Polar Redistribution of the Sialoglycoprotein CD43: Implications for T Cell Function1

Nigel D. L. Savage,* Stephanie L. Kimzey,* Shannon K. Bromley,† Kenneth G. Johnson,† Michael L. Dustin,‡ and Jonathan M. Green2*†

Contact between T cells and APCs results in the orchestrated segregation of molecules at the cell-cell interface and formation of a specialized structure termed the immunological synapse. This model predicts the topological seclusion of large molecules such as CD43 from the site of closest contact between the T cell and APC, allowing for the close apposition of cell membranes and effective TCR engagement. Similarly, during T cell migration segregation of CD43 to the uropod is thought to aid integrin adhesion at the leading edge of the cell by removing steric hindrance. We show in this work that CD43 distribution on T cells is regulated by a membrane proximal ezrin binding site and that failure to displace CD43 from the immunological synapse has no inhibitory effects on primary T cell activation. We also report that CD43 expression at the contact zone between T cells and matrix does not negatively regulate motility but may regulate LFA-1 de-adhesion. These results suggest that the steric barrier model of CD43 is inadequate and that alternative mechanisms account for the negative regulatory properties of CD43. The Journal of Immunology, 2002, 168: 3740–3746.

he interaction of T cells with APCs results in the forma- surface (10, 11). CD43-deficient cells are more responsive to Ag tion of a specialized structure termed the immunological and are hyperadhesive, presumably due the reduced steric hin- synapse, which consists of a TCR-rich central supramo- drance in the absence of the extracellular domain of CD43 (Refs. T 3 lecular activation complex (cSMAC) surrounded by a ring-shaped 12 and 13 and N. D. L. Savage and J. M. Green, unpublished data). peripheral supramolecular activation complex (pSMAC) contain- During locomotion, T cells form a distinct cellular structure at ing adhesion molecules such as LFA-1. This unique organization the trailing edge of the cell termed the uropod. Many proteins, of surface proteins provides a molecular basis for both sustained including CD43, localize to the uropod. The leading edge of the adhesion and signaling necessary for successful T cell activation cell has significantly increased sensitivity to Ag, despite similar (1–5). The formation of the immunological synapse is thought to densities of TCR (14). Relocalization of CD43 to the uropod is, there- require the segregation of cell surface proteins based in part on fore, assumed to support both T cell adhesion and activation by min- size. Proteins that extend from the cell surface a distance similar to imizing steric hindrance at the leading edge. However, this model of the TCR, including CD2 and CD28, are maintained in the cSMAC CD43 function, as well as the requirement for exclusion of CD43 while larger proteins, including LFA-1 and CD22, are in the from the T cell-APC contact zone, has not been formally tested. pSMAC (reviewed in Ref. 6). CD45 and CD43, which are among The localization of CD43 to the cellular uropod in migrating the most abundant and largest proteins expressed on T cells, are lymphocytes has been thought to be dependent on the interaction effectively excluded from the immunological synapse, suggesting of cytoplasmic domain of CD43 with the adapter protein ezrin, a that the presence of large molecules within the cSMACs and member of the ezrin, radixin, and moesin (ERM) family (15, 16). pSMACs could inhibit effective receptor-ligand interactions (7, 8). ERM proteins link transmembrane proteins to actin by either direct CD43, in particular, has been implicated in negatively regulating T or indirect interactions (17, 18). Thus, the anchoring of actin fil- cell function (reviewed in Ref. 9). The extracellular domain of aments to CD43 may be important in regulating cellular processes CD43 is highly glycosylated and sialylated. This results in a neg- such as the establishment of cell polarity or cell motility, or in atively charged, rigid molecule, making CD43 an ideal candidate forming an initiating nucleus for the assembly of signal-transduc- for nonspecific inhibition of protein-protein interactions at the cell ing complexes (19). We formally evaluated the requirement for CD43 redistribution for T cell activation, proliferation, and motility. Reconstitution of Departments of *Medicine and †Pathology and Immunology, Washington University CD43-deficient primary T cells by retroviral gene transfer with School of Medicine, St. Louis, MO 63110; and ‡Program in Molecular Pathogenesis, specific mutants of CD43 demonstrates that the failure of CD43 to Skirball Institute of Biomolecular Medicine, New York, NY 10016 redistribute from the T cell-APC contact zone does not inhibit Received for publication December 11, 2001. Accepted for publication February formation of an immunological synapse or proliferation in re- 11, 2002. sponse to Ag. In addition, the redistribution of CD43 to the cellular The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance uropod is not required for T cell locomotion. Taken together, our with 18 U.S.C. Section 1734 solely to indicate this fact. findings suggest a function of CD43 other than steric hindrance in 1 This work was supported by National Institutes of Health Grant HL58444 (to inhibition of T cell function. J.M.G.). N.D.L.S. is a fellow of the Cancer Research Institute. 2 Address correspondence and reprint requests to Dr. Jonathan M. Green, Washington Materials and Methods University School of Medicine, 660 South Euclid Avenue, Box 8052, St. Louis, MO Mice 63110. E-mail address: [email protected] 3 Abbreviations used in this paper: cSMAC, central supramolecular activation com- CD43-deficient mice were backcrossed to the DO11.10 TCR-transgenic plex; pSMAC, peripheral SMAC; ERM, ezrin, radixin, and moesin; Cy5, CyChrome background (provided by K. Murphy, Washington University, St. Louis, 5; GFP, green fluorescent protein. MO (20)) for seven generations. BALB/c mice were purchased from The

Jackson Laboratory (Bar Harbor, ME). Animals were maintained in spe- Statistical analysis cific pathogen-free housing at Washington University School of Medicine. All protocols have been approved by the Animal Studies Committee at Statistical significance of the data obtained from motility studies and flu- Washington University School of Medicine. orescence intensity was determined by an unpaired two-tailed t test using Excel software (Microsoft, Seattle, WA). Antibodies Results and Discussion Anti-CD43 hybridoma (S7, rat IgG2a) was provided by J. G. Frelinger (Rochester University, Rochester, NY). Anti-TCR␤ hybridoma (H57-597, A membrane proximal cytoplasmic domain (KRR) regulates Armenian hamster IgG) was provided by P. M. Allen (Washington Uni- localization of CD43 versity). All other mAb were purchased from BD PharMingen (San Diego, Ј Ј To determine the domain within CD43 that regulates its redistri- CA). F(ab )2 were generated using Pierce Immunopure F(ab )2 kit (Pierce, Ј bution upon T cell polarization, we reconstituted primary T cells Rockford, IL). Abs and F(ab )2 were conjugated to fluorochromes (Cy- Chrome 3, CyChrome 5 (Cy5), and Alexa 488; Molecular Probes, from CD43-deficient mice with mutant CD43 protein using retro- Eugene, OR). viral gene transduction. Wild-type or mutant CD43 constructs (Fig. 1a) were cloned into the retroviral vector GFPRV, which Constructs encodes for a bicistronic message allowing for expression of green Full-length murine CD43 cDNA was cloned into the retroviral vector fluorescent protein (GFP) and CD43. Flow cytometric analysis GFPRV (21) (provided by W. Sha, University of California, San Francisco, demonstrated that the expression of the retrovirally encoded CD43 CA) or tailless human CD4RV (hCD4RV, provided by K. Murphy). All mutations were introduced by oligonucleotide site-directed mutagenesis protein was at similar levels to the endogenous protein, with the using the Quickchange kit (Stratagene, La Jolla, CA). The CD43/CD102 exception of the CD43⌬cyto (Fig. 1b). The CD43⌬cyto construct chimera was made by overlap extension PCR (CD102 cDNA provided by consistently had lower levels of expression, suggesting that the T. Springer, Center for Blood Research, Boston, MA). Sequences were stability of the expressed protein may be decreased due to deletion confirmed by automated fluorescent sequence analysis (Big Dye; Applied of the cytoplasmic domain. The retrovirally expressed CD43 was Biosystems, Lincoln, CA). recognized normally by a variety of anti-CD43 mAb (Fig. 1 and Retroviral infections data not shown), some of which recognize carbohydrate-dependent epitopes, suggesting normal glycosylation of the retrovirally en- Retroviral vectors containing CD43 were transiently transfected into the Phoenix Eco packaging cell line (provided by G. Nolan, Stanford Univer- coded CD43. In addition, as we used primary T cells derived from sity, Palo Alto, CA) and T cells infected with retroviral supernatant as mice in which the CD43 gene has been deleted by homologous previously described (22). Expression of CD43 was confirmed in all ex- recombination, it is unlikely that there are defects in the glycosyl periments by flow cytometry. When indicated, T cells retrovirally infected transferase enzymes that mediate posttranslational modification of with the hCD4RV constructs were enriched using immunomagnetic cell sorting and anti-human CD4 beads with an AutoMACS sorter (Miltenyi CD43. Therefore, it is likely that the retrovirally expressed CD43 Biotec, Auburn, CA). is glycosylated and sialylated in a manner similar to endogenous CD43. APC-T cell interaction To determine the localization of CD43, retrovirally infected T DO11.10 CD43Ϫ/Ϫ T cells were infected with retrovirus containing CD43 cell blasts from CD43-deficient mice were adhered to supported with the indicated mutations. L cells transfected with I-Ad and ICAM-1 (provided by A. S. Shaw, Washington University) were cultured in com- ␮ plete medium with 10 m OVA323–339 peptide on coverslips for 24 h. Heated (37°C) FCS2 units (Bioptechs, Butler, PA) were assembled with coverslips to which L cells were adhered, and retrovirally infected T cells Ј stained with conjugated Abs and/or F(ab )2 were injected into the units. Digitized three-color fluorescence images were obtained using a Zeiss Ax- iovert LSM 510 confocal microscope (Zeiss, Thornwood, NY) as described previously (23). Z-stacks were performed by acquiring 30 images at opti- mal confocal planes. False color image analysis was performed using LSM 510 software (Zeiss). Quantification of the fluorescence in T cell:APC contacts was done using National Institutes of Health ImageJ software (Na- tional Institutes of Health, Bethesda, MD). Nine independent readings of fluorescence intensity were taken over both the area of the T cell:L cell contact and over noncontact areas of the T cell. Three separate cells expressing each contact were analyzed. Presented is the mean fluorescence of the contact and noncontact areas for each cell Ϯ SEM.

Proliferation assays DO11.10 CD43Ϫ/Ϫ T cells were infected with retrovirus encoding for either full-length CD43 or mutant CD43 proteins and 2.5 ϫ 105 T cells FIGURE 1. Generation of CD43 mutants and identification of a mem- 6 cocultured with 1.25 ϫ 10 irradiated BALB/c splenocytes in complete brane proximal site responsible for CD43 distribution. a, Diagram repre- medium. Graded doses of OVA323–339 peptide were added and prolifera- senting the intracellular domain of CD43 constructs used in these studies. tion was determined by tritiated thymidine incorporation for the final8hof Premature stop codons (CD43⌬cyto, CD43 STOP1–4) and three amino a 42-h culture. All conditions were plated in quadruplicate and the mean Ϯ SD of the quadruplicate wells was presented. acid substitutions (CD43NGG) as well as a chimeric molecule consisting of CD43 extracellular and transmembrane domains with the intracellular T cell motility assay domain of CD102 (CD43/CD102) were generated by PCR and cloned into the bicistronic retroviral vectors GFPRV and hCD4RV. The darkened box Supported planar lipid bilayers containing Cy5-conjugated GPI-ICAM-1 depicts the mutated ERM binding site. The numerals represent the amino were prepared as previously described (1). Ab- and F(abЈ) -stained retro- 2 acid number. b, Flow cytometric analysis of CD43 expression on retrovi- virally infected T cells were injected into a heated FCS2 chamber (Bioptechs) and allowed to adhere, and data were acquired for 360 s using rally infected T cells. T cells from CD43-deficient mice were infected with the Zeiss LSM 510 confocal microscope. Data analysis of trajectory and the indicated constructs and stained for CD43 expression with PE-conju- velocity of T cells was performed using National Institutes of Health Im- gated S7 mAb. Retrovirally infected cells expressing GFP were gated on ageJ analysis software. Two-tailed t tests were performed to determine the and presented is a histogram of the CD43 expression of these cells. Wild- statistical significance of any differences. type cells were mock infected and stained for endogenous CD43. 3742 CD43 REDISTRIBUTION IN T LYMPHOCYTE FUNCTION lipid bilayers containing GPI-anchored ICAM-1 as previously de- nonredistributing mutants (Fig. 2a). In both the CD43NGG- and scribed (1). Examination by confocal microscopy revealed that de- CD43⌬cyto-expressing cells, there is some suggestion of nonuni- letion of the entire cytoplasmic domain prevented redistribution of form clustering of CD43; however, the significance of this is un- CD43, whereas cells expressing the C-terminal deletion mutants clear. Expression of a chimeric protein consisting of CD43 extra- CD43 STOP1–4 redistributed the receptor to the uropod (Fig. 2a). cellular and transmembrane domain fused with the cytoplasmic In these cells, the entire amount of surface CD43 is concentrated portion of ICAM-2 (CD102), which also contains a membrane in the uropod, leading to a higher local density of CD43. Within proximal ERM-binding site, restored the polar localization of the the membrane proximal region, the amino acid sequence KRR, at chimeric receptor. Analysis of the percentage of cells that form position 276–278, has been demonstrated to mediate interaction of uropods revealed no difference in the ability of the cells to form a CD43 with the cytoskeletal adapter protein ezrin (24). Consis- uropod based on the expression of mutant CD43. However, virtu- tently, mutation of the KRR motif to NGG within the full-length ally all of the cells that did form uropods redistributed CD43, with cytoplasmic tail of CD43 abrogated the redistribution of CD43 to the exception of the CD43⌬cyto and CD43NGG constructs, in the uropod. False color image analysis of the fluorescence intensity which essentially none of the cells redistributed CD43 (Table I). demonstrated high expression of CD43 throughout the cell in the The number of cells forming uropods in cells expressing the CD43

FIGURE 2. Redistribution of mutant CD43 constructs to the uropod. a, T lymphocytes from CD43Ϫ/Ϫ mice were retrovirally infected with CD43 mutants in GFPRV vector (identiﬁed by GFP expression) and stained with CyChrome 3-conjugated anti-CD43 Ab (S7, red) before settling onto a GPI-ICAM-1-containing lipid bilayer for 10 min to initiate uropod formation. Infected T cells were analyzed by confocal microscopy for cell surface show uropod formations. False color image (ء) distribution of CD43. Yellow areas depict overlapping areas of red (CD43) and green (GFP) and the asterisks analysis is shown in the right panels to allow for a relative quantiﬁcation of CD43 expression. b, Confocal images of retrovirally infected T cells with nonredistributing CD43 mutants in GFPRV, labeled with anti-CD43 (S7, red) and anti-CD102 Ab (blue) to detect endogenous CD102, before placing cells on supported lipid bilayer containing GPI-anchored ICAM-1 for 10 min to initiate uropod formation. Cells expressing nonredistributing mutants of CD43 are still able to localize endogenous CD102 to the uropod. The Journal of Immunology 3743

Table I. Uropod formation and CD43 redistributiona

CD43FL CD43/CD102 CD43NGG CD43⌬cyto Mock (n ϭ 21) (n ϭ 28) (n ϭ 19) (n ϭ 22) (n ϭ 40)

Forming uropods (%) 71 75 89 63 65 Redistributing (%) 100 95 6 0

a CD43-deﬁcient T cells were retrovirally infected with the indicated constructs and adhered to ICAM-1-containing planar lipid bilayers as described in Materials and Methods. The percentage of infected cells that formed a uropod was determined for each group. Of those that formed uropods, redistribution of CD43 was determined. n ϭ number of cells analyzed. Data were obtained from three independent experiments.

STOP1–4 constructs were similar to wild type (data not shown). To determine whether cells expressing the nonredistributing Thus, the redistribution of CD43 on the cell surface is an active CD43 mutants were capable of localizing other proteins to the process requiring a speciﬁc interaction of the cytoplasmic domain uropod, endogenous CD102 was examined (25). Tight clustering of CD43 with intracellular proteins. of CD102 (Fig. 2b, blue staining) at the uropod indicates that the

FIGURE 3. Nonexclusion of CD43 from T cell-APC contact does not affect the immunological synapse formation or proliferation. a, Confocal analysis of immunological synapse formation between CD43Ϫ/Ϫ T lymphocytes expressing CD43 proteins (CD43FL, CD43/CD102, CD43⌬cyto, and CD43NGG), ␮ d Ј and peptide-loaded (10 m OVA323–339) L cells expressing I-A and ICAM-1. Labeled T lymphocytes with anti-CD43 (S7, red) and anti-TCR F(ab )2 (H57, green) were incubated for 20 min with L cells at 37°C. Transmitted light (TL) panels show contact between T cell and APC. TCR panels demonstrate presence of a TCR (green)-rich cSMAC at the site of T cell-L cell contact. CD43 panels show exclusion of CD43 (red) in CD43FL- and CD43/CD102- expressing cells but partial or no exclusion of CD43NGG or CD43⌬cyto proteins, respectively, from the immunological synapse. b, National Institutes of Health ImageJ software was used to quantify the intensity of CD43 fluorescence along the area of the T cell:L cell contact or the noncontact area of the T cell. Nine readings were taken at each region of the cell and meaned. Three independent cell:cell contacts from each construct were analyzed and the p Ͻ 0.01; N.S., not significant ,ء .mean fluorescence of the contact and noncontact areas of the three cells were averaged. Presented is the mean Ϯ SEM by two-tailed t test. c, DO11.10 CD43Ϫ/Ϫ T lymphocytes were retrovirally infected with CD43 constructs in hCD4RV. Infected cells were purified by immunomagnetic separation selecting for human CD4 Ag and restimulated using irradiated syngeneic peptide-loaded splenocytes for 42 h. Proliferation was assessed by tritiated thymidine incorporation. The experiment was repeated three times and representative data from one experiment are presented. 3744 CD43 REDISTRIBUTION IN T LYMPHOCYTE FUNCTION failure to redistribute CD43 was not the result of a global defect in dephosphorylation occurred in response to TCR signaling and led surface protein reorganization. to the release of CD43 from the cytoskeleton, which permitted an initial passive exclusion of the molecule from the contact. Re- Presence of CD43 in the T cell-APC contact zones does not anchoring of CD43 to the cytoskeleton was then required for com- affect T cell proliferation plete exclusion. However, the initial release required engagement We evaluated whether expression of nonredistributing CD43 mu- of the TCR, suggesting that effective TCR engagement occurs be- tants would prevent the formation of an immunological synapse fore redistribution of CD43. Furthermore, TCR-induced Ca2ϩ flux and/or inhibit the T cell proliferative response to Ag. CD43 con- was normal in cells expressing a nonredistributing mutant of structs were expressed in DO11.10 TCR-transgenic CD43-defi- CD43. Similarly, Roumier et al. (27) found redistribution of ezrin cient T cells by retroviral gene transfer. Infected cells were sorted to occur in response to TCR signaling and to depend upon Lck by immunomagnetic selection and CD43 expression was con- activity. Allenspach et al. (29) found the predominant ERM pro- firmed by flow cytometry (data not shown). Sorted T cells were tein interacting with CD43 to be ezrin. Expression of a GPI-linked cocultured with ICAM-1- and I-Ad-transfected L cells previously CD43 ectodomain was not excluded from the T cell:APC contact, loaded with OVA323–339 peptide. T cell-APC contact and CD43 yet CD69 was normally up-regulated, indicating T cell activation. exclusion were monitored by confocal microscopy. T cells infected Interestingly, both groups demonstrate a reduction in IL-2 secre- with virus encoding full-length CD43 (CD43FL) excluded CD43 tion by cells expressing nonredistributing mutants of CD43, from cell-cell contacts, as did CD43ϩ/ϩ controls (Fig. 3a and data whereas we find that proliferation is normal. Thus, it may be that not shown). Cells expressing CD43/CD102 also redistributed the while initial engagement is normal in the absence of CD43 redis- chimeric protein to areas of no cellular contact, consistent with tribution, prolonged signaling is not maintained. However, our ezrin redistribution (Fig. 3a and data not shown). The CD43NGG and CD43⌬cyto mutants failed to exclude CD43 from the site of contact (Fig. 3a) without affecting the ability of the T cell to gen- erate sustained cSMACs. All cells expressing CD43FL or CD43/ CD102 that formed a stable contact with the APC redistributed CD43, whereas all CD43NGG and CD43⌬cyto cells failed to redistribute. Quantification of the intensity of CD43 staining at both the T cell:L cell contact and the region of the T cell not in contact with the L cell confirmed a failure to redistribute CD43 in the CD43⌬cyto-expressing cells (Fig. 3b). In addition, there was no difference in the intensity of CD43 staining in the cSMAC as compared with the pSMAC. Interestingly, there was partial exclusion of CD43 from the contact region in cells expressing CD43NGG. Previous studies have demonstrated that there is a second ERM protein binding site in the tail of CD43 (24). This site is preserved in the CD43NGG mutant and may mediate the movement of this protein from the contact. Nonetheless, neither the CD43⌬cyto nor the CD43NGG constructs were excluded to the same degree as the full-length CD43 or CD43/CD102 constructs, which were virtually completely redistributed away from the T cell:L cell contact. Both the CD43⌬cyto- and CD43NGG-expressing cells formed a stable immunologic synapse, suggesting that, despite the large size and negative charge of the protein, redistribution away from the region of cell:cell contact is not required for formation of this structure. Interestingly, these observations are in contrast to data obtained by Wild et al. (26) which determined that extension of another protein, CD48, can inhibit T cell recognition of Ag, presumably by disrupting close apposition of cell membranes, thus disrupting long-term protein-ligand interaction, which would otherwise lead to cellular activation. In addition to formation of a stable cSMAC, the proliferative response of cells expressing nonredistributing CD43 was comparable to cells expressing wild-type CD43 (Fig. 3c). Thus, the presence of CD43 at the site of cell-cell contact does not inhibit func- tional TCR engagement. Consistent with the finding that large molecules can be resident among smaller-sized proteins without necessarily disrupting formation of an immunological synapse, FIGURE 4. Presence of CD43 at cell-matrix interface is not an inhibitor CD45, another large molecule, was found to actively migrate into of LFA-1/ICAM-1 interaction. a, Six confocal images of retrovirally in- cSMACs during immunological synapse formation (23). fected T lymphocytes with CD43FL and CD43NGG constructs placed on ICAM-1-containing bilayers for 10 min to initiate uropod formation. Trac- The ability of TCR engagement to occur in the absence of CD43 ing the movement of cells for a period of 6 min, we established that the redistribution is supported by a recent series of papers examining trajectory between constructs was comparable and there was no difference the role of ERM proteins in the segregation of T cell surface pro- observed between constructs tested. b, The velocity of the cells on ICAM-1 teins during T cell activation (27–29). Delon et al. (28) demon- bilayers at 37°C (rate ϭ distance/time) was calculated for each construct. strated that CD43 interacted with moesin and that dephosphoryla- There was no significant difference in the velocity of the cells expressing tion of moesin resulted in the release of CD43. The CD43FL, CD43⌬cyto, or CD43NGG by two-tailed t test (p Ͼ 0.05) The Journal of Immunology 3745 data suggest that this defect is not sufficient to lead to a decrease tors with ligand. To test this hypothesis, primary T cells expressing in proliferative responses. either wild-type or mutant CD43 were placed on a supported pla- These data suggest that cell-cell interactions should not be re- nar lipid bilayer containing GPI-anchored ICAM-1. Cross-sec- garded purely as flat and rigid membrane interactions but rather as tional reconstituted images (Z-stack) obtained by confocal micros- highly active undulating processes in which differently sized mol- copy confirmed the presence of CD43 at the cell-bilayer interface ecules can be in relative proximity without sterically hindering one (Fig. 4). Time lapse analysis of migrating cells demonstrated that another. Alternatively, molecules like CD43 may display consid- expression of nonredistributing mutant of CD43 did not impair T erable flexibility at physiological ion concentrations, such that they cell trajectory (Fig. 4a) and/or velocity (Fig. 4b) compared with are accommodated in 15-nm contact regions formed at the site of redistributing constructs. Thus, the redistribution of wild-type TCR:MHC interactions. The electron microscopy studies that CD43 to the uropod, while a consequence of the movement of show CD43 as a rigid rod were performed at low ionic strength, ezrin to the uropod (16), which itself may or may not have func- where the low dielectric constant reduces the shielding between tional consequences, is not required for effective engagement of sialic acids. The sialic acids then strongly repel each other, result- adhesion receptors by their ligands. ing in a fully extended conformation where the distance between In contrast to these observations, expression of the CD43NGG sialic acids is maximal (10). At physiological ionic strengths the mutant did seem to affect LFA-1 disengagement (Fig. 5). Motile dielectric constant is higher and the repulsion between sialic acids cells expressing CD43NGG had a striking phenotype of long re- is lower. Under these conditions CD43 should be much more com- sidual strands of membrane trailing the cell that remained adherent pressible and may coexist with the TCR contacts where the rela- to the bilayer which was not observed in CD43⌬cyto-expressing tively slow (seconds to minutes) contact formation process allows cells. These stained heavily for CD43, and the persistent adherence time for conformational changes in CD43 leading to interdigitation of the strands did not inhibit migration velocity over the 6-min of CD43 chains in the 15-nm contact area (30). The negative role observation period. IRM images obtained in conjunction with Z- of CD43 in adhesive interactions with endothelial cells in flow stack reconstitution of retrovirally infected cells revealed another conditions (31) may be more related to the very rapid kinetics of striking observation. The uropods of cells expressing CD43NGG these interactions that do not allow time for the CD43 to move out and CD43⌬cyto were in full contact with the bilayer compared of the way of interacting receptors. with endogenous CD43-, CD43FL-, and CD43/CD102-expressing These studies demonstrate that during a physiologically relevant cells, which clearly elevated uropods from the bilayer (Fig. 5, ar- interaction, i.e., T cell with an APC, the presence of CD43 in the rowheads in Z-stack and IRM panels). cSMAC does not inhibit T cell proliferation. However, it remains Both the CD43⌬cyto and the CD43NGG mutations resulted in a formal possibility that the kinetics of cell activation could be contact of the uropod with the bilayer. However, the trailing altered by the presence of CD43 in the cSMAC. Changes in the strands were observed only in the CD43NGG mutation. Thus, the time course of effective TCR engagement would not be evident by specific loss of the ezrin binding site in CD43, while keeping the measurement of proliferation. However, these data do establish remainder of the cytoplasmic interactions intact, resulted in this that failure to exclude CD43 from the T cell-APC contact does not phenotype. This suggests that the binding of a distinct cytoplasmic preclude effective T cell activation. protein to CD43 in the absence of ezrin binding to the proximal tail is responsible or, alternatively, that the introduction of the NGG Presence of CD43 in the adhesion contact zone does not inhibit mutation resulted in new protein-protein interaction with CD43. T cell motility but affects de-adhesion The precise function of CD43 remains controversial (9). The Polarization of T cells occurs during cell migration, resulting in the most consistent observation has been that CD43-deficient cells redistribution of CD43 to the uropod. The leading edge, which is have increased adhesion. This has been demonstrated both in vitro depleted in CD43, is the site at which initial adhesion receptor and in vivo (12, 13, 31, 33). The inhibitory function of CD43 on engagement occurs. The activity of both adhesion receptors and cell adhesion has been presumed to be mediated by the structural the TCR is increased in this area of the cell (14, 32). This has been features of the extracellular domain, resulting in the steric hin- thought to be due in part to the removal of large proteins such as drance of receptor ligand interactions. The data presented in this CD43, which may sterically inhibit the interaction of these recep- work suggest that this mechanism is not a tenable explanation. Disruptions in cytoskeletal associations or other signaling path- ways could result in the alterations in the regulation of cell adhesion molecule activity. These and other possibilities remain to be explored as we move beyond the steric barrier model of CD43 function.

Acknowledgments We thank Blair Ardman for providing CD43-deﬁcient mice, Terry Wood- ford-Thomas, Andy Chan, and Andrey Shaw for access to the Zeiss confocal microscope, Arup Chakraborty for discussions on polymer dynamics, and Robert Arch for many discussions and for critical review of the manuscript.

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