BKV-associated nephropathy (BKVAN) represents a serious complication of the posttransplant period in kidney transplant recipients (KTRs) leading to organ loss in 50% of all cases. Being widespread in the normal population, BK virus (BKV) can reactivate in KTRs causing BKVAN. Previous studies suggested that monitoring of BKV-specific immunity is of a special importance for assessment of clinical course in patients with BKV reactivation and BKVAN (1, 2).
BKV is a relatively small virus with genome encoding six proteins: early regulatory proteins (large and small tumor antigens [LT, st]), late structural viral proteins (VP1, VP2, and VP3), and late nonstructural agnoprotein (3). To date, studies correlating the BKV-specific immune responses and parameters of viral infection have been restricted to the T-cell responses against two selected BKV proteins, LT and VP1 (4–9). Our recent data demonstrated, however, that not only LT and VP1 but also three other BKV-related proteins, VP2, VP3, and st are immunogenic and elicit T-cell responses (10). Thus, analysis of only VP1- and LT-specific T-cell responses can lead to underestimation of BKV-specific immunity. Therefore, all five BKV proteins should be evaluated in terms of BKV-specific immunity.
However, analysis of five proteins performed separately is time- and cost-intensive, and requires large amount of blood. The aim of the present study is to establish a fast and sensitive method for the analysis of BKV-specific CD4- and CD8-positive T cells and apply it for phenotypic and multifunctional T-cell analysis in KTRs.
Here, we implement a new approach for analysis of BKV-specific T cells using a mixture of overlapping peptide pools (MOPP) that covers both the late structural viral proteins and the early regulatory proteins. Single overlapping peptide pool (SOPP) stimulations were performed in parallel. Multiparameter flow cytometry was used to analyze the BKV-specific T-cell responses according to the expression of interferon gamma (IFN-γ), interleukin (IL)-2, tumor necrosis factor-alpha (TNF-α), and IL-17 alone and in combination to identify polyfunctional T cells. The analyses were performed in kidney transplant patients divided into different groups according to the diagnosis and severity of BKV infection.
Group 1 included 11 KTRs with a recovery from severe, prolonged BKV reactivation (defined as 3 or more consecutive detections of high-level [>30 copies/mL] BKV load within at least 3 months). Group 2 included 16 KTRs with a fast recovery from BKV reactivation (defined as the clearance of BKV reactivation within <3 months). Group 3 included 12 BKV-seropositive KTRs without any reactivation of infection after transplantation. This group did not have PCR-detectable BKV-DNA in their plasma at any time of screening for BKV after transplantation that was carried out every 3 months.
Additionally, five KTRs (group 4) with an active high-level BKV replication were monitored during the clinical course of BKV infection. The clinical characteristics of the KTRs are summarized in Table 1. There were no statistical differences between the analyzed groups regarding patient age, transplant age, and regimen of immunosuppressive therapies.
Improved Sensitivity of BKV-Specific CD4+T-Cell Detection Can Be Achieved by Simultaneous Stimulation With MOPP
Comparison of responder rate against BKV proteins obtained by SOPP versus MOPP stimulation, demonstrated a much higher sensitivity in samples stimulated by MOPP. The superior sensitivity of the new method was observed for BKV-specific T cells producing IFN-γ, IL-2, and TNF-α. Thus, on stimulation with SOPP (VP1, VP2, VP3, LT, or st separately), the incidence of patients with detectable IFN-γ-producing CD4+ T cells against any of BKV proteins was 0% to 55% in group 1, 56% to 69% in group 2, and 50% to 67% in group 3, respectively. In contrast, stimulation by MOPP showed detectable BKV-specific IFN-γ+CD4+T cells in 100% of patients in all groups (Fig. 1A).
Similar to IFN-γ, 100% of patients in analyzed groups showed BKV-specific IL2-producing CD4+ T cells on MOPP stimulation. In contrast, stimulation by SOPP revealed lower number of patients with detectable CD4+IL-2+T cells (18%–91% in group 1; 56%–100% in group 2, and 50%–92% in group 3; Fig. 1C). Analysis of TNF-α response showed also 100% of study patients with positive BKV-specific CD4+T cells after MOPP stimulation, whereas stimulation by SOPP demonstrated low incidence of patients with detectable TNF+CD4+T cells ranging from 27% to 91% in group 1, 69% to 75% in group 2, and 67% to 92% in group 3 (Fig. 1E).
In addition to showing a higher percentage of patients with detectable BKV-specific CD4+T cells, simultaneous stimulation by MOPP demonstrated also a higher magnitude of T-cell response. So, significantly higher frequencies of BKV-specific IFN-γ+CD4+T cells were observed after MOPP stimulation in comparison with SOPP in groups 1 and 2 (P<0.05; Fig. 1B). In group 3, statistically significant differences in T-cell frequencies were achieved only between MOPP and VP2- and LT-SOPP stimulations, respectively (P<0.05; Fig. 1B).
The frequencies of IL-2+CD4+T cells and TNF-α+CD4+T cells were also significantly higher on stimulation by MOPP versus stimulation by each SOPP for all three study groups, respectively (P<0.05; Fig. 1D,F).
Not surprisingly, the theoretical summated frequencies of T cells specific to all SOPP (sum) were higher as compared with MOPP, because several identical epitope sequences were shared within the structural and regulatory proteins, respectively (see Figures S1-S3, SDC 2, http://links.lww.com/TP/A543).
In addition, MOPP stimulation approach enabled much more efficient monitoring of BKV-specific cellular kinetics during the clinical course of BKV reactivation compared with SOPP stimulations. Thus, high number of BKV-specific cytokine (IFN-γ, IL-2, and TNF-α) producing T cells were observed using the new stimulation protocol with MOPP at the time points of the first BKV load decline and BKV clearance, whereas no low frequencies of BKV-specific CD4+ and CD8+ T cells were seen by SOPP stimulation protocol (see Figure S4, SDC 3, http://links.lww.com/TP/A544).
Dominance of IFN-γ+ T Cells Within BKV-Specific Cytokine-Producing CD8+ Memory T Cells
The sensitivity of T-cell detection using new stimulation approach was also significantly improved for CD8+T-cell compartment. Thus, all study KTRs demonstrated IFN-γ+CD8+T cells after stimulation by MOPP. In contrast, SOPP stimulation revealed lower number of patients with IFN-γ-producing CD8+T cells, in which the response to VP2 was higher for all groups (72%, 88%, and 67% in groups 1, 2, and 3, respectively). The percentage of patients with detectable IFN-γ+CD8+T cells was less than or equal to 50% for all other antigens in all study groups (Fig. 2A).
In addition, the frequencies of IFN-γ+CD8+T cells were also higher on stimulation with MOPP versus all SOPP for all groups, although no statistical significance were achieved versus MOPP and VP2-SOPP stimulation in groups 2 and 3, respectively (Fig. 2B).
Analysis of activated BKV-specific CD8+T cells demonstrated that IFN-γ-producing T cells dominated within the compartment of the analyzed cytokine-producing CD8+ T cells. So, after stimulation with MOPP, the incidence of patients with detectable IL-2+CD8+T cells were observed in 36%, 100%, and 25% patients in group 1, 2, and 3, respectively (Fig. 2C). Similarly, MOPP stimulation revealed 36%, 44%, and 67% patients in group 1, 2, and 3, respectively, with detectable TNF-α+CD8+T cells (Fig. 2D). In contrast, using SOPP stimulation IL-2 and TNF-α-producing BKV-specific CD8+T cells were hardly detectable in all study KTRs.
Rapid BKV Clearance Is Associated With a Higher Magnitude of Polyfunctional BKV-Specific CD4+T Cells
Because the magnitude of the T-cell responses as measured by a single parameter does not reflect the full functional potential, we analyzed the multifunctional capacity of BKV-specific T cells on stimulation with MOPP encompassing five BKV antigens in comparison with SOPP stimulation. Using the new stimulation protocol with MOPP, we were able to observe IL-2/TNF-α-double and IFN-γ/IL-2/TNF-α-triple-producing populations as the most representative. Triple producers were found in 91% of patients in group 1 (median T-cell frequencies, 44/106; range, 32/106 to 87/106), in 100% of KTRs in group 2 (median T-cell frequencies, 170/106; range, 35/106 to 270/106), and 100% in group 3 (median T-cell frequencies, 100/106; range, 18/106 to 140/106) (Fig. 3A,B). Magnitude comparison of polyfunctional triple-cytokine producers between the analyzed groups demonstrated that the patients with a history of fast BKV clearance had a significantly higher frequencies of BKV-specific polyfunctional IFN-γ/IL-2/TNF-α-producing CD4+T cells (P=0.048).
In addition, multifunctional IL-2/TNF-α-positive T cells were detected after MOPP stimulation in 91% of patients in group 1 (median frequencies, 131/106; range, 65/106 to 219/106), in 100% of KTRs in group 2 (median, 150/106; range, 54/106 to 360/106), and in 92% of group 3 (median, 100/106; range, 17/106 to 385/106) (Fig. 3C, D).
In contrast, stimulation by SOPP showed lower incidences of patients with detectable multifunctional IL-2/TNF-α-double and IFN-γ/IL-2/TNF-α-triple-producing BKV-specific CD4+T cells and the significantly lower magnitude of the T-cell responses. At the same time, polyfunctional CD8+T cells were rarely detected within studied patients neither by SOPP nor by MOPP stimulation (data not shown).
Lack of IL-17-Producing T Cells in BKV Seropositive Patients With Resolved BKV Reactivation
To test the possible contribution of IL-17 in the pathogenesis of BKV infection, we measured the content of this cytokine in all groups of KTRs with and without BKV reactivation. IL-17-secreting T cells were not detected in any of the subjects (data not shown).
Interplay between viral reactivation and immune response is known to determine the outcome of viral infections, and impaired BKV-specific immunity is a recognized risk factor for BKVAN development (11, 12). Thus, assessment of BKV-specific immunity may serve as a predictive factor for the clinical course of BKV infection. Herein, we established a sensitive and valid method for detection of BKV-specific CD4+ and CD8+T cells using a new approach for ex vivo cell stimulation. Rather than focusing on SOPP-specific T cells as a representation of the BKV-specific cellular immunity, we examined T-cell responses to the mixture of overlapping 15-mer peptide pools that cover the late structural proteins VP1, VP2, VP3, and the early regulatory proteins LT and st. Our data show that MOPP-activated peripheral blood mononuclear cells (PBMCs) unraveled to us BKV-specific CD4+ and CD8+T-cell responses and their phenotypic and multifunctional characteristics that were not consistently detectable with single antigen stimulation only.
Previous studies demonstrated an important role of BKV VP1- and LT-specific T cells in the clinical course of BKV infection in renal transplant patients (7, 13, 14). In addition, our recent data revealed that also three other BKV antigens (VP2, VP3, and st) are able to elicit an antigenic response with variable immunogenic dominance and analysis of VP1- and LT-specific T cells as done previously might lead to underestimation of BKV-specific cellular immunity (10). Although testing of BKV immunity using all five single BKV proteins provides more comprehensive analysis, the method is time, labor, and cost intensive, and requires large amounts of patients' blood. In addition, the detection threshold is low with the most of the healthy BKV-seropositive blood donors showing no detectable BKV-specific T cells. In this study, we show that stimulation by MOPP results in the magnitude of CD4+ and CD8+ BKV-specific T-cell response, which is significantly higher compared with any frequencies detected by single BKV antigen stimulation. In addition to eliciting higher frequencies of BKV-specific CD4+ and CD8+T cells, the MOPP stimulation significantly increased the incidence of patients with detectable CD4+ and CD8+T cells. In particular, MOPP stimulation facilitated the detection of BKV-specific CD8+T cells, which were detected in all analyzed patients (100%). In contrast, only marginal CD8-positive BKV-specific T cells were detected previously in renal transplant patients by flow cytometry using five single BKV antigens (10), and only in 33% of healthy donors (15).
Another important finding of the study is the preferential detection of polyfunctional memory/effector BKV-specific T cells in patients with a history of rapid BKV clearance in comparison with patients with a history of long-lasting BKV reactivation. Thus, we observed significantly higher frequencies of polyfunctional IFN-γ/IL-2/TNF-α T cells and multifunctional IL-2/TNF-α-double producing CD4+T cells in patients with a history of rapid BKV clearance. These data are in line with our previous observation demonstrating higher percentage of patients with detectable polyfunctional IFN-γ/IL-2/TNF-α BKV-specific CD4+T cells within the group of the patients with clinically unapparent BKV reactivation in comparison with the patients with a history of BKVAN (10). However, no statistical significances between these two groups could be observed in the previous study, because the magnitude of these polyfunctional T cells was low. The advantageous approach applied in the present study allowed for significantly higher sensitivity in the detection of polyfunctional T cells, known to have the most protective function in antiviral immune response (16, 17). In fact, improved control of HIV infection was observed in patients with increased frequencies of multifunctional CD4+T cells producing two or more different cytokines (18). In addition, multifunctional CD4+T cells were found at a higher percentage in patients after clearance of viral reactivations such as EBV, CMV, or VZV (16). Furthermore, animal studies demonstrated that measuring T cells producing single cytokines such as IFN-γ was not sufficient to predict protection; and assessment of multifunctional T cells was required for the outcome estimation (19). Assuming that the found T-cell population frequencies reflects the pattern of T-cell immunity at the time point of BKV clearance, our observations confirm the previous data stating that protective multifunctional T cell mediate an efficient control of virus replication in the human chronic viral infections (20–23) and suggest that detection of multifunctional T cells might predict clinical outcome of BKV reactivation. Taken together, these data suggest an important role of our T-cell assay for the clinical application. The new cell stimulation approach allowed monitoring of BKV-specific T cells for their qualitative and quantitative characteristics during the clinical course of BKV reactivation. The introduction of a frequent T-cell monitoring applied in our study enabled targeted guidance of immunosuppression in patients with BKV reactivation (reduction in case of insufficient BKV-specific T-cell immunity, or readjustment of the immunosuppressive therapy after T-cell recovery and BKV clearance, especially in patients with a high risk for allograft rejection).
Of interest, multiple cytokine production by BKV-specific memory T cells was restricted to CD4+T-cell compartment. These data are in agreement with our previous results obtained by single protein stimulation (10) and might suggest an assumption of effector functions by CD4+T cells.
Further observation of our study includes lack of IL-17-producing BKV-specific memory T cells in patients recovered from BKV reactivation. Emerging data suggest that the IL-17+T-cell population bridges innate and adaptive immunity to produce a robust antimicrobial inflammatory response. Although most evidences on involvement of Th17 response in host defense have been reported for extracellular pathogens, particular bacteria, and fungi (24), some recent studies demonstrate elevated frequencies of IL-17+T cells during viral infections such as vaccinia virus, HSV, and HIV (25–27). However, it is not clear so far whether IL-17 provides a protective antiviral response or contributes to detrimental inflammation. In this study, BKV-specific IL-17+T cells were analyzed in KTRs with different severity of BKV reactivation and in patients without detectable BKV reactivation. BKV-specific IL-17-producing T cells were not detected in any study groups. Because the analysis of IL-17+T cells was performed at the time point of resolved infection, involvement of Th17 response in the course of BKV infection can not be excluded. However, IL-17+T cells are known to persist after resolution of other infections (26, 28); therefore, disappearance of IL-17+T cells after resolution of BKV reactivation seems to be rather unlikely. Further prospective analyses of IL-17 response in patients with BKV reactivation are needed to determine the role of this cell population in the pathogenesis of BKV infection.
Our study is limited by the individually different immunosuppressive therapy that might affect ex vivo T-cell activation within the investigated groups. Because of the therapeutic changes of the immunosuppressive drugs in patients with severe long-lasting BKV infection, no matches in immunosuppressive medication between the groups were possible at the time point of sample acquisition. However, stimulation with third-party antigen showed comparable results in different patient groups (data not shown), suggesting that the observed differences between the groups were not the results of different immunosuppressive regimen but rather BKV specific. Additionally, differences in gender may bias the data. However, statistical analysis of T-cell frequencies with regard to patient gender did not reveal any associations between gender and BKV-specific immunity.
In conclusions, our novel stimulation approach using MOPP encompassing 5 BKV antigens is an easy-to-use, sensitive, and reliable method, which can be applied to monitor BKV-specific immunity. Results obtained by this method provide the most comprehensive assessment of the total T-cell responses to BKV performed to date and offer a new platform for further prospective studies.
MATERIALS AND METHODS
This study was approved by the local ethical review committee in compliance with the declaration of Helsinki, and informed consent was obtained from all patients. To address the relationship between severity and duration of BKV infection and specific cellular immunity, KTRs were divided into different groups according to the diagnosis and severity of BKV infection (detailed group characteristics is presented in Results). To avoid false-positive results, only BKV-seropositive individuals were included into the study.
Determination of BK Viral Load
BKV-DNA analysis was performed by TaqMan Real Time PCR as described previously (29). Briefly, DNA was isolated from serum using a QIAamp DNA Mini Kit (Qiagen Corp., Hilden, Germany) according to manufacturer's instructions. Primers and probes were designed to amplify the VP1 region of BKV. A plasmid standard containing the VP1 coding region was used to determine the copy number per milliliter. Samples exceeding detection level (>1000 copies/mL) were considered positive.
Design of BKV Overlapping Peptide Pools
Lyophilized overlapping peptide pools representing BKV proteins VP1, VP2, VP3, st, and LT antigens were created according to primary amino acid sequences (SWISS-Prot Accession No. P14996, P03094, P09034-2, P03082, and P14999, respectively), synthesized by JPT (Berlin, Germany), diluted in DMSO, and used at final concentration of 1 μg/mL for each peptide. The MOPP were derived from the same original peptides as the SOPP. For MOPP, VP1, VP2, VP3, st, and LT antigens were mixed and used at final concentration of 1 μg/mL for each single protein, respectively (total of 5 μg/mL). The final concentration of DMSO in MOPP was 0.5% that is below recommended 1% to avoid toxicity in the biological system.
PBMCs were isolated from heparinized whole blood in a standard Ficoll-Hypaque (PAA Laboratories) density gradient and cultured in RPMI 1640 medium supplemented with 100 IU/mL penicillin/streptomycin (both PAA, Austria), 2 mmol/L l-glutamine, 50 μM β-mercaptoethanol (Sigma, Germany), and 10% (vol/vol) heat-inactivated human AB serum (Lonza, Switzerland).
BKV-Specific Stimulation and Multiparameter Flow Cytometry Analysis
A total of 5×106 PBMCs were stimulated using MOPP encompassing five BKV proteins VP1-, VP2-, VP3-, st-, and LT-antigen (1 μg each) and SOPP stimulation in a total volume of 1 mL of medium in the presence of 1 μg/mL CD28-specific antibody (BD Pharmingen, USA) for 6 hr at 37°C. Superantigen Staphylococcus enterotoxin B (Sigma, Germany) was used as a positive control and DMSO as negative control (Fig. 4). Brefeldin A (Sigma, Germany) was added for the last 4 hr. Then, the cells were fixed and permeabilized using commercial kit (BD Pharmingen) according to the manufacturer's instructions. Cells were stained as previously described (30) with the following fluorescence-conjugated mAbs: CD4-ECD (SFCI12T4D11) (Beckman Coulter), CD154-APC (5C1) (Miltenyi Biotec), CD3-APC-Cy7 (SK7), CD8-PeCy7 (RPA-T8), CD69-FITC (FN50), IL-2-PE (5344.111), TNF-α-Alexafluor700 (MAb11) (all from BD Pharmingen), IL-17A-PerCPCy5.5 (eBio64DEC17), and IFN-γ-eFluor450 (4S.B3) (both eBioscience, USA). Live/dead cells were discriminated using aqua-fluorescent fixable dead cell stain kit (Invitrogen, Germany). Isotype-matched control antibodies (BD Pharmingen; eBioscience, USA) were used to distinguish nonspecific “background” stainings (see Figure S5, SDC 4, http://links.lww.com/TP/A545).
Data acquisition was performed on BD LSRII flow cytometer and analyzed using FlowJo software version 6.4.7 (Tree Star, USA).
Calculations were performed using SPSS software version 18 (SPSS Inc., Chicago, IL). Two-sided Mann-Whitney U test and two-sided Wilcoxon signed rank sum test for nonparametric independent samples were used for calculating the P value as appropriate. The contribution of various variables as risk factors to BKV replication was evaluated by chi square or Fisher exact tests. Two-sided P values of less than 0.05 were considered significant.
Contributions were made possible by DFG funding through the Berlin-Brandenburg School for Regenerative Therapies GSC 203.
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