T-helper Cell-Mediated Proliferation and Cytokine Responses against Recombinant Merkel Cell Polyomavirus-Like Particles

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.

section_content: Merkel cell polyomavirus (MCPyV) discovered by Feng et al in 2008, is responsible for a rare, yet aggressive neuroendrocrine neoplasia, Merkel cell carcinoma (MCC) [1, 2, 3]. The virus has been shown to be present in 24-89% of MCCs in populations of varied geographic origins [4] [5] [6] [7]. It has been shown to be integrated clonally into the MCC genome [1, 8]. Antibodies recognizing MCPyV tumor associated antigens appear to be a relatively specific MCC marker [9]. Recently, an association of MCPyV infection with chronic lymphocytic leukemia (CLL) was reported [10] [11] [12], yet the causal association remains to be proven. Serological studies have shown that 50-80% of adults display MCPyV-specific antibodies [13] [14] [15]. Very recently, the presence of MCPyV DNA sequences was reported in buffy coats of healthy blood donors pointing to latency/persistence in peripheral blood leukocytes [16, 17]. As MCPyV VLPs can elicit antibody responses, they have been suggested to be potential vaccine candidates [18]. However, as neutralizing MCPyV antibodies occour in high titers among patients, they apparently fail to prevent MCC tumorigenesis [18]. It is therefore possible that cell mediated immunity (CMI) may be involved in protection against MCPyV-induced malignancy. Our aim was to elucidate the strength and polarization of MCPyV-specific T-helper cell immunity among asymptomatic adults. T-helper cell mediated proliferation, interferon-gamma (IFN-c), interleukin-10 (IL-10) and interleukin-13 (IL-13) responses were studied. 

IFN-c is a major antiviral cytokine, produced not only by Th1 cells but also by cytotoxic T-cells and NK cells [19]. It is a critical extrinsic tumor-suppressor factor in immunocompetent hosts and it has several types of antitumor actions [20] [21] [22] [23] [24]. IL-10 is an important anti-inflammatory cytokine [25] and its major sources are T-helper type 2 (Th2) cells and a subset of regulatory T-cells [26]. IL-10 inhibits Th1 cells, NK cells and macrophages. These three cell types are required for optimal pathogen clearance, and they also contribute to tissue damage during infection. In consequence, IL-10 can both impede pathogen clearance and ameliorate immunopathology [25]. The role of this cytokine on the immune response against cancer is controversial. As it can inhibit several key phenomena of adaptive immune responses, it has been considered to allow malignant cells to escape from immune surveillance [27, 28]. By contrast, there is data to suggest that IL-10 might also favour immune-mediated cancer rejection [29] [30] [31] [32]. IL-13 is an important cytokine produced mainly by Th2 cells [33, 34]. It possesses several unique effector functions including regulation of gastrointestinal parasite expulsion, intracellular parasitism, airway hyperresponsiveness, allergic inflammation [35] and class switch to IgE and IgG4 [36]. The role of IL-13 in regulating tumor growth depends on the tumor cell type. In some models inhibition of IL-13 or IL-13 receptors has promoted tumor growth [37, 38] whereas in others tumor growth has been inhibited [38, 39]. In chronic B lymphocytic leukemia (B-CLL) models IL-13 has been shown to block apoptosis of tumor cells [40, 41]. 

In this study we demonstrate that vigorous MCPyV-specific Th cell responses are readily detectable in constitutionally healthy adults.
section_name: Introduction

section_name: Results

section_content: We determined MCPyV-specific T-cell proliferation in 15 MCPyV-seropositive and 15 seronegative subjects. Virus-specific proliferation responses of the MCPyV-seropositive subjects were much stronger than those of the seronegative subjects, both at 0. 25 mg/ml and 2. 5 mg/ml concentrations of antigen (Tables 1 and 2 and Fig. S1 ). 

The proliferation responses became more vigorous when the MCPyV-antigen concentration was elevated from 0. 25 mg/ml to 2. 5 mg/ml. The increase in mean response was,2 fold among the seropositive subjects, and even higher,,5 fold among the seronegative controls (Tables 1 and 2 ). These increases were statistically significant both among the seropositive (P = 0. 011) and seronegative subjects (P = 0. 002). 

Six seropositive and none of the seronegative subjects were responders (having DCPM. 5000) with the lower 0. 25 mg/ml antigen concentration (P = 0. 017). With the higher 2. 5 mg/ml concentration, the corresponding numbers were 12 and three among the seropositive and seronegative subjects, respectively (P = 0. 001) (data not shown). With the control antigens HBoV (Table 3 ) and Candida albicans (Table 4 ) no statistically significant differences in proliferation were found. With the responder criteria the control-antigen specific responses were statistically identical among the MCPyV-seropositive and seronegative subjects, P = 1. 0 with both antigens (data not shown).
section_name: Proliferation responses among MCPyV-seropositive and seronegative subjects

section_content: MCPyV-specific IFN-c, IL-10 and IL-13 responses were readily detectable even with the lower 0. 25 mg/ml antigen concentration among the seropositive subjects, and they were significantly stronger than the corresponding responses among the seronegative subjects (Table 1 ). 

With the 2. 5 mg/ml MCPyV antigen concentration the average cytokine responses were higher than with the 0. 25 mg/ml concentration (Tables 1 and 2 ), and the differences were statistically significant with all the cytokines, both among the seropositive (P#0. 011) and seronegative (P#0. 018) subjects. 

Also with the higher MCPyV antigen concentration the cytokine responses were stronger among the seropositive subjects than among the seronegative ones. The difference was particularly evident with IFN-c, and also significant with IL-10 and IL-13 (Table 2 and Fig. S1 ). 

With the control antigens the cytokine responses were very similar among the MCPyV seropositive and seronegative subjects. The P-values with the HBoV and Candida albicans antigens were $0. 305 and $0. 285, respectively (Tables 3 and 4 ). 

As seen in Tables 1 and 2, IFN-c was the dominant MCPyVassociated cytokine. Also at individual level, a response pattern of IFN-c. IL-10 and IFN-c. IL-13 was often detected with both antigen concentrations (P#0. 014). Among the seronegative subjects this pattern was not borne at MCPyV VLP concentration of 0. 25 mg/ml (P$0. 444), whereas at the higher concentration also the seronegative subjects tended to show higher responses with IFN-c than with IL-10 (P = 0. 069) or with IL-13 (P = 0. 047). 

With the positive control antigen HBoV (Table 3 ) IFN-c responses were also higher than the corresponding IL-10 or IL-13
section_name: IFN-c, IL-10 and IL-13 responses among MCPyVseropositive and seronegative subjects

section_content: To identify the proliferating and cytokine secreting cell populations, the PBMC were depleted either of CD4+ or CD8+ T cells by using monoclonal antibodies (MAbs) attached to magnetic beads. Seropositive subjects P1 to P4 and a seronegative subject N1 who had constantly shown strong responses with MCPyV were studied. MCPyV-specific proliferation, IFN-c, IL-10 and IL-13 secretion was readily detectable after depletion of CD8+ T cells, whereas the removal of CD4+ T cells strongly reduced the responses among all the subjects (Fig. 1 ).
section_name: Identification of the proliferating and cytokine secreting cells

section_content: HLA class restriction of the cytokine and proliferation responses were studied with a class II-specific MAb (which blocks antigen presentation) and with an isotype-matched control MAb. Seropositive subjects (P1, P2, P3 and P5) together with a seronegative subject (N1) were studied. With the isotype control MAb proliferation and cytokine responses were readily detectable, whereas they were invariably reduced with the HLA class IIspecific MAb (Fig. 2 ).
section_name: HLA restriction of cytokines and proliferating cells

section_content: A significant proportion of human population has encountered MCPyV. Because neutralizing MCPyV antibodies occur in high titers in patients with MCC [18], it is likely that infection surveillance is not completely mediated by humoral immunity. Instead, cell mediated immune mechanisms may play a central role, yet they have not been explored so far. The present study is the first to report on cell-mediated immunity against MCPyV. The responses were regarded to be highly specific for MCPyV, as the studies were carried out by using highly purified (Fig. 3 ) VLPs. Tcell subset depletion and HLA class II blocking showed that the main sources of MCPyV-specific proliferation and cytokine responses were CD4+ Th-cells, not the cells of innate immunity, even in a MCPyV seronegative subject (N1) showing strong cellular responses to MCPyV. The concentration of the MCPyV-antigen used in the assay had a significant importance. With the lower concentration a ''classical'' response pattern was observed: Th-cell proliferation and cytokine responses were largely confined within the seropositive subjects. However, with the higher concentration, MCPyV-specific responses were detectable also among some seronegative subjects. This type of responders have been previously denoted as ''immune seronegative'' subjects in a herpes simplex virus model [42, 43]. With both antigen concentrations the seropositive subjects nevertheless had significantly stronger MCPyV-specific Th-cell responses than the seronegative subjects had, whereas Th-cell responses against the control antigens HBoV and Candida albicans were statistically similar in the two groups. The presence of MCPyV-seronegative responders suggests that Bcell immunity against MCPyV is not always persistent, or that a degree of cross-reactivity in the VP1 Th-cell epitopes may exist between MCPyV and some hitherto-unidentified virus. VP1 proteins of other polyomaviruses are possible candidates. For instance, the VP1 protein of a recently discovered trichodysplasia spinulosa-associated polyomavirus (TSPyV) has as high as 50. 6% amino-acid similarity with that of MCPyV [44]. Alternatively, some MCPyV strains might be of aberrant B-cell antigenicity. One such MCPyV strain, termed ''350'', having critical double mutations at VP1 positions 288 and 316, has been described to date [45]. VP1 of strain ''350'' is not recognized by sera strongly reactive with VP1s of MCPyV strains lacking these mutations [45]. 

Furthermore, it remains possible that the MCPyV-reactive Th cells have been originally primed by pathogens possessing largely different T cell epitopes. This is because a very high level of crossreactivity is an essential feature of the T-cell receptor [46] [47] [48] [49]. 

We found that MCPyV specific Th-cells secrete the Th2-like cytokine IL-13, the regulatory-like cytokine IL-10 and the Th1-like cytokine IFN-c. MCPyV-specific IL-10 may have importance in regulation of humoral immunity (Note S1). IFN-c was the most readily detectable cytokine with MCPyV, and the responses were significantly stronger than the corresponding responses with the HBoV positive-control antigen, highlighting the strength of this response. This is the main finding of our study, due to the tumorsuppressing and antiviral functions of this cytokine. Of note, cellular infiltration and cytokine mRNA (including IFN-c) have been studied in MCC biopsies. Arany and Tyring found lacking of IFN-c responses in MCC biopsies [50], whereas Kelly et al found an association between favorable prognosis and the presence of elevated expression of IFN-c mRNA [51]. It should be noted that in these studies the antigen specificity of tumor infiltrating lymphocytes was not determined. 

Taken together, the antigen-specific Th-cell responses of healthy individuals in the present study along with studies showing a lack of Th-cell responses on a general level in patients with MCC suggest a central role for CMI in infection surveillance of MCPyV. The imperative next step is to study MCPyV-related diseases such as MCC or CLL for antigen-specific CMI to get further evidence of the pathogenic importance of MCPyV-specific Th immunity.
section_name: Discussion

section_name: Materials and Methods

section_content: Altogether randomly selected 30 asymptomatic subjects (age range 25-58 years) were studied: 15 were seropositive and 15 seronegative for MCPyV. This study protocol followed the human experimentation guidelines of the US Department of Health and Human Services in the conduct of clinical research and was approved by the ethics committee of the Department of Medicine in Helsinki University Central Hospital. Written informed consent was obtained from all volunteers.
section_name: Study groups

section_content: MCPyV VP1 and HBoV VP2 capsids were expressed with recombinant baculoviruses in Sf9 cells and purified by CsCl gradient ultracentrifugation [52] [53] [54]. After extensive dialysis the protein was concentrated and purified further by using 50 KDa MWCO centrifugal filters (Amicon Ultra, Millipore, Billerica, MA). The antigens were further characterized by silver staining (SilverXpress, Invitrogen, Carlsbad, CA, USA) (Fig. 3 A ) and dot blotting (Fig. 3 B ) with MCVPy seropositive human sera as described earlier [52, 53]. The purity for MCPyV protein was. 90% by densitometry (Gel Doc 2000 Gel Documentation systems with Quantity One Quantitation Software, Bio-Rad). Electron microscopy with negative staining showed virus-like particles (Fig. 3 C ). As a second control antigen, we used in-house prepared and heat inactivated Candida albicans. Endotoxin in the antigen preparations was measured by the Limulus amebocyte lysate assay (QCL-1000; Cambrex Biosciences, Walkersville, MD, USA), and it was less than 2 EU/mg with MCPyV and HBoV antigens.
section_name: Antigens for proliferation and cytokine assays

section_content: We measured MCPyV and HBoV IgG in plasma by EIA, employing as antigen virus-like particles [52, 54].
section_name: Antibody assay

section_content: Blood was drawn to mononuclear cell separation tubes (Vacutainer CPT, Becton Dickinson, Franklin Lakes, NJ, USA). The tubes were centrifuged at 15006g for 30 minutes and washed two times with PBS. PBMC were separated within 2 hrs of blood sampling followed by counting [55].
section_name: Isolation of PBMC

section_content: For lymphocyte culture, isolated PBMC were resuspended in RPMI-1640 (Sigma, St Louis, MO, USA) containing 20 mM HEPES, 2 mM L-glutamine, streptomycin (100 mg/ml), penicillin (100 U/ml), 50 mM 2-mercaptoethanol and 10% human AB serum (Cambrex Biosciences, USA) and were cultured with the antigens [55]. MCPyV VLP were used at 0. 25 mg/ml and 2. 5 mg/ ml and the HBoV VLP and Candida albicans control antigens at 2. 5 mg/ml.
section_name: Lymphocyte culture

section_content: Counted PBMC and antigens in triplicate were placed in 96 well U-bottom plates (Coster, Corning Inc., Corning, NY, USA). Cells (200,000/well) were cultured for 6 days (37uC; 5% CO 2 ) and pulsed for the last 16 hours with 1 mCi of tritiated thymidine (specific activity 50 Ci/mmol; Nycomed Amersham, Buckinghamshire, UK). Thymidine incorporation was measured in a liquid scintillation counter (Microbeta, Wallac,Turku, Finland). The data were expressed as counts per minute (D cpm): D cpm = mean cpm (test antigen)2mean cpm (media) [53, 55]. Cytokine assays PBMC culture supernatants were harvested after 3 days for IFN-c and after 5 days for IL-10 and IL-13, and were stored at 220uC. Cytokine production in the supernatants was analysed by IFN-c, IL-10 (Pharmingen, San Diego, CA, USA) and IL-13 (Invitrogen corporation CA, USA) kits, according to the manufacturers' instructions. Background (media) cytokine production was subtracted from total to yield antigen specific cytokine production. The detection limits for IFN-c, IL-10 and IL-13 were 5, 8 and 6 pg/ml, respectively.
section_name: Proliferation assay

section_content: PBMC were depleted of CD4+ or CD8+ T cells by using magnetic beads coated with CD4-or CD8-specific monoclonal antibodies (Invitrogen Dynal AS, Oslo, Norway), according to the manufacturer's instructions. Then, 200,000 pure CD4+ or CD8+ depleted cells were cultured with the antigens as described [55].
section_name: Depletion of CD4+ or CD8+ cells

section_content: Class restriction of the T-cell responses was further studied by HLA class II-specific MAbs (HLA-DR, DP, DQ) (IgG2a, clone Tu39; BD PharMingen), or isotype control MAb (IgG2a, clone G155-178; BD PharMingen). These antibodies were used at 5 mg/ml, according to the manufacturer's instructions.
section_name: Antibody blocking assays

section_content: Responses among MCPyV seropositive and seronegative subjects were compared by using the Mann-Whitney U test. Paired responses were evaluated by using the Wilcoxon Signed Rank test. The distribution of responders having D cpm. 5000 [42, 43] against each antigen was studied using Fisher's Exact test. P values,0. 05 were considered significant. All analyses were done with a SPSS statistical program version 15. 0.
section_name: Statistical methods

section_name: Supporting Information

section_name: Acknowledgments

section_content: We are grateful to all the volunteers for donating blood samples. We thank Lea Hedman ( Departments of Virology, Haartman Institute, University of Helsinki ) for carrying out the antibody assays for HBoV and Minna S. Vuojolainen ( Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki ) for experimental assistance.
section_name: Acknowledgments

section_content: This study was supported by the Helsinki University Central Hospital Research and Education Fund, the Medical Society of Finland, the Academy of Finland (project 1122539 ), the Clinical Chemistry Research Foundation Grant ( A. Kumar ), the Orion-Farmos Research Foundation Grant ( A. Kumar ), the University of Helsinki Funds Grant ( A. Kumar ), and the Sigrid Juse ´lius Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

section_name: Author Contributions