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*
Neurology Department and
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PML is a demyelinating disease of the CNS caused by a reactivation of JCV in the setting of immunosuppression (7). Although IgG Abs specific for JCV can be detected in 90% of the normal adult population by a hemagglutination inhibition assay or ELISA (8), this humoral immune response is unable to prevent the development of PML. This disease occurs in patients with AIDS, cancer, or in organ transplant recipients, and is usually fatal in less than 1 year. However, approximately 10% of PML patients have a protracted clinical course and a prolonged survival (9). The better clinical prognosis in this subset of patients has been unexplained.
Studies of the cellular immune response against JCV have been limited. PML patients have been shown to have a reduced lymphocyte proliferation in response to PHA or JCV Ag, compared with normal controls (10, 11, 12). We have recently begun exploring JCV-specific cellular immune responses in patients with PML. In these studies, we have shown that JCV Ag-stimulated PBMC of HIV-infected patients who were survivors of PML had detectable JCV-specific CTL activity. However, these findings were not definitive, since the assay employed in this study was not quantitative, and the number of subjects studied was limited (13).
CTL recognize virus-infected cells through the interaction of their TCR with an 8- to 10-aa viral epitope in association with an MHC class I molecule on the surface of the infected cells. Virus-specific CTL can be analyzed with great precision if we have knowledge of the peptide epitope presented by the MHC class I molecules to the cytotoxic effector cells. To facilitate more precise studies of JCV-specific CTL, we have chosen to characterize the JCV epitope peptides presented to CTL by the commonly expressed MHC class I molecule HLA-A*0201 (14). In these studies, we have defined a commonly recognized JCV CTL epitope presented to CD8+ T cells by A*0201, constructed a tetramer-staining reagent with this peptide and A*0201, and used this reagent as a tool to explore the role of JCV-specific CTL in the immunopathogenesis of PML.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To measure the immune response against selected JCV nonamer peptides, a total of 24 HLA-A2+ study subjects was enrolled in this study, including 10 HIV+/PML patients, 3 HIV-/PML patients, 6 HIV+ patients, and 5 healthy HIV- control subjects. The diagnosis of PML was ascertained by clinical and neuroradiological criteria and confirmed by brain biopsy or by positive JCV PCR in the cerebrospinal fluid. Of the 10 HIV+/PML patients, 6 were survivors whose disease had improved or remained stable 2 to 51/2 years after their initial diagnosis of PML. Three HIV+/PML patients had a progressive neurologic disease and a rapid fatal outcome in 7 wk to 5 mo after their diagnosis. One HIV+/PML patient had been diagnosed with PML 6 years before testing, and presented with clinical and radiological evidence of disease progression at the time of testing.
Of the three HIV-/PML patients, one who had a
history of non-Hodgkins lymphoma was neurologically stable 81/2
years after her initial diagnosis. This patient had received Ara C for
treatment of PML. The two other HIV-/PML
patients had progressive disease and a fatal outcome 3–5 mo after the
diagnosis of PML. One of them had a history of autologous
CD30+ stem cell transplant for multiple myeloma,
and the other had received a bone marrow transplant for treatment of
acute myeloid leukemia. Of the six HIV+ patients
without PML, three had a leukoencephalopathy that was clinically and
radiologically consistent with PML, but with negative JCV PCR in the
cerebrospinal fluid or brain biopsy specimen. The other three
HIV+ patients had other neurological diseases,
including a history of CMV polyradiculopathy, HIV encephalopathy, and
thoracic polyradiculitis, respectively. All of these patients had
negative JCV PCR in cerebrospinal fluid samples, and were survivors of
their neurological diseases (Table I
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A computer algorithm (http://bimas.dcrt.nih.gov/molbio/hla_bind/) was used to predict 9-aa peptides from JCV T, t, VP1, VP2, VP3, and agnoprotein for their likely ability to bind to the HLA-A*0201 molecule (15, 16). A total of 11 nonamer peptides from the T (n = 5), VP1 (n = 4), and VP2 (n = 2) proteins was selected and synthesized.
Functional lysis assay
PBMC from HLA-A2-positive individuals were isolated using a Ficoll-diatrizoate gradient. These PBMC were cultured in aliquots of 7 x 106 cells with pools of peptides derived from the T, VP1, and VP2 proteins, at a concentration of 1 µg/ml for each peptide. Cells were cultured in RPMI 1640 at a density of 3.5 x 106 PBMC/ml. After 72 h, an equal volume of RPMI/12% FCS containing 40 U/ml rIL-2 was added to each culture well, and every 2 days thereafter half of the medium was changed. After 11–14 days, the peptide-stimulated cells were analyzed in a 51Cr release assay. EBV-transformed autologous B-lymphoblastoid cell lines (B-LCL) were used as target cells. Aliquots of 106 B-LCL were incubated overnight with either peptide pools or individual peptides at a concentration of 5 µg/ml. An OVA peptide (SIINFEKL) or the peptide p11B (ALSEGCTPYDIN) from the SIV was used as negative control peptides. After a 16-h incubation period, target cells were labeled with 100 µCi 51Cr for 90 min. These cells were washed, and 104 cells were added to the effector cells as targets in 96-well U-bottom plates in a final volume of 200 µl/well. The assays were performed in duplicate. When a positive result was obtained using a peptide pool, the assay was repeated using individual 9-aa peptides.
Construction of the tetrameric HLA-A*0201/VP1p100 complex
The HLA-A*0201 protein was expressed in vitro from the plasmid
HLA-A2/glyser/BirA substrate peptide (17). The HLA-A*0201
protein was then refolded in vitro with human
2-microglobulin in the presence of the peptide
VP1p100, as described (18). The
HLA-A*0201/VP1p100 monomers were purified by gel
filtration and biotinylated with the BirA enzyme (Avidity, Denver, CO).
The biotinylated monomers were then mixed with PE-labeled streptavidin
(Prozyme, San Leandro, CA) at a molar ratio of 4:1 to generate
the HLA-A*0201/VP1p100 tetramers.
Staining and phenotypic analysis of VP1p100-specific CD8+ T cells
The mAbs used for this study were directly coupled
to FITC, allophycocyanin, or PE-Texas Red. The following mAbs were
used: anti-CD8
(SK1)-FITC (Becton Dickinson,
San Jose, CA), anti-CD8 /(2ST8-5H7)-PE-Texas Red, and
anti-CD3(UCHT1)-allophycocyanin (Beckman Coulter, Miami, FL). The
PE-coupled tetrameric HLA-A*0201/VP1p100 and the
three mAbs noted above were used in four-color flow cytometric
analyses. Two hundred nanograms of the PE-coupled tetrameric
HLA-A*0201/VP1p100 were used in conjunction with
the directly labeled mAbs to stain 100 µl fresh whole
blood or 5 x 105 lymphocytes that were
cultured in vitro with the VP1p100 peptide. To
remove RBCs, fresh blood samples were lysed using a Q-Prep Workstation
(Beckman Coulter). The lysed samples were washed with PBS, and
centrifuged for 3 min at 300 x g.
Similarly, the stained cultured lymphocyte samples were washed in PBS and centrifuged for 3 min at 300 x g. The supernatants were decanted, and cells were resuspended in 0.5 ml PBS containing 1.5% paraformaldehyde. Samples were analyzed on a FACSCalibur Flow Cytometry System (Becton Dickinson). Data presentation was performed using WinMDI software version 2.7 (Joseph Trotter, La Jolla, CA) and Microsoft PowerPoint software version 97 (Microsoft, Redmond, WA).
MHC class I typing
The MHC class I alleles expressed by the study were determined using standard serologic tissue-typing procedures. In addition, molecular analyses to determine HLA-A*02 subtypes were performed on five subjects.
DNA extraction from plasma
Extraction of DNA from plasma samples was performed, as previously described (19). Four and a half milliliters of plasma were centrifuged at 2000 rpm for 5 min to remove remaining cells. Viral particles were collected by centrifugation of the supernatant at 214,000 x g, resuspended in Tris-NaCl-EDTA (TNE) buffer (10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA), lysed by adding SDS (1% final concentration), and incubated in the presence of 0.1 mg/ml proteinase K at 60°C for 1 h. DNA was then extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and resuspended in Tris-EDTA buffer.
PCR amplification of JCV DNA
PCR amplification of JCV DNA and detection of the amplified products were performed, as previously described (19), using the primer pair VP11/VP12, which flanks a 181-bp fragment of the VP1 gene (20). The PCR reaction was analyzed by electrophoresis on a 2% agarose gel and transferred onto nylon membranes by Southern blotting, and amplified products were detected by hybridization to the JCV-specific 32P end-labeled oligonucleotide probe IKVP1S, as described. The positive control JCV VP1 gene fragment was obtained as previously described (19). Using these conditions, we could reliably detect as few as 10 copies of JCV DNA. PCR oligonucleotide primer sequences: VP11, 5'-cagatacatttgaaagtgac-3' (nt 1662–1681); VP12, 5'-ccattagagtgcacattcatc-3' (nt 1842–1822); IKVP1S, 5'-ggacatgcttccttgttacagtgtg-3' (nt 1693–1717).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC from HLA-A2+ study subjects were stimulated
with these peptides and assessed as effector cells in a
51Cr release assay using as target cells
autologous B-LCL pulsed with each of these peptides. The JCV
VP1p100–108 nonamer peptide ILMWEAVTL
(VP1p100) was recognized by CTL from three of the
six HLA-A2+ HIV+/PML
survivors that were evaluated (Fig. 1).
No VP1p100-specific CTL activity could be
detected in the PBMC of one HIV-/PML survivor,
six HIV+/PML patients who had progressive
disease, two HIV+ patients without PML, and five
normal control subjects. Interestingly, two
HLA-A2+ HIV+ patients with
leukoencephalopathy resembling PML by clinical and radiological
criteria, but with negative JCV PCR in the cerebrospinal fluid or
negative brain biopsy, had detectable CTL specific for this epitope
(Fig. 1). These results suggested that VP1p100
was indeed an epitope recognized by CTL of
HLA-A2+ PML survivors.
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). The autologous and
A2+, but not the fully allogeneic target cells,
were lysed by the A2+ effector cells. These
experiments, therefore, confirmed that the
VP1100-specific CTL were HLA-A2 restricted in
their target cell recognition.
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). No tetramer staining of CD8+ T cells
from any of the fresh blood samples was observed. However, the
lymphocytes of four of six HIV+/PML survivors had
between 2.1% and 13.5% VP1p100-specific
CD8+ T cells demonstrable after in vitro peptide
stimulation. Tetramer binding was also detected to 1.1% of
CD8+ T cells of one
HIV-/PML survivor, and to 5.7% and 6% of
CD8+ T cells, respectively, from two
HIV+ individuals with leukoencephalopathy of
unknown etiology (Fig. 3).
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, d and i),
yet 2.1 and 1.1% of their CD8+ T cells,
respectively, were stained with the HLA-A*0201/JCV
VP1p100 tetramer complex (Fig. 3, d
and i). PBMC from no study subject had demonstrable
VP1p100-specific functional CTL and an absence of
this tetramer staining.
To assess the correlation between the results of the functional lysis
assay and the tetramer-staining assay, the percentage of
tetramer-staining CD8+ T cells was compared with
the percentage of specific lysis at an E:T ratio of 20:1 in the same
cell population in 12 separate experiments performed on
PBMC of seven patients who had detectable tetramer-staining
CD8+ T cells (Fig. 4). A linear correlation between these
values was seen, suggesting that the functional lysis assay and the
tetramer-staining assay were measuring the same population of
functionally active effector CTL.
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JCV is rarely found in the peripheral blood of normal persons (19, 21, 22, 23). JC viremia occurs in the setting of immunosuppression, and JCV DNA has been detected in the blood of HIV+ patients with or without PML who have CD4+ cell counts below 200/µl (19, 21, 24, 25, 26). We sought to determine whether the presence of VP1p100-specific CTL was positively or negatively correlated with detectable JCV in the blood. In the 24 study subjects, JCV DNA could only be detected in plasma samples from 3 of 6 PML progressors (one HIV+ and two HIV- patients). Overall, PML progressors had lower peripheral blood CD4+ T cell counts (57 ± 68/µl) and higher HIV viral loads (67,800 ± 85,500 copies/ml) than PML survivors (CD4+ T cell counts 467 ± 248/µl, and HIV viral loads <50 copies/ml in five and 3694 copies/ml in one). Thus, the presence of VP1p100-specific CTL, as well as evidence of immune reconstitution demonstrated by high CD4+ T cell counts and suppression of HIV replication, correlated with undetectable JC viremia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3,
d and i). Finally, the
VP1p100 epitope is located at nt position
1766–1792 on the JCV genome, which is conserved among JCV genotypes at
the amino acid level (27).
Since two of the seven PML survivors evaluated in this study did not
have detectable JCV-specific CTL, we sought to determine how these
individuals might differ from the other subjects in this cohort.
Different HLA-A2 subtypes have been shown to be functionally distinct
in their ability to bind and present peptide (28). In
Caucasians, the A*0201 subtype is expressed in 
95%
(29), and 49 other subtypes are expressed by the remainder
of A2+ individuals. The two
HIV+/PML survivors who did not have demonstrable
VP1p100-specific CD8+ T
cells were assessed to determine whether they expressed A2 subtypes
other than the A*0201. Molecular typing showed subtype A*0201 in one
subject, and A*0206 in the other. The A*0206 molecule differs from
A*0201 by only a single amino acid in the floor of the binding groove,
and endogenous peptides eluted from this molecule have been shown to
have a leucine in anchor positions 2 and 9, similar to those eluted
from the A*0201 molecule (30). Therefore, we would expect
the VP1p100 peptide, which has a leucine in
anchor positions 2 and 9, to be able to bind to the groove of the
A*0206 molecule. Hence, the A*02 subtype of these two study subjects
did not appear to account for the absence of detectable
VP1p100-specific CTL in their PBMC.
Although PML is usually a rapidly fatal disease, approximately 10% of affected individuals survive more than 1 year (9). We were interested in determining whether the presence of a detectable cellular immune response against JCV in individuals with PML was associated with their clinical outcome. JCV-specific CTL were detectable in five of seven (71%) PML survivors, but none of the six PML patients with progressive neurologic disease (Fisher exact test, two-tailed, p = 0.02). This observation is consistent with the notion that this cellular immune response plays an important role in the prevention of disease progression (13). Therefore, the detection of JCV-specific CTL in PBMC of individuals with PML appears to be a good prognostic marker of disease evolution.
Two of three HIV+ patients with leukoencephalopathy of unknown etiology had detectable JCV-specific CTL in their PBMC. These two patients were also survivors of their neurologic disease, and were clinically stable 15–20 mo after their diagnosis. These two patients may indeed have PML, but with a JC viral load below 50 copies/ml cerebrospinal fluid, the level of detection of this assay (19). It is conceivable that the brain biopsy tissue specimen came from an area of brain that was totally demyelinated and therefore devoid of JCV-infected oligodendrocytes. It is also possible that these patients were infected with an as yet undescribed polyomavirus that shares CTL epitopes with JCV, yet is sufficiently distinct in sequence from JCV to evade detection by PCR using JCV-specific primers.
To determine whether the presence of JCV-specific CTL in PBMC was associated with the clearance of JCV from the blood, the plasma of all 24 study subjects was subjected to PCR amplification for the detection of JCV DNA. JCV DNA could only be detected in the plasma samples of three subjects, all of them individuals who were PML progressors. These results suggest that the presence of JCV-specific CTL in PBMC of patients with PML is associated with clearance of JCV from the blood. It should be noted that PML progressors also had lower CD4+ T cell counts and higher plasma HIV viral loads than PML survivors. Therefore, detectable JC viremia in PML progressors may also reflect the depressed global immune status of these individuals.
JCV is found in renal tubular epithelial cells, and 30% of healthy individuals shed JCV in the urine (19, 31). JC viremia, however, is usually not detectable in immunocompetent individuals (19, 21, 22). The mechanisms that contain JCV replication in normal individuals are not well understood. It is possible that JCV-specific CTL may prevent JCV spread in the bloodstream, but the CTL responses in these individuals might be below the level of detection of our assays, or they might be directed against other JCV epitopes.
The detection of JCV-specific CTL in survivors of PML suggests that these cells may be instrumental in the prevention of disease progression. The tetramer-staining assay may therefore have direct relevance as a prognostic tool in the clinical management of these patients.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Igor J. Koralnik, Beth Israel Deaconess Medical Center, RE-213B, 330 Brookline Avenue, Boston, MA 02215. E-mail address: ikoralni{at}caregroup.harvard.edu
3 Abbreviations used in this paper: JCV, JC virus; B-LCL, B-lymphoblastoid cell line; PML, progressive multifocal leukoencephalopathy.
Received for publication September 24, 2001. Accepted for publication October 26, 2001.
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