Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo

Abstract

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex-peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8+ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8+ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.

Figure 1

Specificity of the HLA A2–GLCTLVAML tetrameric complex for HLA A2–restricted GLCTLVAML-specific T cell clones. An HLA A2–restricted GLCTLVAML-specific T cell clone (a), an HLA A2–restricted SLYNTVATL-specific T cell clone (b), PBLs from a normal healthy HLA A2–negative individual (c), PBLs from an HLA A2–negative individual with AIM (d), and PBLs from an HLA A2– positive, EBV-seronegative individual (e) were stained with phycoerythrin-conjugated HLA A2–GLCTLVAML tetrameric complex and with an antibody to CD8-conjugated to tricolor.

Figure 2

The frequency of CD8⁺ T cells specific for three epitopes from EBV within PBLs taken from donors suffering from AIM (a) PBLs from the two HLA A2–positive donors, IM74 and IM83, suffering from AIM, were stained with phycoerythrin-conjugated HLA A2–GLCTLVAML tetrameric complex and with an antibody to CD8 conjugated to tricolor. (b) PBLs from HLA B8–positive donors IM70 and IM59, suffering from AIM, were stained with phycoerythrin-conjugated HLA B8– RAKFKQLL tetrameric complex and with an antibody to CD8 conjugated to tricolor. (c) PBLs from HLA B8–positive donors IM63 and IM59, suffering from AIM, were stained with phycoerythrin-conjugated HLA B8–FLRGRAYGL tetrameric complex and with an antibody to CD8 conjugated to tricolor. In each figure, the number of CD8⁺ T cells that stain with the tetrameric complex is shown as percent frequency.

Figure 3

The frequency of CD8⁺ T cells specific for three epitopes from EBV within PBLs taken from donors suffering from AIM and from the same donors at least 6 mo later. PBLs from HLA A2–positive donors (a) and HLA B8–positive donors (b and c) suffering from AIM were stained with the phycoerythrin-conjugated HLA A2–GLCTLVAML tetrameric complex (a), the phycoerythrin-conjugated HLA B8–RAKFKQLL tetrameric complex (3), or the phycoerythrin-conjugated HLA B8–FLRGRAYGL tetrameric complex (c). The frequency of tetramer-positive cells within CD8⁺ PBLs taken from donors suffering from AIM is shown in solid bars, and the frequency of tetramer-positive cells within CD8⁺ PBLs taken from the same donors at least 6 mo later is shown in shaded bars.

Figure 4

Phenotypic analysis of antigen-specific CD8⁺ T cells in an individual suffering from AIM. PBLs taken from donor IM73 while he was suffering from AIM were stained with the phycoerythrin-conjugated HLA A2–GLCTLVAML tetrameric complex, with a tricolor-conjugated anti-CD8 antibody, and with one of a panel of antibodies conjugated to FITC. a shows staining with the HLA A2–GLCTLVAML tetrameric complex on the x-axis and with CD8 on the y-axis. b–h show staining of the CD8⁺ T cells with the HLA A2–GLCTLVAML tetrameric complex on the y-axis and with an antibody specific for HLA DR (b), CD38 (c), CD62L (d), CD45RO (e), CD45RA (f), CD28 (g), or CD57 (h) on the x-axis. In each figure, the number of tetramer–reactive T cells that stain with the particular phenotypic marker is shown as percent frequency.

Figure 5

Comparison of the frequency and phenotype of antigen-specific CD8⁺ T cells within PBLs taken from a donor suffering from AIM and 37 mo later. PBLs taken from donor IM63 while he was suffering from AIM (IM63.1; a–e) and 37 mo later (IM63.3; f–j) were stained with the phycoerythrin-conjugated HLA B8–RAKFKQLL tetrameric complex, with a tricolor-conjugated anti-CD8 antibody, and with one of a panel of antibodies conjugated to FITC. Only CD8⁺ T cells were included in the phenotypic analysis. Staining with the tetrameric complex is shown on the y-axis and staining with normal mouse serum (negative control; a and f), and with an antibody specific for CD38 (b and g), CD45RO (c and h), CD45RA (d and i), or CD28 (e and j) is shown on the x-axis. In each figure, the number of tetramer-reactive T cells that stain with the particular phenotypic marker is shown as percent frequency.