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The Journal of Immunology, 2003, 171: 2825-2831.
Copyright © 2003 by The American Association of Immunologists

Modeling TCR Signaling Complex Formation in Positive Selection 1

Katherine J. Hare2, Judit Pongracz, Eric J. Jenkinson and Graham Anderson

Department of Anatomy, Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell receptor signaling in the thymus can result in positive selection, and hence progressive maturation to the CD4+8- or CD4-8+ stage, or induction of apoptosis by negative selection. Although it is poorly understood how TCR ligation at the CD4+8+ stage can lead to such different cell fates, it is thought that the strength of signal may play a role in determining the outcome of TCR signaling. In this study, we have characterized the formation of an active signaling complex in thymocytes undergoing positive selection as a result of interaction with thymic epithelial cells. Although this signaling complex involves redistribution of cell surface and intracellular molecules, reminiscent of that observed in T cell activation, accumulation of GM1-containing lipid rafts was not observed. However, enforced expression of the costimulatory molecule CD80 on thymic epithelium induced GM1 polarization in thymocytes, and was accompanied by reduced positive selection and increased apoptosis. We suggest that the presence or absence of CD80 costimulation influences the outcome of TCR signaling in CD4+8+ thymocytes through differential lipid raft recruitment, thus determining overall signal strength and influencing developmental cell fate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In addition to its role in T cell activation, TCR signaling plays a crucial role in regulating thymocyte selection at the CD4+8+ stage (1). Thus, high affinity/avidity TCR-MHC interactions lead to negative selection via induction of apoptosis, while low affinity/avidity interactions promote thymocyte survival and maturation (2, 3). This maturation results in the generation of MHC class I-restricted CD4-8+ and MHC class II-restricted CD4+8- cells, which can be activated by foreign peptide/MHC complexes, but are tolerant to self peptide/MHC. How TCR signaling in CD4+8+ thymocytes results in such distinct cellular fates is poorly understood. However, current models emphasize the role of overall signal strength, reflecting the input from costimulatory and accessory molecules as well as the TCR, in determining the developmental outcome of TCR signaling (4, 5).

In mature T cells, TCR signaling is associated with the formation of a multimolecular complex, the immunological synapse, at the T cell/APC interface (6, 7). Association of TCR molecules and other signaling mediators with glycosphingolipid-enriched lipid rafts within these complexes is thought to facilitate the activation of signaling cascades. Thus, raft disruption has been shown to inhibit early signaling events in T cell activation (8). However, the relevance of signaling complex formation to TCR signaling during development is less clear. Recent studies have begun to analyze immunological synapse formation during thymic selection, using either lipid bilayers containing peptide/MHC complexes (9), or in the context of thymocyte responses in a negative selection system (10). However, this issue has not been addressed under conditions known to lead to positive selection, which under physiological circumstances is driven by interactions between thymocytes and cortical epithelium (11).

In contrast to positive selection, negative selection is normally mediated by dendritic cells and/or a subset of medullary epithelium (12, 13, 14). These two cell types differ from positively selecting cortical epithelium in their expression of costimulatory molecules of the B7 family (15). These molecules contribute to immunological synapse formation in mature T cells by generating signals important in the recruitment of lipid rafts, as a result of interactions with ligands such as CD28 (16, 17). Thus, differences in costimulatory molecule expression between positively and negatively selecting stromal cells may be important in determining the composition and duration of signaling complex formation, which may in turn play a crucial role in the developmental outcome of TCR signaling. Such a mechanism would be consistent with the notion that compartmentalization of costimulatory molecules within the thymus (18) is an important factor in controlling the outcome of TCR-driven thymocyte selection.

In this study, we have developed a model to study thymocyte responses as a consequence of interactions with epithelial cells using conjugates generated in vitro. We show that epithelial cell-CD4+8+ thymocyte conjugate formation occurs in a TCR-MHC-dependent manner, initiates a tyrosine kinase-dependent calcium flux, and results in the generation of phenotypically mature CD4+8- and CD4-8+ cells, thus indicating the relevance of this approach to the study of positive selection. By analyzing the distribution of cell surface and intracellular molecules in preselection CD4+8+ thymocytes as a consequence of TCR-mediated interactions with thymic epithelium, we have defined the formation of a multicomponent molecular signaling complex in positive selection. Interestingly, while complex formation in positive selection induction shares some features with immunological synapse formation in T cell activation such as p56lck recruitment (19), strikingly, signaling complex formation in positive selection does not involve accumulation of GM1-containing lipid rafts. In contrast, providing additional costimulatory signals by introduction of CD80 to thymic epithelial cells promotes the recruitment of GM1-marked rafts in CD4+8+ thymocytes. Functionally, this recruitment correlates with a reduction in positive selection and increased thymocyte apoptosis. Thus, our data define for the first time the formation of an active signaling complex during the initiation of positive selection. Moreover, we suggest that the outcome of TCR signaling in thymocytes is determined by the costimulatory signals provided by thymic stromal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c and MHC-deficient mice (Taconic, Germantown, NY) were bred and maintained at the Biomedical Sciences Unit, University of Birmingham. TCR-{alpha}-/- mice were a gift of M. Owen (Imperial Cancer Research Fund, London, U.K.). Adult or neonatal BALB/c or adult MHC-deficient (4–6 wk) and neonatal TCR-{alpha}-/- mice were used as a source of thymocytes. Thymuses from day 15 BALB/c embryos were used as a source of thymic stromal cells by culture for 5–7 days in 2-deoxyguanosine (Sigma-Aldrich, Poole, U.K.). Detection of the vaginal plug was designated day 0 of gestation.

Abs and fluorescence reagents

The following were used for flow cytometry: anti-CD4 PE (GK1.5), anti-CD8 FITC (53-6.7) (both BD PharMingen, San Diego, CA), and anti-human CD80 (MAB140; R&D Systems, Abingdon, U.K.), which was detected using anti-mouse FITC (Caltag, San Francisco, CA). Cells were analyzed using a BD Biosciences (Oxford, U.K.) LSR machine, with forward and side scatter gates set to exclude nonviable cells. Primary Abs for immunofluorescence were: anti-CD3 (KT3; Serotec, Oxford, U.K.), anti-CD45 FITC (I3/2, Sigma-Aldrich), anti-CD4 FITC, anti-CD8 FITC, anti-LAT 3 (linker for activation of T cells) (Upstate Biotechnology, Lake Placid, NY), anti-p56lck (Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated anti-phosphotyrosine (4G10; Upstate Biotechnology), and anti-CD80. The actin cytoskeleton was studied using phalloidin-rhodamine (Molecular Probes, Eugene, OR), and GM1 was detected using FITC-conjugated cholera toxin B (Sigma-Aldrich).

Preparation of thymocytes and thymic epithelium

In some experiments, thymocytes from BALB/c adult or neonatal mice were depleted of CD3+ cells using anti-rat Dynabeads (Dynal, Wirral, U.K.) precoated with rat anti-mouse CD3 (clone KT-3; Serotec), followed by selection of CD8+ cells using anti-CD8-coated Dynabeads. Removal of CD8 beads was conducted using Detachabead (Dynal, Great Neck, NY). Alternatively, as stated in figure legends, thymocytes were obtained from MHC-/- mice, or from TCR-{alpha}-/- mice. Thymic epithelial cells were obtained by disaggregating 2-deoxyguanosine (2-dGuo)-treated 15-day BALB/c thymus lobes, as described (12).

Formation and flow cytometric analysis of thymocyte-epithelial cell conjugates

Thymocytes and thymic epithelial cells were mixed by centrifugation at a ratio of 1:1, and the resultant cell pellet was incubated at 37°C. Analysis of cell pellets immediately following centrifugation was designated time 0. In some experiments, before conjugate formation, thymocytes were labeled with PKH26 (Sigma-Aldrich), according to manufacturers’ instructions, and thymic epithelium was labeled with CFSE (Molecular Probes) at a concentration of 10 nM, for 10 min at 37°C. Conjugate formation was performed, as described above, with cell pellets being resuspended in 200 µl PBS at the indicated time points, with immediate analysis by flow cytometry.

Cytochalasin D treatment of cells

Thymocytes and epithelial cells were labeled with PKH26 and CFSE, respectively, and then treated with 10 µM cytochalasin D (Sigma-Aldrich) for 10 min at 37°C. Cells were then used to study conjugate formation, as described above.

Measurement of changes in cytosolic Ca2+ concentration

Thymocytes were loaded with 5 µM Indo-1 AM (Sigma-Aldrich) at 37°C for 45 min. Thymocytes and epithelial cells were then labeled with PKH26 and CFSE, respectively, and mixed by centrifugation at a ratio of 1:1. Changes in cytosolic Ca2+ levels were monitored in free thymocytes and in thymocytes bound to epithelial cells, by gating on unbound thymocytes and thymocyte-epithelial cell conjugates and plotting the ratio of Ca2+-bound Indo-1 to free Indo-1 against time. Where stated, thymocytes were incubated with 50 µM tyrphostin A9 (Calbiochem, San Diego, CA) for 10 min before mixing with epithelial cells.

Immunofluorescent analysis of thymocyte-epithelial cell conjugates

Thymocyte-Epithelial conjugates were allowed to form over 30-min incubation, and then allowed to adhere to poly(L-lysine)-coated slides (Sigma-Aldrich) at room temperature. Slides were fixed in 3.7% paraformaldehyde, permeabilized in 0.1% saponin, and blocked for 2 h at room temperature or at 4°C overnight in 1% FCS containing 0.1% saponin. Binding of Abs to LAT and p56lck was detected by biotinylated anti-rabbit IgG (Amersham, Bucks, U.K.), followed by streptavidin-FITC (Amersham), while binding of anti-CD3 was revealed using anti-rat FITC (Caltag). Labeling with biotinylated anti-phosphotyrosine was revealed by streptavidin-FITC. For detection of the lipid raft component GM-1, thymocytes were labeled with FITC-conjugated cholera toxin B before incorporation into conjugates with either control or CD80-infected thymic epithelial cells. CD80 expression in conjugates was performed using sequential incubations in mouse anti-human CD80 (R&D Systems) and anti-mouse rhodamine (Chemicon, Temecula, CA). Analysis was performed using a Zeiss (Oberkochen, Germany) Axioplan fluorescence microscope with Digital Scientific (Cambridge, U.K.) camera and software with a total magnification of x1000.

Reaggregate thymus organ cultures

Thymocytes and thymic epithelial cells were mixed together by centrifugation at a ratio of 1:1, and the cell pellet was transferred to the surface of a 0.8-µm filter in organ culture (11, 12). After the specified time period, cultures were teased apart, and recovered thymocytes were analyzed by flow cytometry.

Adenoviral infection of thymic epithelium

Adenoviral supernatant generated from adenoviral vectors containing cDNA encoding either human CD80 or green fluorescence protein (GFP) was obtained from Qbiogene (Carlsbad, CA). Freshly trypsinized 2-dGuo-treated thymuses were used as a source of thymic epithelium. Aliquots (3 x 105) of cells were resuspended in 96-well plates in a volume of 50 µl RPMI with the addition of 0.5 µl of adenoviral supernatant, and then centrifuged for 60 min at 2100 rpm at room temperature. Cells were then recovered and used immediately for the formation of reaggregate cultures. In experiments analyzing the effects of CD80 expression on the kinetics of conjugate formation and lipid raft formation, freshly infected stromal cells were cultured overnight before use to allow expression of the introduced gene.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolated thymocytes and thymic epithelial cells form conjugates in a TCR-MHC-dependent manner

Thymic epithelium promotes positive selection of thymocytes in a TCR-MHC-dependent manner (1). Conjugate formation has been used previously to study membrane-associated molecular redistribution in the interaction of T cells and APC (reviewed in Ref.7). To explore the possibility of using this approach to study immune complex formation in thymocyte selection, we examined conjugate formation between preselection CD4+8+ thymocytes and thymic cortical epithelial cells, the cell type normally responsible for driving positive selection under physiological conditions. To study thymocyte selection on a molecular basis, we sought to recreate thymocyte-epithelial cell interactions in vitro. Thus, equal numbers of fluorescently labeled CD4+8+ thymocytes (PKH26) and thymic epithelial cells (CFSE) were associated by centrifugation, and then conjugate formation was identified by two-color events using flow cytometry. In contrast to cell pellets analyzed immediately following centrifugation (time 0, Fig. 1a), incubation of cell mixtures for 30 min revealed the formation of thymocyte-epithelial cell conjugates appearing as CFSE+PKH26+ events (Fig. 1b), which consisted of a single epithelial cell bound by thymocytes (Fig. 1c). Importantly, immunofluorescence analysis routinely showed that greater than 99% of the thymocytes bound to epithelial cells in conjugates were of a CD4+8+ phenotype (data not shown), ruling out the possibility that thymocytes at other maturational stages were being studied. To investigate the relevance of these thymocyte-epithelial cell interactions to thymic selection events, we assessed the importance of TCR-MHC interactions in conjugate formation, using combinations of MHC-deficient epithelium and wild-type thymocytes or TCR-deficient thymocytes and wild-type epithelial cells. In contrast to conjugate formation between TCR-{alpha}{beta}- and MHC-expressing cells, use of either MHC-deficient thymic epithelium or TCR-{alpha}{beta}-deficient (TCR-{alpha}-/-) thymocytes resulted in abrogation of conjugate formation (Fig. 1d). Thus, conjugate formation between thymocytes and epithelial cells (in this system) is TCR-MHC dependent, demonstrating the potential of this system to study molecular events in TCR-triggered thymocyte selection.



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  		FIGURE 1. CD4+8+ thymocytes form conjugates with thymic epithelial cells in a TCR-MHC-dependent manner. CD4+8+ thymocytes from MHC-/- mice were labeled with PKH26, and thymic epithelial cells with CFSE. Cells were mixed at a ratio of 1:1 and analyzed by flow cytometry immediately (a) or after 30-min incubation at 37°C by flow cytometry (b) or microscopy (c). The proportion of conjugates formed at time points between 0 and 60 min was determined, with PKH26+CFSE+ events representing conjugates (d, diamonds). Alternatively, MHC-/- thymic epithelial cells were mixed with wild-type thymocytes (d, squares), or TCR-{alpha}-/- thymocytes mixed with wild-type thymic epithelium (d, triangles), and conjugate formation between 0 and 60 min was analyzed. Similar data were obtained from three separate experiments.

 
Conjugate formation leads to active signaling in thymocytes

Within lymphocytes, polymerization of the actin cytoskeleton provides the initial cellular polarization necessary for signaling molecule recruitment, and hence intracellular response to the TCR signal (20). Thus, as a functional measure of signaling, we analyzed accumulation of polymerized actin in thymocytes bound to thymic epithelium. First, polymerization of the actin cytoskeleton at the point of epithelial cell contact (Fig. 2a) was detected in thymocytes in response to thymic epithelium. Moreover, disruption of the actin cytoskeleton in either thymocytes or thymic epithelium by prior treatment with cytochalasin D abrogated conjugate formation (Fig. 2b), suggesting that actin redistribution in both cell types is functionally important for stable conjugate formation.



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  		FIGURE 2. Thymocyte-Epithelial cell interactions are dependent upon polymerization of the actin cytoskeleton. CD4+8+ thymocytes from MHC-/- mice were incubated with thymic epithelial cells for 30 min, adhered to slides, and permeabilized to enable analysis of actin cytoskeleton polymerization using phalloidin (a). Analysis of conjugate formation using flow cytometry was used to compare interactions between cytochalasin D-treated thymocytes and untreated thymic epithelial cells (squares), or between untreated thymocytes and cytochalasin D-treated epithelium (triangles), to conjugate formation between untreated thymocytes and untreated epithelial cells (diamonds) (b).

 
In mature T cells, TCR ligation by peptide/MHC complexes triggers a complex signaling cascade leading to the activation of signaling mediators through tyrosine phosphorylation. To further define the signaling response of thymocytes interacting with epithelial cells, we analyzed tyrosine phosphorylation in thymocyte-epithelial cell conjugates. In contrast to unconjugated thymocytes, which showed diffuse staining (data not shown), accumulation of tyrosine phosphorylation at the focus of cell-cell contact was observed in thymocytes interacting with epithelial cells (Fig. 3a, Table I). A change in intracellular calcium levels is also a key downstream indicator of membrane-proximal signaling events, and therefore could be used as an early indicator of thymocyte signaling as a consequence of thymocyte-epithelial cell interactions. Thus, CD4+8+ thymocytes were loaded with Indo-1 before incorporation into conjugates (21). Again, PKH26 (thymocytes) and CFSE (thymic epithelium) labeling was used to detect thymocyte-epithelial cell interactions. As calcium mobilization represents a rapid intracellular response to signal induction, cells were monitored at an early time point following mixing by centrifugation (5 min) (Fig. 3b). As shown in Fig. 3c, gating on unbound thymocytes showed that these cells maintain an unchanging basal level of intracellular calcium in the absence of epithelial cell interactions. In contrast, when calcium levels were measured in thymocytes involved in conjugates, 65% demonstrated a significant and sustained increase in intracellular calcium concentration (Fig. 3d), an early indication of signal transduction. Of note, pretreatment with the tyrosine kinase inhibitor tyrphostin A9 (22) did not affect conjugate formation, but effectively blocked this calcium flux in epithelial-bound thymocytes (Fig. 3f). Thus, calcium elevation as a result of thymocyte-epithelial cell interactions is consistent with a tyrosine kinase-mediated TCR-induced activation of intracellular calcium release.



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  		FIGURE 3. Thymocyte-Epithelial cell interactions increase thymocyte intracellular calcium levels, and induce phosphotyrosine accumulation. CD4+8+ thymocytes from MHC-/- mice were incubated with thymic epithelial cells for 30 min, adhered to slides, permeabilized, and labeled with Abs to phosphorylated tyrosine residues (a). CD4+8+ thymocytes from MHC-/- mice were loaded with Indo-1 and then labeled with PKH26. Thymic epithelial cells were labeled separately with CFSE. Cells were mixed at a ratio of 1:1 and incubated for 5 min, and conjugate formation was analyzed by flow cytometry (b). The cytosolic calcium concentration of free (c) and epithelial cell-bound thymocytes (d) was then analyzed by flow cytometry for 204 s. To see whether the calcium flux was a direct consequence of tyrosine kinase activation, Indo-1/PKH26-loaded thymocytes were incubated with 50 µM tyrphostin A9 before conjugate formation, in which calcium flux was again measured in free (e) and epithelial cell-bound (f) thymocytes.

 

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  		Table I. Polarization of thymocyte cell surface and intracellular signaling molecules

 
Signaling induced in thymocyte-epithelial cell conjugates can lead to positive selection

Although it is clear from the above data that interactions between thymic epithelial cells and thymocytes in conjugates generate a cellular response, in terms of both actin polymerization and intracellular signaling, it was important to clarify the developmental response of thymocytes to this stimulus, because TCR signaling in CD4+8+ cells can lead to either negative selection through the induction of apoptosis or positive selection leading to functional maturation. Thus, conjugates formed between MHC-expressing thymic epithelium and TCR-{alpha}{beta}-expressing thymocytes were purified and reassociated in reaggregate thymic organ cultures, able to support the positive selection process (11, 12). These cultures were harvested after 7 days and analyzed for the generation of mature CD4+8- and CD4-8+ cells. As shown in Fig. 4b, such cultures efficiently support the appearance of single-positive thymocytes derived from CD4+8+ cells associated with epithelial cells in conjugates. Thus, thymocyte-epithelial cell conjugates provide a model in which to study signaling-associated molecular events involved in positive selection driven by thymocyte/epithelial cell interaction.



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  		FIGURE 4. Thymocyte-Epithelial cell conjugates represent positive selection induction. Conjugates between CD4+8+ thymocytes purified from BALB/c mice (a) and wild-type thymic epithelium were purified and placed in reaggregate organ culture. After 7 days, thymocytes were harvested and analyzed for CD4, CD8, and TCR expression by flow cytometry. Shown are the CD4/CD8 profiles for TCR+ cells (b). In the experiment shown, an input of 3 x 105 thymocytes gave a recovery of 6 x 104 thymocytes. Similar data were obtained from three separate experiments.

 
Thymocyte-Epithelial cell interactions induce polarization of key cell surface and intracellular signaling mediators

Numerous reports now indicate that TCR-MHC-mediated activation of mature T cells is associated with the redistribution of the TCR and accessory molecules to form an immunological synapse at the point of T cell contact with APC (7, 23). This signaling complex formation is now known to include accumulation of molecules such as CD45 (24), CD3 (25), and exclusion of CD43 (24). In contrast, little is known about molecular redistribution in thymocytes associated with the delivery of positive selection signals by thymic epithelium. Having established a model to look at individual cell-cell interactions leading to positive selection, we used immunofluorescence labeling to analyze the influence of interactions with thymic epithelial cells on the distribution of cell surface and intracellular molecules in thymocytes in comparison with that seen in signaling complex formation in mature T cell activation. For this purpose, conjugates formed between CD4+8+ thymocytes and thymic epithelial cells after 30 min of incubation, when conjugate formation is optimal (Fig. 1d), were adhered to slides and permeabilized to allow analysis of both cell surface and intracellular molecules. Using fluorescently labeled Abs, we found redistribution of CD3, CD4, CD8, and CD45 to the point of contact with epithelial cells in up to 80% of thymocytes forming conjugates (Fig. 5, a–d; Table I). Thymocytes not involved in interactions with epithelium showed an unbroken ring of staining in the cell membrane (data not shown).



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  		FIGURE 5. Induction of molecular redistribution in thymocytes by thymic epithelium. CD4+8+ thymocytes from MHC-/- mice were incubated with thymic epithelial cells for 30 min, adhered to slides, permeabilized, and labeled with Abs to CD3 (a), CD45 (b), CD4 (c), CD8 (d), p56lck (e), and LAT (f). Polarization to the point of contact with epithelial cells was observed for all molecules examined, while unbound thymocytes did not show evidence of molecular polarization (data not shown).

 
Using this method to study intracellular signaling molecule distribution, we also observed accumulation of the tyrosine kinase p56lck (Fig. 5e) and the adapter protein LAT in thymocytes interacting with thymic epithelial cells (Fig. 5f; Table I), which contrasted with diffuse labeling in unconjugated thymocytes (data not shown). These findings correlate with the evidence of thymocyte signaling activation described above, and further characterize the composition of the signaling structure formed in prepositive selection thymocytes upon interactions with epithelial cells.

Thymocyte-positive selection is not associated with accumulation of GM1-marked lipid rafts

T cell activation involves the localization of cholesterol- and glycosphingolipid-rich membrane microdomains to the site of TCR ligation, forming the now well-studied lipid raft, which is thought to enhance recruitment of key raft-associated signaling molecules (26, 27, 28). Strikingly, using FITC-conjugated cholera toxin B to detect GM1, we found that CD4+8+ thymocytes bound to thymic epithelial cells failed to show accumulation of GM1 at the point of cell-cell contact (Fig. 6a). One possible reason for the lack of GM1 accumulation in thymocytes could be lower levels of expression of this molecule in these cells. To investigate this, we analyzed CD4+8+ thymocytes and mature T cells for levels of expression of GM1. As shown in Fig. 6b, GM1 surface expression in thymocytes and T cells appears to be developmentally regulated, with CD4+8+ cells expressing lower levels than mature peripheral T cells. Thus, the absence of lipid raft accumulation in positive selection initiation could be due to insufficient levels of GM1 expression in CD4+8+ thymocytes, rather than an intrinsic inability to mediate this response. An additional possibility is that the absence of GM1 polarization in thymocytes is due to the lack of appropriate costimulatory molecule expression on thymic epithelium. Costimulation has been shown to play a role in raft accumulation in peripheral T cells (29, 30, 31), although a recent publication indicates that lipid raft accumulation can occur independently of the CD28/B7 system (32). Less is known about requirements for lipid raft accumulation in immature thymocytes, however. Interestingly, Ebert et al. (29) analyzed the responses of thymocytes to Ab-coated beads and reported a lack of GM1 polarization in thymocytes even when CD28 was cross-linked along with TCR and CD4. However, whether such interactions between thymocytes and Ab-coated beads are representative of more physiological cell-cell interaction is unclear. Unlike mature APC, cortical epithelial cells capable of mediating positive selection do not normally express costimulatory ligands CD80 and CD86. Thus, to explore the functional consequences of differences in costimulatory molecule expression in relation to signaling complex formation and lipid raft aggregation, we set out to induce defined modifications in the costimulatory profile of positively selecting thymic epithelium.



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  		FIGURE 6. The initiation of thymocyte-positive selection does not involve accumulation of GM1 lipid rafts. CD4+8+ thymocytes from MHC-/- mice were labeled with cholera toxin FITC, and then incubated with thymic epithelium for 30 min. Cells were adhered to slides for analysis of GM1 polarization in thymocytes (a). CD4+8+ thymocytes and peripheral T cells were labeled with cholera toxin FITC and analyzed by flow cytometry (b).

 
Thymic epithelial cell suspensions from 2-dGuo-treated thymus lobes were infected with adenoviral supernatant encoding cDNA for either human CD80, which has previously been shown to interact with mouse ligands (33), or GFP, which served as a control vector. In the experiment shown, 64% of epithelial cells were found to be GFP+ (Fig. 7a), with 60% of epithelial cells demonstrating cell surface expression of CD80 (Fig. 7b). Moreover, the CD80 molecules introduced into these cells are functionally competent, as adenoviral infection of thymic epithelium with CD80 enables these cells to act as effective APCs in stimulation assays with mature T cells (data not shown). Interestingly, interactions with CD80+ thymic epithelium caused a dramatic accumulation of GM1 in thymocytes at the point of cell contact (Fig. 7c), with a 13-fold increase in the number of thymocytes displaying GM1 polarization compared with thymocytes interacting with control thymic epithelium (Figs. 6a and 7d). Thus, despite their lower levels of GM1 expression, thymocytes are able to accumulate GM1 when appropriate costimulation is available, as would be the case for thymocytes interacting with bone marrow-derived APC expressing CD80/CD86.



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  		FIGURE 7. Enforced expression of CD80 by thymic epithelium promotes lipid raft accumulation and a reduction in thymocyte-positive selection. Thymic epithelial cells from 2-dGuo-treated fetal thymic lobes were infected with CD80 adenoviral supernatant or GFP-only adenoviral supernatant. Infected cells were analyzed by flow cytometry for expression of GFP and CD80 (a and b, respectively). CD4+8+ thymocytes from MHC-/- mice were labeled with cholera toxin FITC, and then incubated with thymic epithelium infected with CD80 adenovirus for 30 min. Cells were adhered to slides, and labeled with anti-CD80, followed by anti-mouse rhodamine. Quantitation of the number of thymocytes showing GM1 polarization in thymocytes bound to CD80+ (c) or CD80- thymic epithelium is shown in Fig. 7d, in which a minimum of 50 conjugates was counted per experiment. Reaggregates were made of 1 x 106 CD4+8+ thymocytes and either GFP-infected epithelial cells or CD80-infected epithelial cells. Thymocytes were harvested after 5 days and analyzed for CD4 and CD8 expression. Average numbers of CD4+8+ (e) and CD4+8- (f) cells were calculated from three separate experiments.

 
To assess the impact of CD80 expression by thymic epithelial cells on positive selection, we compared the generation of CD4+8- cells in reaggregate cultures formed from CD4+8+ thymocytes and either GFP- or CD80-infected thymic epithelium. After 5 days, cultures were harvested and thymocytes were counted and stained for CD4 and CD8 expression. A 2- to 3-fold reduction in the generation of CD4+8- thymocytes was noted in cultures in which thymic epithelial cells expressed CD80, as compared with development in control GFP-infected reaggregates (Fig. 7f). Moreover, this decrease in the generation of CD4+8- cells correlated with a decrease in the number of CD4+8+ precursors in CD80-expressing reaggregate cultures (Fig. 7e). Importantly, the reduction in positive selection observed in CD80-expressing reaggregate cultures was not due to a reduction in the efficiency of thymocyte-epithelial cell interactions, as flow cytometry showed an increase in the frequency of conjugate formation with CD80-infected thymic epithelium when compared with control thymic epithelium (data not shown). Moreover, in cultures harvested at the earlier time point of 2 days, an increased proportion of thymocytes binding annexin V was observed in reaggregates of CD80-expressing thymic epithelium compared with control reaggregate thymic organ culture (data not shown). This correlated with a decrease in overall cell yield at this earlier 2-day time point, consistent with a role for apoptosis in determining CD4+8+ thymocyte numbers.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TCR signaling as a result of interactions between thymocytes and thymic epithelial cells is essential for the induction of positive selection and the development of mature T cells from CD4+8+ cortical thymocytes. In this study, we have recreated the initial cellular interactions involved in thymocyte-positive selection in vitro. The formation of thymocyte-epithelial cell conjugates in this system is dependent upon TCR-mediated recognition, inducing rapid reorganization of the thymocyte actin cytoskeleton, and an increase in levels of intracellular calcium. Importantly, we also show that such interactions lead to thymocyte-positive selection, with CD4+8+ cells within epithelial cell conjugates developing into CD4+8- and CD4-8+ cells in reaggregate culture. Interestingly, evidence shown in this work also supports involvement of the epithelial cell cytoskeleton in positive selection initiation, reminiscent of the requirement for dendritic cell cytoskeletal reorganization in peripheral T cell activation (34). Redistribution of cell surface and intracellular molecules has been shown to be a key feature of mature T cell activation by APC interactions. Thus, recent reports show the polarization of molecules such as CD45 (24), CD3, and CD4 (25) following activation, although the exclusion of CD43 has also been noted (24). Similarly, intracellular signaling mediators such as LAT and protein kinase C-{theta} have also been shown to accumulate at the point of interaction (35, 36). However, little is known about molecular redistribution in thymocytes undergoing selection; thus, our observations that polarization of CD3, CD4, CD8, and CD45, together with key signaling molecules, to the point of thymocyte-epithelial cell contact also takes place during the initial stages of positive selection provide new evidence for similarities between thymocyte selection and T cell activation. This redistribution plays an integral role in the formation of a multimolecular signaling complex at the thymocyte-epithelial cell interface, also involving accumulation of phosphotyrosine. In mature T cells, this synapse formation involves the clustering of a number of molecules in an ordered fashion, to form both a central and peripheral supramolecular activation cluster (6). Whether such segregation occurs during thymocyte/epithelial cell interactions described in this work remains to be determined. Nevertheless, our findings provide direct evidence that thymocyte-epithelial cell interactions leading to positive selection result in the redistribution of cell membrane-associated signaling molecules to the thymocyte-epithelial cell interface in a manner analogous to that seen in mature T cell-APC interactions.

A notable exception to this similarity is that the initiation of positive selection in CD4+8+ thymocytes does not involve accumulation of lipid rafts marked by GM1 labeling. Lipid raft accumulation is thought to be important in enhancing the recruitment of signaling molecules to the TCR complex and is a key feature of TCR signaling in peripheral T cells, although recent studies in human T cells also show that raft recruitment is not required for CD8 activation (37). However, the involvement of costimulatory signals in raft recruitment, delivered as a result of interaction between CD28 and costimulatory ligands of the B7 family expressed on professional APC, is controversial (30, 31, 32). Although we show GM1 levels to be lower on CD4+8+ thymocytes than on peripheral T cells, we conclude that the lack of GM1 lipid raft accumulation during positive selection initiation, either by thymic epithelial cells as shown in this work, or previously by Ab-coated beads (29), is not due to an intrinsic inability of thymocytes to facilitate raft accumulation, because targeting expression of CD80 to thymic epithelial cells does induce lipid raft aggregation in CD4+8+ thymocytes. Interestingly, GM1 accumulation in thymocytes interacting with CD80-expressing thymic epithelium correlates with a reduction in positive selection and increased apoptosis in reaggregate cultures of CD4+8+ thymocytes and CD80-transfected epithelium. We speculate that thymic epithelium engineered to express CD80 converts positive selection of thymocytes to negative selection as a result of enhanced signaling through costimulatory-dependent raft recruitment. In this context, the failure of anti-CD28-coated beads to induce lipid raft accumulation (29) raises the possibility that CD80 may interact with a receptor other than CD28 to promote thymocyte raft aggregation. We are currently investigating the CD80 receptor expressed by CD4+8+ thymocytes, which is involved in the formation of lipid rafts as a result of interaction with CD80-expressing thymic epithelium. Interestingly, Abs to CTLA-4, another receptor for CD80, have been found to inhibit negative selection of thymocytes (38), perhaps implicating CTLA-4 in the experiments performed in this study. In contrast to our observations, some studies have shown that coligation of TCR, CD4, and CD28 can induce apoptosis in the absence of raft accumulation (29, 39). One possible explanation for this difference is that cells bearing very high affinity TCRs may be able to be triggered to undergo apoptosis without the need for signaling enhancement by raft accumulation, while cells with more moderate affinity TCRs may require raft accumulation to generate adequate signaling levels for apoptosis induction. Overall, our observations suggest a possible mechanism whereby the presence or absence of costimulation normally associated with professional APC and a subset of medullary epithelium, and absent from cortical epithelium, can determine the outcome of thymocyte selection through differential lipid raft recruitment, leading to quantitative or qualitative variations in TCR signaling.


   Footnotes
 
1 This work was supported by a Medical Research Council (U.K.) program grant to E.J.J. and G.A. Back

2 Address correspondence and reprint requests to Dr. Katherine Hare, Department of Anatomy, Medical Research Council Centre for Immune Regulation, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. E-mail address: K.J.Hare{at}bham.ac.uk Back

3 Abbreviations used in this paper: LAT, linker for activation of T cells; GFP, green fluorescence protein; 2-dGuo, 2-deoxyguanosine. Back

Received for publication April 4, 2003. Accepted for publication July 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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