BK Polyomavirus-Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination.

BACKGROUND: BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. METHODS: Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. RESULTS: BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1-) and cytotoxic. CONCLUSIONS: Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

JC polyomavirus-specific antibody responses in pediatric kidney transplant recipients.

BKPyV is widely recognized in KTRs, but little is known about rates of primary and secondary JCPyV exposure in pediatric KTRs. We evaluated JCPyV exposure in pediatric KTRs using antibody responses in the first 12 months post-transplant. Of 46 children transplanted between 2009 and 2014, 6 lacked any samples for serologic testing, leaving 40 KTRs for study. JCPyV-specific IgG and IgM antibodies were measured using a normalized VLP ELISA. Significant JCPyV exposure was defined as IgG seroconversion, increasing IgG levels of >0.5 nOD units, or IgM detection. Of 40 recipients (median age 3.2 years), 11 (27.5%) were seropositive, 20 (50%) seronegative for JCPyV-IgG, while 9 (22.5%) had no specimen at the time of transplantation, but were confirmed as seronegative in post-transplant samples. Of 29 (72.5%) at risk, JCPyV-IgG seroconversion occurred in 15/29 (51.7%) including JCPyV-IgM in 6 patients (20.7%). Two patients (6.9%) developed only JCPyV-IgM. Among JCPyV-IgG-positive KTRs, six (12.5%) had significant IgG increases. Altogether 23 of 40 patients (57.5%) had serological evidence of primary or secondary JCPyV exposure. In these patients, kidney function tended to be lower during the 2 years of follow-up, but only one patient lost the graft due to JCPyV nephropathy. Thus, JCPyV exposure is common in pediatric KTR and may present serologically as primary or secondary infection. Although only one case of JC-PyVAN occurred, a trend toward lower renal function was seen. Dedicated studies of larger cohorts are warranted to define impact of JCPyV in pediatric KTR.

BK polyomavirus viremia and antibody responses of pediatric kidney transplant recipients in Finland.

BACKGROUND: BKPyV is an important cause of premature graft failure after KT. Most clinical studies describe BKPyV infection in adult KT patients. We studied the prevalence of post-transplant BKPyV viremia, serology, and graft function in pediatric KT recipients. METHODS: Forty-six pediatric patients transplanted between 2009 and 2014 were followed up for BKPyV DNAemia by plasma PCR for median 2.3 (range: 1-6) years. BKPyV-specific antibodies were retrospectively analyzed using virus-like particle ELISA. GFR was measured annually by 51 Cr-EDTA clearance, and serum samples were screened for DSAs by Luminex assay. RESULTS: BKPyV viremia was demonstrated in nine patients at a median of 6 months post-KT. Early BKPyV viremia at 3 months post-KT associated with decreased concomitant GFR and tendency for decreased subsequent graft function. Three of nine patients with BKPyV viremia developed DSA, all against class II antigens. PyVAN developed to four patients and responded to judicious reduction in IS. One graft was lost later due to ABMR. BKPyV-IgG was found in 18 of 31 patients (58%) tested at transplantation, and seven recipients seroconverted after transplantation with a significant increase in IgG levels with IgM. Finally, BKPyV-IgG was detectable in 31 of 40 patients (78%) at the end of the study. CONCLUSIONS: Post-transplant BKPyV viremia in pediatric KT patients may alter graft function and contribute to progression of chronic allograft injury. BKPyV-IgG predicts past exposure. Low or absent BKPyV-specific antibody levels were seen pretransplant in 42% of tested patients, but were not predictive of prolonged replication or poor outcome.

Comparing effects of BK virus agnoprotein and herpes simplex-1 ICP47 on MHC-I and MHC-II expression.

BACKGROUND: Among human polyomaviruses, only BK virus (BKV) and JC virus (JCV) encode an agnoprotein upstream of VP1 on the viral late transcript. BKV agnoprotein is abundantly expressed late in the viral life cycle, but specific cellular and humoral immune responses are low or absent. We hypothesized that agnoprotein might contribute to BKV immune evasion by downregulating HLA expression, similar to Herpes simplex virus-1 ICP47. METHODS: UTA-6 or primary human renal proximal tubular epithelial cells (RPTEC) were co-transfected with plasmids constitutively expressing agnoprotein, or ICP47, and enhanced green-fluorescent protein (EGFP). EGFP-gated cells were analyzed for HLA-ABC and HLA-DR expression by flow cytometry. HLA-ABC and HLA-DR expression was also analyzed on UTA-6 bearing tetracycline-regulated agnoprotein or ICP47. Effects of agnoprotein on viral peptide-dependent T-cell killing were investigated using (51)Cr release. RESULTS: ICP47 downregulated HLA-ABC without affecting HLA-DR, whereas agnoprotein did not affect HLA-ABC or HLA-DR expression. Interferon- γ treatment increased HLA-ABC in a dose-dependent manner, which was antagonized by ICP47, but not by agnoprotein. In UTA-6 cells, agnoprotein expression did neither impair HLA-ABC or -DR expression nor peptide-specific killing impaired by HLA-matched T-cells. CONCLUSION: Unlike the HSV-1 ICP47, BKV agnoprotein does not contribute to viral immune evasion by down-regulating HLA-ABC, or interfere with HLA-DR expression or peptide-dependent T-cell cytotoxicity.

CMX001 (1-O-hexadecyloxypropyl-cidofovir) inhibits polyomavirus JC replication in human brain progenitor-derived astrocytes.

Polyomavirus JC (JCV) replication causes progressive multifocal leukoencephalopathy (PML), a frequently fatal brain disease in immunodeficient patients, yet antiviral drugs are lacking. We characterized the lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding JCV (Mad-4) replication in human brain progenitor-derived astrocytes (PDA) and the simian virus 40 (SV40) large-T-antigen-expressing COS-7 cells up to 7 days postinfection (dpi). We examined JCV loads by PCR, the infection rate by immunofluorescence, and host cell toxicity by WST-1 and BrdU incorporation assays. Supernatants from CMX001-treated PDA demonstrated a drug concentration-dependent decrease in JCV loads and infectivity. CMX001 had only a modest effect on host cell metabolism but reduced overall BrdU incorporation. In PDA at 7 dpi, the CMX001 50% effective concentration (EC50) was 5.55 nM, the 50% cytotoxic concentration (CC50) was 184.6 nM, and the 50% selectivity index (SI50) was 33.3. The EC90 was 19.7 nM, the CC90 was 5,054 nM, and the SI90 was 256.1. In COS-7 cells, JCV replication was faster and the EC50 and EC90 were 18- and 37-fold higher than those in PDA, i.e., 0.1 µM and 0.74 µM (CC50, 0.67 µM; SI50, 6.7; CC90, 12.2 µM; SI90, 16.5) at 5 dpi. We conclude that CMX001 inhibits JCV replication at concentrations in vitro that can be attained by oral administration without significant side effects in clinical studies.

BK Polyomavirus Evades Innate Immune Sensing by Disrupting the Mitochondrial Network and Promotes Mitophagy.

Immune escape contributes to viral persistence, yet little is known about human polyomaviruses. BK-polyomavirus (BKPyV) asymptomatically infects 90% of humans but causes premature allograft failure in kidney transplant patients. Despite virus-specific T cells and neutralizing antibodies, BKPyV persists in kidneys and evades immune control as evidenced by urinary shedding in immunocompetent individuals. Here, we report that BKPyV disrupts the mitochondrial network and membrane potential when expressing the 66aa-long agnoprotein during late replication. Agnoprotein is necessary and sufficient, using its amino-terminal and central domain for mitochondrial targeting and network disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta expression, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant viruses unable to disrupt mitochondria show reduced replication and increased interferon-beta expression but can be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy occur in allograft biopsies of kidney transplant patients with BKPyV nephropathy. JCPyV and SV40 infection similarly disrupt mitochondrial networks, indicating a conserved mechanism facilitating polyomavirus persistence and post-transplant disease.

Gene silencing of transcription factor Gli2 inhibits basal cell carcinomalike tumor growth in vivo.

Basal cell carcinoma (BCC) belongs worldwide to the most frequent malignancy among Caucasians. The understanding of the molecular mechanisms of BCC formation, which is a prerequisite for the development of efficient new therapies, is still incomplete. The formation of sporadic BCCs in the skin is associated with uncontrolled hedgehog signaling, and the transcription factor Gli2 has been identified as a key mediator or effector of this signaling. There is indication in the literature that preventing Gli2 function may inhibit BCC formation and growth in vivo; however, the mechanism is unclear and difficult to study in humans. Therefore, we used a mouse tumor allograft model to investigate the role of Gli2 in tumor formation. A constitutively Gli2 expressing mouse tumor cell line was stably transfected with Gli2-specific shRNA to induce Gli2 gene silencing or with control shRNA. Injecting the Gli2 gene silenced cells into nude mice for tumor formation we detected a strongly retarded tumor growth compared with control tumor cells. Investigating the mechanisms, we found that Gli2 gene silencing has led to the disruption of the tumor structure as demonstrated by staining tumor sections with hematoxylin. Two main reasons for the tumor destruction were identified. We found that apoptosis was markedly increased while vascularization was strongly decreased in these tumors. Thus, important functions of the transcription factor Gli2 in this tumor model are the prevention of apoptosis and the promotion of microvascularization.

Apoptosis and pathogenesis of melanoma and nonmelanoma skin cancer.

Skin cancers, i.e., basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and melanoma, belong to the most frequent tumors. Their formation is based on constitutional and/or inherited factors usually combined with environmental factors, mainly UV-irradiation through long term sun exposure. UV-light can randomly induce DNA damage in keratinocytes, but it can also mutate genes essential for control and surveillance in the skin epidermis. Various repair and safety mechanisms exist to maintain the integrity of the skin epidermis. For example, UV-light damaged DNA is repaired and if this is not possible, the DNA damaged cells are eliminated by apoptosis (sunburn cells). This occurs under the control of the p53 suppressor gene. Fas-ligand (FasL), a member of the tumor necrosis superfamily, which is preferentially expressed in the basal layer of the skin epidermis, is a key surveillance molecule involved in the elimination of sunburn cells, but also in the prevention of cell transformation. However, UV light exposure downregulates FasL expression in keratinocytes and melanocytes leading to the loss of its sensor function. This increases the risk that transformed cells are not eliminated anymore. Moreover, important control and surveillance genes can also be directly affected by UV-light. Mutation in the p53 gene is the starting point for the formation of SCC and some forms of BCC. Other BCCs originate through UV light mediated mutations of genes of the hedgehog signaling pathway which are essential for the maintainance of cell growth and differentiation. The transcription factor Gli2 plays a key role within this pathway, indeed, Gli2 is responsible for the marked apoptosis resistance of the BCCs. The formation of malignant melanoma is very complex. Melanocytes form nevi and from the nevi melanoma can develop through mutations in various genes. Once the keratinocytes or melanocytes have been transformed they re-express FasL which may allow the expanding tumor to evade the attack of immune effector cells. FasL which is involved in immune evasion or genes which govern the apoptosis resistance, e.g., Gli2 could therefore be prime targets to prevent tumor formation and growth. Attempts to silence these genes by RNA interference using gene specific short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) have been functionally successful not only in tissue cultures and tumor tissues, but also in a mouse model. Thus, siRNAs and/or shRNAs may become a novel and promising approach to treat skin cancers at an early stage.

High-level JCPyV viruria after kidney transplantation-Clinical and histopathological findings.

BACKGROUND: The significance of JC polyomavirus (JCPyV) after kidney transplantation ranges from irrelevant to full-blown nephropathy or PML. OBJECTIVES: To investigate the clinical significance of high-level JCPyV viruria and JCPyV primary infections after kidney transplantation. STUDY DESIGN: JCPyV viruria was detected in routine screening by quantitative real-time PCR in 40/238 kidney transplant recipients and was high-level (>107 copies/ml) in 17 patients. A protocol biopsy at the time of JCPyV viruria was available from 10 patients. RESULTS: Peak urine viral loads were 1.0×107-2.5×109 copies/ml in the 17 high-level viruria patients. 6/15 (40%) patients with high-level JCPyV viruria with pretransplant sera available were JCPyV IgG negative suggesting that JCPyV viruria resulted from the donor graft in most cases. No acute graft dysfunction was associated with JCPyV viruria. No positive SV40 staining was detected in protocol biopsies, and no specific histopathology was associated with high-level viruria; JCPyV nephropathy was not found. No differences were seen in histopathology or graft function at 3 years in patients with high-level viruria compared to non-JCPyV viruric patients transplanted during the same time period, and outcome was similar in patients with presumably primary and reactivated JCPyV. The mean estimated GFR at last follow-up was 44ml/min (range 12-60ml/min). One graft with high-level viruria was lost 9 years posttransplant due to recurrent IgA nephropathy CONCLUSIONS: High-level JCPyV viruria seems to be associated with primary JCPyV infection reflecting the average seroprevalence of 60%, but is not stringently associated with inferior graft function or survival, or histopathological changes.

Simultaneous BK Polyomavirus (BKPyV)-associated nephropathy and hemorrhagic cystitis after living donor kidney transplantation.

BK polyomavirus (BKPyV) commonly reactivates after kidney transplantation, and can cause polyomavirus-associated nephropathy (PyVAN), whereas after allogeneic stem cell transplantation the most frequent manifestation of BKPyV is polyomavirus-associated hemorrhagic cystitis (PyVHC). Despite high-level BKPyV replication in both, the pathogenesis and manifestation of both BKPyV entities appears to differ substantially. We describe an unusual case of simultaneous PyVAN and PyVHC presenting with acute symptoms in a BKPyV-IgG positive recipient eight months after kidney transplantation from a haploidentical living donor, who was BKPyV-IgG negative. Symptoms of cystitis and viremia subsided rapidly after reduction of immunosuppression.

Role of apoptosis in basal cell and squamous cell carcinoma formation.

Long-term ultraviolet-light (UV) exposure of human skin epidermis is associated with an increased risk for the development of skin cancers, such as melanoma, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). UV radiation not only induces DNA damage in epidermal cells, it also interferes with skin homeostasis, which is maintained by a unique distribution pattern of apoptosis-inducing and -preventing molecules. If the DNA damage is not repaired or the damaged cells are not eliminated by apoptosis, the consequence can be cell transformation, uncontrolled proliferation and eventually skin tumor formation. An important "repair" gene is the p53 suppressor gene. Excessive UV exposure can mutate the p53 gene leading to the loss of its repair function and thus apoptosis resistance of the DNA-damaged cell. For BCC formation an additional pathway has been identified. Mutation of genes of the Hedgehog signaling pathway evokes the downregulation of apoptotic genes and upregulation of anti-apoptotic genes preventing the elimination of damaged cells. In addition, BCC and SCC strongly express the apoptosis-inducing Fas-ligand (FasL) which may help the tumor to escape the attack of immune effector cells. Silencing the genes involved in tumor formation by RNA interference might become a promising new approach to treat skin tumors.

Fas ligand downregulation with antisense oligonucleotides in cells and in cultured tissues of normal skin epidermis and basal cell carcinoma.

Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis upon interaction with Fas-receptor-expressing cells. FasL normally plays an important immune regulatory role, but it can also cause severe skin diseases if overexpressed and it may serve some tumors for immune evasion. Thus, in situ inhibition of FasL expression with antisense oligonucleotides in patients may be a novel approach to overcome its pathogenic role. We designed and evaluated 15 phosphorothioate antisense oligonucleotides directed against different regions of the human FasL mRNA. They exhibited different inhibitory activities on FasL expression in HEK293 cells. The most potent antisense oligonucleotide, ASO8, specifically downregulated 90% FasL expression at the protein level and 80% at the mRNA level. FasL downregulation reduced the effector function of HEK293 cells toward Fas receptor positive target cells. Further studies demonstrated that ASO8 efficiently inhibited FasL synthesis in split skin and basal cell carcinoma tissue. Our results show that the modulation of FasL expression by antisense oligonucleotides is possible in cells as well as tissue and indicate that antisense oligonucleotides may provide a promising strategy for the therapy of FasL-mediated disorders.

Enhanced gene silencing by the application of multiple specific small interfering RNAs.

Small interfering RNA duplexes (siRNA) induce gene silencing in various eukaryotic cells, although usually in an incomplete manner. Using chemically synthesized siRNAs targeting the HIV-1 co-receptor CXCR4 or the apoptosis-inducing Fas-ligand (FasL), co-transfection of cells with two or more siRNA duplexes targeting different sites on the same mRNA resulted in an enhanced gene silencing compared with each single siRNA. This was shown in the down-regulation of protein and mRNA expression, and functionally in the inhibition of CXCR4-mediated HIV infection and of FasL-mediated cell apoptosis. Transfection efficiency determined for the FasL-specific siRNAs was dose-dependent and varied among the siRNAs tested, but was not the main reason for the enhanced gene silencing.

Intralesional interferon in basal cell carcinoma: how does it work?

Basal cell carcinoma (BCC) is the most common skin cancer among Caucasians, and its incidence is increasing. Intralesional injection of interferon alpha (IFN alpha) has been shown to provide a safe and effective treatment for BCCs. The predominant mechanism for the effect of IFN alpha on BCC has been partially identified. We have shown that in untreated patients, BCC cells constitutively express CD95 ligand (CD95L), but not the receptor. BCC cells make use of the CD95 ligand to escape from a local immune response by averting the attack from activated CD95 receptor-positive CD4+ T cells. The CD95L of BCC cells is functional as CD95+ target cells incubated on BCC cryosections become apoptotic and are lysed. In IFN alpha-treated patients BCC cells express not only CD95L but also CD95 receptor, and regress by committing suicide or fratricide through apoptosis induction via CD95 receptor-CD95L interaction. Peritumoral infiltrating cells, predominantly CD4+ T cells, may support regression of BCC by the secretion of cytokines such as IFN gamma or interleukin-2 which may also be responsible for the up-regulation of CD95 on BCC cells.

Influence of antiretroviral therapy on programmed death-1 (CD279) expression on T cells in lymph nodes of human immunodeficiency virus-infected individuals.

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Tumor necrosis factor-related, apoptosis-inducing ligand supports growth of mouse mastocytoma tumors by killing tumor-infiltrating macrophages.

TRAIL antisense transfected mastocytoma cells (R56VTas) injected into syngeneic DBA/2 mice demonstrate significantly delayed tumor growth compared to mock transfected cells (R56VTMo). TRAIL expression in R56VTas cells was successfully, albeit not completely, downregulated, as shown by Western blots, flow-cytometric analysis and functionally by loss of cytolytic activity against TRAIL-R-bearing target cells. Immunohistochemic and immunoblotting analyses of ex vivo tumors confirmed the lower expression of TRAIL by the antisense transfection compared to the mock transfection. Investigating the mechanism of the delayed tumor growth, it was found that neither T nor NK cells but activated macrophages infiltrated the tumors. The number of infiltrating macrophages was significantly lower in the mock transfected compared to the TRAIL antisense transfected tumor sections, indicating that TRAIL-expressing tumor cells may lyse macrophages. Indeed, activated macrophages proved to be sensitive to TRAIL-mediated apoptosis. This indicates that, although macrophages can infiltrate the mastocytoma R56VT, they are in part eliminated by TRAIL-expressing tumor cells, allowing the tumor to rapidly grow. Hence, downregulation of TRAIL allows more macrophages to survive and to better attack the tumor cells, slowing down tumor growth. In conclusion, TRAIL expressed on R56VT tumor cells can impair an important innate immune defense mechanism against tumors by eliminating effector macrophages.

Differential expression of viral Bcl-2 encoded by Kaposi's sarcoma-associated herpesvirus and human Bcl-2 in primary effusion lymphoma cells and Kaposi's sarcoma lesions.

Expression of human herpesvirus 8 viral Bcl-2 protein was demonstrated in spindle cells of late-stage Kaposi's sarcoma lesions but not in primary effusion lymphoma cell lines. In contrast, strong expression of human Bcl-2 was found in stimulated primary effusion lymphoma cells, whereas in Kaposi's sarcoma lesions preferential mononuclear cells, and to a lesser extent spindle cells, stained positive.