to assess the ability of B cells to migrate into SILT in the absence of CXCR5. A and B, Lethally irradiated Ly5.1+ wild-type mice received an equal mixture of CXCR5-deficient Ly5.2+ bone marrow and B cell-deficient bone marrow that was isolated from Ly5.1+ µMT-H2B mice lacking B cells. After 8 wk, cryosections from the small intestines were analyzed and SILT were classified as explained for Fig. 4. A, Mixed bone marrow chimeras retained the size spectrum observed in wild-type mice but largely lacked B cells in SILT and no class C and D structures were observed (73 SILT analyzed pooled from four mice). B, Representative image demonstrating Ly5.2 donor cells (green) and B220 (red) staining. Very few B220+ cells were identified in SILT. C, Lethally irradiated CXCR5-deficient Ly5.2+ mice received equal numbers of bone marrow cells from Ly5.1+ wild-type and Ly5.2+ CXCR5-deficient mice. Eight weeks after transplantation, B cell follicles were observed in CXCR5-deficient recipients. B cells (B220, red) were virtually exclusive of the Ly5.1+ (blue) wild-type origin.

 
CXCR5-deficient mice fail to mount efficient humoral responses to intestinal Ags

The general lack of PP and B cell follicles in SILT in CXCR5-deficient intestines prompted us to investigate humoral immune responses elicited by intestinal Ags in these mice. To this end, CXCR5-deficient mice and wild-type controls were orally immunized with a single dose of cholera toxin. After 10 days, the levels of cholera toxin-specific Igs in serum and intestinal washes were determined. In CXCR5-deficient mice, the amount of cholera toxin-specific IgA in the intestine was significantly decreased to ~40% of the wild-type level. Strikingly, Ag-specific Ig levels in the serum almost matched those of wild-type controls (Fig. 7A). To extend this observation, we next tested the immune response of CXCR5-deficient mice toward the attenuated noninvasive Salmonella enterica serovar typhimurium strain SL1344 aroA ssrB. CXCR5-deficient mice and wild-type controls were orally infected with 108 CFU of Salmonella SL1344 aroA ssrB and continuously treated with antibiotics via the drinking water to allow for a persistent colonization of the intestine. Infected mice shedded Salmonella with the feces until sacrifice, indicating persistent colonization (data not shown). Interestingly, in CXCR5-deficient mice, infection with Salmonella yielded significantly decreased levels of pathogen-specific Igs in both, intestinal washes and serum compared with wild-type mice (Fig. 7B). Total levels of serum IgM, IgG, and IgA as well as intestinal IgA were unchanged in CXCR5-deficient mice (data not shown and Ref. 28).


Figure 7
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  		FIGURE 7. Impaired humoral responses in CXCR5-deficient mice. CXCR5-deficient mice ({square}) and wild-type mice ({blacksquare}) were orally immunized with 20 µg of cholera toxin (A) or orally infected with 108 attenuated Salmonella (B). After 10 or 28 days, respectively, serum titers of Ag-specific IgM, IgG, and IgA as well as intestinal IgA were determined by ELISA. A, Following immunization with cholera toxin, CXCR5 deficiency led to a 2-fold reduction of Ag-specific IgA in the intestinal washes but did not change the levels of Ag-specific Igs in the serum (CXCR5–/–: n = 13; wild type: n = 12; results are representative of three individually performed experiments). B, In contrast, oral infection with Salmonella provoked a significantly reduced humoral response in CXCR5–/– mice as evidenced by reduced pathogen-specific Ig in serum and reduced IgA in intestinal washes (CXCR5–/–: n = 5; wild type: n = 6; results have been normalized to the Ig levels observed in wild-type mice; significance was revealed by unpaired Student’s t test: *, p < 0.05 and **, p < 0.01).

 
Impaired IgA responses in face of rescued follicle formation in SILT of CXCR5-deficient mice reconstituted with wild-type bone marrow

As described above, transplantation of irradiated CXCR5-deficient recipients with mixtures of wild-type and CXCR5-deficient bone marrow cells allowed for the formation of B cell follicles in SILT. To test whether restored formation of follicle-sufficient SILT rescues the induction of Ag-specific IgA responses in CXCR5-deficient mice, bone marrow chimeras were generated and immunized orally with cholera toxin. To this end, CXCR5-deficient recipients were lethally irradiated and transplanted with either wild-type or CXCR5-deficient bone marrow cells. Conversely, wild-type recipients were reconstituted with CXCR5-deficient or wild-type bone marrow cells. Systematical analysis of SILT in these chimeras revealed that reciprocal bone marrow transplantation converted the spectrum of SILT observed in unmanipulated CXCR5-deficient mice to that of wild-type mice and vice versa (Fig. 8, A and B). Ten days after oral immunization of bone marrow chimeras with cholera toxin, the amount of cholera toxin-specific IgA was determined in intestinal washes (Fig. 8C). Despite faithful restoration of SILT B cell follicles, we found that CXCR5 mutants reconstituted with wild-type bone marrow did not produce higher levels of IgA than CXCR5 mutants that were reconstituted with CXCR5-deficient bone marrow and thus lack prominent B cell follicles in their SILT. Consistent with this, wild-type recipients of CXCR5-deficient bone marrow that have poor B cell follicles in SILT still show a higher intestinal IgA titer in response to oral immunization with cholera toxin than CXCR5 mutants transplanted with wild-type bone marrow. In contrast, total IgM, IgG, and IgA levels in the serum showed no differences between CXCR5-deficient mice reconstituted with wild-type bone marrow cells and vice versa (data not shown). These results show that bone marrow transplantation is not sufficient to restore the induction of cholera toxin-specific IgA responses, indicating that defects other than the lack of B cells in SILT of CXCR5-deficient mice prevent the efficient induction of intestinal IgA.


Figure 8
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  		FIGURE 8. Transfer of wild-type bone marrow rescues follicle formation in SILT but not efficient production of cholera toxin-specific IgA in CXCR5–/– mice. The spectrum of SILT in bone marrow chimeras was determined as described for Fig. 4. Bone marrow cells from wild-type mice were injected i.v. into lethally irradiated CXCR5–/– mice and vice versa (wild type to CXCR5–/–: n = 12; CXCR5–/– to wild type: n = 8). A, SILT in wild-type mice transplanted CXCR5–/– bone marrow cells resembled that of untreated CXCR5 mutants. (176 SILT analyzed pooled from nine mice). B, Conversely, in CXCR5–/– mice reconstituted with wild-type bone marrow cells, the spectrum of SILT was shifted toward smaller structures containing B cell follicles, thereby resembling the situation in wild-type mice (403 SILT analyzed pooled from 12 mice; cf with Fig. 4B). C, Eight weeks after transplantation, bone marrow chimera were orally immunized with 20 µg of cholera toxin (CXCR5–/– recipients receiving wild-type bone marrow: n = 6; wild-type mice receiving CXCR5–/– bone marrow: n = 6; isogenic transplantations for CXCR5–/–:n = 5; isogenic transplantations for wild-type mice: n = 4; results have been pooled from two independent experiments and normalized to the Ig levels observed in isogenic wild-type transplantations). Ten days after immunization, intestinal washes were collected and the amount of cholera toxin-specific IgA was determined by ELISA. Intestinal washes of CXCR5-deficient mice contained reduced amounts of IgA irrespective of reconstitution with wild-type or CXCR5-deficient bone marrow. The results depicted are representative of two individual experiments; *, p < 0.05.

 
In conclusion, results presented in this study reveal that like PP development, initiation of SILT formation in the endogenous time window crucially depends on CXCR5. However, in contrast to PP development, CXCR5-independent formation of aberrant SILT can be triggered by microbial and other stimuli. Still, B cell homing and follicle formation in both PP and SILT utilize a CXCR5-dependent mechanism. Moreover, CXCR5-deficient mice show reduced humoral responses to T cell-dependent Ags, indicating that SILT do not fully compensate for the lack of PP in these mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The anatomical location and the number of classical secondary lymphoid organs such as lymph nodes and PP are determined during gestation. Therefore, postnatal events may alter the phenotypic appearance of secondary lymphoid organs, but will not affect their location or number. In contrast, tertiary lymphoid tissue may form ectopically at any time in the adult organism once appropriate stimulation occurs, e.g., during chronic infections (recently reviewed in Ref. 14). In this study, we provide direct evidence that the positioning and density of SILT are fixed. Using a newly developed experimental approach, we tracked individual SILT in vivo. We observed that in adult mice >95% of all SILT could be retrieved a second time after a time period of 3 wk. Even though at present we cannot exclude that changes in the pattern of SILT would be detectable after longer time periods, these results argue for a stable position of SILT. A low level of uncertain structures might have been overlooked for technical reasons during the first inspection of the small intestine. In contrast to stable SILT patterns, the phenotypic manifestation of SILT is highly dynamic. In particular, microbial stimulation induces a shift in the spectrum of SILT toward follicle-containing structures (8, 9, 10). This situation resembles the situation in PP, which like SILT are hypoplastic in germfree mice and rapidly recruit mature lymphocytes upon microbial stimulation (29, 30, 31). Based on these observations, we suggest that SILT share decisive features, i.e., fixed positioning and number, with secondary lymphoid organs but not with tertiary lymphoid tissue.

A critical step in lymphoid organogenesis is the interaction of LTIC and mesenchymal organizer cells that allows for the growth of the organ anlage. The chemokine receptor CXCR5 constitutes an essential element in the interaction of LTIC and organizer cells. Consequently, PP as well as inguinal and sporadically cervical lymph nodes are absent from CXCR5-deficient mice (15, 26, 32). However, CXCR5 does not represent a general prerequisite for lymphoid organogenesis since other lymph nodes form normally in these mice. In this manuscript, we report that 4-wk-old CXCR5-deficient mice entirely lack SILT in the small intestine, suggesting that like PP, regular development of SILT depends on CXCR5. However, during aging, SILT develop independent of CXCR5 and adult CXCR5-deficient and wild-type mice have a comparable density of SILT. The CXCR5-independent mechanism is supported by microbial stimulation and the development of SILT is delayed in germfree compared with SPF-kept CXCR5-deficient mice. Still, SILT eventually form in germfree CXCR5-deficient mice, indicating that stimuli other than microbial stimuli can trigger SILT formation. This compensatory mechanism does not involve the chemokine receptor CCR7. Despite the complete absence of all peripheral lymph nodes in CXCR5/CCR7 double-deficient mice (32), the number of SILT in these mutants matches that of the wild-type and single-deficient mouse strains (data not shown). SILT in these double-deficient mice like in CCR7-deficient mice (9) occasionally contains multiple nodules which renders their distinction from PP difficult. The chemokine receptor CCR6 was found to be expressed by the majority of B cells and lin–c-Kit+ cells present in SILT (20). Furthermore, it was shown that bacterial recognition by the innate receptor NOD1 that is expressed on epithelial cells is sufficient to induce ILF formation in a mechanism depending on CCR6 (10). Therefore, high expression of CCR6 on LTIC-like cells in SILT might compensate for the absence of CXCR5 and induce SILT formation in adult SPF CXCR5-deficient mice. Because activation of this pathway requires a bacterial trigger, this scenario is however not suited to explain the presence of SILT in 20-wk-old germfree CXCR5- deficient mice.

One of the most distinguishing features of PP and SILT may be their divergent time of development. Normal development of PP occurs during gestation, whereas SILT develops postnatally. The adoptive transfer of CXCR5-sufficient LTIC into newborn CXCR5-deficient mice leads to the formation of macroscopically visible follicular structures in the small intestine that share features of both PP and SILT (27). lin–c-Kit+ cells in SILT strikingly resemble LTIC in their phenotype and ontogeny. In this light, the results obtained by Finke et al. (27) can be perceived as support to the hypothesis that lin–c-Kit+ cells in SILT might recapitulate lymphoid organogenesis postnatally (19, 33).

Entry of B cells into SILT and thereby B cell follicle formation depend on β7 integrin (34) and as shown here CXCR5 function. In this respect, B cell homing into SILT resembles B cells homing into PP and into omental milky spots (17, 35, 36, 37). We demonstrate here that SILT in CXCR5-deficient mice are devoid of B cells and that this defect can be rescued by transplantation of wild-type bone marrow. Moreover, the spectrum of SILT present in wild-type intestines adapts to the phenotype of SILT observed in CXCR5-deficient mice after transplantation of the wild-type gut tube into CXCR5-deficient recipients. This indicates that a constant influx of CXCR5-sufficient B cells into SILT is required for their homeostasis.

Oral immunization of CXCR5-deficient mice fails to induce a robust production of intestinal IgA. This defect cannot be attributed to an intrinsic defect in B cell function, since bone marrow reconstitution failed to revert this phenotype and CXCR5-deficient B cells significantly contribute to the production of Ig in mixed bone marrow chimera (S. Velaga, unpublished observation). Along the same line of evidence, we can exclude that CXCR5 function on T cells accounts for the observed lack of IgA induction in CXCR5-deficient mice. CXCR5 expression by follicular T cells has been shown to be important for optimal humoral immune responses (38, 39). SILT do harbor a very small population of CD4+ICOS+CD69+ T cells which express CXCR5 and might represent follicular T cells (data not shown). However, like B cells, follicular T cells will be reconstituted in CXCR5-deficient recipients receiving wild-type bone marrow cells. Thus, reconstitution of CXCR5-deficient mice with wild-type bone marrow rescues B cell follicles as well as CXCR5-expressing follicular T cells. Moreover, we observed that in such chimera germinal centers follicular dendritic cells may be present. Thus, bone marrow transplantation of CXCR5-deficient mice with wild-type bone marrow reconstitutes major cellular and architectural traits of SILT but still fails to restore optimal IgA responses. This suggests that an architectural defect might account for the defective humoral immune response in CXCR5-deficient mice. The most obvious of such defects is the complete lack of PP in CXCR5-deficient mice as well as in CXCR5-deficient recipients reconstituted with wild-type bone marrow cells. It is well established that intestinal IgA responses are induced in PP (40, 41). However, wild-type mice that have been modified to lack PP by treatment with antagonistic agents interfering with PP development (PP-deficient mice) generate Ag-specific IgA against soluble protein Ags (42, 43, 44). Therefore, the exclusive role of PP for the induction of IgA has been a matter of debate for a long time. Recent observations indicate that PP appear to be essential for intestinal immune responses to Salmonella (45, 46, 47), even though conflicting results have been obtained regarding the role of SILT in anti-Salmonella responses (48).

Additional complexity is added to the system considering the contribution of B1 cells to the intestinal IgA production. Peritoneal B1 cells express CXCR5 and require this receptor for homing into the peritoneal cavity (37, 49). In consequence, CXCR5-deficient mice as well as mice lacking the CXCR5 ligand CXCL13 harbor largely reduced B1 cell populations in the peritoneal cavity. However, B1 cells are unlikely to contribute to the induction of T cell-dependent anti-cholera toxin responses. We therefore suggest that impaired cholera toxin-specific IgA response in CXCR5-deficient mice might reflect the lack of PP. Interestingly, PP would play a nonredundant role in that other mechanisms, including follicle-sufficient SILT, cannot fully compensate for the lack of PP. A possible explanation for these on first-view contradictory observations might relate to the type of Ag investigated. PP as well as SILT are equipped with M cells, allowing for the efficient uptake of particulate material from the intestinal lumen. However, only PP but not SILT harbor dedicated T cell areas that are likely to be required for the induction of T cell-dependent anti-cholera toxin IgA responses, indicating that SILT might not be able to support T cell-dependent IgA responses (40).

In conclusion, our results indicate that SILT might be unable to fully compensate for the lack of PP during the induction of anti-cholera toxin IgA responses. Instead, PP and SILT might serve nonredundant functions in the intestine and both compartments might be particularly equipped to handle different types of Ags (40). Moreover, we demonstrate a nonredundant function for CXCR5 in the developmentally programmed induction of SILT during early postnatal development as well in SILT homeostasis.


   Acknowledgments
 
We thank Dirk Bumann for providing Salmonella SL1344 aroA ssrB and Sabrina Dähne and Tim Worbs for critically reading this manuscript and immunofluorescent microscopy.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Oliver Pabst, Institute of Immunology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. E-mail address: Pabst.Oliver{at}mh-hannover.de Back

2 Abbreviations used in this paper: PP, Peyer’s patch; CP, cryptopatch; ILF, isolated lymphoid follicle; SILT, solitary intestinal lymphoid tissue; LTIC, lymphoid tissue inducer cell; SPF, specific pathogen free; DAPI, 4',6-diamidino-2-phenylindole. Back

Received for publication April 7, 2008. Accepted for publication December 18, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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