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The Journal of Immunology, 2005, 174: 5720-5728.
Copyright © 2005 by The American Association of Immunologists

Adaptive Immune Responses Are Dispensable for Isolated Lymphoid Follicle Formation: Antigen-Naive, Lymphotoxin-Sufficient B Lymphocytes Drive the Formation of Mature Isolated Lymphoid Follicles 1

Keely G. McDonald, Jacquelyn S. McDonough and Rodney D. Newberry2

Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Isolated lymphoid follicles (ILFs) are recently appreciated members of the mucosal immune system. The architecture, composition, and inducible nature of these structures indicates that these structures are tertiary lymphoid structures. The process leading to the formation of tertiary lymphoid structures, lymphoid neogenesis, has been observed in a number of inflammatory and autoimmune conditions. Given this association, there is considerable interest in identifying the factors promoting lymphoid neogenesis, and understanding the steps in this process. Using murine ILF formation as a model, we have examined the roles of different cellular sources of lymphotoxin (LT) and the adaptive immune response in lymphoid neogenesis. In this study, we report that, although other cellular sources of LT may supplant B lymphocytes in the formation of immature ILFs (loosely organized clusters of B lymphocytes), LT-sufficient B lymphocytes are required for the progression of immature ILFs to mature ILFs (organized lymphoid aggregates with a follicle-associated epithelium). ILF formation occurs in the absence of T lymphocytes and Ag-specific B lymphocyte responses, and ILF B lymphocytes express elevated levels of LT in the absence of antigenic stimulation. Consistent with a role for chemokines inducing LT expression in Ag-naive B lymphocytes, and a chemokine-driven positive-feedback loop driving mature ILF formation, mature ILFs express elevated levels of B lymphocyte chemoattractant in the absence of Ag-specific B lymphocyte stimulation. These observations indicate that ILFs contain Ag-naive lymphocytes, and suggest that events occurring within ILFs shape subsequent immune responses mediated by these lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mucosal immune system is a complex network of lymphoid compartments working together to protect higher organisms from invading pathogens. The presence of aggregates of mononuclear cells resembling Peyer’s patches (PP) 3 or lymph nodes in the human intestine is well documented (1, 2, 3); however, the relationship of these structures to PP, the nature of their formation, and their function has remained unclear. Analogous structures, isolated lymphoid follicles (ILFs), have been identified in the murine small intestine (4). ILFs were found to be composed of predominantly B-2 B lymphocytes and CD4+ T lymphocytes. And, like PP, ILF were found to possess a follicle-associated epithelium (FAE) containing M cells (4). Recently, ILFs were found to be sites for IgA class switching (5).

The initial identification of ILFs used B220+ staining of frozen sections of murine intestine cut at an axis perpendicular to the villi, and did not allow assessment of the macroscopic architecture of the B220+ structures (4). Using whole-mount techniques and dissecting microscopy, we have identified a variety of structures containing B220+ cells, including loosely organized clusters of B220+ cells preferentially positioned at the base of villi, or immature ILFs (iILFs), and well-organized lymphoid structures containing a germinal center and an overlying FAE, or mature ILFs (mILFs). The relationship of iILFs progressing to mILFs is supported by several findings, which include the following: the identical distribution and location of iILFs and mILFs; the requirements for formation of iILFs are shared by mILFs, whereas not all of the requirements for formation of mILFs are shared by iILFs (indicating that iILFs precede mILFs); and the ability to promote the progression of iILFs to mILFs (6).

Several factors required for ILF formation have been identified. These requirements parallel those required for PP and other secondary lymphoid structure formation, but have key distinctions. Like PP formation, ILF formation was found to be dependent on lymphotoxin (LT), LT{beta}R, and NF-{kappa}{beta}-inducing kinase function because ILFs were absent in LT-deficient mice, LT{beta}R-deficient mice, and aly/aly mice (4, 6). Although both ILF and PP formation were found to be dependent on LT{beta}R-sufficient stromal cells, ILF and PP formation were found to differ in the cellular source of LT required for their formation. PP formation requires a LT-sufficient CD4+, CD3–, bone marrow-derived cell, which may be a precursor to NK cells (7), whereas mILF formation was found to be dependent on LT-sufficient B lymphocytes (6).

The requirement for LT-sufficient B lymphocytes is not only a distinction between secondary lymphoid structure formation and ILF formation, but it also provides an opportunity for critical insight into the process of ILF formation. LT expression is induced in B lymphocytes following activation, which occurs in the context of BCR ligation by Ag. Alternatively, LT expression may be induced in Ag-naive B lymphocytes following chemokine receptor ligation (8). Understanding the requirement for Ag exposure in ILF formation not only yields insight into the mechanisms leading to ILF formation, it also yields insight into the roles ILFs play in immune responses. The requirement for previous Ag exposure would suggest that ILFs are less likely to be sites where immune responses to new Ags are shaped, and are more likely sites where Ag-experienced lymphocytes receive ongoing stimulation to perpetuate immune responses. The absence of a requirement for Ag exposure in ILF formation would suggest that ILFs contain Ag-naive lymphocytes, and immune responses to new Ags are shaped within ILFs.

In this study, we demonstrate that, in the absence of LT-sufficient B lymphocytes, ILF formation is arrested at an immature stage, and ILF formation occurs in the absence of T lymphocytes and in the absence of prior Ag exposure by B lymphocytes. We also observed that ILF B lymphocytes have a naive phenotype and express LT in the absence of prior Ag exposure. Therefore, whereas the formation of mILFs is dependent on LT-sufficient B lymphocytes, the formation of ILFs is not dependent on prior Ag-specific stimulation of these B lymphocytes. These findings indicate that ILF formation is driven by innate immune responses, and suggest that a chemokine-driven positive-feedback loop drives the progression of iILFs to mILFs. These observations suggest that ILFs are sites where Ag-naive lymphocytes have initial encounters with Ags, and events occurring within ILFs may significantly shape the mucosal immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice used for this study were housed in a specific pathogen-free facility and fed chow diet not containing hen egg white lysozyme (HEL). Animal procedures and protocols were conducted in accordance with the institutional review board at Washington University School of Medicine. C57BL/6, RAG-1-deficient, B cell-deficient JH–/– mice (9), and MD4 mice (10) on the C57BL/6 background were purchased from The Jackson Laboratory. MD4 mice possess a transgenic rearranged Ig L chain and Ig H chain linked to µ and {delta} C region genes of the IgHa allotype, which is specific for HEL (10). MD4 mice were bred with JH–/– mice to obtain the MD4JH–/– mice, possessing exclusively the transgenic BCR on all B lymphocytes. LT{alpha}-deficient mice (11) were bred onto the C57BL/6 background for >10 generations before use in experiments. Timed pregnant C57BL/6 female mice for use in experiments involving the injection of LT{beta}R-Ig fusion protein, were generated by matings with C57BL/6 male mice. Six- to 10-wk-old LT{alpha}–/– mice were used as recipients for bone marrow transfers.

Bone marrow transfers

Bone marrow transfers were performed after lethal irradiation as previously described (12). A total of 1 x 107 T lymphocyte-depleted bone marrow cells from gender-matched donors were injected i.v. into recipients on the second day of irradiation. Mice receiving bone marrow from multiple donors (LT{alpha}–/– and Rag–/–, LT{alpha}–/–, and JH–/–, or C57BL/6 and JH–/–), received 5 x 106 cells from each donor. Mice were allowed 12 wk for reconstitution with donor bone marrow before use in experiments. Flow-cytometric analysis was performed on splenocytes from recipients at the time of sacrifice to document appropriate lymphocyte reconstitution.

LT{beta}R-Ig treatment

LT{beta}R-Ig production and treatment were performed as previously described (12). Timed pregnant female C57BL/6 mice were injected with 100 µg of LT{beta}R-Ig via tail vein on day 16 postconception. Mice receiving LT{beta}R-Ig in utero were analyzed for the presence of mILF at 6–7 wk of age (see Fig. 2).



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  		FIGURE 2. T lymphocytes and Ag-specific B lymphocyte responses are dispensable for ILF formation. Intestines from C57BL/6 mice, MD4JH–/– mice, TCR{beta}{delta}–/– mice, and C57BL/6 mice receiving LT{beta}R-Ig in utero to ablate PP formation were examined for the presence of iILFs (a), and/or mILFs (b). iILFs and mILFs were present in MD4JH–/– mice, and TCR{beta}{delta}–/– mice, indicating that T lymphocyte responses and Ag-specific B lymphocyte responses were dispensable for the formation of both iILFs and mILFs (a and b). Consistent with our previous observations, iILFs were present in unmanipulated C57BL/6 mice, but significant numbers of mILFs were only formed after manipulations to ablate PP formation. Evaluation of fecal IgA levels revealed that MD4JH–/– mice did not produce intestinal IgA, and that C57BL/6 mice receiving LT{beta}R-Ig in utero were relatively deficient in intestinal IgA production at 6–7 wk of age (c). TCR{beta}{delta}–/– mice had total fecal IgA levels that were significantly less than age-matched C57BL/6 mice (c). Consistent with this, we observed that TCR{beta}{delta}–/– mice had significantly fewer IgA-producing cells, 1329 ± 254 per 106 small intestine lamina propria cells (n = 6), when compared with age-matched C57BL/6 mice, 5863 ± 886 per 106 small intestine lamina propria cells (n = 3); p < 0.05. *, p < 0.05 when compared with results from C57BL/6 mice.

 
Whole mounts of small intestine

Small intestines were removed intact, flushed with cold PBS, and opened along the mesenteric border. Intestines were mounted, lumen facing up and fixed with cold 10% phosphate-buffered formyl saline (Fisher Scientific) for 1 h at 4°C. Intestines were washed three times in cold PBS, incubated in a solution of 20 mM DTT, 150 mM Tris, and 20% ethanol at room temperature for 45 min, washed three times in cold PBS, and incubated in a solution of 1% H2O2 for 15 min at room temperature to block endogenous peroxidases. Intestines were washed three times in PBS, followed by incubation in PBS containing 1% BSA and 0.3% Triton X-100 for 30 min. Intestines were incubated with HRP-conjugated lectin from Ulex europaeus (UEA-I) (Sigma-Aldrich) in PBS, BSA, Triton X-100 solution overnight at 4°C to facilitate the identification of PP and mILF. The following day, intestines were washed three times in PBS, incubated in DAB metal peroxide substrate (Pierce) for 15 min, rinsed twice in distilled water, and returned to PBS for further analysis. Investigators unaware of the treatment groups determined the presence of mILFs. Under low-power microscopy (x25–65) the following criteria were used to determine the presence of mILF: 1) presence of a nodular structure with size equal to or greater than the width of one villus, 2) nodular structure possessing an overlying dome resembling the FAE of PP, and 3) nodular structures occurring singly or in groups of two (three or more nodules of approximately the same size were considered to be PP).

For B220+ staining of whole mounts (modified whole mounts) to determine the numbers of iILFs, intestines were removed intact, flushed with PBS, opened along the mesenteric border, and mounted as above. Intestines were then incubated three times in HBSS (BioWhittaker) containing 5 mM EDTA at 37°C with shaking for 10 min to remove epithelial cells. Intestines were then fixed in 10% phosphate-buffered formyl saline and treated with 1% H2O2 for 15 min at room temperature as above. Intestines were incubated in a solution of 50 mM Tris (pH 7.2), 150 mM NaCl, 0.6% Triton X-100, and 0.1% BSA for 1 h at 4°C to block nonspecific Ab binding and then incubated with rat anti-mouse B220 Ab (BD Pharmingen) diluted in the above solution overnight at 4°C. Intestines were washed three times in the above solution and incubated with a HRP-conjugated goat anti-rat IgG Ab (Jackson ImmunoResearch Laboratories) diluted in the above solution at room temperature for 1 h. Intestines were washed three times and incubated in DAB metal peroxide substrate as above. Investigators unaware of the treatment groups determined the presence of iILFs. The criterion used to determine the presence of iILFs were clusters of B220+ cells not having a nodular appearance, and occurring preferentially at the base of villi (6).

Measurement of fecal IgA

Mice used for the measurement of fecal IgA were 6–7 wk of age. Feces were collected from individual mice, diluted 1/10 wet weight to volume with PBS, vortexed into a uniform suspension, and centrifuged at 12,000 rpm for 10 min in a table top microcentrifuge, and supernatants were removed. Fecal supernatants or IgA standards (Southern Biotechnology Associates) diluted in PBS containing 0.05% Tween 20 (Sigma-Aldrich) were incubated in 96-well Immulon 4 plates (Fisher Scientific) previously coated with goat anti-mouse Ab (Southern Biotechnology Associates) and blocked with PBS containing 5% BSA and 0.05% Tween 20 at room temperature for 2 h. Plates were washed three times with PBS containing 0.05% Tween 20, and then goat anti-mouse IgA alkaline phosphatase-conjugated Ab (Southern Biotechnology Associates) diluted in PBS containing 5% BSA and 0.05% Tween 20 was added to the plate and incubated for 2 h at room temperature. Plates were washed three times with PBS containing 0.05% Tween 20, and p-nitrophenyl phosphate alkaline phosphatase substrate (Sigma-Aldrich) was added. Plates were read at 405 nm using BioTek Instruments Microplate Reader. Each sample was measured in duplicate in at least three dilutions. Data are reported as the mean concentration of IgA in the fecal supernatant prepared as above.

ELISPOT assay

Lamina propria cellular populations were isolated as previously described (12). Ninety-six-well multiscreen-HA plates (Millipore) were coated with goat anti-mouse Ig (Southern Biotechnology Associates) overnight at room temperature. Plates were washed three times in PBS, blocked with PBS containing 5% newborn calf serum (HyClone) for 1 h at 37°C, washed, and lamina propria cellular suspensions in IMDM (BioWhittaker), 5% FCS (HyClone), 2 mM Glutamax I (Invitrogen Life Technologies), 50 U/ml penicillin-50 mg/ml streptomycin (Invitrogen Life Technologies), and 50 mM 2-ME (Fisher Scientific) were added to the plates. Plates were incubated at 37°C, 10% CO2 overnight, washed with PBS containing 0.05% Tween 20, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgA Ab 2(Southern Biotechnology Associates) overnight at 4°C. Plates were washed with PBS and exposed to 5-bromo-4-chloro-3-indolyl phosphatase/NBT substrate (Sigma-Aldrich), and spot-forming cells were counted under a dissecting microscope.

Immunohistochemisty

Paraffin-embedded sections containing mILF from whole-mount intestines (performed as described above) were deparaffinized by sequential treatments with Citrosolv (Fisher Scientific) and isopropyl alcohol, rinsed with tap water, rehydrated in PBS, treated with avidin/biotin blocking kit (Vector Laboratories), washed three times in PBS, and blocked for 15 min at room temperature in PBS containing 1% BSA, and 0.1% Triton X-100. Sections were then incubated with biotin-conjugated lectin from Arachis hypogaea (peanut lectin (agglutinin) (PNA)) (Sigma-Aldrich), and diluted in PBS containing 1% BSA, and 0.1% Triton X-100 overnight at 4°C. For detection of PNA staining, we used biotinyl-tyramide signal amplification (DuPont/NEN) followed by incubation with streptavidin-conjugated cyanine 2 dye (Jackson ImmunoResearch); sections were counterstained with Hoechst dye (Sigma-Aldrich) to visualize nuclei.

Cell isolation from mILFs, PP, and spleen

Spleens were removed and disrupted by mechanical disassociation. Intestines were flushed with cold PBS, opened along the mesenteric border, and mounted with the lumen facing up in cold PBS, as described above. PP were identified, cut out, and disrupted using mechanical disassociation. Using the dissecting microscope, and a blunt-end 26-gauge needle and syringe, the contents of multiple mILFs were aspirated and placed in cold PBS. RBC were lysed from each cellular suspension and then used for flow-cytometric analysis as described below. Average yield of viable mononuclear ILFs cells ranged from 3 to 7 x 105 cells/intestine.

ILF cell populations were enriched for CD19+ cells using anti-CD19 microbeads and magnetic separation columns (Miltenyi Biotec) per the manufacturer’s recommendations. ILF cellular populations enriched in this fashion contained >90% B220+ cells; populations depleted in this fashion contained <20% B220+ cells by flow-cytometric analysis.

Flow-cytometric analysis

Single-cell suspensions were obtained as above, and flow-cytometric analysis was performed as previously described (12). Reagents used for analysis were FITC-conjugated rat anti-mouse CD19, PE-conjugated rat anti-mouse CD19, FITC-conjugated hamster anti-mouse CD69, hamster anti-mouse LT{beta}, biotin-conjugated mouse anti-hamster IgG mixture, streptavidin-PE, streptavidin-FITC, appropriate isotype control Abs (all from BD Biosciences), FITC-conjugated rat anti-mouse MHC class II (MHCII) (Southern Biotechnology Associates), biotin-conjugated rat anti-mouse CD86, and FITC-conjugated rat anti-mouse CD23 (eBioscience). HEL (catalog no. L-6876; Sigma-Aldrich) was conjugated to Alexa Fluor 488 (Molecular Probes) per the manufacturer’s recommendations and incubated with isolated cell populations at a concentration of 500 ng/ml for the determination of Ag binding. Dead cells were excluded based on forward- and side-light scatter. Gates for positive staining were defined such that 1% of the analyzed population stained positive with the appropriate isotype control Ab.

To document modulation of the expression of cell surface markers associated with Ag activation, splenocytes from MD4JH–/– mice were cultured at a density of 1 x 106 cells/ml with hen egg lysozyme (1 µg/ml) in medium consisting of RPMI 1640 (BioWhittaker), 2 mM L-glutamine (Invitrogen Life Technologies), 10 mM HEPES (BioWhittaker), 1 mM sodium pyruvate (BioWhittaker), 5 x 10–5 M 2-ME (Sigma-Aldrich), 1% penicillin/streptomycin (Invitrogen Life Technologies), and 10% FCS (HyClone) at 37°C, 5% CO2 for 12 h.

RNA isolation and real-time PCR

Spleen, PP, and mILFs were isolated from MD4JH–/– mice as described above. mILFs used for RNA isolation contained both bone marrow-derived cell populations and stromal cell populations. Non-PP non-mILF-bearing intestine from the distal small intestine of MD4JH–/– mice was identified using a dissecting microscope and removed. RNA was isolated from the appropriate cell populations and tissues using TRIzol (Invitrogen Life Technologies) and treated with DNase I (Ambion) to remove contaminating DNA, and cDNA was synthesized from 2 µg of total RNA using Superscript II RNase H– reverse transcriptase (Invitrogen Life Technologies). Expression of targets was detected by real-time PCR using ABI Prism 7700 sequence detection system and SYBR Green PCR master mix (Applied Biosystems). The following primers were used for detection of the targets (forward primers are listed first, followed by reverse primers): 18s, 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3'; BLC, 5'-CAGAATGAGGCTCAGCACAGC-3' and 5'-CAGAATACCGTGGCCTGGAG-3'; SLC, 5'-TCCCGGCAATCCTGTTCTC-3' and 5'-CCTTCCTCAGGGTTTGCACA-3'; ELC, 5'-ATGCGGAAGACTGCTGCCT-3' and 5'-GGCTTTCACGATGTTCCCAG-3'; SDF-1, 5'-TGCTCTCTGCTTGCCTCCA-3' and 5'-GGTCCGTCAGGCTACAGAGGT-3'; LT{alpha}, 5'-GAGAGGGTCTACGTTAACATCAGTCA-3' and 5'-TTGCTCAAAGAGAAGCCATGTC-3'; and LT{beta}, 5'-CCCCAGCAAGCAGAACTCA-3' and 5'-CGCCCCGAAGAAGGTCTT-3'. Expression of each target was measured in triplicate. Relative quantitation of target expression was determined using the comparative CT method with 18s expression as a control, as described in the ABI Prism 7700 sequence detection system user bulletin.

Statistical analysis

Data analysis using one-way ANOVA followed by Tukey’s multiple comparison posttest was performed using GraphPad Prism (GraphPad Software). A value of p < 0.05 was used as a cutoff for statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LT-sufficient B lymphocytes are not required for the formation of iILFs, but are required for the progression of iILFs to mILFs

Previous observations have demonstrated that LT and the LT{beta}R play a crucial role in the formation of ILFs, and the requirement for LT and the LT{beta}R occurs early in this process, because both mILFs and iILFs are absent in LT-deficient and LT{beta}R-deficient mice (4, 6). We have previously demonstrated that, in the absence of LT-sufficient B lymphocytes, mILFs do not form (6). However, it is unclear whether LT-sufficient B lymphocytes are required throughout the process of ILF formation, or whether other LT-sufficient cells may suffice to drive ILF formation at earlier stages. To address this question, we reconstituted lethally irradiated LT{alpha}–/– mice with bone marrow from LT{alpha}–/– and RAG–/– mice, LT{alpha}–/– and JH–/– mice, C56BL/6 and JH–/– mice, and LT{alpha}–/– mice, thus reconstituting the LT/LT{beta}R axis with different cellular sources of LT. The recipient’s intestines were examined for the presence of iILFs and mILFs using whole-mount techniques. We observed that reconstitution of LT{alpha}–/– mice with LT{alpha}–/– bone marrow did not induce the formation of iILFs or mILFs. Reconstitution of LT{alpha}–/– recipients with a combination of bone marrow from LT{alpha}–/– mice and RAG–/– mice, such that the recipients would have LT-sufficient NK cells and LT-deficient T and B lymphocytes, induced the formation of iILFs but not mILFs. Reconstitution of LT{alpha}–/– recipients with a combination of bone marrow from LT{alpha}–/– mice and JH–/– mice, such that recipients would have LT-sufficient NK cells and T lymphocytes and LT-deficient B lymphocytes, induced the formation of more iILFs but not mILFs. In comparison, reconstitution of LT{alpha}–/– recipients with a combination of C57BL/6 and JH–/– bone marrow, such that the recipients now have LT-sufficient B lymphocytes, induced the formation of iILFs and mILFs (Fig. 1).



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  		FIGURE 1. LT-sufficient B lymphocytes are not required for the formation of iILFs, but are essential for the progression of iILFs to mILFs. Lethally irradiated LT{alpha}–/– mice were reconstituted with LT{alpha}–/– bone marrow, a combination of LT{alpha}–/– and Rag–/– bone marrow, a combination of LT{alpha}–/– and JH–/– bone marrow, or a combination of C57BL/6 and JH–/– bone marrow as described in Materials and Methods. Following reconstitution, mice were sacrificed, and their small intestines were examined for the presence of iILFs (a) and mILFs (b) as described in Materials and Methods. Representative images of B220-stained whole mounts and UEA-I-stained whole mounts from each group are shown in c–f and g–j, respectively. k shows an enlarged view of a B220-stained whole mount demonstrating a cluster of B220+ cells at the base of a villus. Representative sections of H&E- and PNA-stained (green) sections of mILFs from LT{alpha}–/– mice reconstituted with wild-type bone marrow are shown in l and m, respectively. Consistent with a requirement for LT in the formation of ILFs, no iILFs or mILFs were identified in LT{alpha}–/– mice reconstituted with LT{alpha}–/– bone marrow. iILFs, but not mILFs were formed in LT{alpha}–/– recipients of a combination of LT{alpha}–/– and Rag–/– bone marrow and in LT{alpha}–/– recipients of a combination of LT{alpha}–/– and JH–/– bone marrow, documenting that LT-sufficient NK cells and T cells can supply the LT-dependent signals for iILF formation, but not mILF formation. LT{alpha}–/– recipients of wild-type bone marrow developed both iILFs and mILFs, confirming that LT-sufficient B lymphocytes are essential for the progression of iILFs to mILFs. Black arrows denote structures counted as iILFs, black arrowheads denote structures counted as mILFs, and blue arrowheads (k) outline a villus arising from a B220+ cluster. Original magnification: c–f, x20; g–j, x40; k–m, x200. *, p < 0.05 when compared with LT{alpha}–/– recipients of C57BL/6 and JH–/– bone marrow.

 
These findings are consistent with previous observations of an essential role for LT in ILF formation, because we observed iILF formation only when a cellular source of LT was supplied. Also consistent with this observation, we noted that, as the numbers of cell types expressing LT increased, the numbers of iILFs formed increased. Although the formation of iILFs was not dependent on LT-sufficient B lymphocytes, the progression from iILFs to mILFs was dependent on LT-sufficient B lymphocytes.

ILF formation does not require Ag-specific lymphocyte stimulation

The above findings demonstrate that LT-sufficient lymphocytes are required for the formation of iILFs, and the progression of iILFs to mILFs is driven by LT-sufficient B lymphocytes. LT is largely expressed by activated lymphocytes. In addition to up-regulation of LT expression, lymphocyte activation affects a number of properties that may be important for the formation of ILFs, including the regulation of adhesion molecules and chemokine receptor activity (13, 14, 15). To define the requirement for Ag-specific lymphocyte activation in ILF formation, we examined the intestines of TCR{beta}{delta}–/– mice and the intestines of MD4 BCR transgenic mice on a JH–/– background. Previous observations have demonstrated that T lymphocyte-deficient mice contain ILFs (4); however, these studies could not assess whether both iILF and mILFs were formed in the absence of T lymphocytes. Examination of the intestines of TCR{beta}{delta}–/– mice revealed that these animals possess iILFs and mILFs (Fig. 2), demonstrating that T lymphocytes are dispensable for the formation of ILFs. Consistent with previous reports regarding the presence of ILFs in nu/nu mice (4), we noted that the ILFs in the TCR{beta}{delta}–/– mice were generally smaller than those seen in their wild-type counterparts.

The MD4 mice possess a transgenic rearranged Ig L chain and Ig H chain linked to µ and {delta} C region genes of the IgHa allotype (10). The rearranged Ig gene is specific for HEL, is of the IgM or IgD isotype, and cannot undergo class switching to other isotypes. The MD4JH–/– mice, in comparison to MD4 mice on the wild-type background, express exclusively one BCR on all B lymphocytes. We have observed that MD4 mice on the wild-type background produce intestinal IgA at a level of 20% of that produced by wild-type mice due to endogenously expressed Ig genes (data not shown), whereas, in MD4JH–/– mice, there is no detectable intestinal IgA production (Fig. 2c). Examination of the intestines of MD4JH–/– mice revealed that these animals possess iILFs and mILFs (Fig. 2, a and b).

The mILFs in the MD4JH–/– mice are present in the absence of manipulations to ablate PP formation, whereas in C57BL/6 mice, significant numbers of mILFs are induced only following PP ablation. We feel this is consistent with a protective role for intestinal IgA in preventing inflammatory responses leading to the formation of mILFs. MD4JH–/– mice are unable to produce IgA, and C57BL/6 mice treated with LT{beta}R-Ig in utero, are relatively IgA deficient until 7 wk of age, a time at which mILF formation can easily be appreciated in these animals (Fig. 2c). Consistent with a relative IgA deficiency promoting mILF formation, we observed that TCR{beta}{delta}–/– mice have reduced IgA production when compared with age-matched wild-type mice, and have a reduced number of IgA-producing plasma cells in their small intestine (Fig. 2c).

ILF B lymphocytes have a naive phenotype and express membrane-bound LT in the absence of Ag-specific stimulation

In the absence of exogenous HEL, B lymphocytes in the MD4JH–/– mice do not receive stimulation through their BCR and therefore are Ag naive. Consistent with this, we noted that ILF B lymphocytes from the MD4JH–/– mice expressed the high-affinity receptor for HEL and had a naive phenotype expressing low levels of CD69 and CD86, and expressing high levels of CD23 (Fig. 3). ILF B lymphocytes from the MD4JH–/– mice expressed higher levels of MHCII when compared with splenocytes from the same animals, but this expression was still lower than that seen in splenocytes from MD4JH–/– mice stimulated with Ag (Fig. 3d). Although events independent of Ag stimulation can modulate the expression of these molecules, the overall pattern of expression of these molecules is supportive of an Ag-naive phenotype.



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  		FIGURE 3. ILF B lymphocytes have a naive phenotype and express cell surface LT in the absence of Ag stimulation. Single-cell suspensions from spleens, PP, and mILF were isolated from MD4JH–/– mice and analyzed by flow cytometry as described in Materials and Methods. ILF cellular populations were pooled from two mice for flow-cytometric analysis; splenic and PP populations were analyzed from individual mice. Consistent with an Ag-naive phenotype, B lymphocytes (CD19+ cells) from MD4JH–/– mILFs (filled gray histograms) expressed low levels of CD69 and CD86 and elevated levels of CD23 similar to the expression of these markers by MD4JH–/– splenic B lymphocytes (unfilled dark-line histograms) (a). MD4JH–/– mILF B lymphocytes expressed higher levels of MHCII when compared with freshly isolated MD4JH–/– splenic B lymphocytes; however, MHCII expression in MD4JH–/– splenocytes could be augmented above this level by overnight culture with HEL (unfilled light-line histograms). In a similar manner, culture of MD4JH–/– splenocytes with Ag up-regulated CD69 and CD86 expression and down-regulated CD23 expression on B lymphocytes, consistent with an Ag-activated phenotype (a). B lymphocytes from MD4JH–/– mILFs expressed high levels of a BCR-specific for HEL (b). B lymphocytes from each tissue expressed cell surface LT{beta} (d), a component of the membrane-bound LT complex (LT{alpha}1{beta}2). Notably, B lymphocytes represented the majority of LT-expressing cells in mILFs from MD4JH–/– mice (d). Consistent with this, CD19-enriched MD4JH–/– mILF B lymphocytes demonstrated increased expression of LT{alpha} when compared with CD19-depleted populations (c). Results shown are representative of one of two experiments. *, p < 0.05 when comparing CD19-enriched and -depleted populations.

 
Consistent with our findings of a requirement for LT-sufficient B lymphocytes in driving the progression of iILFs to mILFs, we observed that mILF B lymphocytes from MD4JH–/– mice express cell surface LT{beta} in the absence of HEL, and we observed that LT{alpha} mRNA expression was increased in MD4JH–/– mILF cell populations enriched for B lymphocytes (Fig. 3). We observed a modest increase in LT{beta} mRNA expression in the CD19-enriched population that was not significantly different from the CD19-depleted population (data not shown). These observations are consistent with expression of the membrane-bound LT because the cell surface expression of LT{beta} occurs only in association with LT{alpha} and therefore requires the expression of LT{alpha}, and LT{beta} exhibits constitutive mRNA expression in murine lymphocytes (16, 17).

mILFs express "homeostatic" chemokines in the absence of B lymphocyte Ag stimulation

A basal level of expression of chemokines has been described in secondary lymphoid structures. Due to this constitutive expression, these chemokines have been referred to as homeostatic chemokines and include the following: B lymphocyte chemoattractant (BLC), secondary lymphoid tissue chemokine (SLC), and EBV-induced molecule 1 ligand chemokine (ELC). These chemokines have several properties of interest relative to the formation of ILFs. The production of these chemokines in secondary lymphoid structures is diminished by abrogation of LT{beta}R signaling (18). The overexpression of these chemokines in transgenic animals results in lymphoid neogenesis (19, 20). And notably, BLC induces the expression of cell surface LT in Ag-naive B lymphocytes (8).

We examined the expression of these chemokines, and stromal cell-derived factor-1 (SDF-1) in spleen, mILFs, PP, and non-PP-, non-ILF-bearing intestine from MD4JH–/– mice. SDF-1 is a chemokine induced in response to inflammation, and has a capacity to attract naive T lymphocytes and B lymphocytes (21, 22). In addition, SDF-1 also has a capacity to induce LT expression in naive B lymphocytes (8). We observed that the mRNA expression of BLC and ELC were significantly increased in mILFs when compared with non-PP-, non-mILF-bearing small intestine (Fig. 4). SLC expression was also elevated in mILFs; however, this did not reach statistical significance. Parallel changes in BLC, SLC, and ELC expression were also seen in the spleen and PP. SDF-1 expression was increased in the spleen, but its expression was not increased in ILFs or PP when compared with non-PP-, non-ILF-bearing small intestine (Fig. 4).



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  		FIGURE 4. ILFs express homeostatic chemokines and LT in the absence of Ag-specific B lymphocyte stimulation. RNA was isolated from spleen, PP, mILFs, and non-PP-, non-mILF-bearing small intestine from MD4JH–/– mice, and used to evaluate differential target gene expression as described in Materials and Methods. Consistent with prior observations regarding the expression of homeostatic chemokines in secondary lymphoid structures, the spleen and PP demonstrated elevated expression of BLC, SLC, and ELC. ILFs also demonstrated elevated expression of BLC, SLC, and ELC when compared with non-PP-, non-ILF-bearing intestine. The spleen, but not PP, mILFs, nor non-PP-, non-ILF-bearing small intestine, demonstrated elevated expression of SDF-1, a chemokine that can be induced in response to inflammatory stimuli, and has been demonstrated to up-regulate LT expression in Ag-naive B lymphocytes. Tissues from three mice were pooled for analysis. The data presented are representative of one of two experiments. *, p < 0.05 when compared with results from non-PP-, non-mILF-bearing small intestine.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
ILFs are a recently appreciated member of the mucosal immune system. Previous studies have demonstrated that ILFs are organized, ectopic lymphoid structures that can be formed de novo in the intestine of adult animals (4, 6). These observations indicate that ILFs are tertiary lymphoid structures. Lymphoid neogenesis, the process leading to tertiary lymphoid structure formation, has been observed in a number of chronic inflammatory and autoimmune conditions including rheumatoid arthritis, Sjögren’s syndrome, primary sclerosing cholangitis, chronic hepatitis C infection, myasthenia gravis, multiple sclerosis, and Hashimoto’s thyroiditis (23, 24). Tertiary lymphoid structures have also been observed in a number of inflammatory conditions involving the gastrointestinal tract including inflammatory bowel disease, gastritis, and animal models of intestinal inflammation (2, 25, 26, 27). Although the role ILFs and other tertiary lymphoid structures play in immune responses is unclear, their architecture and composition suggest that these structures allow efficient interactions between Ag, APCs, and lymphocytes. It has been suggested that tertiary lymphoid structures may contribute to inappropriate immune responses by facilitating interactions of Ag, APCs, and lymphocytes in an environment that lacks the "normal" regulatory environment of secondary lymphoid structures (24). Given this hypothesis and the association of tertiary lymphoid structures with chronic inflammatory and autoimmune conditions, there has been considerable interest in understanding the steps leading to lymphoid neogenesis.

Similar to the formation of PP and other secondary lymphoid structures, the formation of ILFs is dependent on LT and LT{beta}R. The requirement for LT and LT{beta}R occurs early in ILF formation as LT- and LT{beta}R-deficient mice lack both mILFs and iILFs. We have previously observed that LT-sufficient B lymphocytes are required for mILF formation (6); whether other LT-sufficient lymphocytes can provide the source of LT for iILF formation has not been previously addressed. In this study, we demonstrate that, although iILF formation is dependent on LT-sufficient lymphocytes, it is not dependent on LT-sufficient B lymphocytes; LT-sufficient T lymphocytes and NK cells can provide the necessary source of LT for iILF formation. However, LT-sufficient B lymphocytes are essential for the progression of iILFs to mILFs. In contrast to mILF formation, LT-sufficient B lymphocytes are not required for secondary lymphoid structure formation, and LT-sufficient B lymphocytes are dispensable for the development of M cells in PP FAE (28). Recent studies have suggested an alternative cellular source of LT inducing the formation of iILFs, the adult counterpart of the fetal lymphoid tissue inducer (LTi) cell. Fetal LTi and their adult counterparts are among the few cell types that express the nuclear hormone receptor ROR{gamma}{tau} (29, 30). In the absence of ROR{gamma}{tau}, these cells do not develop, and consequently lymph nodes, PP, cryptopatches, and ILFs fail to form (29, 30). The fetal LTi cells express LT and are believed to be the cellular source of LT required during embryogenesis for the formation of secondary lymphoid structures. Although the expression of LT on the adult counterparts to the fetal LTi has not been formally investigated, it is felt that these cells may deliver the LT-dependent signal required for cryptopatch and ILF formation. Whether this signal occurs very early, forming cryptopatches or other iILF precursors, or whether it is the signal driving iILF formation is not clear. Our observations of increasing numbers of iILFs as we add increasing cellular sources of LT (NK cells vs NK cells and T lymphocytes vs NK cells, T lymphocytes, and B lymphocytes) would favor this step being driven by mature LT-expressing lymphocytes as opposed to the adult counterparts to LTi cells.

The differential requirements for LT-sufficient B lymphocytes in the formation of PP and ILFs not only provides a distinction between these structures, it also provides a focal point for understanding the events leading to lymphoid neogenesis in the small intestine. LT expression is induced in lymphocytes following activation. In B-2 B lymphocytes, which exclusively comprise the B lymphocyte population of mILFs, this activation occurs in response to CD40 ligation in the context of T cell help. BCR Ag binding, Ag internalization and processing, and cognate interactions of MHCII/peptide complexes with TCR are prerequisites for this process. We observed that ILF formation occurs in the absence of T lymphocytes and therefore in the absence of T cell help. Further supporting this observation, we demonstrate that ILF formation is preserved in the absence of BCR ligation by Ag, and that Ag-naive ILF B lymphocytes express LT. The form of LT, soluble (LT{alpha}3) or membrane-bound (LT{alpha}1{beta}2), driving this step is not known. We have previously demonstrated that TNFRI, a receptor for LT{alpha}3, is required for the transition of iILFs to mILFs (6), suggesting that LT-sufficient B lymphocytes may be the source of TNFRI ligands driving this transition. The findings we present here could be consistent with either soluble LT{alpha}3 or membrane-bound LT{alpha}1{beta}2 driving this step. Therefore, although the process of ILF formation is dependent on LT-sufficient lymphocytes, and specifically LT-sufficient B lymphocytes are essential for the progression of iILFs to mILFs, this process occurs in the absence of adaptive immune responses, and therefore is driven by the innate immune system.

Due to their constitutive expression in secondary lymphoid organs, SLC, ELC, and BLC have been referred to as homeostatic chemokines. These chemokines regulate lymphocyte entry and organization within secondary lymphoid structures (31). SLC and ELC are ligands for CCR7, which is highly expressed by naive T lymphocytes, maturing dendritic cells, and activated B lymphocytes (15, 32, 33, 34). BLC is the only known ligand for CXCR5, which is expressed by B lymphocytes and subsets of T lymphocytes (35, 36). Although the formation of tertiary lymphoid structures in CXCR5-deficient and CCR7-deficient mice has not been evaluated formally, the ectopic expression of BLC, SLC, and ELC in transgenic mice has been shown to induce ectopic organized aggregates of lymphocytes, suggesting that these chemokines play a significant role in lymphoid neogenesis (19, 20). Additional observations supporting a role for these chemokines in lymphoid neogenesis include the demonstration of BLC and/or SLC expression in tertiary lymphoid structures in ulcerative colitis, primary sclerosing cholangitis, Sjögren’s syndrome, and rheumatoid arthritis (23, 37, 38, 39).

LT and LT{beta}R have established roles in lymphoid neogenesis. Animal models demonstrated that overexpression of LT can induce lymphoid neogenesis, that LT- and LT{beta}R-deficient animals do not form tertiary lymphoid structures, and that tertiary lymphoid structure formation can be inhibited by continuous LT{beta}R blockade (6, 20, 40). One role that LT and LT{beta}R may play in this process is the production of homeostatic chemokines, because others have demonstrated that blockade of LT{beta}R signaling in wild-type mice reduces splenic expression of BLC, SLC, and ELC (18). Further evidence supporting a role for the production of these chemokines downstream of LT and the LT{beta}R in lymphoid neogenesis is the observation of increased BLC and SLC expression at sites of lymphoid neogenesis in transgenic mice with ectopic expression of LT{alpha} (40).

Additional studies have implicated that these chemokines also act upstream of the LT{beta}R in the formation of tertiary lymphoid structures. In transgenic mice with ectopic expression of BLC, lymphoid neogenesis was found to be LT{alpha} dependent, suggesting that LT may have a function downstream of BLC (20). Other studies have demonstrated that SLC and ELC can induce the expression of LT{alpha}1{beta}2 in naive CD4+ T lymphocytes (19). And, most relevant to our observations, BLC has been shown to induce LT{alpha}1{beta}2 expression in Ag-naive lymphocytes, setting up a positive-feedback loop in which BLC induces LT{alpha}1{beta}2 expression in Ag-naive B lymphocytes and these B lymphocytes induce further BLC production by ligation of the LT{beta}R on stromal cells (8). In this study, we demonstrate that ILF formation is independent of B lymphocyte Ag exposure and that ILF formation is arrested at an immature stage in the absence of LT-sufficient B lymphocytes. We also demonstrate that Ag-naive ILF B lymphocytes have elevated expression of cell surface LT, and that ILFs have increased expression of homeostatic chemokines in the absence of B lymphocyte Ag exposure. These observations implicate the existence of a BLC-driven positive-feedback loop in ILF formation and specifically in the progression of iILFs to mILFs.

Although mILFs are infrequent in unmanipulated C57BL/6 mice, their formation occurred spontaneously in MD4JH–/– mice and TCR{beta}{delta}–/– mice. Treatment of C57BL/6 mice with LT{beta}R-Ig in utero to ablate PP formation can induce ILF formation in C57BL/6 mice. Consistent with the hypothesis that mILF formation is induced by inflammatory responses precipitated by a relative deficiency of intestinal IgA, we observed that 7-wk-old C57BL/6 mice receiving LT{beta}R-Ig in utero were relatively deficient in intestinal IgA production when compared with unmanipulated C57BL/6 controls. In addition, we observed "spontaneous" formation of mILFs in MD4JH–/– mice, which lack the capacity to produce IgA, and in TCR{beta}{delta}–/– mice, which produce lower intestinal IgA levels when compared with their wild-type counterparts. In the absence of T cell help, the IgA produced is primarily, if not exclusively, derived from B-1 B lymphocytes. In comparison to B-2 B lymphocytes, B-1 B lymphocytes have a restricted V region repertoire, and undergo little-to-no somatic hypermutation, resulting in the production of Ab with relatively low affinity. Therefore, in addition to the reduced amount of IgA, the IgA produced by TCR{beta}{delta}–/– mice may be less effective at preventing an inflammatory response to lumenal bacteria. In support of this hypothesis, studies of mice deficient in activation-induced cytidine deaminase (AID), which have impaired class switch recombination and somatic hypermutation resulting in impaired IgA production (41), revealed that these mice have hyperplastic ILFs and altered bacterial flora (42). The ILF hyperplasia could be reversed with antibiotic treatment to decrease the bacterial flora, or normalization of the IgA production by the generation of parabiotic AID–/– and wild-type mice (43).

Recent studies investigated the ability of lumenal stimuli to influence the gut-associated lymphoid tissues, and suggested varying pathways in which these stimuli mediate their effects. The ability of exogenous stimuli to induce ILFs was demonstrated by the formation of mILFs in germfree mice reconstituted with normal cecal flora (6). Studies of AID-deficient mice support the role of bacterial flora in expanding ILFs in size and cellularity, and have suggested that B cell sensing of lumenal bacterial drives this ILF hyperplasia (42). Studies of BCR-deficient mice expressing limiting levels of the EBV protein LMP2A to rescue B lymphocyte survival in the absence of BCR signaling revealed that these mice developed germinal centers in their PP and mesenteric LN, but not in their spleen (44). The formation of germinal centers in the PP and mesenteric LN was driven by lumenal flora, because treating the mice with antibiotics could abolish these germinal centers (44). In addition, the formation of these germinal centers was dependent on the presence of T lymphocytes because germinal center formation was absent when these animals were bred onto a recombination activation gene-deficient background (44). This is most consistent with TCR-mediated recognition of a bacterial product because other studies demonstrated that TCR transgenic mice have an impaired ability to develop splenic germinal centers following immunization in the absence of their Ag (45). In the present study, we describe the formation of ILFs in the absence of BCR-dependent and TCR-dependent responses, and in conjunction with our previous studies demonstrating that the ILF formation can be driven by lumenal flora, we suggest that innate immune responses to lumenal bacteria are driving this event. The apparent differences between these studies can be reconciled by breaking ILF formation down into a series of events, all of which may be influenced by lumenal bacteria. The studies presented here indicate that the formation of ILFs, or the events leading to the accumulation of lymphocytes into a loosely organized collection with an overlying dome-shaped FAE, can occur independent of adaptive immune responses. Studies on AID–/– mice suggest that ILF expansion, or hyperplasia, can be driven by B lymphocytes "sensing" bacterial stimuli (42, 43). This expansion is consistent with the known responses of B lymphocytes to both cognate Ags, recognized through the BCR, and noncognate stimuli, such as LPS, leading to polyclonal activation and expansion. Although our studies indicate that adaptive immune responses are not required for the formation of ILFs, adaptive immune responses play a role in the function of ILFs. The function of ILFs is still being investigated, but current studies suggest that ILFs are sites of B-2 B lymphocyte stimulation, driving their subsequent differentiation into IgA-producing plasma cells that contribute to the production of intestinal IgA (5, 46). In this context, the development of a germinal center, which facilitates the stimulation and differentiation of B-2 B lymphocytes into plasma cells, would be a later event in ILF formation, and could be driven by cognate interactions of TCR with Ag/MHC complexes. In line with these observations, we have not been able to demonstrate the presence of germinal centers in mILFs from TCR{beta}{delta}–/– mice.

The findings presented here expand the previous model of ILF formation (Fig. 5). ILF formation occurs independent of adaptive immune responses and is driven by the innate immune system. Any LT-sufficient lymphocyte may deliver the necessary signals to allow accumulation of B lymphocytes into clusters at the base of villi and form iILFs; however, the progression of iILFs to mILFs is dependent on LT-sufficient B lymphocytes, is independent of Ag-specific stimulation of B lymphocytes, and occurs in the absence of T cell help. Consistent with previous observations, we propose that the progression of iILFs to mILFs is driven by a chemokine-driven positive-feedback loop.



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  		FIGURE 5. Putative sequence of events in ILF formation. Events elucidated by this study are indicated in bold type. The formation of iILFs and mILFs is driven by innate immune responses because it occurs in the absence of T lymphocytes and in the absence of Ag-specific B lymphocyte responses. The requirement for LT and LT{beta}R occurs early in this process, because LT{alpha}–/– mice and LT{beta}R–/– mice lack both iILFs and mILFs. The formation of iILFs can be rescued in LT{alpha}–/– mice by supplying a cellular source of LT; LT-sufficient T lymphocytes and/or LT-sufficient NK cells are sufficient sources of LT to drive the formation of iILFs. The progression of iILFs to mILFs requires LT-sufficient B lymphocytes, and occurs independent of Ag-specific stimulation of B lymphocytes. The Ag-independent expression of LT by B lymphocytes and the requirement for B lymphocyte LT expression is consistent with a chemokine-driven, positive-feedback loop driving the progression of iILFs to mILFs. Based upon prior observations, the formation of germinal centers is likely dependent on T lymphocyte responses.

 
These observations have several implications for the potential role of ILFs in the immune response. Because ILF formation is driven by the innate immune system, ILF formation should be reproducible in response to the appropriate inducing stimuli and should be able to occur throughout life. ILFs preferentially contain Ag-naive lymphocytes, suggesting that ILFs are sites of initial interactions of lymphocytes with Ag and APCs. Therefore, events occurring within ILFs will shape the phenotype of subsequent immune responses mediated by these lymphocytes.


   Acknowledgments
 
We thank W. Stenson, R. Lorenz, and E. Newberry for assistance with the preparation of this manuscript.


   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 This work was supported in part by National Institutes of Health Grants DK064798 and DK060648, the Broad Medical Research Program IBD-0042, and Washington University School of Medicine Digestive Diseases Research Core Center Grant P30-DK52574. Back

2 Address correspondence and reprint requests to Dr. Rodney D. Newberry, 660 South Euclid Avenue, Box 8124, St. Louis, MO 63110. E-mail address: rnewberry{at}im.wustl.edu Back

3 Abbreviations used in this paper: PP, Peyer’s patch; FAE, follicle-associated epithelium; ILF, isolated lymphoid follicle; iILF, immature ILF; mILF, mature ILF; LT, lymphotoxin; PNA, peanut lectin (agglutinin); HEL, hen egg white lysozyme; MHCII, MHC class II; BLC, B lymphocyte chemoattractant; SLC, secondary lymphoid tissue chemokine; ELC, EBV-induced molecule 1 ligand chemokine; SDF-1, stromal cell-derived factor-1; LTi, lymphoid tissue inducer; AID, activation-induced cytidine deaminase. Back

Received for publication August 16, 2004. Accepted for publication February 24, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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