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• W1980365355 abstract "Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6−/− B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer's patch B lymphocytes and an associated re-duction in the number and size of Peyer's patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development. Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6−/− B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer's patch B lymphocytes and an associated re-duction in the number and size of Peyer's patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development. The mucosal immune system is a complex network of lymphoid compartments generating immune responses that both protect the host and mitigate potential damage due to uncontrolled inflammation. In the gastrointestinal tract this system includes diffuse effector sites, such as the intestinal lamina propria, as well as organized lymphoid structures that collectively are referred to as the gastrointestinal-associated lymphoid tissues. Organized lymphoid structures provide sites for the efficient interactions of antigens, antigen-presenting cells, and lymphocytes in a controlled environment. These structures are essential for the initiation of primary immune responses and the regulated environment they provide is felt to be necessary to prevent inappropriate immune responses. Isolated lymphoid follicles (ILFs) have only recently become appreciated as distinct members of the gastrointestinal-associated lymphoid tissues. ILFs resemble Peyer's patches (PPs) in their architecture, cellular composition, and ability to act as inductive sites for mucosal immune responses.1Hamada H Hiroi T Nishiyama Y Takahashi H Masunaga Y Hachimura S Kaminogawa S Takahashi-Iwanaga H Iwanaga T Kiyono H Yamamoto H Ishikawa H Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.J Immunol. 2002; 168: 57-64PubMed Google Scholar, 2Lorenz RG Chaplin DD McDonald KG McDonough JS Newberry RD Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function.J Immunol. 2003; 170: 5475-5482PubMed Google Scholar, 3Shikina T Hiroi T Iwatani K Jang MH Fukuyama S Tamura M Kubo T Ishikawa H Kiyono H IgA class switch occurs in the organized nasopharynx- and gut-associated lymphoid tissue, but not in the diffuse lamina propria of airways and gut.J Immunol. 2004; 172: 6259-6264PubMed Google Scholar, 4Lorenz RG Newberry RD isolated lymphoid follicles can function as sites for induction of mucosal immune responses.Ann NY Acad Sci. 2004; 1029: 44-57Crossref PubMed Scopus (115) Google Scholar, 5Newberry RD Lorenz RG Organizing a mucosal defense.Immunol Rev. 2005; 206: 6-21Crossref PubMed Scopus (122) Google Scholar Our understanding of how these organized structures develop is rapidly evolving. PP formation, like lymph node formation, is developmentally driven, with the early vital events leading to PP formation occurring only during embryogenesis.6Rennert PD Browning JL Mebius R Mackay F Hochman PS Surface lymphotoxin alpha/beta complex is required for the development of peripheral lymphoid organs.J Exp Med. 1996; 184: 1999-2006Crossref PubMed Scopus (345) Google Scholar On the other hand, ILF formation occurs after birth and may be induced or augmented by luminal stimuli, including normal intestinal flora.1Hamada H Hiroi T Nishiyama Y Takahashi H Masunaga Y Hachimura S Kaminogawa S Takahashi-Iwanaga H Iwanaga T Kiyono H Yamamoto H Ishikawa H Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.J Immunol. 2002; 168: 57-64PubMed Google Scholar, 2Lorenz RG Chaplin DD McDonald KG McDonough JS Newberry RD Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function.J Immunol. 2003; 170: 5475-5482PubMed Google Scholar Further delineation of this process indicates that in the normal animal ILFs are a spectrum of lymphoid aggregates in various stages of formation and that cryptopatches (CPs) are the precursor lymphoid aggregates giving rise to ILFs.2Lorenz RG Chaplin DD McDonald KG McDonough JS Newberry RD Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function.J Immunol. 2003; 170: 5475-5482PubMed Google Scholar, 7Pabst O Herbrand H Worbs T Friedrichsen M Yan S Hoffmann MW Korner H Bernhardt G Pabst R Forster R Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes.Eur J Immunol. 2005; 35: 98-107Crossref PubMed Scopus (152) Google Scholar, 8Eberl G Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway?.Nat Rev Immunol. 2005; 5: 413-420Crossref PubMed Scopus (113) Google Scholar, 9Eberl G Littman DR Thymic origin of intestinal alphabeta T cells revealed by fate mapping of RORgammat+ cells.Science. 2004; 305: 248-251Crossref PubMed Scopus (407) Google Scholar Deficiency of CC chemokine receptor 6 (CCR6) has been demonstrated to adversely affect the development of mucosal immune responses,10Cook DN Prosser DM Forster R Zhang J Kuklin NA Abbondanzo SJ Niu XD Chen SC Manfra DJ Wiekowski MT Sullivan LM Smith SR Greenberg HB Narula SK Lipp M Lira SA CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue.Immunity. 2000; 12: 495-503Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar but the mechanisms resulting in this defect are still being elucidated.11Lügering A Floer M Westphal S Maaser C Spahn TW Schmidt MA Domschke W Williams IR Kucharzik T Absence of CCR6 inhibits CD4+ regulatory T-cell development and M-cell formation inside Peyer's patches.Am J Pathol. 2005; 166: 1647-1654Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Salazar-Gonzalez RM Niess JH Zammit DJ Ravindran R Srinivasan A Maxwell JR Stoklasek T Yadav R Williams IR Gu X McCormick BA Pazos MA Vella AT Lefrancois L Reinecker HC McSorley SJ CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer's patches.Immunity. 2006; 24: 623-632Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 13Varona R Villares R Carramolino L Goya I Zaballos A Gutierrez J Torres M Martinez AC Marquez G CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses.J Clin Invest. 2001; 107: R37-R45Crossref PubMed Scopus (190) Google Scholar, 14Zhao X Sato A Dela Cruz CS Linehan M Luegering A Kucharzik T Shirakawa AK Marquez G Farber JM Williams I Iwasaki A CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells.J Immunol. 2003; 171: 2797-2803PubMed Google Scholar In contrast to most chemokine receptors, CCR6 pairs monogamously with its ligand, CCL20.15Liao F Alderson R Su J Ullrich SJ Kreider BL Farber JM STRL22 is a receptor for the CC chemokine MIP-3alpha.Biochem Biophys Res Commun. 1997; 236: 212-217Crossref PubMed Scopus (86) Google Scholar β-Defensins have also been identified as potential ligands for CCR6; however, these ligands have a significantly reduced affinity for binding CCR6 and a reduced ability to induce CCR6-dependent chemotaxis when compared with CCL20.16Yang D Chertov O Bykovskaia SN Chen Q Buffo MJ Shogan J Anderson M Schroder JM Wang JM Howard OM Oppenheim JJ Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.Science. 1999; 286: 525-528Crossref PubMed Scopus (1516) Google Scholar CCL20 is preferentially produced at mucosal surfaces by a variety of cell types including monocytes, endothelial cells, dendritic cells, fibroblasts, and epithelial cells in the follicle-associated epithelium (FAE).17Tanaka Y Imai T Baba M Ishikawa I Uehira M Nomiyama H Yoshie O Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans.Eur J Immunol. 1999; 29: 633-642Crossref PubMed Scopus (175) Google Scholar Multiple cell types express CCR6, including immature dendritic cells, memory T lymphocytes, and B lymphocytes.15Liao F Alderson R Su J Ullrich SJ Kreider BL Farber JM STRL22 is a receptor for the CC chemokine MIP-3alpha.Biochem Biophys Res Commun. 1997; 236: 212-217Crossref PubMed Scopus (86) Google Scholar, 18Ebert LM McColl SR Up-regulation of CCR5 and CCR6 on distinct subpopulations of antigen-activated CD4+ T lymphocytes.J Immunol. 2002; 168: 65-72Crossref PubMed Scopus (67) Google Scholar, 19Liao F Rabin RL Smith CS Sharma G Nutman TB Farber JM CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3 alpha.J Immunol. 1999; 162: 186-194PubMed Google Scholar Most investigations into the role of CCR6 in mucosal immune responses have revolved around alterations in the function of PP dendritic cells in CCR6-deficient mice.11Lügering A Floer M Westphal S Maaser C Spahn TW Schmidt MA Domschke W Williams IR Kucharzik T Absence of CCR6 inhibits CD4+ regulatory T-cell development and M-cell formation inside Peyer's patches.Am J Pathol. 2005; 166: 1647-1654Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Salazar-Gonzalez RM Niess JH Zammit DJ Ravindran R Srinivasan A Maxwell JR Stoklasek T Yadav R Williams IR Gu X McCormick BA Pazos MA Vella AT Lefrancois L Reinecker HC McSorley SJ CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer's patches.Immunity. 2006; 24: 623-632Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 13Varona R Villares R Carramolino L Goya I Zaballos A Gutierrez J Torres M Martinez AC Marquez G CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses.J Clin Invest. 2001; 107: R37-R45Crossref PubMed Scopus (190) Google Scholar, 14Zhao X Sato A Dela Cruz CS Linehan M Luegering A Kucharzik T Shirakawa AK Marquez G Farber JM Williams I Iwasaki A CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells.J Immunol. 2003; 171: 2797-2803PubMed Google Scholar The contributions of other cell types and the effects of CCR6 deficiency on the development of ILFs have not been previously addressed. In this study, we evaluated the role for CCR6 in the development of ILFs. We observed elevated expression of CCR6 by ILF B lymphocytes when compared with B lymphocytes from other tissues and elevated expression of CCL20 within ILFs. We found that CCR6−/− mice have significantly reduced numbers of ILFs, but the formation of CPs, as well as CPs containing a large population of dendritic cells, was unaffected. Consistent with this block in ILF formation at a stage before the influx of B lymphocytes, we observed that CCR6−/− mice had a significantly reduced population of non-PP intestinal B lymphocytes, and this defect was most pronounced in the distal small intestine where we have observed the majority of ILFs in wild-type mice. Bone marrow reconstitution demonstrated that CCR6-sufficient B lymphocytes are required for the formation of ILFs, and adoptive transfer studies confirmed that CCR6−/− B lymphocytes have a deficiency in their ability to localize to wild-type PPs and CP/ILFs. Expanding previous reports, and paralleling our observations with ILFs, we observed that CCR6−/− mice have a reduced proportion of B lymphocytes within the PP with an associated decrease in the number of follicles within each PP and a decreased surface area of each PP follicular dome. These findings demonstrate a role for CCR6 expression by B lymphocytes in localizing these cells to organized intestinal lymphoid structures and suggest this requirement is particularly important for ILF development. The profound defect we observed in ILF development in CCR6−/− mice, coupled with the inflammatory nature of CCL20 expression, further highlights the dynamic nature of ILF development and suggests that CCL20 expression facilitates the transition of CPs into ILFs. Mice used for this study were housed in a specific pathogen-free facility and fed routine chow diet. Animal procedures and protocols were performed in accordance with the institutional review board at Washington University School of Medicine. C57BL/6 mice, C57BL/6 congenic mice (B6.Cg-IgHa Thy1a Gpi1a/J), and B-cell-deficient JH−/− mice20Gu H Zou YR Rajewsky K Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting.Cell. 1993; 73: 1155-1164Abstract Full Text PDF PubMed Scopus (815) Google Scholar on the C57BL/6 background were purchased from The Jackson Laboratory, Bar Harbor, ME. Lymphotoxin (LT)α-deficient mice21De Togni P Goellner J Ruddle NH Streeter PR Fick A Mariathasan S Smith SC Carlson R Shornick LP Strauss-Schoenberger J Russell JH Karr R Chaplin DD Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.Science. 1994; 264: 703-707Crossref PubMed Scopus (870) Google Scholar (a gift from Dr. Dave Chaplin, University of Alabama, Birmingham, AL) were bred onto the C57BL/6 background for more than 10 generations before use in experiments. CCR6-deficient mice22Kucharzik T Hudson 3rd, JT Waikel RL Martin WD Williams IR CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock-in mouse.Eur J Immunol. 2002; 32: 104-112Crossref PubMed Scopus (86) Google Scholar were bred on to the C57BL/6 background for four generations before use in experiments. Timed pregnant C57BL/6 female mice and CCR6−/− mice for use in experiments involving the injection of lymphotoxin β receptor (LTβR)-Ig fusion protein, were generated by matings with C57BL/6 male mice or CCR6−/− male mice, respectively. Six- to 10-week-old LTα −/− mice were used as recipients for bone marrow transfers. Bone marrow transfers were performed after lethal irradiation as previously described.23Newberry RD McDonough JS McDonald KG Lorenz RG Postgestational lymphotoxin/lymphotoxin beta receptor interactions are essential for the presence of intestinal B lymphocytes.J Immunol. 2002; 168: 4988-4997PubMed Google Scholar A total of 1 × 107 T-lymphocyte-depleted bone marrow cells from gender-matched donors were injected intravenously into recipients on the second day of irradiation. Mice receiving bone marrow from multiple donors (C57BL/6 and JH−/− or CCR6−/− and JH−/−; see Figure 4) received 5 × 106 cells from each donor. Mice were allowed 12 weeks 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 B- and T-lymphocyte reconstitution. LTRβ-Ig was purified from supernatants generated by a Chinese hamster ovary cell line producing the LTRβ-Ig fusion protein (a gift from Dr. W. Yokoyama, Washington University School of Medicine, St. Louis, MO) as previously described.23Newberry RD McDonough JS McDonald KG Lorenz RG Postgestational lymphotoxin/lymphotoxin beta receptor interactions are essential for the presence of intestinal B lymphocytes.J Immunol. 2002; 168: 4988-4997PubMed Google Scholar Timed pregnant female mice were injected with 100 μg of LTβR-Ig or 100 μg of human Ig (Bayer Corporation, Elkhart, IN) via tail vein on day 16 postcoitus. Mice receiving LTβR-Ig or human Ig in utero were analyzed for the presence of ILFs at 7 weeks of age (see Figure 2). Small intestines were removed intact, flushed with ice-cold phosphate-buffered saline (PBS), and opened along the mesenteric border. Intestines were mounted, lumen facing up, and fixed with ice-cold 10% phosphate-buffered formyl saline (Fisher Scientific, Pittsburgh, PA) for 1 hour at 4°C. Intestines were washed three times in ice-cold PBS, incubated in a solution of 20 mmol/L dithiothreitol, 150 mmol/L Tris, and 20% ethanol at room temperature for 45 minutes, washed three times in ice-cold PBS, and incubated in a solution of 1% H2O2 for 15 minutes at room temperature to block endogenous peroxidases. Intestines were washed three times in PBS, followed by incubation in PBS containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 for 30 minutes. Intestines were incubated with horseradish peroxidase-conjugated lectin from Ulex europaeus (UEA-I; Sigma-Aldrich, St. Louis, MO) in PBS, BSA, and Triton X-100 solution overnight at 4°C to facilitate the identification of PPs and mature ILFs (mILFs). The following day intestines were washed three times in PBS, incubated in DAB metal peroxide substrate (Pierce Chemical Co., Rockford, IL) for 15 minutes, rinsed twice in distilled water, and returned to PBS for further analysis. Investigators unaware of the treatment groups determined the presence of mILF. Under low-power microscopy (25 to 65×) previously established criteria were used to determine the presence of mILF2Lorenz RG Chaplin DD McDonald KG McDonough JS Newberry RD Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function.J Immunol. 2003; 170: 5475-5482PubMed Google Scholar: 1) presence of a nodular structure with size equal to or greater than the width of one villus; 2i) nodular structure possessing an overlying dome resembling the FAE of PP; and 3) nodular structures occurring singly or in groups of two. For the analysis of PPs, whole mounts of intestines were stained with UEA-I as above. The following criteria were used for the purpose of enumerating PPs. PPs were defined as nodular structures more than five villi wide, located along the anti-mesenteric border of the small intestine. These criteria overlap with the criteria used to enumerate mILFs but were necessary because some of the PPs of the CCR6−/− mice contained only one follicle. PPs were identified using a dissecting microscope at 32× magnification and photographs obtained of each PP. Photographs of PPs and a scale were enlarged to 10 × 12 inches, and the diameter of each dome was measured. The surface area of each dome was calculated using the following formula: surface area = π (diameter/2).2Lorenz RG Chaplin DD McDonald KG McDonough JS Newberry RD Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function.J Immunol. 2003; 170: 5475-5482PubMed Google Scholar For anti-B220 and anti-CD11c staining of whole mounts to determine the numbers of iILFs and CD11c+ clusters, intestines were removed intact, flushed with PBS, opened along the mesenteric border, and mounted as above. Intestines were then incubated three times in Hanks' balanced salt solution (BioWhittaker, Walkersville, MD) containing 5 mmol/L EDTA at 37°C with shaking for 10 minutes to remove epithelial cells. Intestines were then fixed in 10% phosphate-buffered formyl saline and treated with 1% H2O2 for 15 minutes at room temperature as above. Intestines were incubated in a solution of 50 mmol/L Tris, pH 7.2, 150 mmol/L NaCl, 0.6% Triton X-100, and 0.1% BSA for 1 hour at 4°C to block nonspecific antibody binding and then incubated with rat anti-mouse B220 antibody (PharMingen, San Diego, CA) or biotin-conjugated hamster anti-mouse CD11c (eBiosciences, San Diego, CA) diluted in the above solution overnight at 4°C. Intestines were washed three times in the above solution and incubated with a horseradish peroxidase-conjugated goat anti-rat IgG antibody or streptavidin-conjugated horseradish peroxidase (Jackson Immuno-Research Laboratories, West Grove, PA) diluted in the above solution at room temperature for 1 hour. Intestines were washed three times and incubated in DAB metal peroxide substrate as above. Intestine whole mounts were examined under a dissecting microscope at 25 to 65×. Immature ILFs (iILFs) were counted as clusters of B220+ cells occurring at the base of villi and not containing an overlying dome. Dendritic cell clusters were counted as clusters of CD11c+ cells occurring at the base of villi. Spleens and PPs were removed from unmanipulated C57BL/6 mice and disrupted by mechanical dissociation. Intestines were removed from C57BL/6 mice receiving LTβR-Ig in utero, flushed with ice-cold PBS, opened along the mesenteric border, and mounted with the lumen facing up in cold PBS, as described above. Using the dissecting microscope and a blunt-end 26-gauge needle and syringe, multiple mILFs were aspirated and placed in cold PBS. Red blood cells were lysed from cellular suspensions and then used for flow cytometric analysis as described below. Average yield of viable mononuclear ILF cells ranged from 3 to 7 × 105 cells/intestine. Single-cell suspensions were obtained as above and flow cytometric analysis performed as previously described.23Newberry RD McDonough JS McDonald KG Lorenz RG Postgestational lymphotoxin/lymphotoxin beta receptor interactions are essential for the presence of intestinal B lymphocytes.J Immunol. 2002; 168: 4988-4997PubMed Google Scholar Reagents used for analysis were fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated rat anti-mouse CD19, fluorescein isothiocyanate-conjugated anti-mouse CD45, streptavidin-conjugated phycoerythrin, appropriate isotype control antibodies (all from BD Biosciences, San Diego, CA), and rat anti-mouse CCR6 (R&D Systems, Minneapolis, MN). 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 antibody. Flow cytometric analysis for the expression of CCR6 was performed using directly conjugated anti-CCR6 antibodies in some replicates as well as anti-CCR6 antibodies with an anti-rat IgG secondary antibody (eBiosciences) to augment the fluorescence intensity in some replicates (Figure 1, c and d); both methods gave equivalent results. Paraffin-embedded sections containing PPs from whole-mount intestines (performed as described above) were deparaffinized, treated with antigen-unmasking solution (Vector Laboratories, Burlingame, CA), treated with avidin/biotin blocking kit (Vector Laboratories), washed three times in PBS, and blocked for 15 minutes 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 (PNA) (Sigma-Aldrich) or biotin-conjugated anti-B220 antibody (BD Biosciences) diluted in PBS containing 1% BSA and 0.1% Triton X-100 overnight at 4°C. Biotinyl-tyramide signal amplification (DuPont/NEN, Boston, MA) followed by incubation with streptavidin-conjugated cyanine 2 dye (Jackson ImmunoResearch) was used for detection of PNA staining. Anti-B220 staining was detected using streptavidin-conjugated cyanine 3 dye (Jackson ImmunoResearch). Sections were counterstained with Hoescht dye (Sigma-Aldrich) to visualize nuclei. Immunohistochemistry on frozen sections of intestine was used to enumerate CD90+ cellular clusters and to evaluate the localization of adoptively transferred B lymphocytes. Intestines were embedded in OCT compound and serial 8-μm sections were obtained from each block. Slides were fixed with a 1:1 solution of acetone and methanol for 15 minutes, dried, rehydrated, and blocked with PBS containing 1% BSA. Sections were incubated with primary antibodies overnight at 4°C, washed with PBS, and incubated with fluorescently labeled secondary reagents for 1 hour at room temperature. Sections were stained with Hoescht dye (Sigma-Aldrich) to visualize nuclei. Segments of small intestine of 1.5 cm were embedded in OCT compound, frozen, and cut at an axis perpendicular to the villi into 8-μm sections. The segments comprised more than one-half of the entire intestine, and identical areas of the intestine were obtained from each animal evaluated. Two serial sections of small intestine were stained with anti-CD90 (eBioscience) and anti-CD3 (eBioscience) as above using two-color immunofluorescence. The serial sections were examined at a magnification of 100× or higher, and the number of CD90+CD3− cellular clusters falling within the crypt area on each section was counted and averaged between the serial sections. Additional sections were stained with anti-CD90 and anti-c-kit to confirm that the CD90 clusters are also c-kit+, stained with anti-B220 to assess the numbers of B lymphocytes associating with the CD90 clusters (Figure 2, d and e), and stained with anti-CD11c (BD Biosciences) to assess the clustering of dendritic cells associated with the CD90 clusters. Photomicrographs of the sections taken with a 25× objective were assembled, and the crypt area of each section was determined using AxioVision software (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). The average number of CD90+ clusters/mm2 of crypt surface area was then determined. PPs were removed from unmanipulated C57BL/6 mice. Mature ILFs were isolated from C57BL/6 mice receiving LTβR-Ig in utero using a dissection microscope and 26-gauge needle as described above. Non-PP, non-mILF-bearing intestine from the distal small intestine of C57BL/6 mice was identified using a dissecting microscope and removed. The PP and mILF tissue contained the overlying FAE, stromal elements, and mononuclear cells. RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and treated with DNase I (Ambion, Austin, TX) to remove contaminating DNA, and cDNA was synthesized from 2 μg of total RNA using Superscript II RNase H− reverse transcriptase (Invitrogen). Expression of targets was detected by real-time polymerase chain reaction using ABI prism 7700 sequence detection system and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). 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′, β-actin 5′-GCTTCTTTGCAGCTCCTTCGT-3′ and 5′-ATATCGTCATCCATGGCGAAC-3′, CCL20 5′-TGATGCTTTTTTGGGATGGAA-3′ and 5′-AGCCTTCAACCCCAGCTGT-3′, and CCR6 5′-TGTTCTGCTATCTGTTCATTATCAAGA-3′ and 5′-CACGACTCGGATGGCTCTGT-3′. Samples were measured in triplicate. Relative quantitation of target expression using 18S or β-actin as a housekeeping gene was determined using the comparative crossing threshold method as described in the ABI Prism 7700 sequence detection system user bulletin. To assess the ability of wild-type and CCR6−/− B lymphocytes to localize to the organized intestinal lymphoid tissues, splenocytes isolated from CCR6−/−, C57BL/6 congenic mice (expressing the IgHa allotype) were injected intravenously into JH−/− (B lymphocyte-deficient) recipients. Intestinal B lymphocytes could not be used for these studies because the CCR6−/− mice had few intestinal B lymphocytes (Figure 2f). Recipients received a total 3 × 107 cells from either CCR6−/− mice, C57BL6 congenic mice, or combined CCR6−/− and C57BL/6 congenic mice. Flow cytometric analysis was performed on the cellular populations at the time of transfer to confirm that equivalent numbers of B lymphocytes from each donor were injected into recipients. Mice were sacrificed 1 week later and frozen sections from PPs and small intestines were evaluated for the presence of transferred cellular populations using immunohistoch" @default.
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• W1980365355 date "2007-04-01" @default.
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• W1980365355 title "CC Chemokine Receptor 6 Expression by B Lymphocytes Is Essential for the Development of Isolated Lymphoid Follicles" @default.
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• W1980365355 doi "https://doi.org/10.2353/ajpath.2007.060817" @default.
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