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The Journal of Immunology, 1999, 162: 1367-1375.
Copyright © 1999 by The American Association of Immunologists

TCR, LFA-1, and CD28 Play Unique and Complementary Roles in Signaling T Cell Cytoskeletal Reorganization1

Caitlin E. Sedwick*,{ddagger}, Margaret M. Morgan{dagger}, Lismaida Jusino{dagger}, Judy L. Cannon§, Jim Miller2,{dagger},{ddagger},§ and Janis K. Burkhardt{dagger},§

Departments of * Pharmacology and Physiology, {dagger} Pathology, and {ddagger} Molecular Genetics and Cell Biology and the § Committee on Immunology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells interacting with APCs undergo rearrangement of surface receptors and cytoskeletal elements to face the zone of contact with the APC. This polarization process is thought to affect T cell signaling by organizing a specialized domain on the T cell surface and to direct T cell effector function toward the appropriate APC. We have investigated the contribution of TCR, CD28, and LFA-1 signaling to T cell cytoskeletal polarization by assaying the response of an Ag-specific Th1 clone toward a panel of transfected APCs expressing MHC class II alone or in combination with ICAM-1 or B7-1. We show that polarization of talin, an actin-binding protein, occurs in response to integrin engagement. In contrast, reorientation of the T cell microtubule-organizing center (MTOC) is dependent on and directed toward the site of TCR signaling, regardless of whether integrins or costimulatory molecules are engaged. MTOC reorientation in response to peptide-MHC complexes is sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. CD28 coengagement overcomes this sensitivity, as does activation via Ab cross-linking of the TCR or via covalent peptide-MHC complexes, suggesting that phosphatidylinositol 3-kinase is not required per se but rather plays a role in signal amplification. Engagement of TCR in trans with LFA-1 results in separation of MTOC reorientation and cortical cytoskeletal polarization events, indicating that the two processes are not directly mechanistically linked. These studies show that T cells mobilize individual cytoskeletal components in response to distinct and specific cell surface interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell activation requires the engagement of an APC carrying the correct complement of ligands. In addition to class II molecules bearing the appropriate peptide, adhesive and costimulatory interactions are required to induce T cell proliferation and effector function (1, 2, 3). After the initial encounter with an APC, low affinity adhesive interactions facilitate T cell sampling of the APC surface for the presence of specific peptide-MHC complexes recognized by the clonally distributed TCR. Initial TCR signaling up-regulates integrin affinity and avidity, stabilizing the T cell:APC interaction (4, 5, 6, 7). This in turn facilitates formation of the lower affinity TCR:MHC interactions, allowing for serial engagement of the TCR (8, 9). Costimulatory interactions such as CD28:B7 synergize with TCR signaling to transduce full activating signals to the T cell (3). The combination of TCR and costimulatory signaling culminates in changes in gene regulation, which in turn induce T cell proliferation and differentiation.

In addition to these well-studied events, T cell:APC interactions result in dramatic changes in T cell shape and cytoarchitecture. Before APC binding, migrating T cells have an inherent polarity, with a leading edge that responds readily to an encounter with an APC and with a trailing uropod that is relatively insensitive to APC contact (10, 11). Upon encountering an APC bearing appropriate peptide-MHC complexes, T cell migration is arrested, the uropod is retracted, and APC binding is stabilized by integrin activation (6, 12). These morphological changes are accompanied by the recruitment to the cell:cell interface of a number of cell surface receptors such as the TCR, CD4, and the integrin LFA-1, as well as signaling molecules such as protein kinase C{theta} (13, 14, 15, 16). T cell cytoskeletal elements also rearrange in response to APC binding. Filamentous actin (17, 18) and the actin-binding protein talin (15) accumulate at the T cell:APC interface, and the T cell microtubule-organizing center (MTOC)3, which was previously in the trailing uropod (12), is actively recruited into the portion of the cytoplasm proximal to the APC (14, 15, 19). These polarization events facilitate T cell function at several levels. Interactions with the cortical actin cytoskeleton have been shown to regulate integrin-mediated adhesion (5, 7), and the recruitment of cell surface receptors and signaling molecules to the APC-binding site forms a specialized domain of the T cell surface that may enhance or modulate T cell signaling. Finally, polarization of the MTOC is important for effector function because it establishes polarity of the Golgi complex and associated secretory organelles, thereby directing the secretion of cytokines (20) and lytic granules (10, 21, 22) toward the appropriate APC.

Relatively little is known about the signaling events that induce T cell polarization. In T cell:B cell couples, both talin polarization and MTOC reorientation have been shown to be Ag-dependent processes. MTOC reorientation is clearly downstream of TCR signaling, since this reorganization can be induced by cross-linking of the TCR with Ab-coupled beads (23, 24). This process has been shown to involve the immunoreceptor tyrosine-based activation motifs of the TCR, as well as the TCR-proximal tyrosine kinases Lck and ZAP-70 (24). Expression in T cells of a dominant-negative form of the Rho family GTPase Cdc42, a protein that regulates actin remodeling in fibroblasts (25), disrupts both cortical actin polarization and MTOC reorientation (18). This observation suggests that the two processes are mechanistically linked, with cortical cytoskeletal rearrangements a necessary prerequisite for MTOC reorientation. However, the precise series of events that leads from TCR signaling to cytoskeletal polarization has not been elucidated.

In addition to stimulation via the TCR, it is likely that other receptor-ligand interactions contribute to cytoskeletal reorientation. In this report, we specifically address the role of TCR, LFA-1, and CD28 signaling in cytoskeletal polarization by engaging these receptors with their relevant ligands on transfected cell lines. The use of transfected cells for these studies is important because T cells are known to respond differently to Ag receptor signals derived from Ab cross-linking of the TCR vs signals initiated by peptide-MHC complexes (26). We find that talin polarization is largely integrin dependent, while MTOC reorientation is both dependent on and targeted toward the site of TCR signaling. CD28 engagement alone cannot induce either cytoskeletal rearrangement but can contribute to MTOC reorientation directed toward the TCR through signal amplification. Surprisingly, polarization of the MTOC toward the site of TCR engagement can be separated from detectable cortical (actin and talin) polarization in response to isolated receptor:ligand interactions, indicating that these two cytoskeletal changes may not be mechanistically linked.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and Abs

A20 B cells, 6132 Pro cells transfected with MHC class II alone (ProAd) or in combination with ICAM-1 (ProAd-ICAM) or B7-1 (ProAd-B7) have been previously described (27, 28). To generate ProEk-ICAM cells, 6132 Pro cells were transfected with cDNA clones encoding I-Ek (29) and ICAM-1 (30) by the calcium phosphate method as previously described (27). Cells coexpressing I-Ek and ICAM-1 were sorted using anti-Ig-conjugated magnetic beads (Dynal, Great Neck, NY) and were maintained as a bulk transfected cell line. 6132 Pro cells transfected with MHC class II covalently linked to OVA peptide (residues 323–339) (OVA peptide) (ProAd/OVA) alone or in combination with ICAM-1 were generated in the same manner, and will be described more fully elsewhere.4 Pro line cells were grown in DMEM supplemented with 10% FCS, 2 mM glutamine, 50 mM 2-ME, 0.1 mM nonessential amino acids, 2 mM HEPES buffer, and 40 µg/ml gentamicin (Life Technologies, Gaithersburg, MD). For transfectants, this medium was supplemented with 200 µg/ml G418 and/or 250 µg/ml xanthine, 15 µg/ml hypoxanthine, and 6 µg/ml mycophenolic acid. U937 cells, Jurkat cells, and Jurkat cells transfected with the 2B4 TCR {alpha}- and ß-chains (JBN-ß{alpha}.2.8.24) (31) (gift of Dr. R. Germain, National Institutes of Health, Bethesda, MD) were grown in RPMI supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg of streptomycin. pGL2 and pGL10, Th1 clonal cell lines specific for OVA peptide 323–339 in the context of I-Ad, were maintained by stimulation with Ag and irradiated BALB/cJ splenocytes every 2 wk as already described (27) and rested for 7–14 days before use.

Abs were obtained as follows: rat anti-yeast {alpha}-tubulin (YL 1/2, Serotec, Washington, DC); mouse anti-rat {alpha}-tubulin (Amersham Life Science, Arlington Heights, IL); hamster anti-rat CD3{epsilon}, 2C11 (32) (kind gift of Dr. J. Bluestone, University of Chicago, Chicago, IL); mouse anti-mouse class I MHC, 28.14.8 (33), (gift of Dr. A. Sperling, University of Chicago); mouse anti-rat talin, 8d4 (Sigma); mouse anti-sea urchin acetylated tubulin, 6-11B-1 (Sigma); rat anti-mouse CD11a, M18/2 (PharMingen, San Diego, CA); mouse anti-human CD3, OKT3 (Ortho Biotech, Raritan, NJ); and appropriate fluorochrome-conjugated secondary Abs (Jackson Immunoresearch, West Grove, PA). BODIPY-conjugated phallacidin was from Molecular Probes (Eugene, OR). Rhodamine-conjugated phalloidin and Hoechst stain (No. 33258) were from Sigma. Abs and BODIPY-phallacidin or rhodamine phalloidin were diluted to working concentration in PBS-saponin-gelatin (PBS containing 0.05% w/v saponin and 0.25% v/v fish skin gelatin (both from Sigma).

Cell conjugation for immunofluorescence microscopy

Pro line APCs grown to subconfluence at 37°C in TC flasks were harvested by incubation with trypsin-EGTA (Life Technologies), plated at 35,000 cells/well in Flexiperm 8-well chambers (Heraeus, South Plainfield, NJ) on glass slides, and grown overnight. Pro line APCs (except ProAd/OVA) were pulsed with OVA peptide 323–339 (27) to a final concentration of 2 µg/ml for 2 h at 37°C before conjugation with T cells. T cells were harvested by Ficoll purification at day 7–14 after stimulation and were plated at 200,000 cells/well in APC culture slides. T cells were allowed to interact with APCs for 30 min at 37°C, fixed, and processed for immunofluorescence analysis as described below.

To distinguish A20 B cells from T cells in the immunofluorescence assay, A20 B cells were loaded with the vital dye 7-amino-4-chloromethylcoumarin (CMAC Cell-Tracker Blue; Molecular Probes). To do this, A20 B cells were preincubated in medium or in medium plus 2 µg/ml OVA peptide for 1 h; then A20 cells were incubated in 10 µM CMAC in serum-free medium or in serum-free medium plus 2 µg/ml OVA peptide for 30 min at 37°C, washed, and chased for 30 min in medium or medium plus OVA peptide. A20 B cells (2 x 105) were mixed with an equal number of Ficoll-Paque-purified T cells and centrifuged for 5 min at 200 x g in Falcon 2058 tubes at room temperature, and the conjugates were then gently resuspended using a 1000-µl pipettor and plated onto poly-L-lysine (m.w. 30,000–70,000, from Sigma)-coated slides for 20 min before fixation.

Ab-coated beads were made by incubating 50 x 106 polystyrene, sulfate-coated beads (5 µm, Interfacial Dynamics, Portland, OR) overnight at room temperature with 100 µg of Ab (hamster anti-mouse CD3, 2C11, mouse anti-human CD3, OKT3, or rat anti-mouse Class I MHC, 28-14-8) in 1 ml of 0.2 M carbonate-bicarbonate buffer, pH 9.5. Beads were then washed twice in medium and stored at 4°C for up to 2 wk. To form conjugates, T cells and Ab-coated beads were mixed at a 1:1 ratio in medium and allowed to interact for 5 min at room temperature (RT). Conjugates were then gently resuspended and plated onto poly-L-lysine-coated slides for 20 min before fixation. For three-way (bead:T cell:APC) experiments, T cell:APC conjugates were formed for 20 min as described above, and then 400,000 beads/well were added (2 beads:1 T cell) and allowed to interact for 20 min before fixation.

For conjugation experiments using Jurkat cells, U937 cells (106 cells/ml) were dyed with 10 µM CMAC as described above for A20 cells. U937 cells were then incubated with or without 2 µg/ml OKT3 mAb at 37°C for 30 min, washed in balanced salt solution (BSS, Life Technologies), and resuspended at 106 cells/ml in BSS. Jurkat cells (2 x 105 cells) and an equal number of U937 cells were mixed in BSS, centrifuged at 200 x g for 5 min at RT, and then incubated at RT for 10 min. The cells were gently resuspended and plated onto poly-L-lysine-coated slides for 20 min before fixation. Adherent ProEk-ICAM cells were labeled with 10 µM CMAC and then incubated with or without antigenic peptide (4 µg/ml DASP; moth cytochrome c residues 86–90, 94–103) (34) for 2 h at 37°C. The cells were harvested with trypsin, and ProEk-ICAM cells (2 x 105) were mixed with Jurkat cells (2 x 105) in the presence of antigenic peptide, centrifuged at 200 x g for 5 min at RT, and then incubated at RT for 10 min. The cells were gently resuspended, plated for 10 min at 37°C onto poly-L-lysine-coated slides, and fixed.

Use of inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) in T cell polarization assays

Wortmannin and Ly 294002 were obtained from Calbiochem (San Diego, CA) and stored in DMSO at -20°C. Drugs were diluted in medium before use. T cells were pretreated with the indicated concentration of wortmannin or Ly 294002 for 30 min at 37°C, and the inhibitor was retained at this concentration throughout the experiment. For control experiments, T cells were left untreated while Pro line APCs were pretreated with wortmannin for 30 min, washed, and then allowed to conjugate with T cells. Additional controls were performed where T cells were pretreated with wortmannin and then washed free of drug before conjugation with APCs.

Immunofluorescent labeling of specimens

For all experiments, slides were rinsed in PBS and fixed in 3% (w/v) paraformaldehyde in PBS for 20 min. Specimens were quenched by washing in 50 mM NH4Cl (Sigma) in PBS, permeabilized in 0.3% (vol/vol) Triton X-100 (Sigma) in PBS for 5 min, and then rinsed in PBS and blocked in PBS-saponin-gelatin for 10 min. All subsequent Ab incubations and washes were performed in PBS-saponin-gelatin. Primary and secondary Abs were applied sequentially for 45 min at RT and washed five times after each Ab. In experiments requiring visualization of actin, BODIPY-phallacidin or rhodamine-phalloidin was applied for 15 min at room temperature after application of primary Abs. In adhesion assays, Hoechst stain was applied to samples for 15 min after application of secondary Abs. After labeling, specimens were mounted in Mowiol 4-88 (Hoechst Celanese, Charlotte, NC), with 10% 1,4-diazobicyclo[2.2.2]octane (Sigma) added as antifade. Samples were analyzed using a Zeiss Axioscop microscope equipped with a motorized stage and a Photometrics PXL-cooled CCD camera. Image capture and deconvolution analysis (where appropriate) was performed using Openlab v1.1.7 (Improvision, Coventry, U.K.) software running on a Power Macintosh 9600/300.

Analysis of polarization and conjugation

Conjugates were first identified in differential interference contrast (DIC) images as cases where substantial contact between the T cell and the APC (or bead) occurred; between 50 and 100 events were counted per condition per experiment. T cells were scored as conjugates only if they contacted no more than one APC or Ab-coated bead, and if there were no other T cells touching or near the T cell being scored. For experiments using both Ab-coated beads and APCs, only clear three-way conjugates (bead:cell:APC) were scored for these experiments. To count T cell conjugates with the various APCs, cells were labeled in three colors: mouse anti-rat talin to label both T cells and APCs; rat anti-mouse CD11a to specifically label T cells; and Hoechst stain to label nuclei. The number of T cells adhering to APCs was assessed as the number of interacting T cells per 100 APCs. For talin or actin polarization experiments, conjugates were assessed for polarization by counting the number of conjugates exhibiting notable brightness at the border between the T cell and APC. Conjugates were examined for MTOC reorientation by visually dividing the T cell cytoplasm into thirds: 1) APC-proximal; 2) middle third; and 3) distal third. Only conjugates with MTOCs that were located in the APC-proximal third of the T cell cytoplasm were counted as positive reorientation events. Because not all contacts were rounded but often involved significant flattening of T cell morphology, it was not always possible to divide the T cell into meaningful thirds; therefore, these T cells were divided in half and scored as positive/negative events along the midline. Because the T cell MTOC could theoretically be located anywhere in the cell at the time of fixation, this convention led to a background of 30–50% T cells with the MTOC oriented toward the APC or bead, even in the absence of positive signaling events. Conjugates with T cell MTOCs that could not be located were discarded from the sample.

Statistical analyses

Unless otherwise noted, at least three independent repetitions were performed of each experimental condition, and the results were analyzed using post hoc one-way (Fig. 5GoC) or two-way (Figs. 3Go and 5Go, A and B) analysis of variance with Bonferroni correction. The Bonferroni correction is used to raise the threshold to establish statistical significance, when multiple statistical comparisons are performed. The Bonferroni correction establishes a new, more stringent {alpha}, according to the number of comparisons that were performed, such that, for n comparisons, the new {alpha} = 0.05/n. The new {alpha} values obtained by the Bonferroni correction are reported in the figure legends, where appropriate.



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  		FIGURE 5. Inhibition of T cell MTOC reorientation by wortmannin is dependent on the specific signal received. T cells were pretreated with the indicated concentrations of wortmannin for 30 min and then allowed to interact with either Pro line APCs, anti-CD3 Ab-coupled beads or anti-CD3-coated U937 cells (as indicated) for an additional 30 min in the presence of wortmannin (WM). A, pGL2 murine T cells interacting with Pro line APCs. Note that there is no -Ag condition for ProAd/OVA APCs. B, pGL2 murine T cells interacting with either ProAd cells (ProAd) or Ab-coated beads (Beads). C, Ag-specific JBN-ß{alpha}.2.8.24 Jurkat T cells interacting with ProEk-ICAM cells (JBN:ProEk-ICAM), or NIH10 wild-type Jurkat T cells interacting with anti-CD3 Ab-coated U937 cells (Jurkat:anti-CD3/U937). Data are the means of at least three independent experiments; error bars are SD of the mean for each data set. *, significant differences observed vs no-drug positive controls.

 


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  		FIGURE 3. Talin polarization and MTOC reorientation in response to the Pro panel of APCs. T cells were allowed to interact with the various APCs in the presence () or absence ({blacksquare}) of Ag for 30 min before fixation and immunofluorescent labeling with anti-talin mAb (A) or anti-tubulin mAb (B). Polarization was scored as described in Materials and Methods. Data are the means of four independent experiments; error bars are SD of the mean for each data set. Talin polarization responds most strongly to integrin engagement, with significant Ag-dependent enhancement (p < 0.0083) observed only in T cells interacting with ProAd-ICAM APCs. In contrast, addition of Ag significantly enhances of MTOC reorientation in response to ProAd, ProAd-ICAM, and ProAd-B7 APCs (p < 0.0083). The fraction of polarization observed reflects the percentage of adherent cells demonstrating polarized morphology. Therefore, for APCs to which T cells bound poorly (ProPAR and ProAd), the absolute number of polarization events per sample was far fewer than for APCs which induced good adhesion (ProAd-ICAM and ProAd-B7).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Th1 clones interact differentially with a panel of transfected APCs

T cell interactions with Ag-specific APCs result in the rearrangement of surface receptors and signaling molecules within the T cell to face the APC, and a corresponding rearrangement of actin and tubulin-based cytoskeletal elements. To test the relative contribution of individual receptor:ligand interactions to the polarization of cytoskeletal elements, we used a panel of cells generated by transfection of the mouse fibrosarcoma cell line 6132 Pro (35) (ProPAR), to express MHC class II alone (ProAd) or in combination with ICAM-1 (ProAd-ICAM) or B7-1 (ProAd-B7). This panel of APCs has been previously characterized for interactions required to induce IL-2 expression, T cell activation, anergy, and clonal expansion in Th1 T cell clones and in naïve T cells (27, 28). Briefly, Ag presentation by ProAd cells does not result in the induction of IL-2 gene expression or proliferation but does engage the TCR, inducing anergy in Th1 clones. Ag presentation by ProAd-ICAM cells results in low levels of IL-2 gene expression but still induces anergy in Th1 clones; ProAd-ICAM induces proliferation, but not clonal expansion, in naïve T cells. Finally, Ag presentation by ProAd-B7 cells results in efficient T cell activation, clonal expansion, and long term survival. We used this well-characterized T cell:APC system to investigate the cytoskeletal rearrangements that take place after conjugate formation.

We began by comparing the ability of the Pro panel APCs to form stable conjugates with pGL2 T cells under conditions where cytoskeletal polarization could be studied. Using an assay based on direct visualization of conjugates, distinct differences were observed in the ability of the individual Pro line APCs to stably interact with pGL2 T cells (Fig. 1Go). While very few T cells bound to Ag-pulsed ProPAR or ProAd cells, a large number of T cells bound to Ag-pulsed ProAd-ICAM APCs, reflecting the additional contribution of LFA-1:ICAM interactions to T cell adhesion. Ag-pulsed ProAd-B7 cells were intermediate in their ability to form T cell conjugates, possibly due to the adhesive contacts provided by CD28:B7 interactions (36), or by enhancement of ß1 integrin adhesion through costimulatory signaling (37). Adhesion of Th1 clones to Pro line APCs in the absence of Ag followed the same profile as that shown in the presence of Ag, but fewer conjugates were retained in all cases (data not shown).



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  		FIGURE 1. Adherence of T cells to the different Pro line APCs. Pro line APCs were pulsed with peptide, and were allowed to interact with T cells for 30 min before fixation and labeling with anti-talin Ab to show both APCs and T cells, anti-LFA-1 Ab to show T cells, and Hoechst stain to localize nuclei. Percent conjugation was calculated using the formula: % conjugation = [no. of conjugating T cells/(no. of total nuclei - no. of T cells)] x 100. Data are the average of two experiments.

 
Talin polarization is driven by integrin engagement

The actin-binding protein talin has been shown to colocalize with clustered and occupied integrins, in part via direct interaction with the ß integrin cytoplasmic tail (38, 39, 40, 41, 42, 43), and talin polarization has been used as a marker for identifying Ag-specific T cell:B cell conjugates (15). To determine which receptor:ligand interactions are responsible for triggering talin polarization, conjugates between pGL2 T cells and individual Pro panel APCs were processed for talin immunofluorescence (Fig. 2Go). In the cases in which talin polarization was observed, it occurred as a bright band at the cell:cell interface (Fig. 2GoH). Talin polarization was quantitated by counting the percentage of T cells engaged in stable conjugates in which this bright band was observed. T cell interactions with ProPAR and ProAd cells rarely resulted in talin polarization in either the presence or the absence of Ag (Fig. 3GoA). In contrast, talin polarization was observed in the vast majority of cells bound to ProAd-ICAM. This was further enhanced in the presence of Ag, possibly reflecting the activation of the ß2 integrin LFA-1 by TCR signaling (44). ProAd-B7 cells generated moderate levels of talin polarization. It is not clear whether this recruitment of talin is a direct consequence of CD28 engagement or an indirect effect mediated by CD28 activation of ß1 integrins. Overall, we find that the frequency of talin polarization responses toward the individual APCs mirrors the abilities of these APCs to form stable conjugates (compare Fig. 3GoA with Fig. 1Go). This indicates that talin reorganization is closely linked to adhesion, as expected if both processes are integrin dependent.



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  		FIGURE 2. Representative micrographs of polarization experiments in the presence of specific peptide Ag. Conjugates were formed between pGL2 T cells and Ag-pulsed untransfected Pro cells (ProPAR; A–C), Pro cells transfected with class-II MHC alone (ProAd; D–F), or in combination with ICAM-1 (ProAd-ICAM; G–I) or B7-1 (ProAd-B7; J–L). Cells were permeabilized and immunofluorescently double-labeled for talin (B, E, H, K) and tubulin (C, F, I, L). The elongated fibroblasts and round T cells are easily distinguished in the DIC images. Talin polarization is seen as a bright band at the cell:cell interface in the T cell conjugate with ProAd-ICAM (H). The MTOC is identified as a bright congruence of tubulin staining and is scored as polarized when it is localized in the portion of the cytosol nearest to the APC. In these micrographs, the MTOC is polarized toward ProAd, ProAd-ICAM, and ProAd-B7 (F, I, L), but not ProPAR (C).

 
TCR signaling is necessary and sufficient to induce MTOC reorientation

To analyze the effects of different receptor:ligand interactions on the microtubule cytoskeleton, the orientation of the MTOC in T cells stably interacting with APCs was assessed by immunofluorescent labeling of tubulin and quantified as described in Materials and Methods.Because the T cell MTOC exists within the cell at all times, random orientation will juxtapose the MTOC toward the APC in 30–50% of the cell:cell conjugates, so that a specific response is represented by reorientation above this background. As shown in Figs. 2Go and 3GoB, none of the Pro line APCs could cause MTOC reorientation in the absence of Ag, but all the lines transfected with class II molecules induced MTOC reorientation in the presence of Ag. MTOC reorientation responses were essentially equivalent following Ag presentation by ProAd, ProAd-ICAM, and ProAd-B7 cells. Thus, induction of MTOC reorientation does not require the adhesive and/or costimulatory signals that are required for complete T cell activation. That TCR cross-linking alone is sufficient to induce MTOC reorientation is further demonstrated by the fact that reorientation can be induced in pGL2 T cells by anti-CD3-coated polystyrene beads, but not by anti-class I-coated beads (Refs. 23 and 24; see Fig. 5GoB).

Actin polarization is not required for MTOC reorientation

As previously shown for other T cell:B cell conjugates (17, 18), polarization of actin in T cells interacting with A20 B cells occurred in an Ag-dependent manner and was observed as a bright band of filamentous actin at the site of cell:cell contact (Fig. 4Go, A and B). However, actin polarization was not observed in response to Ag-pulsed ProAd APCs, although MTOC reorientation did occur under these conditions (Fig. 4Go, C and D). Even when Ag was presented by ProAd-ICAM cells, where we consistently observed strong polarization of talin (Fig. 3Go), we noted only occasional, Ag-dependent, actin accumulation (see Fig. 6GoE, below). These results show that MTOC reorientation can occur at high frequency under conditions in which actin polarization was not observed. Therefore, either actin polarization occurs at levels below our threshold of detection or the strong actin accumulations are not a necessary consequence of strong talin accumulations.



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  		FIGURE 4. Large scale actin polarization is not necessary for T cell MTOC reorientation. pGL10 T cells were allowed to interact with A20 B cells or ProAd APCs for 30 min in the presence or absence of Ag before fixation and processing for microscopy. A, B, In T cells (arrows) interacting with A20 B cells, strong actin polarization is observed in the presence (B) but not the absence (A) of peptide Ag. C, D, In T cell conjugates with ProAd APCs in the presence of Ag, actin polarization is not observed (D), even though the T cell MTOC has reoriented to face the APC (C). Representative micrographs from one experiment of three.

 


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  		FIGURE 6. MTOC reorientation is directed toward the site of TCR engagement in spite of integrin-induced talin and actin polarization at a distal site. T cells were allowed to interact for 20 min with ProAd-ICAM cells in the absence of Ag before addition of anti-CD3 Ab-coated beads and then incubated for 20 additional min before processing for microscopy. A–F, Representative micrographs of three-way (anti-CD3-coated bead:T cell:APC) polarization experiments: A–C, Three-way conjugate showing DIC (A), immunofluorescent labeling for talin (B), and immunofluorescent labeling for tubulin revealing the location of the T cell MTOC (C). D–F, Independent three-way conjugate showing DIC (D), phalloidin labeling of filamentous actin (E), and immunofluorescent labeling for tubulin (F). G, MTOC reorientation responses toward ProAd-ICAM cells in the absence of Ag (cells) or anti-CD3-coated beads (beads) incubated separately with T cells or incubated together in three-way conjugation experiments. MTOC reorientation occurs randomly in T cells conjugating individually with ProAd-ICAM cells in the absence of Ag and in three-way experiments proceeds toward the anti-CD3 bead rather than the APC. Data are from one representative experiment of three.

 
CD28 signaling renders MTOC reorientation insensitive to wortmannin inhibition

CD28 engagement synergizes with TCR signaling in the pathways that lead to gene induction and T cell proliferation (3). Although our results show that CD28 engagement is not required to induce MTOC reorientation, they do not rule out an effect on the downstream signaling pathways involved. Because PI 3-kinase has been implicated in the signaling pathways downstream of both TCR and CD28 (45, 46, 47, 48) and in cytoskeletal remodeling in other systems (49, 50, 51, 52, 53, 54), we tested the role of this kinase in MTOC reorientation. pGL2 T cells were pretreated with the PI 3-kinase inhibitor wortmannin and allowed to interact with the individual Pro line APCs. Wortmannin inhibited MTOC reorientation by ~50% following Ag presentation by ProAd or ProAd-ICAM cells (Fig. 5GoA). This inhibition was maximal at 10 nM and was not enhanced by 10-fold higher concentrations of the drug (data not shown). Because the effects of wortmannin are irreversible, control experiments were performed by preincubating either T cells or APCs with wortmannin, but omitting the drug during the conjugation period. Inhibition of MTOC reorientation was observed only when T cells were drug treated, indicating that the effects were specific for the responding T cell (data not shown). A more specific PI 3-kinase inhibitor, Ly 294002, inhibited MTOC reorientation toward ProAd-ICAM APCs to the same extent as wortmannin (50% inhibition at 500 mM), verifying that the observed effects on reorientation are due to inhibition of PI 3-kinase.

In contrast to our findings with ProAd and ProAd-ICAM, wortmannin had no effect on MTOC reorientation toward Ag-loaded ProAd-B7 cells (Fig. 5GoA) or B7-positive A20 B cells (data not shown). Thus, costimulation through CD28 overcomes the sensitivity of MTOC reorientation to wortmannin. It is not clear whether this wortmannin insensitivity occurs because CD28 coligation initiates a PI 3-kinase-independent signaling pathway, or because CD28 coligation induces levels of PI 3-kinase activity that are not readily inhibitable by wortmannin. Nevertheless, these data show that coligation of TCR and CD28 delivers a cytoskeletal signal that is qualitatively different from ligation of TCR alone.

The role of PI 3-kinase in MTOC reorientation is to amplify a weak TCR signal

The role of PI 3-kinase in MTOC reorientation has been examined by others, but with conflicting results: wortmannin inhibited MTOC reorientation in murine T cells interacting with B cell APCs (18) but did not affect MTOC reorientation of Jurkat T cells toward anti-TCR-coated beads (24). The observed differences in wortmannin sensitivity in response to the Pro line APCs suggested an explanation for this apparent discrepancy, i.e., that the sensitivity to wortmannin differs with the signal received. To address this issue, we took advantage of the fact that MTOC reorientation can be induced in pGL2 T cells either by peptide-MHC complexes or by anti-CD3-coated polystyrene beads. While wortmannin was capable of inhibiting MTOC reorientation toward Ag-loaded ProAd APCs, it failed to inhibit MTOC reorientation in response to anti-CD3-coated beads (Fig. 5GoB). Similarly, MTOC reorientation in JBN-ß{alpha}.2.8.24, a Jurkat cell line stably transfected with the {alpha}- and ß-chains of the murine 2B4 TCR (31), was sensitive to wortmannin when tested in response to Ag presentation by I-Ek-transfected APCs (Fig. 5GoC), whereas wild-type Jurkat cells showed no sensitivity to wortmannin when presented with anti-CD3-coated beads (data not shown) or anti-CD3-coated U937 cells (Fig. 5GoC). These results demonstrate that the differential sensitivity of T cell MTOC reorientation to wortmannin is not a result of cell type differences but rather of differences in the TCR signal received.

The inability of wortmannin to inhibit MTOC reorientation in anti-CD3-stimulated T cells could result from either the high affinity (low off-rate) of Ab binding or the high number of TCRs engaged. To address this issue, we used fibroblasts expressing class II molecules bearing a covalently linked OVA peptide fragment (ProAd/OVA) (Fig. 5GoA). ProAd/OVA cells are estimated to express 100-fold higher TCR ligand density than can ProAd APCs loaded with maximal exogenous peptide.4 Stimulation of pGL2 T cells with ProAd/OVA cells induced MTOC reorientation that was not inhibitable by wortmannin (Fig. 5GoA). Taken together, our results show that the requirement for PI 3-kinase can be overcome either by extremely high ligand density (as with anti-CD3-coated beads or ProAd/OVA APCs) or by coengagement of costimulatory molecules (i.e., Ag presentation by ProAd-B7 APCs), suggesting that the role of PI 3-kinase in MTOC reorientation may be to amplify weak TCR signals.

Polarization of cortical cytoskeletal elements and the MTOC can occur on opposite faces of the T cell

Normally, when the full complement of receptor:ligand interactions takes place together at the T cell:APC interface, both cortical cytoskeletal elements and the MTOC polarize toward this site, and it has been assumed that these processes are mechanistically linked. The differences in the abilities of the individual Pro line APCs to induce talin polarization and MTOC reorientation, however, suggested that this might not be the case. Moreover, although we observed MTOC reorientation toward anti-CD3-coated beads, we detected no talin polarization toward the beads (data not shown). Staining with rhodamine-phalloidin also failed to reveal strong actin accumulation at the site of bead binding, although T cells were sometimes observed to have formed a phagocytic cup around the beads (data not shown).

To address the relationship between cortical cytoskeletal rearrangements and MTOC reorientation, we tested the effects of ligating LFA-1 and the TCR on opposite faces of the same T cell. For this, conjugates were first formed between T cells and ProAd-ICAM cells in the absence of Ag. Under these conditions, engagement of LFA-1 is sufficient to recruit talin, but not to reorient the MTOC (see Fig. 3Go). A TCR signal was then supplied at a different site on the T cell by adding anti-CD3-coated beads to the preformed cell:cell conjugates. We reasoned that if the primary function of TCR signaling is to transduce a signal that links the microtubule array to the cortical cytoskeleton, the MTOC should localize toward the ProAd-ICAM cells. In contrast, if the site of TCR engagement also serves to define the locus for MTOC reorientation, then the MTOC should localize toward the anti-CD3-coated beads. We found that the latter possibility was correct (Fig. 6Go). In the bead:T cell:APC conjugates, talin polarization (Fig. 6GoB) (and occasionally actin polarization (Fig. 6GoE)) was detected only at the contact zone between T cells and ProAd-ICAM APCs, while MTOC reorientation was observed toward the anti-CD3-coated beads (Fig. 6Go, C, F, and G). Therefore, MTOC reorientation can be separated from detectable actin and talin polarization, suggesting that any mechanistic link that may exist between these processes is not obligatory.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of cytostructural changes accompany or precede the better characterized transcriptional and translational events brought on by T cell:APC interactions. In this study, we have investigated the contribution of individual receptor:ligand interactions to T cell cytoskeletal polarization and the mechanisms by which these events are achieved. We took advantage of a panel of transfected fibroblasts to present specific signals to T cells as a more physiological alternative to cross-linking of receptors by Abs. Our results show that reorganization of the components of the T cell cytoskeleton is directed by the engagement of specific cell surface receptors, with TCR, CD28, and LFA-1 playing distinct and complementary roles.

Our results show that talin polarization occurs as a direct result of LFA-1 cross-linking by its physiological ligand, ICAM-1, and that talin polarization is not induced by cross-linking of TCR alone. Thus, previous studies showing that talin polarization in T cell:B cell conjugates is an Ag-dependent process (15) probably reflect not the direct involvement of TCR, but rather the requirement for TCR-mediated activation of integrin-based adhesion (44). Our finding that talin polarization occurs toward ProAd-ICAM cells in the absence of Ag may be due to the activation status of the Th1 clones or to the relatively high levels of ICAM presented by ProAd-ICAM cells, since high ligand density can induce integrin activation (55). By analogy with focal adhesions in other cell types, where the cytoplasmic tails of ß1 integrins have been shown to bind talin upon engagement and clustering (39), it is likely that in T cells, ICAM binding initiates direct interaction of talin with the ß2 subunit of LFA-1. This interpretation is also supported by our finding that talin polarizes under conditions in which actin polarization is not observed.

Although we did not detect strong actin polarization in pGL2 cells responding to Pro cells or to anti-CD3-coated beads, we did observe filopodia and actin-containing extrusions in bead-stimulated T cells. We interpret this as the formation of phagocytic cups, since at later time points beads were partially engulfed (data not shown). These actin-containing phagocytic structures resemble the actin polarization events described by Lowin-Kropfe et al. (24), but they bear little resemblance to the more distinct actin clusters observed in T cell:B cell couples in the presence of Ag (Fig. 4Go) (17, 18) or to the strong actin accumulations that we occasionally observed in response to the Pro panel APCs (Fig. 6GoE). Since we were unable to consistently induce strong actin clustering in response to either Pro line APCs or anti-CD3-coated beads, we conclude that TCR signaling alone is insufficient to drive this process and that the receptor-ligand interactions required are not among those we have tested thus far.

While we find that talin polarization responds to integrin ligation, our results show that MTOC reorientation occurs in response to TCR engagement. Our findings confirm previous studies showing that MTOC reorientation can be induced by Ab-mediated cross-linking of the TCR (23, 24) and further show that presentation of Ag by MHC class II molecules has the same effect. The manner in which the MTOC reorientation event is linked to TCR signaling is unknown. Recent work has implicated TCR-associated tyrosine kinases, since Jurkat T cells lacking Lck fail to reorient their MTOCs and since overexpression of a dominant-negative ZAP-70 mutant partially inhibits MTOC reorientation (24). It has been proposed that ZAP-70 phosphorylation of {alpha}-tubulin could cause localized microtubule catastrophe at the site of TCR signaling (24), bringing about MTOC reorientation by shortening microtubules in the proximity of TCR signaling while microtubules elsewhere in the cell continue to lengthen. However, since the ZAP-70 mutant only partially inhibits MTOC reorientation, it must be considered that ZAP-70 might not be solely responsible for this event. Furthermore, since both Lck and ZAP-70 act early in TCR signaling, it is possible that their role is indirect; there may be many intervening steps in between TCR signaling and the effector mechanisms that actually bring about MTOC reorientation.

PI 3-kinase has been implicated in signaling by both TCR and CD28, although there is some disagreement in the literature regarding whether PI 3-kinase activity is essential to CD28 signaling (56, 57, 58). We found that while CD28:B7 interactions alone could not induce MTOC reorientation, they could affect the sensitivity of TCR-induced MTOC reorientation to wortmannin, a specific inhibitor of PI 3-kinase. While wortmannin inhibited MTOC reorientation in response to ProAd cells by ~50%, it had no effect on MTOC reorientation in response to ProAd-B7 APCs. In this context, it is important to note that although Stowers et al. (18) found that wortmannin did inhibit MTOC reorientation using the B cell line CH27 as an APC, the T cell they used as a responder is a hybridoma that is not responsive to costimulatory signals. The effect of CD28 is probably not due to increases in adhesion mediated by CD28:B7 interactions because wortmannin inhibits MTOC reorientation following Ag presentation by ProAd-ICAM cells, where the adhesive effects are greater. Instead, CD28 engagement could transduce a PI 3-kinase-independent signal that enhances TCR-stimulated MTOC reorientation or could activate additional PI 3-kinase isoforms that are insensitive to wortmannin inhibition.

The finding that wortmannin failed to inhibit MTOC reorientation in the presence of costimulatory signaling suggests that the role of PI 3-kinase in MTOC reorientation is one of signal amplification. This conclusion is supported by the observation that wortmannin inhibits MTOC reorientation in response to TCR stimulation by peptide-MHC complexes but is incapable of affecting MTOC reorientation when the TCR is cross-linked by anti-CD3 Ab-coated beads, or when the T cell is presented with exceptionally high levels of peptide-MHC complexes, expressed by ProAd/OVA cells. These observations explain a discrepancy in the literature regarding the role of PI 3-kinase in MTOC reorientation (18, 24). The contradictory observations made by these two groups are likely due to the fact that presentation of the polarizing signal was accomplished by Ab-coated beads in the former case and by peptide-MHC complexes (in the absence of CD28 signaling) in the latter. On the basis of our cumulative results, we conclude that PI 3-kinase likely amplifies suboptimal TCR signaling to produce MTOC reorientation but is not required in the presence of exceptionally strong TCR signaling such as that induced by Ab cross-linking of the TCR.

In many systems, the cortical cytoskeleton and the microtubule cytoskeleton are thought to act in concert to produce the morphological changes that accompany cellular responses to extracellular stimuli. It therefore seemed likely that T cell cortical rearrangements could facilitate MTOC reorientation by creating an anchoring superstructure for the proteins involved. Evidence in favor of an interaction between the cortex and the microtubule cytoskeleton comes from the work of Stowers et al. (18), showing that a dominant negative form of Cdc42 (a member of the Rho family of small GTPases, which are involved in actin remodeling) disrupts both actin polarization and MTOC reorientation in 2B4 T cells. Recent work has demonstrated that PI 3-kinase products and substrates can modify the activity of Vav, an exchange factor for Rho family proteins (59), suggesting that PI 3-kinase may indirectly affect MTOC reorientation via modulation of the cortical cytoskeleton.

Surprisingly, however, we have found that MTOC reorientation, which responds specifically to TCR signaling, can occur in the absence of detectable talin or actin accumulation. Moreover, we found that MTOC reorientation can occur on the opposite face of the cell from detectable cortical polarization events. Although our results do not preclude a contribution by the cortical cytoskeleton to the process of MTOC reorientation, they clearly show that the dramatic accumulations of cortical cytoskeletal elements normally seen during T cell:B cell interactions are not required for TCR-induced MTOC reorientation. TCR engagement alone is capable of defining the site toward which MTOC reorientation occurs. Thus, either TCR signaling itself is capable of recruiting the necessary effector molecules responsible for MTOC reorientation or the cortical cytoskeleton can transmit the TCR signal to the relevant effectors without undergoing obvious reorganization.

The T cell cytoskeleton, far from being a passive observer of signaling events, is directly involved in the mechanisms and consequences of T cell:APC interactions. Therefore, understanding the interplay of these cytoskeletal rearrangements and the signaling events that bring them about is likely to be an area of much interest in the future. The experimental systems described here will provide valuable tools with which to investigate these and related questions.


   Acknowledgments
 
We thank Dr. Jim McIlvain from the Ben May/Pathology Digital Light Microscope Facility at the University of Chicago for technical assistance and Arthur Simen for assistance with statistical analyses. Thanks to Drs. Anne Sperling, David Straus, Tom Gajewski, and Craig Thompson for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant DK-49799 to J.M. and by grants from The Louis Block Fund and the Cancer Research Foundation to J.K.B. C.E.S. was supported by the Pharmacological Sciences Training Grant (GM-07151), and J.L.C. was supported by the Interdisciplinary Training Program in Immunology (AI-107090). Animal care and peptide synthesis were supported by the Cancer Research Center (Grant CA-14599). Back

2 Address correspondence and reprint requests to Dr. Jim Miller, University of Chicago, Department of Molecular Genetics and Cell Biology, 920 E. 58th Street, CLSC 1021, Chicago, IL 60637, E-mail address: Back

3 Abbreviations used in this paper: MTOC, microtubule-organizing center; PI 3-kinase, phosphatidylinositol 3-kinase; CMAC, 7-amino-4-chloromethylcoumarin; OVA peptide, OVA peptide (residues 323–339); BSS, balanced salt solution; DIC, differential interference contrast; RT, room temperature. Back

4 C. Abraham, J. Griffith, and J. Miller. The dependence for LFA-1/ICAM-1 interactions in T cell activation cannot be overcome by high density TCR ligand. Submitted for publication. Back

Received for publication July 30, 1998. Accepted for publication October 19, 1998.


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