Low frequency of cytotoxic T lymphocytes against the novel HLA-A*0201-restricted JC virus epitope VP1(p36) in patients with proven or possible progressive multifocal leukoencephalopathy

Abstract

JC virus (JCV)-specific cytotoxic T lymphocytes (CTL) in peripheral blood are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML). However, the frequency of these cells in the peripheral blood mononuclear cells (PBMC) of PML patients is unknown. To develop a highly sensitive assay for detecting the cellular immune response against this virus, we performed a CTL epitope mapping study of JCV VP1 major capsid protein by using overlapping peptides. A novel HLA-A*0201-restricted epitope, the VP1(p36) peptide SITEVECFL, was characterized. The cellular immune response against JCV was assessed in 32 study subjects. By combining the results of the (51)Cr release assay on pooled peptides and staining with the HLA-A*0201/JCV VP1(p36) tetramer, VP1-specific CTL were detected in 10 of 11 PML survivors (91%) versus only 1 of 11 PML progressors (9%, P = 0.0003). VP1-specific CTL were also detected in two of two patients recently diagnosed with PML and in four of four human immunodeficiency virus-positive patients with possible PML. The frequency of CTL specific for the novel VP1(p36) and the previously described VP1(p100) epitopes was determined. In two patients, the frequency of CTL specific for the VP1(p36) or VP1(p100) epitopes, as determined by fresh blood tetramer staining (FBTS), ranged from 1/6,000 to 1/24,000 PBMC. A CTL sorting technique combining tetramer staining and selection with immunomagnetic beads allowed the detection of epitope-specific CTL in two cases that were determined to be negative by FBTS. The phenotype of these CTL in vivo was consistent with activated memory cells. These data suggest that, although present in low numbers, JCV-specific CTL may be of central importance in the containment of JCV spread in immunosuppressed individuals.

FIG. 1.

CTL recognition of the JCV VP1_p36 epitope SITEVECFL is HLA-A*0201 restricted. Autologous (a) and allogeneic HLA-A*0201-matched (b) target cells, but not fully MHC class I-mismatched target cells (c), pulsed with VP1_p36 were lysed by JCV VP1_p36-stimulated PBMC of an HIV⁺ PML survivor.

FIG. 2.

Staining of JCV VP1_p36-stimulated PBMC from HLA-A*0201⁺ individuals with the HLA-A*0201/JCV VP1_p36 tetramer. The percentage of all CD8⁺ T cells that bind this tetramer is indicated in each panel. Tetramer-positive cells were detected in VP1_p36-stimulated CD8⁺ T lymphocytes of HIV⁺ PML survivors (a and b), HIV⁺ possible-PML patients (e and f), and one HIV⁺ OND patient (h). However, negligible tetramer binding was seen in the CD8⁺ T lymphocytes of two HIV⁺ PML progressors (c and d) and an HIV⁺ OND patient (g). Cells were gated on CD3⁺ CD8⁺ lymphocytes. The results displayed represent staining with an anti-CD8αβ antibody and the HLA-A*0201/JCV VP1_p36 tetramer.

FIG. 3.

Determination of the frequency and phenotype of JCV VP1_p36- and -_p100-specific CTL in an HIV⁺ PML survivor. Fresh blood was stained with either HLA-A*0201/JCV VP1_p36 (Aa) or VP1_p100 (Ba) PE-labeled tetramers. In addition, 5 × 10⁷ fresh PBMC were stained with the same tetramers and sorted by using an AUTOMACS cell sorter with PE-labeled immunomagnetic beads. A positive (Ab and Bb) and a negative (Ac and Bc) fraction were collected and analyzed immediately after being sorted by flow cytometry. Sorted cells were stimulated in vitro in the presence of VP1_p36 or VP1_p100, feeder cells, and rIL-2 and then stained with their respective tetramers after 17 days (Ad and -e) or 14 days (Bd and -e). The percentage of all CD8⁺ T cells that bind the tetramers is indicated in each panel. These cells were then assessed for the presence of functionally active effector cells in a ⁵¹Cr release assay (Af and -g and Bf and -g). (C) Fresh PBMC were costained with the HLA-A*0201/JCV VP1_p100 tetramer, sorted with immunomagnetic PE-labeled beads, and then stained with the activation markers CD62L, CD45RA, and CD49d. A total of 97% of the tetramer-positive cells were also CD49d positive compared to 2.2 and 1.1% that were positive for CD62L and CD45RA, respectively, indicating that these were activated memory cells. Tetr., tetramer.