Control of Α4β7 Integrin Expression and CD4 T Cell Homing by the Β1 Integrin Subunit

Control of α4β7 Integrin Expression and CD4 T Cell Homing by the β1 Integrin Subunit

This information is current as Christopher C. DeNucci, Antonio J. Pagán, Jason S. Mitchell of September 26, 2021. and Yoji Shimizu J Immunol 2010; 184:2458-2467; Prepublished online 29 January 2010; doi: 10.4049/jimmunol.0902407

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Supplementary http://www.jimmunol.org/content/suppl/2010/01/22/jimmunol.090240 Material 7.DC1 http://www.jimmunol.org/ References This article cites 56 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/184/5/2458.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Control of a4b7 Integrin Expression and CD4 T Cell Homing by the b1 Integrin Subunit

Christopher C. DeNucci,* Antonio J. Paga´n,† Jason S. Mitchell,* and Yoji Shimizu*

The a4b7 integrin promotes homing of T cells to intestinal sites. The a4 integrin subunit that pairs with b7 integrin can also pair with b1 integrin. In this paper, we show that the preferential pairing of b1 integrin with a4 integrin regulates the expression of a4b7 on T cells. In the absence of b1 integrin, naive mouse CD4 T cells have increased a4b7 expression, resulting in increased adhesion to mucosal addressin cell adhesion molecule-1 and enhanced homing to Peyer’s patches (PP). In a reciprocal manner, overexpression of b1 integrin causes the loss of a4b7 expression and decreased homing to PP. A similar upregulation of b1 integrin and suppression of a4b7 expression occurs rapidly after CD4 T cell activation. b1 integrin thus dominates b7 integrin for a4 integrin pairing, thereby controlling the abundance of unpaired a4 integrin. Increasing the abundance of a4 integrin relative b a b b to 1 integrin is critical to retinoic acid-mediated expression of 4 7 integrin during T cell activation. In the absence of 1 Downloaded from integrin, endogenous Ag-speciﬁc CD4 T cells uniformly express high levels of a4b7 after Listeria monocytogenes infection. The resulting b1-deﬁcient early memory T cells have decreased localization to the bone marrow and enhanced localization to PP after infection. Thus, the preferential association of b1 integrin with a4 integrin suppresses a4b7 integrin expression and regulates the localization of memory CD4 T cells. The Journal of Immunology, 2010, 184: 2458–2467.

ntegrins are heterodimeric cell surface expressed adhesion of either a4b1ora4b7 promotes altered trafficking properties http://www.jimmunol.org/ molecules composed of noncovalently linked a and b sub- based on the site-specific expression of the a4b1 ligand, VCAM- I units (1). T cells express several integrin family members 1, and the a4b7 ligand, mucosal addressin cell adhesion mole- that are involved in activation, trafficking, and retention in tissue cule-1 (MAdCAM-1). VCAM-1 is expressed at high levels on the (2, 3). On T cells, the a4 integrin subunit associates with either the vasculature of the bone marrow (BM) (2) and the inflamed brain b1 subunit, to form a4b1 integrin, or the b7 subunit, to form (13). Thus, a4b1 expression is critical for effector/memory T cell a4b7 integrin. Both a4b1 and a4b7 are expressed at low levels entry into these sites (10, 14). In contrast, MAdCAM-1 is spe- on naive T cells (4). The b7 integrin subunit can also pair with the cifically expressed at steady state on the venules of the mesenteric aE subunit, which is expressed on naive CD8 T cells (5) and CD4 lymph node (mLN) and Peyer’s patches (PP), and becomes highly regulatory T cells (6) but not naive CD4 T cells. The a4 integrins, upregulated on intestinal venules during inflammation (15, 16). by guest on September 26, 2021 along with aLb2 (LFA-1), promote recirculation through sec- Expression of a4b7 on T cells has been associated with prefer- ondary lymphoid organs at steady state (3, 7). Although a4b1 also ential trafficking to the intestine (17). The role a4 integrins play in localizes to the immunological synapse that forms between a T directing site-specific homing has made them attractive thera- cell and an APC (8), the in vivo relevance of a4 integrins for T peutic targets for treatment of multiple sclerosis and inflammatory cell activation by APCs remains unclear (9, 10). bowel disease (18, 19). During T cell activation, the expression of integrins changes to Recent studies have identified T cell extrinsic factors that control promote the entry of T cells into nonlymphoid sites. In contrast to the expression of a4b7 and the generation of gut homing T cells low levels of both b1 and b7 integrin on naive CD4 T cells, human (20). This work has revealed that retinoic acid (RA) produced by memory CD4 T cells express either high levels of a4b1 or high intestinal dendritic cells (DCs) and/or stromal cells specifically levels of a4b7 integrin (4, 11, 12). This reciprocal high expression promotes expression of a4b7 and CCR9 on T cells (21–23). In contrast, the vitamin D metabolite, 1,25 dihydroxy-VitD3, suppresses RA-driven induction of a4b7 and CCR9 while enhancing *Department of Laboratory Medicine and Pathology and †Department of Microbiol- the expression of skin-homing molecules in human T cells (24, ogy, Center for Immunology, Masonic Cancer Center, University of Minnesota Med- 25). These results suggest that the regulation of homing molecules ical School, Minneapolis, MN 55455 during T cell activation involves the integration of a variety of Received for publication July 24, 2009. Accepted for publication December 28, 2009. both positive and negative signals. This work was supported by National Institutes of Health Grants AI031126 (to Y.S.), The T cell intrinsic factors that regulate the expression of a4 F30 DK082139 (to C.C.D.), T32 AI007313 (to A.J.P.), and T32 CA009138 (to J.S.M.). Y.S. is supported in part by the Harry Kay Chair in Biomedical Research at the integrins on T cells are not known. As both a4b7 and a4b1 share University of Minnesota. a common a subunit, we predict that their expression is interrelated. Address correspondence and reprint requests to Dr. Yoji Shimizu, University of In this study, we show that the loss of b1 integrin on mouse CD4 Minnesota Medical School, Campus Code 2641, 2101 6th Street Southeast, Minne- T cells results in increased a4b7 expression, whereas high-level apolis, MN 55414. E-mail address: [email protected] expression of b1 integrin results in the loss of a4b7 expression. The online version of this article contains supplemental material. Interestingly, alterations in b7 integrin do not produce reciprocal Abbreviations used in this paper: BM, bone marrow; DC, dendritic cell; hCAR, b b human coxsackie adenovirus receptor; iLN, inguinal lymph node; Iso, isotype; MAd- changes in 1 integrin expression. We demonstrate that 1 integrin CAM-1, mucosal addressin cell adhesion molecule-1; mLN, mesenteric lymph node; regulates the expression of a4b7 expression through preferential PP, Peyer’s patches; RA, retinoic acid; TCPM, T cell proliferation media; wt, wild pairing with a4 integrin. In the absence of b1 integrin, CD4 T cells type. aberrantly express high levels of a4b7 in the spleen, resulting in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 enhanced localization to the PP and reduced maintenance in the BM. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902407 The Journal of Immunology 2459

B220, CD45.1, CD45.2, and CD44. The percentage of CD45.2+ and Materials and Methods + + low Mice CD45.1/45.2 CD4 CD44 T cells recovered from each organ was normalized to the percentage recovered from the spleen. b1 integrin floxed mice (26) were backcrossed to the C57BL/6 background for .14 generations and then crossed with CD4-Cre transgenic mice (27). In vitro stimulation assay b7 integrin-deficient mice were purchased from The Jackson Laboratory Purified naive T cells labeled with 5 mM CFSE were stimulated with plate- (Bar Harbor, ME) (28). Human coxsackie adenovirus receptor (hCAR) bound anti-CD3ε (3 mg/ml)/anti-CD28 (3 mg/ml) and maintained in T cell transgenic mice (29) were provided by Dr. C. Weaver (University of proliferation media (TCPM) (RPMI medium 1640 with L-glutamine and Alabama-Birmingham). All mice were housed and bred under specific 25 mM HEPES [Invitrogen] supplemented with 10% FBS [Atlanta Bio- pathogen-free conditions and generally used between the ages of 6–12 wk. logicals, Lawrenceville, GA], 100 mM sodium pyruvate, penicillin/strep- All experimental procedures were approved by the Institutional Animal tomycin, and 55 mM 2-ME). Cells were harvested at 3 d after plating and Care and Use Committee at the University of Minnesota. FACS stained for CD4, b1 integrin (HMb1.1-APC) (Biolegend), a4 in- a b Cell preparation and flow cytometry tegrin (R1-2-PE) (eBioscience), and 4 7 (DATK32-Bio, SA-PECy7) (eBioscience). For the RA treatment experiments, RA (R2625, Sigma- Single-cell suspensions were prepared from the spleen, lymph nodes, BM, Aldrich, St. Louis, MO) was added at 1000 nM in DMSO to the TCPM at and PP by mashing through cell strainers. BM cells were flushed from the start of stimulation and cells were harvested after 2 d. DMSO carrier bilateral, hindleg tibias, and femurs using PBS/2% calf serum. PP were alone was added to “No RA” samples. dissected from the small intestine, mechanically disrupted, and digested in HBSS/HEPES/10% calf serum and 400 U/ml collagenase D (Roche, Basel, Adenovirus production and transduction Switzerland) for 30–45 min at 37˚C. Purified T cells were obtained using b Murine b1 integrin (mCD29) and a4 integrin (mCD49d) cDNA clones negative selection by depleting cells expressing B220, I-A , CD16/32 (Abs (Open Biosystems, Huntsville, AL) were subcloned by PCR into the all from eBioscience, San Diego, CA) using MACS LS columns (Miltenyi Downloaded from pENTR-UP-IT vector using SalI and BamHI restriction sites. The pro- Biotec, Auburn, CA). For naive CD4 T cell purification, anti-CD8a and duction of adenovirus and transduction of T cells was performed as pre- anti-CD44 (both from eBioscience) were also added. For flow cytometry, 6 viously described (29, 32, 33). The isolated lymph node cells from hCAR 1–5 3 10 cells were stained in HBSS/0.2% sodium azide/2% calf serum expressing mice were transduced with either Thy1.1 control or integrin (FACS buffer) for 20–30 min at 4˚C. Samples were collected on a BD virus and cultured for 2–3 d in TCPM plus 10 ng/ml mouse IL-7 (R&D LSRII (BD Biosciences, San Jose, CA) and analyzed with FlowJo software Systems) then FACS stained for CD4, b1 integrin, a4 integrin, and a4b7 (Tree Star, Ashland, OR). integrin as above.

Intracellular integrin staining http://www.jimmunol.org/ Quantitative real-time RT-PCR ∼ After CD4 T cell purification ( 95% purity), cells were resuspended in 3 6 0.01% pronase (EMD Chemicals, Darmstadt, Germany) in PBS for 30–45 The 1–3 10 purified CD4 T cells were homogenized using QIAsh- mins at 37˚C to nonspecifically remove cell surface proteins. Samples were redder, and RNA was isolated with an RNeasy kit (Qiagen, Valencia, CA). washed twice with FACS buffer supplemented with 10% calf serum, and cDNA was produced from equivalent amounts of RNA with the Superscript then fixed with 2% paraformaldehyde for 25 min at room temperature. III Platinum Two-Step qRT-PCR Kit (Invitrogen) and the PCR products Fixed samples were stained once prior to permeabilization with the same were amplified with the Fast Start SYBR Green Master Mix (Roche). Abs to be used for intracellular staining. Cells were permeabilized using Samples were detected on ABI PRISM 7000 (Applied Biosystems, Foster 0.02% Triton-X 100 in PBS/0.2% BSA for 7–9 min at room temperature. City, CA). Hypoxanthine phosphoribosyltransferase was used to normalize Staining for intracellular integrin was performed for 20–30 min at room samples and the comparative CT method was used to quantify relative temperature with anti-b1 integrin (HMb1.1-APC) (Biolegend, San Diego, mRNA expression (34). Primers were designed using Primer Express by guest on September 26, 2021 CA) and anti-b7 integrin (M293-PE) (BD Bioscience) Abs. (Applied Biosystems). The primer sets used were as follows: hypoxanthine phosphoribosyltransferase (forward primer 59-CTTCCTCCTCAGACC- Adhesion assays GCTTT-39, reverse primer 59-ACCTGGTTCATCATCGCTAA-39); a4 integrin (forward primer 59-AATTGGACCAAGTGAGGGACAA-39, reverse Adhesion assays were generally performed as previously described (30). primer 59-TCGCTAGATCCATACACAAATGAAGT-39); b1 integrin (for- Purified T cells labeled with calcein-AM (Invitrogen, Carlsbad, CA) were ward primer 59-AATGCCAAATCTTGCGGAGAA-39, reverse primer added to wells coated with recombinant mouse VCAM-1 (0.6 mg/ml), 59-TCTAAATCATCACATCGTGCAGAAGTA-39); b2 integrin (forward MAdCAM-1 (6 mg/ml), or ICAM-1 (6 mg/ml) (R&D Systems, Minneap- primer 59- GATAACATGTACAAGAGGAGCAATGAGT-39, reverse primer olis, MN). Cells were incubated with anti-CD3ε (2C11) (eBioscience) and 59-CGCAAAGATGGGCTGGAT-39); b7 integrin (forward primer 59-TGC- integrin blocking anti-b1 (Ha2/5, 10 mg/ml), anti-b7 (Fib27, 10 mg/ml), or AGCTCATCATGGATGCTTA-39, reverse primer 59-CCGTCTTCTCAG- anti-aL (M17/4, 10 mg/ml) (all from BD Bioscience) Abs for a 15-min GACCCTTACA-39). binding time at 4˚C, followed by a 15-min stimulation at 37˚C. Wells were washed and the percentage of adherent cells was determined by comparing Listeria infections and peptide:MHC class II-tetramer well-fluorescence postwash to prewash. For adhesion assays with adeno- enrichments virus transduced cells, flow cytometry was used to quantify percentage of 3 7 adherent Thy1.1 high CD4 cells after stimulation with 50 ng/ml PMA. Mice were infected i.v. with 1 10 CFUs of ActA-deficient Listeria monocytogenes expressing 2W1S (A2Lm-2W1S) (35). 2W1S:I-Ab tetra- Short-term in vivo cohoming assays mer production, staining, and T cell enrichment were completed as previously described (36, 37). The enriched fraction of cells was FACS Lymph node cells from b1wt/wt CD4-Cre+ and b1fl/fl CD4-Cre+ mice (or stained with an Ab mixture for CD3ε, CD4, CD8a, and CD44. B220, Thy1.1 and b1 virus transduced T cells) were labeled with either Cell CD11b, CD11c, and F4/80 were used in a dump gate to exclude cells Tracker Green CMFDA (0.25 mM) or Cell Tracker Orange CMTMR (2 binding tetramer nonspecifically. CD11b and F4/80 were not used in the mM) (Invitrogen). Reversing these colors did not alter the experimental dump gate for the PP. Some samples were additionally labeled with anti-b7 outcome. Cells were then equally mixed and i.v. injected at 5–10 3 106 integrin (Fib504-PE) (Biolegend) alone or anti-b1 integrin (HMb1.1-PB) cells/mouse. An aliquot of the mixture was taken prior to injection to serve (eBioscience) and anti-a4b7 (DATK32-PE) (eBioscience). Total tetramer as the input control. At 2 or 24 h postinjection, the indicated organs were positive events were enumerated using CALTAG Counting Beads (In- harvested. Transferred cells were identified by intravital labels and CD4 vitrogen). Assuming that only 20% of total body BM cells are resident in staining by flow cytometry. For the adenovirus transduced T cell cohoming the hindlimb (38), the number of tetramer positive cells in the BM was assays, Thy1.1 staining was additionally used to identify transduced cells. multiplied by five to obtain total BM cell numbers (14). The steady-state The homing index was calculated as previously described (31). percentage of cells in the BM and PP was calculated on a per mouse basis by dividing the number of 2W1S-specific CD4 T cells recovered from Mixed BM chimeras these tissues by the number recovered from the spleen of the same mouse. Mixed BM chimeras were generated by mixing T cell-depleted BM cells Statistical analysis from b1wt/wt CD4-Cre+ (CD45.1/45.2) and b1fl/fl CD4-Cre+ (CD45.2) mice and transferring 5–10 3 106 cells i.v. into irradiated (1000 cGy) B6.SJL All statistical analysis was performed using GraphPad Prism software 5.0 (CD45.1) recipients. Tissues were harvested 8–15 wks post-BM transplant (La Jolla, CA). Two-tailed t test or one-way ANOVA, followed by Tukey’s and single-cell suspensions were FACS stained for CD3ε, CD4, CD8a, multiple comparison test were used to assess significance. 2460 CONTROL OF a4b7 INTEGRIN EXPRESSION BY b1 INTEGRIN

Results subunit or the b7 integrin subunit (Fig. 1C), consistent with the idea Loss of b1 integrin on CD4 T cells results in increased surface that enhanced a4b7 expression is occurring at the level of a/b expression of a4b7 subunit pairing. The enhanced surface expression of a4b7 observed on b12/2 T cells suggests that wild type (wt) CD4 T cells may To evaluate the function of b1 integrin on T cells, we crossed mice contain intracellular stores of b7 integrin that are capable of pairing with a floxed b1 integrin gene (b1fl/fl) (26) with transgenic mice with unpaired a4 integrin. We used pronase to remove cell surface expressing Cre recombinase under the control of the CD4 promoter 2 2 proteins from naive CD4 T cells, followed by permeabilization and (CD4-Cre) (27). In b1fl/fl CD4-Cre+ mice (b1 / mice), there is staining with anti-integrin Abs to assess expression of intracellular a complete loss of b1 integrin on the majority of CD4 T cells integrin subunits. Wt CD4 T cells had clearly detectable levels of collected from peripheral lymph nodes when compared with b1wt/wt intracellular b7 integrin subunit (Fig. 1D). CD4-Cre+ controls (Fig. 1A). However, the surface expression of a4 In contrast to the results obtained with loss of b1 integrin ex- integrin, a major pairing partner for b1 integrin, remained un- 2 2 pression, CD4 T cells from b7 integrin-deficient mice showed no changed on b1 / CD4 T cells. As intracellular a/b subunit pairing increase in b1 integrin and did not maintain a4 integrin expression is required for cell surface integrin expression, this suggests that at wt level (Fig. 1E). These results predict that the majority of b1 enhanced cell surface expression of b7 integrin, the other known integrin is cell surfaced expressed, leaving excess unpaired a4 pairing partner for a4 integrin, is responsible for maintaining a4 2 2 integrin without a pairing partner for cell surface expression when integrin expression on b1 / T cells. Using an Ab that specifically b7 integrin is lost. This is in agreement with the minimal levels of recognizes the a4b7 integrin, we found that a4b7 was significantly 2 2 intracellular b1 integrin observed in wt CD4 T cells (Fig. 1D). elevated on b1 / CD4 T cells (Fig. 1A,1B). The loss of b1 integrin Overall, these results suggest that through competition for a4 Downloaded from did not result in changes in mRNA levels for either the a4 integrin integrin pairing, b1 integrin expression limits the amount of a4b7 that is expressed on the surface of naive CD4 T cells.

b12/2 CD4 T cells have altered adhesion to a4 integrin ligands and trafficking to the BM and PP http://www.jimmunol.org/ To determine the functional consequence of loss of b1 integrin, we analyzed the adhesion of control and b12/2 T cells to VCAM-1 and MAdCAM-1. Control T cells showed low basal levels of adhesion to VCAM-1 that were dramatically enhanced by TCR stimulation (Fig. 2A). Adhesion to VCAM-1 was inhibited by a blocking anti-b1 integrin Ab. In contrast, b12/2 T cells did not adhere to VCAM-1 under any tested stimulation condition. b7- deficient CD4 T cells had normal adhesion to VCAM-1, further supporting that the loss of b7 integrin does not result in enhanced by guest on September 26, 2021 expression or function of a4b1 integrin (Supplemental Fig. 1). The elevated expression of a4b7onb12/2 T cells did result in enhanced adhesion to MAdCAM-1 compared with control T cells (Fig. 2A). Adhesion of both control and b12/2 T cells to MAd- CAM-1 was inhibited by a b7 integrin-specific Ab. The loss of b1 integrin on CD4 T cells did not alter mRNA transcript for b2 integrin, expression of LFA-1 (aLb2), or adhesion to ICAM-1 (Fig. 1C, Supplemental Fig. 2). We next performed short-term in vivo cohoming assays to determine how the loss of b1 integrin expression alters CD4 T cell homing. Control and b12/2 CD4 T cells were differentially labeled with intravital dyes, mixed in equal numbers, and transferred into recipient mice. At 2 h, equivalent numbers of control and b12/2 b FIGURE 1. Loss of 1 integrin on mouse CD4 T cells results in increased transferred CD4 T cells were recovered from the spleen, inguinal surface expression of a4b7. A, Representative FACS staining of iLN cells from b wt/wt + b fl/fl + b 2/2 lymph nodes (iLNs), and mLNs (Fig. 2B). In contrast, a significantly 1 CD4-Cre (wt) and 1 CD4-Cre ( 1 ) mice gated on Thy1.2 and b 2/2 CD4 double positive cells using anti-b1 integrin, anti-a4 integrin, and anti- higher number of 1 CD4 T cells were isolated from the PP of a4b7 integrin Abs. Light gray histograms represent appropriate isotype (Iso) the small intestine. This result is consistent with the elevated levels 2/2 control staining. B, Median fluorescence intensity of a4b7 expression on CD4 of a4b7 integrin on b1 T cells and the high levels of MAdCAM- T cells from wt or b12/2 lymph nodes. Bars show mean with SEM (n =5 1 expressed on venules in the PP. Expression of the gut-homing 2 2 separate mice). pp = 0.0009, two-tailed paired t test. C, Comparison of relative chemokine receptor CCR9 was not different between b1 / and 2 2 abundance of a4, b7, and b2 integrin mRNA levels between b1 / and wt control CD4 T cells (Supplemental Fig. 3). Fewer b12/2 CD4 CD4 T cells. Real-time RT-PCR data are presented as fold change between T cells were recovered from the BM, an area rich in the b1 integrin groups based on the comparative CT method. Bars represent mean with SEM ligand VCAM-1. Transferred CD4 T cell numbers were also ex- b b (n =3).D,Intracellular 1and 7 integrin staining on purified, wt CD4 T cells. amined at 24 h to assess retention (14) at these sites. Differences in The samples in the top panel were treated with pronase (to strip cell surface 2 2 the localization of control and b1 / T cells to the PP and BM that proteins), fixed, and stained for b1andb7 integrin as a control for the effec- tiveness of pronase treatment. Samples in the bottom panel were permeabilized were observed at 2 h were maintained at 24 h (Fig. 2B). b 2/2 after pronase treatment and stained again to identify intracellular stores of To examine the long-term steady-state distribution of naive 1 integrin. b12/2 and b7 integrin-deficient (b7KO) CD4 T cells were used as CD4 T cells, we generated mixed BM chimeras. The percentage of 2/2 a control for nonspecific staining. E, Representative FACS histograms of control and b1 CD4 T cells in various tissues was examined lymph node cells from wt and b7 KO mice. 8–15 wk after marrow transplant. No gross defect in CD4 T cell The Journal of Immunology 2461 Downloaded from FIGURE 2. T cells lacking b1 integrin have altered adhesion to a4 integrin ligands and trafficking to the BM and PP. A,AdhesionofCD4-Cre+ (wt) and CD4- Cre+ (b12/2) purified T cells to plate bound VCAM-1 or MAdCAM-1 after stimulation with soluble anti-CD3ε (2C11) Ab. Integrin blocking Abs were used to demonstrate the specificity of T cell-ligand interactions. The bars represent meanvalues from four replicates in one representative experiment of 3–4. B, b1wt/wt wt and b1fl/fl b12/2 CD4 T cells were differentially labeled, equally mixed, and transferred into recipient mice. Recipient organs were harvested at 2 and 24 h posttransfer and the ratio of CD4 transferred cells recovered was normalized to the transfer ratio from the mixed sample prior to injection (input). Values .1 indicate that b12/2 CD4 T cells were present in higher numbers than wt. Bars represent mean with SEM (n = 3–5 mice from two independent experiments). C, 2/2 + low 2/2

Percentage of b1 CD4 CD44 T cells recovered from the lymphoid organs of wt:b1 mixed chimeric mice. All samples are normalized to the percentage http://www.jimmunol.org/ of splenic b12/2 CD4+CD44low T cells, which was set to 50%. The values represent altered steady-state distribution of b12/2 CD4+CD44low T cells compared with the percentage recovered in the spleen. All experimental groups were compared against input or normalized spleen, ns . 0.05. pp = 0.01–0.05; ppp =0.001– 0.01; pppp . 0.001, one-way ANOVA, followed by Tukey’s multiple comparison test. development was noted with the loss of b1 integrin in these mice Elevated levels of b1 integrin results in loss of a4b7 expression (Supplemental Fig. 4). When compared with normalized T cell and function ratios from the spleen, there were similar numbers of control and b 2/2 After T cell activation, the induction of 1 integrin expression

b by guest on September 26, 2021 1 naive CD4 T cells in the iLN (Fig. 2C). In contrast, there was directly correlates with suppression of a4b7 expression. To de- b 2/2 an increased percentage of 1 naive CD4 T cells in both the PP termine whether high expression of b1 integrin is sufficient to and mLNs and a decreased percentage in the BM. These results suppress a4b7 expression, we overexpressed b1 integrin in naive b suggest that loss of 1 integrin on CD4 T cells results in decreased CD4 T cells. We used recombinant adenovirus expressing localization and retention in the BM, and enhanced localization and a Thy1.1 expression marker and transduced resting naive CD4 retention in the intestinal compartment. T cells isolated from transgenic mice expressing the hCAR (29, 33). Nontransduced cells (no virus) have no Thy1.1 expression a b T cell activation enhances 4 7 integrin expression in the and express b1 integrin, a4 integrin, and a4b7 integrin at wt b absence of 1 integrin levels (Fig. 4A). Transduction of CD4 T cells with a Thy1.1 We next examined changes in a4b7 expression after in vitro control virus did not alter the expression of b1 integrin, a4 in- stimulation of b12/2 CD4 T cells with anti-CD3 and anti-CD28 tegrin, or a4b7 integrin regardless of the level of Thy1.1 ex- Abs. Control and b12/2 CD4 T cells showed an equivalent pression. Strikingly, CD4 T cells transduced with adenovirus number of cell divisions and percentage of CFSE dilute cells after expressing Thy1.1 and b1 integrin exhibited a dramatic loss of 3 d of stimulation in vitro (Fig. 3A, Supplemental Fig. 5A). Anti- a4b7 integrin expression that correlated with increasing b1 in- CD3/CD28 stimulation of control CD4 T cells resulted in the tegrin expression (Fig. 4A). This finding replicates the alterations upregulation of b1 integrin and concurrent loss of a4b7 expres- in b1 integrin and a4b7 integrin expression observed after anti- sion (Fig. 3A). These changes in integrin expression began during CD3/CD28 stimulation of wt CD4 T cells (Fig. 3A). Over- blastogenesis even prior to the first cell division (nonblasting [0- expression of b1 integrin did not alter the level of the a4 integrin NB] to blasting [0-B]) (Fig. 3B, Supplemental Fig. 5B). The in- on the T cell surface. Unlike wt T cells (Fig. 1D), CD4 T cells crease in b1 integrin expression peaked by the second cell di- overexpressing b1 integrin contained clearly detectable levels of vision, which corresponds to the same point at which a4b7 intracellular b1 integrin (Fig. 4B). This indicates that not all ex- expression is completely lost. In contrast, stimulation of b12/2 ogenously expressed b1 integrin is able to be surface expressed. CD4 T cells resulted in dramatically enhanced expression of a4b7 The amount of intracellular b7 integrin was not increased by during blastogenesis that reached a peak of expression by the overexpression of the b1 integrin subunit (Figs. 1D,4B). Although second cell division. Surface expression of a4 integrin on control b1 integrin mRNA levels were elevated ∼20-fold in T cells CD4 T cells also increased prior to the first cell division and overexpressing b1 integrin, mRNA levels for a4, b7, and b2 in- peaked by the second cell division. Similar results were observed tegrin were unaltered (Fig. 4C). These results indicate that b1 with b12/2 CD4 T cells, although the peak of a4 integrin ex- integrin subunit outcompetes b7 integrin for a4 integrin pairing at pression was lower when compared with control. These results the protein level, resulting in the suppression of a4b7 expression. indicate that b1 integrin expression after CD4 T cell activation is In vitro adhesion assays demonstrated that CD4 T cells over- required to suppress a4b7 expression. expressing b1 integrin exhibited enhanced activation-dependent 2462 CONTROL OF a4b7 INTEGRIN EXPRESSION BY b1 INTEGRIN

FIGURE 3. b1 integrin-deficient T cells express high levels of a4b7 after stimulation in vitro. A, Purified naive b1wt/wt CD4-Cre+ (wt) and b1fl/fl CD4-Cre+ (b12/2)CD4 T cells labeled with CFSE were stimulated for 3 d with plate-bound anti-CD3/CD28 Abs. Histograms show CFSE dilution profiles of wt (white) and b12/2 (gray) CD4 T cells overlaid on unstimulated control CD4 T cells (black). Stimulated cells were additionally stained for integrin expression. B, Median florescence intensities of wt and b12/2 CD4 T cells based on CFSE division peaks from one representative experiment of three. Gate 0 (un- divided) is separated into nonblasting (0-NB) and blasting (0-B) CD4 T cells (Supplemental Fig. 5B). Dotted lines represent isotype control staining on stimulated samples. The isotype control for a4b7 overlays the x-axis. Downloaded from

adhesion to VCAM-1 and reduced adhesion to MAdCAM-1 when tegrin were also observed in the mLNs at both time points, but this compared with control T cells (Fig. 4D). In short-term in vivo difference was not statistically signiﬁcant. http://www.jimmunol.org/ cohoming assays, overexpression of b1 integrin did not alter CD4 a a b T cell localization to the spleen or iLN at both time points ex- Abundance of 4 integrin determines expression of 4 7 amined (Fig. 4E). However, localization of CD4 T cells over- Our ﬁndings predict that high b1 integrin expression in T cells expressing b1 integrin to the PP was reduced and localization to suppresses a4b7 integrin expression via competition for pairing with the BM was enhanced at 2 h and even more dramatically at 24 h the a4 subunit. To create a situation in which the amount of the a4 posttransfer. A reduced number of T cells overexpressing b1 in- integrin expressed in T cells is not limiting, we overexpressed a4

FIGURE 4. High expression of b1 integrin results in by guest on September 26, 2021 loss of a4b7 expression and function. A, Integrin staining on hCAR+ CD4 T cells transduced with no virus, a Thy1.1 control virus, or virus expressing Thy1.1 and b1integrin (b1 virus). Gates deﬁne the Thy1.1 high population. B, Intracellular b1andb7integrinstainingonpuriﬁed CD4 T cells transduced with b1 virus for 2 d. Cells treated with pronase alone then stained (pronase) are compared with cells that underwent additional permeabilization and staining to identify intracellular stores of integrin (Pronase + Perm). C, Comparison of relative abundance of b1, a4, b7, and b2 integrin mRNA levels between b1 virus and Thy1.1 control transduced CD4 T cells. Real- time RT-PCR data presented as fold change between

groups based on the comparative CT method. Bars represent the mean. D, Adhesion of Thy1.1 high expressing Thy1.1 control or b1 virus transduced CD4 T cells to plate- bound VCAM-1 or MAdCAM-1 after stimulation with PMA. Bars represent mean values with SEM (n =4in- dependent experiments). pp , 0.05, two-tailed paired t test. E, Cohoming of adenovirus transduced CD4 T cells labeled with Cell Tracker Orange (CTO) (Thy1.1 virus) or Cell Tracker Green (CTG) (b1 virus). Representative FACS dot plots show the percentage of Thy1.1 control and b1 virus transduced Thy1.1 high CD4+ cells recovered from each sample at 2 h posttransfer. The ratio of Thy1.1 high CD4+ transferred cells recovered was normalized to the ratio of Thy1.1 high CD4 T cells transferred (input). Values .1indicatethatb1 virus transduced CD4 T cells were present in higher numbers than Thy1.1 virus control. Bars represent mean with SEM (n = 4 mice in two independent experiments). Experimental groups were compared against input. pp = 0.01–0.05; ppp,0.001, one-way ANOVA, followed by Tukey’s multiple comparison test. The Journal of Immunology 2463 Downloaded from http://www.jimmunol.org/

FIGURE 5. Abundance of a4 integrin controls a4b7 integrin expression. A, Representative FACS histograms showing a4, a4b7, and b1 integrin staining on Thy1.1 control and a4 virus transduced CD4 T cells. a b a Light gray histograms represent appropriate Iso control staining. B, Rep- FIGURE 6. RA enhances 4 7 expression by increasing 4 integrin a resentative FACS histograms showing a4, a4b7, and b1 integrin staining abundance. A, Representative histogram of 4 integrin staining on naive on Thy1.1 control, b1 virus, and b1 and a4 virus cotransduced CD4 CD4 T cells or CD4 T cells stimulated (anti-CD3/CD28) with or without b a T cells. Light gray histograms represent appropriate Iso control staining. C, 1000 nM RA for 2 d. B, Comparison of relative abundance of 1, 4, and b Comparison of relative b1, a4, and b7 integrin levels between a4 virus 7 integrin mRNA levels between purified CD4 T cells stimulated with or and Thy1.1 control transduced CD4 T cells. Real-time RT-PCR data pre- without RA for 2 d. Real-time RT-PCR data presented as fold change by guest on September 26, 2021 sented as fold change between groups based on the comparative C between groups based on the comparative CT method. Bars represent the T b method. Bars represent the mean with SEM (n = 3). mean with SEM (n = 3). C, Representative dot plots showing 1 integrin versus a4b7 integrin expression on naive CD4 T cells or CD4 T cells stimulated without or with RA for 2 d. The percentage of cells in the integrin in naive CD4 T cells. Increasing a4 integrin expression re- a4b7-high, b1 integrin high gate is displayed in the plots. D, Represen- sulted in increased expression of a4b7 integrin without altering b1 tative dot plots showing b1 integrin versus a4b7 integrin on Thy1.1 integrin expression (Fig. 5A). These results are consistent with our control and b1 virus transduced CD4 T cells stimulated without or with finding that there are high amounts of intracellular b7, but not b1 RA for 2 d. The percentage of cells in the a4b7 high, b1 integrin high gate integrin, in naive CD4 T cells (Fig. 1D). mRNA transcript for a4 is displayed in the plots. integrin was increased ∼5-fold after overexpression of a4 integrin, whereas mRNA for b1andb7 integrin was not affected (Fig. 5C). little affected by the addition of RA (Fig. 6C). In contrast, ∼20% of Overexpression of both b1anda4 integrin resulted in suppression of the RA-treated CD4 T cells at 48 h after stimulation expressed high a4b7 similar to that seen with overexpression of just the b1 integrin levels of a4b7 (Fig. 6C). Thus, RA-induced a4b7 integrin ex- subunit (Fig. 5B). Overall, these findings indicate that an increase in pression is likely mediated by upregulation of a4 integrin. a4 integrin subunit expression can result in increased a4b7expres- To directly test the ability of b1 integrin to suppress a4b7integrin sion, but a relative abundance of b1integrintoa4 integrin can sup- upregulation after RA treatment, we stimulated CD4 T cells overex- press this effect. pressing b1 integrin. CD4 T cells transduced with control adenovirus demonstrated similar upregulation of a4b7 as wt cells after stimula- RA enhances a4b7 expression by increasing a4 integrin tion for 2 d (Fig. 6D). In contrast, activated T cells transduced with b1 abundance integrin adenovirus demonstrated no induction of a4b7 expression The presence of RA during T cell activation promotes increased in the presence of RA. This mirrors our findings with coexpression of expression of a4b7 integrin (21). To determine whether RA-in- a4andb1 integrin in naive CD4 T cells (Fig. 5B) and demonstrates duced a4b7 expression is mediated by suppression of b1 integrin that high abundance of b1 integrin can suppress RA-induced en- or enhancement of a4 integrin, we stimulated naive CD4 T cells hancement in a4b7. These results highlight the importance of a4 with anti-CD3/CD28 Abs for 2 d in the presence of RA. RA and b1 integrin stoichiometry in the control of a4b7 expression. treatment resulted in enhanced a4 integrin expression compared b 2/2 with CD4 T cells stimulated without RA (Fig. 6A). The increased a4 Ag-specific activation of polyclonal endogenous 1 CD4 a b integrin expression with RA treatment was accompanied by an ∼4- T cells results in enhanced 4 7 fold increase in a4 integrin mRNA levels, whereas mRNA levels for To determine whether b1 integrin expression is critical for regulating b 1 and b7 integrin remained unchanged (Fig. 6B). CD3/CD28 a4b7 expression after Ag challenge in vivo, we i.v. infected control stimulation resulted in increased b1 integrin expression that was and b12/2 mice with an ActA-deficient strain of L. monocytogenes 2464 CONTROL OF a4b7 INTEGRIN EXPRESSION BY b1 INTEGRIN expressing the 2W1S variant of peptide 52–68 from the I-E a-chain integrin expression (Fig. 7C). This population of a4b7-high cells (A2Lm-2W1S) (35, 39). At various time points after challenge, the resembles a circulating population of gut homing memory CD4 number of 2W1S-specific CD4 T cells was determined using recently T cells previously identified in humans that is b1 integrin low and described peptide:I-Ab MHC class II tetramer-based enrichment and a4b7-high (4, 11). This b7-high T cell population was main- flow cytometry gating approaches (Supplemental Fig. 6) (36, 37). tained long-term and represented ∼15% of the total splenic Endogenous populations of naive 2W1S-specific CD4 T cells from population by 60 d postinfection. In contrast, 2W1S-specific CD4 the spleens of uninfected control and b12/2 mice were equivalent in T cells from the spleens of b12/2 mice became uniformly b7- number (n = 7, wt: 238 6 29; b12/2:2346 39 [mean 6 SEM]) (Fig. high (98 6 0.2% n = 4) rapidly after infection and maintained 7A). Infection of control and b12/2 mice with A2Lm-2W1S resulted high level expression of b7 integrin out to day 120 (Fig. 7B, 7C). in a comparable expansion, contraction, and long-term maintenance Thus, the vast majority of 2W1S-specific b12/2 CD4 T cells in of 2W1S-specific CD4 T cells in the spleen out to 120 d (Fig. 7A). the spleen resemble the small population of a4b7-high CD4 However, the expression of a4 integrins on these activated T cells T cells observed in control mice. was dramatically different. At the peak of the response (day 5), 2 2 Altered maintenance of b1 / earlymemoryCD4T cellsin the BM a surprisingly high percentage of 2W1S-specific control CD4 T cells and PP (50 6 3%, n = 4) in the spleen expressed high levels of b7 integrin (Fig. 7B). By day 18, the majority of 2W1S-specific CD4 T cells To determine whether loss of b1 integrin and the resulting aberrant remaining in the spleen had low b7integrincomparedwithnaive upregulation of a4b7 results in altered early memory CD4 T cell CD4 T cells, but a population of a4b7-high cells (8 6 2%, n =5)was localization, we assessed the number of 2W1S-specific T cells in 2/2 2 maintained. These control 2W1S-specific CD4 T cells demon- the BM and PP of control and b1 mice 18 d after A Lm-2W1S Downloaded from strated a reciprocal relationship between b1integrinanda4b7 infection. This is a time point where we observed similar numbers of 2W1S-specific CD4 T cells in the spleen (Fig. 7A). Yet, in the BM there was a reduced number of 2W1S-specific CD4 T cells in infected b12/2 mice compared with control mice (Fig. 8A, 8B). In contrast, there were increased numbers of 2W1S-specific CD4 b 2/2

T cells in the PP of infected 1 mice compared with control http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Polyclonal endogenous b12/2 CD4 T cells in the spleen have enhanced a4b7 expression after i.v. infection. A, Peptide:MHC class II tetramer enrichment was used to quantify the number of endogenous FIGURE 8. b1 integrin-deficient early memory CD4 T cells have en- 2W1S-specific CD4 T cells in control b1wt/wt CD4-Cre+ (wt) and b1fl/fl hanced localization to the PP and reduced maintenance in the BM. A, CD4-Cre+ (b12/2) mouse spleens after i.v. infection with A2Lm-2W1S. Representative 2W1S:I-Ab tetramer staining of CD4+ T cells isolated from 2 Time 0 represents the number of 2W1S-specific recovered from naive wt or BM and PP 18 d after i.v. infection with A Lm-2W1S. The bold numbers b12/2 mice. Symbols represent mean with SEM (n = 4–11 mice/time below the gate are the total calculated 2W1S-specific CD4 T cells re- point, except wt day 120, n = 2). B, Representative histograms of b7 in- covered from each sample. B, Individual replicates of 2W1S-specific CD4 tegrin expression on splenic 2W1S-specific CD4 T cells from wt and b12/2 T cell numbers recovered from the BM and PP 18 d postinfection. Line mice. The table indicates mean percentage (6 SEM) of b7-high splenic represents mean value. pp , 0.02, two-tailed unpaired t test. C, The 2W1S-specific CD4 T cells (n = 2–5 mice). C, Representative 2W1S:I-Ab steady-state percentage represents the number of 2W1S-specific CD4 tetramer staining of splenic CD4+ T cells from wt and b12/2 mice 18 d T cells recovered in the BM or PP compared with the spleen of the same postinfection. b1 integrin versus a4b7 integrin expression is shown on the mouse. Bars represent mean with SEM (n = 3–7 mice). pp , 0.04; ppp , gated CD44high 2W1S:I-Ab tetramer+ cells. 0.006, two-tailed unpaired t test. The Journal of Immunology 2465 mice. All the 2W1S-specific CD4 T cells in the BM and PP ex- unit expressed in CD4 T cells. Thus, there is a hierarchy of b pressed high levels of CD44, a marker of Ag experience. As subunit pairing to the a4 integrin, with a “dominant” b1 integrin a measure of the steady-state maintenance of Ag-specific CD4 subunit that modulates the expression of the other heterodimer, T cells in the BM and PP, we also analyzed the percentage of 2W1S- a4b7. specific CD4 T cells in these tissues relative to the number of The functional relevance of this integrin subunit pairing hierarchy 2W1S-specific CD4 T cells in the spleen. This analysis revealed is revealed by our analysis of b1 and a4b7 integrin expression after that relative to the spleen, ∼8% of the 2W1S-specific CD4 T cells CD4 T cell activation. Activation of control T cells with anti-CD3/ were found in the BM and only ∼0.1% were found in the PP CD28 Abs for 3 d resulted in increased b1 integrin expression and postinfection of control mice (Fig. 8C). In contrast, postinfection of loss of a4b7 integrin expression even before the first cell division. b12/2 mice, only ∼2% of 2W1S-specific CD4 T cells were found These results are consistent with our overexpression data demon- in the BM relative to the spleen, whereas there was a 15-fold en- strating b1 integrin as the dominant a4 integrin pairing partner. In hancement in Ag-specific CD4 T cells in the PP (∼1.5%). Thus, the contrast, b12/2 T cells exhibit dramatically elevated levels of high level of a4b7 aberrantly expressed by b12/2 CD4 T cells after a4b7 expression. Expression of a4 integrin on T cells is also in- activation is associated with decreased steady-state numbers of creased after activation, although the level of surface a4 integrin early memory CD4 T cells in the BM and increased numbers in was slightly lower on b12/2 T cells. This is likely due to the overall intestinal sites. lower levels of b subunits available for pairing with a4 integrin in b12/2 T cells. These findings suggest that the preferential pairing Discussion of b1 integrin with a4 integrin is critical for the suppression of T cells express two distinct integrin heterodimers that contain the a4b7 expression after T cell activation. Downloaded from a4integrinsubunit,a4b7anda4b1. It is critical to define the Our work supports a model where b1 integrin expression mechanisms that control the expression of a4b7anda4b1on modulates a4b7 expression by controlling the abundance of a4 T cells, as these integrins promote homing to the gut (a4b7) and integrin available to pair with b7 integrin. In a naive T cell, all to extraintestinal sites, such as the BM and brain (a4b1) (3, 17). available b1 integrin pairs with a4 integrin and is expressed on the In this study, we demonstrate that changes in the expression of cell surface as a4b1. Any remaining free a4 integrin is then b1 integrin reciprocally alter a4b7 expression on CD4 T cells. available for pairing with b7 integrin, resulting in a low level of http://www.jimmunol.org/ We show that this regulation occurs at the protein level, where a4b7 integrin expression on the cell surface. The identification of b the a4 integrin subunit preferentially pairs with the b1 integrin an intracellular pool of 7 integrin in naive CD4 T cells suggests b a subunit when both b1andb7 integrin are present. We identify that 7 integrin is expressed in excess of the available 4 integrin. a b the abundance of a4integrinasthemajordriverofa4b7ex- We also demonstrate that 4 7 cell surface expression is en- a pression on CD4 T cells and demonstrate its importance in RA- hanced when the 4 integrin subunit is overexpressed in naive b induced a4b7 upregulation. Finally, by tracking an endogenous T cells, even though mRNA levels for 7 integrin are not altered. b Ag-specific population of CD4 T cells after infection, we dem- This suggests that intracellular 7 integrin serves as a reservoir of b a onstrate that the absence of b1 integrin results in enhanced a4b7 7 integrin available for pairing with free 4 integrin. Our model b by guest on September 26, 2021 expression and altered localization of early memory CD4 is consistent with microarray data that shows human, 7-high memory CD4 T cells have an increase in mRNA transcript for a4 T cells. integrin but not b7 integrin (40). The relative abundance of b1 Naive CD4 T cells express low levels of both the a4b1and integrin to a4 integrin is critical, as overexpression of both a4 and a4b7 integrins. When we crossed mice with floxed alleles of the b1 integrin results in suppression, rather than induction, of a4b7 b1 integrin gene with CD4-Cre transgenic mice, the loss of b1 expression. In this situation, the excess b1 integrin subunits likely integrin on naive CD4 T cells was associated with increased associate with the free a4 integrin subunits, thereby suppressing expression of a4b7. These results are similar to recent findings a4b7 heterodimer formation. Overall, our model predicts that the from an independently derived line of conditional b1-deficient precise ratio of ob1t a4 subunit is critical for controlling the mice (10). We show that this change in a4b7 expression has expression of a4b7. A ratio favoring b1 integrin results in sup- functional significance, as b12/2 T cells exhibit enhanced ad- pression of a4b7, whereas a ratio favoring a4 integrin results in hesion to MAdCAM-1 in vitro and increased localization to PP increased a4b7 expression. in vivo. Because successful cell surface integrin expression re- Previous work has shown that RA produced by intestinal DCs a b quires intracellular / heterodimer formation, we suggest that induces a4b7 expression on T cells (21, 22), but the exact cellular a the availability of the 4 integrin subunit for pairing regulates mechanism for how this occurs is unknown. CD8 T cells activated a b 4 7 integrin expression on T cells. Several lines of evidence by intestinal DCs are reported to have increased mRNA transcript a b suggest that the 4 integrin preferentially pairs with 1 integrin. for a4 integrin but not b1orb7 integrin (41). We find that RA- b First, we do not observe increased 1 integrin expression or treated, activated CD4 T cells have a similar pattern of enhanced b b function on 7-deficient CD4 T cells. Instead, loss of 7 integrin a4 integrin protein and mRNA without alterations in b1orb7 a expression results in decreased cell surface expression of 4in- integrin mRNA. Thus, RA-induced a4b7 expression is driven by tegrin. This suggests that all available b1 integrin associates with increased abundance of a4 integrin. As a result, the b1toa4 a4 integrin, even when b7 integrin is present. Staining of per- subunit ratio favors the a4 integrin. Under these conditions, a4 meabilized cells further supports this hypothesis, as there are integrin is no longer a limiting pairing partner. Thus, b7 integrin abundant levels of intracellular b7 integrin, but not b1 integrin, in can pair with excess a4 integrin not bound to b1, resulting in naive CD4 T cells. The inability of b7 integrin overexpression to increased a4b7 cell surface expression. Our finding that over- reduce b1 integrin expression in another system is also consistent expression of a4 integrin mimics the effects of RA treatment on with this model (4). Second, overexpression of b1 integrin on a4b7 expression supports this model. The precise balance be- naive CD4 T cells results in a dose-dependent decrease in a4b7 tween the b1 and a4 subunits remains critical, as the over- integrin expression without altering a4 integrin expression. Thus, expression of b1 integrin in RA-treated CD4 T cells suppresses increased levels of b1 integrin can effectively outcompete b7 the induction of a4b7. In this study, the addition of excess b1 integrin for association with the limiting amount of the a4 sub- integrin elevates the b1toa4 subunit ratio, resulting in 2466 CONTROL OF a4b7 INTEGRIN EXPRESSION BY b1 INTEGRIN suppression of a4b7 expression. These results highlight the im- of clinical interest with its known involvement in the progression portance of the stoichiometry of the a4, b1, and b7 integrin of IBD (19) and, more recently, as a coreceptor for HIV (56). subunits in determining the relative levels of a4b1 and a4b7 that are expressed on the surface of a T cell. This mechanism of Acknowledgments regulation of integrin expression may also be applicable to other T We thank Dr. M. Jenkins for critical discussion and input, Dr. S. S. Way for cell subsets that express integrin subunits that share a common the ActA-deficient Listeria monocytogenes-2W1S, M. Weinreich for assis- integrin subunit binding partner (42–45). tance with mixed BM chimeras, G. Hart for assistance with primer design To examine changes in integrin expression after T cell activation and real-time RT-PCR, and T. Lee, M. Schwartz, D. Loughran, and in vivo, we used peptide:MHC class II tetramer-based enrichment N. Rahman for mouse genotyping and colony maintenance. approaches to monitor changes in integrin expression on a polyclonal population of Ag-specific CD4 T cells after L. monocytogenes infection (36). This approach avoids possible alterations Disclosures in activation kinetics, homing molecule expression, and memory The authors have no financial conflicts of interest. generation and maintenance that have been reported when using high cell number adoptive transfer (46–49). In the spleen, we References unexpectedly detected high levels of a4b7on∼50% of activated 1. Hynes, R. O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell control CD4 T cells at the peak of the response (day 5). Although 110: 673–687. a b 2. von Andrian, U. H., and C. R. Mackay. 2000. T-cell function and migration. Two splenic DCs have been reported to induce 4 7 on CD8 T cells sides of the same coin. N. Engl. J. Med. 343: 1020–1034. in vitro, this has not been reported for CD4 T cells in vivo (50). A 3. Denucci, C. C., J. S. Mitchell, and Y. Shimizu. 2009. Integrin function in T-cell Downloaded from transient increase in the availability of the a4 subunit after T cell homing to lymphoid and nonlymphoid sites: getting there and staying there. Crit. b Rev. Immunol. 29: 87–109. activation in the spleen may explain the high percentage of 1 4. Schweighoffer, T., Y. Tanaka, M. Tidswell, D. J. Erle, K. J. Horgan, G. E. Luce, integrin expressing a4b7-high CD4 T cells recovered 5 d after A. I. Lazarovits, D. Buck, and S. Shaw. 1993. Selective expression of integrin infection. By day 18, the majority of activated control CD4 T cells a4b7 on a subset of human CD4+ memory T cells with hallmarks of gut-trophism. b a b J. Immunol. 151: 717–729. expressed high levels of 1 integrin and low levels of 4 7, 5. Masopust, D., V. Vezys, E. J. Wherry, D. L. Barber, and R. Ahmed. 2006. whereas a smaller subpopulation had a b1-low a4b7-high gut- Cutting edge: gut microenvironment promotes differentiation of a unique http://www.jimmunol.org/ homing phenotype (∼8%). Our results suggest that CD4 T cells memory CD8 T cell population. J. Immunol. 176: 2079–2083. 6. Lehmann, J., J. Huehn, M. de la Rosa, F. Maszyna, U. Kretschmer, V. Krenn, with this gut-homing phenotype have a mechanism to suppress b1 M. Brunner, A. Scheffold, and A. Hamann. 2002. Expression of the integrin a b + - integrin abundance at the protein and/or mRNA level. In contrast E 7 identifies unique subsets of CD25 as well as CD25 regulatory T cells. to the results obtained with control CD4 T cells, activation of Ag- Proc. Natl. Acad. Sci. USA 99:13031-13036. 2/2 7. Evans, R., I. Patzak, L. Svensson, K. De Filippo, K. Jones, A. McDowall, and specific b1 CD4 T cells resulted in uniformly elevated ex- N. Hogg. 2009. Integrins in immunity. J. Cell Sci. 122: 215–225. pression of a4b7 at all time points examined. This is consistent 8. Mittelbrunn, M., A. Molina, M. M. Escribese, M. Yań˜ez-Mo´, E. Escudero, b A. Ursa, R. Tejedor, F. Mampaso, and F. Sańchez-Madrid. 2004. VLA-4 integrin with our model that 1 integrin expression is critical for sup- concentrates at the peripheral supramolecular activation complex of the immune pressing a4b7 expression and modulating cell surface a4b7 after synapse and drives T helper 1 responses. Proc. Natl. Acad. Sci. USA 101: 11058– CD4 T cell activation. 11063. by guest on September 26, 2021 b 2/2 9. Nguyen, K., N. R. Sylvain, and S. C. Bunnell. 2008. T cell costimulation via the Control and 1 T cells exhibited comparable kinetics of integrin VLA-4 inhibits the actin-dependent centralization of signaling micro- CD4 T cell expansion, contraction, and maintenance in the spleen clusters containing the adaptor SLP-76. Immunity 28: 810–821. after L. monocytogenes infection. Although the number of b12/2 10. Bauer, M., C. Brakebusch, C. Coisne, M. Sixt, H. Wekerle, B. Engelhardt, and R. Fa¨ssler. 2009. b1 integrins differentially control extravasation of in- 2W1S-specific CD4 T cells was lower at all time points examined flammatory cell subsets into the CNS during autoimmunity. Proc. Natl. Acad. than controls, this difference was not statistically significant. Sci. USA 106: 1920–1925. Overall, this indicates that b1 integrin expression is not essential 11. Erle, D. J., M. J. Briskin, E. C. Butcher, A. Garcia-Pardo, A. I. Lazarovits, and M. Tidswell. 1994. Expression and function of the MAdCAM-1 receptor, in- for the expansion or maintenance of a polyclonal population of tegrin a4b7, on human leukocytes. J. Immunol. 153: 517–528. CD4 T cells in the spleen after Ag challenge. However, there were 12. Rott, L. S., M. J. Briskin, D. P. Andrew, E. L. Berg, and E. C. Butcher. 1996. A clear differences in the localization of activated CD4 T cells, with fundamental subdivision of circulating lymphocytes defined by adhesion to 2/2 mucosal addressin cell adhesion molecule-1. Comparison with vascular cell lower numbers of b1 T cells in the BM and higher numbers in adhesion molecule-1 and correlation with b7 integrins and memory differenti- the PP. As a source of IL-7 and IL-15, the BM appears to function ation. J. Immunol. 156: 3727–3736. 13. Koni, P. A., S. K. Joshi, U. A. Temann, D. Olson, L. Burkly, and R. A. Flavell. as a reservoir for the long-term maintenance of CD8 T cells (14, 2001. Conditional vascular cell adhesion molecule 1 deletion in mice: impaired 51, 52). IL-7 and IL-15 are also important for memory CD4 T cell lymphocyte migration to bone marrow. J. Exp. Med. 193: 741–754. survival (53, 54) and recent work suggests that CD4 memory 14. Mazo, I. B., M. Honczarenko, H. Leung, L. L. Cavanagh, R. Bonasio, W. Weninger, K. Engelke, L. Xia, R. P. McEver, P. A. Koni, et al. 2005. Bone T cells reside in the BM and associate with stromal cells that marrow is a major reservoir and site of recruitment for central memory CD8+ 2/2 produce IL-7 (55). Although less memory b1 CD4 T cells are T cells. Immunity 22: 259–270. maintained in the BM, we do not detect a significant loss of 15. Berlin, C., E. L. Berg, M. J. Briskin, D. P. Andrew, P. J. Kilshaw, B. Holzmann, I. L. Weissman, A. Hamann, and E. C. Butcher. 1993. a4b7 integrin mediates peptide-specific CD4 T cells in the spleen out to day 120 post- lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74: 2 2 infection. It is possible that enhanced a4b7 expression on b1 / 185–195. T cells promotes aberrant entry into other sites where similar 16. Briskin, M., D. Winsor-Hines, A. Shyjan, N. Cochran, S. Bloom, J. Wilson, L. M. McEvoy, E. C. Butcher, N. Kassam, C. R. Mackay, et al. 1997. 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