CD2 molecules redistribute to the uropod during scanning: Implications for cellular activation and immune surveillance

Elena V. Tibaldi*†, Ravi Salgia†‡, and Ellis L. Reinherz*†§

*Laboratory of Immunobiology and ‡Division of Adult Oncology, Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, and †Department of Medicine, Harvard Medical School, Boston, MA 02115

Communicated by Stuart F. Schlossman, Dana-Farber Cancer Institute, Boston, MA, April 9, 2002 (received for review February 14, 2002) Dynamic binding between CD2 and CD58 counter-receptors on op- cells, whereas its counter-receptor CD58 is expressed on a posing cells optimizes immune recognition through stabilization of diverse array of nucleated and non-nucleated cells including cell–cell contact and juxtaposition of surface membranes at a distance APCs and stromal cells (reviewed in refs. 11 and 12). CD2 suitable for T cell receptor–ligand interaction. Digitized time-lapse functions in both T cell adhesion and activation processes (13). Ϸ differential interference contrast and immunofluorescence micros- Of note, the weak affinity of the CD2-CD58 interaction (Kd copy on living cells now show that this binding also induces T cell 1 ␮M) is associated with remarkably fast on and off rates that polarization. Moreover, CD2 can facilitate motility of T cells along foster rapid and extensive exchange between CD2 and CD58 antigen-presenting cells via a movement referred to as scanning. Both partners on opposing cell surfaces (14–16). These biophysical activated CD4 and CD8 T cells are able to scan antigen-presenting cells characteristics are reminiscent of the –ligand interactions surfaces in the absence of cognate antigen. Scanning is critically involved in the rolling processes along endothelial cells before dependent on T cell ␤- function, as well as chain extravasation (17). Recent x-ray crystallographic analysis of the kinase. More importantly, surface CD2 molecules rapidly redistribute human CD2-CD58 complex explains this behavior by demon- on interaction with a cellular substratum, resulting in a 100-fold strating a very favorable charge complementarity at the molec- greater CD2 density in the uropod versus the leading edge. In ular interface in the presence of few hydrophobic contacts (18). contrast, no redistribution is observed for CD11a͞CD18 or CD45. The cytoplasmic tail of CD2 is linked to the via a Molecular compartmentalization of CD2, T cell receptor, and lipid rafts variety of adapters that influence cytoskeletal polarization, within the uropod prearranges the cellular activation machinery for adhesion, and activation, raising the possibility that CD2 may subsequent immune recognition. This ‘‘presynapse’’ formation on regulate T cell polarization in a key manner (19–21). Further- primed T cells will likely facilitate the antigen-dependent recognition more, given the clustering of CD2-CD58 counter-receptor pairs capability required for efficient immune surveillance. in the T cell–APC contact zone, which positions cell membranes at a distance of 15 nm suitable for subsequent T cell receptor rrival of antigen-laden dendritic cells into the (TCR)–peptide MHC interaction, it is not surprising that CD2- Aresults in the activation of naive T cells, leading to their CD58 counter-receptors enhance the efficiency of immune proliferation and acquisition of effector functions (1). The subse- recognition (20, 22–24). quent migration of activated T cells into pathogen-invaded body Here we use time-lapse video microscopy and image analysis to tissues is a key feature of immunity and immune surveillance. examine the role of CD2 in T cell motility, polarization, and Diapedesis of T cells through the vascular endothelium, as well as immune surveillance. We demonstrate that CD2 ectodomain their crawling movement on the surface of antigen presenting cells crosslinking results in T cell polarization with attendant redistribu- (APCs), parenchymal cells and extracellular matrix (ECM), re- tion of CD2 to the uropod along with the TCR and lipid raft quires T cell polarization (2). Although the molecular basis of this microdomains. This highly specified redistribution process prear- process is not well understood, certain features have been identi- ranges the activation machinery for subsequent antigen recogni- ͞ fied. Cell polarization is characterized by the formation of a leading tion. The distinctly different behavior of CD11a CD18 on these ␤ edge at the front of the cell and a uropod at its back, allowing same T cells implies that the critical role of 2 in T cell conversion of mechanical forces generated through adhesion to a adhesion, migration, and immune activation are nonredundant with substratum into net cellular locomotion (3). While the actin cy- those of CD2. toskeleton redistributes at the leading edge, the microtubule orga- Materials and Methods nizing center (MTOC) localizes within the uropod allowing a greater deformability of the migrating cells (4–6). Given that the Cell Purification and Culture. Human peripheral blood mononu- MTOC and the colocalize, the secretion of soluble clear cells (PBMC) were purified from leukopaks by using a mediators is directed toward the ligated cell surface (7). Moreover, Ficoll gradient. T cells were isolated using the nylon wool chemokine receptors responsible for the directionality of the mi- method, cultured in RPMI medium 1640 supplemented with gration along a gradient of chemoattractants are concentrated 10% human AB serum (Nabi, Miami), 2 mM L-glutamine ͞ within the leading edge (8). In contrast, the uropod mediates (GIBCO), and penicillin streptomycin (GIBCO; complete me- ͞ important adhesion functions, as suggested by accumulation of a dium), and activated for 72 h in the presence of 25 ng ml PMA notable number of adhesion molecules including ICAM-1, CD44, (Sigma) and 1:200 anti-CD3 mAb (2Ad2, IgM ascites). CD4 and CD43, P-selective ligand 1 (PSGL-1), and L-selectin CD8 T cell subsets were isolated by negative selection with Ͼ during migration (9, 10). The uropod therefore appears to anchor resulting purity 95%. Human were isolated from the cell to the ECM, APCs, or endothelial cells while the leading purified PBMC by using magnetic beads coated with anti-CD14 edge propels the cell forward. It has also been suggested that adhesion molecules within the uropod support binding to other Abbreviations: APC, antigen-presenting cell; CTx, Cholera toxin; DIC, differential interfer- bystander leukocytes, thereby enhancing inflammatory cell recruit- ence contrast; MHC, major histocompatibility complex; MLC, myosin light chain; pMHC, ment (9). peptide complexed MHC; TCR, T cell receptor; CHO, Chinese hamster ovary. Human CD2 is a transmembrane cell surface glycoprotein §To whom reprint requests should be addressed at: Dana-Farber Cancer Institute, 44 Binney expressed in a restricted manner on T cells, , and NK Street, Boston, MA 02115. E-mail: ellis࿝[email protected].

7582–7587 ͉ PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.112212699 Downloaded by guest on September 30, 2021 (Miltenyi Biotec, Auburn, CA; ref. 25). Immature dendritic cells intensity value of the leading edge and of the uropod. The values were obtained by culturing CD14ϩ monocytes with granulocyte͞ obtained were summed and the uropod͞leading edge ratio calcu- ͚ Ϫ ͚͞ Ϫ colony-stimulating factor (GM-CSF) and IL-4 at 2 lated as [I(uropod) I(bkg)]n [I(leading edge) I(bkg)]n. ng͞ml for 9 days. J77, a clone of Jurkat cells, was maintained in RPMI medium 1640 supplemented with 10% FCS (Sigma), 2 Polarization and Change of Morphology of T Cells On CD2 Crosslinking. mM L-glutamine, and penicillin͞streptomycin (GIBCO). Glass cover slips (Clay Adams) were incubated overnight at 4°C CHO58 and additional Chinese hamster ovary (CHO) transfec- with the indicated mAbs at 5 ␮g͞ml in PBS and then washed with tants were produced and maintained as described (26). PBS to remove the excess mAbs. Activated T cells (5 ϫ 106) were resuspended in complete medium and plated on the mAbs-coated Preparation of the APCs and Time-Lapse Experiments. The APCs cover slips. After incubating at 37°C for 1–5 h, the coverslips were were plated on sterile circular cover slips (Fisher Scientific) in 6-well washed with PBS and mounted on microscope slides (Fisher). To plates at Ϸ1 ϫ 106 cells per well and cultured overnight at 37°Cto examine the Golgi apparatus distribution, Jurkat 77 cells at 4 ϫ 106 obtain a 95% confluency. The cell-coated coverslips constituted the per ml in PBS ϩ 5% FCS were incubated for 30 min at 37°Conthe base of a chamber (Micro Video Instruments, Avon, MA) con- mAb-coated cover slips, washed twice, and then fixed with 3.7% nected to a source of 10% CO2 balanced air (BOC Gases, Hingham, paraformaldehyde in PBS for1hat4°C. Following further washing, MA) and a brass flow meter (BOC Gases) to maintain the pressure the cells were permeabilized with 0.1% saponin͞1% BSA in PBS at 130 mm. The chamber was placed on a 37°C heating stage. for30minat4°C and stained with 0.5 ␮M BODIPY TR ceramide Activated T cells (3 ϫ 105) suspended in 3 ml of complete medium (Molecular Probes) for1hat4°C. Subsequently, the cells were were inserted into the chamber and allowed to settle for Ϸ10 min washed twice in permeabilization buffer and mounted on slides before the initiation of recording. The frames were captured using (Fisher) for analysis. a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI) connected to a Nikon Diaphot 300 fluoro-microscope (Micro Results Video Instruments). A user-designed program allowed capture of Polarization of T Cells by CD2 Crosslinking. Previous analysis of T frames of the selected field in differential interference contrast cell–APC interaction showed that CD2 plays an important role in (DIC) or in fluorescence with an interval of 30 s and over a period immune recognition (23, 28). CD2–CD58 interaction contributes of 1 h. All of the images were processed using the public domain NIH directly to stabilized conjugate formation between the T cell and IMAGE program (National Institutes of Health) and the IPLAB CD58-bearing APC, thereby reducing the molar requirement for software (Scanalytics, Fairfax, VA). In blocking experiments, the foreign nominal peptide antigen by Ͼ1 order of magnitude (23). As APCs were preincubated with anti-CD58 (TS2͞9, 200 ␮g͞ml) for shown in Fig. 1, conjugate formation between human T cells and 30 min at 4°C, whereas T cells were preincubated with anti-CD11a hCD58-expressing CHO cells (CHO58) is antigen-independent and (TS1͞22, 50 ␮g͞ml, IgG1; Endogen, Cambridge, MA), anti-CD18 results in redistribution of CD2 to the interface of the cell conju- (TS1͞18, 30 ␮g͞ml, IgG1; Endogen), and anti-CD3 (RW2–8C8, 10 gate. In three T cells at the bottom of Fig. 1B and in the two T cells ␮g͞ml) for 30 min at 4°C. T cells were also preincubated with the interacting with the upper CHO cell, CD2 is redistributed almost kinase inhibitors 50 ␮M PD98059, 10 ␮M SB203520, and 10 ␮M exclusively along the CHO58–T cell junction. In contrast, CD2 ML-7 (Calbiochem). maintains its uniform distribution on the T cell not contacting the APC (upper left). This result is comparable to that reported Transfection of Murine L Cell Fibroblasts. Murine L cell fibroblasts previously between human T cells and CD58-transfected murine were transfected with human GPI-linked CD58 (27) in pSH-SX fibroblasts (23). In the present study, we address whether the vector and human ICAM-1 (CD1.8 plasmid was kindly provided by redistributed CD2 is localized to a specific subcellular compartment T. Springer, Harvard Medical School) subcloned into pIRES2- and whether this reorganization is linked to cell polarization and, EGFP vector (CLONTECH). Cells (106) were plated the day perhaps more importantly, to T cell motility. before the transfection and 20 ␮g of each individual plasmid were To assess whether T cell morphological changes are induced on transfected using the calcium phosphate method. After 48 h, the CD2 crosslinking consistent with a polarized phenotype, activated cells were put under selection in the presence of 1 mg/ml G418 T cells were incubated on cover slips coated with anti-CD2 or (GIBCO) and͞or 1 mg/ml Hygromycin B (Roche Diagnostics unrelated mAbs at 37°Cfor1–5 h. As shown in Fig. 1E, activated GmbH) for 4 days. The positive cells were sorted on the MoFlo human T cells undergo a dramatic change in cell shape on incu- (Cytomation, Fort Collins, MO) and maintained at 0.2 mg/ml G418 bation with anti-CD2 mAbs for1hat37°C. As described below, this and͞or 0.1 mg/ml hygromycin. phenotype is associated with increased locomotion͞migration. Af- ter5hofincubation, polarization becomes even more striking, as Immunofluorescence of T Cells and Analysis of Fluorescence Intensity. demonstrated by formation of a very elongated uropod (trailing Activated T cells (2–3 ϫ 106) were suspended in 50 ␮lofPBSϩ 2% edge) that can extend more than 60 ␮m in length (Fig. 1F). FCS and stained for CD2 (T11–2 Texas red mAb, 1.6 ␮g), for CD45 Polarization appears to be specifically induced by CD2 crosslinking [KC56 (T-200)-FITC (Coulter) 8 ␮l], for CD11a͞CD18 (TS2͞4 because neither anti-MHC class I nor anti-CD3␧ mAb-coated Texas red, 2.4 ␮g), for CD3 (RW2–8C8 Texas red mAb, 4.8 ␮g), surfaces induce a similar effect at1h(Fig.1,C and D, respectively) and for Raft GM-1 ganglioside [Cholera toxin B subunit (CTx)- or any time interval examined (data not shown). To independently biotin (Sigma), 4 ␮g, detected by Streptavidin Texas red (Jackson evaluate the ability of CD2 clustering to induce T cell polarization, Immunoresearch lab 1.8 ␮g]for30minat4°C. After washing, 3 ϫ we further examined the distribution of the Golgi apparatus in T 105 stained cells suspended in 3 ml of complete medium were cells after mAb-mediated crosslinking. For clarity of observation, incubated in the chamber on a monolayer of CHO58 cells. Scanning the larger-size, cytoplasm-rich Jurkat J77 T cells were used. As T cells were followed over a period of 15 min to 1 h, recording DIC shown in the Insets in Fig. 1, anti-CD2 but not anti-MHC class I or and fluorescence images at intervals of 30 s. anti-CD3␧ mAb crosslinking results in Golgi reorganization͞ To evaluate different T cell compartments, regions of 225 pixels polarization. Hence, staining of the Golgi apparatus of J77 cells (2) were selected on the DIC image of the uropod or of the leading after interaction with anti-CD2 mAb-coated cover slips shows a edge of a given cell and overlayed on the corresponding fluorescent clear Golgi polarization toward the center of the cell (Fig. 1E Inset) image. Five areas surrounding the cells of interest were measured compared with its random position in J77 cells bound to anti-MHC- and the intensity values averaged to determine the mean intensity class I or anti-CD3␧ mAb-coated surfaces (Fig. 1, C and D Insets, value of the background [I(bkg)]. For each of the 12 cells analyzed respectively). Both the and Golgi polarization are per molecule, the I(bkg) value was subtracted from the fluorescence observed when cells are incubated with anti-T112 mAb alone. This

Tibaldi et al. PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7583 Downloaded by guest on September 30, 2021 Fig. 2. Representative trajectories of T cells scanning the surface of a cellular substratum. A and B delineate the pathway of two individual T cells along the CD58 CHO epithelial surface. Yellow dots represent the relative position of the analyzed cell at each 2 min interval and arrows show the direction of scanning. Accompanying digitized movies are included (Movie 3 and 4). C and D are graphical representations of the surface area (blue, ␮m2) and distance traversed (pink axis, ␮m) per unit of time (min) during the scanning function of cells in Aand B, respectively. The analysis is performed using NIH IMAGE software.

In this paper we define ‘‘scanning’’ as the migration of T cells on the surface of a cellular monolayer substratum. As examples, we show the trajectory covered by two T cells (Fig. 2; see Movies 3 and 4, which are published as supporting information on the PNAS web site). In Fig. 2A (Movies 3), the cell enters from the bottom of the field as it scans the edges of three CHO58 cells while continuing toward the right of the field. The cell then stops, inverts its direction, Fig. 1. CD2 redistributes to the interface of T cell͞APC conjugates. (A) DIC image and proceeds to scan two other CHO58 cells during the time of of the conjugates between CD58 transfected CHO cells (labeled CHO) and T cells observation interval. In Fig. 2B (Movie 4), the T cell arrives at the (unlabeled). (B) Immunofluorescence staining of CD2 on the same conjugates as top of the field. During its subsequent scanning activity, the uropod in A.(C–F) Polarization of activated T cells induced by mAbs over 1–5 h: (C) anti-MHC class I mAb (W6͞32; ref. 54), 1 h, 37°C; (D) anti-CD3␧ mAb (RW2–8C8; remains attached to one CHO58 cell, thereby tethering the T cell ref. 55), 1 h, 37°C; (E) anti-CD2 (anti-T112 ϩ anti-T113; ref. 13), 1 h, 37°C; (F) and pulling it back; later, the T cell continues the scanning toward anti-CD2 (anti-T112 ϩanti-T113), 5 h, 37°C. (Insets) Staining of the Golgi apparatus the right of the field. Note how the T cells invariably round up on Jurkat 77 cells plated on the above mAbs coated cover slips. (Scale bar, 20 ␮m.) before altering their direction of migration. Analysis of the same two T cells in terms of distance traversed per unit time is given in Fig. 2 C and D. The graphs show that, even in result rules out the possibility that the effects are mediated by a the event of continuous cell motion, the distance covered by the ϩ mitogenic stimulus; anti-T112 alone unlike the anti-T112 anti- centroid of the T cell per unit time varies within a range of 1.5–8 T113 combination does not induce T cell proliferation, as judged by ␮m͞0.5 min, suggesting abrupt acceleration and deceleration in- 3H-TdR incorporation (13). tervals rather than a constant velocity. Furthermore, we measured the surface area of the T cells during scanning and observed a T Cell Scanning on a Cellular Substratum. We next performed positive correlation between the surface area of the cells and the time-lapse video microscopy analysis to examine the role of the distance traversed. This finding suggests that the polarized pheno- CD2–CD58 interaction on T cell migration on a cellular monolayer type is primarily associated with locomotion. In the absence of consisting of CHO58 cells. Several observations are apparent. First, movement or on trajectory inversion, redistribution of intracellular Ϸ50% of substrate-bound T cells scan CHO58, whereas very little components, including the actin cytoskeleton, occurs with atten- or no scanning is observed on a substratum of CHO cells alone or dant reduction in cell surface area and loss of traction forces CHO cells transfected with mutant CD58 molecules defective in required to maintain polarity. The pattern shown for these two cells CD2 binding (see below). Second, individual cell migration behav- is representative of 20 other cells including both continuous vs. ior on CHO58 cells is heterogeneous; some T cells scan continu- intermittent scanning types (Movie 1) and ‘‘top’’ vs. ‘‘under’’ ously along the APC surface, whereas others stop and start, often scanners (Movie 2). The process of cells navigating under cellular reversing direction. In Movies 1 and 2 (which are published as substratum has been termed pseudoemperipolesis (32). supporting information on the PNAS web site, www.pnas.org), we highlight the migration of several cells to document this point. Role of CD2 and CD11a͞CD18 in T Cell Scanning. To directly investi- Third, although not shown, both purified activated CD4 and CD8 gate the role of CD2–CD58 interaction in scanning, we adopted two T cells demonstrate scanning at comparable levels, as do CD45RA different approaches. The first was to preincubate the APC mono- and CD45RO subpopulations (29). Fourth, resting T cells failed to layer with anti-CD58 mAb and then plate T cells onto the pre- mediate scanning activity, indicating a clear requirement for cel- treated CHO58 substratum. One of the parameters affected by this lular activation as might occur in secondary lymphoid organs. Cell treatment is the percentage of scanning T cells bound to the CHO58 motility is a dynamic process involving lamellipodia formation at monolayers. In Fig. 3A, we show that the percentage of scanning T the leading edge, attachment to the substratum (30, 31), generation cells significantly decreases from 50% [n (number of cells ana- of contractile forces and traction (3), and ultimate release of the lyzed) ϭ 282] on the untreated CHO58 substratum (Movie 5) to uropod (6). 30% (n ϭ 88) on anti-CD58 mAb-treated CHO58 cells. The second

7584 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112212699 Tibaldi et al. Downloaded by guest on September 30, 2021 MAPK (39), we investigated the involvement of the ERK pathway in the migration of T cells on the CHO58 surface. Inhibition of MAPK kinase (MEK) upstream of ERK by using an inhibitor (PD98059; ref. 40) reduces the percentage of T cell scanning to 32% (n ϭ 38). Because PD98059 pretreatment blocks CD2-mediated activation of the MAPK without effecting CD2-induced adhesion (41), we assume that this effect is a consequence of impairment in concomitant integrin signaling (42). Moreover, blocking MLCK downstream of ERK by using a specific inhibitor (ML-7) reduces the percentage of T cells scanning to Ϸ18% (n ϭ 61; Fig. 3A). This result is consistent with the hypothesis that the phosphorylation of MLC is essential for the assembly of the contractile migration machinery. We also investigated the involvement of p38 MAPK. On treatment with a specific inhibitor (SB203580; ref. 43), we observe a reduction in the percentage of T cells scanning (29%, n ϭ 34) comparable to that obtained with the MEK inhibitor (Fig. 3A). From these data we conclude that the ERK and p38 MAPK pathways are linked to T cell motility.

Fig. 3. T cell scanning on all APC surfaces is critically dependent on ␤2 integrin CD18 Is Essential for Scanning of APCs by T Cells. The ability of function. Three cellular substrata were examined: CHO (A), human dendritic cells anti-CD18 mAb to affect T cell scanning of CHO58 more than (B), and murine L cell transfectants (C). The percentage of T cells scanning over the anti-CD58 mAb blockade suggests that ␤2-integrins may be essen- total number of APC bound T cells are designated on the y axis. In A, treated or tial for cell locomotion on other cellular monolayers. We therefore untreated T cells were plated on CHO58, mAb treated CHO58, or mutant CHO performed time-lapse experiments using human dendritic cells as cells as described in Materials and Methods.InB, DC were untreated (CTR) or APCs. As shown in Fig. 3B, 76% (n ϭ 104) of T cells are able to pretreated with anti-CD58 or with anti-CD18 mAb. In C, the scanning substratum migrate on dendritic cell monolayers with an average speed of 8 consists of untransfected, hCD58-transfected, or hICAM-1-transfected L cells. **, ␮m͞min (see Movie 6, which is published as supporting information P Յ 0.001; , P Ͻ 0.05; as calculated by one-sided ␹2 test and by Fisher’s exact test. * on the PNAS web site). While the preincubation of the APCs with anti-CD58 mAb only modestly (but with statistical significance) ϭ approach was antibody-independent, using two mutant forms of affects the percentage of scanning T cells (63%, n 82), the preincubation of the T cells with anti-CD18 mAb markedly reduces human CD58 (K34A and K87A) with reduced CD2 binding activity ϭ (33). The analysis of the percentage of T cell scanning shows a the percentage of scanning to 4% (n 135; see Movie 7, which is reduction to 23% on K34A (n ϭ 148) and to 11% on K87A mutants published as supporting information on the PNAS web site). (n ϭ 27; Fig. 3A). Note how CHO transfectants of CHO58 variants, Furthermore, anti-CD58 preincubation does not significantly alter the speed of T cell migration (7 ␮m͞min). As an independent

E76A and K50A, involving mutations located outside the CD2 IMMUNOLOGY confirmation of the importance of ␤2-integrin function in T cell binding site (18), have no influence on scanning activity [E76A scanning, we analyzed T cell movement on monolayers consisting (53%, n ϭ 57); K50A (57%, n ϭ 21)]. Collectively, these findings of untransfected, CD58- or hICAM-1-transfected L cells. In Fig. 3C show that blocking CD2–CD58 interaction significantly impairs the we show that the percentage of T cells scanning on a monolayer of percentage of scanning T cells without affecting the adhesion to the hICAM-1-transfected L cells is Ϸ46% (n ϭ 648; see Movie 8, which CHO58 cells via the uropod, implicating additional molecules in is published as supporting information on the PNAS web site), local adhesion. ͞ ␤ whereas there is no significant difference between the percentage CD11a CD18 is the most abundant 2 integrin on the T lym- of T cells scanning on CD58-transfected L cells (8%, n ϭ 181; see phocyte surface and binds to ICAM-1 and -2 on stromal cells (34, Movie 9, which is published as supporting information on the PNAS 35). Integrins play an important role in the adhesion processes web site) and on untransfected L cells (6%, n ϭ 53). Note that between leukocytes and other cells, as well as to extracellular matrix FACS analysis on L cells shows that neither murine ICAM-1 nor (ECM) proteins (12). The strong and stable interactions between murine CD48 molecules are expressed (data not shown). From integrins and their ligands are primarily responsible for the termi- these results, we conclude that a productive CD11a͞CD18-ICAM-1 nation of leukocyte rolling on endothelial cell surfaces before interaction is critical for migration͞scanning function of T cells on extravasation (36). Blocking experiments using T cells pretreated ␤ cellular monolayers, whereas the CD2–CD58 interaction is impor- with anti-CD18 and anti-CD11a mAbs demonstrate that 2 inte- tant in a more restricted way—i.e., only being key on certain APC ͞ grins and, in particular, CD11a CD18 are indispensable for scan- substratum. ning activity. As shown in Fig. 3A, the percentage of T cells scanning ϭ is reduced to 7% on anti-CD18 mAb treatment (n 106) and to Differential Redistribution of Molecules to the Uropod During T Cell Ϸ ϭ 10% on anti-CD11a mAb treatment (n 39). In contrast, Scanning: Formation of a Preactivation Complex. The requirement ␧ following pretreatment with anti-CD3 mAb, the percentage of for productive CD2–CD58 interaction during cognate antigen- scanning T cells is comparable to that of the untreated control, triggered T cell activation is well established (23, 24, 27). The implying that TCR complex crosslinking does not affect scanning limited role for CD2 in T cell scanning of certain APC, therefore, function. suggests that a linked, yet distinct, CD2 function may be critical for its participation in immune recognition. To this end, we investigated Myosin Light Chain Kinase (MLCK) and T Cell Motility. An important the distribution of surface molecules on T cells during scanning, aspect of cell motility is the myosin-dependent cell contraction (37). analyzing the disposition of CD2, CD3, and lipid rafts, as well as the The modulation of myosin light chain (MLC) phosphorylation tyrosine phosphatase, CD45 (44), and the integrin CD11a͞CD18. through the regulation of MLCK and MLC phosphatase mediated In Movie 10, a single T cell is followed over a period of 30 min. As by GTPases is a key event in the generation of contractile forces. A shown, CD2 molecules become exclusively localized to the uropod. role for mitogen-activated protein kinase (MAPK) in the regulation Fig. 4A (a–d) represents still images derived from the time-lapse of MLCK and in cell migration has been suggested (38). As CD2 sequence. Fig. 4A (e–h) shows the distribution of the CD45 ectodomain clustering on peripheral T lymphocytes activates molecules on various T cells at different stages of polarization. In

Tibaldi et al. PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7585 Downloaded by guest on September 30, 2021 Discussion An important feature of CD2 ectodomain ligation noted in the current study is the induction of T cell polarization and motility. Thus, on anti-CD2 mAb binding or on contact with a CD58- expressing cell monolayer, a T lymphocyte transforms itself from a round cell into an elongated motile counterpart with a clearly delineated leading edge and uropod. Both activated CD4 and CD8 T cells demonstrate this CD2-inducible morphological transformation and manifest accompanying scanning activity. In contrast, polarization͞scanning activity is absent from CD2- triggered resting T cells, indicating that primed but not naive T cells are capable of such directed motile behavior. Consistent with these findings, prior analysis of the interaction between rat CD2 Jurkat transfectants and rodent CD48 on glass-supported lipid bilayers showed that cytoskeletal polarity was linked to CD2 ligation, as evidenced by microtubule organizing center (MTOC) reorganization (19). Cytoskeletal polarity depended on the CD2 cytoplasmic tail and involved CD2AP, one of several recently identified CD2 cytoplasmic tail adaptor proteins (19–21). The reorganization of the Golgi apparatus triggered by CD2 crosslinking herein independently confirms the role of CD2 in cytoskeletal polarization and links CD2 to specialized T cell motility. Using various monolayers (CHO58 cells, murine L cell transfec- tants, and human dendritic cells), we were able to demonstrate that CD2 molecules specifically redistribute to the uropod together with the TCR- and CTx-binding lipid rafts. In contrast, CD45 and CD11a͞CD18 molecules maintain a uniform distribution on the T cell surface. Independent analysis of human T cell interaction with human umbilical vein endothelial cells is also consistent with differential localization of adhesion molecules (10). Moreover, CD11a͞CD18 interaction with ICAM-1 is required for T cell Fig. 4. Redistribution of CD2 molecules to the uropod during scanning and formation of a ‘‘preactivation’’ complex. (A) Cellular localization of surface scanning on all monolayers examined, whereas CD2–CD58 partic- molecules on T cells plated on a monolayer of CHO58 cells. Activated T cells were ipation is selectively relevant on CHO58 and, to a lesser extent, on stained for CD2, CD45, CD11a͞CD18, CD3, or Raft GM-1, plated on a monolayer DC. Analysis of CD58 and hICAM-1 fibroblasts confirms the of CHO58 cells and images of T cells at different stages of polarization were notion that CD2 participation in scanning per se is not essential. recorded. (a–d) Sequential images at the indicated times in minutes following T Whether this result implies that hCD2–CD58 interaction is more cell binding to the cellular substratum. Other panels represent independent cells. critical for certain cellular conjugates because of a differential (B) Graphical representation of the ratio between the fluorescence in the uropod ͞ Ͻ expression and or engagement of adhesion receptors pairs remains and in the leading edge calculated for each of the indicated molecules. **, P to be determined. The difference in subcellular distribution and 0.001, as calculated by two-sided Wilcoxon sum. scanning requirement unequivocally distinguishes CD2 and CD11a͞CD18 molecules and provides additional evidence for their contrast with the discrete localization of CD2 in the uropod, the nonredundant function (45). CD45 is present in a more uniform way over the cell surface and, A role for CD2 in positioning the T cell membrane at a distance Ϸ hence, detected at the leading edge as well as at the uropod. The suitable for TCR recognition of pMHC ( 130 Å) has been same diffuse pattern is shown for CD11a͞CD18 (Fig. 4A, i–l). This previously suggested and confirmed by x-ray crystallographic anal- system also allowed us to investigate the distribution of molecules ysis (18). Consistent with this notion, interaction of CD2 with an important in T cell activation, including the TCR complex itself and elongated form of CD48 significantly inhibits immune recognition lipid microdomains (lipid rafts) before TCR engagement. In a in the rodent system (46). In this regard, the 100-fold enrichment of CD2 in the uropod of APC-conjugated T cells with attendant fashion similar to CD2, CD3 molecules are redistributed primarily concentration of TCR molecules in the same subcellular locale in the uropod (see Movie 11, which is published as supporting preconfigures the T cell surface in an idealized mode for subsequent information on the PNAS web site, and Fig. 4A, m–p). Moreover, pMHC recognition. The rapid on- and off-rates between CD2 and the same uropod-skewed distribution is observed for lipid rafts ͞ its counter-receptor (14) favor an appropriate opposing membrane themselves as revealed by CTx-biotin streptavidin TR (see Movie distance geometry to support TCR–pMHC interaction without 12, which is published as supporting information on the PNAS web impeding TCR diffusion into the critical cell–cell contact surface site, and Fig. 4A, q–t). From these observations, we conclude that area as the uropod scans the opposing APC in search of MHC- the essential activation machinery represented by the TCR com- bound peptides. Furthermore, the elongated shape of the uropod plex, those lipid microdomains binding CTx and associated signal- affords a larger surface contact area to optimize the likelihood of ing molecules as well as CD2, are recruited to the same locale productive immune recognition. Only a fraction of the lamellipodia before TCR engagement. Fig. 4B offers a quantitative representa- preserves contacts with the APC, by contrast, although the impor- tion of the ratio of the fluorescence intensity in the uropod and in tance of such initial cell–cell contact via the leading edge is not to the leading edge for these various structures. CD2 and CD3 be underestimated. molecules concentrate 10–100-fold in the uropod as determined Recently, we demonstrated through subcellular fractionation with specific mAb probes. Likewise, rafts are concentrated Ն10- that a significant number of human CD2 molecules is inducibly fold based on CTx localization. In contrast, CD11a͞CD18 and recruited into lipid rafts on CD2 ectodomain ligation (47). This CD45 show no quantitative difference. translocation is independent of the TCR and requires no tyrosine

7586 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112212699 Tibaldi et al. Downloaded by guest on September 30, 2021 phosphorylation. The uropod-specific relocalization of CD2 and human and mouse lymphocytes during their interaction with lipid rafts observed microscopically appears to be the morpholog- dendritic cells in a three-dimensional collagen matrix (52), we ical counterpart of that inducible CD2-raft association analyzed suspect that surface T cell molecule preconfiguration during T biochemically. This redistribution is important because numerous cell movement noted herein is generally applicable to most, if not signaling molecules localize in lipid rafts. These include Src family all, immune recognition events. protein-tyrosine kinases, heterotrimeric and monomeric Ras-like G Previously, a human CD2 cytoplasmic tail-binding protein proteins, molecules involved in Ca2ϩ flux, and the small signaling termed CD2BP1 was identified and shown to interact via its SH3 molecule phosphatidylinositol bisphosphate (reviewed in ref. 48). domain with aa 300–309 of the CD2 tail (20). CD2BP1 binding to Analysis of T cell interaction with planar bilayers containing CD2 was dramatically augmented as a result of CD2 ectodomain fluorescently labeled pMHC and ICAM-1 molecules has identified crosslinking by anti-CD2 mAb or conjugation to CD58-expressing a specialized junction between T lymphocytes and ‘‘APC’’ referred cells. Hence, it follows that CD2BP1 association with CD2 should to as the immunological synapse (49). This site has been shown to be maximal in the CD2-enriched T cell uropod during scanning. consist of a central cluster of TCRs surrounded by adhesion CD2BP1 also independently binds a second intracellular ligand, the tyrosine phosphatase PTP-PEST, at a site separate from CD2. molecules, most notably integrins. Dynamic transport of TCR– cas pMHC complexes into the center of the synapse has been observed. Because PTP-PEST dephosphorylates p130 , it is probable that Moreover, studies by Krummel et al. (50) demonstrated that the focal adhesion can be readily down-modulated by this means (30, initiation of T cell activation correlates with formation of small yet 53). Transfection analysis in COS cells and Jurkat cells vis-a`-vis unstable clusters of CD3 and CD4 driven by specific pMHC͞TCR CD2-dependent adhesion function are consistent with this view oligomerization, independent of the cytoskeleton. Yet other studies (20). Thus, an important role of focal CD2 redistribution will be to regulate adhesive interactions within the uropod, including but not suggest an important role for CD28 within the immunologic restricted to CD2. In this way, the uropod is able to detach from the synapse as well (51). The present analysis using an entirely cell- cellular substratum without fracture from the cell body because of based system in the absence of specific cognate antigen defines a the adhesive forces applied to the T lymphocyte during scanning. specialized subcellular compartmentalization in the uropod that The emerging picture of T cell adhesion in general, and CD2- precedes MHC-restricted peptide recognition. We suggest that mediated function in particular, is that of a highly dynamic, regu- this geometric configuration may be properly referred to as the lated state of complex molecular and spatial interactions. The ‘‘presynapse’’ because it is likely that such preorganization biology of T cell movement, membrane reorganization, and im- precedes pMHC-dependent TCR engagement and fosters sub- mune recognition are tightly interrelated and worthy of detailed sequent productive interactions particularly important for T cell exploration. Such analysis will lead to a molecular understanding of proliferation and cytokine production. To our knowledge, this the processes involved in immune surveillance. represents the first molecular description using time-lapse meth- odology to correlate T cell surface immune recognition mole- We thank Drs. Martin Hemler and Linda Clayton for thoughtful cules and their subcellular locale during cell–cell interaction. comments on the manuscript. This work was supported by National Nevertheless, as similar T cell motility has been shown for both Institutes of Health Grant AI21226. IMMUNOLOGY 1. von Andrian, U. H. & Mackay, C. R. (2000) N. Engl. J. 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