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The Journal of Immunology, 2001, 166: 5331-5335.
Copyright © 2001 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Distinct Motifs Within CD28 Regulate T Cell Proliferation and Induction of Bcl-XL1

John S. Burr*, Nigel D. L. Savage*, Grace E. Messah*, Stephanie L. Kimzey*, Andrey S. Shaw{dagger}, Robert H. Arch*,{dagger} and Jonathan M. Green2,*,{dagger}

Departments of * Medicine and {dagger} Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
CD28 provides an important costimulatory signal in T cell activation that regulates multiple cellular processes including proliferation and survival. Several signal transduction pathways are activated by CD28; however, the precise biochemical mechanism by which CD28 regulates T cell function remains controversial. Retroviral gene transfer into primary T cells from TCR-transgenic, CD28-deficient mice was used to determine the specific sequences within CD28 that determine function. Discrete regions of the cytoplasmic domain of CD28 were identified that differentially regulate T cell proliferation and induction of the anti-apoptotic protein Bcl-XL. Mutation of C-terminal proline residues abrogated the proliferative and cytokine regulatory features of CD28 costimulation while preserving Bcl-XL induction. Conversely, mutation of residues important in phosphatidylinositol 3-kinase activation partially inhibited proliferation but prevented induction of Bcl-XL. Thus the ability of CD28 to regulate proliferation and induction of Bcl-XL map to distinct motifs, suggesting independent signaling cascades modulate these biologic effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Costimulation through CD28 regulates multiple aspects of T cell function including proliferation, cytokine secretion, and cell survival (1). Several pathways have been implicated in CD28 signaling; however, the precise role of each remains controversial (2). Most mutational analysis of CD28 has been performed on transformed T cell lines, which have intrinsic alterations in the regulation of cell growth and survival. Thus, although these studies have provided important information, the results may not apply to primary T cells.

We have examined the requirement for specific elements of the cytoplasmic domain of CD28 in costimulation of primary T cells. Reconstitution of CD28-deficient T cells specific for the OVA323–339 peptide with retroviral constructs encoding wild-type or mutant CD28 revealed a dissociation between elements required for T cell proliferation and for induction of the anti-apoptotic protein Bcl-XL. Thus, distinct domains within the cytoplasmic tail of CD28 regulate these processes, implying that CD28 activates multiple signaling pathways, which in turn mediate discrete biologic consequences of costimulation.


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

CD28-deficient mice on the DO11.10 TCR-transgenic background were obtained from C. Thompson and S. Reiner (University of Pennsylvania, Philadelphia, PA). D011.10 mice were a gift of K. Murphy (Washington University, St. Louis, MO; Ref. 3). BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Antibodies

Anti-CD3 (145-2C11, hamster IgG) was provided by J. A. Bluestone (University of California, San Francisco, CA). Anti-CD28 (PV-1, hamster IgG) was provided by C. June (University of Pennsylvania). Anti-Bcl-XL Abs (13.6, rabbit polyclonal IgG and clone 7B2, mouse IgG3) were provided by L. Boise (University of Miami, Miami, FL). All other mAbs were purchased from PharMingen (San Diego, CA).

Retroviral infections

Full-length and mutant murine CD28 cDNA was cloned into the retroviral vector GFPRV or CD4RV (provided by W. Sha, University of California, San Francisco and K. Murphy, Washington University) and transiently transfected into the Phoenix Eco packaging cell line (provided by G. Nolan, Stanford University, Palo Alto, CA) as previously described (4, 5). Retroviral supernatants were incubated with activated splenocytes, and expression of green fluorescence protein (GFP)3 and CD28 were determined by flow cytometry. Infection efficiencies ranged from 10 to 20% between experiments, but were similar for all constructs within a given experiment. For Western blotting, the cells were infected with the CD4RV retrovirus (encoding for tailless human CD4 instead of GFP) and sorted by immunomagnetic beading using anti-human CD4 microbeads and an AutoMACS cell sorter (Miltenyi Biotec, Auburn, CA). Expression of CD28 was confirmed in all experiments by flow cytometry.

Proliferation assays

Wild-type or CD28-deficient DO11.10 splenocytes were isolated and infected with retrovirus, and 5 x 104 cells were cocultured with 1.5 x 105 T-depleted, irradiated BALB/c splenocytes. OVA323–339 peptide was added alone or in combination with murine CTLA4Ig (10 µg/ml; provided by Genetics Institute, Cambridge, MA), anti-CD28 mAb (1.0 µg/ml), or control Ig, and proliferation was determined by tritiated thymidine incorporation for the final 8 h of a 72-h culture. All conditions were plated in quadruplicate, and the mean ± SD of the quadruplicate wells presented. Replicate plates were assayed for IL-2 content by CTLL-2 bioassay at 48 h. All experiments have been repeated a minimum of three times, and representative data from one experiment presented.

Bcl-XL expression

Retrovirally infected cells were enriched by immunomagnetic cell sorting and stimulated with immobilized anti-CD3 (10 µg/ml) alone or in combination with soluble anti-CD28 (1.0 µg/ml) for 48 h. The cells were lysed in 0.2% Nonidet P-40 lysis buffer, separated on a 12.5% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane, probed with anti-Bcl-XL anti-sera, and developed by ECL. Membranes were reprobed with anti-actin Ab (Clone C4; Boehringer Mannheim, Indianapolis, IN). For intracellular flow cytometric analysis of Bcl-XL, cells were surface stained with FITC-conjugated anti-CD4 followed by intracellular staining with anti-Bcl-XL mAb 7B2 or an isotype-matched control Ab and analyzed on a FACSCalibur flow cytometer.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
CD28-mediated T cell activation and proliferation depends upon specific C-terminal proline residues

The most profound effects of CD28-mediated costimulation are in the activation of naive T cells (1). We addressed the mechanism by which CD28 regulates proliferative and anti-apoptotic signaling pathways by reconstituting primary T cells from OVA-specific TCR-transgenic, CD28-deficient mice with specific mutants of CD28 by retroviral gene transfer. This approach allowed us to examine CD28 in a physiologic context, activation of primary cells with peptide Ag presented by normal APC.

Retroviral infection of CD28-deficient T cells resulted in expression of CD28 proteins on the cell surface at levels comparable to that of endogenous CD28 expressed on control-infected splenocytes (Fig. 1Go). The expression level of the retrovirally expressed mutant or wild-type CD28 proteins were all similar, and are shown as superimposed histograms labeled CD28-/- + CD28RV.



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  		FIGURE 1. Expression of CD28 on retrovirally infected T cells. A, The amino acid sequence of wild-type and mutant CD28 proteins. Specific substitutions are shown in bold type. B, Splenocytes from CD28+/+ or CD28-/- DO11.10 mice were infected with retrovirus encoding for control (GFPRV) or CD28 protein (CD28RV). Expression of CD28 was determined by staining with PE-conjugated anti-CD28 mAb. Shown is a histogram plot of the PE fluorescence after gating on GFP-positive cells. All CD28 mutants showed expression similar to endogenous CD28 expression on wild-type cells (CD28+/+ + GFPRV) and are shown as overlapping histograms labeled CD28-/- + CD28RV.

 
Analysis of the proliferative response of T cells expressing wild-type or mutant CD28 protein revealed that deletion of the cytoplasmic tail (dcyto) or the C-terminal 16 aa (CD28d16) led to complete loss of CD28-dependent proliferation (Fig. 2Go). Within this region is the sequence, PYAP, which may function to recruit and activate either SH2 or SH3 domain-containing proteins. Substitution of both proline residues (P187, 190A) abrogated CD28-dependent proliferation, whereas mutation of the tyrosine (Y188F) within this motif had less effect, suggesting that proline-mediated interactions with SH3 domains are critical for downstream signaling events. Reconstitution with wild-type CD28 (FLCD28) resulted in a strong proliferative response. The proliferation was B7-dependent, as inclusion of CTLA4Ig in the cultures inhibited the proliferative response.



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  		FIGURE 2. CD28-dependent proliferation requires prolines 187 and 190. Splenocytes from CD28-deficient DO11.10 mice were infected with retrovirus encoding either control (GFPRV), wild-type CD28 (FLCD28), or mutant CD28 constructs. Proliferation was determined following stimulation with OVA323–339 peptide and irradiated T-depleted splenocytes from BALB/c mice. CTLA4Ig or a control Ig (data not shown) was added at the initiation of the culture. Replicate wells were activated with PMA (5 ng/ml) and ionomycin (0.1 µg/ml). Proliferation was determined by tritiated thymidine incorporation for the final 8 h of a 72-h culture. Data from one representative experiment are shown. A control Ig had no effect on proliferation (data not shown).

 
CD28 contains the motif YMNM that following phosphorylation of the tyrosine (Y170) can bind and activate phosphatidylinositol 3-kinase (PI 3-kinase) (6). The role of PI 3-kinase activation in CD28 function remains controversial (6, 7, 8, 9, 10, 11). Reconstitution with a mutant CD28 substituting a phenylalanine for tyrosine (Y170F) in this motif resulted in a moderate defect in CD28-dependent proliferation with responses ranging from 60 to 80% of control in multiple experiments (Fig. 2Go and data not shown).

CD28 regulates IL-2 by both transcriptional and posttranscriptional processes (12, 13). Stimulation of cells expressing the P187, 190A mutant with Ag or Ag plus anti-CD28 led to a marked decrease in IL-2 secretion. In contrast, the Y188F and Y170F mutants had levels comparable to wild type in response to anti-CD28 costimulation, suggesting these residues are not essential for CD28 regulation of IL-2 secretion. The IL-2 production in response to Ag alone was variable between experiments, with some experiments revealing little difference between CD28-deficient or CD28-sufficient T cells (compare Fig. 3Go, A and B). Inclusion of CTLA4Ig had little effect in these circumstances, suggesting that endogenous B7-dependent costimulation was low. However, in all experiments, anti-CD28 mAb augmented IL-2 production from the Y188F and Y170F mutants to levels equivalent to FLCD28, whereas all other mutants had marked reduction in IL-2 secretion (Fig. 3Go). These results were observed at both a low and high dose of Ag (Fig. 3Go, A and B).



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  		FIGURE 3. CD28 regulation of IL-2 requires prolines 187 and 190. Splenocytes from CD28-deficient DO11.10 mice were infected with retrovirus encoding control (GFPRV), wild-type CD28 (FLCD28), or mutant CD28 constructs and stimulated with OVA323–339 peptide alone, with CTLA4Ig (10 µg/ml) or with anti-CD28 mAb (1.0 µg/ml). Culture supernatant was harvested at 48 h and assessed for IL-2 by CTLL-2 bioassay. Shown is the tritiated thymidine incorporation of the CTLL-2 cells. The mean ± SD of quadruplicate wells is plotted. Data from one representative experiment are shown. A, CTLL-2 assay following stimulation with 0.1 µM OVA. B, CTLL-2 assay following stimulation with 0.03 µM OVA.

 
Induction of Bcl-XL requires residues important in PI 3-kinase activation

Activation of PI 3-kinase has been shown to regulate cellular processes important in cell survival and has been implicated in the regulation of Bcl-XL (14, 15, 16). Substitution of a phenylalanine for a tyrosine at position 170, a critical residue in PI 3-kinase activation by CD28 (6, 17), resulted in the failure to induce Bcl-XL after stimulation with anti-CD3 and anti-CD28 (Fig. 4Go). In contrast, cells expressing wild-type CD28 of either endogenous or retroviral origin demonstrated CD28-dependent induction of Bcl-XL protein. IL-2 secretion in response to CD28 cross-linking in the Y170F mutant was equivalent to levels from cells reconstituted with wild-type CD28, suggesting that induction of Bcl-XL by CD28 is not mediated exclusively by secondary effects of IL-2.



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  		FIGURE 4. Induction of Bcl-XL by CD28 requires tyrosine 170. A, Splenocytes from CD28-deficient DO11.10 mice were infected with retrovirus encoding control (CD4RV), wild-type CD28 (FLCD28), or the indicated mutant CD28 constructs and stimulated with plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (1.0 µg/ml) for 48 h. The cells were lysed in 0.2% Nonidet P-40 lysis buffer, and the protein was separated on a 12.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. The membrane was probed with anti-Bcl-XL anti-sera followed by HRP-conjugated goat anti-rabbit Ig and developed by ECL. Subsequently, the blot was stripped and reprobed with anti-actin mAb. B, CD28-deficient splenocytes were retrovirally infected with either wild-type CD28 (FLCD28) or the P187, 190A CD28 mutant and stimulated as in A with or without CTLA4Ig (10 µg/ml), and Bcl-XL expression was assessed as described above.

 
Anti-CD3 stimulation of cells reconstituted with the P187, 190A mutant led to increased Bcl-XL expression. However, there was no apparent increase in expression with anti-CD28. CTLA4Ig effectively blocked the induction of Bcl-XL by anti-CD3 in cells reconstituted with either FLCD28 or the P187,190A mutant, indicating that B7-expressing cells present in the culture were providing endogenous costimulation, thereby demonstrating intact B7-CD28-dependent expression of Bcl-XL (Fig. 4GoB). The preservation of Bcl-XL expression following stimulation of cells expressing the P187,190A mutant is in contrast to the near complete abrogation of proliferation and IL-2 secretion observed in these same cells.

Pagès et al. demonstrated that deletion of the terminal 10 aa of CD28 led to a marked reduction in the binding of the p85 subunit of PI 3-kinase to CD28 (6, 11). Consistent with this data we found that deletion of the C-terminal 16 aa (d16) also led to a loss of Bcl-XL induction. As Bcl-XL induction was preserved in the P187,190A mutant, our data suggest other residues within this region are required. These data are consistent with a cooperative relationship between these two domains of CD28 in both the activation of PI 3-kinase and the induction of Bcl-XL. One model that could account for these observations would be the recruitment or activation of a kinase by a motif within the C-terminal 16 aa of CD28, which then phosphorylates the tyrosine at position 170, allowing for binding and activation of PI 3-kinase at this site.

PI 3-kinase activation of protein kinase B (PKB); the effector of CD28-dependent Bcl-XL induction?

To examine the role of PI 3-kinase in Bcl-XL expression using an approach complementary to the mutagenesis strategy, we examined the effect of pharmacologic inhibition of PI 3-kinase activity. Treatment of wild-type T cells with an inhibitor of PI 3-kinase (LY294002) resulted in a moderate decrease in expression of CD69, but complete blockade of Bcl-XL induction (Fig. 5Go), in agreement with the results reported by Collette et al. using wortmannin (14). Together with the mutational data, these results support the hypothesis the PI 3-kinase pathway is important for induction of Bcl-XL.



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  		FIGURE 5. PI 3-kinase activity is required for induction of Bcl-XL. Splenocytes cells from DO11.10 mice were stimulated with OVA323–339 peptide either in medium alone or in the presence of LY294002 (25 µM) for 24 h. Top, CD69 expression of the CD4-positive population is shown. Bcl-XL expression was determined by staining with CD4-FITC followed by intracellular staining with anti-Bcl-XL mAb. Bottom, Histogram plot of the Bcl-XL expression of the CD4-positive population.

 
Global inhibition of PI 3-kinase activity with pharmacologic inhibitors has been shown to inhibit T cell proliferation and IL-2 production (18, 19). However, these reagents inhibit the activity of the enzyme and cannot selectively prevent activation by CD28 as opposed to other pathways. The decrease in CD69 expression we observed following treatment with LY294002 is in fact consistent with an important role for PI 3-kinase in T cell activation. However, our mutational data implies that activation of PI 3-kinase by CD28 is not absolutely required for costimulation-dependent proliferation and IL-2 production. This does not suggest that PI 3-kinase is entirely dispensable for normal T cell activation and cytokine production.

Although we have not directly demonstrated the binding or activation of PI 3-kinase by the mutant CD28 proteins, several groups have established that the tyrosine at position 170 is required for PI 3-kinase binding and activation by CD28 (6, 17). In addition, a second tyrosine-based motif within the C-terminal 10 aa has also been shown to be involved in PI 3-kinase binding (11). Thus, it is highly likely that these identical mutations in our retroviral constructs also fail to bind PI 3-kinase. However, it remains a formal possibility that these mutations interrupt other protein-protein interactions that are then responsible for the observed loss of Bcl-XL induction.

The serine/threonine kinase PKB (Akt) is a downstream effector of PI 3-kinase that is an important regulator of Bcl-XL expression and is activated following CD28 ligation (15, 20). Thus, our data are consistent with a mechanism in which CD28-dependent activation of PI 3-kinase leads to PKB activation and up-regulation of Bcl-XL expression.

Dahl et al. reported that overexpression of Bcl-XL in CD28-deficient lymphocytes enhanced cell survival, but did not restore proliferation and cytokine secretion (21). Similarly, we find dissociation of CD28-dependent regulation of proliferation and expression of an important factor that effects cell survival. Although we have not directly examined cell survival in this system, several groups have demonstrated that CD28 is a major regulator of Bcl-XL expression in T cells, and that this is an important component in the control of cell survival (22, 23, 24). Because retroviral infection requires activation of T cells for infection, the cells were not truly naive; therefore, it is possible that our results are biased toward seeing an effect on Bcl-XL expression that may not be representative of truly resting cells.

In this analysis, we have presented data from only a single time point for each parameter assayed. We found that peak proliferative responses and the greatest differences between constructs were observed at 72 h. Similar trends were seen at earlier times for both proliferation and Bcl-XL expression (data not shown). At later time points, it is possible that the Y170F mutation in particular might have had more of a proliferative defect, perhaps due to impaired survival given the failure to induce Bcl-XL. Similarly, the P187, 190A mutation might have exhibited defective Bcl-XL expression at later time points.

This analysis enabled us to examine CD28 function on primary T cells following engagement of both CD28 and the TCR with native ligand expressed on normal APC. These data demonstrate that two of the consequences of CD28 ligation, proliferation and induction of Bcl-XL, can be dissociated at the level of the amino acid sequence of CD28. We have previously shown that the proline residues at position 187 and 190 of CD28 can recruit and activate Lck (25). Thus, proliferation induced by CD28 may be critically dependent upon this kinase. In contrast, mutation of a region of the cytoplasmic domain of CD28 known to be essential for PI 3-kinase activation had less effect on proliferation and IL-2 secretion, but prevented induction of Bcl-XL. Pharmacologic inhibition of PI 3-kinase suggests a key role for this enzyme in Bcl-XL regulation, perhaps through activation of PKB/Akt. Thus CD28 engages multiple signaling pathways that may independently regulate the biologic consequences of costimulation.


   Acknowledgments
 
We thank A.C. Chan for helpful discussion and review of this manuscript. We are grateful to L. Boise for valuable discussion and providing the Bcl-XL Abs and M. Collins for providing the CTLA4Ig. We thank C. Thompson, S. Reiner, and K. Murphy for providing mice, and K. Murphy for assistance with cell sorting.


   Footnotes
 
1 This work was supported in part by grants from the National Institutes of Health (to J.M.G. and A.S.S.) and the Glaxo-Wellcome Corporation (to J.S.B.). Back

2 Address correspondence and reprint requests to Dr. Jonathan M. Green, Washington University School of Medicine, 660 South Euclid Avenue, Box 8052, St. Louis, MO 63110. Back

3 Abbreviations used in this paper: GFP, green fluorescence protein; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B. Back

Received for publication January 29, 2001. Accepted for publication March 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Ligation of CD28 Stimulates the Formation of a Multimeric Signaling Complex Involving Grb-2-Associated Binder 2 (Gab2), Src Homology Phosphatase-2, and Phosphatidylinositol 3-Kinase: Evidence That Negative Regulation of CD28 Signaling Requires the Gab2 Pleckstrin Homology Domain
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Combined Deficiency of p50 and cRel in CD4+ T Cells Reveals an Essential Requirement for Nuclear Factor {kappa}B in Regulating Mature T Cell Survival and In Vivo Function
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F. Michel and O. Acuto
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Recruitment of Phosphatidylinositol 3-Kinase to CD28 Inhibits HIV Transcription by a Tat-Dependent Mechanism
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Polar Redistribution of the Sialoglycoprotein CD43: Implications for T Cell Function
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