Background
Human polyomavirus BK (BKV) is a DNA virus belonging to the Polyomaviridae family that also includes human polyomavirus JC (JCV), and Simian virus 40 (SV40) (International Committee on the Taxonomy of Viruses (ICVD))[
1]. The virus is ubiquitous in the human population, establishing latent infections in the kidney and the urogenital tract[
2]. Spontaneous reactivation and low-level replication with shedding into urines is observed in 5–20% of healthy individuals[
3]. Serological evidence indicates that nearly 90% of individuals are infected by early childhood, although a decrease in this rate during the human lifespan may be due to viral seroconversion (70–80%) [
4,
5]. Usually, polyomaviruses cause persistent subclinical infections in humans and BKV infection rarely leads to clinical manifestations. However, when the immune system is compromised, as following transplantation, HIV infection, chemotherapy or pharmacologic immunosuppression, rate and level of BKV replication increase and may lead to organ diseases[
6]. The polyomavirus genome consists of the non-coding control region, the early genes and the late genes. The polyomavirus-encoded early gene product large tumor antigen (LTag) has been identified early on as a key regulatory molecule[
7]. LTag interacts with a number of host cell molecules including the tumor-suppressor gene retinoblastoma family (Rb) products and p53. Initially, the LTag binds to products of the Rb-family (pRb, p107, and p130) thereby interfering with their activity and inducing the infected cell to enter the cell cycle (phase S). Subsequently, and most importantly, the LTag inactivation of p53 allows the re-phosphorylation of pRb through the cyclin-dependent kinase (cdk) pathway and prevents the p53-mediated cell apoptosis of infected cell[
8].
This mechanism is used by the virus to keep the infected cells alive during productive infection but in non-permissive cells it may lead to cell transformation[
9]. In fact, abortive replication may result in oncogenic transformation which has rendered polyomaviruses prototypes of DNA tumor virus well amenable to studies in experimental models. Indeed, p53 mutations and overall inactivation have been implicated in tumor progression.
The role of human polyomaviruses in human cancer is still debated. Recent investigations have associated them with the outgrowth of specific cancer types including colorectal cancer[
10,
11], glioblastomas[
12,
13], mesotheliomas[
14], prostate cancer[
15] and, possibly, lymphomas[
16,
17].
Since LTag mediated inactivation of p53 has been suggested to represent a critical step for viral oncogenicity[
18,
19], this antigen has been identified as an important target of cancer immunity in murine models[
20]. The involvement of BKV LTag in the alteration of critical pathways of the human cell cycle together with the detection of LTag-p53 complexes by immunohistochemical analysis of normal and pathological specimens[
15], prompted us to investigate the role of this viral antigen as target of cellular immune surveillance. The specific aim of this study was to evaluate in HLA-A*0201+ BKV LTag experienced donors immune responsiveness to candidate epitopes from portions of the LTag specifically expressed in long lasting infected cells and possibly playing a role in BKV mediated oncogenisis.
Materials and methods
Large Tag selection and peptide synthesis
The LTag sequence used in this study was selected by comparing 15 BKV LTag sequences available on the net[
21] sharing a homology between 97 to 100% as detectable by using BLAST search[
22]. Two major peptide-algorithms, BIMAS[
23] and SYFPHEITI[
24] were employed to list candidate immune dominant epitopes from LTag. A standardization of peptides' scores was applied to identify best binders according to their final values using the following formula: z
i
= [(x
i-μ) σ] [
25]
Four peptides were used as positive or negative controls: the HLA-A*0201-restricted vaccinia virus H
3L peptide
184–192 (SLSAYIIRV)[
26], the HLA-A*0201-restricted MelanA/Mart1
26–35 (ELAGIGILTV)[
27], the HLA-A*2402-restricted CMV pp65
341–350 (QYDPVALLFF) [
28,
29] and the HLA-A*0201-restricted CMV pp65
495–503 (NLVPMVATV) [
30]. Peptides were synthesized by Princeton Biomolecules (Langhorne, PA) with purity from 90 to 100% as analyzed by HPLC, dissolved in 100% DMSO and stored at -70°C until use.
Peptide binding affinity to HLA-A*0201 processing defective T2 cells
HLA-A*0201+ human antigen processing defective T2 cells were diluted in PBS at a concentration of 2 × 106 cells/ml and were distributed in 96-well U bottom plates (100 μl total) in the presence or absence of peptides at a 250 μg/ml concentration. Plates were subsequently incubated for 16 hours at 37°C in humidified air containing 5% CO2. Cells were stained with FITC-tagged antibodies against human HLA-A*0201 (BD, Bioscience, San Jose, CA) and samples were analyzed by FACS. The fluorescence index (FI) was calculated by the following formula: (mean fluorescence experimental sample-mean fluorescence background)/mean fluorescence background). Results were reported in logarithmic scale with a 2-fold increase cut-off based on the fluorescence intensity in the presence of the irrelevant peptide HLA-A*2402-restricted CMV pp65341–350.
Donor selection and serology
Upon informed consent, HLA-A*0201+ Caucasians (mean age: 36.8 ± 7.6) were selected for this study. Detection of BKV-specific Ab (IgG) in donor sera was performed by ELISA using recombinant LTag and capsid protein 1 (VP1) purified from baculovirus expression system (H.H.H. and R. G., unpublished data). Briefly, the N-terminal part of LTag overlapping with the small T-antigen was expressed as a Glutathione-S-transferase (GST)-fusion protein in SF9-insect cells and purified by gluthathione-sepharose affinity chromatography. The fusion proteins and GST were coated on microtiter plates according to standard procedures. Sera were diluted 1:400 and tested in duplicates at room temperature. After washing 5 times with PBS, peroxidase labeled goat anti-human IgG (1:10'000 dilution in PBS) were added for 30 min before extra washing (5 times with PBS). O-phenyldiamine (0.4 mg/L) was added as substrate for 30 min in the dark. The reaction was stopped with H2O2 and the OD450 nm was read. Reactive sera were defined by OD readings above background subtracted of GST+2SD.
Peptide ex vivo induction and qrt-PCR analysis
Ex vivo induction of peptide-specific responses was attempted as follows. Briefly, PBMCs isolated from venous blood by Ficoll gradient centrifugation (Ficoll-hypaque density gradient), were incubated in 96 U bottom well plates at the concentration of 2 × 10
5 cells in 200 μl total RPMI medium supplemented with 100 μg/mL Kanamycin, 10 mM Hepes, 1 mM sodium pyruvate, 1 mM Glutamax and nonessential amino acids (all from GIBCO Paisley, Scotland) thereafter referred to as complete medium and 5% human serum (Blutspendezentrum, Kantonsspital Basel, Switzerland). After an overnight resting, cells were both peptide-stimulated (1 μM) or left unstimulated and 3 hours after they were harvested for RNA extraction (RNeasy
® Mini Kit Protocol, Qiagen, Basel, Switzerland) and cDNA synthesis (Invitrogen, Carlsbad, CA). Quantitative real-time PCR (qrt-PCR) assays were performed as previously described [
31] and conducted on an ABI prism™ 7500 FAST sequence detection system using TaqMan
® Universal PCR Master Mix Reagents Kit (Applied Biosystems, Rotkreuz, Switzerland) and sets of primers and probes from cytokine genes (IFN-γ, IL-2, IL-4, and IL-10) already extensively utilized[
28]. Beta actin (β-actin) was used as endogenous reference gene. Normalized data were subsequently presented as relative quantification. The 2
-ΔΔCt method [ΔΔC
T = (C
T, cytokine – C
T,β-actin)
induction – (C
T, cytokine – C
T,β-actin)
baseline, where C
T is the mean cycle times of the triplicate well readings] was utilized to compute the fold change of cytokine gene expression after peptide induction relative to baseline (unstimulated cells), normalized to an endogenous reference gene (β-actin)[
32].
Cell collection and culture
Subpopulations of cells (CD14+, CD8+, CD45RA+) were either positively or negatively purified from freshly isolated PBMCs by MACS (Miltenyi Biotech, Bergisch Gladbach, Germany) according to producers' protocols and in relation to different experimental procedures. For dendritic cell (DC) generation, isolated CD14+ were cultured for 5 to 7days in complete medium supplemented with 10% FCS (GIBCO Paisley, Scotland), β-mercaptoethanol 0.004%, recombinant human IL-4 (1000 UI/mL, courtesy of Dr. Lanzavecchia, Bellinzona, Switzerland) and recombinant human GM-CSF (50 ng/mL, Laboratorio Pablo Cassara', Buenos Aires, Argentina) to generate immature DC (iDCs). Maturation of iDCs was induced by exposure to lipopolysaccaride (Abortus Aequi, Sigma-Aldrich, St. Louis, MO) at a concentration of 1 μg/mL. Both whole CD8+ and CD45RA+ purified T cells were subsequently plated in complete medium supplemented with 5% human serum in either 96-well U bottom or 24-well plates for in vitro inductions at various concentrations and time exposures. Mature peptide-loaded DCs (mDCs) were used as APC either for priming or for rounds of restimulations. Recombinant human IL-2 (rhIL-2) was administered to the cultures at different concentrations according to experimental procedures.
Cytotoxic T lymphocytes in vitro expansion and cytotoxicity assay
CD8+ T cells purified from PBMCs of healthy donors (106/mL) were primed with irradiated (30 Gy) autologous mDCs (2 × 105/mL) previously loaded for 2 hours with peptide at a final concentration of 5 μg/mL. On days7 and 14 cultures were restimulated with peptide loaded mDCs. During the in vitro induction the cells were supplemented every other day with rhIL-2 at the final concentration of 20 UI/mL. Cytotoxic activity was tested on day 21 by using a 4-hrs chromium release assay on T2 cells previously labeled with 51Cr (50 μCi of 51Cr for 1 h at 37°C) and pulsed for 2hours with cognate or control peptides at a concentration of 2.5 μg/mL in triplicate wells. Specific lysis of target cells was calculated according to the standard formula: 100 × [(cpm experimental release - cpm spontaneous release)/(cpm maximal release - cpm spontaneous release)]. Following testing, cytotoxic T lymphocytes (CTLs) were expanded by repeated restimulations to generate epitope-specific CTL lines which were used to test functional activities and to analyse natural processing of defined peptides.
Limiting dilution analysis (LDA)
CD8+ T cells were cultured in 96-well plates according to the same conditions as outlined above at 10'000, 5'000 and 2'500 cells/well for total of 32 wells for each concentration. Autologous mDCs were pulsed with peptides, irradiated and used as APC at a final concentration of 2'500 cells/well. On day 7 cells were re-stimulated following the same procedure. On day 3 and day 10, rhIL-2 was administered to cultures at a concentration of 20 UI/ml and 100 UI/ml, respectively. Cytotoxic activity was tested on day 14 against peptide pulsed
51Cr-labeled T2 cells, as reported above. Cytotoxic T cell precursor (CTLp) frequencies were evaluated as previously described[
33]. Wells were considered positive when their cytolysis exceeded 3 standard deviations and at least 12% above average values of negative control lysis. On the basis of the single-hit Poisson model, the frequency of antigen specific CTLp's was estimated from the initial responder cell number at which 37% of the wells were negative for cytotoxicity.
Plasmid generation and transfection of HLA-A*0201 target cells
In plasmid DNA pTRE2LTag, the cDNA coding sequence of LTag of BKV is under control of a tetracycline-controlled transactivator-dependent promoter. The intron of the LTag was removed by 'gene splicing' by overlap extension[
34]. Two DNA fragments were generated by PCR using primer pairs 1 -2: (1) 5'-GAGAGAGCTAGCCACCATGGATAAAGTTCTTAACAGGGAAGA-3' (2) 5'-TTCTGTTCCATAGGTTGGCACCTCTGAGCTACTCCAGGTTCC-3' and 3–4: (3) 5'-GGAACCTGGAGTAGCTCAGAGGTGCCAACCTATGGAACAGAA-3' (4) 5'-GAGAGAATCGATTATTTTGGGGGTGGTGTTTTAGG-3' on DNA extracted from BKV-positive urine as template. The two DNA fragments were subsequently joined in a PCR reaction containing primer pair 1–4. The amplification product was digested with
Cla I and
Nhe I, followed by ligation into
Cla I-
Nhe I digested pTRE2pur (BD Biosciences, San Jose, CA). The integrity of plasmid DNA was verified by automated sequencing on a Beckman CEQ 8000 (Beckman Coulter Inc. Fullerton, CA).
The metastatic melanoma HBL cell line expressing HLA-A*0201 molecule were selected for pTRE2LTag transfection. Cells were routinely passaged in conventional cultures in complete medium supplemented with 10% heat-inactivated FCS. Transfection of HBL cells was performed by liposome-mediated gene transfer in 6 or 12-well plates. Cells at 90–95% confluency were transfected with a plasmid DNA (1.6 and 3.2 μg) to Lipofectamine 2000 (2 and 3 μl) (Invitrogen, Carlsbad, CA) ratio of 1.06 according to the manufacturer's recommendations. For transfection, pTRE2LTag and pTet-Off (BD Biosciences, San Jose, CA) were mixed at a ratio of 1:1. At 24 hours post transfection, the cells were transferred into T75 culture flasks. To determine the peak of protein expression, a variant of wild-type green fluorescent protein plasmid (pEGFP-N1; BD Biosciences, San Jose, CA) was transfected into parallel cell cultures and the enhanced green fluorescent protein (EGFP) expression was monitored by fluorescence microscopy at 24 and 48 hours post transfection. The LTag gene expression was measured by qrt-PCR using the following primers and probe[
15]:
Forward: 5'-TTTTGGAACCTGGAGTAGCTCAGAGGTTT-3'
Reverse: 5'-GCTTGACTAAGAAACTGGTGTAGAT-3'
Probe: 5'-TTGAGTGTTGAGAATCTGCTGTTGCTTCTTCATCACTGGCAAACA-3'
Standard enzyme-linked immunosorbent assay
Release of IFN-γ protein by either in vitro sensitized PBMCs after peptide re-stimulation or expanded CTLs cultured with pTRE2LTag BKV tranfected targets was measured using an ELISA kit (Endogen, Woburn, MA) on culture supernatants collected 18 hours after the last induction. ELISA results were extrapolated from a standard curve generated by linear regression. All assays were performed in duplicate and results were reported as average values.
Cell phenotype and multimer staining analysis
HLA-A*0201 MHC Pro5™ PE-labeled pentamers for LTag579–587, LTag406–414 and Melan-A/MART-126–35 (ProImmune, UK) were used for surface staining of the cells under investigation. Samples were analyzed on a FACSCalibur flow-cytometer equipped with Cellquest software (Becton Dickinson, San Jose, CA). Cells were first stained with 1 μl MHC PE-labeled pentamer for 15 min at 4°C in the dark. Subsequently, they were washed once and stained with 5 μl of CD8-PE-Cy7, CCR7-APC and CD45RA-FITC (BD, Bioscience, San Jose, CA) for 30 min at 4°C (on ice) in the dark. After staining, cells were washed twice and immediately analyzed on a flow cytometer or fixed in 200 μl 4% paraformaldehyde in PBS solution, kept at 4°C in the dark and analyzed later.
Discussion
Polyomavirus large tumor-antigen (LTag) interacts with a number of host molecules involved with cell cycle, including the tumor-suppressor gene product p53 and has been identified as an important target of cancer immunity in murine models[
38]. Early studies in SV40 infected mice have reported on conserved CD8+ T cells immune responses against SV40 LTag[
39]. In humans, however, despite continuous efforts aimed at identifying potential HLA class I restricted LTag epitopes[
40,
41], an epitope-specific cytotoxic immune response against this antigens or its portions is still poorly characterized [
42,
43].
Upon viral entry in non-permissive cells, in the context of an abortive infection, viral DNA is fully integrated in the host genome or resides in host cells as plasmid, thereby favouring the persistence of the virus without production of progeny virions[
8]. Within these modalities of virus-cell interaction, LTag is the most highly expressed BKV antigen in non permissive infected cells and might encompass viral epitopes generated by the endogenous class I restricted antigen processing pathway and, possibly, targeted by specific CTL responses. Thus, the identification of LTag derived antigenic peptides might provide decisive advances for the analysis of BKV specific immune responses against infected non permissive cells.
Importantly, LTag regions required for viral transformation, including pRb domains aa101–118; p53 domains aa351–450 and aa533–626, accounting for about 80% of the entire sequence, are less likely to be mutated or lost, and are thus highly genetically conserved[
44].
HLA-class I restricted immune responses against defined viral antigens can be frequently induced by ex vivo stimulation of peptide-specific T cells from seropositive subjects[
45]. Taking advantage of well studied algorithms, in this study we comprehensively analysed CTL responses against BKV LTag-related epitopes in HLA-A*0201+ BKV LTag experienced individuals. In particular, we focused on antigen triggered cytokine gene expression and lytic capacity in BKV specific CTL.
A first important result of our study is represented by the validation of a rapid, previously characterized[
31], screening technology. Indeed, we found a highly significant correlation between IFN-γ gene expression induced by a 3-hour stimulation of CD8+ T cells and the corresponding epitope specific cytotoxic activity detectable following 2–3 week cultures in the presence of peptides and IL-2.
Most interestingly, since antigen triggered cytokine gene expression is detectable after a 3-hour culture only, it is unlikely to be related to primary "in vitro" sensitization, but rather to the stimulation of antigen experienced specific CD8+ T cells, present in the peripheral blood samples under investigation[
46]. Moreover, this pattern of epitope immunorecognition "ex vivo" and following "in vitro" culture was consistently reproducible in serial tests performed on cells from the same donors for over one year, thus suggesting that these sustained responses are not associated to specific phases of the host-virus interaction.
LTag 406, 410 and 579 emerged as the most immunogenic peptides among those studied. The latter appeared to be naturally processed in non professional HLA-A*0201 positive antigen presenting cells expressing LTag. The relatively weak response detected following co-cultures of peptide specific expanded T cells with LTag transfected targets (only 3 of 5 cultures produced detectable levels of IFN-γ following stimulation) could be ascribed either to: i) low expression of LTag protein (in spite the relevant production of LTag mRNA following transient transfection) and subsequent impaired peptide-processing; or ii) T cell anergy due to the lack of professional antigen presenting cells (i.e. mDC).
Four out of five HLA-A*0201+ BKV LTag seropositive donors responded to LTag 579 and LDA data indicate that the frequency of CTLp specific for this peptide may be comparable to that of CTLp specific for the highly immunogenic HLA-A*0201-restricted influenza matrix 58–66 epitope (Michel Adamina, manuscript in preparation), as detectable in seropositive individuals.
CTL specific for antigens from viruses responsible for persistent infections are frequently characterized by specific phenotypic profiles. For instance, EBV or CMV specific CTL responses are elicited by distinct subsets of memory T lymphocytes reported as early effectors for EBV (CD45RA-(+)/CCR7+) or late effectors for CMV (CD45RA+/CCR7-)[
47]. Our studies indicate that BKV LTag 579 specific CTL responders exquisitely belong to a CD45RA+/CCR7+(-) CD8+ T cell population. Indeed "ex vivo" studies clearly demonstrate CD8+ T cells characterized by a relatively dim positivity for LTag 579/HLA-A*0201 pentamers and displaying this phenotypic profile in the peripheral blood of BKV seropositive donors. In addition 2–3 week cultures of separated cell populations indicate that specific CTL can only be generated from CD45RA+ cells. Importantly, even after these culture times, LTag 579 specific CD8+ T cells retain a CD45RA+/CCR7- phenotype, a relatively infrequent event for CTL recognizing BKV unrelated peptides.
Our study demonstrates for the first time that virtually all HLA-A*0201+ BKV seropositive donors mount a powerful CTL response towards epitopes encompassed by a highly phylogenetically conserved region of the LTag implicated in the viral replicative activity and in the p53 mediated control of the cell cycle of host cells.
These results set the stage for further research aiming at identifying appropriate formulations of LTag immunogenic peptides that might be used to stimulate BKV specific CTL responses in patients at risk of infection reactivation and at clarifying whether this specific immunosurveillance fails in patients bearing tumors potentially associated to BKV mediated oncogenic transformation.
Interestingly, these results may possibly also be extended to JCV-seropositive individuals, as already seen for VP1[
48], due to the high homology that the two LTag-p53 binding domains share among the human polyomavirus BK and JC strains. Clearly, in this context, our data may suggest combined use of epitopes from capsid proteins and LTag leading to innovative approaches in the treatment and prevention of human polyomavirus related diseases.
Competing interests
The author(s) have no financial conflict of interest.