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• W2000951661 abstract "The mouse intestinal epithelium undergoes continuous renewal throughout life. Intraepithelial lymphocytes (IELs) represent a significant fraction of this epithelium and play an important role in intestinal mucosal barrier function. We have generated a germ-free transgenic mouse model to examine the effects of a genetically engineered proliferative abnormality in the principal epithelial cell lineage (enterocytes) on IEL census and on IEL-enterocytic cross-talk. SV40 large T antigen (TAgWt) or a mutant derivative (TAgK107/8) that does not bind pRB was expressed in small intestinal villus enterocytes under the control of elements from the intestinal fatty acid binding protein gene (Fabpi). Quantitative immunohistochemical and flow cytometric analyses of conventionally raised and germ-free FVB/NFabpi-TAgWt,Fabpi-TAgK107/8, and nontransgenic mice disclosed that forced reentry of enterocytes into the cell cycle is accompanied by an influx of thymically educated αβ T cell receptor (TCR)+ CD4+ and αβ TCR+CD8αβ+ IELs and a decrease in intestinally derived γδ TCR+ CD8αα IELs. Real time quantitative reverse transcriptase-PCR studies of jejunal villus epithelium recovered from germ-free transgenic and normal mice by laser capture microdissection and γδ TCR+ jejunal IELs purified by flow cytometry disclosed that the proliferative abnormality is accompanied by decreased expression of enterocytic interleukin-7 as well as IEL interleukin-7Rα and transforming growth factor β3. The analysis also revealed that normal villus epithelium expresses Fms-like tyrosine kinase 3 (Flt3), a known regulator of hematopoietic stem cell proliferation and neuronal cell survival, and its ligand (Flt3L). Epithelial expression of this receptor and its ligand is reduced by the proliferative abnormality, whereas IEL expression of Flt3L remains constant. Together, these findings demonstrate that changes in the proliferative status of the intestinal epithelium affects maturation of γδ TCR+ IELs and produces an influx of αβ TCR+ IELs even in the absence of a microflora. The mouse intestinal epithelium undergoes continuous renewal throughout life. Intraepithelial lymphocytes (IELs) represent a significant fraction of this epithelium and play an important role in intestinal mucosal barrier function. We have generated a germ-free transgenic mouse model to examine the effects of a genetically engineered proliferative abnormality in the principal epithelial cell lineage (enterocytes) on IEL census and on IEL-enterocytic cross-talk. SV40 large T antigen (TAgWt) or a mutant derivative (TAgK107/8) that does not bind pRB was expressed in small intestinal villus enterocytes under the control of elements from the intestinal fatty acid binding protein gene (Fabpi). Quantitative immunohistochemical and flow cytometric analyses of conventionally raised and germ-free FVB/NFabpi-TAgWt,Fabpi-TAgK107/8, and nontransgenic mice disclosed that forced reentry of enterocytes into the cell cycle is accompanied by an influx of thymically educated αβ T cell receptor (TCR)+ CD4+ and αβ TCR+CD8αβ+ IELs and a decrease in intestinally derived γδ TCR+ CD8αα IELs. Real time quantitative reverse transcriptase-PCR studies of jejunal villus epithelium recovered from germ-free transgenic and normal mice by laser capture microdissection and γδ TCR+ jejunal IELs purified by flow cytometry disclosed that the proliferative abnormality is accompanied by decreased expression of enterocytic interleukin-7 as well as IEL interleukin-7Rα and transforming growth factor β3. The analysis also revealed that normal villus epithelium expresses Fms-like tyrosine kinase 3 (Flt3), a known regulator of hematopoietic stem cell proliferation and neuronal cell survival, and its ligand (Flt3L). Epithelial expression of this receptor and its ligand is reduced by the proliferative abnormality, whereas IEL expression of Flt3L remains constant. Together, these findings demonstrate that changes in the proliferative status of the intestinal epithelium affects maturation of γδ TCR+ IELs and produces an influx of αβ TCR+ IELs even in the absence of a microflora. intraepithelial lymphocyte T cell receptor interleukin-7 interleukin-7 receptor SV40 large T antigen mutant TAg with Glu → Lys substitution at positions 107 and 108 fluorescence activated cell sorting laser capture microdissection real time quantitative reverse transcriptase-PCR Fms-like tyrosine kinase 3 receptor ligand for Fms-like tyrosine kinase 3 receptor bromodeoxyuridine phosphate-buffered saline fluorescein isothiocyanate phycoerythrin transforming growth factor The adult mouse small intestine is a complex, spatially diversified ecosystem that maintains distinctive cephalocaudal differences in its various functions. This regional variation in function is accompanied by regional differences in the differentiation programs of its four continuously renewing epithelial cell lineages, in the composition of its mucosal immune system, and in the composition of its resident society of commensal/symbiotic microorganisms (reviewed in Refs. 1Falk P.G. Hooper L.V. Midtvedt T. Gordon J.I. Microbiol. Mol. Biol. Rev. 1998; 62: 1157-1170Crossref PubMed Google Scholar, 2Hooper L.V. Gordon J.I. Science. 2001; 292: 1115-1118Crossref PubMed Scopus (1823) Google Scholar, 3McCracken V.J. Lorenz R.G. Cell Microbiol. 2001; 3: 1-11Crossref PubMed Scopus (253) Google Scholar). A full understanding of how this ecosystem is organized and functions in health and how it is reorganized or disorganized in various disease states requires knowledge about the nature and regulation of interactions between its microflora, epithelium, and gut-associated lymphoid tissue (1Falk P.G. Hooper L.V. Midtvedt T. Gordon J.I. Microbiol. Mol. Biol. Rev. 1998; 62: 1157-1170Crossref PubMed Google Scholar, 4Gordon J.I. Hooper L.V. McNevin M.S. Wong M. Bry L. Am. J. Physiol. 1997; 273: G565-G570PubMed Google Scholar, 5Hooper L.V. Wong M.H. Thelin A. Hansson L. Falk P.G. Gordon J.I. Science. 2001; 291: 881-884Crossref PubMed Scopus (1731) Google Scholar). The molecular nature and significance of the signals exchanged between these components have been difficult to decipher because of the dynamic quality and complexity of the system. One way of approaching this problem is to simplify the ecosystem using inbred strains of mice with defined microbiological status (gnotobiotic animals). For example, comparative functional genomics studies of mice containing no resident microorganisms (germ-free), conventionally raised mice harboring an complete microflora, and germ-free animals that have been colonized with a single species from the normal microflora (ex-germ-free) have shown that indigenous commensal bacteria play an important role in regulating host nutrient processing, fortifying the epithelial barrier, and organizing/educating the mucosal immune system (5Hooper L.V. Wong M.H. Thelin A. Hansson L. Falk P.G. Gordon J.I. Science. 2001; 291: 881-884Crossref PubMed Scopus (1731) Google Scholar, 6Hooper L.V. Mills J.C. Roth K.A. Stappenbeck T.S. Wong M.H. Gordon J.I. Sansonetti P.J. Zychlinsky A. Methods in Microbiology: Molecular Cellular Microbiology. Academic Press, London2002: 559-589Google Scholar). The intestine contains a large population of intraepithelial lymphocytes (IELs),1equivalent in size to the population of peripheral lymphocytes that resides in the spleen (7Kaufmann S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2272-2279Crossref PubMed Scopus (283) Google Scholar). IELs are distributed throughout the epithelium that overlies small intestinal villi (average of one IEL for every 6–10 epithelial cells (8Beagley K.W. Husband A.J. Crit. Rev. Immunol. 1998; 18: 237-254Crossref PubMed Google Scholar)). Virtually all small intestinal IELs are T cells, but they are heterogeneous with respect to their surface phenotype. The majority are CD3+ and can be divided into αβ T cell receptor-positive (TCR+) and γδ TCR+ subsets (8Beagley K.W. Husband A.J. Crit. Rev. Immunol. 1998; 18: 237-254Crossref PubMed Google Scholar). They can be further subdivided based on expression of CD8 (αα homodimer or αβ heterodimer) or CD4 coreceptors (i.e. (i) γδ TCR+CD8α− CD8β−; (ii) γδ TCR+CD8α+ CD8β− (abbreviated γδ+ TCR+ CD8αα); (iii) αβ TCR+ CD4+; (iv) αβ TCR+CD8α+ CD8β− (αβ TCR+CD8αα); and (v) αβTCR+ CD8α+CD8β+ (αβTCR+ CD8αβ)). Studies of Rag1 −/− mice injected with bone marrow from nude mice or peripheral lymph node T cells from euthymic mice demonstrated that generation of αβ TCR+CD4+ and CD8+ IELs is thymus-dependent, whereas γδ TCR+CD8αα+ IELs appeared in the absence of a thymus (9Rocha B. Vassalli P. Guy-Grand D. Immunol. Today. 1992; 13: 449-454Abstract Full Text PDF PubMed Scopus (157) Google Scholar). One site of extrathymic maturation may be the crypts of Lieberkuhn. These distinct mucosal invaginations surround the base of each villus and contain long-lived multipotent stem cells (10Wong M.H. Saam J.R. Stappenbeck T.S. Rexer C.H. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12601-12606Crossref PubMed Scopus (113) Google Scholar) that give rise to the four epithelial lineages of the small intestine: enterocytes, goblet, and enteroendocrine cells, which differentiate as they migrate from the crypt up adjacent villi; and Paneth cells, which differentiate and remain at the crypt base (11Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 537-561Crossref PubMed Scopus (1120) Google Scholar, 12Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 461-479Crossref PubMed Scopus (545) Google Scholar, 13Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 503-519Crossref PubMed Scopus (247) Google Scholar, 14Nichols D.B. Cheng H. Leblond C.P. J. Histochem. Cytochem. 1974; 22: 929-944Crossref PubMed Scopus (22) Google Scholar, 15Cheng H. Merzel J. Leblond C.P. Am. J. Anat. 1969; 126: 507-525Crossref PubMed Scopus (76) Google Scholar). Crypts possess structures (cryptopatches) that contain clusters of c-Kit+interleukin-7 receptor (IL-7R)+ Thy1+lymphocytes (16Kanamori Y. Ishimaru K. Nanno M. Maki K. Ikuta K. Nariuchi H. Ishikawa H. J. Exp. Med. 1996; 184: 1449-1459Crossref PubMed Scopus (376) Google Scholar). Mice with a truncated mutation of the common cytokine receptor chain (17Ohbo K. Asao H. Kouro T. Nakamura M. Takaki S. Kikuchi Y. Hirokawa K. Tominaga A. Takatsu K. Sugamura K. Int. Immunol. 1996; 8: 951-960Crossref PubMed Scopus (3) Google Scholar) lack these cryptopatches and do not have γδ TCR+ CD8αα+ IELs but contain thymus-dependent αβ TCR+ CD4+and αβ TCR+ CD8αβ+ IELs, suggesting a role for cryptopatches in maturation of extrathymically derived γδ TCR+ IELs (18Oida T. Suzuki K. Nanno M. Kanamori Y. Saito H. Kubota E. Kato S. Itoh M. Kaminogawa S. Ishikawa H. J. Immunol. 2000; 164: 3616-3626Crossref PubMed Scopus (66) Google Scholar, 19Suzuki K. Oida T. Hamada H. Hitotsumatsu O. Watanabe M. Hibi T. Yamamoto H. Kubota E. Kaminogawa S. Ishikawa H. Immunity. 2000; 13: 691-702Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20Lambolez F. Azogui O. Joret A.M. Garcia C. von Boehmer H., Di Santo J. Ezine S. Rocha B. J. Exp. Med. 2002; 195: 437-449Crossref PubMed Scopus (88) Google Scholar). The epithelium also appears to play a direct role in regulating IEL development. Epithelial cells produce stem cell factor (21Godfrey D.I. Zlotnik A. Suda T. J. Immunol. 1992; 149: 2281-2285PubMed Google Scholar), a ligand for the c-Kit receptor expressed on the surface of γδ TCR+ IELs (22Puddington L. Olson S. Lefrancois L. Immunity. 1994; 1: 733-739Abstract Full Text PDF PubMed Scopus (117) Google Scholar). Mice deficient in either stem cell factor or c-Kit have reduced numbers of γδ TCR+ IELs (22Puddington L. Olson S. Lefrancois L. Immunity. 1994; 1: 733-739Abstract Full Text PDF PubMed Scopus (117) Google Scholar). Furthermore, thyrotropin-releasing hormone stimulation of enterocytes results in local release of thyroid-stimulating hormone, which interacts with IEL-based thyroid-stimulating hormone receptor to promote IEL development (23Wang J. Whetsell M. Klein J.R. Science. 1997; 275: 1937-1939Crossref PubMed Scopus (117) Google Scholar) (e.g. hyt/hyt mice, which have a loss-of-function thyroid-stimulating hormone receptor mutation, have disrupted IEL maturation) (24Stein S.A. Zakarija M. McKenzie J.M. Shanklin D.R. Palnitkar M.B. Adams P.M. Thyroid. 1991; 1: 257-266Crossref PubMed Scopus (21) Google Scholar, 25Stein S.A. Oates E.L. Hall C.R. Grumbles R.M. Fernandez L.M. Taylor N.A. Puett D. Jin S. Mol. Endocrinol. 1994; 8: 129-138Crossref PubMed Scopus (188) Google Scholar). Epithelium-based IL-7 provides another regulatory signal for IEL proliferation (26Fujihashi K. McGhee J.R. Yamamoto M. Peschon J.J. Kiyono H. Eur. J. Immunol. 1997; 27: 2133-2138Crossref PubMed Scopus (43) Google Scholar). Studies of mice that lack IL-7 or the IL-7R have demonstrated that IL-7R-mediated signaling is essential for γδ TCR+ IEL development (26Fujihashi K. McGhee J.R. Yamamoto M. Peschon J.J. Kiyono H. Eur. J. Immunol. 1997; 27: 2133-2138Crossref PubMed Scopus (43) Google Scholar, 27Maki K. Sunaga S. Komagata Y. Kodaira Y. Mabuchi A. Karasuyama H. Yokomuro K. Miyazaki J.I. Ikuta K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7172-7177Crossref PubMed Scopus (288) Google Scholar). Moreover, Laky et al. (28Laky K. Lefrancois L. Lingenheld E.G. Ishikawa H. Lewis J.M. Olson S. Suzuki K. Tigelaar R.E. Puddington L. J. Exp. Med. 2000; 191: 1569-1580Crossref PubMed Scopus (130) Google Scholar) used transcriptional regulatory elements from the rat intestinal fatty acid-binding protein (Fabpi) to express IL-7 in the villus enterocytes of Il-7−/−mice. γδ TCR+ IELs were restored in the intestinal epithelium but remained absent from all other tissues, indicating that local production of IL-7 was sufficient for proper development/survival of this IEL subset. Interactions between intestinal epithelial cells and IELs are reciprocal; IELs can influence epithelial cell biology. One illustration of this reciprocity is provided by TCRδ subunit-deficient mice. These animals have reduced numbers of dividing cells in their crypts of Lieberkuhn and reduced crypt cellularity (29Komano H. Fujiura Y. Kawaguchi M. Matsumoto S. Hashimoto Y. Obana S. Mombaerts P. Tonegawa S. Yamamoto H. Itohara S. Nanno M. Ishikawa H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6147-6151Crossref PubMed Scopus (317) Google Scholar) and exhibit more severe intestinal epithelial damage following infection with the parasite Eimeria vermiformis (30Roberts S.J. Smith A.L. West A.B. Wen L. Findly R.C. Owen M.J. Hayday A.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11774-11779Crossref PubMed Scopus (211) Google Scholar). γδ TCR+ IELs produce keratinocyte growth factor, which affects epithelial cell growth and repair (31Boismenu R. Havran W.L. Science. 1994; 266: 1253-1255Crossref PubMed Scopus (569) Google Scholar). These findings raise the question of whether γδ TCR+ IELs form part of a homeostatic surveillance mechanism that can detect and respond to perturbations in intestinal epithelial proliferation in order to maintain steady state cellular census in crypts and their associated villi. Some workers have proposed that IELs are key elements in a “mucosal intranet,” where they function to control epithelial integrity and immunologic homeostasis (32Yamamoto M. Fujihashi K. Kawabata K. McGhee J.R. Kiyono H. J. Immunol. 1998; 160: 2188-2196PubMed Google Scholar). Recent comparative DNA microarray-based studies of gene expression in γδ TCR+ IELs harvested from the small intestines of conventionally raised adult C57Bl6/J mice and αβ TCR+ cells harvested from their mesenteric lymph nodes have provided a list of candidate factors, preferentially expressed by γδ TCR+ IELs, that may support this mucosal intranet (33Fahrer A.M. Konigshofer Y. Kerr E.M. Ghandour G. Mack D.H. Davis M.M. Chien Y.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10261-10266Crossref PubMed Scopus (140) Google Scholar, 34Shires J. Theodoridis E. Hayday A.C. Immunity. 2001; 15: 419-434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In the present study, we examine the cross-talk between IELs and epithelium using transgenic mice that express simian virus 40 large T antigen (TAgWt) in their villus-associated enterocytes. The rationale for our experimental approach was as follows.Fabpi-directed expression of TAgWt produces a proliferative abnormality restricted to villus enterocytes:Fabpi-reporter transgenes are not expressed in the IELs. Expression of the viral oncoprotein in postmitotic enterocytes induces their reentry into the cell cycle (35Chandrasekaran C. Coopersmith C.M. Gordon J.I. J. Biol. Chem. 1996; 271: 28414-28421Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and an associated p53-independent apoptosis (36Coopersmith C.M. Chandrasekaran C. McNevin M.S. Gordon J.I. J. Cell Biol. 1997; 138: 167-179Crossref PubMed Scopus (20) Google Scholar) but is not accompanied by evidence of dysplasia during the 1–2-day interval that they take to complete their migration to the cellular extrusion zone located at the villus tip (36Coopersmith C.M. Chandrasekaran C. McNevin M.S. Gordon J.I. J. Cell Biol. 1997; 138: 167-179Crossref PubMed Scopus (20) Google Scholar,37Falk P. Roth K.A. Gordon J.I. Am. J. Physiol. 1994; 266: G987-G1003Crossref PubMed Google Scholar). Fabpi-directed expression of a mutant TAg containing a Glu → Lys substitution at residues 107 and 108 (TAgK107/8) disrupts pRB binding that does not produce this proliferative abnormality. Thus, a three-way comparison of FVB/NFabpi-TAgWt andFabpi-TAgK107/8 transgenic mice and their age-matched nontransgenic littermates would allow direct assessment of whether a proliferative abnormality limited to the predominant intestinal epithelial lineage is accompanied by changes in the fractional representation of extrathymically educated or thymically derived IEL subsets. By performing this analysis in conventionally raised and germ-free mice, we could also determine whether the microflora contributed to any observed changes in IELs. Finally, by using laser capture microdissection (LCM) of small intestinal cryosections to harvest villus epithelium, flow cytometry to retrieve their IELs, and the DNA microarray-based data sets of IEL gene expression to direct quantitative reverse transcription-PCR measurements of the levels of specified mRNAs in each cell population, we could use this environmentally well defined system to identify enterocytic gene products affected by proliferative status that may impact on IEL development/survival. Our results show that the engineered proliferative abnormality is accompanied by a microflora-independent reduction in extrathymically educated γδ TCR+ CD8αα+ IELs. This change is accompanied by coordinate changes in the expression of enterocytic and γδ TCR +IEL gene products that probably help legislate the observed change in IEL representation. FVB/N mice hemizygous for a transgene containing nucleotides −1178 to +28 of rat Fabpi linked to TAgWt or TAgK107/8 are described in earlier reports (35Chandrasekaran C. Coopersmith C.M. Gordon J.I. J. Biol. Chem. 1996; 271: 28414-28421Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 36Coopersmith C.M. Chandrasekaran C. McNevin M.S. Gordon J.I. J. Cell Biol. 1997; 138: 167-179Crossref PubMed Scopus (20) Google Scholar, 38Kim S.H. Roth K.A. Moser A.R. Gordon J.I. J. Cell Biol. 1993; 123: 877-893Crossref PubMed Scopus (97) Google Scholar). Conventionally raised animals were maintained in microisolators in a specified pathogen-free state. Normal and transgenic mice were rederived as germ-free by Caesarian section of transgenic mothers and transfer of their embryonic day 19 fetuses to plastic gnotobiotic isolators (Standard Safety Equipment Co.) containing germ-free foster mothers. The protocol used for this rederivation is described in a recent publication (6Hooper L.V. Mills J.C. Roth K.A. Stappenbeck T.S. Wong M.H. Gordon J.I. Sansonetti P.J. Zychlinsky A. Methods in Microbiology: Molecular Cellular Microbiology. Academic Press, London2002: 559-589Google Scholar). Both conventionally raised and germ-free mice were given sterilized BeeKay Autoclavable Diet (B & K Universal Inc.) ad libitum. All animals were maintained under a strict light cycle (lights on at 0600 h and off at 1800 h). Animals were genotyped using primers, tail DNA, and PCR conditions described in Ref. 36Coopersmith C.M. Chandrasekaran C. McNevin M.S. Gordon J.I. J. Cell Biol. 1997; 138: 167-179Crossref PubMed Scopus (20) Google Scholar. Some mice received an intraperitoneal injection of an aqueous solution of 5-bromo-2′-deoxyuridine (BrdUrd; 120 mg/kg) and 5-fluoro-2′-deoxyuridine (12 mg/kg) (Sigma) 90 min prior to sacrifice. Only male mice were studied. FVB/N transgenic mice and their wild type littermates were sacrificed at 6–8 weeks of age (n = 3 conventionally raised or germ-free animals/genotype/experiment;n = 3 independent experiments). The middle third of their small intestine (arbitrarily defined as jejunum) was immediately flushed with PBS and subdivided into five equal length segments. All were segments placed together in a tissue cassette, overlaid with OCT (Miles Scientific), and frozen in Cytocool II (Stephens Scientific). 100 serial 5-μm thick sections were cut parallel to the cephalocaudal axes of the segments. For each antibody surveyed, every 10th section was fixed for 20 min in methanol (at −20 °C), washed three times in PBS (3 min/cycle), and treated with PBS-blocking buffer (1% bovine serum albumin, 0.05% Triton X-100 in PBS) for 2 h at room temperature. Sections were subsequently treated three times with TNT wash buffer (0.1 m Tris, pH 7.5, 0.15 mNaCl, 0.05% Tween 20; three cycles; 5 min/cycle) and then incubated overnight at 4 °C with each of the following monoclonal antibodies (all from BD PharMingen, each diluted 1:1000 in TNB-blocking buffer (0.1 m Tris (pH 7.5), 0.15 m NaCl, and 0.5% blocking reagent from PerkinElmer Life Sciences)): (i) rat anti-mouse CD4 (clone H129.19); (ii) rat anti-mouse CD8α (clone 53-6.7); (iii) rat anti-mouse CD8β (clone Ly-32); (iv) hamster anti-mouse αβ TCR (β chain; clone H57-597); (v) hamster anti-mouse γδ TCR (δ chain; clone GL3); and (vi) hamster anti-mouse CD103 (integrin αIEL chain; clone 2E7). Following incubation with these primary antibodies, sections were washed in TNT buffer (three cycles, each 5 min). Biotin-conjugated mouse anti-rat IgG1/IgG2a (BD PharMingen) or biotin-conjugated mouse anti-hamster IgG mixture (BD PharMingen) was added (final dilution of each = 1:100 in TNB blocking buffer). After a 30-min incubation with the secondary antibodies at room temperature, sections were treated three times with TNT wash buffer (5 min/wash cycle). The sections were then incubated for 30 min at room temperature with streptavidin-horseradish peroxidase (PerkinElmer Life Sciences; 1:1000 in TNB) followed by three washes of 5 min each in TNT buffer. The final steps consisted of (i) adding biotinyl-tyramide (PerkinElmer Life Sciences; diluted 1:100 in 1× amplification diluent from the same manufacturer) for 10 min; (ii) washing three times with TNT buffer (5 min/cycle); (iii) incubating the section with indocarbocyanine (Cy3)-conjugated streptavidin (PerkinElmer Life Sciences; diluted 1:500 in TNB) for 30 min, and (iv) performing three final rinses in TNT buffer. Two controls were performed to verify the specificity of the signals produced: (i) direct amplification of endogenous peroxidase activity alone without the addition of primary or secondary antibodies but with the addition of biotinyl-tyramide; (ii) direct amplification of endogenous peroxidase activity followed by omission of each primary antibody but with inclusion of all other steps and reagents. Only well oriented jejunal crypt-villus units were scored. “Well oriented” was defined as sectioned parallel to the crypt-villus axis with an unbroken epithelial column extending from the crypt base to the villus tip. The data were compiled as the number of IELs of a particular type per 1000 villus epithelial cells or per 100 crypt epithelial cells. A minimum of 100 jejunal crypt-villus units were scored per mouse. Data obtained with each antibody from all mice of a given genotype (germ-free or conventional) were averaged. Multilabel immunohistochemical studies were performed on sections of normal and transgenic mouse jejunums using rabbit anti-TAg (1:1000 in PBS-blocking buffer; kindly provided by Doug Hanahan, University of California, San Francisco, CA) and goat anti-BrdUrd (1:1000) (38Kim S.H. Roth K.A. Moser A.R. Gordon J.I. J. Cell Biol. 1993; 123: 877-893Crossref PubMed Scopus (97) Google Scholar, 39Cohn S.M. Lieberman M.W. J. Biol. Chem. 1984; 259: 12456-12462Abstract Full Text PDF PubMed Google Scholar). Antigen-antibody complexes were detected with Cy3-labeled donkey anti-rabbit Ig and fluorescein isothiocyanate (FITC)-labeled donkey anti-goat Ig (1:500; Jackson ImmunoResearch). 6–8-week-old transgenic mice and their normal littermates were sacrificed, and their jejunums were recovered (n = 3 germ-free and 3 conventionally raised mice/genotype/experiment; three independent experiments). Peyer's patches were identified by inspecting the serosal surfaces of the jejunal segment and were then excised. Each jejunal segment was subsequently opened with a longitudinal incision, washed in PBS, and cut into 1-cm pieces that were placed in 40 ml of ice-cold sterile PBS. The pooled segments from all three animals/genotype/experiment were washed five times in PBS (vigorous shaking), allowed to settle by gravity, and resuspended in 25 ml of R2 medium (RPMI 1640 buffer containing 5% fetal calf serum (Sigma), 1 mm sodium pyruvate, 1 mm sodium bicarbonate, 1% nonessential amino acids (Sigma), and 0.1% 2-mercaptoethanol). The mixture was shaken gently for 30 min at 37 °C and then rigorously for 2 min at room temperature. The intestinal segments were allowed to settle by gravity, and the supernatant was collected and passed through a Nytex filter (Becton Dickinson). The flow-through, containing IELs and epithelial cells, was passed over a column of dimethyldichlorosilane-treated glass wool fiber (0.5 g/10-ml syringe) preequilibrated in R2 medium. The flow-through was spun at 1500 × g for 5 min, and the resulting cell pellet, highly enriched for IELs, was resuspended in 10 ml of R2 medium. The suspension was centrifuged at 1500 ×g for 5 min, and the pellet resuspended to a final concentration of 107 cells/ml of FACS staining buffer (RPMI, 1% bovine serum albumin (Sigma), 1 mg/ml human IgG (Sigma)). IELs were stained with the following antibodies in various combinations (all from BD PharMingen; all diluted 1:100 in FACS staining buffer): (i) phycoerythrin (PE)-conjugated hamster anti-mouse αβ TCR (β chain; clone H57-597); (ii) PE-conjugated hamster anti-mouse γδ TCR (δ chain; clone GL3); (iii) PE-conjugated rat anti-mouse CD8β.2 (clone 53-5.8); (iv) FITC-conjugated rat anti-mouse CD8α (clone 53-6.7); (v) FITC- or PE-conjugated rat anti-mouse CD4 (clone RM4-5); (vi) FITC-conjugated rat anti-mouse CD45 (clone 30-F11); and (vii) biotinylated hamster anti-mouse CD103 (integrin αIEL chain; clone 2E7). Biotinylated primary antibodies were visualized with FITC-streptavidin or PE-streptavidin (BD PharMingen). Idiotype as well as secondary antibody alone controls were also performed. Following incubation with these reagents (60–90 min on ice), cells were spun for 5 min at 1500 × g, washed with sterile ice-cold PBS, and examined by flow cytometry (FACScalibur; Becton Dickinson). γδ TCR+ IELs were isolated from jejunal segments that had been removed from 6-week-old germ-free male transgenic mice and their normal littermates. The γδ TCR+, CD103+ lymphocyte population was sorted (FACS Vantage; Becton Dickinson), collected in sterile cold PBS, and recovered by centrifugation (1000 ×g for 5 min at room temperature). RNA was isolated using the RNAeasy kit (Qiagen) (5 mice/IEL preparation; n = 10 preparations/genotype). RNA was also isolated from intact jejunal segments (n = 10 germ-free mice/genotype). LCM was conducted using jejunal cryosections that had been stained briefly with eosin Y and methyl green. Dissection of villus epithelium was restricted to well oriented crypt-villus units and was accomplished using the PixCell II system (Arcturus; 7.5-μm diameter laser spot), CapSure HS LCM Caps (Arcturus), and protocols described in Ref. 40Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 31: 559-589Google Scholar. ∼10,000 jejunal villus epithelial cells were harvested from each germ-free normal and TAg mouse (n = 3 animals/group). RNA was prepared from captured cells from each mouse in each group using the PicoPure RNA Isolation Kit (Arcturus). The concentration of each preparation was defined (RiboGreen RNA quantitation kit; Molecular Probes, Inc., Eugene, OR), and equally sized aliquots from members of a group of animals were pooled. Data sets of gene expression profiles from γδ TCR+ IELs and the αβ TCR+ cells were a generous gift from Aude Fahrer and Y-H. Chien (Dept. of Microbiology and Immunology, Stanford University) (33Fahrer A.M. Konigshofer Y. Kerr E.M. Ghandour G. Mack D.H. Davis M.M. Chien Y.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10261-10266Crossref PubMed Scopus (140) Google Scholar). These data sets were obtained using an early manufactured version of a high density, oligonucleotide-based DNA microarray containing probe sets representing 6352 mouse genes or expressed sequence tag clusters (Mu6K GeneChip; Affymetrix). We used GeneChip software (version 4.0; Affymetrix) to compute an average fluorescence intensity across all probe sets on the GeneChips prior to conducting pairwise chip-to-chip comparisons (41Mills J.C. Gordon J.I. Nucleic Acids Res." @default.
• W2000951661 created "2016-06-24" @default.
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