Movement Within Lymph Nodes T Lymphocytes

Impaired Trafficking of Gnai2+/− and Gnai2− /− T Lymphocytes: Implications for T Cell Movement within Lymph Nodes This information is current as Il-Young Hwang, Chung Park and John H. Kehrl of September 23, 2021. J Immunol 2007; 179:439-448; ; doi: 10.4049/jimmunol.179.1.439 http://www.jimmunol.org/content/179/1/439 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2008/03/14/179.1.439.DC1 Material References This article cites 35 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/179/1/439.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology :Impaired Trafficking of Gnai2؉/؊ and Gnai2؊/؊ T Lymphocytes Implications for T Cell Movement within Lymph Nodes Il-Young Hwang, Chung Park, and John H. Kehrl1 Signals generated by the engagement of chemoattractants with their cognate receptors orchestrate lymphocyte movements into and out of lymphoid organs and sites of inflammation. Yet, the role of chemokines in organizing lymphocyte movements in lymphoid organs is controversial. Recent evidence suggests that the extensive network of fibroblastic reticular cells within the T cell areas helps guide T cells. The expression of adhesion molecules and chemokines by fibroblastic reticular cells most likely facilitates their influence on T cell movements. Consistent with this hypothesis, CD4 T cells with defective chemokine receptor signaling move very differently within lymph nodes than do normal cells. For the imaging studies, we used CD4 T ؊/؊ cells prepared from Gnai2 mice, which lack G␣i2 expression. We first demonstrate that CD4 as well as CD8 T cells from ؊/؊ these mice are markedly defective in chemokine receptor signaling. Gnai2 T cells have profound defects in chemokine- Downloaded from .induced intracellular calcium mobilization, chemotaxis, and homing, whereas Gnai2؉/؊ T cells exhibit modest defects Intravital imaging revealed that within the inguinal lymph nodes Gnai2؊/؊ CD4 T accumulate at the cortical ridge, poorly accessing the lymph node paracortex. They also lack the customary amoeboid-like cell movements and active membrane projections observed with normal CD4 T cells. These results demonstrate the importance of G␣i2 for T lymphocyte chemokine receptor signaling and argue that local chemoattractants regulate the movement of CD4 T cells in lymph nodes. The Journal of Immunology, 2007, 179: 439–448. http://www.jimmunol.org/ roper functioning of the immune system depends upon thelial venules (HEVs), and decreased B cell motility within lymph changing intracellular contacts and cellular localization node follicles (8). P both within immune organs and within the body. Che- The impact of Gnai2 deficiency on T cell chemotaxis and T cell moattractants act as signposts to recruit and position lymphocytes trafficking has not been reported. Yet defective T cell function has and dendritic cells in lymphoid organs and inflammatory sites (1, been documented. Gnai2Ϫ/Ϫ mice develop a Th1-mediated in- 2). Most chemoattractants and chemokines signal through G pro- flammatory colitis reminiscent of human ulcerative colitis, whose tein-coupled receptors (GPCRs)2 that use the heterotrimeric G pro- penetrance depends upon the genetic background of the mice (10). Ϫ Ϫ / by guest on September 23, 2021 tein Gi to activate downstream effectors (3, 4). The binding of Gnai2 CD4 T cells exhibit augmented responses to TCR sig- ligand activates receptors triggering G␣i subunits to exchange GTP naling with enhanced intracellular calcium release and cytokine Ϫ/Ϫ for GDP, resulting in the dissociation of the G␣ subunit from its production; in contrast, Gnai3 T cells respond normally to associated G␤␥ heterodimers (5, 6). The release of Gi-associated TCR signaling (9). A relative increase in mature thymocytes in the Ϫ/Ϫ G␤␥ subunits is necessary for triggering directional migration (3, 4, thymus has also been noted in the Gnai2 mice (10). 7). Because G␣ subunits possess an intrinsic GTPase activity, GTP To characterize the impact of Gnai2 deficiency on T cell che- hydrolysis leads to the reassembly of heterotrimeric G protein, motaxis and T cell trafficking, we have examined CD4 and CD8 T causing signaling to cease (5, 6). Lymphocytes strongly express cells from wild-type, Gnai2ϩ/Ϫ, and Gnai2Ϫ/Ϫ mice. We find that Ϫ/Ϫ Ϫ/Ϫ two members of the Gi subfamily, G␣i2 and G␣i3 (8). Gnai3 Gnai2 T cells exhibit significant defects in chemokine receptor mice are reportedly without a phenotype (9); however, Gnai2Ϫ/Ϫ signaling and lymphocyte trafficking, suggesting that signals that mice exhibit defective B cell chemokine receptor signaling, as ev- modulate the level of G␣i2 present in lymphocytes directly affect idenced by depressed B cell chemotaxis, defective B cell homing the capacity of lymphocytes to respond to chemokines. The to lymph nodes, poor B cell adherence to lymph node high endo- Gnai2Ϫ/Ϫ mice have pronounced defects in chemokine receptor signaling, indicating that Gnai3 and Gnai1 poorly compensate for the loss of Gnai2 and that CXCR5 and CCR7 predominantly cou- ple to Gi␣2 in T lymphocytes. Finally, these results argue that Laboratory of Immunoregulation, National Institute of Allergy and Infectious Dis- GPCR signaling significantly impacts the movement of T cells eases, National Institutes of Health, Bethesda, MD 20892 within the lymph node. Received for publication September 25, 2006. Accepted for publication April 16, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Mice 1 Address correspondence and reprint requests to Dr. John H. Kehrl, Laboratory of Ϫ/Ϫ Immunoregulation, National Institute of Allergy and Infectious Diseases, National The generation of Gnai2 mice has been previously described (10). The Institutes of Health, Building 10, Room 11B08, 10 Center Drive MSC 1876, Be- mutation was backcrossed onto a C57BL/6 background six times. Mice thesda, MD 20892. E-mail address: [email protected] heterozygotic or homozygotic for the mutation were produced by crossing heterozygotic mice. Wild-type littermates were used as controls. C57BL/6 2 Abbreviations used in this paper: GPCR, G protein-coupled receptor; 3-D, three- 2ϩ 2ϩ mice were purchased from The Jackson Laboratory. All mice used in this dimensional; [Ca ]i, intracellular Ca concentration; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and 6-)(((4-chloromethyl)benzoyl) amino)tetram- study were 8–14 wk of age. Mice were housed under specific pathogen- ethylrhodamine; FRC, fibroreticular cell; HEV, high endothelial venule; S1P, free conditions and used in accordance with the guidelines of the Institu- sphingosine 1-phosphate. tional Animal Care Committee at the National Institutes of Health. www.jimmunol.org 440 DEFICIENCY OF G␣i2 INHIBITS CHEMOKINE RECEPTOR SIGNALING Reagents cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star). Forward and side scatter parameters were used to gate Abs against mouse CD11a, CD11c, GR-1, CD49d, CXCR4, CXCR5, on live cells. Alternatively, the labeled cells were injected into the footpad CCR5, CD4, CD8a, B220, and CD62L were purchased from BD Pharm- of recipient mice, and 12 h later the ipsilateral popliteal and inguinal lymph ␣ ␣ ␣ ingen; CCR7 from BioLegend; and G i1,G i2, and G i3 from Santa Cruz nodes and the contralateral popliteal lymph node were removed and pro- Biotechnology. Streptavidin conjugated to PE was purchased from BD cessed, as above. In some instances, the T cells were pre incubated with Pharmingen. Pertussis toxin was purchased from Calbiochem. The 5-(and 100 ng/ml pertussis toxin for2hat37°C before transfer. 6-)(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine (CMTMR) and 5-chloromethylfluorescein diacetate (CMFDA) were purchased from Lymph node transit assay Molecular Probes. Murine CCL19, CXCL12, and CXCL13 were purchased from R&D Systems. The assay was performed as previously described (8). Splenic CD4 T cells from wild-type or Gnai2Ϫ/Ϫ mice were labeled with either 2 ␮M CMFDA Cells or CMTMR for 15 min at 37°C, and 7–20 million cells of each population were injected i.v. to recipient mice. Two hours later, the mice were injected Splenic T cells were isolated by negative depletion using biotinylated Abs i.v. with either PBS or anti-L-selectin Ab (100 ␮g/mouse). After 12 h, to B220, GR-1, and CDllc and Dynabeads M-280 streptavidin (Dynal Bio- inguinal and popliteal lymph nodes were removed and gently dissociated tech), as previously described (11). The addition of a biotinylated Ab to into single-cell suspensions. Flow cytometric analysis was performed on a CD4 or CD8 allowed isolation of CD8 or CD4 T cells, respectively. The FACSCalibur flow cytometer (BD Biosciences), and the data were ana- cell purity was greater than 95%. Cells were placed in complete RPMI lyzed using the FlowJo software (Tree Star). Forward and side scatter 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml parameters were used to gate on live cells. penicillin, 100 ␮g/ml streptomycin, 1 mM sodium pyruvate, and 50 ␮M 2-ME.

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