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The Journal of Immunology, 1999, 162: 1237-1245.
Copyright © 1999 by The American Association of Immunologists

Antigen-Induced Coreceptor Down-Regulation on Thymocytes Is Not a Result of Apoptosis1

Maureen A. McGargill and Kristin A. Hogquist2

Department of Lab Medicine and Pathology and the Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The various stages of T cell development are typically characterized by the expression level of the two coreceptors, CD4 and CD8. During the CD4+CD8+ (double-positive, DP) stage of development, thymocytes that perceive a low avidity signal through the TCR go on to differentiate (positive selection), and ultimately down-regulate one coreceptor to express either CD4 or CD8. Alternatively, thymocytes that perceive a high avidity signal down-regulate both coreceptors and are induced to die via apoptosis (negative selection). However, it has recently been suggested that positively selected thymocytes may also partially down-regulate both coreceptors before up-regulating the one coreceptor that is ultimately expressed. This would imply that coreceptor down-regulation (dulling) is not a consequence of commitment to the death pathway. To explore this possibility, we have utilized an in vitro assay to demonstrate that dulling occurred in response to both positive and negative selecting ligands in vitro, was not a result of nonspecific membrane perturbation, was not dependent on the type of APC, and occurred before death in vitro. Furthermore, when thymocyte apoptosis was blocked, CD4 and CD8 were down-regulated in response to TCR stimulation. These data suggest that dulling in response to TCR ligation is distinct from death, and support a model in which DP dulling occurs during both positive and negative selection. The biological implications of this phenomenon are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The processes of positive and negative selection in the thymus ensure that a clonally diverse, yet specific repertoire of mature T cells is generated (1). These events occur during specific developmental stages that are typically characterized by the expression levels of the TCR, and the coreceptor molecules, CD4 and CD8. Before positive and negative selection, thymocytes express both CD8 and CD4, and therefore are referred to as double-positive (DP3) thymocytes. DP thymocytes that do not receive a signal through the TCR will not develop further, and will die by neglect. Alternatively, DP thymocytes that express TCRs that are able to recognize self MHC molecules (potentially useful) receive a signal through the TCR. These cells are positively selected, and ultimately down-regulate one coreceptor to become either a CD4 or CD8 single-positive (SP) mature T cell. However, if this signal is too strong, those thymocytes may become self destructive, and are therefore induced to die via apoptosis (2). These thymocytes down-regulate both CD4 and CD8 before completing apoptosis, giving rise to a DP dull intermediate phenotype.

This DP dull phenotype is not obvious in vivo because apoptotic cells are efficiently engulfed by phagocytic cells present in the thymus (3). However, when the amount of apoptosis was vastly increased by either breeding TCR transgenic mice to negative selecting backgrounds (4, 5, 6), injecting TCR transgenic mice with the antigenic peptide (7), or injecting nontransgenic mice with Abs to CD3 (5), the phagocytic cells were overwhelmed, and an apoptotic population accumulated that was DP dull. Additionally, TCR transgenic thymocytes cultured in vitro with APC and the negative selection ligand dramatically down-regulated both CD4 and CD8 (6, 8, 9). Furthermore, if thymocytes were cultured overnight in media alone, a significant percentage became DP dull, and separation of these DP dull cells from the DP bright cells indicated that only the DP dull subset contained apoptotic cells (8, 10). Because of this data, CD4 and CD8 down-regulation has commonly been used as an assay to indicate negative selection.

However, recent evidence suggests that during positive selection, DP thymocytes also partially down-regulate both coreceptors, and transit through a DP dull stage before expressing only CD4 or CD8. This evidence includes the fact that the small percentage of DP dull cells that are present in vivo do not have the same forward scatter/side scatter profile as apoptotic cells (11). In fact, these cells express high levels of CD69 and TCR (12), a phenotype that correlates with positive selection. Analysis of the production kinetics of the various thymic subsets after sublethal radiation demonstrated that DP dull cells appeared after DP bright cells, but before mature T cells (12). Additionally, pronase stripping assays that identify lineage-committed cells revealed that CD4- and CD8-committed cells were present in the DP dull population (12, 13).

Further evidence that positively selected cells transit through a DP dull stage has been derived from experiments in which the generation of DP dull cells by anti-TCR mAb stimulation was accompanied by events that also occur during positive selection (14, 15, 16). These events include increased expression of CD5 and CD69, increased intracellular levels of Bcl-2, and decreased expression of RAG-1, TdT, and the {alpha}-chain of the pre-TCR. Interestingly, TCR stimulation also decreased the level of CD4 and CD8 mRNA (15, 16). Moreover, CD69+ thymocytes generated by reaggregate cultures also had decreased levels of RAG-1, RAG-2, CD4, and CD8 mRNA (17), which provides further evidence of a correlation between positive selection and down-regulation of CD4 and CD8. However, many of these events also occur during negative selection, and in some systems anti-TCR stimulation induced apoptosis (18, 19, 20). Therefore, these data alone do not exclude the possibility that DP dull cells are in the process of being negatively selected.

The most compelling evidence that positively selected cells down-regulate both CD4 and CD8 is that DP dull cells transferred intrathymically do not necessarily undergo apoptosis. In fact, anti-TCR-generated DP dull cells produced mature SP thymocytes (14). Likewise, DP dull cells from transgenic mice that overexpress Bcl-2 generated mature SP T cells upon transfer (21, 22).

These data suggest that the DP dull phenotype may represent an intermediate stage of positive selection, as well as negative selection. Thus, we tested whether CD4 and CD8 down-regulation (dulling) induced by peptide/MHC ligands was an incidental result of cells going through apoptosis. We modified an in vitro dulling assay utilizing the OT-I TCR transgenic system to demonstrate that DP dulling in response to peptide/MHC is not simply a consequence of apoptosis, rather it is a biochemically distinct event, and may reflect an intermediate stage of positive selection.


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

C57BL/6 (B6) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). TAPo is a (129 x C57BL/6)F2 strain with a targeted disruption of the TAP-1 gene on both chromosomes (gift of Anton Berns, Netherlands Cancer Institute, Amsterdam, The Netherlands). OT-I is a C57BL/6 TCR transgenic strain, expressing a receptor that is specific for the OVA257–264 peptide (OVAp) in the context of the H-2Kb MHC class I molecule (23). CD8tg is a C57BL/6 strain overexpressing CD8ß and CD8{alpha} under the control of the CD2 promoter (24).

Cells and cell lines

The thymic stromal cell line, 1308.1, was derived from SV40 T Ag transgenic (B6 x SJL)F2 mice, and displays surface Ags similar to medullary cells (25). They were grown in RP10 media (RPMI with 10% FCS). 5A.Kb (a gift of F. Carbone, Monash University, Melbourne, Australia) is a transformed L cell line that has been transfected with H-2Kb (26). TAPo FB (a gift of A. W. Goldrath and M. J. Bevan, University of Washington, Seattle, WA) is a fibroblast cell line derived from TAPo embryos that was transfected with SV40.

Peritoneal exudate cells were extracted from B6, TAPo, or bm8 mice that were injected 5 days prior with thioglycolate. Macrophages were enriched by adherence to 96-well flat-bottom microtiter plates for 1 h at 37°C, and washed to remove any nonadherent cells.

B cells were enriched from spleen cells, as described (27). In brief, 100 µg/ml goat anti-mouse IgG (Sigma, St. Louis, MO) was adhered to tissue culture-treated plates. Spleen cells isolated from TAPo or B6 mice were cultured on the coated plates for 30 min at room temperature, washed four times with PBS, incubated for 1 h in RP10 at 37°C, and removed from the plate. The suspensions contained greater than 75% B cells.

DP dulling assay

The DP dulling assay was performed as described (28) with a few modifications. APC (3 x 105/well) were adhered to 96-well flat-bottom microtiter plates for 1 h at 37°C.

The monolayers were labeled with 500 nM CellTracker Green CMFDA (Molecular Probes, Eugene, OR) in serum-free media (RPMI) for 15 min, washed twice, and incubated at 37°C for an additional 30 min in RP10, and then washed two more times. B cells were labeled and washed in suspension according to the same procedure. Thymocytes were isolated from OT-I, TAPo or OT-I, TAPo, CD8tg mice and cocultured at 5 x 105/well with the labeled APC, with or without 10–100 nM OVAp (SIINFEKL). For anti-Fas Ab treatment, 20 µg/ml of soluble anti-Fas (Jo2; PharMingen, San Diego, CA) was added to suspensions of thymocytes with no APC. In dexamethasone-treated cultures, 1 µM of dexamethasone was added to suspensions of thymocytes with no APC.

To inhibit apoptosis, the thymocytes were preincubated for 2 h at 37°C with 100 µM of the tripeptide, caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl(o-methyl)-fluoromethylketone (Z-VAD.fmk; Enzyme Systems Products, Dublin, CA), or DMSO as a solvent control. The thymocytes and inhibitor were then added to the labeled APC, in the presence or absence of OVAp. Likewise, to block new protein synthesis, thymocytes were preincubated for 2 h, at 37°C with 100 µg/ml cycloheximide (CHX; Sigma), or equal amounts of ethanol as a solvent control, and cocultured with the labeled APC, in the presence or absence of OVAp.

After 16–20 h at 37°C, the cells were harvested, stained with mAbs, and analyzed by flow cytometry using a FACScaliber (Becton Dickinson, San Jose, CA). The APC were gated out of the analysis by CellTracker exclusion. The mAbs that were used were specific for CD4 (L3T4, RM4-5), CD69 (H1.2F3), CD5 (Ly-1), CD25 (7D4), CD2 (RM2-5) (PharMingen), endogenous CD8 (2.43; American Type Culture Collection, Manassas, VA), or transgenic CD8 (1116, a gift from M. J. Bevan).

The amount of dulling reflects the loss of thymocytes from the CD4+CD8+ DP bright gate. Specific dulling activity is the inverse of the loss of thymocytes from the DP bright gate, and was calculated using the equation (1 - (percentage of DP bright cells in experimental cultures/percentage of DP bright cells in control cultures)) x 100.

Fetal thymic organ culture (FTOC)

Fetal thymic lobes were taken on day E16 and cultured for 2 days. On day 2, either 20 µM P815(HIYEFPQL), 20 µM V-OVA(RGYNYEKL), or 20 nM OVAp was added to the cultures. The lobes were harvested after 18 h and analyzed by flow cytometry with CD8 APC, CD4 PerCP, and B20-FITC Abs. The dead cells were gated out of this analysis via Annexin V staining (see below).

Apoptosis analysis

Thymocytes were harvested and stained with mAbs for 30 min, washed twice, and then stained with 100 µg/ml 7-amino actinomycin D (7AAD; Sigma) for 20 min, and analyzed by flow cytometry. The 7AAD dye stains dead cells brightly, the cells in the process of apoptosis partially, and is excluded from viable cells (29). Because apoptotic thymocytes can be engulfed by APC, we compared the number of live thymocytes remaining after culture. We used the cytometer to count the number of remaining thymocytes by standardizing the volume of each sample analyzed. This was accomplished by adding 40,000 latex beads (5 µM; Interfacial Dynamics, Portland, OR) to each sample before collection, and setting the cytometer to collect 10,000 beads. Therefore, a quarter of each sample was collected, and the amount of specific death was calculated by comparing the number of thymocytes remaining after culture with TAPo APC alone, with the number remaining in the presence of OVAp. The following equation was used: (1 - (number of thymocytes remaining in experimental cultures/the number of thymocytes remaining in control cultures)) x 100.

Phycoerythrin-conjugated Annexin V (PharMingen) was used to confirm apoptosis by detecting aberrant phosphatidylserine in the outer leaflet of the plasma membrane. After culture, thymocytes were harvested, washed in PBS, and resuspended in binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2). The thymocytes were incubated with 5 µl of Annexin V-phycoerythrin and 100 µg/ml 7AAD for 15 min at room temperature, and then analyzed by flow cytometry.

DiOC6 (3) (Molecular Probes) was used to detect mitochondrial membrane permeability transition (PT). After culture, thymocytes were harvested and resuspended in PBS, washed, incubated in 20 nM DiOC6 (3) for 15 min at 37°C, washed twice, incubated at 37°C for an additional 30 min, and then analyzed by flow cytometry. As a positive control, PT was induced by preincubation with the protonophore, carbonyl cyanide m-chlorophenylhydrazone (mCICCP; 50 µM; Sigma) for 15 min at 37°C.

Reverse-transcriptase PCR

A total of 1.5 x 106 thymocytes was incubated in a dulling assay with 20 µM p815, 20 nM OVA, or 20 µM V-OVA, and harvested after 18 h. The RNA was isolated from these cells with a Micro RNA Isolation kit (Stratagene, La Jolla, CA), reverse transcribed with oligo(dT) primers (Superscript; Life Technologies, Gaithersburg, MD), and amplified by PCR at 60°C annealing temperature for 27–33 cycles with CD8ß and HPRT primers (17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dulling occurs in response to positive and negative selection ligands in vitro

To investigate CD4 and CD8 down-regulation, we modified an in vitro assay that has historically been used to mimic negative selection (6, 8, 28, 30). This approach builds on the observation that during development, thymocytes are induced to die when exposed to the antigenic peptide. Thus, to mimic negative selection, thymocytes were cultured with adherent APC in the presence of the antigenic peptide, and then the amount of death (via 7AAD uptake) and CD4/CD8 down-regulation (dulling) was determined using flow cytometry. The OT-I TCR transgenic system allowed us to extend this assay to also include positive selection ligands. OT-I thymocytes express a transgenic TCR that is specific for OVAp in the context of the H-2Kb MHC class I molecule (23). Thus, in vivo, OT-I thymocytes are positively selected in the presence of Kb and self peptides, and negatively selected in the presence of Kb and OVAp. Therefore, in vitro, the positive selection ligand was mimicked by B6 APC that express Kb and endogenous self peptides, and the negative selection ligand was supplied by APC that express Kb along with exogenously added OVAp. APC from TAPo mice provided a control for any nonspecific activity that may occur as a result of overnight culture or interactions with cell surface ligands other than class I MHC. TAPo mice do not stably express Kb on the cell surface (31), and therefore cannot stimulate OT-I T cells. However, Kb expression can be induced with the addition of exogenous peptide. Thus, by adding OVAp to TAPo APC, we can directly study the effect of the peptide/MHC by comparing the amount of activity in thymocytes cultured with TAPo APC and no peptide, with the activity in those cultured with TAPo APC and OVAp.

In addition, to study only the relevant preselection DP thymocytes, OT-I mice were bred to TAPo mice. Positive and negative selection of OT-I thymocytes does not occur in these mice due to the lack of the appropriate TCR ligand, and therefore thymocytes accumulate at the DP stage (data not shown).

We used this assay to investigate whether dulling was solely the result of negative selection, or if it also occurred in response to a naturally occurring positive selection ligand. B6 APC were used as a source of the OT-I TCR ligand for positive selection (Kb molecules bearing self peptides), and TAPo APC + the antigenic peptide, OVAp, were used as a source of the OT-I TCR ligand for negative selection. OT-I, TAPo thymocytes were cultured in vitro, with or without APC that had been previously labeled with a fluorescent CellTracker dye. After 18 h of culture, the thymocytes were harvested and stained with Abs to CD4 and CD8. The APC were excluded from this analysis via CellTracker exclusion. The percentage of DP bright thymocytes remaining in response to each ligand was determined (Fig. 1Go). Interestingly, both positive (B6 APC) and negative (TAPo APC + OVAp) selection ligands induced significant dulling, while the controls did not. The positive selection ligand induced less dulling than the negative selection ligand, which is consistent with the notion that low affinity ligands mediate positive selection, while higher affinity ligands mediate negative selection (32). Additionally, the level of dulling seen in OT-I, TAPo thymocytes exposed to B6 APC in vitro is similar to the level of dulling seen in vivo on the small percentage of CD69+ DP thymocytes that were presumed to be precursors of mature T cells (12). As additional negative controls, neither TAPo APC pulsed with irrelevant peptides, nor Kbm8 APC, which have normal levels of MHC molecules, but do not positively select OT-I thymocytes in vivo (33), induced dulling ((28) and data not shown). Therefore, the dulling seen in this study only occurred in response to specific TCR interactions, including the positive selection ligand. It should be noted that some dulling was seen even in cultures with no APC, the specificity of which is addressed later.



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  		FIGURE 1. Both positive and negative selection ligands induce dulling in vitro. A, OT-I, TAPo thymocytes were cultured for 18 h with no APC, TAPo macrophages, B6 macrophages, or TAPo macrophages with 80 nM OVAp. The plots represent total thymocytes with a forward scatter/side scatter gate to eliminate debris. The number indicates the percentage of DP bright thymocytes that remained after culture. The data are representative of at least 10 separate experiments. B, Fetal thymic lobes were taken on day E16 and cultured for 2 days. On day 2, the indicated peptide was added to the cultures, and the lobes were harvested 18 h later. The dead cells were gated out of the FACS analysis via Annexin V staining, and the percentage of DP bright cells (number in the lower left corner) was determined for the remaining live cells.

 
To confirm that this dulling also occurs in the intact thymic environment, we analyzed the level of CD4 and CD8 on thymocytes during positive and negative selection in FTOC. As in the OT-I, TAPo mouse, positive or negative selection of CD8 T cells does not occur in OT-I, TAPo FTOC. However, if a peptide with a low affinity for the OT-I TCR, such as V-OVA, is added to the culture, CD8 T cells will be synchronously positively selected. Conversely, if a peptide with a high affinity is added, OT-I T cells will be deleted (23). Therefore, to analyze dulling in the thymic environment, thymic lobes were taken from day 16 OT-I, TAPo embryos, and cultured in vitro for 2 days. On day 2, either 20 µM P815 (a peptide that binds to Kb, but does not stimulate the OT-I TCR), 20 µM V-OVA, or 20 nM OVAp was added to the cultures. The lobes were harvested the following day, and analyzed by flow cytometry. For this analysis of CD4 and CD8 down-regulation, the dead cells were eliminated via Annexin V staining. As shown in Fig. 1GoA, both V-OVA and OVA induced dulling in FTOC, while P815 did not. Again, the positive selection ligand induced less dulling than the negative selection ligand. This indicated that dulling occurred in response to the naturally occurring positive selection ligands in vitro (B6 APC), and a positive selection ligand in the intact thymus (V-OVA in FTOC). Additionally, Barnden et al. showed that OT-I, B6 adult mice exhibit considerable DP dulling, while OT-I, bm8 mice do not (33). Together this suggests that dulling occurs in response to both positive and negative selection ligands.

Dulling is not dependent on the type of APC

The role of costimulators on APC in positive and negative selection is not well characterized, and it is possible that dulling reflects the interaction of a particular costimulator. Thus, to investigate whether dulling was dependent on differentially expressed costimulators, we tested CD4 and CD8 down-regulation in response to a variety of APC. Macrophages were purified from the peritoneal exudate of TAPo mice. B cells were enriched from the spleen of TAPo mice by panning with goat anti-mouse IgG-coated plates. The source of thymic epithelial cells was a transformed cell line, 1308.1, which expresses H-2Kb and markers of medullary epithelial cells (25). The fibroblasts, 5A.Kb cells, were derived from an L cell line that had been transfected with Kb (26). All of these APC were labeled with CellTracker dye, and cultured with OT-I, TAPo thymocytes, in the presence or absence of OVAp, for 18 h. Table IGo lists the percentage of OT-I, TAPo DP bright thymocytes that remained after culture with the various APC. Each type of APC was able to induce a significant decrease in the amount of DP bright thymocytes, specifically in response to OVAp. Additionally, B6 B cells and macrophages were able to induce dulling to a greater extent than their TAPo controls (Fig. 1Go and data not shown).


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  		Table I. Dulling is induced by a variety of cell types1

 
Dulling is not a result of nonspecific membrane perturbation

While these experiments revealed that positive selection ligands can induce dulling, they do not rule out that the cells were dull because a limited amount of apoptosis was induced in vitro. Therefore, we tested whether dulling in response to Ag was a nonspecific event that may occur with all cell surface ligands as a result of apoptosis. OT-I, TAPo thymocytes were cultured with TAPo fibroblasts, with and without OVAp. After 18 h, the thymocytes were harvested and analyzed for the expression of a variety of cell surface ligands (Fig. 2Go). The shaded histograms show the expression level of several cell surface proteins on live thymocytes in the control cultures (TAPo APC), and the solid line represents the level on total thymocytes in response to OVAp. As seen previously, OVAp induced down-regulation of both CD4 and CD8. However, the level of other molecules such as CD69, CD5, CD2, and CD25 actually increased, while the level of {alpha}6 and ß1 integrins did not change (data not shown). Additionally, the amount of surface TCR was also down-regulated during this time course (data not shown). This is similar to TCR internalization seen with T cell clones in response to TCR stimulation (34). This demonstrates that CD4 and CD8 dulling are not the result of a generalized internalization of proteins from the cell surface that might occur as a result of membrane perturbation during apoptosis.



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  		FIGURE 2. Dulling is not a result of nonspecific membrane perturbation. OT-I, TAPo thymocytes were cultured with TAPo fibroblasts, with and without 10 nM OVAp for 18 h, and analyzed by flow cytometry for cell surface expression of the indicated molecules. The shaded area represents the expression level on the remaining live thymocytes cultured with TAPo fibroblasts alone. The solid line represents the total remaining thymocytes after culture with TAPo fibroblasts and 10 nM OVAp. The data are representative of four separate experiments.

 
The kinetics of thymocyte response to Ag

If CD4 and CD8 down-regulation are a consequence of apoptosis, we would expect them to become detectable only after the cells are committed to the apoptotic pathway. Therefore, we looked at the kinetics of dulling and various apoptotic events that occur in response to OVAp. OT-I, TAPo thymocytes were cultured with TAPo APC, in the presence or absence of OVAp, and harvested at various time points. CD4 and CD8 dulling, CD69 up-regulation, mitochondrial membrane PT (an early event thought to be central in initiating apoptosis (35, 36)), and altered plasma membrane permeability (indicative of cells late in apoptosis) were measured (Fig. 3Go). After 3 h in culture with OVAp, the thymocytes had reached maximal CD69 up-regulation, and had just begun to initiate dulling and PT. At 6 h, there was an increase in both the percentage of thymocytes that had down-regulated CD4 and CD8, and the percentage of cells that had undergone PT. However, there was not an increase in the number of cells that had an altered plasma membrane permeability. After 12 h, the amount of dulling was maximal, and there was a further increase in the number of PT+ cells. On the other hand, there was only a small increase in the percentage of 7AAD+ cells, which did not reach maximal levels until 24 h (data not shown). Note that at 12 h not all of the DP dull cells have incurred PT. Thus, dulling occurred concurrent with, or before the earliest detectable apoptotic events.



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  		FIGURE 3. Dulling in response to Ag occurs before altered plasma membrane permeability. OT-I, TAPo thymocytes were cultured with TAPo fibroblasts, with and without 10 nM OVAp for the indicated time points. The thymocytes were harvested and analyzed by flow cytometry for CD4, CD8, and CD69 expression. Separate fractions were stained with DiOC6 (3) to measure mitochondrial PT, or 7AAD to measure altered plasma membrane (PM) permeability. The percentage of cells positive for CD69 expression, 7AAD uptake, and DiOC6 (3) uptake is plotted. The percentage of cells positive for dulling is the inverse of the amount of live DP remaining after culture (see Materials and Methods). In all cases, the amount of activity that occurs on freshly isolated thymocytes is subtracted out. These data are representative of two separate experiments.

 
Dulling occurs in the presence of apoptosis inhibitors

Caspases, a family of cysteine proteases, are important mediators of cell death induced by a variety of stimuli, and are required for apoptosis during negative selection (37, 38). If Ag-induced dulling occurs only as a consequence of apoptosis, then caspases would also be required for dulling. Thus, we next asked whether caspase activation was required for DP dulling. We took advantage of a caspase inhibitor, Z-VAD.fmk, which inhibits thymocyte apoptosis induced by a variety of stimuli (37, 38). OT-I, TAPo thymocytes were preincubated with various concentrations of Z-VAD.fmk or DMSO (as a solvent control), and then cultured with TAPo APC, with and without OVAp. After 18 h of culture, the thymocytes were harvested and analyzed for the amount of death and dulling (Fig. 4Go). The amount of specific death and dulling was calculated by subtracting the amount of activity that occurred when thymocytes were cultured with TAPo APC alone. Thus, any activity above 0% is Ag specific. As seen in Fig. 4GoA, increasing concentrations of Z-VAD.fmk completely blocked Ag-specific death (as measured by 7AAD). Z-VAD.fmk also inhibited the nonspecific death, and therefore generates negative values in Fig. 4GoA. However, even at the highest concentrations of inhibitor, significant dulling was seen in response to OVAp (Fig. 4GoB). The absence of apoptosis under these conditions was confirmed by analysis of mitochondrial PT, as measured by DiOC6 (3) (data not shown). Thus, even in the presence of apoptosis inhibitors, CD4 and CD8 are significantly down-regulated in response to Ag, and therefore dulling is not simply a downstream consequence of cells going through apoptosis.



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  		FIGURE 4. Dulling occurs in the absence of death. OT-I, TAPo thymocytes were preincubated with various concentrations of Z-VAD.fmk or DMSO (a solvent control) for 2 h and then cultured with TAPo macrophages and 10 nM OVAp for 18 h, and analyzed by flow cytometry for CD4, CD8, and 7AAD uptake. The amount of death (A) or dulling (B) that occurred when thymocytes were cultured with TAPo macrophages alone was subtracted out, so that 0% sp. act. includes any nonspecific death and dulling. Z-VAD.fmk effectively blocks nonspecific death, as well as Ag-specific death, and thus generates negative values in graph A. The data are representative of three separate experiments.

 
Only Ag-induced dulling occurs in live cells

CD4 and CD8 down-regulation occurs in thymocytes in response to many distinct apoptotic stimuli (8, 29). To examine dulling in response to other apoptotic stimuli, OT-I, TAPo thymocytes were cultured overnight with either anti-Fas mAb, dexamethasone, TAPo APC + OVAp, or media alone. Each of these stimuli was able to induce both death and dulling in the DP thymocytes (Fig. 5GoA and data not shown). However, dulling in response to anti-Fas mAb, dexamethasone, or overnight culture was not as pronounced as dulling in response to Ag. Furthermore, exclusive analysis of the remaining live cells revealed that only OVAp could induce dulling in the living cells (Fig. 5GoB).



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  		FIGURE 5. In living cells, dulling occurs only in response to Ag. A, Apoptosis was induced in OT-I, TAPo thymocytes by either overnight culture in media alone, TAPo fibroblasts + 10 nM OVAp, 20 µg/ml anti-Fas mAb, or 1 µM dexamethasone (DEX). After 18 h, the thymocytes were harvested and analyzed by flow cytometry for CD4 and CD8 expression. The numbers indicate the percentage of total DP bright cells that remained after culture. B, In the same cultures, the apoptotic cells were gated out of the analysis via 7AAD staining. The number indicates the percentage of viable cells that are DP bright. C, OT-I, TAPo thymocytes were preincubated with 100 µM Z-VAD.fmk or DMSO for 2 h, and then cultured with the indicated apoptotic stimuli for 18 h. The numbers indicate the percentage of total DP that remained after culture. The data are representative of three separate experiments.

 
To further examine whether there was a difference in Ag-induced dulling and the dulling that occurred in response to the other stimuli, we asked whether the latter also occurred independently of caspases. OT-I TAPo thymocytes were preincubated with Z-VAD.fmk and then cultured with the various stimuli. Z-VAD.fmk blocked apoptosis in response to all stimuli (data not shown). Again, the inhibitor did not block OVAp-induced dulling, but it did block dulling in response to anti-Fas mAb, dexamethasone, and overnight culture (Fig. 5GoC). Thus, coreceptor dulling can occur in thymocytes as a consequence of apoptosis triggered by a variety of stimuli. However, dulling on viable cells is an exclusive consequence of TCR activation, and this suggests that a distinct biochemical mechanism is involved.

Only OVAp-induced dulling was blocked by CHX

To further distinguish Ag-induced dulling from the nonspecific dulling that occurs as a result of other forms of apoptosis, we tested whether the mechanism in each situation was dependent on new protein synthesis. OT-I, TAPo thymocytes were preincubated with CHX, an inhibitor of new protein synthesis, and then cultured with the various apoptotic stimuli (Table IIGo). CHX stimulates a slight down-regulation of CD4 on both the DP and the CD4 SP subset; therefore, only the CD8 mean fluorescence intensity (MFI) was analyzed. As in the previous experiment, OVAp decreased the CD8 MFI on both the live and dead cells, while Fas only induced dulling on the dead cells. Interestingly, CHX inhibited CD8 down-regulation on the live cells stimulated by OVAp (from 117 to 239 with CHX), but it did not block the dulling seen on the dead cells in response to any of the stimuli. This demonstrates that Ag-induced dulling is distinct from dulling in response to anti-Fas or overnight culture in that it requires new protein synthesis, whereas the latter does not.


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  		Table II. Only OVAp-induced dulling can be blocked by cyclohexamide1

 
Dulling may be transcriptionally regulated

To further investigate the mechanism of dulling, we tested whether CD4 and CD8 down-regulation in response to Ag was due to increased internalization or transcriptional regulation. To accomplish this, we took advantage of transgenic mice that express allelic forms of the CD8 {alpha} and ß genes under the control of the CD2 promoter (24). If dulling is due to increased CD8 internalization, we would expect to see down-modulation of both the endogenous and transgenic CD8. Conversely, if dulling is transcriptionally regulated, only the endogenous CD8 should be down-regulated. CD8 transgenic mice were bred to OT-I, TAPo mice and backcrossed to generate homozygosity at the TAP locus. Thymocytes from OT-I, TAPo, CD8 transgenic mice were cultured with TAPo fibroblasts, with and without OVAp, and then analyzed for the level of endogenous and transgenic CD8 using allele-specific Abs (Table IIIGo). OVAp induced down-regulation of endogenous CD8 from an MFI of 313 to 177. However, the level of the transgenic CD8 under the control of the CD2 promoter did not significantly change (159 versus 158). This suggests that Ag-induced dulling is transcriptionally regulated.


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  		Table III. Dulling may be transcriptionally regulated1

 
Because CD2 is slightly up-regulated in response to Ag (Fig. 2Go), it is possible that this up-regulation is compensating for a decrease of the transgenic CD8 expression. Therefore, we directly analyzed the amount of CD8 mRNA in thymocytes after exposure to OVAp. OT-I, TAPo thymocytes were cultured with TAPo APC, and either P815, OVAp, or V-OVA. After 18 h, the thymocytes were harvested and analyzed by reverse-transcriptase PCR with primers specific for CD8, or HPRT as a standard between samples. Fig. 6Go indicates that the level of CD8 mRNA decreased significantly in response to OVAp, and decreased only slightly in response to V-OVA compared with P815. This confirms that CD8 down-regulation is transcriptionally regulated.



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  		FIGURE 6. The level of CD8 mRNA decreases in response to OVAp. OT-I, TAPo thymocytes were cultured with TAPo APC for 18 h in the presence of 20 µM p815, 20 nM OVAp, or 20 µM V-OVA. The thymocytes were harvested and analyzed by reverse-transcriptase PCR with primers specific for CD8ß or HPRT, as a control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The surface level of CD4 and CD8 coreceptors reflects the developmental stage of thymocytes during maturation in the thymus. Before positive and negative selection, thymocytes express both of these coreceptors. During negative selection, thymocytes down-regulate both coreceptors before completing apoptosis. In contrast, positively selected thymocytes complete maturation and permanently down-regulate only one of the coreceptors. However, it has recently been suggested that positively selected thymocytes transit through a stage in which both coreceptors are partially down-regulated before the single coreceptor is ultimately expressed. Therefore, we tested whether CD4 and CD8 down-regulation on DP thymocytes was a consequence of the apoptotic process, and if it occurred in vitro in response to the TCR ligand for positive selection, Kb molecules presenting self peptides.

Using a modified in vitro dulling assay, we demonstrated that CD4 and CD8 down-regulation occurred in response to both positive and negative selection ligands. We previously showed that thymocytes that overexpress a CD8 transgene can undergo dulling in response to such ligands (28), but the data presented in this study demonstrate that this phenomenon can occur in cells with normal levels of CD8. It was not noted in the previous work because the assays were performed at a suboptimal APC to thymocyte ratio. Thus, the data in this study are the first demonstration of the use of an in vitro assay that does not rely on CD8 overexpression to detect positive selection ligands. This suggests that such an assay should be applicable for defining relevant self interactions using any TCR transgenic strain. Many groups have used such an in vitro dulling assay to detect negative selection without reporting an effect with cells that bear the positive selection ligand. In most cases, preselection thymocytes were not directly tested with both APC bearing the positive selection ligand, and those without it (6, 8, 9). Thus, the relatively weak dulling that occurs in response to positive selection ligands might have gone unnoticed in the presence of the often significant dulling that occurs in any overnight culture of thymocytes. Moreover, an optimal thymocyte to APC ratio is critical for detecting self MHC peptide interactions (data not shown).

To further distinguish death and dulling, we demonstrated that dulling in response to Ag was not the result of a nonspecific down-regulation of all cell surface molecules that might occur because apoptotic cells have an altered plasma membrane. In fact, the surface expression of certain molecules actually increased in response to Ag, while the level of other molecules remained constant. Additionally, dulling occurred before, or at the same time, as one of the earliest events of apoptosis (mitochondrial membrane PT), and at least 6 h before apoptosis was detected by an altered plasma membrane permeability.

To definitively show that dulling was not simply a consequence of apoptosis, we blocked the death pathway with a caspase inhibitor, and stimulated the thymocytes with Ag. While the caspase inhibitor effectively blocked apoptosis, it did not block dulling, even at the highest concentration. This demonstrates that coreceptor down-regulation in response to Ag is not a consequence of apoptosis.

Dulling occurs in thymocytes undergoing apoptosis in response to a variety of apoptotic stimuli. In contrast to Ag-induced dulling, dulling in response to these other stimuli only occurred on apoptotic cells, and could be blocked with the caspase inhibitor. Thus, only Ag was able to induce dulling in live thymocytes. Based on this, it would seem that dulling in response to Ag is distinct from the nonspecific dulling that occurs as a result of apoptosis. To support this distinction, we showed that only Ag-induced dulling required new protein synthesis. In addition to the in vitro dulling assay, we also demonstrated that CD4 and CD8 down-regulation occurs during positive and negative selection in intact thymic lobes. Together, these data demonstrate that DP dulling is not simply a consequence of apoptosis, and that it occurs in response to positive selection ligands. Along with previous data, this suggests that DP dull cells may be intermediates of positive selection.

It is not clear how a signal through the TCR initiates coreceptor down-regulation. This signal may increase CD8 and CD4 internalization, decrease translation, or decrease transcription of these genes. Our data suggest that Ag-induced coreceptor down-regulation is transcriptionally regulated. Using TCR transgenic thymocytes that expressed a CD8 transgene under the control of the CD2 promoter, we demonstrated that the endogenous CD8 dulled in response to OVA, while the transgenic CD8 did not. This suggests that the endogenous CD8 promoter is required for Ag-induced down-regulation. However, it is possible that the modest increase in CD2 expression seen after stimulation (Fig. 2Go) was due to increased transcription from the CD2 promoter. Such an increase might mask a posttranslational decrease in the transgenic CD8 protein in response to Ag. Thus, we directly assessed the levels of CD8 mRNA in thymocyte cultures and found that the level of CD8 mRNA decreased specifically in response to OVAp (Fig. 6Go). This is consistent with the work of Merkenschlager et al., which showed that reaggregation of thymocytes with MHC+ cells caused a decrease in the level of CD4 and CD8 mRNA (17). Our data also suggest that if a posttranslational mechanism for decreasing CD8 exists, such as class I-induced endocytosis, it does not contribute significantly to DP dulling.

These data support a developmental model in which DP thymocytes receive a signal through the TCR, and down-regulate transcription of both CD4 and CD8. In turn, this causes a decrease in the surface levels of the coreceptor proteins, and produces the DP dull phenotype. The signal that induces this may be either a positive or negative selection ligand. Negatively selected thymocytes continue this surface down-regulation until apoptosis is complete, and they are engulfed. Positively selected thymocytes, on the other hand, begin up-regulation of the coreceptor that is to be ultimately expressed. This is consistent with a hypothesis in which transcription of the coreceptors during the DP stage utilizes different genetic regulatory elements than at the SP stage. Indeed, recent evidence showed that the cluster III DNase I-hypersensitive region of the CD8 locus imparts expression only in the mature CD8+ T cells, and not in DP cells (39, 40). Presumably, an element within the CD8 locus (presently unidentified) controls expression at the DP stage. Thus, the CD8 dull phenotype may occur when the cells have begun extinction of CD8 expression via one control element, but expression via the other control element has not yet initiated.

The physiologic relevance of DP dulling may be linked to the fact that during the DP stage, thymocytes are more sensitive to low affinity ligands than mature SP thymocytes (41). This is true despite the higher level of TCR on the latter subset. Therefore, a DP thymocyte that has encountered a low avidity ligand may down-regulate both CD4 and CD8 coreceptors to decrease the overall avidity between the selected thymocyte and the presenting cells. This provides a mechanism for positively selected cells to avoid apoptosis until they have had time to differentiate into less sensitive mature thymocytes. Negative selection ligands, on the other hand, are so strong that CD4 and CD8 down-regulation is not sufficient to decrease the overall avidity, and the thymocyte is deleted.

This model is supported by the fact that TCR transgenic thymocytes that would normally be positively selected at the DP stage were negatively selected in mice that overexpressed a CD8 transgene under the control of the CD2 or CD3{delta} promoter (42, 43). Since the transgene is not down-regulated in response to Ag (Table IIIGo), the overall avidity cannot be reduced sufficiently to allow differentiation, and thymocytes that would have normally differentiated are induced to undergo apoptosis. Alternatively, it is possible that DP dulling is an epiphenomenon of having separate transcriptional regulatory mechanisms operating at the immature and mature stages, and serves no biologically relevant role.

In conclusion, our experiments demonstrate that DP dulling is not simply a consequence of apoptosis, but that it is a biochemically distinct event that indicates that the thymocyte has received a signal through the TCR. Along with previous data, this advocates that positively selected cells transit through a DP dull stage before completing maturation.


   Acknowledgments
 
We thank Lisa Rogers for technical assistance, Steve Jameson and our lab colleagues for critical reading of the manuscript, and Ellen Robey for providing the CD8.1 transgenic mice.


   Footnotes
 
1 This work was supported by the National Institutes of Health Grants AI39560 and AI35296. Back

2 Address correspondence and reprint requests to Dr. Kristin A. Hogquist, Department of Laboratory Medicine and Pathology, University of Minnesota, Box 334 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455. E-mail address: Back

3 Abbreviations used in this paper: DP, double positive; 7AAD, 7-amino actinomycin D; CHX, cycloheximide; FTOC, fetal thymic organ culture; HPRT, hypoxanthine phosphoribosyltransferase; MFI, mean fluorescence intensity; PT, permeability transition; RAG, recombinase–activating gene; SP, single positive. Back

Received for publication May 18, 1998. Accepted for publication October 8, 1998.


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