Direct ex vivo measurement of CD8(+) T-lymphocyte responses to human parvovirus B19

Abstract

Parvovirus B19 is a common human pathogen which can cause severe syndromes, including aplastic anemia and fetal hydrops. The mapping of the first parvovirus B19-derived CD8(+) T-lymphocyte epitope is described. This HLA-B35-restricted peptide derives from the nonstructural (NS1) protein and is strongly immunogenic in B19 virus-seropositive donors.

FIG. 1

Mapping of an immunodominant epitope in parvovirus B19 virus NS1 protein. (A) Chromium-51 release assay using CTL lines from donor 1. Peptide-stimulated PBMC lines were tested for lytic activity against autologous BCL prepulsed with pools. Each pool contained 10 15-mers overlapping by 5 except for pool 15, which contained 6 peptides. (B) The same PBMC from positive pool 8 were tested against the 10 individual peptides tested from the pool in an identical assay against autologous BCL. (C) Fresh PBMC from donor 1 were stimulated using only peptides 9138 and 9139 (in a 1:1 mix at 10 μM each) from pool 8, and after 8 days, cytolysis was tested against autologous and two different HLA-B35-matched targets. (D) PBMC from two HLA-B35-positive, B19 virus-seropositive donors were restimulated for 8 days with optimized peptide QPTRVDQKM, and cytolysis was tested as previously against HLA-B35-matched peptide-pulsed and control targets at various effector-to-target cell (E:T) ratios.

FIG. 2

Ex vivo detection of parvovirus (PARVO)-specific CTL responses by Elispot and tetramer staining. (A) Fresh PBMC from donor 1 were tested for overnight release of IFN-γ after peptide stimulation. Peptides were used at 10 μM, and cells were plated at 300,000 per well. Results are expressed as SFC/10⁵ PBMC. FLU-A2 and EBV-A2 are HLA-A2-restricted peptides derived from influenza virus (GILGFVFTL) and Epstein-Barr virus (GLCTVAML), respectively. Donor 1 is HLA-A2 positive. (B) A CTL line from donor 1 specific for the optimized peptide QPTRVDQKM was stained with the HLA-B35 QPTRVDQKM tetramer and anti-CD8 (left-hand panel). CD8 staining (FL3) is on the y axis, and tetramer staining (FL2) is on the x axis. Staining of peptide-stimulated PBMC from a separate HLA-B35-positive B19 virus-seronegative donor, where no peptide-specific responses were obtained, is shown for comparison (right-hand panel). PE, phycoerythrin. (C) Fresh PBMC from donor 1 were stained with tetramer and CD8 as in panel B, but in addition phenotypic markers were conjugated with FITC, as shown. Representative results from assays of samples from three individuals stained at separate time points are shown. Tetramer staining is on the y axis, and the population shown is that contained within the CD8 high gate.