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Immature CD4⁺CD8⁺ Thymocytes Do Not Polarize Lipid Rafts in Response to TCR-Mediated Signals¹

Department of Biology, Haverford College, Haverford, PA 19041

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4⁺CD8⁺ thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4⁺CD8⁺ thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mature CD4⁺8^- or CD4^-8⁺ (single-positive or SP³) T cells develop in the thymus from immature CD4⁺CD8⁺ (double-positive or DP) thymocyte precursors. Like their descendants, CD4⁺CD8⁺ thymocytes respond to TCR stimulation by activating a cascade of signaling events. However, the consequences of TCR stimulation differ markedly between immature and mature T cells. Whereas mature T cells respond to TCR signals by dividing, immature CD4⁺CD8⁺ thymocytes do not proliferate after engagement of TCR. Rather, depending on both the avidity of TCR engagement and the specific involvement of costimulatory or coactivating molecules, CD4⁺CD8⁺ thymocytes undergo either maturation (positive selection) or apoptosis (negative selection) (1, 2).

TCR signals, alone, are not sufficient to induce T cells to divide, differentiate, or die. Instead, they must be accompanied by second or costimulatory signals, of which CD28 is one example. CD28 has been found to act as a costimulatory signal in both mature and immature T cells. In mature cells, TCR/CD28 costimulation results in activation, sustained IL-2 production, and proliferation (3, 4). In immature CD4⁺CD8⁺ thymocytes, however, TCR/CD28 costimulation results in apoptosis (5, 6, 7, 8). Although CD28 is not unique in its ability to cooperate with TCR to induce thymocyte death, in vivo (9, 10), it is a potent costimulator of TCR-induced apoptosis in vitro. Purified subpopulations of cells can be stimulated by discrete receptor/ligand interactions either 1) via cells designed to present MHC/Ag and the CD28 ligand B7, or 2) via immobilized mAbs specific for TCR and CD28 (5, 6, 7, 8). Because TCR/CD28 costimulation results in such different outcomes in immature and mature T cells, in vitro stimulation can be a useful probe for developmental differences in TCR signaling.

Viola and colleagues have recently shown that in vitro TCR/CD28 costimulation of mature T cells induces the recruitment of cholesterol- and glycosphingolipid-enriched membrane microdomains (lipid rafts) to the site of stimulation (11). Lipid rafts are unique substructures in the plasma membrane enriched for a variety of signaling molecules, including GPI-linked proteins (12, 13, 14), lipid-modified proteins (15, 16), and specific lipid subsets, including phosphatidylinositols (17, 18).

In T cells, lipid rafts carry particularly precious signaling cargo, including the src kinases lck and fyn (19, 20), the adaptor molecule linker for activation of T cells (LAT) (21), and phosphatidylinositol-4,5-bisphosphate (PIP₂), a lipid substrate for PLC and phosphoinositide 3-kinase (PI3K) (22). By recruiting such key signaling molecules to the site of signal initiation, rafts have been shown to quantitatively enhance (11) and could qualitatively influence signaling routes initiated by TCR/CD28 costimulation.

Lipid raft polarization occurs concomitantly with reorganization of the actin cytoskeleton (23, 24, 25, 26) and the establishment of an elaborate T cell/APC contact structure called the immunological synapse (25, 27, 28, 29, 30). During the interaction between APC and T cell, TCR complexes colocalize with lipid rafts (31, 32, 33) and the TCR -chain associates with actin (34, 35). How lipid raft recruitment is coordinated with actin reorganization and receptor clustering is unclear.

Because lipid rafts have the potential to regulate signals, they are attractive targets for investigation into developmental differences in TCR signaling between immature and mature T cells. In the present study, we assess the ability of immature CD4⁺CD8⁺ thymocytes to recruit lipid rafts in response to TCR/CD28 costimulation. We show that raft polarization in mature T cells is actin dependent, and, although CD4⁺CD8⁺ thymocytes polarize actin efficiently, they do not polarize lipid rafts in response to TCR/CD28 costimulation. Furthermore, we offer evidence that PI3K activity coordinates polarization of lipid rafts with reorganization of the actin cytoskeleton in mature T cells, and discuss the potential relevance of these findings to developmental differences in immature vs mature T cell responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Surfactant-free polystyrene beads were purchased from Interfacial Dynamics (Portland, OR). LY294002 and cytochalasin D were purchased from Calbiochem (San Diego, CA), and biotinylated cholera toxin B subunit from Sigma (St. Louis, MO). mAbs, anti-TCR- (H57-597), anti-CD28 (37.51), anti-CD4 (GK1.5), and fluorescein (FITC)-conjugated anti-CD69 were purchased from PharMingen (San Diego, CA), and Alexa 488 streptavidin, Alexa 488 phalloidin, and Alexa 488 goat anti-rabbit Ig were purchased from Molecular Probes (Eugene, OR).

Mice

C57BL/6 mice were purchased from Taconic Laboratories. The 4- to 8-wk-old mice were used as a source of CD4⁺CD8⁺ thymocytes, and 8-wk- to 6-mo-old mice were used as a source of mature T cells.

Cell isolation

CD4⁺CD8⁺ thymocytes were purified by plating freshly isolated thymocyte suspensions onto anti-CD8 (83-12-5, 1 µg/ml)-coated petri dishes (6, 8). After 1 h at 4°C, nonadherent cells were washed away, and adherent cells were recovered and plated for a second time on anti-CD8 (83-12-5)-coated petri dishes. Recovered adherent cells were >95% CD4⁺CD8⁺. Mature T cells were enriched from spleen by plating isolated spleen suspensions on petri dishes coated with rat monoclonal anti-mouse IgG (200 µg/ml) to remove B cells. Nonadherent cells were recovered after 1 h at room temperature and contained >85% CD4⁺ or CD8⁺ T cells. Cells were either stimulated immediately after isolation or after 4-h culture at 37°C/5% CO₂ in complete medium (RPMI, 10% FCS, 2 mM l-glutamine, 1 mM penicillin/streptomycin, and 5 x 10^-5 2-ME).

Cell stimulation

Beads (10⁷ beads/ml) were incubated with 1 µg/ml anti-H57, 5 µg/ml anti-CD28, and/or 1 µg/ml anti-CD4 in PBS for 1.5 h at 37°C, then washed three times in serum-containing medium before use. Beads were incubated at 37°C with cells at a ratio of 2 beads per cell and at a cell concentration of 2 x 10⁷ cells/ml for 20 min (for evaluation of actin and lipid raft polarization) or 1 x 10⁶ cells/ml for 5 h (for evaluation of CD69 up-regulation and cell/bead conjugation). For immunofluorescence, cell/bead mixtures were fixed by a 10-min incubation with 3.7% paraformaldehyde, followed by addition of an excess of RPMI before staining. For flow cytometry, cell/bead mixtures were stained immediately after stimulation.

Drug treatment

Mature T cells were incubated in the presence of 10 µM cytochalasin D in 0.05% DMSO, or 50 mM Ly294002 in 0.05% DMSO for 10 min before stimulation and for the duration of stimulation. Control cells were treated with the vehicle, 0.05% DMSO, alone.

Cell staining

A total of 5 x 10⁵ fixed or fresh cells was distributed in each well of a 96-well plate and washed three times with staining medium (1% BSA, 15 mM NaN₃ in balanced salt solution (HBSS)). For actin and GM1 staining, fixed cells (from 20-min stimulations) were stained for actin or GM1 by 40-min incubation with 10 µg/ml phalloidin-Alexa 488, or 10 µg/ml biotinylated cholera toxin B subunit, respectively. Cells were washed three times in staining medium. For GM1 staining, cells were subsequently incubated for 30 min in the presence of 10 µg/ml avidin-Alexa 488, and then washed three times in staining medium. For CD69 staining, unfixed cells (after 5-h stimulations) were stained by 40-min incubation with 1 µg/ml FITC anti-CD69, then washed three times in staining medium. For LAT staining, fixed cells were washed three times in PBS containing 10% FBS, permeabilized with 0.1% Triton X-100 for 4 min at room temperature, washed, and incubated for an additional 45 min in 10% FBS. Cells were then incubated at room temperature for 45 min with 10 µg/ml rabbit anti-mouse LAT primary, washed three times, incubated for 45 min with 10 µg/ml Alexa 488-conjugated goat anti-rabbit IgG1, and washed again. After staining, cells were either analyzed by flow cytometry or resuspended at roughly 1 x 10⁸ cells/ml concentration and plated onto hydrophobically coated slides (Carlson Scientific, Peotone, IL) for visualization by immunofluorescence microscopy.

Microscopy

Stained cell/bead mixtures were visualized at x1000 magnification using a Nikon Labophot immunofluorescence microscope, with an Omega Optical (Brattleboro, VT) XF68 NS332 filter to observe Alexa 488 fluorescence. Conjugates were identified under bright field before their pattern of fluorescence was assessed. Bright field and immunofluorescent images were recorded digitally using Adobe Photoshop 4.0 (Adobe Systems, Mountain View, CA) with an AV Macintosh computer. Unless stated otherwise, 20–30 cell/bead conjugates were counted and scored as either negative or positive for accumulation of Alexa 488 fluorescence intensity at the cell/bead interface.

Flow cytometry

Stained cell/bead mixtures were acquired on a Becton Dickinson (Mountain View, CA) FACScalibur and analyzed with CellQuest software. For CD69 and GM1 staining, live cells were identified based on forward scatter (FSC)/side scatter (SSC) profile and their FITC intensity measured.

Quantitation of percentage of cell/bead conjugates

Cell/bead conjugates can be distinguished by their FSC and SSC profile. Whereas T cells exhibited a high FSC and low SSC profile and hugged the x-axis, beads displayed a low FSC and high SSC profile and hugged the y-axis. In contrast, stable cell/bead conjugates appeared as a single event with both high FSC and high SSC intensities. Percentage of cell/bead conjugates was calculated by dividing the percentage of cells falling into the cell/bead conjugate gate by the total percentage of live cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coengagement of TCR and the costimulatory molecule, CD28, induces lipid raft aggregation in mature T cells (11). We wished to determine whether immature CD4⁺CD8⁺ thymocytes, which respond very differently to TCR/CD28 costimulation, also polarize lipid rafts.

Conditions for optimal acute stimulation of CD4⁺CD8⁺ thymocytes

Whereas freshly isolated mature murine T cells mobilize Ca²⁺ (36) and up-regulate CD69 in response to TCR-mediated stimulation (Fig. 1a), freshly isolated CD4⁺CD8⁺ thymocytes do not (36) (Fig. 1b). The inability of CD4⁺CD8⁺ cells to respond to acute stimulation is thought to be attributable to the sequestration of lck by CD4, which is engaged in vivo by thymic MHC class II (37, 38). This defect in signaling can be readily corrected by either 1) preincubating cells in suspension, thereby disrupting TCR/class II interactions, allowing lck to redistribute, or 2) simultaneously coengaging CD4 with TCR and CD28 to directly recruit lck to the complex (37, 38). Indeed, both mature and immature T cells will up-regulate CD69 in response to simultaneous TCR/CD28/CD4 engagement (Fig. 1, c and d) and after preincubation for 4 h (data not shown), indicating that these are optimal conditions for evaluating early activation events in immature T cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Optimal conditions for acute TCR stimulation of immature CD4+CD8+ thymocytes. Freshly isolated, purified mature T cells (SP T cells, a and c) and CD4+CD8+ immature thymocytes (DP thymocytes, b and d) were stimulated for 5 h with beads coated with indicated combinations of Abs (anti-TCR/anti-CD28-coated beads (T/28, a and b) or anti-TCR/anti-CD28/anti-CD4-coated beads (T/28/4, c and d)). Cells were stained for CD69 expression and analyzed by flow cytometry. Overlay histograms are displayed. The dark line in each panel indicates cells that were stimulated with TCR/CD28, while the light line indicates the profile of unstimulated cells. CD69 up-regulation could also be restored among CD4+CD8+ thymocytes by preincubating them for 4 h at 37°C before bead stimulation (data not shown).

Using the stimulatory conditions that gave optimal TCR signaling in CD4⁺CD8⁺ cells, we compared the ability of mature T cells and immature thymocytes to polarize lipid rafts upon TCR-mediated costimulation. We stimulated cells with beads coated with the indicated Abs for 20 min and visualized lipid rafts with fluorochrome-conjugated cholera toxin B subunit, which binds the raft-resident ganglioside, GM1 (39). As expected, mature T cells exhibited efficient lipid raft capping in response to TCR/CD28 costimulation (Fig. 2, a and c). The majority (70–85%) of mature T cell/bead conjugates showed a distinct accumulation of fluorescence at the cell/bead interface after TCR/CD28 costimulation (Fig. 2, a and c), but not after TCR stimulation alone (data not shown). In contrast, immature CD4⁺CD8⁺ thymocytes did not polarize lipid rafts in response to TCR/CD28 costimulation (Fig. 2, b, d, and e), even when lck activity was restored after preincubation (Fig. 2d) or via recruitment of CD4 (Fig. 2e). Surface expression of GM1 was comparable between resting immature and mature T cell populations (Fig. 3), indicating that observed differences in GM1 distribution are not a consequence of differences in staining efficiency between mature SP and immature CD4⁺CD8⁺ populations.

View larger version (61K): [in this window] [in a new window]   FIGURE 2. Immature CD4+CD8+ thymocytes do not polarize membrane microdomains in response to TCR stimulation. Purified SP T cells (a and c) and DP thymocytes (b, d, and e) were stimulated for 20 min with beads coated with the Abs indicated (anti-TCR and anti-CD28 (T/28) or anti-TCR, anti-CD28, and anti-CD4 (T/28/4)). Cells were then fixed and stained for lipid rafts using fluorochrome-conjugated cholera toxin B subunit, which binds to the raft resident ganglioside, GM1. Where indicated (d), cells were cultured for 4 h just before stimulation. Two representative pairs of images are shown for each stimulatory condition. The top portion of each represents the bright field image of a single cell/bead conjugate, and the bottom represents the immunofluorescence image of the same conjugate. Although image details can differ by the choice of focal plane and by changes in background staining from experiment to experiment, polarization is nonetheless consistently identifiable by the accumulation of fluorescence intensity at the site of cell/bead contact. Numbers displayed next to each stimulatory condition indicate the percentage of cell/bead conjugates scored positive for lipid raft polarization.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Total levels of surface GM1 are comparable in SP and DP T cells. SP and DP cells were purified, stimulated, and stained with biotinylated cholera toxin, followed by streptavidin-Alexa 488 for analysis on FACS. The overlay histograms represent resting levels of GM1 on purified SP T cells (dashed line) and DP thymocytes (solid line). Mean fluorescent intensity for each profile is shown in parentheses.

View larger version (48K): [in this window] [in a new window]   FIGURE 4. Immature CD4+CD8+ thymocytes do not localize LAT to site of bead-cell contact in response to TCR stimulation. SP and DP cells were purified and stimulated as in Fig. 2. Cells were fixed, permeabilized, and stained internally for LAT, a raft-resident adaptor molecule, as described in Materials and Methods. Percentage of cell-bead conjugates showing LAT capping in SP cells was identical with the percentage of cell-bead conjugates exhibiting GM1 recruitment.

It was possible that the inability of immature CD4⁺CD8⁺ thymocytes to recruit lipid rafts reflected an inability to polarize the actin cytoskeleton in response to TCR stimulation. To confirm that lipid raft polarization was, indeed, actin dependent, we stimulated mature T cells in the presence of cytochalasin D, which both actively destabilizes existing actin microfilaments and prevents further actin polymerization (Fig. 5). As expected, in the presence of vehicle alone, TCR/CD28 coengagement induced lipid raft polarization among the majority of mature T cell/bead conjugates (Fig. 5b). However, in the presence of cytochalasin D, only 19% of cell/bead conjugates exhibited lipid raft polarization (Fig. 5d). The few cells that did score positive for polarization tended to exhibited only a small, local accumulation of rafts (Fig. 5d, right panel). Cytochalasin D also significantly inhibited the formation of cell/bead conjugates, which are distinguished by their FSC and SSC profiles (Fig. 5, a vs c). These data indicate that both lipid raft recruitment and the formation of stable cell/bead conjugates require TCR-induced cytoskeleton reorganization.

View larger version (78K): [in this window] [in a new window]   FIGURE 5. Disruption of the actin cytoskeleton abrogates lipid raft movement in mature T cells. Purified SP T cells were stimulated with beads coated with anti-TCR and anti-CD28 (T/28) for 20 min (for immunofluorescence studies) or 5 h (for flow cytometry) in the presence of 10 µM cytochalasin D in 0.05% DMSO (a and b) or vehicle alone (c and d). FSC and SSC profiles of stimulated cell/bead mixtures are shown in the panels at the left (a and c), and percentage of bead/cell conjugates is indicated, as described in Materials and Methods. Microscopy images of cells stained for GM1 are shown in panels at the right, as described under Fig. 2. Forty or more events were counted for these experiments.

Given that lipid raft recruitment requires a functional actin cytoskeleton, we addressed the possibility that immature thymocytes fail to polarize lipid rafts because of their inability to reorganize actin in response to TCR stimulation. We compared patterns of actin organization after stimulation of mature and immature T cells by staining with phalloidin, which binds polymerized actin. As expected, mature T cells responded to acute TCR stimulation (in the presence or absence of CD28 (data not shown)) by accumulating actin at the site of cell/bead contact (42) (Fig. 6, a and c). TCR stimulation also induced formation of lamellipodia and actin collars, which appeared to increase the area of cell/bead contact, resulting in establishment of a greater number of stable cell/bead conjugates (Fig. 6, a and c).

View larger version (70K): [in this window] [in a new window]   FIGURE 6. Immature CD4+CD8+ thymocytes reorganize their actin cytoskeleton in response to costimulation. Purified SP T cells (a and c) and DP thymocytes (b and d) were stimulated for 20 min (for immunofluorescence studies) or 5 h (for flow cytometry) with beads bound to indicated Ab combinations. Frequency of cell/bead conjugation was assessed by flow cytometry, and percentage of conjugates (of total live cells) is indicated in each FSC and SSC profile. Actin polarization was measured by staining fixed cells with phalloidin, and microscopy images are displayed, as described in Fig. 2. These results are respresentative of two distinct experiments.

Lipid raft polarization is dependent on PI3K activity

The inability of CD4⁺CD8⁺ thymocytes to mobilize lipid rafts, despite their ability to reorient their cytoskeleton, suggests that immature thymocytes are unable to couple lipid rafts with actin in response to TCR/CD28 costimulation. Because CD28 costimulation is required for raft movement, but not for TCR-driven actin polymerization (42), it seemed reasonable to suppose that signals downstream of CD28 may be responsible for coupling actin reorganization with lipid raft movement in mature T cells.

PI3K has been implicated as one downstream effector of CD28 (43). To determine whether PI3K were involved in lipid raft movement in mature T cells, we stimulated T cells in the presence of the PI3K inhibitor, LY294002 (44). CD69 up-regulation was abrograted in the presence of LY294002 within the 5-h time frame examined, indicating that the inhibitor abrogated efficient TCR signaling (Fig. 7, a and d). More importantly, the PI3K inhibitor also significantly reduced the frequency of cells exhibiting lipid raft polarization. Whereas lipid raft accumulation was evident in 70% of cell-bead conjugates formed in the absence of LY294002, lipid raft recruitment was observed in only 20% of cell/bead conjugates formed in the presence of the PI3K inhibitor (Fig. 7f). Any lipid raft capping that was observed in the presence of LY294002 was loose and disperse, indicating that even among the few positive events, polarization was not optimal (Fig. 7f).

View larger version (46K): [in this window] [in a new window]   FIGURE 7. Inhibition of PI3K inhibits raft polarization, but not actin accumulation in mature T cells. Purified SP T cells were stimulated with anti-TCR/CD28-coated beads for 20 min (for immunofluorescence studies) or 5 h (for flow cytometry) in the presence of 50 µM PI3K inhibitor Ly294002 (Ly) in 0.05% DMSO (d, e, and f) or vehicle alone (a, b, and c). Overlay histograms of CD69 expression (a and d), FSC/SSC plots (b and e), and microscopy images of lipid raft staining (c and f) are displayed, as described under Figs. 2 and 3. Forty or more events were counted for this set of results, which is representative of two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we demonstrate a developmental difference in the ability of T lymphocytes to recruit lipid rafts to the site of TCR/CD28 costimulation and provide evidence for a mechanism underlying TCR-mediated raft polarization. Specifically, we show that immature CD4⁺CD8⁺ thymocytes reorganize their actin cytoskeleton, but do not polarize lipid raft components (specifically, both the ganglioside GM1 and the adaptor protein LAT) in response to TCR/CD28 costimulation. In addition, we show that a functional actin cytoskeleton is required for lipid raft recruitment in mature T cells and implicate PI3K in mediating the coupling of lipid rafts with the actin cytoskeleton.

These data have several implications concerning the biology and developmental significance of lipid rafts. First, they indicate that lipid raft polarization is not absolutely required for TCR-mediated signaling. Despite the inability of TCR/CD28 costimulation to induce lipid raft mobilization in CD4⁺CD8⁺ thymocytes, TCR/CD28 coengagement initiates robust signals that induce CD69 up-regulation and apoptosis (36). These observations raise the possibility that, rather than being an obligatory event in TCR signaling, lipid raft polarization may be one regulatory option available to a T cell.

Concomitant with the recruitment of lipid rafts is the formation of an immunological synapse (25, 27, 28, 29, 30) that serves to segregate and concentrate signaling components at the interface between T cell and APC. While the synapse is thought to separate molecules based on their size and avidity for their ligands, lipid rafts allow the cell to sort signaling machinery based on its affinity for a unique lipid microenvironment. Thus, both processes may act to quantitatively enhance and sustain TCR signals, while at the same time each can regulate distinct subsets of signaling molecules that can be included in those signaling pathways. Our results indicate that immature CD4⁺CD8⁺ thymocytes cannot take full advantage of one regulatory option available to mature T cells.

These data also suggest a potential mechanism for raft movement in mature T cells. Our results confirm that TCR-mediated lipid raft polarization relies upon an intact actin cytoskeleton. Data showing that 1) actin polarization can be induced by TCR signals, alone (42), but 2) lipid raft recruitment requires costimulatory signals (11) imply that a costimulation-dependent process links rafts to the actin cytoskeleton. The results presented in this study indicate that PI3K activity mediates the establishment of this link. Based on these data, we propose one possible model for costimulation-dependent raft polarization, illustrated in Fig. 8a.

View larger version (34K): [in this window] [in a new window]   FIGURE 8. One model for costimulation-dependent lipid raft polarization. a, Mature T cells. TCR-associated and CD28-associated signals would be generated by coengagement of these receptors through interaction with MHC/peptide and B7 ligands on APCs. CD28-associated PI3K activation would alter the lipid environment of rafts by phosphorylating phosphoinositides at the D-3 position and converting, for instance, PIP2 to PIP3. TCR activation and resulting kinase activity and docking of adaptor molecules to phosphorylated residues of the TCR/CD3 complex would inspire the recruitment of molecules with PH domains, such as vav (46 ). PH domains would then provide a physical link between lipid rafts and TCR complexes, which interact directly with the actin cytoskeleton via TCR- (34 ). b, Immature CD4+CD8+ thymocytes. Although TCR signals result in recruitment of PH domain-containing proteins and the polarization of actin by immature T cells, TCR/CD28 costimulation does not activate PI3K activity and does not convert PIP2 to PIP3 in lipid rafts. In the absence of the link between PIP3 and recruited PH domain-containing proteins, lipid rafts do not connect with polarized TCR complexes. Thus, signals generated by TCR/CD28 costimulation are neither amplified nor accompanied by unique signaling cascades that could be initiated by raft-specific molecules.

This model suggests that the uncoupling of lipid raft recruitment and actin polarization seen in immature CD4⁺CD8⁺ thymocytes could be due to their inability to efficiently stimulate a PI3K activity after TCR/CD28 costimulation, as illustrated in Fig. 8b. Interestingly, this possibility is supported by our previous observation that TCR/CD28 coengagement generates apoptotic signals in CD4⁺CD8⁺ thymocytes in a PI3K-independent manner (8).

It is important to recognize that CD28 is not the only costimulatory molecule that can cooperate with the TCR to induce lipid raft recruitment. TCR/CD48 and TCR/CD5 coengagement can also efficiently polarize lipid rafts in mature T cells (33, 47) (J.F.B., unpublished observations). Notably, consistent with our indications that PI3K is required for raft recruitment, CD5 engagement can activate PI3K activity (48). (Because CD48 is a raft-resident protein, TCR/CD48 coengagement may recruit raft in a more passive manner.) It is also important to note that the role of PI3K in costimulation of primary T cells, particularly its requirement for IL-2 production, is controversial (49, 50). While our data show that PI3K activity is required for lipid raft recruitment, our observation that signaling can take place in the absence of recruitment introduces the possibility that lipid raft recruitment is not strictly required for all aspects of T cell stimulation.

Interestingly, biochemical studies assessing the redistribution of signaling molecules into lipid rafts indicate that TCR/CD3 stimulation, alone, can recruit TCR components to the lipid raft fraction of both T cells and thymocytes (32, 33). In addition, very recent studies indicate that positive selection signals also result in recruitment of signaling molecules to the raft fraction in thymocytes (51, 52). These observations, combined with our immunofluorescence studies (in which TCR signals do not mediate lipid raft polarization in immature CD4⁺CD8⁺ thymocytes, and, in agreement with Viola et al. (11), costimulatory activity is required for raft polarization in mature T cells), raise the possibility that biochemical and immunofluorescence approaches can reveal different aspects of lipid raft involvement. It is possible, for instance, that TCR signals induce a reorganization of components, perhaps in raft microaggregates, which may be below the level of detection using conventional immunofluorescent microscopy. It is also important to consider the influence of different cell preparation protocols on results. Because even small numbers of APCs can provide costimulatory signals, it may be particularly important to purify mature T cell subpopulations away from APC when examining the effect of TCR signals, alone. In addition, interpretation of data from unseparated thymocyte populations can be confounded by the robust activity of mature SP thymocytes, which represent at least 15% of an unseparated preparation. Nonetheless, the ability to detect important yet subtle changes in lipid raft components is an advantage of biochemical approaches, while the ability to examine gross changes in raft behavior at the single cell level is an advantage offered by immunofluorescence. Our immunofluorescence results demonstrate a clear difference in the extent to which TCR costimulation induces polarization of lipid raft components in mature SP T cells vs immature DP thymocytes.

Collectively, our findings raise the possibility that differences in the consequences of TCR-mediated costimulation in mature and immature T cells may be related to developmental differences in the ability to recruit lipid rafts in response to TCR signals. We speculate that without the ability to fully polarize rafts, CD4⁺CD8⁺ thymocytes may be denied access to specific signaling routes that confer survival advantages. Consequently, TCR-mediated costimulation may induce apoptotic signals that run unopposed in immature CD4⁺CD8⁺ thymocytes, but not in mature T cells.

	Acknowledgments

 
We are grateful to Dr. Karl Johnson for his expertise and advice with immunofluorescence. We thank Selin Somersan and Sam Floyd for helping us initiate these studies, and Jeff Bolcavage, Mariam Rahman, Sarah Hanck, Shawn Alexander, Simone Nish, and Katie Brinner for facilitating the experiments. Finally, we thank Drs. David Allman, David Izon, Terri Laufer, Bob Loudon, and Judy Owen for critically reviewing the manuscript.

	Footnotes

 
¹ This work was supported by National Science Foundation Grant NSF-RUI MCB 9728332. Support for P.J.R.E. was also provided by a grant to Haverford College from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program.

² Address correspondence and reprint requests to Dr. Jennifer Punt, Department of Biology, Haverford College, Haverford, PA 19041.

³ Abbreviations used in this paper: SP, single positive; DP, double positive; FSC, forward scatter; LAT, linker for activation of T cells; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PIP₂, phosphatidylinositol-4,5-bisphosphate; PIP₃, phosphatidylinositol-3,4,5-phosphate; SSC, side scatter.

Received for publication May 10, 2000. Accepted for publication August 22, 2000.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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