Mucosal T Lymphocyte Numbers Are Selectively Reduced in αE (CD103)-Deficient Mice

This information is current as Michael P. Schön, Anu Arya, Elizabeth A. Murphy, of September 24, 2021. Cassandra M. Adams, Ulrike G. Strauch, William W. Agace, Jan Marsal, John P. Donohue, Helen Her, David R. Beier, Sara Olson, Leo Lefrancois, Michael B. Brenner, Michael J. Grusby and Christina M. Parker

J Immunol 1999; 162:6641-6649; ; Downloaded from http://www.jimmunol.org/content/162/11/6641

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Mucosal T Lymphocyte Numbers Are Selectively Reduced in ␣ 1 Integrin E (CD103)-Deficient Mice

Michael P. Scho¨n,2* Anu Arya,* Elizabeth A. Murphy,* Cassandra M. Adams,* Ulrike G. Strauch,* William W. Agace,* Jan Marsal,* John P. Donohue,* Helen Her,† David R. Beier,† Sara Olson,§ Leo Lefrancois,§ Michael B. Brenner,* Michael J. Grusby,*‡ and Christina M. Parker3* ␣ ␤ The mucosal lymphocyte integrin E(CD103) 7 is thought to be important for intraepithelial lymphocyte (IEL) localization or ␣ ␣ function. We cloned the murine integrin encoding E, localized it to 11, and generated integrin E-deficient ␤ ␣ ؊/؊ ␣ mice. In E mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of E 7 to E-cadherin

expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E- Downloaded from cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer’s patch, and splenic T lym- ␣ ␣ ␤ phocyte numbers were not reduced in E-deficient mice. Thus, E 7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated orga- ␣ nized lymphoid tissues. Finally, the impact of E deficiency upon intestinal IEL numbers was greater at 3–4 wk of life than in .younger animals, and affected the TCR ␣␤؉ CD8؉ T cells more than the ␥␦ T cells or the TCR ␣␤؉ CD4؉CD8؊ population ؉ ؉␤␣ ␤ ␣ These findings suggest that E 7 is involved in the expansion/recruitment of TCR CD8 IEL following microbial coloni- http://www.jimmunol.org/ ␣ ␣ ␤ ␣ ␤ zation. Integrin E-deficient mice will provide an important tool for studying the role of E 7 and of E 7-expressing mucosal T lymphocytes in vivo. The Journal of Immunology, 1999, 162: 6641–6649.

ucosal T lymphocytes appear to be functionally dis- mune surveillance and immune responses to mucosal pathogens tinct from those in peripheral blood. Indeed, intestinal under normal conditions. In addition, intestinal T lymphocytes M intraepithelial lymphocytes (IEL)4 have been found to have been implicated in the pathogenesis of inflammatory bowel differ from PBL in their Ag-recognition specificity (1–3), acces- diseases, based upon the development of intestinal inflammation in sory costimulatory molecule expression (4), TCR ␣␤ and ␥␦ type, animals with defects in T lymphocyte regulation (7). Thus, it will and thymus-dependent vs -independent T cell development (re- be important to understand the mechanisms whereby mucosal lym- by guest on September 24, 2021 viewed in Ref. 5). There are estimated to be as many T lympho- phocytes selectively localize and function. cytes in the intestinal immune system as in the spleen (6). Fur- Following primary stimulation in organized lymphoid tissues, thermore, many infectious agents invade via mucosal epithelia, such as mesenteric lymph nodes and Peyer’s patches, some acti- emphasizing the importance of mucosal T lymphocytes for im- vated intestine-derived lymphocytes recirculate and then preferen- tially return to the intestinal tract. The selective expression of che- *Division of Rheumatology, Immunology, and Allergy, and †Division of Genetics, mokine receptors and adhesion molecules are thought to contribute Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; to T cell homing (8). Once in the intestine, lymphocyte subpopu- ‡Department of Immunology and Infectious Disease, Harvard School of Public § lations localize to particular microenvironments. For example, the Health, Boston, MA 02115; and Department of Medicine, University of Connecticut ϩ Health Center, Farmington, CT 06030 CD8 T cells are found preferentially within the epithelium, where ϩ Received for publication January 12, 1999. Accepted for publication March 17, 1999. they comprise 90% of the resident population, whereas CD4 T The costs of publication of this article were defrayed in part by the payment of page cells predominate in the lamina propria, where they constitute charges. This article must therefore be hereby marked advertisement in accordance more than half of the T lymphocytes (9). Targeted migration along with 18 U.S.C. Section 1734 solely to indicate this fact. chemokine gradients and selective adhesion to extracellular matrix 1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to or to cellular counterreceptors may account for the localization and M.P.S. and U.G.S.), grants from the National Institute of Health (NIH) and the Mathers Foundation (to M.J.G., who is a Scholar of the Leukemia Society of Amer- retention of T cell subsets within mucosal microenvironments. ica), National Research Service institutional training grants from the NIH (to C.M.A. One candidate to mediate the selective localization or retention and J.P.D), a fellowship from the Swedish Foundation for International Cooperation ␣ ␤ of intraepithelial T lymphocytes is the integrin E(CD103) 7. This in Research and Higher Education (STINT) (to W.W.A), grants from NIH (to Ͼ M.B.B.), grants from NIH, the Crohn’s and Colitis Foundation, the Cancer Research integrin is expressed selectively on 90% of intestinal IEL and on Institute, and a Pilot and Feasibility grant from the Center for the Study of Inflam- 45–50% of lamina propria T lymphocytes (9–11) in both mice and matory Bowel Diseases (to C.M.P), Grant RO1HD29028 from National Institute for humans. It is also found on T lymphocytes in some other mucosal Child Health and Human Development, and Grant ROHG00951 from the National Center for Research (to D.R.B.). epithelia, such as the genitourinary epithelium (12), on ϳ40% of 2 Current address: Department of Dermatology, Heinrich-Heine-University, Mooren- bronchioalveolar lavage T cells obtained from normal humans (13) str. 5, D-40225 Du¨sseldorf, Germany and on some cells of dendritic morphology in rats (14). Further- 3 ␣ ␤ Address correspondence and reprint requests to Dr. Christina M. Parker, Brigham more, E 7 expression can be induced on T lymphocytes and mu- and Women’s Hospital, Smith-552B, 1 Jimmy Fund Way, Boston, MA 02115. E-mail rine mast cells by culture in the presence of TGF-␤1 (15), a cy- address: [email protected] tokine produced by intestinal epithelial cells (16) as well as other 4 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; Itgae, integrin ␣ ␤ Ͻ ␣ cell types. In contrast, E 7 is expressed on 5% of PBL in hu- gene encoding E; SSCP, single strand conformation polymorphism; SPF, specific pathogen-free; MFI, mean fluorescence intensity. mans (10), on only 15% of splenic T lymphocytes in mice (17),

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 ␣ 6642 INTEGRIN E-DEFICIENT MICE and has not been found on B lymphocytes, underscoring its selec- matings between chimeric animals and BALB/c mice were intercrossed to ϫ tive expression on mucosal T cells. generate F2 (129/Sv BALB/cJ) offspring, which were utilized in these The ␣ ␤ integrin mediates T cell adhesion to epithelial cells experiments except where otherwise indicated. Two independent clones E 7 were used to generate chimeric animals that expressed similar phenotypes. (18) through its binding to E-cadherin (19–21), a member of the All animals were housed under specific pathogen-free (SPF) conditions. cadherin family of adhesion molecules that is expressed selectively on epithelial cells. Cadherins are characterized by their tissue-spe- Genotyping cific distribution and are known to mediate homophilic adhesion of Genomic DNA was isolated from tail biopsies by proteinase K digestion, cells within tissues (22). In addition, evidence has suggested that followed by phenol extraction. Southern blot analysis was performed using ␣ ␤ 10 ␮g genomic DNA, Hybond-N (Amersham, Arlington Heights, IL) fil- E 7-dependent adhesion is regulated by inside-out signals, based ␣ ␤ ters and digoxigenin-labeled probes (Nonradioactive DNA labeling and upon the observation that E 7 function is enhanced following ␣ ␤ detection ; Boehringer Mannheim, Indianapolis, IN) according to stan- stimulation through the TCR (21). Integrin E 7 also appears to dard procedures. ␣ transmit an intracellular signal, as anti-human E mAbs enhance T PCR was performed on genomic DNA using primer sequences flanking cell proliferation in response to suboptimal anti-CD3 stimulation the inserted neomycin resistance gene to yield products of 1100 bp from (9, 23), and as an anti-murine ␣ mAb induces T cell-mediated disrupted and 900 bp from wild-type alleles (primers used: 5Ј-GCA ACA E ACG CAT CGT TCA TAT GGA-3Ј and 5Ј-GTG CTC TGT CTA TTG lysis of FcR-bearing target cells in the absence of a signal from the TTC CCC TCC TT-3Ј; conditions: anneal at 62°C for 1 min, extend at ␣ ␤ TCR (17). Thus, the E 7 integrin may be important in the local- 72°C for 2 min, and denature at 94°C for 1 min for 40 cycles, with a final ization or function of T cells, dendritic cells, and/or mast cells. extension at 72°C). To determine the role of ␣ ␤ in in vivo immune responses, the E 7 Cells and culture conditions ␣ Downloaded from murine integrin E-encoding gene (Itgae) was cloned and localized ␣ ␮ to chromosome 11. Then, integrin E-deficient mice were gener- A single cell suspension of splenic leukocytes was stimulated with 5 g/ml ated. Study of these animals revealed an altered distribution of T Con A (Sigma, St. Louis, MO) and cultured for 5 days. A total of 5 ng/ml human recombinant TGF-␤1 was added to the medium, and the cells were lymphocytes within epithelia and in the intestinal lamina propria, grown for an additional 5 days to induce ␣ expression. in the absence of experimentally induced infection or inflamma- E ␣ ␤ tion. Thus, these studies demonstrate an important role for E 7 in Purification of intestinal IEL, flow cytometry, and estimation of

modulating the homeostasis of T lymphocyte numbers in selected Peyer’s patch size http://www.jimmunol.org/ tissue sites. Intestinal IEL were isolated as described (28) with minor modifications. Briefly, small intestines were flushed with HBSS, trimmed to remove Pey- Materials and Methods er’s patches, opened longitudinally, and cut into 3- to 5-mm fragments. The Integrin ␣ -encoding gene (Itgae) cloning fragments were incubated in a shaking water bath at a rate of 120 shakes E per minute in medium (RPMI 1640 containing 104 U/ml penicillin/strep- A murine 129/Sv-derived ␭-Fix II library (Stratagene, La Jolla, CA) was tomycin, 20 ␮g/ml gentamicin, 2% bovine calf serum, and 20 mM HEPES ␣ screened by hybridization with an E cDNA probe incorporating nucleo- buffer) at 37°C for 20–30 min. The intestinal pieces were shaken manually ␣ tides 1088–1253 of the murine E cDNA sequence (15). Inserts were sub- for three cycles of 15 s each to release intestinal IEL. The resulting cell cloned and partial sequence determined by deletion cloning, followed by suspensions were collected, pooled, passed through a glass wool column,

dideoxy chain termination analysis. The nucleotide sequence within iden- and the IEL separated from epithelial cells using 44/66% Percoll density by guest on September 24, 2021 ␣ tified exons was identical to nucleotides 14–2771 of the murine E cDNA gradient centrifugation. To estimate the size of Peyer’s patches, their length reported previously with the exception of only 4/2757 bases (nucleotide and width were multiplied to estimate their relative cross-sectional area in changes t411c, g668a, c1057g, and g1058c; amino acid changes I207E and mm2. To isolate Peyer’s patch lymphocytes, the Peyer’s patches were me- R337A). chanically disrupted by rubbing the patches between two frosted glass slides. Localization of the Itgae gene For FACS analysis, 105 cells were stained with saturating concentra-

tions of mAb (buffer: 2% BSA, 0.05% NaN3 in PBS; blocking reagents: Primers were designed to amplify a region corresponding to intronic se- ␮ quence of Itgae to test for single-strand conformation polymorphisms (SS- 10% mouse serum (for rat Abs) or 10% goat serum and 20 g/ml mAb CPs) between mouse strains. These were analyzed as previously described 2.4G2 (for hamster Abs)). In some experiments, dead cells were excluded (24). Briefly, oligonucleotides were radiolabeled with [32P]ATP using by propidium iodide staining. Samples were analyzed using a FACScan polynucleotide kinase, and genomic DNAs from a series of mouse strains and FACScalibur for four-color analysis and a FACSort and the Cell Quest were amplified using standard protocols (anneal at 55°C for 1 min, extend software (Becton Dickinson, San Jose, CA) for one color analysis. at 72°C for 2 min, and denature at 94°C for 1 min for 40 cycles, with a final Cell surface iodination with 125I and immunoprecipitation extension at 72°C). A total of 2 ␮l of the amplified reaction was added to 8.5 ml USB (United States Biochemical) stop solution, denatured at 94°C A total of 4 ϫ 107 TGF-␤1-stimulated splenocytes was surface iodinated for 5 min, and immediately placed onto ice. A total of 2 ␮l of each reaction and then solubilized in TBS/0.5% Triton X-100 (Sigma). Then, 1 ϫ 107 is loaded on a 6% nondenaturing acrylamide sequencing gel and electro- cell equivalents were precleared with G-Sepharose resin (Pharma- phoresed in 0.5ϫ TBE buffer for 2–3 h at 40 W in a 4°-cold room. A primer cia, Piscataway, NJ), immunoprecipitated with 5 ␮g of purified mAb fol- pair based upon sequence in the intron between exons 2 and 3 (5-AAG lowed by protein G-Sepharose, washed, and the immunoprecipitated pro- GTCAGATGAGCAATATGT-3Ј (forward) and 5Ј-GCCAGCAGACT teins were analyzed by SDS-PAGE using 7% gels under reducing CAGCATTACT-3Ј (reverse)) identified a polymorphism between conditions (29). C57BL/6J and Mus spretus. This primer pair was used to analyze DNA ϫ ϫ prepared from the BSS ((C57BL/6JEi SPRET/Ei)F1 SPRET/Ei) back- Abs cross (25). The strain distribution pattern was analyzed using the Map Manager Program (26). The following mAbs were used as controls: rat IgG1 (R59-40; PharMin- gen, San Diego, CA), rat IgG2A (R35-95; PharMingen), rat IgG2B (SFR3- ␣ Construction of the E targeting vector and generation of DR5; American Type Culture Collection (ATCC), Manassas, VA), and ␣ Ϫ/Ϫ mice hamster IgG (UC8-4B3; PharMingen). The following Ags were detected E by mAbs: CD3⑀ (500A2; PharMingen, and 145-2C11; ATCC), CD4 A targeting construct was generated (Fig. 1a) and transfected by electro- (RM4-5; PharMingen), CD8␣ (53-6.72; ATCC), CD8␤ (53-5.8; ATCC), ␣ ␤ poration into the 129/Sv-derived embryonic stem cell line ES-D3. Trans- CD11b ( M integrin, Mac-1, M1/70; ATCC), CD18 ( 2 integrin, 2E6; ␣ ␤ fected cells were selected for G418 and gancyclovir resistance, as reported ATCC), CD25 (IL-2R -chain, 3C7; PharMingen), CD29 ( 1 integrin, previously (27), and cloned. Clones that incorporated a single copy of the Ha2/5; PharMingen), CD45R/B220 (RA3-6B2; PharMingen), CD45RB ␣ construct into the genome by homologous recombination were identified (MB23G2; ATCC, and 16A; PharMingen), CD49a ( 1 integrin, Ha31/8; Ј Ј ␣ ␣ ␣ by Southern blot analysis of genomic DNA using a 5 probe (Fig. 1a),a3 PharMingen), CD49b ( 2 integrin, HM 2; PharMingen), CD49d ( 4 inte- ␣ ␣ probe, and the neomycin resistance gene. Such clones were injected into grin, P/S2, or R1-2; ATCC), CD49e ( 5 integrin, HM 5; PharMingen), ␣ ϩ/Ϫ ␣ ␤ BALB/c blastocysts to generate chimeric animals. The E progeny of CD49f ( 6 integrin, GoH3; Dianova, Hamburg, Germany), CD49d/ 7 The Journal of Immunology 6643 Downloaded from http://www.jimmunol.org/

␣ ␣ FIGURE 1. Genomic organization and targeted disruption of the murine integrin E-encoding gene. a, Schematic representation of the E-encoding gene (top), the targeting construct (middle), and the disrupted allele (bottom). Exons (thick lines), introns (thin lines), and restriction endonuclease sites are shown ␣ (n, NcoI; B, BamHI; p, PstI; X, XhoI; H, HindIII; for PstI and NcoI, selected cleavage sites are indicated). b, Schematic representation of the E polypeptide Ј ␣ indicating the location of the 5 untranslated region (crosshatched fill pattern), the extra region unique to E (speckled fill pattern), cleavage site (arrow), ␣ I domain (grey fill pattern), divalent cation binding domains (black fill pattern), transmembrane domain (diagonal line fill pattern), the regions of E encoded c, Southern blot analysis after NcoI digestion (top panel) and PCR analysis .(ء) by each exon (below the schematic), and the location of the targeted exon ϩ Ϫ ϩ ϩ Ϫ Ϫ ␣ / ␣ / ␣ / by guest on September 24, 2021 (bottom panel) of DNA isolated from offspring of E mouse intercrosses. d, Intestinal IEL freshly isolated from E and E mice were stained ␣ ϩ/Ϫ ␣ ϩ/ϩ ␣ Ϫ/Ϫ ␤ as indicated. e, Splenocytes derived from E , E and E mice were stimulated with Con A for 5 days, and then cultured for 5 days with TGF- 1 ␣ ␣ to up-regulate E expression, surface radiolabeled, solubilized, and E expression analyzed by immunoprecipitation and SDS-PAGE.

␣ ␤ ␣ ( 4 7, DATK32 (30); LeukoSite, Cambridge, MA), CD51 ( v integrin, For evaluation of T lymphocyte numbers within lung tissue, the left lung H9.2B8; PharMingen), CD54 (ICAM-1, YN1/1.7.4; ATCC), CD62L (L was frozen in OCT, the average number of anti-CD3 mAb stained cells in , MEL-14; ATCC), CD90 (Thy-1, AT15.E; R. MacDonald Ludwig at least seven randomly selected high power (40ϫ) fields was determined ␣ 2 Institute for Cancer Research, Epalinges, Switzerland), CD103 ( E inte- and used to calculate the number of T lymphocytes/mm . In addition, the grin, M290 (11); P. Kilshaw, Department of Immunology, AFRC Babra- average number of CD3ϩ cells per bronchus was determined evaluating all ham Institute, Cambridge, U.K. or 2E7 (17); L. Lefrancois, Department of of the bronchi within these randomly located tissue sections. ␤ Medicine, University of Connecticut Health Center, Farmington, CT), 7 (M293; P. Kilshaw), CD106 (VCAM-1, M/K-2.7; ATCC), CD32/CD16 Statistical analysis (Fc-␥II/IIIR, 2.4G2; ATCC), MHC class I (M1/42.3, rat IgG2A; ATCC), anti-TCR ␣␤ (H57-597; PharMingen), and anti-TCR ␥␦ (GL3; PharMin- Statistical analysis was performed using an unpaired, two-tailed Student’s ϭ Ͼ gen). FITC-conjugated goat anti-hamster and mouse anti-rat secondary Abs t test when n 3 and the Mann-Whitney nonparametric U test when n used in FACS or direct immunofluorescent staining of intestinal sections 3, unless otherwise indicated. Analysis was performed using the Instat were purchased from Jackson ImmunoResearch (West Grove, PA), and software (GraphPad Software, San Diego, CA). biotinylated goat-anti-hamster and mouse adsorbed rabbit-anti-rat serum were obtained from Vector Laboratories (Burlingame, CA). Results Histochemistry Itgae was cloned, and its chromosomal localization was determined For histochemistry, tissue samples were embedded in JB-4 plastic resin ␣ (Polysciences, Warrington, PA) and 3-␮m sections stained with hematox- Genomic clones encoding murine E were identified by hybrid- ␣ ylin-eosin. For immunohistochemistry, tissue samples were frozen in OCT ization with PCR products encoding fragments of the murine E and 5- to 10-␮m cryostat-cut sections stained using the ABC (avidin/biotin cDNA (GenBank accession nos. AF133070-AF133085). Based complex)-immunoperoxidase kit according to the manufacturer’s instruc- upon comparison with the murine ␣ cDNA sequence (15) and tions (Vector Laboratories). For analysis of lamina propria T cell numbers, E a cell was counted as a lamina propria lymphocyte if it did not overlap the identification of the conserved consensus splice sites (Ref. 31, and ␣ basement membrane and was contained within villi rather than crypts. This data not shown), a partial map of the murine E intron/exon struc- criteria may account for the unusually high ratio of CD4ϩ/CD8ϩ T cells ture was generated (Fig. 1a). To determine the chromosomal lo- observed within the lamina propria in these studies, as some cells were cation of Itgae, SSCP analysis was used as previously described counted as IEL that were largely within the lamina propria. For analysis of T cell numbers within vaginal tissue sections, the number of intraepithelial (24). Primers corresponding to intronic sequence between exons 2 T lymphocytes within an entire tissue section derived from the middle third and3ofItgae were analyzed and found to identify an SSCP be- of the vagina was determined and expressed per mm basement membrane. tween inbred mouse strains (see Materials and Methods). The ␣ 6644 INTEGRIN E-DEFICIENT MICE

BSS-interspecific backcross was genotyped and the allele distri- bution pattern analyzed using the Map Manager program. Itgae was found to map to chromosome 11 with a logarithm of odds likelihood score of 27.4. No recombinants were found in 91 prog- eny between Itgae and the marker D11Abb1. The position of Itgae with respect to flanking microsatellite markers was: D11 Mit4 Ϫ 5.5 Ϯ 2.4 cm Ϫ Itgae, D11Abb1 Ϫ 1.1 Ϯ 1.1 cM Ϫ D11 Mit7, D11 Mit32, D11 Mit34 (mapping data submitted to the Mouse Genome Database). ␣ Integrin E-deficient mice were generated ␣ To generate integrin E-deficient mice, exon 10 within the integrin ␣ FIGURE 2. The proportion of splenocytes expressing CD3, CD4, and E-encoding gene was replaced with a neomycin resistance gene ␣ CD8 increased slightly by E deficiency. Single cell splenocyte popula- by homologous recombination (Fig. 1, a–c). FACS analysis con- ϩ ϩ Ϫ Ϫ tions were prepared from ␣ / or ␣ / mice and stained with directly ␣ Ϫ/Ϫ ␣ E E firmed that intestinal IEL isolated from E mice lacked E conjugated anti-CD3, CD4, or CD8 mAbs, as indicated. Flow cytometry ␣ Ϫ/Ϫ expression (Fig. 1d). In addition, splenocytes from E mice was performed to determine the proportion of cells that expressed each were cultured in the presence of TGF-␤1, a cytokine that up-reg- marker, analyzing the total splenocyte population. After subtracting the ␣ ␣ ulates E expression on wild-type T cells. Integrin E was not background staining with isotype-matched noncell binding control mAbs, detected on the surface of TGF-␤1-treated splenocytes derived the percentage of of the total splenic leukocyte population that was stained Downloaded from ␣ Ϫ/Ϫ with each mAb was determined. Then, the total number of stained cells/ from E mice by immunoprecipitation or FACS analysis. In contrast, it was readily detected on the surface of cells derived spleen was calculated by multiplying the total number of leukocytes/ ␣ ϩ/ϩ spleen ϫ the percentage that expressed the analyzed marker, as determined from E animals, and was detected at reduced levels on cells from ␣ ϩ/Ϫ animals (Fig. 1e, and data not shown). Thus, ␣ Ϫ/Ϫ by flow cytometry. The bars represent the average number of positive ;p Ͻ 0.05 ,ء .(E E splenocytes/spleen for each marker (n ϭ 3 for each group mice lacked expression of the ␣ polypeptide, while heterozygous .p Ͻ 0.01 ,ءء E

␣ ␤ http://www.jimmunol.org/ animals expressed intermediate levels of E 7 both in immuno- precipitation (Fig. 1e) and in FACS analysis (data not shown). mice, with a trend toward increased numbers of CD4ϩ and CD8ϩ ␣ Ϫ Ϫ Integrin E deficiency did not alter fecundity, morphogenesis, or splenic T cell numbers in ␣ / animals on the BALB/c genetic Ͼ E overall weight gain, and most animals survived for 18 mo in a background. SPF facility (data not shown). ␣ Integrin E-deficient mice had fewer T cells diffusely distributed ␣ Integrin E-deficient mice had normal or increased numbers of within the intestinal and vaginal epithelia splenic T lymphocytes ␣ Ϫ/Ϫ ␣ ϩ/ϩ The number of intestinal IEL in adult E and E littermates ␣ Because E is selectively expressed on leukocytes, studies were also was compared by immunohistology, with the expectation that performed to determine the impact of ␣ deficiency upon the his- ␣ by guest on September 24, 2021 E intestinal IEL numbers would be reduced by E deficiency due to tologic appearance of organized lymphoid tissues. In this initial ␣ ␤ the loss of the E 7/E-cadherin-mediated adhesion. Indeed, the evaluation, ␣ deficiency had no apparent effect on the size of the ␣ Ϫ/Ϫ E number of jejunal IEL was reduced by 54% in the E progeny thymus or on the immunohistologic appearance of the thymus, ϫ ␣ ϩ/ϩ of F2 (BALB/c 129/Sv) mice as compared with E mice of peripheral lymph nodes, or spleen after staining with anti-CD3, similar genetic background ( p Ͻ 0.002, Mann-Whitney U test; anti-CD4, and anti-CD8 mAbs. In addition, there were no changes ϭ ␣ ϩ/ϩ ␣ Ϫ/Ϫ n 5 E and 8 E mice) (Figs. 3 and 4a). In these studies, ␣ Ϫ/Ϫ in the serum levels of IgM, IgG isoforms, or IgA in E as it was apparent that the number of intestinal IEL was influenced by ␣ ϩ/ϩ compared with E mice (data not shown). genetic background and/or environment, as there were more intes- To further evaluate the impact of ␣ ␤ expression upon the ␣ ϩ/ϩ ϫ E 7 tinal IEL in E mice on the (129/Sv BALB/c) or the number and subset composition of splenic T lymphocytes, addi- C57BL/c backgrounds than on the BALB/c background. However, tional studies were performed. First, the overall number of leuko- ␣ Ϫ/Ϫ E mice had reduced intestinal IEL numbers in all genetic ␣ Ϫ/Ϫ ␣ ϩ/ϩ cytes/spleen after RBC lysis was similar in E and E mice backgrounds examined, including groups of mice housed in two three to four generations backcrossed toward the C57BL/6 strain independent animal facilities. Thus, reduced intestinal IEL num- and then intercrossed (N )(␣ Ϫ/Ϫ mice: 1.07 Ϯ 0.15 ϫ 108 ␣ 3–4 E bers represents a consistent feature of E-deficient mice. cells/spleen; ␣ ϩ/ϩ mice: 1.14 Ϯ 0.16 ϫ 108 cells/spleen; n ϭ 3; ␣ ␤ E Because integrin E 7 is also expressed on T cells in or adjacent p ϭ 0.55). In addition, flow cytometry was used to determine the to other mucosal epithelia, such as the lung and genitourinary tract, total number of splenocytes expressing CD3, CD4, or CD8 using the number of T lymphocytes in these other sites was compared in the formula: (the total number of splenocytes) ϫ (the proportion of ␣ Ϫ/Ϫ ␣ ϩ/ϩ E and E mice. While vaginal IEL numbers were reduced the total splenocyte population that expressed each marker). In this by 60% ( p Ͻ 0.01; n ϭ 3) (Fig. 4b)in␣ Ϫ/Ϫ mice of mixed ϩ ϩ ϩ E analysis, the number of CD3 , CD4 , and CD8 splenocytes was (129/Sv ϫ BALB/c) genetic background, the number of T cells ␣ not altered significantly by E deficiency. In contrast, when mice within the lung parenchyma and adjacent to the bronchi was not N to the BALB/c strain were evaluated, the total number of ␣ 10 affected by E deficiency (Fig. 4, c and d). Thus, under conditions ␣ splenic leukocytes was increased by 24% in E-deficient mice where there was not an experimentally induced disease process, (␣ Ϫ/Ϫ mice: 1.07 ϫ 108 Ϯ 0.11 cells/spleen; ␣ ϩ/ϩ mice: 0.86 ϫ ␣ Ϫ/Ϫ E E E mice had reduced numbers of T cells within some epithelia, 108 Ϯ 0.04 cells/spleen; n ϭ 3; p ϭ 0.04). In addition, the numbers including the intestine and vagina, but not in others, such as in the ϩ ϩ ϩ of CD3 , CD4 , and CD8 splenocytes were increased by 14%, peribronchial regions in the lung. ␣ Ϫ/Ϫ 28%, and 13%, respectively in E mice (Fig. 2), while ϩ ϩ ␣ CD4 CD8 double positive splenic T cells were not detected in Integrin E-deficient mice have reduced numbers of lamina ␣ ϩ/ϩ ␣ Ϫ/Ϫ ϭ propria T cells but not of Peyer’s patch T cells either E or E mice (n 1, pooling cells isolated from three mice of each genotype, data not shown). Overall, peripheral It appeared that the number of lamina propria T lymphocytes was ␣ Ϫ/Ϫ ␣ ϩ/ϩ lymphocyte compartments appeared similar in E and E reduced in immunohistochemical analysis of tissue sections from The Journal of Immunology 6645 Downloaded from http://www.jimmunol.org/

FIGURE 3. Diffusely distributed T cells are diminished in intestinal ␣ mucosa of E-deficient mice. Cryostat-cut sections of proximal jejunum ␣ ϩ/ϩ ␣ Ϫ/Ϫ from E and E adult mice backcrossed two generations to the ␣ C57BL/6 strain were stained with anti- E (M290), anti-CD3 (500A2), anti- ␣ ␮ ␣ CD4 (RM4-5), and anti-CD8 (53-6.72), as indicated. Scale bar, 20 m. FIGURE 4. The number of intestinal and vaginal IEL is reduced by E deficiency. a, Proximal jejunal tissue sections were stained with anti-CD3 (500A2 or 145-2C11), and the number of stained cells/1000 villus epithe- (129/Sv ϫ BALB/c) mice. This was not an expected finding be- lial cells was determined by microscopic evaluation. For (BALB/c ϫ 129/ by guest on September 24, 2021 ␣ ␤ ϭ ␣ ϩ/ϩ ␣ Ϫ/Ϫ ϭ cause E-cadherin, the known E 7 counterreceptor, is not ex- Sv)F2, n 5 E and 8 E mice, for BALB/c (N1–5), n 9 mice. b, pressed in the lamina propria (32). To quantitate this difference, the Vaginal tissue sections derived from the middle third of the vagina were number of T lymphocytes within the villus lamina propria that did stained with anti-CD3 (500A2), and the average number of stained ␣ ϩ/ϩ ␣ Ϫ/Ϫ ϭ not appear to be in contact with either the basement membrane or IEL/mm epithelium was determined for E or E mice (n 3 animals for each genotype). c, Pulmonary sections from ␣ ϩ/ϩ or ␣ Ϫ/Ϫ epithelium was determined per 0.5-mm villus length. In this anal- E E mice were stained with anti-CD3 (500A2), and the average number of ysis, the number of lamina propria lymphocytes was significantly ␣ ϫ stained cells per bronchiolus was determined. d, Pulmonary sections were diminished by E deficiency in animals of the (129/Sv BALB/c) 2 ϩ stained with anti-CD3, and the number of stained cells/mm lung paren- background (CD3 lamina propria lymphocytes/0.5-mm villus chyma was determined. Results from ␣ Ϫ/Ϫ mice are indicated as Ϫ/Ϫ and ϩ/ϩ Ϫ/Ϫ E length in ␣ mice: 76 Ϯ 6vs39Ϯ 9in␣ mice; p Ͻ 0.01; ␣ ϩ/ϩ ϩ ϩ ϭ ␣ ϩ/ϩ ␣ Ϫ/Ϫ E E from E mice are indicated as / (n 5 E and 6 E mice). n ϭ 3). In a second group of mice partially backcrossed toward the Bars indicate the average Ϯ SD. Strains shown in bold were housed in a ␣ BALB/c strain, integrin E deficiency had a similar impact upon SPF facility at Harvard University, and strains shown in normal text were Յ ء ϩ/ϩ Ϯ ␣ lamina propria T lymphocyte numbers (in E mice, 70 7T housed in the SPF facility at the Dana-Farber Cancer Institute. , p 0.05; .p Յ 0.01 ,ءء Ϯ ␣ Ϫ/Ϫ ϭ cells/0.5-mm villus length vs 30 6in E mice; p 0.002; ϭ ␣ Ϫ/Ϫ n 3). Thus, E mice had significantly fewer lamina propria ␣ ϩ/ϩ T lymphocyte numbers than E mice in these two groups. ␣ However, when additional groups of mice were evaluated, E de- ficiency did not appear to have an impact upon lamina propria T facility where the cages were changed once/wk, and the microiso- lymphocyte numbers (Fig. 5a). In these two additional groups, it lator cages were not vented. In the setting of this less-stringent ␣ ϩ/ϩ was notable that the E mice had fewer lamina propria T lym- environment, there were more lamina propria T lymphocytes in ␣ ϩ/ϩ ␣ phocytes than those in the groups initially studied (Fig. 5a). This E mice, and E deficiency had a significant impact upon lam- could be due to differences in genetic background, as these later ina propria T lymphocyte numbers. groups of mice were backcrossed toward either the C57BL/6 or In contrast to the observed decrease in intestinal IEL and lamina ␣ Ϫ/Ϫ BALB/c strains. However, it seems more likely that environmental propria T lymphocyte numbers in E mice, the size of Peyer’s factors may have had an effect. Of note with respect to this pos- patches was increased in the first cohort of mice examined. How- sibility, the two more fully backcrossed groups of mice were ever, Peyer’s patch size was not altered in three additional groups housed in an animal facility where the cages were changed twice/ of mice of different genetic backgrounds, and the proportion of wk, and the microisolator cages were vented with an air circulation Peyer’s patch cells expressing CD3, CD4, or CD8 was not altered system. These animals had relatively few lamina propria T lym- (Fig. 5, b and c). Finally, the organization of Peyer’s patches ap- ␣ ␣ Ϫ/Ϫ ␣ ϩ/ϩ phocytes and E deficiency had no effect on lamina propria T cell peared normal in E as compared with E mice, based numbers. In contrast, the other groups of mice were housed in a upon immunohistochemistry after staining with anti-CD3 mAbs. ␣ 6646 INTEGRIN E-DEFICIENT MICE

␣ FIGURE 6. The impact of E deficiency upon IEL numbers is greater in mice Ͼ25 days of life than in neonatal animals. Proximal jejunum sections ␣ ϩ/ϩ ␣ Ϫ/Ϫ from E (squares) and E (circles) mice of increasing age were stained with anti-CD3 (500A2). When more than one mouse was sacrificed at a given time point, the number of animals is indicated, and the average number of intestinal IEL is graphed. As the number of intestinal IEL de- termined in animals Ͼ35 days of age did not appear to change with in- creasing age, the mean intestinal IEL number in animals Ͼ40 days was graphed. Mice were derived from intercrosses of 129/Sv ϫ BALB/c ani-

mals. Error bars indicate SD. Statistical analysis was performed using the Downloaded from Mann-Whitney U test.

␣ Ϫ/Ϫ only a 20% reduced number of intestinal IEL in E mice. Fi- nally, the intestinal IEL number reached a plateau in animals of

both genotypes at 4–5 wk of age. In mice older than 5 wk of age, http://www.jimmunol.org/ ␣ Ϫ/Ϫ the number of proximal jejunal IEL in E mice was only 46% ␣ ϩ/ϩ of that seen in E mice (Fig. 6). Overall, this kinetic analysis ␣ suggested that E might be more important in the localization of ␣␤ ␥␦ ␣ than of T cells and that the impact of E deficiency upon intestinal IEL numbers becomes apparent at a time when TCR ␣␤ FIGURE 5. The number of lamina propria lymphocytes is reduced by cells are increasing in number, presumably due to bacterial ␣ deficiency, while there was not a consistent effect upon Peyer’s patch colonization. E ␣ ␣␤ size or composition. a, Proximal jejunal tissue sections were stained with If integrin E is more important in the localization of TCR

␥␦ ␣ by guest on September 24, 2021 anti-CD3 (500A2 or 145-2C11), and the average number of stained cells than of T cells, then one would predict that adult E-deficient wholly enclosed within the lamina propria lymphocytes per 0.5-mm villus mice would have an intestinal IEL TCR repertoire skewed toward length was determined by microscopic evaluation. b, The average small a lower proportion of TCR ␣␤ϩ cells. To test this possibility, the intestinal Peyer’s patch size was estimated based upon the calculation: proportion of IEL expressing each TCR was evaluated by flow 2 estimated size ϭ length ϫ width in mm . For the C57BL/c (N ) exper- ϩ/ϩ ϩ/Ϫ Ϫ/Ϫ 3–4 cytometry in populations isolated from ␣ , ␣ ,or␣ ϭ E E E imental group, n 6. c, Peyer’s patch lymphocytes were stained with mice. Mice N to the C57BL/6 strain were used for this study anti-CD3, anti-CD4, and anti-CD8, and the proportion of stained cells 3–4 because ␣ deficiency had a greater impact upon IEL number in within the lymphocyte scatter gate was determined by flow cytometry. E ␣ Ϫ/Ϫ Ϫ Ϫ ␣ ϩ/ϩ the C57BL/6 than in the BALB/c genetic background and because Results from E mice are indicated as / and from E mice are indicated as ϩ/ϩ. Bars indicate the average Ϯ SD. Unless otherwise stated, the (129/Sv ϫ BALB/c) mice were no longer available. In these ␣ ϩ/Ϫ ␣ ϩ/ϩ n ϭ 3 for each experimental group. Strains shown in bold were housed in studies, TCR gene segment usage was similar in E and E ␣ a SPF facility at Harvard University and strains shown in normal text were mice (data not shown), despite the reduced levels of E expressed Յ ␣ ϩ/Ϫ ءء Յ ء housed at the Dana-Farber Cancer Institute. , p 0.05; , p 0.01. on E T lymphocytes (Fig. 1e). In contrast, while the propor- ␣␤ϩ ␣ ϩ/Ϫ ␣ ϩ/ϩ tion of TCR intestinal IEL was 54% in E or E mice, ␣ Ϫ/Ϫ Ͻ ϭ ␣ Ϫ/Ϫ it was only 35% in E mice ( p 0.01; n 4 E and 6 ␣ ϩ/ϩ ␣ ϩ/Ϫ ␣␤ϩ These findings demonstrate that Peyer’s patch size and T lympho- E or E ) (Fig. 7a). The decreased proportion of TCR ␣ ␣ Ϫ/Ϫ cyte composition are not, on average, altered by E deficiency. IEL E mice was accompanied by a corresponding increase in ␣ ␥␦ However, it is possible that E deficiency results in enlarged Pey- the relative proportion of IEL that expressed the TCR. Thus, in ␣ Ϫ/Ϫ er’s patch size when present in a given genetic background or E mice, the number of intestinal IEL that expressed the under some environmental conditions. TCR-␣␤ was reduced more than that of ␥␦ TCR-expressing cells. ␣ ␣␤ ␣ ϩ ␣␤ϩ Integrin E disruption has a preferential effect upon T cell Integrin E disruption reduced the numbers of CD8 TCR localization IEL subsets but had a lesser impact upon the number of ϩ The ␥␦ T cells populate the intestinal epithelium at an earlier age CD4 IEL ␣␤ ␣ ␤ Ͼ than T cells (33, 34) and the recruitment/expansion of TCR Within the intestinal epithelium, E 7 is expressed by 90% of ␣␤ϩ intestinal IEL depends upon microbial colonization of the the CD8ϩ T cells but by only half of the CD4ϩ T cells (9, 11). ␣ ␤ intestinal tract (33). Thus, studies were performed to determine at Thus, it seemed likely that E 7 disruption might affect the num- ␣ ϩ ϩ what age reduced numbers of intestinal IEL were detected in E- ber of CD8 IEL more than the number of CD4 IEL. Thus, deficient animals. In immunohistochemical analyses, intestinal three-color FACS analysis was performed to evaluate the subsets ␣ ϩ/ϩ ␣␤ϩ ␣ Ϫ/Ϫ ␣ ϩ/Ϫ IEL were not observed until 5 days of age in either E or of TCR intestinal IEL in E as compared with E or ␣ Ϫ/Ϫ ␣ ϩ/ϩ E mice. By 3 wk of age, the intestinal IEL number increased E mice backcrossed toward the C57BL/6 strain, in which the ␣ ϩ/ϩ ␥␦ ␣␤ to 40–50 intestinal IEL/1000 epithelial cells in E mice, with TCR /TCR cell ratio was altered (Fig. 7a). In this analysis, The Journal of Immunology 6647

While the expression of many adhesion molecules on IEL was ␣ not altered by E deficiency, the expression of CD44 was increased ␣ ␤ Ͼ Given the expression of E 7 on 80% of intestinal IEL, it was surprising that the intestinal IEL numbers were reduced by only 30% in mice partially backcrossed to the BALB/c strain. Thus, additional FACS analyses were performed to determine whether ␣ Ϫ/Ϫ E IEL derived from mice N3–4 to BALB/c express higher levels of an adhesion molecule that might functionally compensate ␣ for E deficiency in IEL localization, and thus reduce the impact of ␣ E deficiency on intestinal IEL number. Importantly, the expres- ␣ ␤ ␤ sion of 4 7, the other 7 integrin, was not altered on intestinal IEL or Peyer’s patch lymphocytes (Fig. 8, and data not shown). While there were some interindividual variations in cell surface expression of other adhesion receptors, many adhesion molecules were expressed, on average, at similar levels on intestinal IEL ␣ Ϫ/Ϫ ␣ ϩ/ϩ isolated from E or E mice. These included CD18 (LFA-1 ␤ ␤ 2-chain), CD54 (ICAM-1), CD62L (L-selectin), CD29 ( 1 inte- ␣ ␣ Downloaded from grin subunit), CD49d ( 4), and CD49a ( 1 integrin subunit) (Fig. 8). Other adhesion molecules were not detected on intestinal IEL ␣ Ϫ/Ϫ ␣ ϩ/ϩ ␣ isolated from either E or E mice, including CD49b ( 2), ␣ ␣ ␣ CD49e ( 5), CD49f ( 6), and CD51 ( V) (data not shown). Over- all, these studies emphasize the relatively limited expression of ␤ other adhesion molecules, such as the 1 , on intestinal ␣ Ϫ/Ϫ http://www.jimmunol.org/ IEL. On IEL derived from E mice, the only consistent shift in adhesion molecule expression was increased expression of CD44 (average mean fluorescence intensity (MFI) 118 on cells derived ␣ Ϫ/Ϫ ␣ ϩ/ϩ ϭ from E mice vs 78 on cells derived from E mice; n 4; p Ͻ 0.03), a hyaluronate receptor (35, 36). This molecule could participate in mucosal T cell adhesion within the intestinal mucosa, as hyaluronate is expressed within the lamina propria (37). In ad- FIGURE 7. Integrin ␣ deficiency preferentially reduces the number of E dition, CD45RB was expressed a slightly higher levels on IEL ␣␤ TCR -expressing intestinal IEL on the C57BL/6 background. Intestinal derived from ␣ Ϫ/Ϫ than on IEL derived from ␣ ϩ/ϩ mice (aver- IEL isolated from mice backcrossed for two to three generations to the E E by guest on September 24, 2021 age MFI ϭ 890 vs 780; p ϭ 0.0005; n ϭ 3, not visible in the C57BL/6 strain were reacted with the indicated mAb and analyzed for expression by FACS, evaluating the total lymphocyte population (a), the profile due to the logarithmic scale). This finding, while highly TCR ␣␤ϩCD8␣ϩ lymphocytes (b), or the TCR ␣␤ϩ T cells (c). Profiles reproducible, was of unclear functional significance. from one mouse of each genotype are shown, that are representative of ␣ Ϫ/Ϫ ␣ ϩ/ϩ ␣ ϩ/Ϫ results from four E and six E or E mice. Discussion ␣ Overall, the organization of the E-encoding gene was strikingly similar to that of other I-domain containing integrins, ␣ and ␣ ␣␤ϩ ␣␣ M X the proportions of intestinal TCR IEL that expressed CD8 , (38, 39), with a high degree of conservation in the intron/exon ␣␤ CD8 , or both CD4 and CD8 were not consistently altered in boundaries and exon spacing. In addition, an exon was identified ␣ Ϫ/Ϫ ␣ ϩ/Ϫ ␣ ϩ/ϩ E as compared with E or E mice (Fig. 7, b and c). between exons 5 and 6 that was not homologous to exons within ␣␤ϩ ϩ Ϫ However, the proportion of TCR IEL that were CD4 CD8 other integrins, suggesting that it may have resulted from a gene- ␣ Ϫ/Ϫ ␣ ϩ/ϩ ␣ ϩ/Ϫ Ϯ was greater in E than in E or E mice (7.3 0.9% of insertion event. This exon encoded 44 amino acids, corresponding ␣ ϩ/ϩ ␣ ϩ/Ϫ Ϯ the IEL in E or E mice vs 12.9 1.0% of the IEL in ␣ Ϫ Ϫ to the X-(extra)-domain identified within the predicted human E ␣ / mice; n ϭ 4; p ϭ 0.028). As the total number of IEL was ␣ E (40) and murine E subunits (15) (Fig. 1, a and b). The exons were reduced to ϳ50% of normal, an apparent doubling of the numbered based upon homology with the other I domain-contain- CD4ϩCD8Ϫ subset suggests that CD4ϩCD8Ϫ T cell numbers ␣ ing integrins, and the exon unique to integrin E was designated E were not increased but rather remained unchanged, while the other (Fig. 1). SSCP analysis was used to localize Itgae to mouse chro- ␣ subsets were reduced in number in E-deficient mice. In contrast, mosome 11, tightly linked to Abr, and was consistent with the ␣ in the BALB/c strain, where E deficiency had relatively little localization of sequence-tagged sites derived from the human ho- impact upon IEL numbers (Fig. 4a), the proportion of IEL that mologues of both Itgae (SHGC-12572, WI-22591) and Abr (WI- ␣ expressed CD4 was not altered in E-deficient mice (Fig. 8). Fi- 19743) to 17p13 on the human transcript map (http://www.ncbi. nally, within the lamina propria of mice backcrossed toward the nlm.nih.gov/genemap98 (41)). ϩ ϩ ␣ Ϫ/Ϫ C57BL/6 strain, the CD4 and CD8 populations both were re- In the E mice, there were reduced numbers of intestinal duced in number with a slightly greater impact upon the number of IEL in three different genetic backgrounds, evaluating mice housed ϩ ␣ CD4 cells (Fig. 5a). Overall, E deficiency resulted in dimin- in two different animal facilities. Thus, reduced intestinal IEL ϩ ϩ ␣ ished numbers of CD8 IEL but not of CD4 IEL in backgrounds numbers was a consistent feature of E deficiency, although the ␣ where E deficiency had a dramatic impact upon IEL numbers, but magnitude of this effect varied with genetic background and/or resulted in reduced numbers of both CD4ϩ and CD8ϩ lamina pro- environment. IEL numbers were also reduced in the vaginal epi- pria subpopulations. thelium, while the number of lymphocytes in lung parenchyma or ␣ 6648 INTEGRIN E-DEFICIENT MICE Downloaded from

␣ Ϫ/Ϫ ␣ ϩ/ϩ FIGURE 8. Intestinal IEL isolated from E mice N3–4 to the BALB/c strain express higher levels of CD44. Intestinal IEL isolated from either E ء Ϫ/Ϫ ␣ (grey fill pattern) or E (black line) mice were evaluated by FACS analysis for expression of T cell and adhesion molecules as indicated. indicates ␣ Ϫ/Ϫ ␣ ϩ/ϩ http://www.jimmunol.org/ Abs that reproducibly stained E intestinal IEL with a different average MFI intensity than E intestinal IEL. Histograms shown are representative of three experiments.

adjacent to the bronchiolar epithelium was not affected. While suggested one of two major possibilities, either that intestinal IEL ␣ lamina propria T lymphocyte numbers also were diminished by E play a role in maintaining lamina propria lymphocyte numbers or ␣ ␤ deficiency in some strains and/or environmental conditions, the that E 7 mediates another adhesive interaction that results in the number of T lymphocytes in Peyer’s patches and spleen was not homing or retention of lymphocytes within the lamina propria.

␣ ␣ ␤ by guest on September 24, 2021 diminished by disruption of the E-encoding gene. Thus, E de- While integrin 7-deficient mice had markedly reduced numbers ficiency appeared to function selectively in the generation/local- of lamina propria T lymphocytes, we found that intestinal IEL and ization of IEL and lamina propria T lymphocytes but not in the lamina propria T lymphocytes were present in normal numbers in generation of organized lymphoid tissues in the mucosa. While RAG-2-deficient mice that were reconstituted with bone marrow ␣ Ϫ/Ϫ other explanations may exist, these data are most consistent with a from 4 fetuses (our unpublished observations). Taken to- ␣ ␤ role of E 7 in modifying T lymphocyte localization to the intes- gether with the reduced lamina propria lymphocyte number ob- ␣ Ϫ/Ϫ ␣ ␤ tinal mucosa, but suggest that other receptors, such as CD44 and served in E mice, these studies suggest that E 7 participates ␣ ␤ ␣ ␤ possibly L 2 (42), may partially compensate for the loss of E 7. in the localization of lamina propria T lymphocytes. Additional It was notable that the proportion of intestinal IEL that ex- studies will be required to define the cellular and molecular basis ␣␤ ␣ pressed the TCR- was reduced in E-deficient mice and that the for this interaction. ␣ impact of E deficiency upon IEL number was greater in animals By comparing the impact of targeted integrin gene disruption, ␣ ␤ ␣ ␤ that were 5 wk of age or older than in younger mice. Thus, the insight is gained into the relative roles of the 4 7 and E 7 in- ␣ ␤ impact of E deficiency upon intestinal T lymphocyte numbers tegrins. In integrin 7-deficient mice, there are markedly reduced appeared during the time period when microbial colonization re- numbers of T and B cells in Peyer’s patches, intestinal lamina sults in increased numbers of TCR ␣␤-expressing intestinal IEL. propria, and in the intestinal epithelium (43). In addition, in mice ␣ ␣ Finally, the impact of E deficiency upon IEL/lamina propria lym- whose T lymphocytes lack the 4 subunit, there are reduced num- phocyte (LPL) numbers may have been influenced by environmen- bers of T and B lymphocytes in Peyer’s patches, while the numbers tal factors, as LPL numbers were reduced in some groups but not of IEL and of lamina propria T lymphocytes were not reduced ␣ ␤ others. Overall, these findings suggested that integrin E 7 may (Ref. 44, and our unpublished observations). These findings sug- ␣␤ϩ ␣ ␤ play a role in the recruitment/expansion of TCR IEL that is gest that 4 7 is essential for B and T cell localization to Peyer’s triggered by microbial colonization of the intestine. Based upon patches, but not for T lymphocyte localization to the intestinal ␣␤ ␣ Ϫ/Ϫ the flow cytometry analysis of IEL, it appeared that the T cell epithelium or lamina propria. Finally, in the E mice described ϩ Ϫ ␣ subset that was CD4 CD8 was not affected by E deficiency. In in this report, the number of IEL and lamina propria T lympho- contrast, the CD8␣␣ϩ, CD8␣␤ϩ, and CD4ϩCD8ϩ subsets were cytes was partially reduced, without an impact on the localization ␣ ␤ diminished to a similar extent, consistent with the expression of of B or T cells to Peyer’s patches. Thus, it appears that both E 7 ␣ ␤ ϩ ϩ ␣ ␤ E 7 on a larger proportion of CD8 than of CD4 intestinal IEL. and 4 7 can function in the localization of T lymphocytes to the ␣ ␤ While reduced IEL numbers were consistent with the known intestinal lamina propria and epithelium, while E 7 does not me- ␣ ␤ function of E 7, we were surprised to find reduced numbers of diate T lymphocyte localization to Peyer’s patches. The integrin ϩ ␣ Ϫ/Ϫ ␣ CD3 cells in the intestinal lamina propria of E mice, as E-deficient mice described herein will provide an important re- ␣ ␤ E-cadherin is not detected within the lamina propria by immuno- agent with which to further define the in vivo functions of E 7, histology (Ref. 32, and our unpublished observations). This finding and of the cells that express it, in health and disease. The Journal of Immunology 6649

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