 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
BASIC SCIENCE |
*Laboratory of Transplant Immunology and Pediatric Hematology/Oncology, IRCCS Policlinico S. Matteo, Pavia, Italy; Department of Public Health, University of Firenze, Firenze, Italy; Department of Clinical & Experimental Medicine and Surgery, Second University of Napoli, Napoli, Italy; and Department of Transplantation Surgery, S. Martino Hospital, Genova, Italy; ¶Department of Pediatric Nephrology, Istituto G. Gaslini, Genova, Italy
Correspondence to Dr. Patrizia Comoli, Laboratorio Sperimentale di Trapianto di Midollo Osseo e Oncoematologia Pediatrica, IRCCS Policlinico S. Matteo, P.le Golgi 2, 27100 Pavia, Italy. Phone: 39-0382-502716; Fax: 39-0382-527976; E-mail: pcomoli{at}smatteo.pv.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
+/CD3+ displaying MHC class I unrestricted cytotoxicity, was also displayed. Application of this culture system may allow a preemptive therapy approach to BKV-related complications in transplant recipients, based on CTL treatment guided by BKV DNA levels. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Current therapeutic options are limited, and control of viral replication in kidney allograft recipients is tentatively obtained by means of reduction of immunosuppression (4–8). However, most patients with nephropathy caused by BKV slip into a disheartening cycle, alternating between viral interstitial nephritis and rejection, precipitated by the lowered dose of immunosuppressive drugs (8). Preliminary data on the use of the antiviral agent cidofovir have been reported (9); however, the drug used at the doses recommended for cytomegalovirus (CMV) infection is nephrotoxic, and its efficacy at the lower dosage proposed for BKV-related nephropathy remains to be confirmed in randomized, controlled trials. Thus, there is a need to develop alternative therapeutic tools that are able to control the infection before establishment of nephropathy.
Host immune response is of central importance in limiting primary viral infection and in controlling the virus carrier state (10). On the basis of the evidence that herpesvirus-related pathology in immunocompromised hosts originates from a deficiency of virus-specific cytotoxic T cells (11,12), clinical studies demonstrated how infusion of lymphocytes (13) or, better, virus-specific cytotoxic T lymphocytes (CTL) expanded in vitro could safely and effectively prevent or treat Epstein-Barr virus (EBV)-related lymphoproliferative disease or CMV-associated interstitial pneumonia occurring in hematopoietic stem cell transplantation or solid-organ transplant recipients (14–17). Application of this approach to the prevention and treatment of other virus-related pathologies affecting allograft recipients, such as BKV-related interstitial nephropathy, although appealing, rests on the possibility of demonstrating that cellular immunity has a central role in the control of BKV infection and that immunogenic viral antigens that are capable of eliciting CD8+ responses can be presented correctly to potentially reactive T cells.
The crucial role of T-cell immunity in the control of BKV infection is suggested by the increased incidence of reactivation and clinical disease linked to the degree of immunocompromise (5). Furthermore, it has been demonstrated that CTL are critical for the clearance of acute polyomavirus infection in the mouse model (18–20), and a recent work has suggested a role for polyomavirus JC-specific CTL in the containment of progressive multifocal leukoencephalopathy in affected humans (21). However, at the present time, there are no available data in the literature on ex vivo induction of human cellular immune response to BKV.
In the past few years, dendritic cells (DC) generated from peripheral blood monocytes and cultured in vitro in the presence of antigenic proteins or peptides have been increasingly used to induce enhanced tumor-specific or virus-specific cellular responses in vitro and in vivo, because of their optimal capacity to process and present the antigen (22–24). Recently, it has been shown that polyomavirus-infected DC induce antiviral CD8+ T-cell responses in the mouse (25).
The aim of this study was to assess the feasibility of activating autologous BKV-specific cellular immune response from the peripheral blood of seropositive healthy individuals and kidney allograft recipients by delivering inactivated virus to DC in vitro and using DC thus pulsed as stimulators for generation of CTL. We found that virus-pulsed DC are good stimulators of BKV-specific cytotoxic T-cell responses and are also able to reactivate BKV-specific immunity in immunocompromised subjects, such as renal allograft recipients.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BKV Antigen
BKV antigen was obtained from human fibroblast cell cultures infected with a wild-type BKV strain isolated at the Institute of Microbiology, University of Florence, from a bone marrow transplantation patient. For virus stock production, the viral suspension was diluted 1:100 and inoculated on a confluent monolayer of MRC-5 cells; infected cells were incubated in the presence of MEM, plus 2% FCS. After 2 wk of incubation at 37°C, the cells were subcultured and incubated again in the same conditions, until a cytopathic effect appeared and the hemagglutination assay resulted positive. The virus in the supernatant was inactivated by treatment with 0.1% 
-propiolactone (26). Residual BKV infectivity was assessed by inoculation of the treated supernatant in MRC-5 cells, as described above; after 10 passages without development of cytopathic effect, the cultures were discarded. Supernatant from MRC-5 cell culture was used as mock antigen.
DC
DC were generated from peripheral blood monocytes as described previously (24). PBMC were suspended at the concentration of 1 x 106/ml in X-VIVO 20 medium, and 1-ml aliquots were plated in 24-well plates. After 90 min at 37°C, nonadherent cells were discarded, and human recombinant IL-4 (rIL-4; R&D Systems, Minneapolis, MN) at a final concentration of 500 U/ml and human recombinant granulocyte-monocyte colony-stimulating factor at a final concentration of 800 U/ml (Sandoz Pharmaceuticals, Basel, Switzerland) were added. After 6 to 7 d of incubation, cells were recovered, phenotyped to assess the degree of maturity, and cryopreserved for later use or pulsed with inactivated BKV (equivalent of final MOI 0.01).
ELISPOT Assay
ELISPOT assays to determine the frequency of IFN-–secreting PBMC were performed following a method modified from one previously described (27). In detail, 96-well multiscreen filter plates (MAIPS 4510; Millipore, Bedford, MA) were coated with 100 µl of primary antibody (IFN-; Mabtech, Nacka, Sweden) at 2.5 µg/ml and incubated overnight at 4°C. Responder cells were then seeded at 1 x 105/well in the absence or in the presence of irradiated DC pulsed with BKV antigen (DC-BKV; effector:stimulator ratio 40:1). Controls included wells plated with DC-BKV alone. After incubation for 24 h at 37°C, 100 µl of biotinylated secondary antibody (Mabtech; 0.5 µg/ml) was added, and plates were then processed according to standard procedure. IFN-–producing spots were counted using an ELISPOT reader (Bioline, Torino, Italy). The number of spots per well was calculated after subtraction of assay background, quantified as the average of 24 wells containing only sterile complete medium, and specific background, quantified as the sum of cytokine spots associated with responders alone, DC-BKV alone, and responders plated with unpulsed DC.
Preparation of BKV-Specific Cytotoxic T-Cell Lines
BKV-specific CTL were prepared from fresh or frozen PBMC, according to the following method: 2 x 106 PBMC were co-cultured with 5 x 104 BKV antigen-pulsed DC in 2-ml volumes of X-VIVO 20 medium, supplemented or not with 10 ng/ml IL-7 and 10 pg/ml IL-12 (both R&D Systems). After 8 d, cultures were restimulated with 5 x 104 BKV-pulsed autologous DC. On day +11, IL-2 (20U/ml) was added to the cultures. CTL were then expanded with weekly rounds of restimulation with autologous stimulators in the presence of rIL-2. CTL were examined for immunophenotype, and for specificity in a standard 51Cr-release assay against a panel of targets including autologous mock-pulsed or BKV-pulsed DC.
T-Lymphocyte Subset Depletion
CD3+/TCR+ cells were removed from cell suspension by negative selection with anti-TRC mAb (Becton Dickinson, Mountain View, CA) and magnetic microspheres coated with goat-anti-mouse IgG (Dynabeads M-450; Dynal, Lake Success, NY), according to a previously described method (28).
Flow Cytometry
mAb used to characterize cultured cells were CD3 FITC and PE, anti HLA-DR PE, CD8 FITC and PE, CD56 PE, anti-TRC FITC, CD4 PE, CD19 FITC, CD20 PE, CD45 FITC (Becton Dickinson). Appropriate isotype-matched controls were included. Cytofluorimetric analysis was performed by means of direct immunofluorescence on a FACScan flow cytometer (Becton Dickinson).
Target Cell Lines and Cytotoxicity Assay
EBV-transformed B-cell lines (EBV-LCL) were generated and maintained following a protocol previously described (17). Tubular epithelial cells (TBE) were established from cortical portions of normal kidneys using a previously published technique (29). HSV-1–infected phytohemagglutinin blasts were obtained according to a previously described method (28). Cytotoxic activity was measured as described previously (17). Spontaneous release from the target cells was consistently <25%. Results were expressed as percentage of specific lysis. For HLA class I blocking experiments, target cells for cytotoxicity assays were incubated with 30 µl of the murine anti-human HLA class I mAb W6/32 (Dako, Glostrup, Denmark) for 30 min at room temperature. Cytotoxicity was then performed as described above.
T-Cell Receptor CDR3 Spectratyping
CDR3 size length analysis on preculture PBMC and on BKV-specific CTL was performed according to a previously described method (30). In detail, PCR amplification was performed under nonsaturating condition, after titration of cDNA obtained from the different samples. In each PCR tube, we used a panel of TCR-BV family-specific primers and a constant TCR-chain primer labeled with a fluorochrome at the 5' end with 5'-6 carboxyfluorescein. Each reaction also contained a -actin specific primer pair amplifying a product as internal control to check the efficiency of the single PCR (31). Spectratyping was generated by loading, after denaturation, an equivalent volume of PCR product and loading dye on a 5% acrylamide denaturing gel and run into a 377 Fluorescence based DNA sequencer (Applied Biosystems, Foster City, CA) in the presence of Rox-labeled size markers. The data were analyzed using Applied Biosystems Genescan software that allows assignment of size and peak areas to different PCR products. The data for each TCR-BV family were visualized as chromatograms. By this technique, a normal repertoire is visualized as a series of bands, separated by 3 bp, having gaussian distribution; alterations, either in intensity or in distribution of the bands, reflect a turbative of repertoire.
Statistical Analyses
Data were expressed as mean ± SD. Immunologic parameter correlation was evaluated by t test. P < 0.05 was considered statistically significant. Statistical analysis was performed using the SAS System (SAS, Cary, NC).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Production
DC were incubated with inactivated BKV (equivalent of MOI 0.01) overnight, and cell surface phenotypic analysis was then performed. We found that upon pulsing with BKV antigen, immature DC did indeed display an activated phenotype, with upregulation of MHC and co-stimulatory molecules (data not shown). The PBMC from healthy BKV-seropositive donors and kidney transplant recipients with BKV reactivation were tested for their reactivity to autologous DC pulsed with BKV antigen by measuring specific proliferation in a standard 3H-thymidine incorporation assay. IFN- secretion was measured on 10-d CTL culture cells by means of an ELISPOT assay. In all subjects tested, the 6-d proliferation observed after stimulation with BKV-DC was always higher than that observed with mock-pulsed DC (10.8 ± 3.7 x 103 cpm versus 5.4 ± 2.6 x 103 cpm; P < 0.05; Figure 1A). Moreover, T cells from both control subjects and patients showed a measurable IFN- production in response to the virus (patients, 11 mean spots/105 PBMC; healthy donors, 17 mean spots/105 PBMC; Figure 1B).
|
TCR and 3 (±2)% showing a CD3+/CD8+/CD56+ phenotype. Unmanipulated cells recovered from the cultures showed a low although sizable cytotoxicity against autologous BKV-pulsed DC (11 ± 4%), with absent or low activity against unpulsed DC (3.5 ± 5%).
In an attempt to increase virus-specific cell-mediated lysis, we implemented the reactivation protocol by adding rIL-12 and rIL-7 to the cultures at day 0, based on previous observations from our group in the setting of leukemia-directed CTL activation (25). With this new protocol, we obtained a mean fivefold expansion of T cells in the cultures and a significant increase of CD8+ lymphocytes and activated T cells in CTL lines (44 ± 5% CD8+ cells, with 33 ± 12% CD4+ lymphocytes, and 73 ± 6% HLA-DR+ cells). The specific lysis mediated by cytokine-treated CTL was consistently higher than cytotoxicity observed in untreated cultures (treated, 33 ± 17%; untreated, 10 ± 4%; P < 0.05). Data on the phenotype and cytotoxicity of CTL reactivated in the absence or in the presence of IL7/IL-12 are reported in Figure 2. The CTL lines obtained with the latter reactivation protocol, in addition to a strong lysis of the autologous BKV-pulsed DC, showed little or no reactivity against mock-pulsed autologous DC, unpulsed HLA-mismatched EBV-LCL, and HSV-1–infected autologous phytohemagglutinin blasts (Figure 3). Moreover, antibody inhibition studies showed that specific cytotoxicity was mostly HLA class I–dependent (Figure 3). Because depletion of CD3+/+ T cells in the effector population caused a moderate decrease of cytotoxicity against autologous BKV-pulsed targets (
20% of total cytotoxicity), it is likely that specific activity was mediated by both CD3+/CD8+/- and CD3+/+ subsets.
|
|
|
+ and CD3+/CD56+ cells endowed with MHC class I unrestricted cytotoxic activity. In particular, the percentage of CD3+/+ T cells was found increased in T-cell lines reactivated in the presence of IL-7/IL-12 compared with untreated cultures (untreated, 9 ± 2%; treated, 51 ± 17%; P < 0.005). Depletion experiments, performed on BKV-specific CTL lines reactivated from kidney allograft recipients with anti-TCR monoclonal antibody and goat–anti-mouse IgG-coated magnetic beads, confirmed that the majority of cytotoxic activity against allogeneic BKV-infected targets was mediated by the CD3+/+ T-cell subset (Figure 5).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our results demonstrate that it is feasible to reactivate BKV-specific CTL in humans by stimulation with BKV-pulsed autologous DC. The method described allows CTL expansion under GLP conditions, as no animal serum is required in the culture medium and the use of inactivated virions, grown on human cells, minimizes the risk of virus transfer from BKV-specific T cells, as demonstrated by the failure to obtain evidence of a cytopathic effect in BKV infection–permissive cell cultures after incubation with BKV-specific CTL. The use of whole virus to pulse DC is at present mandatory, as the T-cell response to BKV is poorly characterized and immunogenic T-cell epitopes have not been defined. However, this seeming limitation may indeed represent an advantage in the context of BKV disease, as not only can this technique be used for patients of any HLA type, but it also will reactivate CTL with multiple specificities and allow expansion of HLA-unrestricted T-cell subsets.
Data obtained in the setting of human CMV (HCMV)–specific CTL reactivation indicated that DC pulsed with inactivated virus were able to induce T-cell lines that included a variable, although generally low, number of CD8+ lymphocytes (23). The percentage of CD3+/CD8+/TCR- and CD3+/CD8+/TCR+ T cells in our cytokine-treated cultures was decidedly augmented in comparison with what was observed in the absence of cytokines. The CD8+ T-cell increase could have been due to an IL-12–dependent activation of DC (24), which in turn boosted antigen-specific CD8 CTL induction, coupled with CD8+ T-cell proliferation and survival promotion mediated by IL-7 (33), whereas the high number of TCR+ T lymphocytes observed might be ascribable to IL-7, which is known to be a factor involved in early development and in prolonging the life-span of mature TCR+ T cells (34). This finding, reproducible in the individuals tested, may be relevant to a cellular immunotherapy approach for BKV-related disease, because, as already described for polyomavirus JC (21), CD8+ T-cell population may be necessary to contain infection. Moreover, most important in view of a possible cell transfer program, the protocol used to reactivate BKV-specific T cells and, in particular, the use of IL-12 and IL-7 in the early culture phase allows the expansion of sufficient CTL for infusion requirements.
The technique reported can also be exploited to evaluate BKV-specific cellular immunity. Indeed, the same stimulators used to reactivate BKV-specific CTL can be used in an ELISPOT assay to measure the frequency of IFN-–producing T cells. This method could highlight high-risk situations, such as absent or decreased ability to mount a cellular immune response upon development of active BKV infection, and consequently help in identifying subsets of patients who are prone to progression from active infection to BKV-related disease. Thus, serial evaluation of BKV-specific, IFN-–secreting T-cell populations, used in combination with BKV DNA determination, may allow application of a preemptive approach to treatment and represent a useful tool to monitor treatment efficacy, as recently proposed for EBV-related posttransplantation lymphoproliferative disease (35).
The demonstration of the feasibility of reactivating BKV-specific CTL from samples of patients receiving immunosuppression after kidney transplantation could have immediate therapeutic implications. Although in patients with late-phase BKV nephropathy interstitial inflammation may be present, it has been suggested that renal dysfunction can be primarily a consequence of virus-mediated necrosis of tubular cells (36). In addition, a recent report showed that renal allograft with BKV infection is characterized by a decrease in cytotoxic T cells (37). Thus, induction or restoration of protective immunity to BKV through a cellular immunotherapy approach could reduce the incidence of active BKV infection and related interstitial nephropathy in kidney transplant recipients.
BKV-related nephropathy in kidney allograft recipients mostly originates from viral reactivation in the graft. Hence, a major drawback in devising a cell therapy strategy in this heterologous setting is that viral antigens on HLA-mismatched donor renal cells could escape recognition by HLA-mismatched recipient CTL. However, because treatment of established BKV nephropathy does not seem to have substantially changed the prognosis (38), in view of a preemptive immunotherapeutic strategy, lysis of donor tubular epithelial cells harboring BKV might not be essential to avoid progression from active viral infection to nephropathy. Indeed, it has been suggested that invasive renal disease may depend on the virus’s gaining access to the blood via tubular fluid leaking into the interstitium and, hence, gaining access to peritubular capillaries (5). Consequently, MHC-restricted CTL of patient origin could limit viral spread to renal parenchyma and the consequent graft damage, through lysis of autologous circulating B lymphocytes carrying BKV.
In addition, the CTL lines reactivated from our patients included in most cases a high percentage of CD3+/TCR+ cells, which could also contribute to the killing of autologous BKV-infected cells. TCR+ T-lymphocyte subsets have been shown to proliferate in response to virus-infected cells; mediate lysis through a TCR-dependent, MHC-unrestricted antiviral effector function (39,40,28); and play a protective role against viral infections (41,42). Indeed, our CTL lines reactivated from kidney graft recipients mediated an MHC-unrestricted, BKV-directed killing, which was abrogated by depletion of CD3+/TCR+ T cells. Therefore, it is reasonable to hypothesize that these MHC-unrestricted cytotoxic populations could control BKV reactivation from the allograft urothelium. Moreover, it cannot be excluded that in some patients, subsets of cytotoxic CD8+/TCR
+ CTL could also recognize and lyse infected cells in a BKV-specific, MHC-unrestricted manner (43), thus contributing to control BKV reactivation from the graft.
Our data support the feasibility of an adoptive immunotherapy approach to BKV-associated nephropathy occurring in kidney transplant recipients. In particular, a preemptive strategy may be valuable even when immunosuppression discontinuation is prevented by a high risk of rejection. A phase I/II clinical trial will have to be undertaken to evaluate whether early infusion of autologous BKV-specific CTL, following a strategy of preemptive therapy guided by BKV DNA levels (44), could induce control of BKV infection in vivo without increasing the probability of graft rejection or graft damage.
|
Acknowledgments |
|---|
We thank the patients who contributed and the Kidney Transplant Unit in Genova for cooperation and Roberta Oleggini for TBE cultures.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or T-cell receptor: Role of accessory molecules on HLA-unrestricted killing of virus-infected targets. Immunology 85: 49–56, 1995[Medline]
cells during fetal and adult life. J Immunol 170: 4087–4094, 2003 CTL. J Immunol 153: 5133–5140, 1994[Abstract]
T lymphocytes expand, suggesting a protective antiviral role. J Infect Dis 184: 533–541, 2001[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  A. Egli, S. Binggeli, S. Bodaghi, A. Dumoulin, G. A. Funk, N. Khanna, D. Leuenberger, R. Gosert, and H. H. Hirsch Cytomegalovirus and polyomavirus BK posttransplant Nephrol. Dial. Transplant., September 1, 2007; 22(suppl_8): viii72 - viii82. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Li, J. Melenhorst, N. Hensel, K. Rezvani, G. Sconocchia, Y. Kilical, J. Hou, B. Curfman, E. Major, and A. J. Barrett T-cell responses to peptide fragments of the BK virus T antigen: implications for cross-reactivity of immune response to JC virus. J. Gen. Virol., October 1, 2006; 87(Pt 10): 2951 - 2960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Krymskaya, M. C. Sharma, J. Martinez, W. Haq, E. C. Huang, A. P. Limaye, D. J. Diamond, and S. F. Lacey Cross-Reactivity of T Lymphocytes Recognizing a Human Cytotoxic T-Lymphocyte Epitope within BK and JC Virus VP1 Polypeptides J. Virol., September 1, 2005; 79(17): 11170 - 11178. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
), one BKV-negative healthy individual (
), and two BKV-positive kidney transplant recipients (•); the results for each subject are represented linked. (B) IFN-
) is stronger in treated cultures (B), at all effector-to-target (E:T) ratios, in comparison with untreated cultures (A). Moreover, the percentage of CD8+, HLA-DR+, and TCR
). Data refer to a representative experiment and are the mean of triplicate wells.
) in the presence of strong lysis of autologous BKV-pulsed DC (
). Absence of alloreactivity is testified by absent cytotoxicity against allogeneic EBV-transformed B-cell lines (EBV-LCL; 
). Lytic activity of cultures from Peripheral blood mononuclear cells (PBMC) stimulated with mock-pulsed DC, against autologous BKV-pulsed DC (
), is also included. Data represent cytotoxicity at a 10:1 E:T ratio.
