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The Journal of Immunology, 2006, 177: 6098-6107.
Copyright © 2006 by The American Association of Immunologists, Inc.

CD28 Ligation Costimulates Cell Death but Not Maturation of Double-Positive Thymocytes due to Defective ERK MAPK Signaling1

Daniel B. Graham*, Michael P. Bell*, Catherine J. Huntoon*, Matthew D. Griffin{dagger}, Xuguang Tai{ddagger}, Alfred Singer{ddagger} and David J. McKean2,*

* Department of Immunology and {dagger} Department of Nephrology, Mayo Clinic College of Medicine, Rochester, MN 55905; and {ddagger} Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The differentiation of double-positive (DP) CD4+CD8+ thymocytes to single-positive CD4+ or CD8+ T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The developmental program of immature thymocytes is regulated by APCs as the thymocytes migrate from the cortex through the medulla of the thymus. During thymocyte differentiation, a series of receptor-initiated differentiation events positively select thymocytes expressing Ag receptors that can potentially bind foreign Ag fragments bound to self-major histocompatibility proteins on APCs (reviewed in Ref. 1). Thymocytes that express self-reactive Ag receptors are eliminated by receptor-initiated apoptosis, and cells that fail to receive TCR-initiated signals die by neglect. T lymphocytes that survive this selection process exit the thymus and migrate to peripheral lymphoid tissues. To differentiate into effector T lymphocytes in peripheral tissues, naive T cells must receive signals resulting from coengagement of TCR and CD28 or other costimulatory receptors. Signals from TCR alone are insufficient to stimulate the maturation of effector T lymphocytes (2). To assure appropriate negative selection of self-reactive double-positive (DP)3 thymocytes, the receptors that regulate the activation of naive T cells also regulate negative selection of DP thymocytes (3, 4, 5, 6, 7, 8, 9). Consistent with this prediction, previous in vitro studies have indicated that the TCR-stimulated differentiation of DP thymocytes to single-positive (SP) thymocytes can be augmented by coengagement of CD28 or a variety of other costimulatory receptors (CD2, CD5, CD24, CD49d, CD81, and thymic shared Ag-1) (5, 7, 8, 9). Similarly, apoptotic responses of TCR-engaged DP thymocytes can be selectively initiated by signals initiated through CD28 receptors (5, 6, 7, 8, 9). The outcome (maturation or apoptosis) of TCR plus CD28 coengagement of DP thymocytes has been reported to be regulated by the intensity of CD28 engagement (10). The in vitro identification of a role for CD28 costimulation of thymic development has been supported by multiple in vivo studies (11, 12, 13, 14, 15). In addition, CD28 costimulatory signals also have been shown to initiate the T regulatory (Treg) cell differentiation program in the thymus (16). Previous studies evaluating the role of costimulatory receptor coengagement in thymocyte development have largely relied on stimulating purified DP thymocytes with anti-receptor Abs bound to a solid phase matrix rather than using APCs expressing biologically relevant ligands (5, 6, 7, 8, 9, 10, 16). This approach permitted selectivity in defining the specific receptor-initiated responses but may not reproduce biologically relevant interactions that occur between ligands on APCs and receptors on DP thymocytes.

This report characterizes receptor-initiated signaling events that are necessary to stimulate death of DP thymocytes by coengaging TCR and CD28 receptors. These analyses comparatively evaluate Ab-initiated responses with those initiated by artificial APCs produced by stably transfecting Chinese hamster ovary (CHO) cells with a TCR agonist (single-chain anti-TCR CD3{epsilon} or Ad covalently linked to the stimulating peptide for DO11.10 transgenic TCR) plus costimulatory receptor ligands CD80 (B7-1), CD86 (B7-2), or CD48. Our results demonstrate that, as a result of a selective defect in CD28-dependent ERK signaling, CD28 ligation does not directly signal differentiation in TCR-activated DP thymocytes to become CD4low, CD8low, and CD69+ thymocytes. However, ligation of CD2 receptors by CD48 in TCR-activated thymocytes does costimulate thymocyte maturation. Consistent with previous studies (5, 6, 7, 8, 9, 10), ligation of CD28 receptors in TCR-activated DP thymocytes signals a death response. Mutation analysis of the cytoplasmic domain demonstrates that CD28-initiated costimulatory signals that enhance cell death responses can be regulated by multiple signaling motifs in the CD28 cytoplasmic domain. Thus, CD28 costimulation in thymocytes initiates distinct responses from those initiated in peripheral T lymphocytes and selectively regulates downstream signaling pathways rather than simply amplifying existing TCR-initiated signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Female C57BL/6 (B6), DO11.10, and CD28–/– mice were purchased from The Jackson Laboratory and maintained in the Mayo Clinic animal facilities. All procedures involving mice were approved by the Institutional Animal Care and Use Committee at the Mayo Clinic.

Cell preparations

CHO cells were obtained from American Type Culture Collection and stably transfected with a single-chain 2C11 Ab (17) ± B7-1 or B7-2 or Adova beta-chain (12) plus Ad{alpha}± B7-1 and sorted by flow cytometry to obtain cells expressing low levels of the TCR agonists and high levels of B7-1 or B7-2. CHO cells were cultured in CHO-S-SFM11 (Invitrogen Life Technologies), 5% FCS, 10 µg/ml adenosine, 10 µg/ml thymidine, 10 µg/ml deoxyadenosine, and 800 µg/ml G-418. CD4+CD8+ DP thymocytes were purified from 4- to 6-wk-old female mice by panning on anti-CD8 (mAb 83-12-5)-coated plates as described previously (5). For flow cytometry assays, the purified thymocytes were cultured for 18 h in a CO2 incubator in culture medium consisting of RPMI 1640 supplemented with 5 x 10–5 M 2-ME, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM nonessential amino acids (Invitrogen Life Technologies), and 10% FCS. Thymocytes (3 x 106/ml) were stimulated in 24-well plates that had been precoated by overnight culture with anti-TCR Ab, washed, and cultured for an additional 5–6 h with anti-CD28 Ab. Alternatively, thymocytes were stimulated with CHO APCs. Stimulation assays were performed as reported previously (10). DO11.10 secondary T cells were prepared by culturing splenocytes in RPMI 1640 for 2 days in 1 µg/ml OVA peptide aa 327–339, followed by culture in rIL-2 (20 U/ml) in RPMI 1640 for one additional day.

Abs and reagents

Anti-CD28 (mAb 37.51), anti-CD2, anti-CD69, anti-CD4, and anti-CD8 were purchased from BD Pharmingen. Anti-TCR (H57-597) was purified from hybridoma culture supernatant with Affigel protein A MAPS II buffer kit (Bio-Rad). Anti-Bim (SC11425) was purchased from Santa Cruz Biotechnology. Rabbit anti-IP90 (calnexin) was described previously (18).

Transfection procedures

DP thymocytes or DO11.10 secondary T cells were transfected as described previously (19). Briefly, cells were mixed with DNA and electroporated in a BTX Electro Square Porator TB20 (BTX). Transfection efficiencies (GFP+) of viable thymocytes after 18 h of in vitro culture ranged from 21 to 42%. For reporter gene analysis, cells were transfected with a firefly luciferase reporter gene and Renilla luciferase DNA. After 18 h, the cultured cells were assayed for luciferase activity using the Dual Luciferase luminometer Assay System (Promega) and a Model LB9501/16 Lumat luminometer (Berthold Systems). Renilla luciferase values were used as internal controls to which expression of the luciferase reporter gene was normalized.

Phenotype analysis and flow cytometry

Cultured cells were incubated with saturating concentrations of FITC- or PE-labeled Abs in staining medium (PBS, 0.2% BSA, and 0.02% NaN3) for 30 min at 40C. Dead cells were identified by staining with propidium iodide (PI). Labeled cells were analyzed using CellQuest software on a FACScan flow cytometer (BD Biosciences). All experimental results presented are representative of the number of independent experiments identified in the figure legends (n).

Immunoprecipitations and Western blot analysis

Thymocytes were incubated in RPMI 1640 and 0.5% BSA for 4 h before stimulating with immobilized Abs or paraformaldehyde-fixed CHO APCs. The thymocytes were lysed in 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 30 mM Na4P2O7, 2 mM Na2VO4, 10 µg/ml leupeptin, 0.036 U/ml aprotinin, 2.5 µM pepstatin A, 40 mM C3H7O6PNa2, and 1% Triton X-100 (pH 7.5). Centrifuged lysates were incubated with Abs bound to protein G agarose under conditions of Ab excess. Immunoprecipitated proteins were analyzed on SDS-PAGE and transferred to Immobilon-P membrane (Millipore). Blots were probed with Abs, and immunoreactive protein was detected using ECL reagents (Amersham Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ab-dependent thymocyte activation responses

A large number of in vitro and in vivo studies have focused on identifying the receptor-initiated signals that regulate positive and negative selection of DP thymocytes (reviewed in Ref. 1). DP thymocytes have been stimulated to undergo maturation to SP thymocytes in an in vitro culture system using anti-receptor Abs that initiate selective receptor-initiated responses (5). In this assay, purified DP thymocytes were cultured with plate-bound anti-TCR ± anti-costimulatory receptor Abs for 18 h. During this stimulation culture, DP thymocytes initiated many of the phenotypic changes observed as DP thymocytes mature in vivo to SP thymocytes. The activated cells rapidly gained expression of CD69 and decreased expression of both CD4 and CD8. If the 18-h-stimulated thymocytes were transferred to nonstimulatory cultures for an additional 24 h, the activated cells matured into CD4+CD8– SP thymocytes (5). The fraction of DP thymocytes that underwent maturation was dependent on the amount of TCR engagement and was synergistically increased by engagement of a variety of costimulatory receptors (5, 10). Importantly, cell death responses were minimally initiated by even high amounts of TCR cross-linking but were costimulated in a concentration-dependent manner by the addition of anti-CD28 Abs to TCR-activated DP thymocytes (7, 10). Fig. 1A shows an example of the maturation response initiated by B6 DP thymocytes cultured for 18 h with anti-TCR ± anti-CD28. The maturation responses from DP to CD4lowCD8low thymocytes are summarized in Fig. 1B, and the resulting cell viability is summarized in Fig. 1C. When B6 DP thymocytes were stimulated with a low concentration of anti-TCR, minimal maturation or cell death was observed. The addition of either low (10 µg/ml) or high (30 µg/ml) amounts of anti-CD28 stimulated increased maturation to CD4lowCD8low cells, and the number of dead cells was greater in cultures containing the higher amount of anti-CD28. As expected, when CD28–/– DP thymocytes were similarly stimulated in the assay, no CD28-mediated costimulation of TCR-initiated maturation or cell death was observed. When the CD28–/– DP thymocytes were transfected with wild-type (WT) CD28, the responding cells exhibited both CD28-dependent maturation and cell death responses that also were observed from B6 thymocytes. Surprisingly, CD28–/– DP thymocytes reconstituted with a truncated CD28 construct that contains no cytoplasmic domain (TR CD28) responded to CD28 coengagement with a maturation response comparable to WT CD28 cells, but no cell death response was observed. These results suggest that the in vitro maturation response resulting from anti-CD28 Ab engagement of TCR-signaled DP thymocytes is not dependent on CD28 signaling, whereas the CD28-dependent cell death response depends on signals from the CD28 cytoplasmic domain.


Figure 1
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  		FIGURE 1. A, Ab coengagement of TCR and CD28 stimulates differentiation and cell death of DP thymocytes. Flow cytometric analysis of CD4 and CD8 expression on viable (PI-gated) DP thymocytes from B6, CD28–/–, and CD28–/– transfected with WT CD28 or TR CD28, stimulated with immobilized anti-TCR (0.5 µg/ml) ± anti-CD28 (10 or 30 µg/ml), and cultured for 18 h in an in vitro stimulation assay (10 ). Numbers in each panel identify the percentage of cells recovered in the gated box (n = 3). B, Summary of the differentiation response of DP thymocytes stimulated with anti-TCR ± anti-CD28. Data were taken from the lower left quadrant of the flow cytometric profiles shown in A. C, Summary of the viability of DP thymocytes after in vitro culture with anti-TCR ± anti-CD28. The percentages of viable thymocytes were calculated by comparing the number of PI-negative cells in each stimulated culture with the number of PI-negative cells in the parallel culture of untreated cells.

 
Ligand-dependent activation responses stimulated by artificial APCs

The CD28 cytoplasmic domain-independent maturation response could occur as a consequence of increased adhesion between the Ab-coated plastic tissue culture well and the DP thymocytes. Rather than directly initiating signals through the cytoplasmic domain, the function of the anti-CD28 Ab could be to increase the level of TCR engagement by the plastic-bound anti-TCR Abs and thereby augmenting TCR-initiated signals. As an alternative approach to evaluate CD28-dependent effects on thymocyte maturation, we produced two different types of artificial APCs (Fig. 2A). A pan-TCR agonist APC was produced by stably transfecting CHO cells with a single-chain anti-CD3{epsilon} Ab that was tagged on its C terminus with GFP. CHO cells expressing low levels of the single-chain 2C11-GFP and a high level of B7-1, B7-2, or CD48 (CD2 ligand) ligand also were produced. The levels of B7 expression on these CHO APCs is similar to the levels of B7 expressed on LPS-activated dendritic cells (data not shown). A second type of APC (Adova) was produced by transfecting CHO cells with WT Ad{alpha} and an Adbeta construct to which the peptide reactive to DO11.10 TCR transgenic T cells was covalently linked to the beta-chain amino terminus (20). Another Adova CHO cell line coexpressing B7-1 also was produced (Fig. 2B).


Figure 2
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  		FIGURE 2. Phenotypic characterization of CHO APCs and analysis of their functional ability to stimulate an IL-2 response from DO11.10 T lymphocytes. A, Flow cytometric analysis of CHO APCs stably transfected with a single-chain anti-CD3{epsilon} (GFP-2C11) ± CD28 ligands B7-1 or B7-2, or the CD2 ligand CD48. B, Flow cytometric analysis of CHO APCs stably transfected with the DO11.10 TCR ligand Ad covalently linked to OVA peptide aa 327–339 (20 ) plus Ad{alpha} (Adova). C, IL-2 reporter gene analysis of DO11.10 secondary T lymphocytes stimulated with immobilized Abs or CHO APCs. DO11.10 T cells were stimulated in vitro with immobilized Abs or with CHO APCs (150,000 or 400,000 cells) expressing single-chain 2C11 ± B7-1 or B7-2, Adova± B7-1 or untransfected CHO cells (CHO) for 18 h and analyzed for luciferase activity (n = 4).

 
To evaluate whether these different CHO cell lines would function as artificial APCs for normal T cells, we used the CHO APCs to stimulate an IL-2 reporter gene response from DO11 secondary T lymphocytes. The IL-2 luciferase response stimulated by 2C11 CHO cells was greatly augmented when the 2C11 stimulator cells also expressed either B7-1 or B7-2 (Fig. 2C). Similarly, the Adova B7-1 CHO cells stimulated an augmented IL-2 luciferase response as compared with the Adova CHO-stimulated response. Together, these results demonstrate that the CHO cells expressing 2C11 ± B7-1 or B7-2 or Adova± B7-1 are potent APCs that stimulate CD28 costimulatory receptor-dependent activation responses from normal T lymphocytes.

Using the in vitro assay conditions shown in Fig. 1, the 2C11 CHO cells stimulated a low level of maturation of DP to CD4lowCD8low thymocytes (Fig. 3A) without stimulating cell death (Fig. 3B). When the CHO APCs expressed 2C11 plus either B7-1 or B7-2, 50–60% of the thymocytes were dead 18 h after stimulation, a level comparable to that stimulated by anti-TCR plus anti-CD28 plate-bound Abs. Importantly, although immobilized anti-TCR plus anti-CD28 stimulated a high level of maturation of DP to CD4lowCD8low thymocytes, the presence of B7-1 or B7-2 on the 2C11 CHO APCs did not augment the maturation response stimulated by 2C11 CHO APCs. Similarly, Adova CHO APCs stimulated the maturation of DP to CD4lowCD8low thymocytes, and Adova B7-1 CHO APCs stimulated a similar maturation response. Although Adova CHO APCs did not stimulate apoptosis, comparable levels of cell death were stimulated by the Adova B7-1 CHO APCs, 2C11 B7-1 APCs, or 2C11 B7-2 APCs. In contrast, CHO APCs that expressed 2C11 plus CD48 stimulated a higher level of maturation of DP to CD4lowCD8low thymocytes than 2C11 CHO cells while minimally stimulating cell death.


Figure 3
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  		FIGURE 3. Coengagement of TCR agonists 2C11 ± B7-1 or B7-2 or Adova± B7-1 stimulates cell death but not differentiation in DO11.10 DP thymocytes. A, Summary of the activated CD4lowCD8low thymocytes stimulated in vitro by plate-bound Abs as compared with CHO APCs. The data were taken from flow cytometric analysis of DO11.10 DP thymocytes stimulated for 18 h as shown in Fig. 1 (n = 3). B, Viable thymocytes after 18 h of in vitro culture with immobilized Abs or CHO APCs. C, The number of DO11.10 CD69+ thymocytes stimulated by 50,000, 150,000, or 400,000 CHO APCs as compared with immobilized Abs (upper left panel) (n = 4). D, CD69 MFI of Bim-positive thymocytes after stimulation by CHO APCs or immobilized Abs. E, Bim MFI stimulated by CHO APCs or immobilized Abs in DO11 CD69+ or CD69– DO11 thymocytes. F, Viability of DO11.10 DP thymocytes stimulated by CHO APCs or immobilized Abs.

 
CD69 and Bim expression rapidly increases (within 3–4 h) in DP thymocytes stimulated by anti-TCR plus anti-CD28 (10, 21). Previous studies have shown both increased CD69 expression and decreased CD4 and CD8 expression are markers of activated DP thymocytes (5, 7, 10), whereas augmented Bim expression is associated with increased apoptosis (21). Fig. 3C shows that Ad CHO APCs stimulated DO11.10 DP thymocytes to express CD69, and the number of CD69+ cells did not increase if the Ad CHOs also expressed B7-1. Although CD28 costimulation does not increase the number of TCR-activated CD69-positive thymocytes, the amount of CD69 expressed per cell (mean fluorescence intensity; MFI) is increased by CD28 costimulation (Fig. 3D). In contrast, the addition of anti-CD28 Abs costimulated an increase in both the number of CD69-positive cells (Fig. 3C) and the amount of CD69 expressed per cell (MFI; Fig. 3D). Although a large fraction (80–90%) of DP thymocytes constitutively express Bim intracellularly (data not shown), the amount of Bim expressed per cell (MFI) was increased in activated (CD69+) but not unactivated (CD69–) DP DO11.10 thymocytes that were stimulated with Adova B7-1 CHO APCs (Fig. 3E). In addition, Bim expression was also increased by costimulating anti-TCR-activated thymocytes with high amounts of anti-CD28 Abs. Fig. 3F shows that DO11.10 thymocytes stimulated in this assay responded with CD28-dependent cell death responses similar to that shown in Figs. 1C and 3B. These results demonstrate that CD28 engagement by its ligand does not increase the number of TCR-activated DP thymocytes (maturation of DP to CD4low, CD8low, or CD69+) but does costimulate increased CD69 expression, Bim expression, and cell death in TCR-activated (CD69+) thymocytes. This indicates that CD28 costimulation does not increase the number of TCR-activated thymocytes but rather selectively alters the outcome of the activation response (increased Bim expression and cell death but not maturation to SP thymocytes).

Ligand-dependent stimulation of intracellular signaling pathways

The failure of CD28 to costimulate maturation of DP thymocytes while activating a cell death response, suggests that CD28 may selectively increase some but not all of the TCR-initiated signaling cascades that regulate thymocyte activation responses. The CD28-dependent signaling pathways that are responsible for costimulatory effects in mature T lymphocytes are incompletely understood. However, synergistic phosphorylation of TCR-proximal signaling molecules has been demonstrated in TCR-activated peripheral T cells costimulated with CD28 agonists (22, 23). To determine whether DP thymocytes exhibit global defects in CD28 costimulatory signaling, we examined whether CHO APCs stimulate the inducible tyrosine phosphorylation responses of Vav1 and phospholipase C (PLC){gamma}1, two TCR-proximal signaling intermediates that regulate a variety of downstream signaling events in T lymphocytes. The Western blot analysis in Fig. 4A shows that CHO APCs that express the Adova stimulated only a weak induction in the phosphorylation of Vav and PLC{gamma}1 in DO11.10 DP thymocytes. In contrast, Adova B7-1 CHO APCs stimulated a high level of phosphorylation of Vav1 and PLC{gamma}1. Thus, CD28 costimulation increases the TCR-inducible phosphorylation of TCR-proximal signaling molecules, and, importantly, these results demonstrate the absence of a global CD28-dependent signaling defect in DP thymocytes.


Figure 4
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  		FIGURE 4. CD28 costimulates TCR-proximal phosphorylation events but not ERK MAPK in DO11.10 DP thymocytes. A, DO11.10 DP thymocytes were stimulated by CHO APCs expressing Adova, Adova plus B7-1, or untransfected CHO cells for the indicated times. Cell lysates were immunoprecipitated with anti-Vav1 or anti-PLC{gamma}1, and Western blots were sequentially immunoblotted with anti-phosphotyrosine (pTyr) and either anti-Vav1 or anti-PLC{gamma}1 (n = 3). B, DO11.10 DP thymocytes were stimulated by CHO APCs or immobilized Abs for the indicated times. Western blots of cell lysates were immunoblotted with anti-pERK and with a loading control, anti-IP90 (n = 3). C, Summary of the densitometry analysis of the Western blot shown in B. D, Summary of an Elk reporter gene analysis of CD28–/– DP thymocytes either transfected with control DNA, WT CD28, or TR CD28, and then stimulated for 18 h with immobilized Abs or CHO APCs (n = 2).

 
Multiple studies have demonstrated the importance of the ERK MAPK pathway for stimulating the maturation of DP thymocytes (24, 25, 26, 27, 28, 29). Because we did not identify a global defect in CD28-dependent signaling responses in DP thymocytes, we tested for the presence of a selective defect in CD28-dependent activation of the ERK MAPK response. The Adova CHO APCs rapidly stimulated the phosphorylation of ERK, and the kinetics (but not the magnitude) of this phosphorylated ERK (pERK) response was only slightly increased when the DP thymocytes were stimulated by Adova B7-1 CHO APCs (Fig. 4, B and C). In contrast, anti-CD28 Abs did costimulate an increase in the pErk response of anti-TCR-stimulated DP thymocytes. These pERK results are consistent with the ability of anti-CD28 costimulation but not CD28 ligation to augment maturation responses in DP thymocytes. To further investigate potential CD28-dependent effects on ERK MAPK, we evaluated CD28 costimulation of the Elk luciferase response in CD28–/– DP thymocytes transfected with WT or truncated CD28 (Fig. 4D). The Elk luciferase response in DP thymocytes is directly regulated by ERK MAPK because the activation of the Elk transcription factor is completely inhibited by the selective MEK inhibitor PD98059 (10). TCR engagement by either anti-TCR Ab or 2C11 CHO APCs stimulated low levels of the Elk luciferase response in CD28–/– DP thymocytes or in CD28–/– thymocytes transfected with either WT CD28 or TR CD28. Anti-CD28 Ab did not costimulate increased levels of the Elk luciferase response in anti-TCR-activated CD28–/– thymocytes, but anti-CD28 stimulated a large increase in Elk luciferase activity in CD28–/– DP thymocytes that expressed WT CD28. Importantly, anti-CD28 Abs costimulated a large increase in Elk luciferase activity in CD28–/– thymocytes expressing truncated CD28. Thus, CD28 Ab-dependent maturation of DP thymocytes (Fig. 1) and the CD28 Ab-dependent Elk luciferase reporter gene response (Fig. 4D) can occur independently of the CD28 cytoplasmic domain. In contrast, no augmented Elk luciferase response was stimulated by CHO APCs expressing 2C11 plus the natural CD28 ligands in DP thymocytes that express either WT CD28 or truncated CD28. Thus, although anti-CD28 and B7-expressing APCs costimulated similar levels of thymocyte death, only anti-CD28 costimulated Elk activation and maturation. These results demonstrate that although CD28 costimulates an increase in phosphorylation of TCR-proximal signaling molecules and costimulates a cell death response, it is ineffective in costimulating an increased ERK MAPK response. In addition, the results also show that Ab engagement of CD28 does not necessarily stimulate the same biological responses that are stimulated by engagement of CD28 by its natural ligands.

Mapping CD28-signaling motifs that signal death responses

Currently, there is no consensus concerning the signaling mechanism that mediates receptor-initiated cell death in activated DP thymocytes. To identify the signaling motifs in the CD28 cytoplasmic domain that are required for signaling CD28-dependent cell death, we constructed CD28 expression vectors containing mutations in four regions of the CD28 cytoplasmic domain (Fig. 5A). Three of these motifs associate with multiple downstream signaling intermediates, and the C-terminal tyrosine is a potential motif for Src homology 2 domain-containing signaling proteins. These different CD28 constructs were transiently expressed in CD28–/– DP thymocytes along with a GFP expression vector, which was used as a surrogate marker to identify transfected thymocytes. Five hours after transfection, the thymocytes expressed levels of cell surface CD28 proteins that were equal to or greater than CD28 levels expressed by normal B6 mice (data not shown). CD28–/– thymocytes that were transfected with the different WT or mutant CD28 constructs expressed cell surface CD28 at comparable levels (data not shown). After the thymocytes were stimulated for 18 h with different 2C11 CHO APCs, the GFP-positive thymocytes were analyzed for cell viability. Cell death was not stimulated in CD28–/– thymocytes or in CD28–/– thymocytes transfected with CD28 lacking the cytoplasmic domain truncated (TR) by any of the different 2C11 CHO APCs (Fig. 5B). As previously shown in Fig. 3B, little or no cell death was observed in any of the CD28–/–-transfected thymocytes stimulated by 2C11 CHO APCs or 2C11 CD48 CHO APCs. In contrast, 40–50% of the B6 control thymocytes or the CD28–/– thymocytes transfected with WT CD28 died after being stimulated by 2C11 B7-1 CHO APCs or 2C11 B7-2 CHO APCs. The CD28–/– thymocytes transfected with CD28 mutants containing alterations in either one or two of the cytoplasmic domain signaling motifs responded with CD28-dependent cell death. However, the 2C11 B7-1 CHO APCs or 2C11 B7-2 CHO APCs did not elicit a cell death response from CD28–/– thymocytes that expressed CD28 containing mutations in all of the four signaling motifs. These results indicate that no single signaling motif in the CD28 cytoplasmic domain is responsible for initiating a cell death response in TCR-signaled DP thymocytes. Thus, there is functional redundancy in the signaling motifs in the CD28 cytoplasmic domain.


Figure 5
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  		FIGURE 5. CD28 mutational analysis demonstrates functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. A, Summary of the mutations introduced into the CD28 cytoplasmic domain expression vectors. Point mutations are identified by the amino acid single-letter code, and a deletion that removes the C-terminal PXXP motif is identified by parenthesis. The TR mutant deleted the cytoplasmic domain except for the membrane-proximal basic residues. B, Results from representative experiments in which B6 control mice or CD28–/– DP thymocytes were transiently transfected with one of the CD28 expression vectors, stimulated for 18 h with CHO APCs, and analyzed by flow cytometry for viable thymocytes (PI–). (Individual constructs were analyzed 2–27 times.)

 
As an alternative approach to using transient transfection assays to map the CD28-signaling motifs required for initiating a cell death response, we assayed DP thymocytes isolated from CD28–/– mice that express CD28 transgenes containing mutations in each of three cytoplasmic domain signaling motifs (Fig. 6A). These transgenic mice were previously used to map the CD28-signaling motifs required to regulate Treg development in the thymus and IL-2 secretion in peripheral T cells (16). Thymocytes from the mice express transgenic CD28 molecules at levels comparable to CD28 expressed by B6 thymocytes. Thymocytes from these transgenic mice were stimulated for 18 h with the different 2C11 CHO APCs, and cell viability was quantitated (Fig. 6B). As expected from previous assays, 2C11 CHO APCs did not stimulate cell death in DP thymocytes from any of the transgenic mice. In contrast, 55–70% of the CD28-expressing thymocytes died after stimulation with either 2C11 B7-1 or 2C11 B7-2 CHO APCs. DP thymocytes that expressed a CD28 transgene containing mutations in any one of the three cytoplasmic domain signaling motifs and thymocytes from WT mice exhibited comparable death responses after they were stimulated by 2C11 B7-1 or 2C11 B7-2 CHO APCs. In contrast, thymocytes that expressed the TL CD28 transgene (containing no cytoplasmic domain) responded with little cell death when stimulated with the 2C11 B7-1 or 2C11 B7-2 CHO APCs. All of the DP thymocytes tested, WT or mutant, responded with ~20% cell death when stimulated with 2C11 plus CD48 CHO APCs. Thus, as shown in Fig. 5, the analyses of transgenic DP thymocytes that express mutant CD28 in any one of the three CD28-signaling motifs do not exhibit defective CD28-dependent apoptotic responses. Only CD28 proteins that exhibited defective CD28-dependent cell death responses contained mutations in all of the cytoplasmic signaling motifs or had no cytoplasmic domain.


Figure 6
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  		FIGURE 6. DP thymocytes isolated from CD28 transgenic mice expressing mutations in the CD28 cytoplasmic domain demonstrate functional redundancy in the CD28-signaling motifs that initiate a thymocyte death response. A, Summary of point mutations or a truncation (TR) in the CD28 cytoplasmic domain of CD28 transgenic mice. B, Results from representative experiments in which DP thymocytes were isolated from B6 control mice or WT or mutant CD28 mice, stimulated for 18 h with CHO APCs, and analyzed by flow cytometry for viable thymocytes (PI–) (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Costimulatory receptors such as CD28 have been shown to synergistically augment differentiation and antiapoptotic activation responses of TCR-stimulated peripheral T lymphocytes (30, 31). Ab-stimulated CD28 also has been reported to affect the activation program of TCR-stimulated DP thymocytes in opposing directions, to augment both positive selection (maturation of DP to SP thymocytes) and negative selection (apoptosis) (3, 4, 5, 6, 7, 8, 9, 10). The mechanistic basis for the differential effects of CD28 costimulation in thymocytes vs mature T cells is not well understood, and the literature is contradictory. Nevertheless, it is clear that CD28 signaling in thymocytes and T cells differs with regard to specificity toward the activation Bcl-2 family members and MAPKs such as ERK (1, 32, 33). In contrast to previous reports, the experiments described in this study demonstrate that ligand-dependent CD28 costimulation regulates thymocyte death in TCR-activated thymocytes but does not contribute to initiating the maturation of DP to CD4low, CD8low, CD69+ thymocytes. The inability of CD28 to costimulate thymocyte maturation does not occur as a result of detectable alterations in costimulation of TCR-proximal signaling events. Although ligation of CD28 synergistically costimulates the inducible phosphorylation of Vav1 and PLC{gamma}1, it does not costimulate the TCR-stimulated ERK MAPK response needed for thymocyte maturation. The CD28-dependent signals that regulate thymocyte cell death can be initiated through different signaling motifs in the CD28 cytoplasmic domain. Unlike CD28-dependent costimulation of IL-2 production or production of Treg cells, which have been reported to be regulated by the CD28 C-terminal PXXP motif (16), cell death can be initiated in CD28 mutants in which any one of the three principle CD28-signaling motifs remains intact. Together, these results indicate that ligand-dependent engagement of CD28 regulates selective responses in the thymocyte activation program, which are distinct from the CD28-dependent responses that regulate the activation program of peripheral T lymphocytes. In addition, the results demonstrate that CD28 costimulation of DP thymocytes activates selective downstream signaling pathways rather than simply amplifying TCR-initiated signals.

These experiments were originally initiated to express mutated CD28 molecules in CD28-deficient DP thymocytes in an attempt to map the signaling motifs responsible for death vs maturation. Surprisingly, a truncated CD28 lacking a cytoplasmic tail was capable of costimulating thymocyte maturation in response to immobilized anti-TCR plus anti-CD28. Although it is possible that CD28 can signal without its cytoplasmic tail, it is more likely that anti-CD28 augments TCR signals by facilitating adhesion between thymocytes and the anti-TCR-coated tissue culture plates. Consistent with our data, enhanced TCR signaling resulting from increasing amounts of anti-TCR coated on plastic will promote thymocyte maturation but not death, because TCR signals are sufficient to induce maturation but not death of DP thymocytes in vitro (7). In addition to maturation responses mediated by truncated CD28, Ab engagement of CD4 or Ly9 containing complete truncations in their cytoplasmic domains also costimulated biological responses associated with thymocyte maturation such as down-regulation of CD4 and CD8, inducible expression of CD69, or activation of Elk (data not shown). Although others have reported different T cell responses costimulated by anti-CD28 and CD28 ligation (34), most studies evaluating CD28 costimulatory effects used anti-CD28. In attempt to study thymocyte costimulation in a more physiologic model, while maintaining the ability to systematically study individual receptors, we produced artificial CHO APCs expressing a TCR ligand with and without specific costimulatory ligands. In this model, adhesion mediated through costimulatory molecules appears to be negligible because B7-expressing APCs did not enhance initiation of DP thymocyte activation (decreased expression of CD4 and CD8; inducible CD69 expression) in TCR-activated DP thymocytes. However, B7-expressing APCs were capable of delivering CD28-dependent signals that costimulated activated thymocytes (CD4low, CD8low, CD69+) to increase expression of CD69 and Bim, which has been shown to be required for negative selection (33, 35), and initiate thymocyte cell death.

The roles of costimulatory molecules during thymocyte development remain obscured by conflicting data and technical obstacles. Studies performed in vitro using immobilized Abs to stimulate the TCR and costimulatory receptors on DP thymocytes suggest that numerous costimulatory receptors have the capacity to costimulate thymocyte maturation, whereas CD28 is unique in that it can costimulate thymocyte maturation at low doses of anti-CD28 and cell death at higher doses (7, 10). This in vitro model of thymocyte development involves the selective engagement of specific receptors using mAbs. However, presumed agonistic Abs may not faithfully reproduce the same biological effects as natural ligands anchored in a dynamic cell membrane. In fact, CD28 superagonist Abs have been shown to activate T cells in the absence of TCR stimulation, whereas B7 stimulation alone cannot (36). Our data suggest that stimulation of the TCR and CD28 with immobilized Abs stimulates DP thymocyte maturation and cell death, whereas artificial APCs expressing a natural TCR ligand as well as B7-1 or B7-2 augment thymocyte death but not maturation. However, ligand-dependent CD2 costimulation regulates maturation but not death of DP thymocytes. Thus, engagement of different costimulatory receptors by their natural ligands can initiate distinct activation responses (maturation and cell death) in TCR-activated DP thymocytes. Positive selection of DP thymocytes has been reported to occur in the thymic cortex in response to TCR signals provided by thymic epithelial cells (4, 37, 38). This initial activation response may not require CD28 because B7-1 and B7-2 are primarily expressed on epithelial cells and bone marrow-derived APCs in the thymic corticomedullary junction and medulla rather than in the cortex (39, 40, 41). As the maturing thymocytes migrate from the cortex to the thymic medulla, they may encounter APCs that express CD28 ligands and possibly undergo negative selection. Thus, the restricted availability of negative-selecting agonists such as B7 in the thymic medulla and the selective CD28-dependent signals that augment cell death but not differentiation both may contribute to the sequential thymic developmental program where positive selection is initiated in the thymic cortex, whereas negative selection occurs primarily in the medulla. Previous studies performed in vivo support our finding that CD28 plays a role in TCR-induced cell death/negative selection, whereas evidence that CD28 is involved in maturation/positive selection is lacking (11, 12, 13, 14, 15). The lack of an obvious defect in the thymic development of CD28–/– mice (42) suggests that there is redundancy in the costimulatory-initiated signals that regulate negative selection.

In pursuit of the mechanisms of CD28-mediated death in TCR-stimulated DP thymocytes, we used a mutagenesis approach to map the signaling motifs in CD28 that regulate thymocyte cell death. We found that mutation of all three CD28-signaling motifs or truncation of the cytoplasmic tail renders CD28 incapable of costimulating thymocyte cell death in response to Ab stimulation or artificial APCs. However, disruption of any one or two of the three CD28-signaling motifs had no effect on the ability of CD28 to costimulate thymocyte death, indicating that any single motif is sufficient and that CD28 signaling exhibits functional redundancy. Accordingly, the C-terminal PXXP motif of CD28 can recruit Lck (23) and potentiate TCR signaling at the earliest stages by phosphorylating CD3 ITAMs, Zap-70, and Vav. In addition, the N-terminal PXXP domain of CD28 can enhance TCR-proximal signaling by recruiting Itk (43), which has been shown to phosphorylate PLC{gamma} and adaptors that recruit Vav, thus enhancing TCR-proximal signals. The third CD28-signaling motif, YxNM, has been shown to interact with PI3K (44); however, CD28 does not costimulate thymocyte apoptosis in a PI3K-dependent manner, because wortmannin does not inhibit this process (data not shown). Alternatively, the CD28 YxNM motif can also recruit Grb2, which interacts constitutively with Vav (45). Consistent with previous reports (46, 47), Vav1–/– thymocytes exhibit defects in ligand-dependent CD28 costimulation of thymocyte death (our unpublished results). Thus, the observed functional redundancy of the CD28-signaling motifs may be attributable to the fact that each signaling motif can activate Vav-dependent signals that mediate apoptosis. Although CD28-signaling motifs that regulate cell death have not been previously mapped, the transgenic mouse lines used in some of our experiments were previously used to map the signaling motif that is required for IL-2 production and Treg cell generation (16). Unlike the functional redundancy in signaling motifs we observed for regulating cell death, both IL-2 production and Treg cell generation mapped to the C-terminal PXXP motif in the CD28 cytoplasmic domain. Controversy exists over how CD28 signals in mature T cells. The conflicting data may be reconciled by the fact that different groups used different means of expressing CD28 mutants in T cells (transgenics, retroviruses, or transient transfection resulting in very different levels of CD28 expression) and different approaches to stimulate these cells (Abs or Ag-loaded APCs) (16, 48, 49, 50, 51).

In contrast to the strong CD28-dependent increase in Vav1 and PLC{gamma}1 phosphorylation, phosphorylation of the downstream ERK MAPK was minimally enhanced by B7 expression on artificial APCs. Similarly, CD28 costimulation of an ERK-dependent Elk reporter gene was not observed. Although this observation is inconsistent with a report showing that strong transient ERK signals are associated with negative selection in TCR-activated DP thymocytes (52), other publications have shown that ERK is required for positive selection but does not regulate thymocyte negative selection (24, 25, 26, 27, 28). The results shown in Fig. 3, C and D, indicate that TCR signals, which stimulate ERK MAPK, are needed to get DP thymocytes into the maturation program (down-regulate CD4 and CD8, inducibly express CD69). CD28 signals do not participate in this initiation of the maturation response. In contrast, CD28 costimulation affects the outcome of TCR-initiated maturation in part by increasing CD69 and Bim expression as well as initiating a cell death program in CD4low, CD8low, CD69+ thymocytes. It is not clear how CD28 engagement can activate TCR-proximal signals without activating ERK MAPK. In peripheral T cells, CD28 increases ERK activity via a Vav-dependent pathway (47, 53). Several reports have indicated that thymocytes primarily activate ERK via Ras-GRP rather than Grb/2-SOS signaling pathways (54, 55). PLC{gamma}1 catalyzes the production of diacylglycerol, a Ras-GRP activator. Because PLC{gamma}1 was inducibly phosphorylated by CD28 costimulation, a CD28-dependent mechanism may attenuate the activation signal between PLC{gamma}1 and ERK. Others have previously reported that anti-CD28 costimulation can activate MAPK phosphatase-6 (56) and PP2A can physically associate with CD28 (57). If signal attenuation occurs as a result of negative regulation of the signaling cascade (e.g., phosphatase), our data indicate that all three CD28-signaling motifs regulate this potential signal attenuation mechanism that inhibits ERK activation while permitting costimulation of thymocyte death. We cannot rule out the possibility that the ERK response is down-regulated as a result of B7 engagement of CTLA-4 rather than CD28 (58). However, we have not been successful in altering B7-initiated DO11.10 DP thymocyte cell death responses by treating the cells with varying amounts of anti-CTLA-4 (mAb UC10-4F10-11). Whether B7-mediated costimulation of DP thymocytes actively inhibits ERK or it merely fails to activate it, the death pathway initiated by CD28 costimulation may be independent of or dominant over ERK.

It is currently unknown how the signals regulating positive and negative selection are integrated by developing thymocytes. Our results demonstrate that CD28-mediated costimulation by B7 fails to promote significant ERK activation or thymocyte maturation but rather initiates a death response in TCR-activated DP thymocytes. Furthermore, we found that the three known signaling motifs in the CD28 cytoplasmic domain play redundant yet critical roles in costimulating thymocyte death.


   Acknowledgments
 
We thank Jeff Bluestone for providing us with the 2C11 single chain construct, Philippa Marrack and John Kappler for the Adova construct, and Richard Bram for reviewing the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Mayo Foundation and National Institutes of Health (AI44959; to D.J.M.). Back

2 Address correspondence and reprint requests to Dr. David J. McKean, Department of Immunology, Mayo Clinic, 301 Guggenheim Building, Rochester, MN 55905. E-mail address: mckean.david{at}mayo.edu Back

3 Abbreviations used in this paper: DP, double positive; SP, single positive; Treg, T regulatory; CHO, Chinese hamster ovary; PI, propidium iodide; WT, wild type; MFI, mean fluorescence intensity; PLC, phospholipase C; pERK, phosphorylated ERK; TR, truncated. Back

Received for publication May 25, 2006. Accepted for publication August 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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