Protein Kinase C - The Journal of Immunology

The Journal of Immunology

Protein Kinase C« Regulates Proliferation and Cell Sensitivity to TGF-1b of CD4+ T Lymphocytes: Implications for Hashimoto Thyroiditis Prisco Mirandola,*,† Giuliana Gobbi,*,† Elena Masselli,*,‡ Cristina Micheloni,*,† Daniela Di Marcantonio,* Valeria Queirolo,* Paolo Chiodera,‡ Tiziana Meschi,x and Marco Vitale*,† We have studied the functional role of protein kinase C« (PKC«) in the control of human CD4+ T cell proliferation and in their response to TGF-1b. We demonstrate that PKC« sustains CD4+ T cell proliferation triggered in vitro by CD3 stimulation. Transient knockdown of PKC« expression decreases IL-2R chain transcription, and consequently cell surface expression levels of CD25. PKC« silencing in CD4 T cells potentiates the inhibitory effects of TGF-1b, whereas in contrast, the forced expression of PKC« virtually abrogates the inhibitory effects of TGF-1b. Being that PKC« is therefore implicated in the response of CD4 T cells to both CD3-mediated proliferative stimuli and TGF-1b antiproliferative signals, we studied it in Hashimoto thyroiditis (HT), a pathology characterized by abnormal lymphocyte proliferation and activation. When we analyzed CD4 T cells from HT patients, we found a significant increase of PKC« expression, accounting for their enhanced survival, proliferation, and decreased sensitivity to TGF-1b. The increased expression of PKC« in CD4+ T cells of HT patients, which is described for the first time, to our knowledge, in this article, viewed in the perspective of the physiological role of PKC« in normal Th lymphocytes, adds knowledge to the molecular pathophysiology of HT and creates potentially new pharmacological targets for the therapy of this disease. The Journal of Immunology, 2011, 187: 4721–4732.

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rotein kinase C (PKC) is a family of phospholipiddependent serine/threonine kinases that regulate a wide variety of cellular functions (1). The PKC family consists of at least 11 members that have been categorized into three groups based on their structure and biochemical properties, and named conventional, novel, and atypical PKCs. Conventional PKCs (a, bI, bII, and g) require Ca2+ and diacylglycerol (DAG) for their activation, novel PKCs (d, ε, h, u) are dependent on DAG but not Ca2+, whereas atypical PKCs (z, l/i) are independent of both Ca2+ and DAG (1). T lymphocytes contain up to eight different species of PKC isotypes. In the last few years, PKCu has become the most interesting isotype as far as T cell activation, proliferation, and survival are concerned (for review, see Refs. 2, 3). However, previous studies have reported that PKCε, which localizes at the cell membrane of T lymphocytes after stimulation with PMA or antiCD3 Abs (4), has a role in the expression of the transcription *Department of Anatomy, Pharmacology and Forensic Medicine, University of Parma, 43126 Parma, Italy; †Center for Body Composition, University of Parma, 43126 Parma, Italy; ‡Department of Internal Medicine and Biomedical Science, University of Parma, 43126 Parma, Italy; and xDepartment of Clinical Sciences, University of Parma, 43126 Parma, Italy Received for publication October 1, 2010. Accepted for publication August 21, 2011. This work was supported by Italy-Ministry of Health (Strategic Program: Ricerca Finalizzata 2007-2008 to G.G.; Ordinary Project: Ricerca Finalizzata 2007–2009 to M.V.; Finaziamento Italiano Ricerca di Base: Accordi di Programma RBAP10KCNS_ 002 2011-2014 to M.V.). Address correspondence and reprint requests to Dr. Marco Vitale, Department of Anatomy, Pharmacology and Forensic Medicine, University of Parma, Via Gramsci 14, 43126 Parma, Italy. E-mail address: [email protected] Abbreviations used in this article: DAG, diacylglycerol; HC, healthy control donor; HT, Hashimoto thyroiditis; PI, propidium iodide; PKC, protein kinase C; siRNA, small interfering RNA; Treg, regulatory T cell. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003258

factors NF-AT and AP-1, which are involved in the regulation of IL-2 expression (5). The inhibition of PKCs by blocking Abs interferes with CD3- and TCR-induced IL-2 secretion, CD25 cell surface expression, and DNA synthesis of human lymphocytes (6). In particular, anti-PKCε Abs inhibit IL-2 synthesis. Of note, Castrillo et al. (7) reported that mice carrying homozygous disruption of the PKCε locus show unchanged T cell development: mature T cells have a wild type surface phenotype and are present at the expected numbers. However, T cell proliferation of PKCε2/2 animals, stimulated by anti-CD3 or anti-TCR Abs, was reduced (7). Although positively modulated by mitogenic cytokines or factors, T cell proliferation is inhibited by several classes of molecules, like ILs, nucleosides (8, 9), and cytokines (10–12), resulting in the protection from autoimmune disorders. TGF-1b is a critical cytokine that preserves immune homeostasis (13). TGF-1b–deficient mice, or mice expressing dominant-negative TGF-1bRs on T cells, soon develop spontaneous autoimmune diseases (14, 15). Furthermore, TGF-1b–induced T cells can educate naive T cells to develop suppressive properties, generating regulatory T cells (Tregs) (16) that are critical for maintaining peripheral tolerance (17) and secrete different antimitogenic ligands such as adenosine, IL-10, and also TGF-1b itself (18, 19). Tregs inhibit multiorgan autoimmunity induced by the transfer of CD4+CD252 T cells into nude mice (20), and their depletion in vivo permits induction of autoimmunity, including thyroiditis, in otherwise resistant mouse strains (20–22). Autoimmune thyroid diseases are the most common organspecific autoimmune disorder, affecting 1–2% of the population, with a 5- to 10-fold increase among women. Autoimmune thyroid diseases comprise two main clinical entities: Graves’ disease and Hashimoto thyroiditis (HT) (23). HT has been recently viewed as a genetic disorder of cell-mediated immunity (23, 24). After initiation of the autoreactive immune response by CD4+ T cells, the

4722 expansion of autoreactive T cell populations, prolonging the inflammatory response, results in a massive lymphocyte accumulation in patients’ thyroids (23, 24). It has recently been demonstrated that the serum concentration of TGF-1b is decreased in HT (25), and that in patients with HT and Graves’ disease, some Treg subsets apparently exert a defective suppressive function (26). Given this complex background, in this study, we have investigated the functional role of PKCε in the control of human CD4 T cell proliferation and sensitivity to TGF-1b. PKCε expression levels were transiently modulated in primary human T cells induced in vitro by anti-CD3 and anti-CD28 mAb stimulation. Overall, we show that PKCε promotes T cell proliferation in vitro, whereas it is spontaneously expressed at significantly higher levels in CD4+ T cells from HT patients, strongly suggesting a role of this kinase in the molecular pathophysiology of Hashimoto disease.

Materials and Methods Patients Twenty-three HT patients (7 female and 16 male patients; mean age, 46 6 13 y) were selected at the Internal Medicine, Vascular and Metabolic Diseases Unit of the Parma University Hospital (Table I). Selected patients were characterized by high serum levels of anti-thyreoperoxidase (.100 U/ml) and anti-thyreoglobulin Abs (.300 U/ml). Patients with anti-insulin and anti-islet Abs were excluded. Twenty-seven healthy control donors (HC; 4 female and 23 male subjects; mean age, 37 6 9 y) without previously diagnosed autoimmune diseases, viral or bacterial infection, or metabolic syndrome were used as control subjects.

Cell isolation Naive CD4+ Th cells were isolated from human PBMC by using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec) and the VarioMACS cell Separator, as previously described (27–30). Purity of cells was immediately checked by anti–CD4-FITC and anti–CD8-PE staining and flow cytometry analysis. Only samples with a purity exceeding 97% were used.

Cell treatment Cells were activated by seeding into anti-CD3 and/or anti-CD28 mAbcoated plates, as previously described (29, 30). In particular, 1 3 106/ml freshly isolated CD4+ T cells (2.5 3 105/well) were resuspended in 10% FBS-enriched RPMI 1640 medium containing 30 U/ml IL-2 and seeded in 96 flat-well plates. IL-2 was readded to the culture medium at the same final concentration after 2 d. TGF-1b was added to cell culture medium at a concentration of 10 ng/ml 24 h after cell seeding. Anti-CD3 (clone x35; Beckman Coulter) and/or anti-CD28 (clone 28.2; Beckman Coulter) mAb coating was performed for 24 h at 4˚C: the two mAbs, alone or in combination, were diluted at 4 mg/ml (or 2 and 1 mg/ml in some experiments) in 50 ml RPMI and added to each well of a 96 flatwell plate. Before cell seeding, mAb solution was removed and plates gently washed with complete culture medium. Control samples were cultured exactly in the same conditions, in the absence of IL-2 and CD3/ CD28 mAb stimulation. In some experiments, CD4+ T cells were stimulated with 5 mg/ml PHA (Sigma-Aldrich), 500 ng/ml ionomycin (Sigma-Aldrich), and 50 mg/ml PMA (Sigma-Aldrich) for 24 and 48 h. TGF-1b signaling pathway was studied in the Jurkat T cell line and in CD4+ T lymphocytes. Jurkat cells were grown in serum-free medium for 18 h to generate a total inactivation of Smad2 (31), then 10 ng/ml TGF-1b was added for 2 h to induce Smad2 phosphorylation. Because Smad3 is not detectable in Jurkat cells, to study its activation, we used CD4+ T lymphocytes grown in complete medium (RPMI 1640 + 10% FBS + IL2) in anti-CD3 and anti-CD28–coated plates. After 36 h of culture, CD4 cells were starved for 8 h and then stimulated with TGF-1b for 2 h. Cell were harvested and total protein lysate was analyzed by Western blot.

Cell proliferation Cell proliferation was studied analyzing by flow cytometry the number of cell duplications. As previously described (30), cells were labeled with 10 mM CFSE, extensively washed, counted, and resuspended in complete medium. Resting (controls) and activated CFSE-labeled cells were ana-

PKCε IN HUMAN CD4 LYMPHOCYTES lyzed after 4 d of culture. To exclude dead cells from analysis, we stained samples with propidium iodide (PI; 2.5 mg/ml) and CFSE fluorescence was analyzed gating on PI2 cells.

Flow cytometry Staining with anti–CD71-CY5, anti–CD95-PE, anti–CD25-PE (Beckman Coulter) was performed according to manufacturer’s instructions. Control samples were stained with appropriate irrelevant isotype-matched control (CY5 and PE-labeled) Abs. Analysis was performed with an EPICS-XL flow cytometer, followed by the EXPO32 software analysis (Beckman Coulter), collecting at least 10,000 events/sample.

Small interfering RNA design and transfection The respective sense and antisense RNA sequences of double-stranded small interfering RNA (siRNA), designed to target human PKCε mRNA (siRNAPKCε), were synthesized by Silencer siRNA Construction Kit (Ambion, Austin, TX) as previously described (32, 33). Nonspecific siRNA duplexes containing the same nucleotides, but in irregular sequence (i.e., scrambled PKCε siRNA), were prepared according to manufacturer’s protocol and used as controls. The GFP-PKCε expression and control plasmid were kindly provided by Prof. Peter Parker (Cancer Research UK, London Research Institute). To maximize transfection efficiency, we delivered siRNAs (100 nM each) and GFP-PKCε plasmids (1 mg) using the Amaxa nucleofection technology (Amaxa, Koeln, Germany), according to the manufacturer’s protocols. In particular, resting CD4+ T cell cultures were transfected by the human T cell nucleofection kit for unstimulated cells (Amaxa) with the U-014 nucleofection program. Jurkat cell cultures were transfected with GFP-PKCε and GFP-PKCεm plasmids (1 mg) by CD34 cell nucleofection kit (Amaxa) with the U-014 nucleofection program.

Design of a PKC« cDNA resistant to siRNAPKC« To generate proper controls to our RNA silencing experiments, we mutated siRNA-target sequences of mouse PKCε to obtain an siRNA-resistant PKCε cDNA (Table II). Specifically, we used the mouse PRKCE fulllength cDNA (722/737 conserved amino acids versus human PKCε protein) fused in-frame with the N-terminal GFP tag (34) by PCR using the start primer 59-GCGCAGATC TACCATGGT AGTGTTCAA TGGC-39 and the end primer 59-GCGCGTCGA CGCTTGGGA GTATCTCAA CACCG-39. Mutations were introduced in the siRNA-target sequences by overlap extension PCR. Two PCR products bearing the mutations in overlapping complementary ends were produced from the GFP-PKCε with the following primers: siRNA4sense, 59-GAAATTGGCG GCGGGCGCGG AAtccccaca gccggcttctg-39; siRNA4antisense, 59-TTCCGCGCCC GCCGCCAATT TCttcctcct ttggccactg ttgg-39; siRNA3sense, 59-TTATAAAGTG CCCACGTTTt gtgaccactg tgggtc-39; siRNA3antisense, 59AAACGTGGGC ACTTTATAAt tgtggatccc gaacttgtg-39; siRNA2sense, 59-AATTGAACTC GCGGTGTTCc acgacgctcc tatcggc-39; siRNA2antisense, 59-GAACACCGCG AGTTCAATTt tgcgcccatt gcacacatc-39; siRNA1sense, 59-cttcttaaaA TTAAGATTT GTGAAGCGg tgagcttga-39. The overlapping sequence, containing a mutated version of the siRNA target sequence of mouse PKCε cDNA, is reported by using uppercase letters. In particular, as described in Fig. 5D, the PCR products obtained with siRNA4sense+end primers (lane 1; 1322 bp) was hybridized with the product obtained with siRNA4antisense+start primers (lane 2; 977 bp). The overlapping product was amplified by primers start and end (lane 3; 2297 bp). This PCR product is the full-length PKCε cDNA resistant to the siRNAPKCε number 4 (PKCε4R). PKCε4R amplicon was purified on agarose gel and amplified by PCR using the primers siRNA3sense+end (lane 4; 1538 bp) and siRNA3antisense+start (lane 5; 778 bp). The overlapping product was amplified by primers start and end (lane 6; 2297 bp) to yield the full-length PKCε cDNA resistant to the siRNAPKCε numbers 3 and 4 (PKCε34R). The same procedure was repeated in lanes 7 (PCR by siRNA2sense and end primers; 2051 bp), 8 (PCR by siRNA2antisense and start primers; 265 bp), and 9 (PCR by start and end primers; 2297 bp) to obtain the full-length PKCε cDNA resistant to the siRNAPKCε 2, 3, and 4 (PKCε234R). Then, siRNA1sense+siRNA3antisense product (lane 10; 747 bp) was hybridized with the start+siRNA2antisense amplicon (lane 11; 265 bp), both obtained using PKCε234R as template. The overlapping product was amplified by the primers start and siRNA3antisense (lane 12; 778 bp). This last PCR product was hybridized with siRNA3sense+end amplified region of PKCε234R (lane 13; 1538 bp). The overlapping product was amplified by primers start and end (lane 14; 2297 bp) to yield the fulllength PKCε cDNA resistant to our four siRNAPKCε (PKCε1234R). DNA was amplified by 10 cycles of 60 s at 94˚C, 30 s at 55˚C, and 1 min at 72˚C. Then, additional 15 cycles were performed for 60 s at 94˚C and 2 min at 72˚C (extension time was incremented by 3 s/cycle). Hybridization

The Journal of Immunology and extension of overlapping PCR products were obtained in PCR mix by 15 cycles of 60 s at 94˚C, 60 s at 55˚C, and 3 min at 72˚C. Agarose gel purification of PCR products was performed with the GenElute agarose spin columns (Sigma-Aldrich). PKCε1234R was digested with BglII and SalI and ligated to the corresponding sites of the pEGFP-C1 vector. The sequence integrity of siRNAresistant PKCε was tested by DNA sequencing. Table II shows the alignment of nucleotide sequence of human, wild type mouse, and siRNA-resistant PKCε cDNAs.

RT-PCR CD4+ T cell cultures were grown for 48 h, and 2 3 106 cells were collected by centrifugation. Total RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). Total RNA (1 mg) was reverse transcribed, and progressive dilutions (1/10, 1/100, 1/1000) were then subjected to PCR amplification. cDNAs of IL-2R chains were amplified using the primers previously described by Hughes-Fulford et al. (35). IL-2Ra sense 59ATCCCACACGC CACATTCACA AAGC-39 and antisense 59-TGCCCCACCA CGAAATGAT AAAT-39 primers generated an expected PCR product of 347 bp (NM-000417 GenBank locus, GI:4557666). IL-2Rb sense 59-GCCCCCATCT CCCTCCAAGT-39 and antisense 59AGGGGAAGGG CGAAGAGC-39 primers give the expected PCR product of 529 bp (NM-000878 GenBank locus, GI:23238195). By the sense 59GGAAGCCGTG GTTATCTCTG TT-39 and the antisense 59-GGTGGGTTGA ATGAAGGAAA GT-39 IL-2Rg cDNA primer, we obtained two expected amplicons of 386 bp (NM-000206 GenBank locus sequence, GI:4557881) and 407 bp (HUMIL2RGA GenBank locus, GI:349631). By the sense 59-TGACGGGGTC ACCCACACTG TGCCCATCTA-39 and antisense 59-CTAGAAGCAT TTGCGGTGG ACGATGGAG GG-39 primers, we amplified an expected 649-bp region of b-actin mRNA (E00829 GenBank locus sequence, GI:2169090), as previously described (30, 32). The 387-bp region of the human PKCε cDNA, NM005400 GenBank locus (GI: 47157326), was amplified by the sense 59-CAATGGCCTTC TTAAGATCAA AA-39 and antisense 59-CCTGAGAGAT CGATGATCA CATAC-39 primers, as previously described (32). NF-kB1 was amplified by 59-AGATGTGGTGG AGGATTTGC-39 and the antisense GCCAATGAGA TGTTGTCGTG-39 primers; NF-kB2 by 59-GATCGAGGTG GACCTGGTAA-39 and the antisense 59-TGGCTCTAAG GAAGGCAGAG-39; RelA by 59-CTATCAGTCA GCGCATCCAG-39 and antisense 59-CTGGTCCCGT GAAATACACC-39; RelB by 59-ACATCAAGGA GAACGGCTTC-39 and antisense 59-CTTCCTCACA CACTGGATGC-39; cRel by 59-ATTGAACAAC CCAGGCAGAG39 and antisense 59-ATTGGGTTCG AGACAACAGG-39. The lengths of the expected amplified regions were 318, 332, 300, 518, and 488 bp (GenBank locus NM003998 GI:34577121; NM001077494 GI:117320530; NM001145138 GI:223468680; NM006509 GI:35493877; and NM002908 GI:56550118). Temperature and time were modified from the original methodology (35): cDNA was amplified by 35 cycles of 30 s at 94˚C, 30 s at 60˚C, and 30 s at 72˚C. The extension time of the last 15 cycles was incremented by 3 s/cycle. Moreover, to improve specificity and efficiency, we added 3% of DMSO to the PCR mix reaction. To obtain a relative quantification of gene expression, 1/10, 1/100, and 1/1000 of total cDNA was amplified, PCR products were separated in 2% agarose gel, stained with ethidium bromide, and visualized under an UV transillumination by KODAK Image Station 4000MM. Band intensity of each PCR was quantified by KODAK Molecular Imaging Software 4.0 and corrected for b-actin amplified cDNA intensity. The expression levels of the markers of polarization to Th1, Th2, Th17, and Treg were analyzed by RT-PCR. T-bet cDNA (217 bp) was amplified by 59-CCACCAGCCA CTACAGGATG-39 and 59-GGACGCCCCC TTGTTGTTT-39 primers; GATA-3 (205 bp) by 59-GTGCTTTTTA ACATCGACGG TC-39 and 59-AGGGGCTGAG ATTCCAGGG-39; Rorgt (348 bp) by 59-AAATCTGTGG GGACAAGTCG-39 and 59-TGAGGGTATC TGCTCCTTGG-39; and Foxp3 (771 bp) by 59-GCACCTTCCC AAATCCCAGT-39 and 59-TAGGGTTGGA ACACCTGCTG-39 (36). Temperatures and timing were modified from the original methodology: cDNA was amplified by 35 cycles of 30 s at 94˚C, 30 s at 60˚C, and 60 s at 72˚C. The extension time of the last 15 cycles was incremented by 3 s/cycle.

Western blot Cells were resuspended in a cell lysis buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF) supplemented with fresh protease inhibitors and protein concentration was determined using bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Fifty mg proteins from each sample were then migrated in 5% SDS-acrylamide gels and

4723 blotted onto nitrocellulose filters. Blotted filters were blocked and incubated with specific primary Abs diluted as previously described (32, 33). Specifically, rabbit polyclonal anti-PKCε and anti–b-actin Abs were used at the concentration of 1 mg/ml and diluted 1:5000, respectively. PKCu expression was revealed by anti-PKCu (Cell Signaling Technology) rabbit antiserum. Activation of human PKCε (phosphoserine 729) was detected in Western blot by rabbit antiserum (Upstate) (37). Immunoblots were incubated by anti-rabbit (1:2000) or anti-mouse (1:2000) peroxidase-conjugated secondary Abs, and revealed by ECL Western blotting detection reagent (Amersham Biosciences). To obtain absolute quantification of protein levels, we compared PKCε signals obtained by biological samples, corrected for b-actin, with those obtained with purified human recombinant PKCε (5, 2.5, and 1.25 ng; GenWay Biotech, San Diego, CA). TGF-1b signaling was studied by detecting the phosphorylation levels of Smad-2 and Smad-3 with anti–phospho–Smad-2 (ser465/467; Cell Signaling Technology) and anti–phospho–Smad-3 (ser423/425; Cell Signaling Technology) rabbit polyclonal Abs, as previously reported (31). The expression levels of total Smad2 and Smad3 were studied by immunoblotting with anti-Smad2/3 rabbit serum (Cell Signaling Technology) diluted 1:1000, as described by the manufacturer.

Results PKC« and CD4+ T cell proliferation To evaluate the role of PKCε in CD4+ T lymphocyte proliferation, we transfected freshly purified cells with siRNA to PKCε (siRNAPKCε) or with control siRNA, and then seeded them in antiCD3– and/or anti-CD28–coated wells in the presence of IL-2. Cell proliferation was analyzed 4 d later, quantifying by flow cytometry the number of duplications of the viable cells. As shown in Fig. 1, control (resting) cell cultures did not proliferate at all (zero duplications), whereas 40% of stimulated cells did two or more duplications (one representative example is reported in Fig. 1A). Inhibition of PKCε expression by specific siRNA transfection did not affect the number of duplicating cells in the control (unstimulated) cultures (,5%), whereas it significantly impaired the proliferation of CD3/CD28-stimulated T cells. As shown in Fig. 1B, siRNAPKCε-transfected stimulated cultures show a significant increase of cells with zero duplications. Because different donors responded differently to the mitogenic stimulus, the number of cells with zero, one, two, four, and five duplications has been reported as percentages of siRNA-transfected control cultures (Fig. 1C). In particular, analyzing four representative donors, the relative percentage of cells with zero duplications (Fig. 1C, black bars) in siRNAPKCε-treated cultures was 161 6 19% of normalized (100%) controls (siRNActrl-transfected cultures), whereas the number of cells with one duplication was 99 6 34% of controls. The number of cells with two or more duplications (two, three, four, and five duplications) in siRNAPKCε-treated cultures was 46 6 32, 29 6 21, 35 6 14, and 58 6 20% of controls, respectively. Because nucleofection is a traumatic procedure, it significantly increased cell death of CD4+ T cells (Fig. 1D). However, viability of siRNAPKCε-transfected cells was similar to those of control siRNA-transfected cultures (49.7 6 18.2 versus 60.3 6 10.7; p = 0.354, Student t test; Fig. 1E, black bar versus gray bar). There was no significant difference in cell death of the nonproliferating cell subset (zero duplications) between control and siRNAPKCεtransfected cultures (36.6 6 9.8 and 44.2 6 16.7, respectively; p = 0.462, Student t test; Fig. 1F, empty bars). Similarly, no difference was observed in cell death between control and siRNAPKCεtransfected cultures in the cell subsets doing two or more duplications (9.5 6 8.4 and 17.4 6 8.0, respectively; p = 0.222, Student t test; Fig. 1F, solid gray bars). This means that, in the absence of PKCε, CD4+ T cells stimulated with CD3/CD28 show a significant reduction of the number of cells able to do two or more duplications, which is not caused by increased cell death.

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PKCε IN HUMAN CD4 LYMPHOCYTES duplicate (138% of control cell cultures). Similar results were observed in PHA-treated CD4+ T cell cultures. These observations suggest that PKCε likely takes part in a proliferative signaling cascade that is common to both the TCR-dependent and -independent pathways. Because CD4+ T cells still duplicated, although to a minor extent, in the absence of PKCε, we asked whether PKCε also promoted cell activation. Fig. 2C shows that activation markers like CD71 and CD95 are equally expressed on the cell surface in the presence or absence of PKCε, whereas CD25 is clearly downregulated. These data support the hypothesis that PKCε likely has a role in the signaling promoting CD4 T cell duplication. In particular, the percentage of CD25+ cells in the siRNAPKCεtransfected cultures decreased from 78 to 67%, and the cell surface density of CD25, calculated as molecules of equivalent soluble fluorophore, decreased to 84.5 6 6.2% as compared with the control siRNA-transfected cultures (Fig. 2D, 2E). PKC« and TGF-1b activity

FIGURE 1. Functional role of PKCε in CD4+ T cell proliferation. A, CD4+ T cell cultures labeled with CFSE and grown for 4 d in anti-CD3 (4 mg/ml) and anti-CD28 (4 mg/ml) mAb-coated wells or in uncoated plates (Resting). Before seeding, cells had been transfected with siRNA specific for PKCε (siRNAPKCε) or with control siRNA (siRNActrl.). The number of duplications is indicated above the histograms (from 0–5). A representative experiment is shown. B, siRNAPKCε-transfected CD4 T cells: percentage of cells doing zero, one, two, three, four, or five duplications (siRNActrl: control cultures transfected with scrambled nucleotides). Means of four independent experiments are reported 6 SD. *p , 0.05, analysis by t test. C, Antiproliferative effects of PKCε siRNA. Results obtained with four donors and means 6 SD. x-Axis reports the number (0–5) of cell duplications. Values (y-axis) are expressed as percentages of control siRNA (siRNActrl)-treated cell cultures (normalized at 100% and omitted for clarity). *p , 0.05 versus control siRNA-treated cell, analysis by t test. D, Flow cytometry analysis of PI-stained CD4+ T cell cultures labeled with CFSE and grown 4 d in 96-well plates coated with anti-CD3+ anti-CD28 mAbs. Duplication (CFSE fluorescence) and cell death (PI fluorescence) were analyzed in untransfected (Untransf.), control siRNA (siRNActrl.), and siRNA to PKCε (siRNAPKCε) transfected cells. E, Cell viability of siRNA-transfected CD4+ T cell cultures. Means 6 SD from three independent experiments are shown. *p , 0.05 versus untransfected cell cultures (white bar); #p , 0.05 versus control siRNA-transfected cultures (gray bar), ANOVA by Dunnett’s test. F, Cell death of nondoubling cells (zero duplications) and of proliferating cells (two or more duplications). Means 6 SD from three independent experiments are shown.

Because it is known that both TCR-dependent and -independent stimulation of CD4+ T cells activate PKCε (4), to better discriminate the role of PKCε in the proliferation of CD4+ T cells, we stimulated cells either with anti-CD3 alone or in a TCRindependent way. When we analyzed the expression levels of PKCε and PKCu in CD4+ T cells stimulated in a TCRindependent way, we observed that although PHA and PMA promoted a rapid upregulation of both PKCε and PKCu expression, ionomycin stimulation took 24 h to increase both PKCs expression (Fig. 2A). As shown in the case reported in Fig. 2B, siRNAPKCε reduced to 66% the number of cells doing two or more duplications and increased the number of cells that did not

Because PKCε appears to be involved in the control of cell proliferation, we then investigated its role in the presence of antiproliferative signals as those generated by TGF-1b. As preliminary experiments, we started evaluating the TGF-1b effects on CD4+ T lymphocytes proliferation. As shown in Fig. 3A, 10 ng/ml TGF-1b did not impair the proliferation of cell cultures stimulated with high doses of anti-CD3 and anti-CD28 mAbs. In agreement with Kehrl et al. (10) and Wolfraim et al. (11), however, TGF-1b was, on the contrary, able to inhibit proliferation in the absence of anti-CD28 costimulation. However, CD4 T lymphocyte proliferation induced by low doses of anti-CD3/CD28 was partially inhibited by TGF-1b. In particular, in the donor shown in Fig. 3A, when cells were stimulated with anti-CD3 alone at high dose, TGF-1b reduced the number of proliferating cells (i.e., those with two or more duplications) to 64% of the control; when anti-CD28 was used together with low dose (1 ng/ml) of anti-CD3, TGF-1b reduced the number of duplicating cells (i.e., those with two or more duplications) to 42% of the control (Fig. 3B). On the basis of these observations, we then studied the effects of PKCε in this cell system. Fig. 4 shows that siRNAPKCε increased the sensitivity of CD4+ T cells to TGF-1b both when cultures were maximally stimulated with anti-CD3/CD28 or with anti-CD3 alone. The ability of siRNAPKCε to boost TGF-1b effects is more evident in anti-CD3/CD28–stimulated cells (Fig. 4A) because they are not sensitive to TGF-1b. In fact, TGF-1b treatment of control cultures (transfected with siRNActrl) did not interfere with cell proliferation (105 6 14% cells with two or more duplications), whereas in the absence of PKCε, TGF-1b could reduce the number of doubling cells to 55 6 17%. To formally prove that PKCε negatively regulates TGF-1b signaling, we transfected CD4+ T cells with the expression vectors CMV-PKCε (expressing wild type PKCε) and CMV-PKCεm (expressing a mutated PKCε lacking enzymatic activity). As expected, the best results were obtained with CD4+ T cell cultures activated by anti-CD3 alone (Fig. 4B). In fact, anti-CD3–stimulated cells were sensitive to TGF-1b inhibition (68 6 11% of CVM-PKCεm–transfected control cells), but the forced upregulation of PKCε expression completely restored their proliferation rate (98 6 10% of control cell cultures). Similar experiments were performed using a TCRindependent stimulation. Fig. 4C shows that TGF-1b impaired proliferation of PHA-stimulated CD4 T cells, and the inhibition of PKCε expression increased TGF-1b antiproliferative effects, whereas on the contrary, the upregulation of PKCε expression blocked TGF-1b effects. The analysis of cell death in TGF-1b– treated cell cultures showed that their low duplication rate after


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FIGURE 2. PKCε controls cell proliferation, but not cell activation, of T CD4+ lymphocytes. A, Western blot analysis of PKCε and PKCu expression in stimulated CD4+ human T cells. A representative experiment is shown. Resting (Control), PHA (5 mg/ml), PMA (50 mg/ml), ionomycin (ionom.; 500 ng/ ml), and anti-CD3+ anti-CD28 (both at 4 mg/ml) mAb-stimulated CD4+ T cell cultures were grown for 24 or 48 h. Activation of PKCε was also studied using specific anti–phospho-PKCε rabbit serum (pPKCε). B, CD4+ T cells were labeled with CFSE and transfected with control siRNA (siRNActrl.) or with siRNA to PKCε (siRNAPKCε). After 4 d of stimulation with CD3 alone (4 mg/ml), with CD3 and CD28 (both at 4 mg/ml) mAbs, or with PHA (5 mg/ml), the number of cell duplications was quantified by flow cytometry. The number of duplications is indicated above the histograms (from 0–5). A representative experiment is shown. C, Flow cytometry analysis of cell surface expression of CD25, CD95, and CD71. siRNActrl and siRNAPKCε transfected cells were activated with CD3/CD28 and grown for 4 d. The fraction of positive cells is reported in each dot plot. The appropriate irrelevant isotype-matched control (Irrl-CY5 and Irrl-PE) is shown. D, Quantification (as molecules of equivalent soluble fluorophore [MESF]) of cell surface expression of CD25 and CD95 from three independent experiments. Cells were activated with CD3 (4 mg/ml) and CD28 (4 mg/ml) for 4 d. Phenotype of resting cultures is also reported. E, Effects of PKCε downmodulation (siRNAPKCε) on CD25 and CD95 expression. Values are calculated as percentage of the control siRNA-treated cell cultures and are reported as mean 6 SD of three independent experiments (the same shown in C). *p , 0.05 versus control siRNA-treated cultures, analysis by t test.

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PKCε IN HUMAN CD4 LYMPHOCYTES rescue experiments were performed transfecting CD4+ T cells with siRNA-resistant PKCε (PKCε1234R) cDNA. As shown in Fig. 5C, the transfection of PKCε1234R, but not that of the wild type gene (CMV-PKCε), prevented siRNAPKCε-mediated sensitization to TGF-1b antiproliferative effects in CD4+ T cells. PKC« and IL-2R expression

FIGURE 3. TGF-1b impairs CD4+ T cell proliferation. A, CFSE-labeled CD4+ T cells were stimulated with the indicated doses of anti-CD3 and anti-CD28 for 24 h, then treated with 10 ng/ml TGF-1b. Flow cytometry analysis was performed 3 d after TGF-1b addition. The number of duplications is indicated above the histograms (from 0–5). B, proliferation of TGF-1b–treated cell cultures is reported as the number of cells with zero duplications (empty bars) or with more than one duplication (black bars). Values are expressed as percentage of control untreated cultures.

siRNAPKCε transfection was not due to any significant increase of cell death (Fig. 4D). siRNA target specificity and PKC« rescue experiments To test the specificity of our siRNAPKCε, we choose the Jurkat cell model where, at variance with primary CD4 T cells, PKCε and PKCu are constitutively expressed. siRNAPKCε selectively impaired PKCε expression, not affecting PKCu protein levels (Fig. 5A, 5B; *p , 0.05, ANOVA by Dunnett’s test). In parallel,

FIGURE 4. PKCε reduces TGF-1b antiproliferative effects. Residual proliferation of CD4+ T cell cultures treated with 10 ng/ml TGF-1b. Cells were transfected either with siRNA to PKCε (siRNAPKCε) to inhibit PKCε expression or with the wild type murine PKCε (CMV-PKCε). Respective negative controls were represented by scramblednucleotides siRNA (siRNActrl.) and by a vector expressing a mutated PKCε lacking enzymatic activity (CMV-PKCεm). The number of cells with two or more duplications (white bars) or with zero duplications (black bars) is reported as percentage of control cultures (TGF-1b–untreated cells). Cell cultures were stimulated with anti-CD3 mAb (4 ng/ ml) alone (B), with anti-CD3 (4 mg/ml) and CD28 (4 mg/ml) mAbs (A), or with 5 mg/ml PHA (C). Means 6 SD of four independent experiments are reported. *p , 0.05 versus control culture, analysis by t test. D, Flow cytometry analysis of cell death of CD4+ T cell cultures transfected with control siRNA or with siRNAPKCε and stimulated for 4 d with antiCD3 and anti-CD28 mAbs with or without TGF-1b. Cell death is reported as number of PI+ cells. Means 6 SD of four independent experiments are reported.

Because we know that the percentage of activated CD4 T cells that expresses CD25 decreases when PKCε is downregulated (Fig. 2B), to explore the molecular mechanism activated by PKCε in the promotion of cell proliferation, we focused on the transcriptional levels of IL-2R chains. Fig. 6A shows that we were indeed able to detect IL-2Ra–, b- and g-chain mRNAs by RT-PCR from PHAactivated PBLs. Fig. 6B shows that, as expected, CD4+ resting cells do not express detectable levels of IL-2Ra– or b-chains, whereas g-chain transcripts (the 407- and 386-bp PCR products) were detectable at low levels only when a high amount of cDNA (lane 2) was tested (1/10 total cDNA). Cell activation by CD3 and CD28 stimulation promoted the expression of IL-2Ra–, b-, and g-chains. Coherently with our flow cytometry data, inhibition of PKCε expression by siRNAPKCε prevented the anti-CD3/CD28– dependent upregulation of IL-2Ra–, b-, and g-chains, reducing the amount of amplifiable cDNA to the level of resting cells. It is well-known that NF-kB is a key transcription factor involved in IL-2R chain expression in T cells and, more in general, in T cell activation and proliferation (38–41). Using PHAactivated PBLs, we could detect the expression of NF-kB1 and NF-kB2, but not of RelA, RelB, or cRel (Fig. 7A). Interestingly, we found that NF-kB1 and NF-kB2 gene expression was downregulated in siRNAPKCε-transfected cells, suggesting that an interference with NF-kB expression might be at the basis of all the observed effects of PKCε inhibition in T cells (Fig. 7B). PKC« expression in HT Because PKCε levels modulate CD4+ T cell proliferation and sensitivity to TGF-1b, we finally hypothesized that PKCε over-


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FIGURE 5. siRNAPKCε effects were target selective. A, Western blot analysis of PKCε and PKCu expression in Jurkat cell treated with siRNAPKCε. Total protein lysates of untransfected (Control) cell cultures and of siRNAPKCε and control siRNA treated cells (siRNACtrl.), harvested 48 h after nucleofection, were blotted for PKCε, PKCu, and b-actin. A representative experiment is reported. B, Densitometric analysis of PKCε and PKCu expression: mean values of three independent experiments are reported. PKCε and PKCu expression were normalized to b-actin. *p , 0.05 versus untransfected cells (lane 1); analysis was made by ANOVA followed by Dunnett’s test. C, siRNA-resistant PKCε abolished siRNA effects. CFSE-labeled CD4+ T cell cultures, nucleofected with siRNAPKCε and with GFP-PKCε1234R (PKCε1234R) or GFP-PKCε (PKCε wt), were grown in anti-CD3 + anti-CD28 mAb-coated wells for 4 d with or without 10 ng/ml TGF-1b. siRNA-resistant PKCε (PKCε1234R) prevented siRNAPKCε-induced sensitization to antiproliferative effects of TGF-1b, restoring cell proliferation. Flow cytometry analysis was performed 3 d after TGF-1b addition. The number of duplications is indicated above the histograms (from 0–5). A representative experiment is reported. D, Construction of siRNA-resistant PKCε: 0.8% agarose gel, stained with ethidium bromide and visualized under UV exposure. CMV-PKCε (lane P1) was used as template of the PCRs 1 and 2, whose amplicons are shown (lanes 1, 2). The fusion product of PCRs 1 and 2 (PKCε resistant to the siRNA number 4) was obtained (lane 3). Amplicon 3 was used as template of PCRs 4 and 5 (lanes 4, 5). The fusion product of PCRs 4 and 5 (PKCε resistant to the siRNAs number 3 and 4) was obtained (lane 6). With the same procedure, amplicon of the PKCε resistant to the siRNAs numbers 2, 3, and 4 was shown (lane 9). The PKCε resistant to our 4 siRNAs (lane 14) was obtained by fusion of 13 with 12. The amplicon shown in 12 was obtained by fusing PCR 10 to PCR 11, both obtained using 9 as template. For a better description of primers used, see Materials and Methods.

expression could be involved in CD4+ T cell-mediated autoimmune diseases, like HT. Thus, we studied PKCε protein expression levels in peripheral blood-derived resting CD4+ T cells from 23 HT patients and 27 HC (Table I). PKCε expression showed an individual variability (Fig. 8) ranging from 0.00 to 0.58 ng/50 mg cell lysate. In preliminary experiments, we noted that several male subjects in the HC group showed undetectable or very low levels of PKCε (,0.1 ng/50 mg cell lysate). We therefore decided to increase the number of male subjects in both groups. HT subjects showed significantly (p = 0.026) higher mean values of PKCε levels (0.38 6 0.29 ng/50 mg) than HC (0.22 6 0.20 ng/50 mg). We subsequently tried to correlate the expression levels of PKCε with TGF-1b sensitivity of CD4 cells of HT patients. Primary CD4+ T cell cultures were stimulated with antiCD3 mAb. As shown in Fig. 8C, CD4+ T cells from HT patients (solid gray bars) were less sensitive to TGF-1b than those from HC. The transfection of siRNAPKCε (Table II) strongly impaired cell proliferation of HT-derived CD4+ T cells, which acquire a phenotype similar to that of HC (*p , 0.05 ANOVA-Dunnett’s test versus HT siRNActrl-transfected cells with more than two duplications).

PKC« impaired TGF-1b signaling It has been reported that the activation of PKCu by TCR agonists is necessary and sufficient to inhibit TGF signaling on TCR activation (42). Moreover, it is known that TGF inhibits IL-2 and antiCD3/CD28–induced T cell proliferation in a Smad3-dependent manner. By contrast, Smad3 is not essential for TGF-1b–mediated inhibition of IL-2–induced T cell proliferation of lymphocytes activated by ConA (43). Looking for TGF-1b signaling intermediates potentially targeted by PKCε, we analyzed pSmad2 and pSmad3 levels in PKCε-overexpressing Jurkat cells and in primary activated CD4 cell cultures treated with TGF-1b for 2 h. Of note, PKCε overexpression significantly reduced TGF-1b– induced pSmad-2 levels in Jurkat cells (Fig. 8D, 8E; lane 5 versus lane 6) and in anti-CD3/anti-CD28–stimulated CD4 cells (Fig. 8D, 8E; lane 11 versus lane 12). Although pSmad3 was not detectable in Jurkat cells, TGF-1b increased pSmad3 levels in CD4 cells (Fig. 8E, gray bars; *p , 0.05, ANOVA-Dunnett’s test versus 1). PKCε overexpression did not affect TGF-1b–induced phosphorylation of Smad3. TGF-1b has been shown to play an essential role in inhibiting Th1 polarization, as well as inducing both Treg and Th17

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PKCε IN HUMAN CD4 LYMPHOCYTES

FIGURE 6. PKCε modulates mRNA levels of IL-2R chains. A, Analysis by RT-PCR of IL-2R chain expression. RNA from PHA + IL-2–activated PBLs (3 d of cell culture) was reversed transcribed and amplified by PCR. Amplicons were separated in 2% agarose gel, stained with ethidium bromide, and visualized under UV exposure. We were able to detect mRNA of the three IL-2R chains (two transcripts were observed for IL-2Ra and IL-2Rg). B and C, Analysis of IL-2R chain expression by RT-PCR in resting CD4+ T cells (lanes 2–4), activated and untransfected cells (Untr.; lanes 5–7), control siRNAtransfected cell cultures (siRNActrl; lanes 8–10), and siRNAPKCε-transfected CD4+ T cells (siRNAε; lanes 11–13) after 24 h of cultures. 1/10, 1/100, and 1/1000 cDNA, obtained by reverse transcription of 1 mg total RNA, was amplified and PCR products were separated on agarose gel and visualized under UV illumination after ethidium bromide staining. Length of PCR product is also reported. Lanes 2, 5, 8, and 11: PCR with 1/10 cDNA. Lanes 3, 6, 9, and 12: PCR with 1/100 cDNA. Lanes 4, 7, 10, and 13: PCR with 1/100 cDNA. Lane 1: negative control PCR (Blank); M.W., 100-bp DNA ladder. PCR products were quantified by densitometric analysis and plotted in the graph as described in Materials and Methods. *p , 0.05 versus untransfected and activated cells, analysis by ANOVA and Dunnett’s test.

differentiation (16). In particular, the polarization of naive T cells to Th1, Th2, and Th17 T cell subpopulation is mediated by specific agonist combinations: IL-12/anti–IL-4 for Th1; IL-4/anti–IL-

12 for Th2; TGF-1/IL-6 or IL-21 for Th17 (44). Although in our experiments TGF-1b was added 24 h after T cell stimulation and only IL-2 was used as a mitogenic cytokine, the knocking down of

FIGURE 7. PKCε modulates mRNA levels of NF-kB. A, Analysis by RT-PCR of NF-kB family expression. RNA from PHA + IL-2–activated PBLs (after 3 d of cell culture) was reversed transcribed and amplified by PCR. Amplicons were separated in 2% agarose gel, stained with ethidium bromide, and visualized under UV exposure. We found detectable amount of cDNA of NF-kB1 and NF-kB2, but not of RelA, RelB, and cRel. B and C, Analysis of NFkB-1 and NF-kB-2 expression by RT-PCR in resting CD4+ T cells (lanes 1–3), activated and untransfected cells (Untr.; lanes 4–6), control siRNAtransfected cell cultures (siRNActrl; lanes 7–9) and siRNAPKCε-transfected CD4+ cells (siRNAε; lanes 10–12) after 24 h of cultures. 1/10, 1/100, and 1/1000 cDNA, obtained by reverse transcription of 1 mg total RNA, was amplified and PCR products were separated on agarose gel, visualized under UV illumination after ethidium bromide staining. Lanes 1, 4, 7, and 10: PCR with 1/10 cDNA. Lanes 2, 5, 8, and 11: PCR with 1/100 cDNA. Lanes 3, 6, 9, and 12: PCR with 1/100 cDNA. PCR products were quantified by densitometric analysis and plotted in the graph as described in Materials and Methods. *p , 0.05 versus untransfected and activated cells, analysis by ANOVA and Dunnett’s test.


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Table I. Western blot analysis of PKCε expression levels in CD4+ T cells from peripheral blood of adults with HT and of HC

Description

HC HC HC HT HT HT

Sex

n

F M F+M F M F+M

4 23 27 7 16 23

PKCε, Mean ng/ 50 mg 6 SD (Range)

0.24 0.22 0.22 0.42 0.28 0.38

6 6 6 6 6 6

0.24 (0–0.55) 0.20 (0–0.58) 0.20* (0–0.58) 0.17 (0.14–0.97) 0.25 (0–0.84) 0.29* (0–0.97)

*p = 0.026 (t test).

PKCε expression did not induce T-bet, GATA-3, Rorgt, or Foxp3 mRNAs, although it primed the TGF-mediated in vitro Treg polarization of human T CD4+ cells (Fig. 8F).

Discussion PKCs, in particular, PKCu, have a critical role in the activation and proliferation of lymphocytes (3), which, however, express several PKC isoforms that are upregulated and activated on T cell stimulation. In vivo experiments demonstrated that PKCε plays a key role in inflammation and immunity. In fact, using both knockout mice and PKC peptide modulators, Hucho et al. (45) demonstrated that PKCε inhibition profoundly suppresses the acute and chronic inflammatory pain responses, whereas Aksoy et al. (46) suggested that PKCε has a role in inflammation and immune-mediated disorders. Indeed, infiltration of macrophages and T cells into cardiac grafts, as well as parenchymal fibrosis, was decreased in animals treated with PKCε inhibitors (47). Nevertheless, the role of PKCε in lymphocyte activation and proliferation is not yet clear, and even contrasting opinions can be found in the current literature (4–

FIGURE 8. PKCε expression and signaling. A, Western blot analysis of CD4+ T cells from PBLs of four HC adults (HC, lanes a–e) and from eight HT patients (HT, lanes 1–8). Filters were immunoblotted with anti-PKCε and anti–b-actin Abs. Signals obtained from lysates were compared with those produced by 5, 2.5, and 1.25 ng human recombinant PKCε (hrPKCε). Protein expression signals recovered were analyzed and reported in B as PKCε/ b-actin densitometric values. C, Residual proliferation of CD4+ T cell cultures from three HT patients (HT, gray bars) and three healthy controls (HC, white bars) treated with 10 ng/ml TGF-1b. Cells were either transfected with siRNA to PKCε (siRNAPKCε) to inhibit PKCε expression or with scrambled nucleotides siRNA (siRNActrl.). The number of cells with two or more duplications or with zero duplications is reported as percentage of control cultures (TGF-1b–untreated cells). Cell cultures were stimulated with anti-CD3 mAb (4 ng/ml). Means 6 SD are reported. *p , 0.05 versus control culture (siRNActrl-transfected and TGF-1b–untreated cultures), analysis by ANOVA, followed by Dunnett’s test. D and E, PKCε impairs TGF-1b signaling. Western blot analysis of total and activated Smad2 and Smad3 in Jurkat cells (lanes 1–6) and in CD4 lymphocytes (lanes 7–12). Cells were transfected with GFP-PKCε or with the negative control GFP-PKCεm. Transfected Jurkat cultures were starved for 24 h before being treated for 2 h with TGF-1b (10 ng/ ml). CD4+ lymphocytes were grown for 36 h in the presence of IL-2 and anti-CD3 + anti-CD28 stimulation; then cultures were starved for 8 h before being treated for 2 h with TGF-1b (10 ng/ml). Protein expression signals recovered from three experiments were analyzed and reported in E as pSmad/Smad densitometric values. Values were normalized to control cultures (untransfected and TGF-untreated cells). *p , 0.05 versus lane 7 (untransfected and TGFuntreated control lymphocytes); #p , 0.05 versus lane 10 (untransfected TGF-treated lymphocytes); xp , 0.05 versus lane 4 (untransfected TGF-treated Jurkat), analysis by ANOVA, followed by Dunnett’s test. F, PKCε impaired TGF-mediated T cell polarization. Resting CD4+ T cells were nucleofected with siRNAPKCε or with control siRNA (siRNActrl.) and then grown for 4 d on anti-CD3 and anti-CD28 mAb-coated wells. At day 1 of growing, cultures were treated with or without 10 ng/ml TGF-1b. Analysis of T-bet, GATA-3, Rorgt, and Foxp3 expression by RT-PCR in untransfected cells (lanes 1, 4), control siRNA-transfected cell cultures (siRNActrl; lanes 2, 5), and siRNAPKCε-transfected CD4+ cells (siRNAPKCε; lanes 3, 6) grown in absence (lanes 1– 3) and in the presence of TGF-1b (lanes 4–6). cDNA, obtained by reverse transcription of 1 mg total RNA, was amplified and PCR products were separated on agarose gel, visualized under UV illumination after ethidium bromide staining.

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PKCε IN HUMAN CD4 LYMPHOCYTES

Table II. Alignment of siRNAPKCε to target sequence of wild type mouse, of siRNA-resistant mouse, and of human PKCε cDNA sequences Sequence

siRNA#1 WT R H Translation siRNA#2 WT R H Translation siRNA#3 WT R H Translation siRNA#4 WT R H Translation

Alignment

G . A . K G . A . K C . T . N A . G . K

ATC ... ..T ... I ATC ... ..T ... I TAC ... ..T ... Y AAG ... ..A ... K

AAA ... ..G ... K GAG ... ..A ... E AAG ... ..A ... K CTC ... T.G ... L

ATC ... ..T ... I CTG ... ..C ... L GTC ... ..G ... V ATT GC. GCG ... A

TGC ... ..T ... C GCT ... ..G ... A CCT ..C ..C ... P GCT ... ..G ... A

GAG ... ..A ... E GTC ... ..G ... V ACC ..G ..G ... T GGT ... ..C ... G

GCC ... ..G ... A TTT ... ..C ... F TTC ... ..T ... F GCC ..T ..G ... A

GAG ... ..A ... E

Conserved base is reported by a dot. H, siRNA target sequences of human PKCε; R, siRNA target sequences of PKCε1234R; siRNA#1 (#2, #3, and #4), sequence of the number 1 (2, 3, and 4) siRNAPKCε; translation, amino acid sequence of siRNAPKCε target sequence; WT, siRNA target sequences of wild type mouse PKCε.

7, 48). For this reason, and for its potential implications in autoimmune disease pathophysiology, we decided to study the role of PKCε in the activation and proliferation of human CD4+ T cells. Our data demonstrate that PKCε sustains human CD4+ T cell duplication in vitro. In fact, suppression of PKCε expression impaired CD3-mediated activation and proliferation: cell cultures transfected with siRNAPKCε showed more cells that did not duplicate at all, and an overall decrease of the proliferating fraction of the cell culture, without affecting cell viability. In contrast, upregulation of PKCε expression in CD4+ cells transfected with wild type PKCε promoted cell proliferation. This effect was specific to PKCε because it could not be observed in cells transfected with a mutated, kinase-inactive PKCε isoform (PKCεm) and was rescued by siRNA-resistant PKCε (PKC1234R) expression. Searching for a mechanistic explanation for these observations, we found that, although the cell surface expression of activation markers like CD71 and CD95 was not affected, the expected upregulation of CD25 expression was prevented in stimulated CD4+ T cells by the suppression of PKCε expression. Using multiple TCR-independent T cell activators (PHA, PMA, and ionomycin), we observed that PKCε expression and activation were upregulated in stimulated CD4+ cells. Ionomycin, an activator of classical PKCs, activated PKCε as well and promoted its late upregulation, in agreement with recent data reported by P. J. Parker’s group showing that PKCε can be downstream of PKCa (49). Functionally, PHA-induced CD4+ T cell duplication was found to be mediated, in part, by PKCε activation. These data suggest that PKCε is not involved in the early events after T cell activation, but rather acts later, promoting cell proliferation. This conclusion is in agreement with Szamel et al. (6), who compared different PKC isoforms, showing that PKCε is activated later (90 min after TCR ligation) than PKCa and PKCu. We show that PKCε decreases IL-2R expression in stimulated CD4+ T cells both at protein (CD25 cell surface expression) and mRNA level (RTPCR). Szamel et al. (6), on the contrary, did not observe effects of PKCε on CD25 expression. This different result might be possi-

bly explained by the different method of PKCε inhibition (intracellular mAb instead of siRNA). In line with this concept of a late role of PKCε in cell duplication, it should be noted that it has been recently reported in different models that PKCε, by interacting with 14-3-3 protein and RhoA, promotes cytokinesis (50). NF-kB, originally identified in B cells (38, 39), is a central transcription factor in both innate and adaptive immune responses. NF-kB is activated by a plethora of proinflammatory cytokines, chemokines, adhesion molecules, and immunoregulatory mediators. Deregulation of NF-kB has been associated with a number of disorders including arthritis, asthma, and inflammatory bowel disease (38, 51). At least two NF-kB signaling pathways exist (38, 39): the classical pathway, which is dependent on IkBb and is involved in inflammatory responses and innate immunity; and the alternative pathway, which is dependent on IkBa, RelB, and p52 (involved in development, homeostasis, and activation of adaptive immunity) (38, 39). Currently, five mammalian NF-kB family members have been identified and cloned; these include NF-kB1 (p50/p105), NF-kB2 (p52/p100), RelA (p65), RelB, and c-Rel (52, 53). In our hands, activated human PBLs do not express detectable amounts of RelA, RelB, or c-Rel; thus, in our model system of activated human CD4+ T cells, we analyzed the expression of NFkB1 and NF-kB2 only. We observed that in the absence of PKCε, CD4+ T cells failed to upregulate NF-kB1 and NF-kB2 transcription after CD3 and CD28 stimulation. Because IL-2R chain transcription is under the control of NF-kBs (38–41), we believe that PKCε upregulation is a critical event for IL-2R chain expression in activated CD4+ T cells, which operates by promoting NF-kB1 and NF-kB2 transcription. Gruber et al. (48) suggest a redundant function of PKCε in mouse T cell proliferation, and this opinion is in apparent contrast with our findings. However, first, PKC isoforms have different roles and cellular distribution in humans and mice. Perhaps the best example of this is the reciprocal expression of PKCε and PKCd in mouse and human platelets, suggesting species-specific functions of individual PKC isoforms (54). Second, it is not possible to exclude that some PKCε functions, in the case of germinal deletion of the PRKCE as in knockdown mice, may be compensated by other PKCs, such as PKCu, PKCa, and PKCb, expressed in T cells that have relevant roles in the control of cell activation and proliferation. A good example of this is the upregulation of PKCd expression in the heart of PKCε2/2 mice (55). In some instances, indeed, PKCs modulate each other’s expression and activity: the overexpression of PKCa in breast cancer cells, for example, increases PKCb and decreases PKCd and PKCh expression (56); specifically, the overexpression of PKCε induces the recruitment of PKCbII in the development of cardiac hypertrophy (57) by promoting RACK1 expression and PKCbII– RACK1 interaction; and part of the PKCa effects can be mediated by PKCε (49). In contrast, Castrillo et al. (7) reported that lymphocytes in PKCε2/2 mice show a 20% reduction of activation and proliferation in response to classical mitogenic stimuli when compared with lymphocytes from wild type mice. Our data in vitro show that PKCε silencing reduces the proliferation of purified human CD4+ T cells by 50%. T cell proliferation is finely tuned by activating and inhibiting cytokines. In agreement with previously reported data (11), the inhibiting factor TGF-1b is more active on CD3-stimulated CD4+ T cell cultures, in the absence of CD28 costimulation. We have demonstrated that the TGF-1b antiproliferative signaling, undetectable when CD4+ T cells are stimulated with CD3 and CD28, is enhanced by the downregulation of PKCε expression levels in CD3/CD28 and in PHA-stimulated cells. To formally prove that

The Journal of Immunology PKCε interferes with TGF-1b signaling, we upregulated PKCε expression in CD4+ T cells stimulated with CD3 alone, inducing resistance to TGF-1b. We also studied TGF-1b downstream signaling using Jurkat cells and CD4+ T lymphocytes as model systems, focusing on Smad2 and Smad3. Differently from PKCu, the overexpression of PKCε reduced Smad2, but not Smad3, phosphorylation. In line with previous data, demonstrating that both Smad2 and Smad3 contribute to TGF-1b–induced inhibition of IL-2–sustained T cell proliferation (43), it looks reasonable that pSmad2 is downstream PKCε signaling. However, because it is well-known that addition of IL-2 can override the growth arrest of T cells mediated by TGF-1b (10), we cannot exclude that the ability of PKCε to regulate IL-2R expression levels might also explain the increased TGF-1b sensitivity of siRNAPKCε-transfected cells. TGF-1b has been shown to play an essential role in inhibiting Th1 markers, as well as inducing both Treg and Th17 differentiation. Furthermore, Foxp3 expression can suppress Rorgt expression and consequent conversion to Th17 phenotype. Moreover, recently it has been demonstrated that TGF-1b–mediated induction of Foxp3 is also dependent on both Smad2 and Smad3 signaling, which, conversely, are not involved in the TGF-1b– mediated induction of Rorgt (58). In this article, we show that the inhibition of PKCε expression levels does not affect CD4+ T cell polarization in the absence of TGF-1b, whereas increasing Foxp3 expression in TGF-1b–treated cells. On the contrary, siRNAPKCε downregulated Rorgt expression (a Th17 marker) in TGF-1b– treated cultures, probably as a consequence of Foxp3 overexpression. HT has recently been viewed as a genetic disorder of cellmediated immunity (23, 24). After initiation of the autoreactive immune response by CD4+ T cells, the expansion of T cell populations results in a massive lymphocyte accumulation in patients’ thyroids (23, 24). Moreover, it has recently been demonstrated that the serum concentration of TGF-1b is decreased in HT (25), and that some Treg subsets apparently exert a defective suppressive function in these patients. Being that PKCε is implicated in the response of CD4 T cells to both CD3-mediated proliferative stimuli and TGF-1b antiproliferative signals, we were not surprised to find significantly higher expression levels of PKCε in HT patients than in HC. CD4+ T cells from HT patients were resistant to TGF-1b antiproliferative effects, and the downregulation of PKCε expression restored their sensitivity to TGF-1b on anti-CD3 stimulation. The increased expression of PKCε in CD4+ T cells of HT patients, described in this article for the first time, to our knowledge, viewed in the perspective of the physiological role of PKCε in normal Th lymphocytes, adds knowledge to the molecular pathophysiology of HT and creates potentially new pharmacological targets for the therapy of this disease.

Acknowledgments We are grateful to the Instrumentation Laboratory for scientific support and to Enzo Palermo and Luciana Cerasuolo for technical support.

Disclosures The authors have no financial conflicts of interest.

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