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The Journal of Immunology, 2006, 177: 5122-5128.
Copyright © 2006 by The American Association of Immunologists, Inc.

The p110{delta} Isoform of Phosphoinositide 3-Kinase Controls Clonal Expansion and Differentiation of Th Cells1

Klaus Okkenhaug2,*, Daniel T. Patton*, Antonio Bilancio{dagger}, Fabien Garçon*, Wendy C. Rowan{ddagger} and Bart Vanhaesebroeck{dagger},§

* Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge, United Kingdom; {dagger} Ludwig Institute for Cancer Research, London, United Kingdom; {ddagger} GlaxoSmithKline, Stevenage, United Kingdom; and § Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110{delta} isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110{delta} activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110{delta}D910A/D910A T cells is impaired, but that ERK phosphorylation and NF-{kappa}B nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110{delta}D910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110{delta}D910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110{delta} contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The TCR signaling cascade is initiated by tyrosine kinases that regulate the assembly of a complex of adapter proteins and other signaling molecules (1, 2). The transmembrane adapter protein, linker for activation of T cells (LAT),3 nucleates this complex and controls recruitment and activation of phospholipase C{gamma}, which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to produce inositol trisphosphate and diacylglycerol (3). Inositol trisphosphate triggers the cytoplasmic release of Ca2+, which induces the translocation of NF-AT transcription factors to the nucleus, while diacylglycerol activates PKC and Ras guanyl-releasing protein, which in turn activate the Ras-ERK1/2 pathway and AP-1 nuclear translocation (4, 5). PKC also regulates a signaling pathway that leads to the nuclear translocation of NF-{kappa}B subunits (6). The activation of NF-AT, AP-1, and NF-{kappa}B downstream of phospholipase C{gamma} is both necessary and sufficient for connecting the TCR to transcriptional activation of key target genes, including IL-2. However, other signaling pathways, including those triggered by costimulatory receptors, can affect the outcome of T cell activation by amplifying or complementing the signals triggered by PIP2 hydrolysis.

Class I-A PI3Ks are heterodimers of a p85{alpha}, p85beta, or p55{gamma} regulatory subunit and a p110{alpha}, p110beta, or p110{delta} catalytic subunit. One of the earliest detectable signaling events that follows cognate recognition by the TCR of a peptide/MHC complex is the PI3K-mediated phosphorylation of PIP2 to yield the second messenger molecule phosphatidylinositol-3,4,5-trisphosphate (PIP3) (7, 8, 9, 10). PIP3 triggers signaling pathways by binding to the pleckstrin homology domains of the protein kinases Pdk1, Akt, Itk, and other signaling proteins, hence recruiting these to the plasma membrane. The mechanism through which the TCR is coupled to PI3K activation remains poorly defined, but is thought to depend on a direct or indirect association between the adapter protein LAT and the p85 regulatory subunit of PI3K (3, 10, 11, 12, 13, 14). p85 has two Src homology 2 domains, which bind to proteins at the plasma membrane that contain phosphorylated YxxM motifs. LAT lacks such a motif, but may recruit PI3K indirectly via Grb2 or Gads-containing protein complexes (3, 11, 12, 13). The costimulatory receptor CD28 can interact directly with PI3K via a YMNM phosphorylation motif in the cytoplasmic domain; however, this interaction is not essential for costimulation of IL-2 production or proliferation (15, 16, 17, 18).

To further investigate the role of PI3K in T cells, we have generated gene-targeted mice in which p110{delta}, thought to be the main class I-A PI3K catalytic isoform in T cells, is inactivated by a point mutation in the catalytic domain (19). T cells from these p110{delta}D910A/D910A mice show reduced TCR-dependent proliferation as determined by the reduced DNA synthesis in bulk cultures. However, anti-CD3 + anti-CD28-stimulated proliferation and IL-2 production were normal, and another study that made use of p110{delta} knockout mice concluded that p110{delta} is not required for T cell activation (20). In addition, p85{alpha}-deficient mice show a normal T cell phenotype, whereas p85beta-deficient mice show increased proliferation coupled with increased resistance to apoptosis (21, 22, 23). In light of these conflicting results, we analyzed the contribution of p110{delta} to T cell signaling and function in more detail.

In this study, we have investigated signaling downstream of the TCR and CD28 and found that the phosphorylation of Akt and Foxo in p110{delta}-deficient T cells was attenuated. In vitro and in vivo proliferative responses in response to Ag and differentiation along the Th1 and Th2 lineages were impaired. The latter could not be rescued by the provision of exogenous cytokines. These results establish that p110{delta} is a key component of the signaling machinery that promotes clonal expansion and differentiation of Th lineages, and that physiologically relevant costimulation and exogenous cytokines cannot compensate for these defects.


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

p110{delta}D910A/D910A, DO11.10, and OT-II mice have been described (19, 24, 25) and had been backcrossed to the C57BL/6 or BALB/c genetic backgrounds for 8–10 generations. Similar results were obtained with the two TCR transgenic models, and the level and frequency of transgenic TCR expression were indistinguishable between wild-type (WT) and p110{delta}D910A/D910A mice. Mice were maintained under specific pathogen-free conditions. All protocols involving live animals were approved by the United Kingdom Home Office and institutional ethical review committees.

Reagents

All chemicals not described specifically were from Sigma-Aldrich. Abs used for FACS were from BD Pharmingen or eBioscience, unless otherwise noted. The following clones were used: anti-CD4 (L3T4), anti-CD8 (YTS105.18.10), anti-CD69 (H1.2F3), anti-B220 (RA3-6B2), anti-CD49b (DX5), anti-MHC class II (M5/114.15.2), anti-Thy-1.2 (TS; Sigma-Aldrich), anti-DO11 TCR (KJ1-26; Caltag Laboratories), anti-V{alpha}2 (B20.1), and anti-Vbeta5 (MR9-4). Abs for biochemical studies were as follows from Cell Signaling Technology: anti-pAkt Ser473 (catalog 9271), anti-Akt (total) (catalog 9272), and anti-pFoxo1 Thr24/pFoxo3a Thr32 (catalog 9464). Anti-pERK1/2 Tyr204 was from Santa Cruz Biotechnology (catalog sc7383). Anti-GAPDH was from Abcam (catalog ab8245).

Cell purification

T cells were purified from lymph nodes by magnetic sorting (Miltenyi Biotec). Total T cell populations were isolated by negative selection using anti-MHC class II beads. For isolation of TCR transgenic T cells, non-CD4+ T cells were labeled with FITC-conjugated anti-CD8, anti-CD69, anti-B220, anti-CD49b, and anti-MHC class II, followed by negative selection with anti-FITC MACS beads (Miltenyi Biotec). The resulting cells were >95% CD4+ and had similar levels of TCR transgene expression in WT and p110{delta}D910A/D910A backgrounds (as determined by FACS analysis of cells stained with KJ1-26 for DO11.10 cells, or anti-TCR-V{alpha}2 and anti-TCR-Vbeta5 for OT-II cells). APCs were purified from spleen cells after T cell depletion with anti-mouse Thy-1.2 (Sigma-Aldrich) and rabbit complement (Cedarlane Laboratories), followed by purification of viable cells using Lympholyte-M (Cedarlane Laboratories) and 30 Gy irradiation.

PI3K assay

Proteins from 500 mg of total lysate of lymph node T cells were immunoprecipitated using the phosphotyrosine pYVPMLG peptide matrix, essentially as described (19).

Signaling studies

T cells (5–10 x 106) were stimulated for 30 min at 37°C with polystyrene beads (Polysciences) coated with 1 µg/ml anti-CD3 and/or 10 µg/ml anti-CD28 and lysed in ice-cold lysis buffer, essentially as described (19). Proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with the following Abs: pAkt, pFoxo and pERK, total Akt, or GAPDH.

NF-{kappa}B staining

CD4+ T cells were stimulated for 30 min with anti-CD3 or anti-CD3 + anti-CD28 (each used at 10 µg/ml) plus goat anti-hamster IgG (Jackson ImmunoResearch Laboratories), or with different concentrations of OVA323–339 peptide (Southampton Polypeptide), loaded on T cell-depleted spleen cells, fixed with 3.7% paraformaldehyde, and adhered to poly(L-lysine) glass coverslips. Cells were permeabilized with 0.2% Triton X-100 in PBS for 4 min, blocked with 5% BSA in PBS for 30 min, and then stained with anti-p65/RelA (Santa Cruz Biotechnology) and Alexa-488-conjugated goat anti-rabbit Ig (Invitrogen Life Technologies). The nuclei were labeled with 7-aminoactinomycin D (7-AAD; Invitrogen Life Technologies). The slides were mounted with FluorSave Reagent (Calbiochem), and the images were collected using a Zeiss LSM510 META system consisting of a Zeiss Axiovert 200 microscope fitted with a Zeiss Plan-Achromat x63/1.4 numerical aperture oil objective. The percentages of positive cells were obtained by counting >100 cells per experimental condition from 5 to 10 random fields using ImageJ software (<http://rsb.info.nih.gov/ij/>).

Cytokine ELISA

The supernatants from purified DO11.10 T cells, stimulated for 48 h with 1 µM OVA323–339 peptide and irradiated WT APCs, were collected and analyzed by ELISA (BD Pharmingen).

In vitro proliferation of CFSE-labeled cells

Purified CD4+ T cells were washed in HBSS, resuspended at 107 cells/ml in HBSS, and mixed with an equal volume of 2 µM CFSE (Invitrogen Life Technologies) for 7 min in the dark at room temperature. FCS was added to a final concentration of 10%, and the cells were washed twice in complete RPMI 1640 culture medium. CFSE-labeled CD4+ cells and APCs were added to wells of a 24-well plate at 5 x 105 cells/well, with different concentrations of OVA323–339 peptide. After 3 days, the cells were stained with CD90.2-allophycocyanin, KJ1-26 biotin-streptavidin-PE (D011.10), or anti-TCR V{alpha}2-PE (OT-II) and 7-AAD. The cells were acquired on a FACSCalibur (BD Biosciences), and the data were analyzed using the FlowJo (TreeStar) software package.

In vitro differentiation of CD4+ cells

T cells were incubated with 20 ng/ml IL-12 (Peprotech) and anti-IL-4 (R&D Systems) for Th1 skewing, or 20 ng/ml IL-4 (R&D Systems), 1:100 anti-IL-12 (clone C17.8; a gift from G. Trinchieri Schering-Plough Research Institute, Dardilly, France), and 1:100 anti-IFN-{gamma} (American Type Culture Collection) supernatants for Th2 skewing. After 6 days, the cells were restimulated with 50 ng/ml phorbol-12,13-dibutyrate (PdBu) and 1 µM ionomycin for 6 h, with 10 µg/ml brefeldin A added for the final 2 h. The cells were stained with KJ1-26 biotin, followed by streptavidin-PerCP, fixed, permeabilized, incubated with anti-IL-4 PE, anti-IFN-{gamma} allophycocyanin, or anti-IL-2 allophycocyanin, and analyzed by FACS.

Adoptive transfer experiments

These experiments were based on previously published protocols (26). Briefly, lymph node cells from WT or p110{delta}D910A/D910A DO11.10 mice were labeled with CFSE and injected into the tail vein of normal BALB/c recipients (4 x 106 CD4+KJ1-26+ T cells per recipient). The next day, the mice were either killed (to determine the homing of cells to the lymph nodes) or immunized by s.c. injection of 1 mg of OVA and 25 µg of LPS. Draining lymph nodes (inguinal, axial, and brachial) were harvested 3 or 6 days later, and the KJ1-26+ population was quantified and analyzed by FACS for different markers, as indicated. The number of KJ1-26+ T cells was calculated by multiplying the number of lymph node cells recovered by the percentage of KJ1-26+CD4+ T cells. Cells harvested after 6 days were also restimulated with 50 ng/ml PdBu and 1 µM ionomycin for 6 h, with 10 µg/ml brefeldin A added for the final 2 h and stained with anti-IFN-{gamma}, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PI3K signaling is impaired in p110{delta}D910A/D910A T cells

Of the three class I-A PI3Ks expressed in T cells, p110{delta} appears to be the main isoform involved in TCR signaling (19). With the aim to quantitate the relative contribution of p110{delta} to the total pool of class I-A PI3K activity, p85 was purified from WT and p110{delta}D910A/D910A T cells, and the associated p110 PI3K subunits were subjected to an in vitro lipid kinase assay using PIP2 as a substrate. Total class I-A PI3K activity in p110{delta}D910A/D910A cells was reduced by ~50% relative to WT cells (Fig. 1A). Although this shows that p110{delta} indeed contributes to a large fraction of PI3K activity in T cells, these data also indicate that significant levels of p110{alpha} and p110beta activity are still available in p110{delta}D910A/D910A T cells. This raised the possibility that the observed rescue of p110{delta}D910A/D910A T cell proliferation by coligation of CD3 and CD28 in vitro (19) might be the result of CD28-mediated recruitment and activation of p110{alpha} and/or p110beta.


Figure 1
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  		FIGURE 1. p110{delta}-dependent signaling. A, Purified WT or p110{delta}D910A/D910A T cells were lysed, and p85/p110 complexes were subjected to an in vitro kinase reaction using PIP2 as a substrate. The amount of associated PI3K activity (±SD) is represented relative to that of WT cells (which was normalized to 100 arbitrary units). B, Purified WT or p110{delta}D910A/D910A T cells were stimulated for 5, 15, 30, or 60 min with beads coated with anti-CD3 and anti-CD28 mAbs, followed by Western blot analysis of Akt, Foxo, and ERK phosphorylation. Total Akt was used as a loading control. C, Purified WT or p110{delta}D910A/D910A T cells were stimulated for 30 min with beads coated with anti-CD3 and/or anti-CD28 mAbs or with control beads, followed by Western blot analysis of the phosphorylation status of Foxo and ERK. A 1/3 dilution of the lysate loaded in lane 4 was loaded in lane 5. In lanes 6 and 11, cells had been preincubated with 10 µM LY294002. GAPDH was used as a loading control. D, T cells were stimulated as in C and scored for the frequency of nuclear localization of the p65/RelA subunit of NF-{kappa}B by confocal imaging. Data shown are the average of three independent experiments (±SD) with >100 cells per experimental condition in each experiment. E, OT-II WT or p110{delta}D910A/D910A T cells were stimulated with APCs loaded with 1 or 10 µM OVA323–339 peptide, and the percentage of T cells in conjugates was scored for p65/RelA NF-{kappa}B nuclear translocation. In the absence of peptide, conjugates were not found, in which case T cells were picked randomly and scored. Data shown are the average of three independent experiments (±SD) with >100 cells per experimental condition in each experiment.

 
To investigate whether CD28 could activate PI3K in p110{delta}D910A/D910A T cells, we stimulated purified T cells with Abs to CD3 and CD28 and used Akt and/or Foxo phosphorylation as surrogate markers for PI3K activation. In WT T cells, anti-CD3 and anti-CD28 synergistically stimulated Akt phosphorylation, but this was not seen in p110{delta}D910A/D910A T cells (Fig. 1B). In contrast, the Akt substrates Foxo1 and/or Foxo3a were found to be phosphorylated in anti-CD3- and anti-CD28-costimulated p110{delta}D910A/D910A T cells, albeit at much reduced levels in comparison with WT cells (Fig. 1, B and C). To determine whether the observed Foxo phosphorylation was PI3K dependent, the experiment was repeated, but cells were stimulated either with anti-CD3, anti-CD28, or the combination, in the presence or absence of the pan-PI3K inhibitor LY294002. In this experiment, we detected overall minimal Akt phosphorylation in WT cells (data not shown); Foxo proteins, however, were phosphorylated in a PI3K-dependent manner. The level of phosphorylation in p110{delta}D910A/D910A T cells was about one-third of that observed in WT T cells in response to anti-CD3 + anti-CD28 (compare lanes 5 and 10 in Fig. 1C; lane 5 contains a 1/3 dilution of the lysate loaded in lane 4). Taken together, these results indicate that p110{delta} is the predominant class I-A PI3K isoform in T cells, yet some residual PI3K activity is contributed by p110{alpha} and/or p110beta.

ERK and NF-{kappa}B signaling defects can be rescued by anti-CD28 costimulation

We have shown previously that in a time course of anti-CD3-stimulated T cells, ERK phosphorylation was attenuated (19). However, in CD3 + CD28-costimulated T cells, ERK phosphorylation was normal (Fig. 1B). Moreover, anti-CD3 + anti-CD28-stimulated ERK phosphorylation was resistant to inhibition with LY294002 (Fig. 1C). Hence, anti-CD28 enhances ERK phosphorylation independently of PI3K activation.

To investigate the effect of the D910A mutation on NF-{kappa}B signaling, we examined activation-dependent translocation of the p65/RelA subunit to the nucleus. Whereas translocation of p65/RelA was impaired in anti-CD3-stimulated p110{delta}D910A/D910A T cells, anti-CD28 costimulation induced levels of translocation equivalent to those in WT cells (Fig. 1D). The differential impact of p110{delta} inactivation in CD3- vs (CD3 + CD28)-dependent p65/RelA nuclear localization led us to consider whether Ag-dependent p65/RelA nuclear translocation in the presence of natural costimulatory ligands is also affected in p110{delta}D910A/D910A T cells. OT-II TCR transgenic WT or p110{delta}D910A/D910A T cells were exposed to APCs loaded with the OVA323–339 peptide, and p65/RelA nuclear localization was analyzed. Under these conditions, p110{delta} deficiency had no impact on the frequency of p65/RelA nuclear localization. Moreover, NF-{kappa}B nuclear translocation in WT T cells was unaffected by pretreatment with LY294002 (Fig. 1E). These data indicate that while Akt and Foxo phosphorylation are attenuated in the absence of p110{delta} activity, CD28 can use PI3K-independent pathways to enhance activation of the ERK and NF-{kappa}B pathways in p110{delta}D910A/D910A T cells.

Reduced T cell division and Th differentiation in the absence of p110{delta} catalytic activity

The Akt pathway has been implicated in survival and Th differentiation (27), and we therefore set out to investigate whether these parameters may be affected in p110{delta}D910A/D910A T cells. T cells from OT-II TCR transgenic p110{delta}D910A/D910A mice were labeled with CFSE to allow for concurrent analysis of cell division and apoptosis, two criteria that were not distinguished by the [3H]thymidine incorporation assays that we published previously (19). OT-II TCR transgenic p110{delta}D910A/D910A T cells proliferated less extensively than WT cells in response to OVA323–339 peptide stimulation, as determined by CFSE dilution (Fig. 2A). Importantly, we found no evidence for increased cell death of p110{delta}D910A/D910A cells in response to stimulation (Fig. 2, B and C). The reduced cell division observed may in part reflect compromised sensitivity to the dose of Ag, as p110{delta}D910A/D910A T cells stimulated with a high dose of peptide (1.0 µM) showed equivalent proliferation as WT cells stimulated with a lower dose (0.1 µM) of peptide (Fig. 2A).


Figure 2
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  		FIGURE 2. In vitro T cell proliferation and death. A, Purified and CFSE-labeled WT or p110{delta}D910A/D910A CD4+ OT-II TCR transgenic T cells were incubated with irradiated T cell-depleted spleen cells from WT mice and the indicated concentrations of OVA323–339 peptide. The CFSE profile of CD4+, V{alpha}2+, 7-AAD– cells is shown. The FlowJo-calculated cell division profiles are shown superimposed. B, Death as a function of cell division history was revealed by 7-AAD labeling of nuclei from total CD4+, V{alpha}2+ T cells. Boxed cells were considered apoptotic, as indicated in C below. C, The percentage of cell death as determined by the fraction of cells labeled with 7-AAD in B above.

 
We next determined the role of p110{delta} in CD28-dependent and CD28-independent cytokine production in purified DO11.10 TCR transgenic T cells stimulated with OVA and WT APCs. Consistent with previous results (28), IL-2 and IL-4 production in WT TCR transgenic cells were highly dependent on CD28 costimulation, as determined by their sensitivity to inhibition with CTLA4-Ig (Fig. 3A), in contrast to IFN-{gamma} production, which was less dependent on CD28 costimulation. p110{delta}D910A/D910A T cells produced substantially reduced levels of both effector cytokines IFN-{gamma} and IL-4, but were less affected in their capacity to secrete IL-2 (Fig. 3A). Similar results were obtained when cells were stimulated either with anti-CD3 + anti-CD28 or with Con A (data not shown). These results indicate the functional requirement for p110{delta} activity does not correlate directly with the requirement for CD28 costimulation, and that Th1 and Th2 cytokine production is regulated by additional PI3K-dependent processes.


Figure 3
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  		FIGURE 3. T cell differentiation in vitro. A, Purified DO11.10 T cells were stimulated with 1 µM OVA323–339. Culture supernatants were collected after 48 h and analyzed for IL-2, IL-4, and IFN-{gamma} content by ELISA. B, Purified, CFSE-labeled WT or p110{delta}D910A/D910A DO11.10 T cells were stimulated with 1 µM OVA323–339 alone (unskewed), or in combination either with IL-12 and anti-IL-4 (Th1-skewed) or IL-4, anti-IL-12, and anti-IFN-{gamma} (Th2-skewed). After 6 days, the cells were restimulated with PdBu and ionomycin for 6 h and stained with KJ1-26 and the indicated anti-cytokine mAbs. Gates were set up using isotype control mAbs, which stained <0.5% of the cells (data not shown). C, The percentage of cells producing each cytokine plotted as a function of their division history.

 
Whereas high level IL-2 production occurs before cell division, optimal transcription of the IL-4 and IFN-{gamma} gene loci requires epigenetic remodeling, which takes place after several rounds of cell division (29, 30). Therefore, the failure to produce IL-4 and IFN-{gamma} could correlate with the impaired proliferation of p110{delta}D910A/D910A T cells. To investigate this possibility, we established cultures in which CFSE-labeled OT-II T cells were stimulated by OVA323–339 peptide, with or without combinations of cytokines and Abs that promote differentiation along either Th1 or Th2 pathways. After 6 days, the T cells were restimulated with PdBu and ionomycin, and analyzed for cytokine production by intracellular FACS. Again, proliferation of p110{delta}D910A/D910A T cells was reduced compared with WT T cells (Fig. 3B). Under unskewed conditions, the IL-2 secretion profiles were not dramatically different between WT and p110{delta}D910A/D910A cells (Fig. 3B). The proportion of D910A T cells producing IFN-{gamma} was reduced by half both under unskewed and Th1-skewed conditions. IL-4-producing cells were only observed under Th2-skewed conditions and were also less abundant in p110{delta}D910A/D910A cultures. When cytokine production was analyzed as a function of each cell’s division history (Fig. 3C), it was apparent that the proportion of WT T cells that produced IL-4 or IFN-{gamma} increased gradually with each cell division, whereas the fraction of p110{delta}D910A/D910A T cells that produced IL-4 or IFN-{gamma} remained low, in contrast to IL-2, in which production was similar in WT and p110{delta}D910A/D910A T cells. These results indicate that T cells are less susceptible to undergo the epigenetic changes required for Th1 and Th2 lineage differentiation in the absence of p110{delta} activation.

p110{delta} controls clonal T cell expansion in vivo

Clonal expansion in vivo appears to be governed by factors that are in part distinct from those that apply in vitro. For instance, whereas IL-2 is a critical growth factor for T cells in vitro, in vivo proliferation of adoptively transferred T cells is not dependent on IL-2 signaling (26). Therefore, to investigate whether the impaired capacity of T cells to be stimulated in vitro also applied in vivo, DO11.10 T cells were injected into syngeneic hosts. WT and p110{delta}D910A/D910A T cells populated peripheral lymph nodes with similar efficiencies (Fig. 4A, day 0 time point), consistent with a nonessential role for p110{delta} in T cell migration (31). In response to immunization with OVA in combination with LPS, clonal expansion of p110{delta}D910A/D910A T cells was severely reduced in comparison with WT cells (Fig. 4A). Consistent with this impaired T cell expansion, CFSE staining revealed that the p110{delta}D910A/D910A T cells had undergone fewer divisions than WT T cells (Fig. 4B). This was particularly apparent 6 days after immunization, when many of the p110{delta}D910A/D910A T cells had only divided one to four times. In both genotypes, cells having undergone more than seven rounds of division were sparse, suggesting that these may have migrated out of the lymph nodes or died. One important role of adjuvants such as LPS is to increase the amount of costimulatory signals, principally, but not exclusively, through CD28 (26). Therefore, while anti-CD28 can deliver a sufficiently strong signal in vitro to rescue the proliferative defect (19), the provision of physiological costimulation in vivo is insufficient to rescue the proliferative response in p110{delta}D910A/D910A T cells. Consistent with the data shown in Fig. 3B, a lower fraction of adoptively transferred p110{delta}D910A/D910A T cells produced IFN-{gamma} than the corresponding WT T cells (Fig. 4C).


Figure 4
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  		FIGURE 4. Clonal expansion of T cells in vivo. Adoptively transferred CFSE-labeled DO11.10 T cells were harvested just before (day 0) or 3 or 6 days after immunization of mice with OVA and LPS. A, Total number of KJ1-26+CD4+ T cells recovered from the draining lymph nodes before or 3 or 6 days postimmunization. Three mice of each genotype were analyzed on day 0, and four of each genotype on days 3 and 6. Error bars indicate SD. B, The division history revealed by CFSE dilution profiles of CD4+KJ1-26+ T cells recovered from the draining lymph nodes 3 or 6 days postimmunization. C, The fraction of CD4+KJ1-26+ T cells producing IFN-{gamma} 6 days postimmunization. The percentage of divided cells that produced IFN-{gamma} is indicated (±SD; n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we provide the first direct assessment of the role of p110{delta} isoform of PI3K in Th cell differentiation and clonal expansion in vivo. Whereas anti-CD3 and anti-CD28 triggered normal proliferation in vitro, p110{delta}D910A/D910A T cells proliferated less extensively in response to physiological costimulation provided by APCs in vitro and in vivo. These differences could relate to several factors. Anti-CD28 ligation using Abs in vitro can stimulate signals that cannot be induced by the physiological ligands (32). Although the capacity of CD28 to rescue some signals appears to be PI3K independent, the PI3K signaling pathway may be required by other costimulatory receptors such as ICOS and OX40, and these can contribute to clonal expansion and differentiation in vivo (33, 34). A failure of p110{delta}D910A/D910A T cells to form functional conjugates with APCs or a state of anergy in a population of p110{delta}D910A/D910A T cells may also help explain the reduced response to stimulation with APCs. Each of these possibilities is currently under investigation.

In the presence of physiological costimulus and exogenous cytokines in vitro, p110{delta}D910A/D910A T cells showed reduced differentiation along the Th1 and Th2 lineages. A number of PIP3-dependent kinases have also been implicated in the regulation of Th1 and Th2 differentiation: Itk and Pdk1 were reported to be required for Th2 differentiation (17, 35), whereas Akt has been shown to promote both Th1 and Th2 differentiation (36, 37). Our results are most consistent with PI3K signaling being required for both Th1 and Th2 differentiation, without preferential skewing toward either differentiation pathway.

In Caenorhabditis elegans, a mutation of the PI3K gene age-1 prevents the worm from exiting the dauer stage, a state of low metabolism that is actively maintained by daf-16, an ortholog of the Foxo transcription factors in mammals. Akt phosphorylates Foxo, leading to the sequestration and inactivation of these transcription factors in the cytoplasm. The longevity and low metabolism that characterize the dauer stage are reminiscent of naive or anergic T cells in the G0 stage of the cell cycle (38). Foxo3a-deficient T cells have been described recently, and their phenotype contrasts sharply with that of p110{delta}D910A/D910A T cells. Indeed, Foxo3a–/– T cells proliferate extensively and produce excessive amounts of Th1 and Th2 cytokines (39). Hence, the PI3K signaling pathway may in part be an evolutionary conserved switch that permits cells to exit from a resting state and undergo differentiation to more metabolically active, terminally differentiated states.

In summary, the data presented in this work demonstrate that p110{delta} controls a critical checkpoint in peripheral T cell differentiation and clonal expansion. These observations indicate that p110{delta} may be a good target for pharmaceutical intervention of T cell-dependent autoimmune pathologies.


   Acknowledgments
 
We thank Juliet Emery, Adam Hales, and Emma Peskett for expert assistance; Dr. Giorgio Trinchieri for the anti-IL-12 hybridoma; and Robert Salmond, Khaled Ali, Francesco Colucci, and Martin Turner for critical reading of 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 grants from the Biotechnology and Biological Sciences Research Council (to K.O. and B.V.), the Ludwig Institute for Cancer Research, and European Union Framework V (to B.V.). K.O. is a Biotechnology and Biological Sciences Research Council David Phillips Fellow. D.T.P. was supported by a Medical Research Council and GlaxoSmithKline Co-Operative Award in Science and Engineering. Back

2 Address correspondence and reprint requests to Dr. Klaus Okkenhaug, Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Babraham Research Campus, Cambridge, CB2 4AT, U.K. E-mail address: klaus.okkenhaug{at}bbsrc.ac.uk Back

3 Abbreviations used in this paper: LAT, linker for activation of T cells; 7-AAD, 7-aminoactinomycin D; PdBu, phorbol-12,13-dibutyrate; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; WT, wild type. Back

Received for publication October 12, 2005. Accepted for publication August 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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