Cloning, sequencing and characterization of lentiviral-mediated expression of rhesus macaque (Macaca mulatta) interleukin-2 receptor alpha cDNA

Developmental and Comparative Immunology 29 (2005) 989–1002 www.elsevier.com/locate/devcompimm

Cloning, sequencing and characterization of lentiviral-mediated expression of rhesus macaque (Macaca mulatta) interleukin-2 receptor alpha cDNA* Josh D. Silvertowna, Jagdeep S. Waliaa, Jeffrey A. Medina,b,* a

Division of Experimental Therapeutics, Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ont., Canada M5G-2M1 b Department of Medical Biophysics, University of Toronto, Toronto, Ont., Canada M5G-2M9 Received 24 November 2004; revised 22 February 2005; accepted 28 February 2005 Available online 18 April 2005

Abstract The rhesus macaque CD25 (RhCD25) cDNA isolated from rhesus PBMCs was found to share 95.5 and 91.9% homology with the human orthologue at the nucleotide and amino acid levels, respectively. Comparative sequence analyses suggest that both human CD25 (HuCD25) and RhCD25 share identity for most of the critical amino acids previously identified to be essential for viable folding and IL-2 ligand binding. The human leukemic cell line, HH, deficient for IL-2Ra was transduced with a lentiviral vector (LV) engineered to express RhCD25 (HH-RhCD25). RhCD25 was characterized for expression by flow cytometric analyses, ELISA, Western blotting, functional signalling, and biological assays in comparison to HuCD25. In summary, vectors expressing the RhCD25 cDNA can be used as a tool to aid in the characterization of soluble CD25 in non-human primate studies, and to provide a tempting alternative as an autologous cell surface marker in rhesus macaque gene therapy and bone marrow transplantation studies. q 2005 Elsevier Ltd. All rights reserved. Keywords: CD25; Lentivirus; Soluble CD25; HH cells; Stat5; MMP-9; Rhesus macaque; Macaca mulatta

1. Introduction

* GenBank submission: nucleotide sequence data reported is available in the GenBank database under the accession number AY693777. * Corresponding author. Address: University Health Network, Canadian Blood Services Building, 67 College Street, Room 406, Toronto, Ont., Canada M5G 2M1. Tel.: C1 416 340 4745; fax: C1 416 340 3644. E-mail address: [email protected] (J.A. Medin).

0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2005.02.007

The hallmark function of the interleukin-2 receptor (IL-2R) is to mediate antigen-triggered T-cell expansion and promote effector function [1]. However, a diversity of downstream signals activated by the IL-2R have been reported, including events leading to antigen-activated T-cell development and homeostasis [2], production and expansion of CD4CCD25C T regulatory cells [3], upregulation of intermediary metabolic enzymes [4], and effects on other

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molecules, including matrix metalloproteinase-9 (MMP-9) [5,6]. Stat5 is one of the main downstream signalling molecules implicated in the cellular pathways stimulated by IL-2 binding [reviewed in [7]]. The IL-2R is a trimeric receptor, consisting of IL-2Ra (CD25), IL-2Rb (CD122) and IL-2Rgc (CD132) chains. IL-2Rgc is also partly shared by other interleukin receptors, including IL-4, 7, 9, and 15 [8]. IL-2Ra confers low affinity binding of IL-2 (Kdz10K8 M), but in association with IL-2Rb and IL-2Rgc, the resulting trimeric complex synergizes to generate an enhanced ligand-binding affinity in the range of (Kdz10K10 to 10K11 M). While the IL-2Ra subunit offers no direct signalling consequences or IL-2 internalization itself, the IL-2Rb and IL-2Rgc subunits independently or jointly can signal in the absence of IL-2Ra. Due to its rapid turnover, the steady-state expression of IL-2Ra controls the amount of high-affinity IL-2R present [9]. However, the importance of IL-2Ra for IL-2 affinity was demonstrated when the knockout and abrogation of IL-2Ra from human T cells resulted in reduced responsiveness to IL-2 [10,11]. Expression of IL-2Ra in T cells, B cells and monocytes depends on induction by specific stimuli (antigen, IL-1, TNFa, concanavalin A, IL-2, etc.) and is transcriptionally regulated by NF-kB [12]. Lastly, in addition to the intact receptor found on the cell surface, T-cells produce a form of the receptor that is detectable as a soluble, glycosylated protein of approximately 45 kDa [13]. The secretion of this soluble form of IL-2Ra (sIL-2Ra) has been associated as a correlative marker for a number of neoplastic, autoimmune and hematological disorders [14]. We have previously demonstrated the use of human IL-2Ra (HuCD25) as a cell surface marker in gene therapy experiments [15]. In that study, we generated a bicistronic oncoretroviral vector that engineered the expression of HuCD25 and a therapeutic transgene. This strategy not only facilitates titering of viral stocks and detection of infected target cells, but can be used to pre-select functionally transduced cells prior to transplantation [16]. In this regard, use of an endogenous marker will reduce immune responses to foreign antigens such as been seen for enhanced green fluorescent protein (eGFP), for example [17]. This is especially relevant for the use of surrogate models, such as rhesus macaques,

for the development and advancement of gene therapy. In view of the importance of CD25 in the immune system and as a serum cytokine marker in a number of pathophysiological conditions, along with potential uses in gene therapy experiments employing large animal models, we sought to clone out the cDNA of the rhesus macaque CD25 (RhCD25). Since, the rhesus macaque (Macaca mulatta) is anatomically, physiologically, and genetically similar to the human, it is a suitable model to provide biological understanding in the human. Therefore, the goal of this study was to clone out the RhCD25 cDNA and characterize its sequence and function in vitro. This will provide both a relevant tool to explore the role of RhCD25 in rhesus macaque studies, and also a means for comparative studies of IL-2Ra function with other known species orthologues.

2. Methods and materials 2.1. Cell lines and culture conditions All cell lines were obtained from the ATCC (Rockville, MD, USA). The human embryonic kidney cell line 293T (stably transfected with SV40 large T-antigen), was cultured in DMEM supplemented with 10% fetal calf serum (FCS, NorthBio, Inc., Toronto, Ont., Canada). The human leukemic T-cell line, HH, which is deficient for human CD25 (HuCD25), was cultured in RPMI medium (Sigma; Oakville, Ont., Canada), supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 50 mM of b-mercaptoethanol (Sigma). Cells were incubated at 37 8C in a humidified atmosphere with 5% CO2. All molecular subcloning was performed with E. coli XL-10 Ultracompetent cells (Stratagene, La Jolla, CA, USA). 2.2. Cloning of RhCD25 cDNA Peripheral blood mononuclear cells (PBMCs) were harvested from a 5-year-old male rhesus macaque (Chinese origin) and isolated by the Ficoll gradient centrifugation method (Roche, Laval, Que., Canada). Cells were seeded at a density of 5!106 cells in 20 ml of RPMI culture medium (Sigma), supplemented with

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10% FCS, and maintained at 37 8C and 5% CO2. To stimulate endogenous CD25 expression, PBMCs were treated with Con A (5 mg/ml; Sigma) for 48 h. Following stimulation, cells were harvested and total RNA was collected using a Trizol extraction method (Invitrogen; Burlington, Ont., Canada). Reversetranscription PCR was performed on approximately 100 ng of total RNA using random hexamer primers (Superscripte First-Strand Synthesis System for RTPCR; Invitrogen). b-actin was amplified to confirm RNA integrity and efficiency of the reaction, as described previously [18]. Primers to amplify RhCD25 were created based on the published HuCD25 mRNA sequence (Accession Number: NM_000417), targeting the 5 0 and 3 0 untranslated regions. Forward primer: 5 0 -gtcccaagggtcaggaagat-3 0 ; Reverse primer: 5 0 -aagcacaacggatgtctcct-3 0 . The expected RhCD25 amplicons of approximately 900 bp were obtained after amplification for 30 cycles (denaturing at 94 8C, 30 s; annealing at 50 8C, 30 s; elongation at 68 8C, 60 s) using Platinum Taq DNA Polymerase High Fidelity reagents (Invitrogen). The RhCD25 cDNA amplified product was subcloned into pPCR-Script using the PCR-Scripte Amp Cloning Kit (Stratagene) and sequenced using both T7 and T3 primers for forward and reverse sequencing. DNA sequencing was performed using a Model 377 ABI sequencer from Applied Biosystems (ACGT Corp., Toronto, Ont., Canada). Three individual subclones were propagated and sequenced to ensure integrity of the final coding nucleotide sequence. 2.3. Lentiviral construction and infection of HH cells Lentiviral vectors (LV) were constructed to express the RhCD25 cDNA driven by an elongation factor-1 alpha (EF1a) promoter, analogously to that described by Yoshimitsu et al. [19]. Briefly, the 935 bp RhCD25 cDNA fragment was amplified by PCR with primers containing AscI restriction enzyme recognition sites at the 5 0 ends. Forward primer: 5 0 gtggcgcgccgtcccaagggtcaggaag-3 0 ; Reverse primer: 5 0 -gtggcgcgccaagcacaacggatgtctc-3 0 . The RhCD25 cDNA was amplified for 30 cycles (denaturing at 94 8C, 30 s; annealing at 55 8C, 30 s; elongation at 68 8C, 60 s) using Platinum Taq DNA Polymerase High Fidelity reagents (Invitrogen). The cDNA fragment was resolved on a 1% agarose gel, extracted

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using the GeneClean II Kit (Qbiogene, Inc., Montreal, Que., Canada), and digested with AscI (New England Biolabs, Mississauga, Ont., Canada), according to manufacturer’s recommended conditions. The AscIdigested RhCD25 fragment was ligated into the AscI site. Positive clones (pHR-cPPT-EF1a-RhCD25WPRE) were identified by diagnostic restriction enzyme digestion analysis on DNA mini-preparations (Roche) and subsequent DNA sequencing analysis (ACGT Corp., Toronto, Ont., Canada). LVs were produced by a transient triple-transfection method using 293T monolayers [19]. Viral supernatants were titered on 293T cells by serial dilutions and analyzed 72 h later by flow cytometry (FACS Calibur, Becton Dickson; San Jose, CA, USA) for RhCD25 expression (see Section 2.4). Unconcentrated LV titers ranged from 5!106 to 1!107 293T infectious units/ml. Cell cultures of the leukemic human cell line, HH, deficient for the IL-2Ra chain [20] were transduced with viral supernatant [19] for 24 h, followed by a change in culture medium. Rhesus bone marrow (BM) was collected by perfusion method and CD34C cells were isolated as described [21,22]. Cells were cultured for 24 h in StemSpane SFEM Serum-Free Expansion Medium (Stem Cell Technologies; Vancouver, BC) supplemented with rhSCF, rhFLT3, rhIL-6 and rhTPO (all 50 ng/ml) at 37 8C and 5% CO2. Following incubation, cells were transferred to Retronectin-coated (2 mg/cm2; Takara, NY, USA) 6-well plates and transduced with concentrated LV-RhCD25 in fresh media as described previously [19]. After 72 h, cells were analyzed for RhCD25 and CD34 expression by flow cytometry (see Section 2.4). 2.4. Anti-CD25 antibodies and flow cytometry The Con A-stimulated rhesus macaque PBMCs were tested for cross-reactivity with different antihuCD25 antibodies. To detect the expression of RhCD25, the following antibodies were used: PE anti-human CD25 (Clone: MA251, BD Pharmingen; San Diego, CA, USA), Coulter clone IL2R1-FITC (Coulter Corporation; Miami, FL, USA), anti-huIL2R CD25 RPE (Biosource International; MediCorp, Inc., Montreal, Que., Canada), and mouse anti-human CD25 RPE (Southern Biotechnology Association, Inc., Birmingham, AL, USA). To detect CD34C

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rhesus BM cells, we used anti-human CD34 PE-conjugated antibodies (clone 563; BD). Isotypes employed were mouse IgG1k PE (BD) and mouse IgG2ak FITC (BD). Samples were analyzed by FACSCalibur and Cell Quest software (BD). All plots were gated on live cells after 7-AAD exclusion of dead cells. 2.5. ELISA for detection of soluble CD25 ELISA Non-transduced HH cells (HH-NT), HH cells transduced with LV-EF1a-RhCD25 (HH-RhCD25), and HH cells transduced with a Moloney murine leukemia-based virus expressing HuCD25 (HHHuCD25) [15] were seeded in 1 ml at varying cell densities ranging from 0.31!105 to 2.5!105 cells/ml in a 24-well tissue culture plate. Cell culture medium was harvested from 24 to 48 h samples. Supernatant was collected after pelleting cells at 200 g for 5 min. Soluble CD25 (sCD25) in the supernatant was detected using a commercially available human IL-2sRa ELISA set (BD Biosciences). The procedure was carried out according to the manufacturer’s protocol using NUNC Maxisorp 96-well plates (VWR; Mississauga, Ont., Canada). Three independent experiments for the ELISA were performed in triplicate. 2.6. Western blotting for CD25 Cell lysates were generated from approximately 5!106 HH-NT, HH-RhCD25, HH-HuCD25 cells using a cell lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl2; 0.5 mM MgCl2; 0.2 nM EGTA; 1% Triton X-100) and treated with the protease inhibitor cocktail (P8340; Sigma) as done previously [23]. Samples were run on a 10% polyacrylamide gel under non-reducing conditions and transferred to Immobilon-P PVDF membranes (Millipore; Bedford, MA, USA). Membranes were incubated in blocking buffer overnight (5% Blotto; 0.15% Tween-20, TBS); and incubated with a mouse anti-human IL2-Ra antibody (clone 22722; R&D Systems; Minneapolis, MN) for 1 h at room temperature, followed by 3!15 min washes with TBS. Blots were incubated with a secondary mouse IgG antibody conjugated to HRP for 1 h at room temperature, followed by 3!15 min washes with TBS. The ECLe Analysis System

(Amersham Biosciences) was used for detection of banding patterns. Chemiluminescence was detected with the Fluor-Sw Max Multimager (BioRad; Mississauga, Ont., Canada) and analyzed with Quantity One quantitation software (BioRad, Burlington, Ont., Canada). 2.7. Gelatin zymography and protein concentration assay HH-NT and HH-RhCD25 cells were washed three times in PBS and seeded in triplicate at a density of 1.4!106 cells/well of a 24-well culture vessel in 1 ml of serum-free RPMI medium. Following a resting period of 24 h, cells were stimulated with huIL-2 (0, 250, 1000 U/ml; gift from Daniel Fowler; NIH, Bethesda, MD, USA). After 48 h, conditioned medium was collected, frozen at K80 8C for 24 h, and then lyophilized. Samples were resuspended in 1/10th the original volume in sterile water. Zymographic analyses were performed as described previously [6]. To confirm that the observed gelatinolytic bands were due to MMP activity, gelatin zymographies were incubated in incubation buffer supplemented with 10 mM EDTA (Sigma). Quantity One software was employed to measure band densitometry (Quantity One, Bio-Rad) on the zymogram. A Bradford assay was conducted to determine the relative protein concentrations present in the conditioned medium from each sample of stimulated cells. Samples and Bradford Reagent (BioRad) were combined and read at 450 nm on an ELX-800 BioTek Microplate reader. All samples were analyzed in triplicate in a 96-well dish and compared against a standard curve generated using dilutions of bovine serum albumin, ranging from 0.02 to 0.7 mg/ul. 2.8. Immunoprecipitation (IP) and Western blotting of Stat5 To ascertain IL-2-mediated signaling events, Stat5 phosphorylation was examined. HH-NT, HHRhCD25 and HH-RhCD25 cell lines were washed three times in PBS, and seeded at a density of 2!106 cells/well of a 24-well dish. Cells were equilibrated for 1 h in the incubator before IL-2 stimulation (1000 U/ml). At each time point, cells were removed, spun at 1400 rpm for 4 min, resuspended, and

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incubated at 4 8C for 1 h in IP buffer (1% Triton-X 100; 0.5% NP-40; 150 mM NaCl; 10 mM Tris–HCl pH 7.4; 1 mM EDTA; 1 mM EGTA; 0.2 mM Na 3VO 4; 0.2 mM PMSF; 10 mg/ml aprotinin; 2 mg/ml leupeptin; 2 mg/ml pepstatin A; 100 mM Na2PO7; Sigma). Protein A/G (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) and anti-Stat5 antibodies (4 mg/sample; Santa Cruz Biotechnology, Inc., sc-835) were added to each sample and incubated overnight at 4 8C. After samples were washed three times in fresh IP buffer, protein A/G sepharose beads were boiled in reducing SDS sample buffer. IP samples were analyzed by 4–10% SDS-PAGE, and transferred to Immobilon-P PVDF membranes as above. Western blotting was done as described earlier, except membranes were blocked and stained with antibodies in TBS-T containing 3% bovine serum albumin. For detection of phosphorylated and unphosphorylated Stat5, blots were incubated overnight with anti-phosphotyrosine (1:6000, clone 4G10; Upstate Cell Signaling Solutions, Lake Placid, NY) or anti-Stat5 (1:5000; Santa Cruz Biotechnology, Inc., sc-835) antibodies. Secondary antibodies and chemiluminescence detection were carried out as above. When needed, blots were stripped for 10 min (7 M guanidine hydrochloride; 50 mM glycine, pH 10.8; 100 mM KCl, 50 mM EDTA, pH 8.0; 20 mM 2mercaptoethanol) and rinsed extensively in TBS-T buffer prior to reprobing.

3. Results 3.1. Cloning and sequencing of RhCD25 Three individual clones harboring the putative RhCD25 cDNA were isolated and sequenced in forward and reverse reactions. Consistent sequencing among the three clones confirmed a 935 bp cDNA that includes an open reading frame (ORF) of 819 bp and a partial sequence of the 3 0 UTR. The ORF (1–819) encodes a predicted protein of 272 amino acids with a calculated molecular weight of 30.8 kDa. The sequence was submitted to GenBank and given the accession number AY693777. Fig. 1 illustrates the nucleotide (Fig. 1(a)) and translated amino acid sequence (Fig. 1(b)) alignments between the published HuCD25 sequence (Accession number:

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NM_000417) and the RhCD25 sequence. The rhesus macaque and the human receptors share 95.5 and 91.9% sequence identity at the nucleotide (893/935) and amino acid (250/272) levels, respectively. When including conservative amino acid changes (9 out of 22 changes; shaded in Fig. 1(a)), amino acid similarity between the HuCD25 and RhCD25 receptors becomes 95.2%. HuCD25 is characterized to possess N-and O-linked carbohydrate, sulfation, and phosphorylation sites [24]. Therefore, to identify putative post-translational processing motifs (i.e. glycosylation and phosphorylation sites) within the RhCD25 protein, the amino acid sequence was submitted to a number of protein analysis programs and algorithms. Table 1 summarizes a list of the motifs identified, their nucleotide and amino acid locations, the program used to generate the identities, and putative homologies with HuCD25. For example, when submitted to the PFAM database (http://www.sanger.ac.uk/Software/Pfam/; Wellcome Trust Sanger Institute), rhesus IL-2Ra is predicted to share similar sushi domains (amino acids: 24–82 and 125–184) with those encoded in the human form. Sushi domains are known to be involved in many recognition processes, including binding of several complement factors to fragments C3b and C4b [25]. According to the Signal 3.0 Server program (http://www.cbs.dtu. dk/services/SignalP/; Center for Biological Sequence Analysis, Technical University of Denmark, DTU), the RhCD25 cDNA encodes a signal sequence that is cleaved between residues 21 and 22, similar to the human form, resulting in a 251 amino acid mature receptor subunit with a calculated molecular weight of approximately 28.5 kDa. RhCD25 protein, on the basis of the TMHMM Server v. 2.0 algorithm predicting transmembrane helices in proteins (http:// www.cbs.dtu.dk/services/TMHMM-2.0/; Center for Biological Sequence Analysis, Technical University of Denmark, DTU), contains a 219-residue extracellular domain (22–240), a 19-residue hydrophobic transmembrane domain (241–259), and a 13-residue intracytoplasmic segment (260–272). The mapped domains within the RhCD25 structure are analogous to those present in the HuCD25 receptor [26]. Numerous studies employing in vitro mutagenesis have delineated the critical residues important for HuCD25 conformational folding and IL-2 binding [27–31]. Sequence analyses indicate that the HuCD25

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Fig. 1. Nucleotide and amino acid alignments of RhCD25 and HuCD25. (a) The rhesus macaque and the human orthologues share 95.5% sequence identity at the nucleotide (893/935) level. Bracketed numbers next to human and rhesus nucleotide sequences indicate base number as per accession numbers NM_000417 and AY693777, respectively. (b) The rhesus macaque and the human orthologues share 91.9% sequence identity at the amino acid (250/272) level. Bracketed numbers next to human and rhesus amino acid sequences are numbered starting with the start codon, as per accession numbers NM_000417 and AY693777, respectively. Conserved amino acid changes are darkened.

J.D. Silvertown et al. / Developmental and Comparative Immunology 29 (2005) 989–1002 Table 1 Putative motifs present in RhCD25 and homologies with HuCD25 Feature

RhCD25 nucleotide #

RhCD25 amino acid #

RhCD25/ HuCD25 homology

Sushi domaina

70–201 373–552 103–105 268–270 406–408 631–633 811–813 310–312

24–82 125–184 35 90 136 211 271 104

Yes Yes Yes Yes Yes No No Yes

208–210 265–267 220–222 646–648 130–132 613–613 – – – –

70 89 74 216 44 205 24 to 67 51 to 82 125 to 168 152 to 184

Yes c [20] Yes c [20] Yes Yes Yes No Yes Yes Yes Yes c [27]

PKC phosphorylation siteb

cAMP phosphorylation siteb Asn glysosylation siteb Casein II phosphorylation siteb N-myristoylation siteb Disulfide bridges (within sushi domains)d

A summary of putative post-translational modification sites within the RhCD25 cDNA. A list of corresponding locations in the nucleotide and predicted amino acid sequences and their homologies to HuCD25 is provided with the algorithms that were employed for predicting motifs. a Protein families database of alignments and HMMs (PFAM), Wellcome Trust Sanger Inst. (http://www.sanger.ac.uk/Software/ Pfam/). b PPSearch, European Bioinformatics Institute (EMBL-EBI) (http://www.ebi.ac.uk/ppsearch/). c Predicted in literature to be homologous to human interleukin-2 receptor alpha. d The ExPASy (Expert Protein Analysis System) proteomics server, Swiss Institute of Bioinformatics (http://expasy.org/prosite/).

and RhCD25 cDNAs share high homology for posttranscriptional and post-translational processing, along with analogous regions for IL-2 ligand binding. All of the residues present at the junctions for exon– exon boundaries in the RhCD25 cDNA are conserved with the human sequence [28]. Exons 2 and 4 of HuCD25, predicted to be the result of intra-gene duplication [29,30], are reported to contain critical amino acids for proper folding and ligand binding. Although amino acid changes (four non-conserved changes in exon 2; one non-conserved change in exon 4) are observed within these regions of RhCD25, they are likely not the essential residues that contribute to the integrity of the receptor. Rusk et al. [27] showed

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that all but two cysteine residues (Cys 213 and Cys 246) encoded in the full-length HuCD25 are important for proper conformational folding and bioactivity [31]. Therefore, the Arg 213 encoded in the RhCD25 (as compared to Cys 213 in HuCD25) will likely not compromise proper post-translational modifications, folding, and affinity for IL-2 binding. Robb et al. (1988) identified two short segments within exon 2 (residues 22–27 and 56–64) and one amino acid within exon 4 (His 141) that significantly augment binding of both IL-2 and selected monoclonal antibodies to CD25 [27]. Probable secondary structures and folding classes were predicted by two independent servers for the Leu-63-Pro alteration from human to rhesus. Despite this change being a conserved variation of a hydrophobic residue, the prediction servers were unable to provide any firm conclusions for this change, in terms of confidence/ probability regarding similarity of secondary structure (data not shown) [32,33]. 3.2. Characterization of RhCD25 expression Use of an anti-HuCD25 monoclonal antibody to detect and quantify IL-2Ra expression in PBMCs from rhesus macaques has been reported before [34]. Considering the relatively high homology between the rhesus and human orthologues, we wanted to determine whether a selection of anti-HuCD25 antibody clones could cross-react with endogenous RhCD25 from Con A-activated PBMCs from the rhesus macaque. Having a variety of antibody clones to use for detection of RhCD25 enhances the practical application of this cDNA in future studies. Fig. 2(a) shows a flow cytometry histogram of positive staining for RhCD25 presented on stimulated rhesus PBMCs detected by PE anti-human CD25 from BD Pharmingen compared to unstained and isotype controls. Endogenous RhCD25 was also detected using other anti-huIL-2R antibodies (anti-human IL-2R CD25 RPE; Biosource International), Coulter clone IL2R1 FITC (Coulter Corporation), and mouse anti-human CD25 RPE (Southern Biotechnology Association) with comparable staining to the antibody from BD Pharmingen (data not shown). Differential CD25epitope affinity of each of the antibodies exhibited negligible differences on staining patterns from activated rhesus macaque T-cells. The PE-conjugated

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Fig. 2. Flow cytometric analyses of CD25 expression. (a) Stimulated rhesus macaque PBMCs stained for RhCD25. (b) Non-transduced HH cells (HH-NT); (c) HH cells transduced with LV-EF1a-RhCD25 (HH-RhCD25); (d) HH cells transduced with a Moloney murine leukemia-based virus expressing HuCD25 (HH-HuCD25); Black line (open), unstained cells; Dotted line (open), Isotype; Black (filled), anti-human CD25 antibody.

anti-human CD25 antibody was selected to stain recombinant RhCD25 for the balance of the study. In contrast to undetectable staining for CD25 on HH-NT cells (Fig. 2(b)), recombinant LV-transduced HH cells (a single transduction) expressing RhCD25 (HHRhCD25; Fig. 2(c)) or oncoretrovirally transduced HH cells expressing HuCD25 (HH-HuCD25; Fig. 2(d)) [15] were stained to be approximately 83 and 80% positive, respectively. To confirm that RhCD25 can be expressed on primary cells, rhesus BM cells were successfully transduced by LVRhCD25. Whereas negligible CD25 expression was

detected from isotype-stained (Fig. 3(a)) and nontransduced cells (Fig. 3(b)), RhCD25 expression measured by flow cytometry was detected on approximately 80% of BM cells including CD34C cells and their differentiated progeny (Fig. 3(c)). Western blotting analysis confirmed that RhCD25 is expressed as an approximate 50 kDa protein from HH-RhCD25 cells, similar to the recombinant HuCD25 expressed from oncoretroviral-transduced HH-HuCD25 cells (Fig. 4). CD25 has been characterized by immunoblotting to fall within a range of molecular weights. This has been attributed to varying

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Fig. 3. Transduction of primary rhesus bone marrow cells with LV-EF1a-RhCD25. (a) Transduced rhesus bone marrow cells stained with isotype; (b) Non-transduced rhesus bone marrow cells; (c) Transduced rhesus bone marrow cells stained for RhCD25 expression.

patterns of post-translational processing (i.e. glycosylation and phosphorylation) dictated by the cell type [24]. Even though there is some non-specific crossreactivity as detected in the HH-NT cell lysate sample, some differential processing of the human and rhesus forms of CD25 may occur in HH cells. Further experiments will be needed to examine this result.

cellular metabolic activity [4]. Therefore, in the first assay, we sought to quantify protein concentration from conditioned media of huIL-2-stimulated HH-NT and HH-RhCD25 cell cultures. Relative increases of protein concentration that were significant (P!0.05) were observed in the hIL-2-treated HH-RhCD25 cell cultures compared to HH-NT cells (Fig. 6). HH-NT and HH-RhCD25 samples treated with 250 and 1000 U/ml huIL-2 exhibited relative percent increases

3.3. Detection of soluble CD25 by ELISA To determine if the subcloned and expressed rhesus macaque form of CD25 can be cleaved to a soluble form, transduced-HH cell cultures were subjected to a sCD25 ELISA. HH-RhCD25 and HHHuCD25 displayed a time-and cell number-dependent cleavage of CD25 compared to non-transduced HH cells at 24 (Fig. 5(a)) and 48 (Fig. 5(b)) hours. After 48 h, transduced HH cells had an approximate doubling of sCD25 in culture supernatant at each cell density compared to 24 h cell samples. 3.4. Biological activity and signalling due to LV-mediated expression of RhCD25 To determine if the cloned RhCD25 cDNA encodes a biologically active protein, three bioassays were employed. Human IL-2 has been reported to stimulate enhanced protein production, an indicator of

Fig. 4. Western blot analysis of CD25 as detected in cell lysates of transduced HH cells. Lane 1: HH-NT; Lane 2: HH-HuCD25; Lane 3: HH-RhCD25. An approximately 50 kDa immunoreactive band is apparent in the transduced HH cells expressing CD25.

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Fig. 5. Soluble RhCD25 levels in conditioned medium from HH cell cultures seeded at varying cell densities, as measured by ELISA. (a) Soluble CD25 levels after 24 h of cell culture; (b) Soluble CD25 levels after 48 h of cell culture. ,, HH-RhCD25; $, HH-HuCD25; 6, HHNT. Data points are taken from an average of three independent experiments. (Standard error bars: P!0.05.)

of approximately 3 and 21% for HH-NT cells and 21 and 42% for HH-RhCD25 cells compared to untreated controls. In the second assay, the effects of huIL-2 on the upregulation of a gelatinase (MMP-9) from HH-NT and HH-RhCD25 were observed after a semi-quantitative analysis by gelatin zymography. Upon stimulation of transduced and non-transduced HH cells with huIL-2, a subtle, yet consistent and significant increase in MMP-9 activity was observed in the media from

Fig. 6. Human IL-2 stimulates HH cells expressing RhCD25 (HHRhCD25) to exhibit a significant secretion in protein concentration, compared to non-transduced controls (HH-NT). The Bradford assay was employed to quantify total protein concentration in conditioned medium from HH cell cultures after 48 h of IL-2 treatment (0, 250, and 1000 U/ml). Values are the averaged data points from each of the three independent experiments. Data were analyzed with a paired t-test (Microsoft Excel). Asterisks at error bars indicate significance (P!0.05).

the HH-RhCD25 cell line, compared to the nontransduced HH cell line (Fig. 7(a)). Although both cell lines exhibited increases in MMP activity with increasing huIL-2 doses, densitometric analyses

Fig. 7. Human IL-2 stimulates HH cells expressing RhCD25 (HHRhCD25) to upregulate MMP-9 secretion compared to nontransduced controls (HH-NT). (a) Gelatin zymography was employed to visualize MMP-9 activity present in concentrated conditioned medium harvested at 48 h after IL-2 treatment (0, 250, and 1000 U/ml). Zymogram shown is a representative Figure of three independent experiments. (b) Densitometry values are the averaged data points from each of the three experiments. Data were analyzed with a paired t-test (Microsoft Excel). Asterisks at error bars indicate significance (P!0.05).

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Fig. 8. Western blot analysis of Stat5 (total Stat5 and phosphorylated Stat5) from cell lysates collected from HH-NT, HH-RhCD25 and HHHuCD25 cell cultures (2!106 cells/well) stimulated with IL-2 (1000 U/ml) for 0, 5, 15, and 30 min.

showed that the HH-RhCD25 cell cultures secreted greater levels of MMPs relative to untreated controls, compared to the HH-NT samples (Fig. 7(b)). HH-NT and HH-RhCD25 cells stimulated with 250 and 1000 U/ml huIL-2 had relative increases of approximately 1.1-fold and 1.3-fold for HH-NT cells and 1.3-fold and 1.6-fold for HH-RhCD25 cells compared to untreated controls, respectively. To further explore the biological consequences that result due to RhCD25 expression, IL-2-mediated Stat5 phosphorylation was detected (Fig. 8). It was found that IL-2 (1000 U/ml) induced a significant (1.4-fold) increase in Stat5 phosphorylation in the HH-NT cell line at 5 and 15 min, leading to an almost complete abrogation by 30 min. However, IL-2-stimulated HH-RhCD25 and HH-HuCD25 cells sustained Stat5 phosphorylation (1.3-fold) up to 30 min in both cases.

4. Discussion This study reports a high sequence homology between rhesus macaque and human forms of CD25, and provides preliminary evidence that the two orthologues share some similar functional activity. The rhesus and human CD25 share 95.5 and 91.9% homology at the nucleotide and amino acid levels, respectively. Within the predicted encoded region of RhCD25, there are 22 amino acid changes, nine of which are conserved, giving an overall homology of 95% between the human and rhesus form. Most of the key amino acids identified to be critical for proper folding and ligand binding by in vitro mutagenesis studies are conserved between the human and rhesus orthologues [27–31]. In our study, the HH cell line was engineered to constitutively express RhCD25

(HH-RhCD25) as determined by flow cytometric analyses, Western blotting, and in vitro detection of sRhCD25 by ELISA. A number of different antibodies were employed to successfully detect recombinant RhCD25 expressed on and within LV-transduced HH cells, suggesting that the proposed secondary structure for RhCD25 encodes a receptor with a viable conformation. The present study also confirms that the RhCD25 chain can act as a surrogate unit for HuCD25 in the human HH cell line by partially augmenting its bioactivity observed from increased IL-2-induced signalling. Two bioassays were performed to confirm function: a gelatin zymography and a protein assay. In each assay, addition of huIL-2 had an effect on both the nontransduced HH cell line and the transduced HHRhCD25 cell line, however, the effect was more pronounced in the CD25-expressing HH cells compared to the parental cell samples. This disparity is expected because the HH cell line possesses the b and g chains and has a low to moderate affinity for IL-2 binding. However, restoration of the CD25 subunit completes the trimeric IL-2R complex, resulting in higher affinity for IL-2. Similar results for the ability of IL-2 to stimulate an increase in metabolic activity as indicated by increases in detectable protein concentration in lymphocyte cell cultures has been reported before [4]. Moreover, the function of IL-2 to promote the upregulation of MMP-9 from T-cells has also been observed [5,35]. To corroborate these findings, IL-2 signaling was measured by detecting Stat5 phosphorylation. It appears that the expression of RhCD25 or HuCD25 on the HH cell line permits a longer period of IL-2 signaling compared to the HHNT cell line. This difference could be attributed to both the greater number of available IL-2R binding sites and enhanced receptor affinity for IL-2 presented

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by HH-RhCD25 and HH-HuCD25 cells. Therefore, a longer sustained signalling cascade could lead to a greater stimulation of downstream biological effectors. In each case, HH-RhCD25 and HH-HuCD25 cells responded more profoundly to doses of IL-2 compared to non-transduced HH cell cultures. Taken together, these findings suggest that the cDNA sequence reported in this study does encode a functional RhCD25 chain that shares analogous functions to its human orthologue. The newly cloned RhCD25 cDNA undergoes cleavage at the cell surface as a soluble form. It has been reported that endogenous rhesus IL-2Ra forms can be detected in the periphery of monkeys infected with simian immunodeficiency virus [36] and in a primate model exhibiting rejection of xenograft transplantations [37]. This parallels the extensive reports in the literature that human CD25 can be detected as a soluble form in in vitro and in vivo studies [9,13,14]. In this current study, a sCD25 ELISA kit specific for the human form can detect sRhCD25, despite the minor differences in amino acid sequence with HuCD25. Considering that sCD25 have been characterized in a number of pathophysiological conditions, this finding may be of significant relevance. Soluble CD25 is reported to be a serum cytokine marker for malignant, autoimmune and inflammatory conditions, such as in rheumatoid arthritis, progressive systemic sclerosis, multiple sclerosis and diabetes [reviewed in [14]]. Levels of sCD25 appear to accurately reflect cancer progression and tumour burden, thereby permitting clinicians with a simple and useful non-invasive strategy for the management of both cancers, and the above mentioned diseases [38]. Consequently, a RhCD25specific ELISA does not need to be generated: commercially available kits for human sCD25 appear to be equally efficacious and sensitive to the rhesus form. The role of rhesus sCD25 in the immunopathogenesis and treatment of a number of diseases has been investigated. Targeted IL-2R treatment using humanized anti-CD25 (anti-TAC; HAT) has been employed as an effective therapeutic approach for autoimmune uveoretinitis [39], graft survival of a xenograft transplantation [36], and reduction of joint inflammation and erosion in a human rheumatoid arthritis [40] in rhesus macaque models. Laboratories

wishing to investigate the role of RhCD25 in hematological and autoimmune disorders as an analogous system to the human can use the RhCD25 cDNA described in this paper as a reagent for preclinical studies in rhesus macaque models. In addition, a LV engineering the expression of RhCD25 as described herein will allow for studies to delineate IL-2 signaling cascades in cells or tissues of rhesus macaques, for example. Aside from applications for the use of sRhCD25 as a marker for neoplastic and autoimmune conditions, the cDNA for RhCD25 can also be used as a cell surface marker in gene therapy studies involving nonhuman primate models. In this study, we confirmed proof-of-principle that primary rhesus BM cells can be efficiently transduced with lentivectors to engineer RhCD25 expression. In applications where the transgene is not readily detectable by fluorescence, enzymatic, chromogenic, or immunological means, investigators have opted to use bicistronic expression cassettes exploiting prokaryotic internal ribosomal entry site elements to dually express both the transgene and a reporter gene under the control of the same promoter. A problem with fluorescent protein-based markers is that sorting and/or enrichment of transduced cells requires use of high-speed flow cytometric sorting; a technology that is not yet feasible for broad clinical application [16]. Another limitation with current heterologous reporter genes, e.g. eGFP and LacZ, in the gene therapy field is that they are immunogenic [17]. This could be a problem since numerous studies have observed immune responses to reporter genes in non-human primate studies. For example, rhesus macaques that underwent myeloablative irradiation followed by infusions with CD34C cells transduced with a retroviral vector expressing eGFP exhibited a vigorous eGFP-specific cytotoxic T lymphocyte response [41]. Moreover, the LacZ gene encoding b-galactosidase has also been associated with immunogenic responses [42]. Although these reporter genes have been widely used, it is becoming evident that autologous or nonxenogenic reporter genes are preferable. Therefore, gene therapy studies utilizing non-human primate models, such as the rhesus macaque can effectively utilize the non-signaling CD25 subunit as a nonimmunogenic cell surface marker that has a limited expression range.

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Taken together, this study provides evidence that the rhesus macaque orthologue of HuCD25 retains high nucleotide and amino acid sequence homology and exhibits similar biological function. The utility of vectors expressing the RhCD25 cDNA can be used as a tool to further elucidate the hematological role of the IL-2/IL-2R system, to aid in the characterization of sCD25 in non-human primate models, and to provide a better cell surface reporter marker in gene therapy and transplantation studies.

Acknowledgements The authors would like to thank Drs Chris Siatskas and Makoto Yoshimitsu for their help with delineating some of the more technical aspects of the study, and Melissa Brierley and Joanna Zorzitto for their assistance with the Stat5 immunoprecipitation studies. This work was partially funded by the Ontario Cancer Institute—Clinical Research Program in Gene Therapy. Salary for J.D. S. was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). Salary for J.S.W. was funded by Canadian Prostate Cancer Research Initiative (CPCRI).

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