JC virus induces a vigorous CD8+ cytotoxic T cell response in multiple sclerosis patients.

We sought to compare the ongoing CD8+ cytotoxic T lymphocytes (CTL) immune response of MS patients to self and viral antigens. Using 51Cr release and tetramer staining assays, we found that the CTL response against VP1, the major capsid protein of the polyomavirus JC (JCV), was significantly higher than the one against epitopes of MBP and PLP. The JCV-specific CTL response was also significantly stronger in MS patients than healthy control subjects. These findings may shed a new light on the recent events related to the development of progressive multifocal leukoencephalopathy in three natalizumab-treated patients.

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

A prospective study demonstrates an association between JC virus-specific cytotoxic T lymphocytes and the early control of progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS of immunosuppressed individuals caused by the polyomavirus JC (JCV). In previous studies, we showed that JCV-specific cytotoxic T lymphocytes (JCV-specific CTL) were associated with a favourable outcome in patients with PML. However, these CTL had been assessed in PML survivors more than 1 year after the onset of disease and we could not determine whether this immune response was only a surrogate marker for a general recovery of the patient's immune system or a causal factor in the patient's neurological improvement. In this study, we assessed the relationship between JCV-specific CTL detected early in the course of PML and the subsequent course of disease activity. We enrolled 26 patients with possible or proven PML, including 21 HIV+ patients, less than 10 months after the onset of their neurological symptoms (3.7 +/- 2.5 months, median +/- interquartile range). JCV-specific CTL were detected by either 51Cr release or tetramer staining assay. Patients were then followed prospectively and the clinical course of PML was determined. At the time of their first immune evaluation, we found that 15 patients had detectable JCV-specific CTL. HIV+ patients with JCV-specific CTL had a higher CD4+ T-cell count (215 +/- 103/microl) and a lower HIV viral load (144 +/- 431 copies/ml) than those without JCV-specific CTL (32 +/- 59/microl, P = 0.004 and 43 100 +/- 54 778 copies/ml, P = 0.01). Thirteen of these 15 patients with JCV-specific CTL developed clinically quiescent PML, while only two out of 11 without detectable CTL controlled their neurological disease. Therefore, the early detection of JCV-specific CTL had an 87% predictive value for subsequent control of PML, while the absence of such CTL had an 82% predictive value for subsequent active PML (P = 0.0009). Fifteen patients were evaluated less than 4 months after the onset of PML (1.9 +/- 1.3 months). Of nine patients with JCV-specific CTL, seven (78%) demonstrated subsequent control of disease, whereas six out of six (100%) without JCV-specific CTL developed progressive PML (P = 0.007). Two to ten CTL assays were performed on PBMC of 11 patients. Of these patients, one had an increase in JCV-specific CTL preceding a significant clinical improvement. In another patient with otherwise stable immune parameters, a decline in JCV-specific CTL preceded an exacerbation of PML. We conclude that JCV-specific CTL can be detected early in PML and can predict control of this disease. Fluctuations of JCV-specific CTL in the blood are associated with variation in disease manifestations. These results indicate that JCV-specific CTL are associated with the control of PML.

Stable expression of the avian retroviral oncoprotein v-Rel in avian, mouse, and dog cell lines.

Overexpression of the retroviral oncoprotein v-Rel can rapidly transform and immortalize a variety of avian cells in culture. However, mammalian models for v-Rel-mediated oncogenesis have been compromised by the fact that high-level expression of v-Rel has been reported to be toxic in many mammalian cell types, including mouse 3T3 cells, Rat-1 cells, and mouse bone marrow cells. In this article, we demonstrate that 3T3 cells can support expression of v-Rel for at least 24 days when infected with a mouse stem cell virus (MSCV) retroviral vector containing v-rel. In retrovirus-infected 3T3 cells, v-Rel is located in the nucleus and can bind to DNA, but does not transform the cells. On the other hand, 3T3 and Rat-2 cells do not express v-Rel after stable transfection with a pcDNA-based v-Rel expression vector. We also show that infection of the IL3-dependent mouse B cell line BaF3 with the MSCV-v-rel vector results in expression of v-Rel, but does not convert these cells to growth factor independence. In contrast to 3T3 cells, the dog osteosarcoma D17 cell line can support a high level of v-Rel expression, after either transfection or infection with a retroviral vector. That is, v-Rel can be stably expressed as a nuclear, DNA-binding protein in D17 cells to approximately the same level as in chicken embryo fibroblasts. These results suggest that the restriction to v-Rel expression in rodent fibroblasts is generally absent in D17 cells and that the type of v-rel expression vector determines whether 3T3 cells can support stable expression of v-Rel. The findings reported here are an essential first step in the development of mammalian systems to study Rel-mediated oncogenesis.