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Molecular and Cellular Biology, January 2007, p. 79-91, Vol. 27, No. 1
0270-7306/07/$08.00+0     doi:10.1128/MCB.00799-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and Pre-T-Cell Receptor Signaling{triangledown}

Jr-Wen Shui,1 Mickey C.-T. Hu,2 and Tse-Hua Tan1*

Department of Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030,1 Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 770302

Received 5 May 2006/ Returned for modification 18 June 2006/ Accepted 4 October 2006


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ABSTRACT
 
Okadaic acid-sensitive serine/threonine phosphatases have been shown to regulate interleukin-2 transcription and T-cell activation. Okadaic acid inhibits protein phosphatase 4 (PP4), a novel PP2A-related serine/threonine phosphatase, at a 50% inhibitory concentration (IC50) comparable to that for PP2A. This raises the possibility that some cellular functions of PP2A, determined in T cells by using okadaic acid, may in fact be those of PP4. To investigate the in vivo roles of PP4 in T cells, we generated conventional and T-cell-specific PP4 conditional knockout mice. We found that the ablation of PP4 led to the embryonic lethality of mice. PP4 gene deletion in the T-cell lineage resulted in aberrant thymocyte development, including T-cell arrest at the double-negative 3 stage (CD4– CD8– CD25+ CD44–), abnormal thymocyte maturation, and lower efficacy of positive selection. PP4-deficient thymocytes showed decreased proliferation and enhanced apoptosis in vivo. Analysis of pre-T-cell receptor (pre-TCR) signaling further revealed impaired calcium flux and phospholipase C-{gamma}1-extracellular signal-regulated kinase activation in the absence of PP4. Anti-CD3 injection in PP4-deficient mice led to enhanced thymocyte apoptosis, accompanied by increased proapoptotic Bim but decreased antiapoptotic Bcl-xL protein levels. In the periphery, antigen-specific T-cell proliferation and T-cell-mediated immune responses in PP4-deficient mice were dramatically compromised. Thus, our results indicate that PP4 is essential for thymocyte development and pre-TCR signaling.


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INTRODUCTION
 
PP4 (previously named PPX) is a novel, PP2A-related, okadaic acid-sensitive, serine/threonine protein phosphatase that shares 65% amino acid identity with PP2A. Many studies have shown that okadaic acid-sensitive protein phosphatases are involved in the regulation of T-cell signaling and activation. Phorbol myristate acetate- and ionomycin-induced interleukin-2 (IL-2) promoter activity is inhibited by okadaic acid, suggesting that an okadaic acid-sensitive serine/threonine phosphatase is required for IL-2 gene activation in T cells (23). Sp1, a transcription factor involved in T-cell growth and gene regulation, is regulated by PP2A in dividing human T lymphocytes (18). Furthermore, okadaic acid-sensitive protein phosphatases have been implicated in the upregulation of STAT3 activity and IL-1{alpha} gene expression in T cells (26). PP2A is also implicated in the regulation of IL-4-induced STAT6 signaling, which is involved in T helper 2 (Th2) differentiation and B-cell isotype switching to immunoglobulin E (IgE) (25). Results derived from the above-described studies seem conclusive; however, all of these T-cell-related studies have relied upon the use of the PP2A inhibitor okadaic acid, which has recently been shown to inhibit PP4 with a 50% inhibitory concentration (0.01 nM in vitro) comparable to that for PP2A (9). Therefore, it is plausible that some of these identified functions assigned to PP2A by using okadaic acid may in fact be functions of PP4.

Recent studies indicate that PP4 has various cellular functions that are distinct from PP2A activity. PP4 interacts with c-Rel/RelA, stimulates the DNA-binding activity of c-Rel, and activates NF-{kappa}B-mediated transcription (12). Furthermore, PP4 dephosphorylates RelA at Thr-435, and this dephosphorylation is required for NF-{kappa}B activation induced by cisplatin (28). PP4 interacts with and downregulates insulin receptor substrate 4 following tumor necrosis factor alpha stimulation (21) and is involved in tumor necrosis factor alpha-induced activation of the Jun N-terminal protein kinase signaling pathway (31). Furthermore, PP4 physically associates with, dephosphorylates, and inhibits histone deacetylase 3 activity (29). Saccharomyces cerevisiae strains mutated for Pph3 (PP4 ortholog), but not Pph22 (PP2A ortholog), become hypersensitive to the cancer drug cisplatin, suggesting a role for PP4 in DNA damage signaling (5). In yeast, Pph3, but not Pph22, dephosphorylates {gamma}H2AX, whose posttranslational modification is an early hallmark following a double-strand break (15). PP4 interacts with and regulates HPK1, a mitogen-activated protein kinase upstream regulator, in a T-cell receptor (TCR)-dependent manner (30), indicating that PP4 may be a novel serine/threonine phosphatase involved in T-cell signaling.

By conditional inactivation of PP4 in the T-cell lineage, aberrant thymocyte development, including double-negative 3 stage (DN3) cell arrest and decreased positive selection, was detected. Further analysis showed cell accumulation in both the early (E) (small-cell) and the late (L) (large-cell) stages of DN3, which suggests impaired E DN3 differentiation and L DN3 proliferation, respectively. We also found that PP4 ablation led to impaired calcium and phospholipase C-{gamma}1 (PLC-{gamma}1)-extracellular signal-regulated kinase (ERK) signaling and decreased thymocyte survival (less proliferation and more apoptosis) in vivo. In PP4 conditional knockout mice, anti-CD3-induced thymocyte apoptosis was enhanced, accompanied by increased proapoptotic Bim but decreased antiapoptotic Bcl-xL protein levels. PP4 gene deletion also resulted in impaired antigen-induced T-cell proliferation and T-cell-dependent immune responses. Taken together, our results demonstrate that PP4 is a novel regulator of thymocyte development and pre-TCR signaling.


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MATERIALS AND METHODS
 
Generation of T-cell-specific conditional PP4 knockout mice. To generate PP4+/flox mice, we used the gene-targeting vector with which the two-loxP and two-frt strategy was employed to obtain homologous recombination in embryonic stem (ES) cells. After successful germ line transmission, PP4+/flox-frt-neo mice were crossed with ß-actin-driven Saccharomyces cerevisiae enhanced FLP1 recombinase variant (FLPe) deleter mice (The Jackson Laboratory) to facilitate in vivo frt-neo deletion. The PP4flox/flox (F/F will be used herein to indicate PP4flox/flox for simplicity) mice were further crossed with Lck-Cre transgenic mice (The Jackson Laboratory) to generate Lck-Cre; PP4flox/flox mice. All mice were genotyped by Southern blotting using a PP4-specific probe, indicated in Fig. 1. The OT-II transgenic mice were obtained from The Jackson Laboratory. All animals were maintained in accordance with the Baylor College of Medicine guidelines for laboratory animal research.


Figure 1
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  		FIG. 1. PP4 gene conditional deletion in the T-cell lineage. (A) Generation of neo-free PP4-floxed mice by the two-loxP, two-frt strategy. The frt-flanked neo marker was removed from PP4+/flox-frt-neo mice by crossing them with FLPe deletion mice. Lck-Cre transgenic mice were used to generate T-cell-specific PP4 conditional knockout mice. Genomic DNA fragments after EcoRI digestion are indicated. E, EcoRI; H, HindIII; A, ApaI; EV, EcoRV; Nh, NheI; N, NdeI; P, PstI; DTA, diphtheria toxin A. (B) Efficient Cre-mediated PP4 gene deletion in thymuses of T-cell-specific conditional knockout mice. Genomic DNA was prepared and subjected to Southern blotting with EcoRI digestion. Wild-type alleles (WT; 6.2 kb), null alleles (deleted; 3.6 kb), and floxed alleles (flox; 2.8 kb) are indicated. (C) Loss of PP4 protein expression in PP4-deficient thymocytes. Lysates were prepared from thymocytes and analyzed by Western blotting using the PP4-specific antibody. WB, Western blotting. (D) Generation of a reading frame shift and a stop codon in exon 4 of PP4 after exon 3 deletion mediated by loxP-mediated DNA recombination. aa, amino acid.

Flow cytometry analysis. Single-cell suspension samples were prepared by depletion of red blood cells. Cells were stained with different fluorescence-labeled and isotype control antibodies in phosphate-buffered saline (PBS) containing 3% fetal bovine serum. After being stained for 20 min, cells were washed twice and fixed in PBS containing 1% paraformaldehyde. Labeled cells were subsequently analyzed on a FACSCalibur cytometer using Cellquest software (Becton Dickinson). Anti-CD3{varepsilon} (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-Vß5 (MR9-4), anti-B220 (RA3-6B2), and anti-TCRß (H57-597) antibodies were purchased from BD Pharmingen.

T-cell proliferation assay and in vivo BrdU labeling. For in vitro T-cell proliferation, 3 x 105 lymph node T cells were placed in each well of a 96-well plate in Click's medium (Sigma) containing 0.5% mouse serum. Anti-CD3{varepsilon} (145-2C11) was used to stimulate the T cells. Cells were cultured for 72 h, pulsed with 1 µCi/well [3H]thymidine for the final 16 h, harvested, and counted. For cell division assays, cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; 3 µM; Molecular Probes) for 15 min; labeling was stopped by adding fetal calf serum, and the cells were washed with PBS. Cells were then treated with anti-CD3{varepsilon} (1 µg/ml) alone or anti-CD3{varepsilon} plus anti-CD28 (1 µg/ml) antibodies and analyzed by flow cytometry 2 days later. For analysis of thymocyte proliferation, mice received one intraperitoneal injection of bromodeoxyuridine (BrdU; 1 mg) and were sacrificed 1 h later. Cells were harvested, fixed, permeabilized, and stained with anti-BrdU, anti-CD4, and anti-CD8 antibodies (BrdU flow kits; BD Pharmingen).

Calcium flux assay. Thymocytes were loaded with 1 µM Fluo-4 and 2 µM Fura Red in serum-free RPMI medium containing 0.2% Pluronic F-127 (Molecular Probes) for 30 min at room temperature. Cells were then washed twice, allowed to rest in the dark for 15 min, and incubated on ice with biotin-conjugated anti-CD3 (15 µg/ml) and biotin-conjugated anti-CD4 (8 µg/ml) antibodies (BD Pharmingen) for 15 min. Labeled cells were washed, stimulated by adding streptavidin, and analyzed by flow cytometry to obtain the ratio of Fluo-4 to Fura Red.

In vivo and in vitro apoptosis assay. Six- to 8-week-old control and PP4-deficient mice (four mice per group) were injected intraperitoneally with 10 µg PBS or 30 µg of anti-CD3{varepsilon} antibody (145-2C11; BD Pharmingen). After 48 h, mice were sacrificed and thymocytes were prepared and subjected to flow cytometry analysis. For activation-induced cell death, thymocytes were stimulated with anti-Fas (Jo2), plate-bound anti-CD3 (145-2C11), or dexamethasone (Sigma) for 24 h to induce apoptosis. Cell viability was determined using annexin V and a 7-amino-actinomycin D staining kit (BD Pharmingen).

Immunoblotting. Cells were lysed in radioimmunoprecipitation buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 0.1% deoxycholate, 0.1% sodium dodecyl sulfate, and mixtures of protease and phosphatase inhibitors. Proteins were transferred to nitrocellulose, and the membrane was probed with specific antibodies. Where indicated, membranes were stripped and reprobed with specific antibodies to show equal loadings of lysates. Anti-ß-actin, anti-{gamma}-tubulin, anti-ERK, anti-Src, anti-ZAP-70, anti-PLC-{gamma}1, anti-Bax, anti-Bcl-xL, anti-A1, and anti-PP4 antibodies were purchased from Santa Cruz. Anti-Bim, anti-phospho-Src, anti-phospho-ZAP-70, and anti-phospho-ERK antibodies were purchased from Cell Signaling Technology. Anti-Bad antibody was purchased from BD Transduction Laboratories. Anti-Bcl-2 antibody was purchased from Upstate Biotechnology.

Antigen immunization and enzyme-linked immunosorbent assay analysis for antibody. For in vivo antigen-specific T-cell proliferation, control and PP4-deficient mice (eight mice per group) were immunized subcutaneously at the base of their tails with keyhole limpet hemocyanin (KLH; 150 µg) emulsified in complete Freund's adjuvant (CFA; Sigma). Enlarged draining lymph nodes were isolated 7 days later and restimulated with the concentrations of antigen indicated in Fig 9. [3H]thymidine incorporation was performed to measure antigen-induced T-cell proliferation. For T-cell-mediated humoral immune responses, mice (10 mice per group) were immunized by intraperitoneal injection of alum-precipitated dinitrophenyl (DNP)-KLH in PBS. Serum samples were collected at day 14 after immunization. Nitrophenol-specific antibodies were detected by enzyme-linked immunosorbent assay using different isotypes of horseradish peroxidase-conjugated goat anti-mouse antibodies (Sigma). Horseradish peroxidase activity was visualized using a 3,3',5,5'-tetramethylbenzidine peroxidase substrate kit (Bio-Rad).


Figure 9
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  		FIG. 9. PP4 is essential for maintenance of peripheral T-cell cellularity and T-cell-dependent immune functions. (A) Significant reductions in CD3+, CD4+, and CD8+ T cells were detected in peripheral T-cell compartments of Lck-Cre; F/F mice (representative results for 6 mice). SP, spleen; LN, lymph node. (B and C) Analysis of anti-CD3-induced cell proliferation with [3H]thymidine incorporation assay and CFSE labeling. For CFSE analysis, splenic T cells were labeled with CFSE before stimulation. CD4+ T cells were gated and analyzed for fluorescence intensity after 48 h. (D) Decreased antigen-specific T-cell proliferation in PP4-deficient mice. Mice were immunized subcutaneously with KLH-CFA. Sensitized draining lymph node T cells were restimulated with KLH, and proliferation was determined by the [3H]thymidine incorporation assay. (E) Decreased humoral immune responses in PP4-deficient mice. Primary immune response was evaluated by measuring various nitrophenol (NP)-specific antibody titers at 14 days after immunization with alum-precipitated DNP-KLH. The results are representative of two independent experiments.


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RESULTS
 
PP4 ablation at the early developmental stage resulted in a T-cell developmental block and impaired thymocyte maturation. Okadaic acid-sensitive protein phosphatases, including PP2A and PP4, are known to regulate T-cell activation and signaling. To gain more insight about how PP4 is involved in T-cell development and signaling, we generated PP4 knockout mice by homologous recombination in mouse ES cells. We found that the loss of PP4 led to the embryonic lethality of mice at the early embryonic stage, and we could not identify any PP4-deficient embryos when they were dissected as early as embryonic day 9.5 (data not shown). This indicates that PP4 is an gene essential for embryo development. Since the early lethality of PP4-deficient embryos impeded further analysis, we generated mice carrying a germ line-transmitted PP4-floxed allele using Cre-loxP and FLP-frt gene recombination systems (Fig. 1A). To avoid gene interference and genetic ambiguity caused by the presence of the neomycin selection marker in the PP4-floxed allele, PP4+/flox-frt-neo mice were crossed with FLP transgenic mice to generate neo-free PP4+/flox mice (Fig. 1A). Next, PP4+/flox mice were crossed with Lck-Cre transgenic mice to facilitate PP4 gene deletion in the T-cell lineage under the control of the T-cell-specific Lck promoter (7). In Lck-Cre; PP4flox/flox mice, efficient Cre-mediated PP4 gene deletion was detected in thymocytes, as determined by Southern blotting (Fig. 1B, lanes 6 to 8) and Western blotting (Fig. 1C). Based on the genomic organization of the mouse PP4 gene (11), deletion of PP4 exon 3 after Cre-mediated DNA recombination of the PP4-floxed allele will create a stop codon in exon 4 due to a reading frameshift, resulting in a 50-amino-acid, PP4-truncated mutant protein (Fig. 1D).

We examined thymocyte development in T-cell-specific PP4 conditional knockout mice. We first compared thymocyte populations in Lck-Cre, Lck-Cre; PP4+/flox (Lck-Cre; +/F), PP4flox/flox (F/F), and wild-type mice but did not find any significant differences (data not shown). Next, we found that in Lck-Cre; F/F mice, PP4 ablation led to a significant decrease in unfractionated CD4+ CD8+ (double-positive [DP]), CD4+ CD8– (CD4 single-positive [CD4 SP]), and CD4– CD8+ (CD8 single-positive [CD8 SP]) thymocyte numbers compared to those of F/F littermates (Fig. 2A). In contrast, total numbers of CD4– CD8– (double-negative [DN]) cells in Lck-Cre; F/F mice remained comparable to those of control littermates, which led to higher percentages of DN populations in PP4-deficient mice (Fig. 2A). Thus, PP4 ablation resulted in a T-cell developmental block specifically at the DN stage, which was accompanied by an approximately 70% decrease in DP cell numbers compared to those of control mice.


Figure 2
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  		FIG. 2. Aberrant thymocyte development in T-cell PP4 conditional knockout mice. (A) Thymocytes were prepared from mice (>15 per group), stained with anti-CD4 and anti-CD8 antibodies, and analyzed by flow cytometry. Absolute cell numbers were calculated to determine cellularity. (B) Developmental arrest of thymocytes at the DN3 stage in T-cell PP4 conditional knockout mice. DN thymocytes were gated and analyzed by CD25/CD44 expression. Absolute cell numbers and flow cytometry results are shown. PP4 ablation led to developmental arrest at DN3 (CD4– CD8– CD25+ CD44–).

The most immature CD4– CD8– DN thymocytes can be subdivided into four developmental stages in the following order: CD25– CD44+ (DN1), CD25+ CD44+ (DN2), CD25+ CD44– (DN3), and CD25– CD44– (DN4). When DN thymocytes were gated and analyzed, a significant accumulation of DN3 thymocytes (approximately a threefold increase) and a corresponding decrease of DN4 cells were identified in Lck-Cre; F/F mice (Fig. 2B and 3B). Although the percentage of DN4 cells among DN cells in Lck-Cre; PP4flox/flox mice decreased by about 20% as a result of the increase in the percentage of DN3 cells (Fig. 2B, right-panel), we did not observe a dramatic reduction in the numbers of DN4 cells, due to the fact that Lck-Cre; PP4flox/flox mice consistently have more total DN cells (Fig. 2A, left-panel). Therefore, this observation indicates a developmental block specifically at the DN3 stage in PP4 conditional knockout mice. The DN3 cell population can be further subdivided into E and L cells based on cell size, determined by their forward versus side scatter parameters (8, 10). Successful TCR gene rearrangement and functional pre-TCR signaling are required for E DN3 cell maturation into L DN3 cells (TCR ß selection), which will undergo proliferation and proceed to the DN4 stage (10, 14). In Lck-Cre; F/F mice, where Cre-mediated DNA recombination was shown to end at the DN3 stage (27), we found that E DN3 cell numbers in the CD44low CD25+ DN3 cell population increased (about 3.3-fold), further suggesting a cell arrest at this stage which is likely due to an impaired differentiation of E DN3 cells and, potentially, dysregulated pre-TCR signaling (Fig. 3A and B). Surprisingly, the L DN3 cell populations and their corresponding percentages among DN3 cells were significantly increased in Lck-Cre; F/F mice (Fig. 3A and B). The increase in L DN3 cell numbers (approximately 5.2-fold) also indicates a developmental block which is likely due to a proliferation defect between the L DN3/DN4 and DP stages (14). Taking these findings together, we conclude that PP4 ablation in thymocytes leads to cell arrest at the DN3 stage (Fig. 3B) which is associated with impaired differentiation of E DN3 cells and defective proliferation of L DN3 cells.


Figure 3
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  		FIG. 3. DN3 developmental block and aberrant thymocyte maturation in T-cell-specific PP4 conditional knockout mice. (A) Cell arrest at E DN3 and L DN3 in T-cell PP4 conditional knockout mice. The CD44low CD25+ cells (DN3) in the CD4– CD8– population were gated to show their forward versus side scatter for 20,000 events. E represents the small DN3 thymocytes and L represents the large DN3 thymocytes. (B) A bar graph comparing cell numbers (knockout/wild type [KO/WT]) of different thymocyte subpopulations is shown to indicate cell arrest at DN3. (C) Impaired thymocyte maturation in the absence of PP4. Thymocytes were stained by anti-CD3 and anti-TCRß antibodies and analyzed by flow cytometry. Mature thymocytes (TCRßhi CD3hi) are indicated. (D) TCR surface expression levels of each thymocyte subpopulation. Thymocytes were isolated from mice and stained for the TCRß chain and CD4 and CD8 expression. TCRß expression levels are indicated as mean fluorescence intensity (MFI).

Besides a DN3 developmental block, we also found that Lck-Cre; F/F mice have a decreased percentage of TCRßhi CD3hi mature thymocytes (5 to 6%, compared to 9 to 12% for control littermates) as well as decreased TCRßhi CD3hi cell numbers (Fig. 3C). We further analyzed and compared the TCRß expression levels of different thymocyte subpopulations in Lck-Cre; F/F and control mice but did not find any significant differences (Fig. 3D). Thus, the possibility that altered surface TCR levels in PP4-deficient thymocytes may affect pre-TCR signaling is excluded. Taken together, our results indicate that PP4 plays an essential role in early T-cell development, specifically at the E DN3-to-L DN3 and L DN3/DN4-to-DP transition stages.

Impaired cell survival of PP4-deficient thymocytes. The reduced thymus cellularity, developmental DN3 arrest, and impaired DN-to-DP transition detected in PP4-deficient mice prompted us to further examine the cell survival of Lck-Cre; F/F thymocytes. In vivo BrdU labeling was used to determine cell proliferation (19). We found that Lck-Cre; F/F thymocytes showed a decrease in overall BrdU incorporation (Fig. 4, BrdU panels). When thymocytes were gated by CD4 and CD8 expression, a significant decrease in BrdU signals was identified in the DP cell populations of Lck-Cre; F/F mice compared to those of control mice, indicating that PP4-deficient thymocytes were less proliferative in vivo. The reduced percentages of BrdU+ DN cell populations in Lck-Cre; F/F mice also support the notion that L DN3 cells are impaired in cell proliferation, resulting in their accumulation during development (Fig. 3A). In addition, increased percentages of annexin V+ cells were identified in DP and CD4 SP populations of Lck-Cre; F/F mice, suggesting that PP4-deficient thymocytes were more apoptotic in vivo (Fig. 4, annexin V panels). Thus, we conclude that PP4 gene deletion in thymocytes led to impaired cell survival by affecting both cell proliferation and apoptosis, which may contribute to the perturbation of thymocyte development and maturation leading to developmental cell arrest.


Figure 4
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  		FIG. 4. PP4-deficient thymocytes were less proliferative in vivo. (BrdU panels) Mice (4 per group) received one intraperitoneal injection of BrdU (1 mg) and were sacrificed 1 h later, and thymocytes were stained with anti-BrdU, anti-CD4, and anti-CD8 antibodies. Numbers are the percentages of BrdU-positive cells in gated DN, DP, CD4 SP, and CD8 SP subpopulations. (Annexin V panels) PP4-deficient thymocytes were more apoptotic in vivo. Thymocytes were isolated and stained with anti-annexin V, anti-CD4, and anti-CD8 antibodies. Numbers are the percentages of annexin V-positive cells in gated DN, DP, CD4 SP, and CD8 SP subpopulations. Representative data are shown. FC, fluorescein isothiocyanate.

Impaired positive selection and pre-TCR signaling of thymocytes in the absence of PP4. The reduction in DP and SP thymocyte numbers in Lck-Cre; F/F mice suggests potential defects in thymocyte selection. Because the levels of TCRß chain and CD69 are upregulated with positive selection (4), we monitored the percentage of cells undergoing positive selection by gating CD69+ TCRßhi cells. We found that the percentages of CD69+ TCRßhi cells in Lck-Cre; F/F mice decreased by approximately 60% compared to those in F/F littermates (Fig. 5A). This finding correlated with the reduction in the numbers of mature CD4 and CD8 thymocytes (CD4+ TCRßhi and CD8+ TCRßhi, respectively) in Lck-Cre; F/F mice (Fig. 5B). In addition, the CD4/CD8 ratio was not significantly affected in the absence of PP4 (Fig. 5B). Taken together, the reductions in the numbers of CD69+ TCRßhi subpopulations and mature CD4+ TCRßhi and CD8+ TCRßhi cells indicate that PP4 ablation leads to decreased efficacy of positive selection.


Figure 5
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  		FIG. 5. PP4 gene deletion leads to impaired positive selection. (A) Decreased CD69+ TCRßhi cell populations in T-cell-specific PP4 conditional knockout mice. Thymocytes were stained with anti-CD69 and anti-TCRß antibodies and analyzed by flow cytometry. (B) Decreased numbers of mature CD4+ TCRßhi and CD8+ TCRßhi thymocytes in T-cell-specific PP4 conditional knockout mice. Thymocytes were stained with anti-CD4, anti-CD8, and anti-TCRß antibodies, and the percentages of CD4+ TCRßhi and CD8+ TCRßhi cell populations are shown. Absolute CD4+ TCRßhi and CD8+ TCRßhi cell numbers as well as CD4/CD8 ratios were calculated. FC, fluorescein isothiocyanate; PE, phycoerythrin; TC, tricolor or PE-cyanine dye 5.

To further analyze thymocyte-positive selection in T-cell-specific PP4 conditional knockout mice, we crossed Lck-Cre; F/F mice with OT-II (ovalbumin-specific) transgenic mice that expressed the class II-restricted V{alpha}2/Vß5 TCR transgene (22). Like Lck-Cre; F/F mice, OT-II; Lck-Cre; F/F mice showed aberrant thymocyte development compared to their control littermates, with significant decreases in the numbers of unfractionated, DP, and SP thymocytes, but comparable numbers of DN cells (Fig. 6A). The functional TCR transgene expressed on thymocytes in OT-II mice favors positive selection of OT-II TCR-expressing CD4+ cells (22). We found that OT-II; F/F mice showed a significant increase in the percentage of CD4 SP cells (about 26%, compared to the usual 7 to 9% in control mice), which is likely due to more positively selected OT-II CD4 SP cells (Fig. 6B, left panel). However, compared to OT-II; F/F mice, OT-II; Lck-Cre; F/F mice showed only a marginal increase in the percentage of CD4 SP cells (Fig. 6B) (11.5%, versus 26.1% for control mice). To determine whether the reduction in the numbers of CD4 SP thymocytes is due to a decreased efficacy of the positive selection of OT-II cells or simply due to a decreased number of DP thymocytes in PP4-deficient mice, a CD4 SP/DP ratio was calculated such that the numbers of CD4 SP cells, after normalization to the numbers of DP cells, would more accurately reflect the efficacy of positive selection. Consistently, a decrease in positive selection of OT-II CD4 SP cells in OT-II PP4-deficient mice was observed, with a CD4 SP/DP ratio of 0.29, versus 0.46 for OT-II control mice (Fig. 6B). This result indicates a reduced positive selection of OT-II cells in OT-II PP4 conditional knockout mice. We further used the anti-Vß5 antibody to monitor OT-II-expressing (or Vß5-expressing) thymocytes for positive selection in OT-II mice (22). Virtually all CD4 SP thymocytes in both OT-II; F/F and OT-II; Lck-Cre; F/F mice express the Vß5 transgene (Fig. 6C, lower panels). Again, OT-II; Lck-Cre; F/F mice showed less positive selection of CD4 SP Vß5-expressing cells (Fig. 6C, upper panels) (8.9% Vß5+ cells versus 24.4% for control mice). After normalization to CD3+ cells, we found that in OT-II PP4 conditional knockout mice, CD4 SP Vß5+ cells were less-positively selected, with a Vß5/CD3 ratio of 0.45, compared to 0.86 for control mice (Fig. 6C, upper panels). Taken together, our results lead us to conclude that PP4 gene deletion leads to a decreased efficacy of positive selection during T-cell development.


Figure 6
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  		FIG. 6. Analysis of positive selection in OT-II PP4-deficient mice. (A) Decreased cellularity of thymuses in OT-II PP4-deficient mice. (B) Decreased efficacy of positive selection of OT-II thymocytes in OT-II T-cell-specific PP4 conditional knockout mice. Thymocytes from indicated mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. The ratio of CD4 SP cells to DP cells (with normalization of CD4 SP cell numbers to DP cell numbers) was calculated to determine the efficacy of positive selection. (C) OT-II (Vß5+) thymocytes are less-positively selected in OT-II T-cell-specific PP4 conditional knockout mice. Thymocytes from indicated mice were stained with anti-CD3, anti-CD4, anti-CD8, and anti-Vß5+ TCR antibodies and analyzed. The ratio of Vß5 to CD3 cells was calculated to determine the efficacy of positive selection for Vß5+ cell populations, with normalization to total DP cell numbers. PE, phycoerythrin; FC, flucytosine.

To investigate at the molecular level how PP4 is involved in positive selection, we examined pre-TCR signaling, which is known to play a major role in positive selection, in PP4-deficient thymocytes stimulated with anti-CD3 plus anti-CD4 antibodies. While activation/phosphorylation of Lck and ZAP-70 was unaffected in PP4-deficient thymocytes (Fig. 7A and B), we found that tyrosine phosphorylation of PLC-{gamma}1, an upstream regulator of calcium mobilization, was dramatically decreased after pre-TCR cross-linking (Fig. 7C). Subsequent analysis confirmed that PP4-deficient thymocytes failed to flux calcium after anti-CD3 and anti-CD4 cross-linking but that ionomycin treatment resulted in a comparable calcium release (Fig. 7D). Since ERK activity has been shown to play essential roles in positive selection during T-cell development (4), we probed ERK activation in stimulated PP4-deficient thymocytes. Consistent with the reduction in CD69+ TCRßhi cell numbers and in PLC-{gamma}1 tyrosine phosphorylation, we found that ERK activation also decreased after anti-CD3 and anti-CD4 treatment of PP4-deficient thymocytes (Fig. 7E). Therefore, our results indicate that PP4 ablation in thymocytes results in impaired calcium flux and reduced ERK activation during pre-TCR signaling that may subsequently disrupt positive selection of thymocytes during T-cell development.


Figure 7
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  		FIG. 7. PP4 gene deletion leads to impaired pre-TCR signaling. Tyrosine phosphorylation of Lck (A) and ZAP-70 (B) was not affected in PP4-deficient thymocytes stimulated with anti-CD3 and anti-CD4. Cell lysates from stimulated thymocytes were analyzed by Western blotting, and the membranes were probed with the anti-phospho-Lck and anti-phospho-ZAP-70 antibodies, respectively. (C) Decreased tyrosine phosphorylation of PLC-{gamma}1 in PP4-deficient thymocytes. Lysates prepared from stimulated thymocytes were immunoprecipitated with the anti-PLC-{gamma}1 antibody, and tyrosine phosphorylation was measured by probing the membrane with antiphosphotyrosine antibody. IP, immunoprecipation. (D) Impaired calcium mobilization in response to TCR cross-linking in PP4-deficient thymocytes. Thymocytes were labeled with calcium chelators, and calcium flux was measured after anti-CD3 and anti-CD4 cross-linking. (E) Decreased ERK activation in stimulated PP4-deficient thymocytes. Cell lysates were analyzed by Western blotting, and the membranes were probed with the anti-phospho-ERK antibody, followed by reprobing of the membrane with the anti-total ERK antibody. p, phospho; WB, Western blotting.

PP4 plays a role in thymocyte apoptosis. The decrease in thymocyte cell numbers, defective thymocyte proliferation, and the increase in the annexin V+ cell percentage found in PP4 conditional knockout mice may suggest that PP4-deficient thymocytes are more susceptible to apoptosis. We tested this possibility by treating wild-type and PP4-deficient thymocytes with different apoptosis-inducing stimuli and subsequently measuring cell viability after 24 h. We found that PP4-deficient thymocytes showed an increase in apoptosis in response to in vitro anti-CD3 or anti-Fas antibody treatment, while similar responses to dexamethasone were detected in both wild-type and PP4-deficient thymocytes (Fig. 8A). To extend our analysis, we examined in vivo thymocyte viability after apoptosis induction by administration of anti-CD3 antibody, which is known to induce cell death of immature thymocytes, to the mice (24). In PP4-deficient mice, we found that in vivo anti-CD3-induced thymocyte depletion was more vigorous, in a dose-dependent manner, than that in control mice (Fig. 8B). We further investigated whether any antiapoptotic or proapoptotic proteins are involved in PP4-regulated thymocyte apoptosis, since several proteins, such as Bim, Bcl-xL, and A1, are known to play a role in thymocyte apoptosis. After anti-CD3-induced apoptosis, we found that PP4-deficient thymocytes, compared to control thymocytes, upregulated proapoptotic Bim protein levels but showed comparable levels of two other proapoptotic proteins, Bad and Bax (Fig. 8C). Furthermore, PP4-deficient thymocytes showed lower antiapoptotic Bcl-xL protein levels but had comparable levels of two other antiapoptotic proteins, Bcl-2 and A1 (Fig. 8D). Taken together, our results indicate that PP4 regulates thymocyte apoptosis in a signaling pathway involving the proapoptoic protein Bim and the antiapoptotic protein Bcl-xL.


Figure 8
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  		FIG. 8. PP4 regulates thymocyte apoptosis. (A) Susceptibility of thymocytes to cell death induced by different stimuli. Thymocytes were treated with the indicated concentration of anti-Fas antibody, plate-bound anti-CD3 antibody, or dexamethasone (Dex) for 24 h. Cell viability was calculated based on the percentages of the annexin V– and 7-amino-actinomycin D– double-negative populations. The results are representative of two independent experiments. (B) In vivo susceptibility of DP thymocytes to cell depletion induced by anti-CD3 antibody administration. Mice (three per group) were injected intraperitoneally with PBS or the indicated amount of the anti-CD3 antibody. After 20 h, CD4+ CD8+ populations were analyzed by flow cytometry to determine their viability. (C) Upregulation of the proapoptotic Bim protein levels in PP4-deficient thymocytes isolated from anti-CD3-injected mice. Thymocyte lysates were prepared from anti-CD3-injected mice and subjected to Western blotting with different proapoptotic antibodies, including anti-Bim, anti-Bad, and anti-Bax antibodies. BimEL, extralong Bim; BimL, long Bim; BimS, short Bim. (D) Decreased antiapoptotic Bcl-xL protein levels in PP4-deficient thymocytes isolated from anti-CD3-injected mice. Lysates were prepared and analyzed as described for panel C but with different antiapoptotic antibodies, including anti-Bcl-xL, anti-Bcl-2, and anti-A1 antibodies. {alpha}, anti.

PP4 is essential for peripheral T-cell mitogenic proliferation and T-cell-mediated immune responses. With a role for PP4 in T-cell development determined, we further investigated whether aberrant T-cell development in PP4-deficient mice could lead to any abnormalities in peripheral T-cell compartments and T-cell functions. Although T-cell-specific PP4 conditional knockout mice showed body sizes comparable to those of control littermates, we found that numbers of CD3+ T cells, as well as CD4+ and CD8+ T cells, were decreased by 50 to 60% in both the spleens and lymph nodes of PP4-deficient mice (Fig. 9A). Therefore, PP4 appears to be essential for the maintenance of T-cell populations in the periphery. We further tested the role of PP4 in T-cell proliferation and found that PP4-deficient T cells were significantly less proliferative in response to anti-CD3 stimulation, as determined by [3H]thymidine incorporation as well as CFSE labeling assays (Fig. 9B and C). Therefore, it is likely that reduced cell proliferation is one defect leading to decreased T-cell numbers in PP4 T-cell-specific conditional knockout mice.

We examined in vivo antigen-specific T-cell activation by immunizing mice with a T-cell-dependent antigen, KLH, using CFA as an adjuvant. In mice immunized with KLH-CFA, PP4-deficient T cells showed a dramatic decrease in antigen-specific T-cell proliferation (Fig. 9D). The unresponsive phenotype of PP4-deficient T cells to antigen challenge prompted us to further investigate the biological roles of PP4 in T-cell-mediated immune responses. To evaluate this, we assessed primary humoral immune responses by measuring antigen-specific antibody production in mice 14 days after immunization with alum-precipitated DNP-KLH, which is known to induce antibody production in a T-cell-dependent manner. We detected significant decreases in both IgG1 and IgG2b, but not IgM, antibody production in immunized PP4-deficient mice compared to levels in control mice (Fig. 9E). This result indicates that IgG antibody production, which requires class switching and T-cell help, was compromised in PP4-deficient mice (Fig. 9E). Therefore, in addition to having a role in T-cell development, PP4 is also involved in T-cell activation and T-cell-dependent immune responses in vivo.


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DISCUSSION
 
PP4 is differentially expressed in mouse embryos at different developmental stages, suggesting that PP4 is a developmentally regulated protein phosphatase (11) and is essential for embryogenesis. Indeed, we generated PP4-deficient ES cells using the inducible Cre-loxP strategy and found that PP4 ablation in ES cells led to increased cell apoptosis after extracellular stimulation (data not shown). This observation may partially explain why PP4-deficient embryos failed to develop at a very early stage. In addition, this lethal phenotype is different from that of PP2A-deficient embryos; PP2A-deficient embryos with impaired cell differentiation can be recovered at embryonic day 13.5 (6). This further suggests that the function of PP4 during early embryogenesis cannot be complemented by PP2A. Notably, we frequently found PP4+/– heterozygous embryos showing growth retardation (data not shown), suggesting a partial haploinsufficiency effect leading to a decreased survival of PP4+/– embryos.

By disrupting the PP4 gene specifically in the T-cell lineage, we bypassed the embryonic lethality of PP4-null mice and showed that PP4 is essential for thymocyte development at the DN3 stage, with defects in the maturation of E DN3 cells and also in the proliferation of L DN3 cells, resulting in their accumulation. This observation correlated with an increase in the numbers of DN3 cells and a decrease in DP cells. In addition, the fact that pre-TCR signaling is essential for the development of DN3 thymocytes suggests that PP4 plays a role in pre-TCR signaling. Our results also indicate that PP4 ablation leads to impaired positive selection, presumably due to disrupted pre-TCR signaling. In support of this conclusion, we found that PP4-deficient thymocytes showed defects in calcium flux and PLC-{gamma}1-ERK activation, which are known to be important in positive selection. Thus, we have identified PP4 as a novel serine/threonine protein phosphatase that is essential in thymocyte development and pre-TCR signaling. Since PP4 is a serine/threonine phosphatase, further studies to identify the target(s) of PP4 during pre-TCR signaling are required to reveal the molecular mechanism by which PP4 regulates the pre-TCR signaling pathway.

We observed that PP4-deficient thymocytes were more apoptotic than control thymocytes in vivo and in vitro. We also found that thymocytes in anti-CD3-injected mice underwent enhanced deletion in vivo, accompanied by upregulated proapoptotic Bim and decreased antiapoptotic Bcl-xL protein levels. These results indicate an antiapoptotic role for PP4 in thymocyte apoptosis. Previous studies have shown that {alpha}4 protein (a PP4-interacting protein) functions as a negative regulator of apoptosis and is required for sustaining cell survival (16). The absence of {alpha}4 leads to the death of multiple cell types. Decreased proliferation of thymocytes and increased apoptosis of embryonic fibroblasts isolated from {alpha}4-deficient mice have been described previously (13, 16). Since {alpha}4 has no enzymatic activity, it is likely that the phenotypes detected in {alpha}4-deficient mice are caused by dysregulated PP4 function. In this regard, the roles of Bim and Bcl-xL in PP4-regulated thymocyte apoptosis may need further investigation. In addition, the characterization of PP4-interacting proteins during pre-TCR or TCR signaling may further elucidate the functional roles of PP4 in T-cell development, apoptosis, and activation. Specifically, our previous studies have demonstrated that PP4 interacts with, dephosphorylates, and activates HPK1 (30), an upstream kinase known to regulate T-cell activation and apoptosis (1, 20). Moreover, PP4 associates with, dephosphorylates, and inhibits histone deacetylase 3 (29), which was originally cloned from phytohemagglutinin-activated T cells and whose mRNA is upregulated in peripheral blood mononuclear cells cultured with anti-CD3 antibody (3). PP4 also associates with its regulatory subunit {alpha}4 to regulate mammalian target-of-rapamycin (mTOR) signaling, which has been shown to be required for anti-CD3- and anti-CD28-induced, IL-2-independent T-cell proliferation and is also important for T-cell survival (2, 17). Thus, the interplay between PP4 and its different regulatory subunits may switch the activity mode of PP4 toward different targets, leading to various PP4-mediated signaling pathways.

Multiple cellular events, including cell survival/apoptosis; centrosome maturation; spliceosomal assembly; mTOR, Jun N-terminal protein kinase, and NF-{kappa}B signaling; cisplatin resistance; DNA damage checkpoint control; and histone deacetylation are regulated by PP4 and its associated proteins. Our study presented here reveals a novel function of PP4 in the regulation of thymocyte development and pre-TCR signaling. Our results also suggest that PP4 may serve as a novel regulator of thymocyte selection and central tolerance, which may be important in therapeutic strategies for the treatment of autoimmune disorders in humans.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grants R01-AI066895 and R01-CA087076 (to T.-H. Tan) and T32-AI07495 (to J.-W. Shui). We have no conflicting financial interests.

We thank Kelly Stehling for technical assistance, Denise A. Guzman and Robin Cuthbert for secretarial assistance, and members of the Tan laboratory for helpful suggestions on the manuscript. We also thank Francesco J. DeMayo and the Baylor transgenic core facility for ES cell microinjection.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Immunology, M929, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-4665. Fax: (713) 798-3033. E-mail: ttan{at}bcm.edu. Back

{triangledown} Published ahead of print on 23 October 2006. Back


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Molecular and Cellular Biology, January 2007, p. 79-91, Vol. 27, No. 1
0270-7306/07/$08.00+0     doi:10.1128/MCB.00799-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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