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Cutting Edge: T Cell Requirement for CD28 Costimulation Is Due to Negative Regulation of TCR Signals by PTEN¹

Jodi L. Buckler^*, Patrick T. Walsh^*, Paige M. Porrett^*, Yongwon Choi and Laurence A. Turka^2,*

^* Department of Medicine and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104

	Abstract

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Recent studies suggest that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays a critical role in the maintenance of self-tolerance. Using T cell-specific PTEN knockout mice (PTENT), we have identified a novel mechanism by which PTEN regulates T cell tolerance. We found that TCR stimulation alone, without CD28 costimulation, is sufficient to induce hyperactivation of the PI3K pathway, which leads to enhanced IL-2 production by naive PTENT T cells. Importantly, as a result of this increased response to TCR stimulation, PTENT CD4⁺ T cells no longer require CD28 costimulation for in vitro or in vivo expansion. In fact, unlike wild-type T cells, PTENT CD4⁺ T cells are not anergized by delivery of TCR stimulation alone. These data suggest that by negatively regulating TCR signals, PTEN imposes a requirement for CD28 costimulation, thus defining a novel mechanism for its role in self-tolerance.

	Introduction

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T cell activation is initiated when the TCR interacts with a peptide-MHC expressed on the surface of an APC. TCR engagement results in the activation of a number of signaling cascades, including the MAPK and PI3K pathways (1). However, TCR signals alone fail to induce effective T cell activation. Optimal cytokine production, induction of cell survival genes, and avoidance of anergy requires the additional delivery of costimulatory signals. Principal among these costimulatory molecules, CD28 activates a number of signaling pathways, including those that are shared with (PI3K and MAPK) and independent of (NF-B) the TCR (2).

Activation of class I_A PI3K in T cells results in the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP₂)³ on the 3' position to generate phosphatidylinositol 3,4,5-triphosphate (PIP₃) (3, 4). PIP₃ serves as an important second messenger by recruiting and activating molecules containing pleckstrin homology domains, including 3-phosphoinositide-dependent protein kinase 1 and Akt (5). Ultimately, this pathway triggers a number of downstream signaling molecules, such as protein kinase C, glycogen synthase kinase, and mammalian target of rapamycin, and plays a critical role in driving cytokine production, proliferation, and survival. While it has long been appreciated that PI3K is coupled to a number of T cell surface receptors, including the TCR, costimulatory molecules, and common -chain cytokine receptors, most of our understanding of PI3K in the context of T cell signaling has derived from studies focused on its activation downstream of CD28 (6, 7, 8). Either by using chemical inhibitors to block PI3K or by overexpressing an active form of Akt, PI3K has been demonstrated to be critical for the proliferative and cell survival responses mediated by CD28 (9, 10).

Given its role in promoting proinflammatory T cell responses, PI3K activation is tightly controlled. A number of molecules, such as Src homology region 2 domain-containing phosphatase 1 and Cbl-b, act directly on p85, the regulatory subunit of PI3K, to terminate its activity (11, 12). An additional level of regulation is provided by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (13), which does not act on PI3K directly, but rather dephosphorylates PIP₃ on the 3' position to regenerate PIP₂, thus limiting the amount of available PI3K product (14, 15). Mice hemizygous at the Pten locus develop autoimmune disorders and possess T cells that are resistant to activation induced cell death (16). Conditional deletion of PTEN within the T cell compartment leads to lymphoproliferative disorders as well as autoimmunity (17). Taken together, these results suggest that the balance between PI3K and PTEN is critical for regulating appropriate T cell activation. However, given the broad range of receptors that activate PI3K, it remains unclear which dysregulated signals contribute to the breakdown in tolerance observed in these mice.

In this study, we examined the role that PTEN plays in regulating PI3K activity directly induced via the TCR. In the absence of PTEN, TCR stimulation alone is sufficient to induce high levels of IL-2 production and proliferation. Interestingly, this increased responsiveness relieves T cells of the requirement for CD28 costimulation to divide both in vitro and in vivo and renders them refractory to anergy induction. In light of these data, we propose a model in which PTEN sets a threshold for activation such that TCR signals alone are insufficient to activate PI3K to a degree necessary for full T cell activation, and CD28 costimulation is required to overcome negative regulation by PTEN. However, in the absence of PTEN, T cells acquire effector functions, such as the ability to produce proinflammatory cytokines, in response to TCR signals alone. Therefore, we suggest that by negatively regulating TCR signals, PTEN imposes a requirement for costimulation and, in doing so, plays a critical role in the maintenance of self-tolerance.

	Materials and Methods

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Mice

Pten^flox/flox mice (H-2^b background) were provided by Dr. T. Mak (University of Toronto, Toronto, Ontario, Canada) and were bred with CD4-Cre to generate PTENT mice. Pten^flox/flox mice were also bred with OT-II TCR transgenic mice (18). Mice were genotyped as described previously (17). BALB/c, CD4-Cre, and ER-Cre mice were purchased from The Jackson Laboratory. All colonies were maintained at the animal facilities of the University of Pennsylvania.

Abs and flow cytometry

The following mAbs were used: anti-CD4 (FITC, PerCP, or allophycocyanin conjugated), anti-CD69-FITC, anti-CD25-PE, anti-CD3-allophycocyanin, anti-CD28-allophycocyanin, anti-CD86-PE, anti-CTLA-4-PE, and anti-V8.1/8.2-FITC (BD Pharmingen). 7-Aminoactinomycin D (BD Pharmingen) was used to measure viability. Cells were analyzed with a FACSCalibur using CellQuest software (BD Biosciences).

In vitro stimulation

CD4⁺ T cells were purified from the spleen and lymph nodes of wild-type (WT) and PTENT mice using MACS purification with routinely >95% purity (Miltenyi Biotec). T cells were cultured in 96-well plates coated with various concentrations of anti-CD3 (0.1, 1.0, 5.0, and 10.0 µg/ml) alone or in combination with anti-CD28 (5 µg/ml). Alternatively, cells were stimulated with soluble anti-CD3 (1 µg/ml) in the presence of APC. Supernatants were collected at 24 h, and IL-2 or IFN- production was measured by ELISA according to the manufacturer’s instructions (BD Pharmingen). Alternatively, cells were harvested at 72 h, and proliferation was measured either by dilution of CFSE or by incorporation of [³H]thymidine.

In vivo administration of tamoxifen

Mice were injected i.p. with 1 mg of tamoxifen (Sigma-Aldrich) for 6 consecutive days and sacrificed 2 days following the final injection. Lymph nodes were harvested, and CD4⁺ T cells were isolated and stimulated as described.

Western blotting

Western blotting was conducted as described previously (19). The following Abs were used: anti-phosphorylated (p)-Akt, anti-p-GSK (Cell Signaling Technology), and anti-PTEN (Cascade Biosciences).

Popliteal lymph node assay

WT and PTENT mice were injected in the footpad with 20 x 10⁶ BALB/c splenocytes. Half of the mice also received a dose of CTLA4Ig (250 µg) via i.p. injection. Five days after injection, draining and nondraining popliteal lymph nodes were harvested and analyzed by flow cytometry.

In vitro tolerance induction

Purified CD4⁺ T cells from WT or PTENT mice were cultured for 72 h in the presence of an excess number of APC and soluble anti-CD3 (1 µg/ml) alone or in combination with CTLA4Ig (10 µg/ml). Cells were then washed and rested for 24 h. Following the rest, cells were restimulated with anti-CD3 (2.5 µg/ml)- and anti-CD28 (5 µg/ml)-coated beads. Supernatants were collected after 24 h.

In vivo tolerance induction

Staphylococcal enterotoxin B (SEB) was used to induce tolerance as described previously (20). Briefly, WT or PTENT mice were treated with 100 µg of SEB (Toxin Technology). After 7 days, spleens were harvested, and CD4⁺V8⁺ T cells were purified using magnetic bead separation. Purified T cells were restimulated in vitro with SEB. OVA-specific T cells in OT-II TCR transgenic mice were anergized as described previously (20).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion Disclosures References

 
PTEN-deficient CD4⁺ T cells are hyperresponsive to TCR stimulation

To study the role of PTEN as a regulator of TCR signals, we have generated mice with a T cell-specific deficiency in PTEN (PTENT). As previously reported and confirmed in our experiments (data not shown), mice with a deficiency of PTEN targeted to the T cell compartment develop both lymphomas and autoimmune disorders and die by 17 wk of age (17). The earliest sign of disease is an accumulation of activated CD4⁺ T cells in the periphery, evident by 6–8 wk of age. To avoid contaminating our experiments with activated CD4⁺ T cells, all mice were used at 2–3 wk of age. At this age, there are normal numbers and percentages of CD4⁺ T cells in both the spleen and lymph nodes of PTENT mice (data not shown). Additionally, the expression of all T cell markers measured, including CD3, CD25, CD28, CD69, CD86, and CTLA-4, was comparable on CD4⁺ T cells from young PTENT and WT mice (Fig. 1a), and CD4⁺ T cells isolated from young PTENT mice do not produce IFN- in response to primary stimulation in contrast with preactivated (primed) T cells (Fig. 1b). Taken together, these results suggest that CD4⁺ T cells from 2- to 3-wk-old PTENT mice are phenotypically and functionally naive.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. T cells from young PTENT mice are phenotypically and functionally naive. a, Expression of T cell markers on CD4+ T cells from WT (solid gray histogram) and young PTENT (thick black line histogram) mice. Thin gray line represents isotype control. b, Naive CD4+ T cells from WT or PTENT mice were stimulated with plate-bound anti-CD3 (1 µg/ml) alone or in combination with plate-bound anti-CD28 (5 µg/ml). IFN- was measured by ELISA. WT CD4+ T cells preactivated with plate-bound anti-CD3 and anti-CD28 Abs for 48 h were used as a positive control. n.d., IFN- levels were below our level of detection.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. PTEN-deficient CD4+ T cells are hyperresponsive to TCR stimulation. a, CD4+ T cells from WT or PTENT mice were stimulated with plate-bound anti-CD3 for 24 h, and IL-2 production was measured by ELISA. b, CD4+ T cells were isolated, CFSE labeled, and stimulated as in a. Cells were harvested after 72 h and analyzed by flow cytometry. Number of mitotic events per 25,000 live events represented in each CFSE plot are as follows: top row (left to right), 548, 4,517, 12,588, and 13,841; bottom row (left to right), 3,655, 14,496, 15,846, and 16,150. Mitotic events were calculated as described previously (24 ). c, CD4+ T cells were isolated from WT or PTENT mice and stimulated with soluble anti-CD3 in the presence of APC with or without CTLA4Ig. d, Purified CD4+ T cells from WT and PTENT mice were either left unstimulated or were stimulated with plate bound anti-CD3 for 30 min. Cell lysates were prepared and analyzed by Western blotting. e, WT or PTEN-deficient T cells were stimulated as in a for 72 h, and viability was measured at various time points using 7-aminoactinomycin D. f, iCre-expressing and nonexpressing mice were injected for 6 consecutive days with tamoxifen. Two days following the final injection, CD4+ T cells were purified from the lymph nodes. PTEN protein levels were measured by Western blot analysis. g, CD4+ T cells from mice treated as in f were stimulated for 72 h with plate-bound anti-CD3 alone or in combination with anti-CD28.

Augmented response of PTENT T cells is correlated with enhanced activation of the PI3K pathway and is not a result of enhanced survival or developmental defects

We next asked whether TCR stimulation of PTEN-deficient T cells results in enhanced activation of targets in the PI3K pathway. WT T cells fail to significantly activate the PI3K pathway in response to anti-CD3 stimulation alone, as evidenced by a lack of induction of phosphorylated PI3K targets, such as Akt and GSK (Fig. 2d). In contrast, there is enhanced phosphorylation of PI3K targets in TCR-stimulated PTENT T cells. These data suggest that PTEN deficiency results in hyperactivation of the PI3K pathway in response to TCR signals alone. Additional experiments using chemical inhibitors demonstrate that activation of both PI3K and Akt is required for the augmented response of PTENT CD4⁺ T cells (data not shown).

Previous reports suggest that PTENT CD4⁺ T cells have enhanced survival in response to certain apoptotic stimuli, including anti-Fas Abs and gamma irradiation (17). However, we found no difference in viability between WT and PTEN-deficient CD4⁺ T cells in response to anti-CD3 stimulation alone or in combination with CD28 signals (Fig. 2e and data not shown). These data suggest that the enhanced responsiveness of PTENT T cells to TCR signals is not simply a result of enhanced survival.

Additionally, although our initial phenotypic and functional assays suggested that CD4⁺ T cells from young PTENT mice were naive, it remained possible that T cells that develop in the absence of PTEN have uncharacterized defects that otherwise account for TCR hyperresponsiveness. To determine whether altered T cell development contributed to the phenotype of PTENT T cells, we generated mice in which PTEN could be conditionally deleted within the mature T cell compartment by crossing PTEN^flox/flox mice with ER-Cre transgenic mice. Once generated, both ER-Cre expressing and nonexpressing mice were injected with tamoxifen for 6 consecutive days. This treatment was sufficient to greatly reduce PTEN protein levels in the CD4 T cell compartment of mice expressing the ER-Cre transgene (Fig. 2f). In accordance with the response of PTENT CD4⁺ T cells from young mice, induced deletion of PTEN in mature CD4⁺ T cells resulted in an enhanced proliferative response to TCR signals either alone or in the presence of costimulation (Fig. 2g). These data suggest that altered development of T cells does not account for the enhanced responsiveness of PTEN-deficient T cells.

CD4⁺ T cells that lack PTEN have a diminished requirement for costimulation

The fact that PTENT T cells proliferated extensively in vitro without CD28 costimulation led us to hypothesize that PTEN deficiency results in a diminished requirement for CD28 costimulation in vivo. To test this hypothesis, we injected either WT or PTENT mice (B6 background) with allogeneic (BALB/c) splenocytes in the footpad and enumerated cells in the draining popliteal lymph node 72 h later. Consistent with prior reports, the 8-fold expansion observed in response to alloantigen immunization is almost completely dependent on CD28 signals, being blocked by treatment of mice with CTLA4Ig (21). In contrast, the expansion/accumulation of PTEN-deficient CD4⁺ T cells is virtually unaffected by a lack of CD28 signals (Fig. 3a). These data strongly support our hypothesis that PTEN deficiency relieves the requirement for CD28 costimulation.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. PTEN imposes a requirement for CD28 costimulation and regulates anergy induction. a, WT and PTENT mice were injected in the footpad with 20 x 106 BALB/c splenocytes. Half of the mice also received an i.p. injection of CTLA4Ig (250 µg). Five days later, the draining and nondraining popliteal lymph nodes were harvested, and the number of CD4+ T cells was quantified using flow cytometry. Data represent the fold increase in the number of CD4+ T cells in the draining popliteal lymph node vs the nondraining contralateral popliteal lymph node in the same mouse and are representative of four experiments. b, WT and PTENT CD4+ T cells were stimulated with soluble anti-CD3 and APC either with or without CTLA4Ig. After 3 days, the cells were washed and rested in complete medium for 24 h, then restimulated with anti-CD3- and anti-CD28-coated beads. After 24 h of secondary stimulation, supernatants were assayed for IL-2 by ELISA. c, WT and PTENT mice were injected i.v. with SEB (100 µg). Seven days later, spleens were harvested, and V8+CD4+ T cells were isolated and restimulated in vitro for 72 h with SEB (10 µg/ml). [3H]Thymidine was added to the wells for the final 16 h of the stimulation. d, Following treatment with either OVA-peptide or PBS, spleens from WT and PTENT OT-II transgenic mice were harvested, and V8+CD4+ T cells were isolated and restimulated in vitro. IL-2 production was measured by ELISA.

Given the diminished requirement for costimulation that we observed in T cells that lack PTEN, we next asked whether PTENT CD4⁺ T cells could be anergized. For in vitro anergy induction, we stimulated WT or PTEN-deficient CD4⁺ T cells with soluble anti-CD3 and APCs either in the absence or presence of CTLA4Ig. Interestingly, in contrast to WT cells, which are unable to produce significant quantities of IL-2 following initial stimulation under anergizing conditions, CD28 blockade did not induce a state of unresponsiveness in PTEN-deficient T cells (Fig. 3b). In fact, PTENT CD4⁺ T cells initially subjected to anergizing conditions produced as much IL-2 upon restimulation as preactivated WT CD4⁺ T cells.

To test susceptibility to anergy induction in vivo, we first used a model of superantigen-induced tolerance. In vivo treatment of mice with superantigen results in a massive expansion of V-specific T cells, followed by a contraction phase (22). Those V-specific T cells that remain in the host following contraction are anergic and consequently do not respond to restimulation with superantigen. Previous studies have shown that peripheral deletion is defective in PTENT mice following SEB-induced expansion. However, the functionality of the PTEN-deficient cells that remain in the host is not known. To determine whether PTEN-deficient CD4⁺ T cells are susceptible to superantigen-induced tolerance, both WT and PTENT mice were injected with SEB. On day 7 following treatment, V8-specific CD4⁺ T were harvested and restimulated in vitro with SEB. V8⁺CD4⁺ T cells isolated from SEB-treated WT mice exhibit a reduced proliferative response to restimulation with SEB compared with the same population of cells isolated from untreated WT mice (Fig. 3c). In contrast, PTEN-deficient CD4⁺V8⁺ T cells from SEB-treated mice proliferated robustly in response to restimulation. Thus, loss of PTEN in CD4⁺ T cells can prevent appropriate induction of anergy in vivo in response to superantigen.

Anergy can also be induced in vivo by treating TCR transgenic mice with a high dose of soluble peptide. For this experiment, we bred the PTENT mice (H-2^b background) with OT-II TCR transgenic mice, which recognize a peptide derived from chicken OVA in the context of the MHC class II molecule I-A^b. Ten days following treatment with OVA (500 µg), OVA-specific V5⁺ T cells were isolated and restimulated in vitro. CD4⁺ T cells from tolerized, OVA-treated WT mice failed to produce IL-2 in response to restimulation (Fig. 3d). OVA treatment, however, failed to anergize PTENT T cells, confirming our previous finding that anergy induction is inhibited in T cells that do not express PTEN.

In conclusion, there is a large body of literature which demonstrates the critical role that PI3K plays in CD28-mediated proliferation and survival (6, 7, 8). Furthermore, previous studies, using knock down or over expression techniques, have demonstrated the essential role of PTEN in regulating T cell activation, although the mechanism for this was not well defined (9, 10). Our data suggest a novel role for PTEN in maintaining self-tolerance through its regulation of TCR signals. The phosphatase SHIP-1 may also play a critical role in regulating PIP₃ levels in T cells, particularly in situations where PTEN is not expressed (23). SHIP-1-mediated generation of phosphatidylinositol 3,4-biophosphate may help direct the signaling pathway away from phosphatidylinositol 3,4,5-triphosphate-dependent effectors, thereby not simply dampening the strength, but also changing the quality of the PI3K signal. By regulating both the strength and direction of the PI3K pathway, PTEN and SHIP-1 may play a critical role in shaping the fate of the T cell response.

Overall, our data support a model in which PTEN-mediated regulation of PI3K imposes a requirement for CD28 costimulation for full T cell activation. Under WT conditions, PTEN-negative regulation ensures that PI3K will only be minimally activated in response to TCR stimulation alone. This regulatory checkpoint limits the activation of T cells in the periphery in the absence of costimulation. However, without PTEN, TCR ligation alone leads to effective cytokine production and proliferation. These data provide a potential mechanism for the development of autoimmunity observed in PTENT mice and suggest a novel role for PTEN as a regulator of peripheral T cell tolerance.

	Disclosures

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P. T. Walsh and L. A. Turka have a pending patent on PTEN and T cells, and the intellectual property is jointly owned by the indicated authors as well as the University of Pennsylvania.

	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 National Institutes of Health Grant AI-43620 (to Y.C. and L.A.T.) and by a grant from the Cancer Research Institute (to J.L.B.).

² Address correspondence and reprint requests to Dr. Laurence A. Turka, University of Pennsylvania, 700 CRB, 415 Curie Boulevard, Philadelphia, PA 19104-6144. E-mail address: turka{at}mail.med.upenn.edu

³ Abbreviations used in this paper: PIP₂, phosphatidylinositol 4,5-bisphosphate; PIP₃, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; WT, wild type; p, phosphorylated; SEB, staphylococcal enterotoxin B.

Received for publication March 20, 2006. Accepted for publication August 2, 2006.

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Top Abstract Introduction Materials and Methods Results and Discussion Disclosures References

 

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