Lymphocytes Adhesion Molecules on Human T Redistribution of Antigen

Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes This information is current as of September 26, 2021. Jason S. Mitchell, Oguz Kanca and Bradley W. McIntyre J Immunol 2002; 168:2737-2744; ; doi: 10.4049/jimmunol.168.6.2737 http://www.jimmunol.org/content/168/6/2737 Downloaded from

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes1

Jason S. Mitchell, Oguz Kanca, and Bradley W. McIntyre2

The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular ␤ interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell 1 integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an ␤ enrichment of 1 integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of Downloaded from molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is en- ␤ riched in TCR and CD59, while 1 integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, ␤ CD59, CD43, and 1 integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the

TCR is now dissociated. These ﬁndings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the http://www.jimmunol.org/ normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated. The Journal of Immunology, 2002, 168: 2737Ð2744.

he lymphocyte cell surface membrane consists of micro- as Ras, Lck, Grb-2, phosphatidylinositol-3 kinase, and Fyn (2, scopically and biochemically distinguishable lipid mi- 4–7). Lipid rafts/microdomains are also believed to have a func- T crodomains that result from the assembly of sphingolipids tional significance, because cells with cholesterol depleted from and cholesterol into laterally mobile rafts (1, 2). Sphingolipids lipid microdomains have decreased levels of basal adhesion and contain predominately large, saturated acyl chains that allow them decreased CD3-induced TCR␨ phosphorylation (8, 9). Further- to pack tightly together. Furthermore, phase separations between more, lipid raft/microdomain patching can induce Ca2ϩ flux, by guest on September 26, 2021 lipid microdomains and loosely ordered membrane glycerophos- Z-chain associated-70 kDa protein/linker for activation of T cells/ pholipids give lipid microdomains a high degree of lateral mobility extracellular signal-regulated kinase-2 phosphorylation, and within the membrane (3). These lipid microdomains are also NFAT stimulation (10). The partitioning of these signaling mole- known as lipid rafts or glycolipid-enriched membrane domains cules to laterally mobile sphingolipids has been proposed as a and, due to their insolubility in nonionic mild detergents, they are means for signaling molecules to specifically “traffic” to areas of also called detergent-insoluble glycolipid-enriched domains receptor engagement and initiate a variety of different signaling (DIGs)3 or detergent-resistant membranes (4). Lipid microdomains pathways (1, 3). have a low density, and sucrose gradient ultracentrifugation of Lipid microdomains can be detected with the B-subunit of chol- mild detergent cell lysates can isolate these microdomains into era toxin (CTB), the membrane-binding subunit that binds a major low-density fractions along with the molecules that are associated component of lipid rafts, GM1 gangliosides (11, 12). Costimula- with them. Western blot analysis of these low-density fractions has tion of naive T lymphocytes with anti-CD3 and anti-CD28 beads revealed the presence of many GPI-anchored proteins, such as results in redistribution of GM1 lipids from a diffuse distribution to Thy-1, Ly-6, and CD59, and intracellular signaling proteins such a concentration at the bead contact site, as visualized by FITC- CTB. It was also determined that cross-linking GPI-anchored CD59, a raft/microdomain-associated lymphocyte surface protein, Department of Immunology, University of Texas, M. D. Anderson Cancer Center, or, more importantly, cross-linking of GM1 directly with immo- Houston, TX 77030 bilized CTB provided efficient T cell costimulation (13). The im- Received for publication July 10, 2001. Accepted for publication January 14, 2002. plication of these studies was that costimulation is mediated by The costs of publication of this article were defrayed in part by the payment of page lipid microdomains. Furthermore, the integrin LFA-1 was shown charges. This article must therefore be hereby marked advertisement in accordance to colocalize to membrane rafts/microdomains, and high-avidity with 18 U.S.C. Section 1734 solely to indicate this fact. adhesion could be induced by clustering these membrane microdo- 1 This work was supported by National Institutes of Health Grant CA62596 and Predoctoral Cancer Immunobiology Training Program Grant CA09598. mains (8). Therefore, lipid microdomains may also regulate lym- 2 Address correspondence and reprint requests to Dr. Bradley W. McIntyre, Depart- phocyte intercellular interactions needed for efficient adhesion to ment of Immunology University of Texas, M. D. Anderson Cancer Center, 1515 APCs during immune recognition or for adhesion to endothelial Holcombe Boulevard, Box 180, Houston, TX 77030. E-mail address: bmcintyr@ cells to mediate lymphocyte migration and recirculation. The bio- mail.mdanderson.org chemical evidence available so far has substantially implicated 3 Abbreviations used in this paper: DIG, detergent-insoluble glycolipid-enriched domain; CTB, cholera toxin B-subunit; HPB-ALL, human peripheral blood acute lym- lipid rafts/microdomains in the normal function of T cells (14–16). phocytic leukemia; IODO-GEN, 1,3,4,6-tetrachloro-3␣,6␣,diphenylglycoluril. However, the information from this type of analysis may be

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 2738 REORGANIZATION OF LYMPHOCYTE SURFACE MICRODOMAINS limited because the use of detergents in these experiments may sucrose gradients and the collected fractions were immunoprecipitated with ␤ partially solubilize some lipid microdomains even at low temper- protein G-agarose beads (Pierce) precomplexed with the anti- 1 integrin atures, disrupting low-affinity interactions and transient associa- mAb 18D3. Polypeptides were eluted by boiling in reducing Laemmli sample buffer and separated by 7.5% SDS-PAGE. Dried gels were exposed to tions (3, 15). Because lipid microdomain trafficking is based on Kodak XR film (Kodak, Rochester, NY) at Ϫ80°C. molecular compartmentalization, fluorescence microscopy has become an invaluable tool to visualize lipid microdomain associa- Fluorescence staining and image analysis tions and movements. In this paper we have investigated the spatial In the GM1 internalization experiments, HPB-ALL T cells were stained in relationships of various T cell adhesion and activation molecules complete RPMI 1640 medium with FITC-conjugated CTB (Sigma-Al- in the context of Ab cross-linking of GM1 lipid microdomains. drich) at 25 ␮g/ml for1hat37°C. The cells were then washed twice with Using CTB to follow lipid microdomains and specific fluorescent complete medium, fixed for 45 min with 4% paraformaldehyde, permeabilized gently with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, mAbs to follow surface molecules, we have found that clustering and stained with specific monoclonal (10 ␮g/ml) and AlexaFluor 594 goat lipid microdomains at physiological temperatures causes a dy- anti-mouse Abs. Finally, the cells were mounted to glass slides with Pro- namic rearrangement of normal T cell microdomain organization long Antifade reagents (Molecular Probes). and the formation of a novel supramolecular complex. The forma- In the lipid microdomain cross-linking experiments, HPB-ALL T cells tion of new microdomains is significant in that it brings molecules were incubated with specific mAbs and unconjugated CTB (Calbiochem) at 25 ␮g/ml. After washing twice with medium, the lipid microdomains together that were otherwise not associated, allowing for the ini- were cross-linked with a goat anti-CTB polyclonal Ab at 1/100 for 1 h at tiation of signaling cascades and the production of novel extracel- 37°C. The cells were then washed twice in medium and the lipid microdo- lular receptor architecture. mains and surface molecules were capped by staining with donkey anti-

goat AlexaFluor 488 and rabbit anti-mouse AlexaFluor 594 at 1 ␮g/ml Downloaded from Materials and Methods (Molecular Probes) at 37°C for 1 h. Finally, the cells were washed three times in medium, fixed with 4% paraformaldehyde for 20 min, and Cell culture mounted to slides with Prolong Antifade reagents. Images of the cells were The human peripheral blood acute lymphocytic leukemia (HPB-ALL) T taken on an Olympus IX70 fluorescent microscope (Olympus, Melville, cell line was maintained in RPMI 1640 medium supplemented with 100 IU/ml NY). Exposure times and settings were done within limits where no bleed- penicillin, 100 ␮g/ml streptomycin, and 10% FBS at 37°Cin5%CO. through was apparent between the red, green, and blue filters. Image anal- 2 ysis and merging of images was done with Adobe PhotoShop 5.5 software Antibodies (Adobe Systems, Mountain View, CA). http://www.jimmunol.org/ ␤ The mAbs T40/25 (anti-TCR), 33B6 (anti- 1 integrin), and IB7 (anti- Sucrose gradient ultracentrifugation CD43) were obtained from hybridomas produced in the laboratory. The mAbs anti-CD59, anti-CD45, and antitransferrin receptor were purchased The sucrose gradient procedure was modified from previously published from BD PharMingen (San Diego, CA), Caltag Laboratories (Burlingame, protocols (4, 7, 19, 20). Briefly, 3 ϫ 108 HPB-ALL T cells/sample were CA), and Zymed Laboratories (San Francisco, CA), respectively. The anti- treated with 125I-labeled mAb 33B6 with or without CTB for 1 h at 37°C. LFA-3 mAb was obtained from the clone TS2/9 (17). FITC-conjugated After washing twice with medium, the CTB-treated samples were cross- isotype-specific IgG1 and IgG2a Abs were purchased from Cappel/ICN linked with a goat anti-CTB polyclonal Ab for1hat37°C. The cells were Pharmaceuticals (Costa Mesa, CA) and Caltag Laboratories, respectively. then washed twice in medium and treated with rabbit anti-mouse IgG to ␤ Goat anti-CTB was purchased from Calbiochem (La Jolla, CA). Donkey cross-link 1 integrins and donkey anti-goat IgG to cross-link lipid mi- anti-goat AlexaFluor 488, rabbit anti-mouse AlexaFluor 594, and goat anti- crodomains. After cross-linking, the cells were washed in PBS and lysed in by guest on September 26, 2021 mouse AlexaFluor 594 were purchased from Molecular Probes ice-cold gradient buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) (Eugene, OR). containing 1% Brij-97 (Sigma-Aldrich). The lysed cells were kept on ice for 1 h, vortexed vigorously every 15 min, and spun at 800 ϫ g at 4°C for Labeling of Abs 5 min. One milliliter of the postnuclear supernatant was then mixed with 1 ml of 80% sucrose in gradient buffer and placed at the bottom of an ul- Purified 33B6 was radioiodinated using 1,3,4,6-tetrachloro-3␣,6␣,diphe- tracentrifuge tube. Six milliliters of 34% sucrose and 4 ml of 5% sucrose nylglycoluril (IODO-GEN; Pierce, Rockford, IL) (18). In brief, 25 ␮gof was then carefully overlaid on top of the sample and the gradient was then 33B6 in 100 ␮l of PBS was added to an IODO-GEN-coated 12 ϫ 75 mm spun in a SW41 centrifuge tube at 200,000 ϫ g at 4°C for 20 h. One- glass test tube (10 ␮g of IODO-GEN per tube). The reaction was initiated milliliter fractions were then collected from the top of the gradient and each by the addition of 1 mCi of Na125I (New England Nuclear, Boston, MA) fraction and the pellet were counted in a gamma counter where the per- and allowed to proceed for 20 min at room temperature. The reaction was centages of total counts were determined. Fractions 4 and 5 represent the stopped by removing the 33B6 from the reaction vessel. Radioiodinated 5/34% sucrose interface and contain the majority of the DIGs (7). 33B6 was separated from free iodine by gel filtration on Sephadex G-25 (Sigma-Aldrich, St. Louis, MO) equilibrated in PBS containing 1% BSA . AlexaFluor 350 and 594 protein labeling kits were purchased from Mo- Results ␤ lecular Probes. T40/25 and 33B6 Abs were concentrated to 2 mg/ml and Cross-linking GM1 microdomains enhances 1 integrin DIG incubated with the AlexaFluor 350 and 594 succinimidyl ester dyes, re- associations spectively, stirring at room temperature for 1 h. Previous labeling of 33B6 ␤ showed the 1-h incubation did not sufficiently label 33B6, so the reaction Although not found in DIGs in one study with epithelial cells, 1 was continued overnight at 4°C. The reactions were stopped with hydrox- integrins from fibroblasts and CD36-transfected melanoma cells ylamine and unlabeled dye was separated from the labeled Abs with a have been shown to be insoluble in detergents such as Triton sizing column supplied by the kit. The degree of labeling was then determined by taking the absorbances of the labeled Abs at 280/346 nm for X-100, Brij 96, and Brij 97, and move to low-density fractions in ␤ AlexaFluor 350 and 280/590 nm for AlexaFluor 594. T40/25 labeled with a sucrose gradient, suggesting 1 integrins could be associated ␤ AlexaFluor 350 was labeled at 5.74 mol of dye/mol of Ab, and 33B6 with lipid microdomains (20–22). To examine 1 integrin associ- labeled with AlexaFluor 594 was labeled at 6.25 mol of dye/mol of Ab. ations with lipid microdomains in human T cells at physiologic Iodination of cell surface proteins and immunoprecipitation temperatures, HPB-ALL T cells were treated with radioiodinated anti-␤ integrin Ab 33B6 with or without treatment with CTB. The Cell surface proteins were labeled by lactoperoxidase-catalyzed radioiodi- 1 nation as described in a previous publication (18). Briefly, 3 ϫ 108 cells cells were then lysed in 1% Brij 97 and centrifuged in a layered were washed three times in PBS and once in PBS with 1 ␮M potassium sucrose gradient to isolate the DIGs, and radiolabeled 33B6 was ␮ ␮ ␤ iodide (KI). Cells were resuspended in 500 l of PBS with 1 M KI, and used to follow quantitatively where 1 integrin moved in the su- 10 ␮g of lactoperoxidase (Sigma-Aldrich) and 0.2 IU of glucose oxidase crose gradient (19, 20). Without CTB treatment, cross-linked ␤ (Sigma-Aldrich) were added. Next, 3 mCi of Na125Iin500␮l of PBS with 1 1 ␮M KI and 10 mM glucose was added and the sample was incubated at integrins in these T cells are not significantly found in the DIG room temperature. After 15 min the cells were washed three times in ice- fractions (Fig. 1, No CTB), and this is similar to the findings with cold PBS containing 5 mM KI. 125I-labeled cell lysates were processed in the epithelial cells. When CTB is added to the HPB-ALL T cells, The Journal of Immunology 2739

compared with cells with no CTB treatment. CTB exists as a pen- tamer and it binds the oligosaccharide portion of GM1 sphingolipids cooperatively (23). This pentavalency action of CTB may ␤ cross-link GM1, promoting a mild 1 integrin-lipid microdomain ␤ interaction and increasing the buoyancy of 1 integrin in the gradient. However, treatment with CTB alone does not seem to be ␤ signiﬁcant enough to cause 1 integrin enrichment in the DIG fractions (fractions 4–5).

Cross-linking GM1 lipid microdomains redistributes GM1 sphingolipids Another way to observe lipid microdomains is by ﬂuorescent microscopy. To visualize GM1 lipid microdomains HPB-ALL T cells were treated with FITC-labeled CTB. When the staining was done at 4°C the GM1 was localized diffusely over the cells with no clear localization pattern (Fig. 2A). But when the staining was done at 37°C to see GM1 sphingolipid localization at physiological temperatures, the GM1 appeared to cluster and was localized centrally on the cell (Fig. 2A). Previous studies have found that in resting T Downloaded from cells most of the GM1 is intracellular but in T cell blasts a higher proportion of the GM1 is found on the cell surface (13). Further- more, hippocampal neuronal cells have been shown to endocytose CTB and target it to the Golgi complex, and COS-7 cells have been shown to continuously circulate GM1 sphingolipids between the

plasma membrane and Golgi pools (24, 25). To determine whether http://www.jimmunol.org/

␤ FIGURE 1. Cross-linking CTB enhances the association of 1 integrin ␤ with DIGs. Human HPB-ALL T cells were treated with anti- 1 integrin

with or without CTB at 37°C for 1 h. Rabbit anti-mouse IgG with or by guest on September 26, 2021 without goat anti-CTB Abs were added at 37°Cfor1htocross-link the integrin and CTB, respectively. Donkey anti-goat Abs were added to appropriate samples and incubated for 37°C for 1 h. The cells were lysed in 1% Brij 97 and DIGs were isolated by sucrose gradient ultracentrifugation. ␤ A, The anti- 1 integrin mAb was radiolabeled and the fractions collected were counted in a gamma counter and percentages of counts were determined. The graph is representative of four separate experiments. B, HPB- ALL T cell lysates from radiolabeled cells are subjected to sucrose gradient ␤ ultracentrifugation, and then the 1 integrin is immunoprecipitated from the different fractions and resultant polypeptides are resolved by SDS-PAGE.

␤ but not cross-linked, 1 integrin localization to the DIG fractions is not significantly changed (Fig. 1, CTB). In contrast, when HPB- ALL T cells are treated with CTB and cross-linked with anti-CTB Abs at 37°C, the lipid microdomain fractions now contain almost ␤ ␤ 10% of the 1 integrins (Fig. 1, CTB X-linked). This shift of 1 integrins from the pellet to the DIG fractions suggests that cross- ␤ linking of CTB promotes the association of 1 integrins with lipid ␤ microdomains. Furthermore, this is the first demonstration that 1 integrins can be triggered to localize into DIGs in T lymphocytes. FIGURE 2. The distribution of GM1 sphingolipids on T cells. A, HPB- To confirm that the DIG counts detected actually correspond to ALL T cells stained with FITC-CTB at 4°C and 37°C show different dis- ␤ tribution patterns of GM1 sphingolipids. Fixed and permeabilized cells the presence of 1 integrin, the Fig. 1A experiments were repeated using cell surface radioiodinated HPB-ALL T cell lysates. The show a similar distribution pattern to 37°C FITC-CTB-treated cells. B,T fractions collected were then immunoprecipitated with the anti-␤ cells stained with FITC-CTB at 37°C and then cross-linked with goat anti- 1 CTB and anti-goat Alexa 594 Abs at 4°C show a central GM1 sphingolipid integrin mAb 18D3 bound to protein G beads, and the eluted endocytic vesicle (upper panels), while T cells stained with FITC-CTB and polypeptides were resolved by SDS-PAGE. As seen in Fig. 1B, goat anti-CTB/anti-goat Alexa 594 at 37°C show the GM1 sphingolipids only the cross-linked CTB cells show a significant enrichment of clustered at the cell surface (lower panels). C, Kinetic analysis of FITC- ␤ ␣ integrin 1 and the associated 4 subunit in the DIG fractions. In CTB being internalized (upper panels) or being clustered on the cell sur- the CTB alone-treated cells, there seemed to be a slight movement face with anti-CTB Abs (lower panels). Fluorescent images taken through of integrins to the higher gradient fractions (fractions 7–11), as the appropriate filters and computer-generated overlays are shown. 2740 REORGANIZATION OF LYMPHOCYTE SURFACE MICRODOMAINS the altered staining seen at 37°C is CTB in endocytic vesicles, HPB-ALL T cells were treated with FITC-CTB at 37°C for 1 h and then stained with anti-CTB and AlexaFluor 594 secondary Abs at 4°C for 1 h each (Fig. 2B, upper panels). The shift to 4°C stops further endocytosis and membrane movements and, if the large central cluster of GM1 was located on the cell surface, both the FITC-CTB and anti-CTB would bind and the overlays would colocalize. However, Fig. 2B, upper panels, shows that the anti-CTB did not bind the centrally clustered GM1 but rather bound the periphery of the cell, indicating that the centrally clustered GM1 is in fact inside the cell. The clustering of GM1 does not appear to be CTB dependent as HPB-ALL T cells fixed, permeabilized, and stained with FITC-CTB also showed the centrally clustered GM1 (Fig. 2A). To examine the localization of GM1 during cross-linking of GM1 lipid microdomains at physiologic temperatures, HPB-ALL cells were treated with FITC-CTB for1hat37°C and cross-linked with an anti-CTB Ab and AlexaFluor 594 secondary Abs at 37°C for 1 h each (Fig. 2B, lower panels). Instead of seeing the char- Downloaded from acteristic CTB-defined GM1 endocytic vesicle, the Ab was able to FIGURE 3. Cointernalization of T cell surface molecules with GM1 capture and retain the GM1 at the cell surface and form distinct sphingolipids. HPB-ALL T cells were treated with FITC-CTB at 37°C for patches or caps of GM1 sphingolipids. We have concluded from 1h,fixed, permeabilized, and stained with specific mAbs and AlexaFluor ␤ this that CTB binding of GM1 allows us to follow the GM1 being 594 secondary Abs for the T cell surface molecules TCR (A), 1 integrin recycled and that CTB cross-linking Abs can prevent this recycling (B), CD43 (C), CD59 (D), and LFA-3 (E). Brightfield, fluorescent images by capturing and clustering the GM1 at the cell surface. The dis- taken through the appropriate filters and computer-generated overlays are http://www.jimmunol.org/ appearance of the GM1 endocytic vesicle is not believed to be due shown. to degradation of the CTB, because when HPB-ALL T cells are treated with FITC-CTB at 37°C for 1 h, washed of excess CTB, ␤ and then incubated at 37°C for an additional 3 h, the GM1 endo- 3, A and D). However, 1 integrin, CD43, and LFA-3 (Fig. 3, B, cytic vesicle was still apparent (data not shown). C, and E) were not internalized with the GM1 endocytic vesicle, as To assess the dynamics of the GM1 sphingolipid endocytosis seen by the diffuse and peripheral staining. These internalization and CTB cross-linking, the kinetics of these events were next ex- experiments were also done with some classically defined non- amined. Cell surface GM1 sphingolipids on HPB-ALL T cells raft-associated molecules such as transferrin receptor and CD45 (2, were labeled with FITC-CTB at 4°C for 1 h. The cells were then 4, 10, 26). We found that CD45 was not internalized with GM1 by guest on September 26, 2021 washed extensively at 4°C and then either placed at 37°C (CTB- and, similar to the findings from the Lippincott-Schwartz labora- treated cells) or incubated with anti-CTB Abs at 4°C for 1 h and tory (25), transferrin receptor was internalized with GM1 endo- then placed at 37°C (CTB cross-linked cells). The cells were then cytic vesicles (data not shown). fixed in 3.2% paraformaldehyde at specific time intervals. Fig. 2C ␤ integrin, CD43, and LFA-3 colocalize in a microdomain shows the general cell population phenotype at each time interval. 1 independent of CD59/TCR/GM1 For the CTB alone-treated cells (Fig. 2C, upper panels), at 0 and ␤ 1 min it can be clearly seen that GM1 sphingolipids are evenly The results in Fig. 3 indicate that 1 integrin, CD43, and LFA-3 do dispersed over the cells. However, by 2 min traces of CTB are seen not colocalize with the GM1 and, by inference, suggest no colo- inside the cells as GM1 sphingolipid cycling resumes. By 3 min calization with TCR and CD59. Because the spatial relationships ␤ the endocytic GM1 pools are clearly evident in the cells, and this within the group of CD43, LFA-3, and 1 integrin were not de- ␤ phenotype is also readily visible at 10 min. For the cross-linked termined, 1 integrin was visualized with a directly conjugated CTB cells (Fig. 2C, lower panels) at 0 min the GM1 sphingolipids AlexaFluor 594 mAb, while CD43 and LFA-3 were visualized are evenly dispersed over the cells, similar to CTB alone-treated with mAbs and FITC-conjugated secondary Abs. Each Ab step cells. As the cells warm, the GM1 sphingolipids are not endocy- was done at 37°C for 1 h. As shown in Fig. 4, CD43 and LFA-3 ␤ tosed as seen in the CTB alone-treated cells, but rather they begin are associated with 1 integrin in the same region of the cell. These ␤ to cluster on the cell surface, forming multiple small clusters at the results indicate that 1 integrin, CD43, and LFA-3 have the ca- early time points (1–2 min), then forming larger clusters (3 min), pacity to colocalize to a region of the cell separate from the TCR/ and eventually forming large caps (10 min). CD59/GM1 microdomain. This previously undescribed organization of surface proteins defines a novel microdomain. ␤ Exclusion of 1 integrin, CD43, and LFA-3 from the TCR, CD59, and GM1 lipid microdomains Cross-linking the GM1 microdomain results in the redistribution The endocytosis of GM1 could be viewed as the endocytosis of a of molecules from two different microdomains into a specific lymphocyte microdomain containing GM1 and GM1-as- supramolecular complex sociated molecules. To determine which lymphocyte surface mol- The Ab cross-linking of GM1 through CTB was shown in Fig. 2 ecules are being internalized with the GM1, HPB-ALL T cells to redistribute the GM1 into defined caps or patches. It is possible were stained with FITC-CTB at 37°C (Fig. 3, CTB), fixed and that these dynamic events may be accompanied by the lateral permeabilized with paraformaldehyde and Triton X-100, and movement of membrane molecules into novel associations. HPB- ␤ stained with mAbs to TCR, 1 integrin, CD43, CD59, and LFA-3 ALL cells were incubated with CTB and then cross-linked with a (Fig. 3, mAb). The GM1 endocytic vesicles are highly enriched goat anti-CTB polyclonal Ab and then with a donkey anti-goat with TCR and CD59 as clearly demonstrated in the overlay (Fig. AlexaFluor 488, where each step is done for1hat37°C. This The Journal of Immunology 2741

calization of CD45 with lipid rafts was not absolute, as small regions of colocalization are seen, but this is not surprising, as it has been reported that CD45 can localize to lipid rafts (27). ␤ Cytochalasin D does not inhibit colocalization of 1 integrin and GM1 sphingolipids Actin polymerization in T cells has been shown to be important in the normal function of both lipid rafts/microdomains and integrins ␤ (9, 19, 28–31). To investigate whether the colocalization of 1 integrin with GM1 sphingolipids could be inhibited by disrupting ␤ ␤ FIGURE 4. CD43 and LFA-3 colocalize with 1 integrin. HPB-ALL T actin polymerization, GM1 sphingolipids and 1 integrins were ␤ cells were stained at 37°C with 1 integrin-specific mAb 33B6 directly cross-linked, as in Fig. 5B, in the presence of 0.1 mM cytochalasin conjugated with AlexaFluor 594, and with mAbs to CD43 (A) and LFA-3 D. This concentration of cytochalasin D totally inhibits HPB-ALL (B), which were visualized with an isotype-specific rabbit anti-mouse FITC cellular spreading on fibronectin (data not shown). Inhibition of conjugate. Brightfield, fluorescent images taken through the appropriate actin polymerization by cytochalasin D did not appear to affect the filters and computer-generated overlays are shown. ␤ ability of 1 integrin to colocalize with GM1 sphingolipids under cross-linking conditions, as can be seen in the overlay (Fig. 6A). However, cytochalasin D did appear to have an effect on the pat-

␤ Downloaded from causes 1 integrin and CD43 to localize into the GM1 microdo- tern of the colocalization, as cells treated with cytochalasin D main (Fig. 5, B and C), while CD59 remained in the GM1 mi- stained with small patches, while cells stained in control medium crodomain (Fig. 5D). LFA-3 was always seen to localize to a close generally showed single large caps (Fig. 6A). These different pat- proximity of the GM1 microdomain, but the overlays show that terns were quantitated by classifying the colocalized patterns into LFA-3 and the GM1 microdomain never completely colocalize three categories: one large cap, two or three medium-sized patches, (Fig. 5E). The TCR was typically seen to also stay in the GM1 and multiple small patches. The cells were stained in three differ- microdomain, as seen by the arrow in the overlay (Fig. 5A); how- ent treatments: medium, medium and 1/100 ethanol/DMSO (cy- http://www.jimmunol.org/ ever, there were some occasions where the TCR and GM1 mi- tochalasin D solvent), and in medium 1/100 ethanol/DMSO and crodomains were not seen to cocluster together as seen by the 0.1 mM cytochalasin D, where the total number of cells counted distinct localization of the TCR at the right periphery of the cell ␤ (Fig. 5A). Therefore, some of the components ( 1 integrin and CD43) from the previously deﬁned novel microdomain have merged with the TCR/CD59/GM1 microdomain upon GM1 cross- linking. Transferrin receptor and CD45 were also examined under these cross-linking conditions. Upon CTB cross-linking, transferrin receptor is still seen to colocalize with GM1 sphingolipids at by guest on September 26, 2021 the cell surface while the majority of CD45 is not colocalized with GM1 sphingolipids (data not shown). The ﬁdelity of the noncolo-

FIGURE 6. The effect of cytochalasin D treatment on GM1 sphingo- ␤ FIGURE 5. Cross-linking GM1 microdomains causes redistribution of lipid and 1 integrin colocalization. A, HPB-ALL T cells were treated with surface molecules. GM1 gangliosides were cross-linked on HPB-ALL T or without cytochalasin D and GM1 gangliosides were cross-linked by cells by CTB followed by a goat anti-CTB Ab and then a donkey anti-goat CTB followed by a goat anti-CTB Ab and then a donkey anti-goat Alex- ␤ ␤ AlexaFluor 488 Ab. TCR (A), 1 integrin (B), CD43 (C), CD59 (D), and aFluor 488 Ab. 1 integrin was visualized with a specific mAb and a rabbit LFA-3 (E) were each visualized with specific mAbs and a rabbit anti- anti-mouse AlexaFluor 594 Ab. B, The resultant patterns with and without mouse AlexaFluor 594 Ab. Brightfield, fluorescent images taken through cytochalasin D were quantitated as one large cap, two to three medium the appropriate filters and computer-generated overlays are shown. sized patches, or multiple small patches. 2742 REORGANIZATION OF LYMPHOCYTE SURFACE MICRODOMAINS

FIGURE 7. Dissociation of the TCR from the GM1 lipid microdomain. A, HPB-ALL T cells were stained with FITC-CTB to show the internalization ␤ of the GM1 microdomain. TCR was visualized with a TCR-specific mAb directly conjugated with AlexaFluor 350 and 1 integrin localization was shown ␤ with a 1 integrin mAb directly conjugated with AlexaFluor 594. B, GM1 gangliosides were stained and cross-linked on HPB-ALL T cells by adding CTB followed by a goat CTB Ab then a donkey anti-goat AlexaFluor 488 Ab. The TCR was visualized with a TCR-specific mAb directly conjugated with ␤ ␤ AlexaFluor 350 and 1 integrin localization was shown with a 1 integrin mAb directly conjugated with AlexaFluor 594. Brightfield, fluorescent images taken through the appropriate filters and computer-generated overlays are shown. Downloaded from

for each treatment was 884, 388, and 921, respectively. As can be gated caps on T cells but may not be as important for the associ- ␤ seen in Fig. 6B, the predominant phenotype was one large cap in ation of 1 integrin with GM1. the medium (51.9%) and medium/DMSO/ethanol (51.2%) treat- http://www.jimmunol.org/ ments, while in the cytochalasin D-treated cells the predominant Triple-color stain shows lymphocyte microdomain phenotype was small patches (70.9%). These data suggest that ac- reorganization tin polymerization is necessary for the formation of large aggre- As presented in Fig. 5, cross-linking GM1 sphingolipids through CTB with secondary Abs results in a reorganization of microdo- ␤ mains such that 1 integrins can become associated with the GM1- defined microdomain into a supramolecular cluster. To further visualize this rearrangement a triple-color stain using FITC-CTB and directly conjugated Abs was used to simultaneously follow the ␤ by guest on September 26, 2021 movements of GM1, TCR, and 1 integrin under GM1 cross- linked and noncross-linked conditions. In Fig. 7A, HPB-ALL T ␤ cells were treated with FITC-CTB, anti- 1 integrin mAb Alexa 594-33B6, and anti-TCR mAb Alexa 350-T40/25 for1hat37°C. The overlay of the images clearly shows the CTB endocytic ves- ␤ icle is enriched with TCR and the 1 integrin is localized away in a separate microdomain. Upon cross-linking of GM1 sphingolip- ␤ ids, the overlay in Fig. 7B shows 1 integrin localizing to the GM1 cap, but the TCR was always found away from the GM1 supramolecular cluster. Dissociation of the TCR from the GM1 supramolecular complex was also seen in Fig. 5A, but not to the extent that it is seen under the conditions in Fig. 7B. The difference between the two conditions is that the TCR in Fig. 5A was visualized with secondary Abs that may be clustering the TCR, while the TCR in Fig. 7B was visualized with a directly conjugated fluorescent mAb. This suggests that through cocapping the TCR can be held in the FIGURE 8. Quantitation of molecular reorganization. HPB-ALL cells GM1 cap, while an unrestrained TCR is able to dissociate from the were stained under conditions as described in previous figures. These con- GM1 lipid microdomain after GM1 cross-linking. ditions are as follows: 1) GM1 endocytosis is allowed to proceed and then Quantitation of many of the events concerning integrin, TCR, cells are fixed, permeabilized, and stained for either the integrin (A)orTCR and GM1 are presented in Fig. 8. Under conditions such as in Figs. (B); 2) GM1 and integrin (C) or TCR (D) are treated with primary reagents, 2 and 3, where GM1 internalization is followed, in 78% of the cells ␤ cross-linked with secondary Abs at physiologic temperatures and then 1 integrins are not associated with GM1 and in none of these cells fixed; or 3) at physiologic temperature, GM1 is treated with cholera toxin were these molecules found to be completely colocalized (Fig. and cross-linked with Abs but the primary anti-TCR mAb alone is added 8A). In contrast, in Ͼ80% of the cells, the TCR completely colo- (E). Results are presented as the percentage of cells with no colocalization, calized with GM1 and Ͻ2% of the cells showed no colocalization partial colocalization, or complete colocalization of the integrin or TCR of these two molecules (Fig. 8B). Under conditions such as in Fig. with GM1. Partial colocalization indicates that the molecules in the caps ␤ are Ͼ50% colocalized even though parts of an individual cap could be 5, where both molecules are cross-linked, most of the 1 integrin segregated into GM1-, TCR-, or integrin-enriched zones. In contrast, com- (Fig. 8C) and the TCR (Fig. 8D) are found to be completely co- plete colocalization means Ͼ95% of the molecules in the cap are colocal- localized with GM1. Finally, under conditions where GM1, but not ized. The number of cells analyzed for each condition are as follows: A, TCR, is cross-linked such as in Fig. 7B, the TCR and GM1 do not 107; B, 106; C, 108; D, 105; and E, 1030. colocalize (Fig. 8E). The Journal of Immunology 2743

Discussion ence of the TCR, CD59, and GM1 sphingolipids and the other is ␤ The physical relationship of lipid microdomains with other surface a previously undefined microdomain consisting of 1 integrin, proteins has been explored in a variety of experimental conditions. CD43, and LFA-3. When GM1 sphingolipids are cross-linked with One of these is the use of sucrose gradients to isolate detergent secondary Abs, a different novel microdomain is detected that con- ␤ insoluble glycolipids and their associated molecules from low-den- tains GM1 sphingolipids, 1 integrin, CD43, and CD59. Thus, it ␤ sity fractions. The tight packing from the long acyl chains of sphin- appears that 1 integrin and CD43 can assemble free from the LFA-3 region and become stabilized with the GM1/CD59 mi- golipids in the liquid-ordered (lo) and the gel-like phases leads to the detergent insolubility of the lipid rafts/microdomains because crodomain, which in turn is dissociated from the TCR, forming a these lipid-lipid interactions are more stable than lipid-detergent separate microdomain. These observed rearrangements imply a ␤ highly dynamic set of microdomains whose molecular composi- interactions (3). 1 integrin is a type I transmembrane molecule; therefore, it is predicted to be poorly associated with lipid mi- tion is greatly influenced by the ligation of surface molecules. An crodomains in intact cells and in DIGs in cell lysates, as it is understanding of the dynamics of these microdomain rearrange- lacking a GPI anchor or closely spaced myristate and palmitate ments will make clear which molecules are anchoring and which chains, which are found on classical DIG-associated molecules may be laterally mobile during critical events in lymphocyte such as CD59, linker for activation of T cells, p56Lck, and p59Fyn function. (3, 32–36). The functional significance of the distribution of surface pro- In other investigations of ␤ integrin associations with lipid mi- teins in microdomains is only now becoming realized. Many dif- 1 ferent signaling molecules have been shown to be associated with crodomains there have been conflicting reports on the degree of lipid microdomains such as Ras, Lck, Grb-2, phosphatidylinosi- Downloaded from DIG/␤ integrin associations, which could be due to differences in 1 tol-3 kinase, and Fyn (2, 4–7). Because different surface proteins cell types, detergents, and cell treatments (20–22). The function of are associated with characteristic intracellular signaling molecules, ␤ integrin in T cells is notably different from nonhematopoietic 1 it is intriguing to speculate that novel combinations of signaling cells. Besides serving roles in adhesion and migration, T lympho- ␤ molecules could activate or inhibit a wide array of biological pro- cyte 1 integrins have been found to be potent costimulatory mol- ␤ ␤ cesses. When the novel supramolecular complex of TCR/GM1/ 1 ecules (37). In our analysis to determine whether T cell 1 inte- integrin/CD43/CD59 is formed, proteins such as focal adhesion http://www.jimmunol.org/ grins associated with lipid microdomains in DIGs, we clustered ␤ 1 kinase, vinculin, paxillin, Ras, and c-Src may be brought in by ␤ integrin and GM1 sphingolipids with Abs at 37°C. The cells were 1 integrin, which could create a new molecular configuration of cy- then lysed in 1% Brij 97, a detergent that is less hydrophobic than toskeletal proteins, molecular scaffolds, and signaling proteins and Triton X-100, which would help maintain lower-affinity interac- allow novel signaling to occur (43, 44). Because p56Lck has been tions, which are nevertheless still highly specific (20). As the re- found to be enriched in lipid microdomains, and Lck has been sults showed, clustering ␤ integrin alone did little to stabilize ␤ 1 1 shown to be a key regulator in the affinity of ␣ ␤ integrins for integrin-DIG associations. However, by simultaneous cross-link- 4 1 VCAM-1 (45), an intriguing possibility is that the movement of ␤ ing of ␤ integrins and GM1 lipid microdomains, the ␤ integrin 1 1 1 integrins into lipid microdomains may be a way of directing inte- content in DIG fractions increased 5-fold. The enrichment of ␤ by guest on September 26, 2021 1 grin affinity state or controlling cellular avidity (2, 6, 45). Thus, a ␤ integrin in DIG fractions through simultaneous cross-linking of 1 mechanism of selective redistribution of surface receptors into dif- integrins and lipid microdomains may be viewed as either a sta- ferent functional domains could provide a system for regulating bilization of existing molecular associations or a dynamic activa- lymphocyte activation, homing, and recirculation. Furthermore, ␤ tion event where 1 integrin is recruited into lipid microdomains. ␤ when the TCR leaves this GM1/ 1 integrin/CD43/CD59 complex, We have also found that CD43 from detergent-solubilized cells the TCR contribution to this signaling complex would be termi- subjected to ultracentrifugation on sucrose gradients has a similar nated. Immune responses could be ended or enter a new phase as ␤ DIG distribution pattern to 1 integrin. Under conditions where the TCR moves from its original location to other areas of the cell. only CD43 is cross-linked with Abs, 2% of CD43 is DIG associ- The identification of different organized regions on the T lym- ϳ ated, but upon cross-linking both CD43 and GM1, 13% of CD43 phocyte surface further illustrates the complexity of cellular be- becomes DIG associated (data not shown). Because cross-linking havior. The spatial and temporal dynamics of these microdomains T cell GM1 lipid microdomains at physiologic temperatures has indicate their formation probably depends on a variety of factors been previously described to stimulate various T cell activation including proximity, lateral diffusion, molecular recognition and ␤ pathways, it will be interesting to follow the time course of 1 exclusion, cytoskeleton association, and activation modality. Fu- integrin, CD43, and GM1 colocalization in relationship to the for- ture studies should help unravel the possible combinations of sur- mation of an immunological synapse (8, 10). face molecules that are driven by different activation processes or In the study of microdomain organization many different models associated with various differentiation states. When compiled, a have been used to determine where molecules spatially position descriptive catalog of possible microdomains will be a useful tool themselves on T cells. Some of these focus on T cell contact sites in studies to identify the physical, molecular, and biological forces with other cells, microbeads, or planar membranes, while other that establish microdomain composition and the rules that control studies have used models that rely on the addition of soluble pro- molecular segregation between domains. These studies will be es- teins such as specific Abs to induce the capping of surface proteins sential in our understanding of T lymphocyte biology. (13, 38–42). We have chosen this latter approach to explore the dynamics of microdomains because we wanted to eliminate the Acknowledgments constraints imposed by an opposing surface, thus allowing the ex- We thank Dr. Bernard Andruss for critically reviewing the manuscript. amination of the inherent propensity of surface molecules to redistribute and colocalize into domains when triggered with soluble reagents. References The results of our localization experiments demonstrate that ini- 1. Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes. Nature 38:569. tially at least two distinct molecular regions or microdomains can 2. Rogers, W., and J. K. Rose. 1996. Exclusion of CD45 inhibits activity of p56lck be distinguished on the T cell surface. One is defined by the pres- associated with glycolipid-enriched membrane domain. J. Cell Biol. 135:1515. 2744 REORGANIZATION OF LYMPHOCYTE SURFACE MICRODOMAINS

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