CD2 molecules redistribute to the uropod during T cell 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 selectin–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 ␤-integrin function, as well as myosin light 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 cytoskeleton 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 lymph node (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 integrins 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 Golgi apparatus 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 glycoprotein 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 monocytes 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, thymocytes, 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 macrophage 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).