Article Figures & Data
Figures
- FIG. 1.
Tetramer staining of PBMCs from HLA-A*0201-positive IM patients. (A) IM13, sampled during acute primary infection (IM13.1) and about 14 years later (IM13.2). (B) IM140, sampled during acute primary infection (IM140.1) and at intervals up to 14 months later (IM140.5) as indicated. FACS profiles from cells dually stained with a Tricolor-conjugated anti-CD8 MAb and a PE-labeled tetramer specific for the YVL, GLC, or TLD epitope are shown. Numbers in the upper-right quadrant refer to the percentages of CD8+ T cells that stained with the tetramer.
- FIG. 2.
IL-7Rα statuses of YVL-, GLC-, and TLD-specific responses over time post-IM. (A) FACS profiles of IL-7Rα and either YVL- or TLD-tetramer staining from the blood samples of an HLA-A*0201-positive IM patient (IM179) taken during acute infection (IM179.1), and after a further 4 (IM179.2), 10 (IM179.3), and 27 (IM179.4) months. Data from a healthy EBV carrier are shown for comparison. Profiles are gated on CD8+ T cells. Numbers in the upper-right quadrants refer to the percentages of tetramer-positive, CD8+ cells that express IL-7Rα. (B) Summary of data from another four prospectively analyzed HLA-A*0201-positive IM patients (IM113, IM119, IM140, and IM146), showing the percentages of YVL-, GLC-, and TLD-specific tetramer-positive cells that expressed IL-7Rα, plotted against time from the acute disease. In each case, the last blood sample taken before disappearance of the TLD-specific population is marked with a dagger. Comparing values from the last available blood sample in which all three epitope-specific populations were detectable for the five patients analyzed plus IM123 (see the text), the difference between the percentages of TLD-specific cells versus YVL/GLC-specific cells that were IL-7Rα positive is significant (P = 0.001).
- FIG. 3.
PD-1 staining of PBMCs from healthy donors. (A) PD-1 expression on PBMCs from an HLA-A*0201, B*0801-positive EBV/CMV carrier. The FACS profiles show distribution of PD-1 staining (filled area) versus staining with an irrelevant isotype control (open area) for total CD8+ T cells and for CD8+ tetramer-stained T cells specific for the HLA-B*0801-restricted EBV latent cycle epitope FLR, for the HLA-A*0201-restricted EBV lytic cycle epitope YVL, and for the HLA-A*0201-restricted CMV epitope NLV. For each cell population, the level of PD-1 staining is expressed as a ratio of the MFI of PD-1 antibody staining relative to that of isotype control antibody staining; isotype control MFI values were between 11 and 29, with a mean of 19.2. (B). Scatter plot showing composite PD-1 data from blood samples from 11 healthy EBV and/or CMV carriers stained as described above. Results, expressed as MFI ratios as described above, are shown for total CD8+ T cells in PBMCs and for CD8+ tetramer-stained cells specific for panels of EBV latent cycle, EBV lytic cycle, and CMV epitopes. Individual symbols refer to individual results from each donor, and horizontal lines show the median levels. Statistically significant differences in levels of PD-1 expression were observed between EBV latent cycle and CMV epitopes (P = 0.002) and EBV lytic cycle and CMV epitopes (P = 0.001) but not between EBV latent cycle and EBV lytic cycle epitopes (P = 0.09).
- FIG. 4.
PD-1 staining of YVL-, GLC-, and TLD-specific T cells in the blood of HLA-A*0201-positive IM patients studied over time. (A) IM140 blood samples, taken during acute primary infection (IM140.1) and after a further 8 (IM140.3) and 10 (IM140.4) months. (B) IM113 blood samples, taken during acute primary infection (IM113.1) and after a further 14 (IM113.2) and 17 (IM113.3) months. At each time point, the FACS profiles show distribution of PD-1 staining (filled area) versus staining with an irrelevant isotype control (open area) on the tetramer-positive, CD8+ population. For each cell population, the level of PD-1 staining is expressed as a ratio of the MFI of PD-1 antibody staining relative to that of isotype control antibody staining; isotype control MFI values were between 8.9 and 26.5, with a mean of 14.9.
- FIG. 5.
Histograms showing levels of PD-1 expression on total CD8+ T cells and on the YVL, GLC, and TLD epitope-specific populations in PBMCs from patients IM140, IM113, IM119, and IM123 during acute IM and at later times. Results are expressed as MFI ratios as described in the legend to Fig. 4. Note that the IM140.4, IM113.3, IM119.2, and IM123.2 samples are the last samples obtained in which a TLD-specific response was detectable; where PD-1 staining on TLD-specific cells exceeds the vertical scale, the actual MFI ratios for these TLD-specific populations are shown above the relevant columns. In a comparison of values from the last available blood sample in which all three epitope-specific populations were detectable for the four patients analyzed, the difference between PD-1 expression on TLD-specific and YVL/GLC-specific cells was significant (P = 0.028).
- FIG. 6.
Analysis of the functional capacity of GLC- and TLD-specific populations within the IM140.4 blood sample, taken 10 months post-IM, at a time when the TLD-specific cells had upregulated PD-1 expression prior to the disappearance of that response. Cells were stained with tetramer and then exposed to epitope peptide in the presence of appropriately conjugated anti-CD107a MAb and then (after fixation and permeabilization) to appropriately conjugated MAbs to CD8 and to the intracellular cytokines IFN-γ (IFN-g), TNF-α (TNF-a), and IL-2. Data are shown as FACS profiles (gating on tetramer-labeled CD8+ T cells) that plot the IFN-γ response against each of the other response markers. Responses induced by epitope peptide stimulation are compared with those seen for the unstimulated (US) cells (the unstimulated control values illustrated are for the TLD-specific population; the GLC-specific population gave similar control data). Numbers refer to the percentages of tetramer-positive cells that, in dual staining for IFN-γ and a second marker (CD107a, TNF-α, or IL-2), respond by IFN-γ alone (bottom-right quadrant), by the second marker alone (top-left quadrant), or by both IFN-γ and the second marker (top-right quadrant).
Tables
- TABLE 1.
Summary of phenotypic and functional analysis of GLC- versus TLD-specific CD8+ T cellsa
Sample (no. of mo) and T-cell epitope % Tetramer-positive cells with indicated phenotype/marker MFI ratio for PD-1b CCR7+ CD45RA− CCR7− CD45RA− CCR7− CD44RA+ CD38 Ki67 Bcl2 IL-7Rα CD107α IFN-γ TNF-α MIP-1β IL-2 IM140.1 GLC 0.4 88.4 10.9 98.8 74.1 4.5 5.8 NT NT NT NT NT 17.5 TLD 1.5 73.2 24.6 100 50.0 9.5 5.8 87.9 51.1 51.2 7.8 11.5 11.5 IM140.3 (8) GLC 1.3 37.2 59.7 12.5 5.8 52.4 12.0 57.8 31.5 27.9 8.2 9.5 6.9 TLD 1.9 19.5 76.5 19.5 6.1 52.8 36.0 71.7 14.8 26.4 7.0 6.0 3.9 IM140.4 (10) GLC 1.7 26.2 70.9 40.5 7.5 54.3 24.0 64.2 31.9 24.7 8.8 10.0 4.5 TLD 3.2 14.3 80.8 57.9 9.0 50.8 64.0 73.2 14.9 22.7 7.7 6.9 103.5 IM119.2 (4) GLC 4.6 73.7 20.1 71.4 11.1 46.7 11.6 50.0 52.1 61.7 17.0 4.9 7.4 TLD 1.4 75.9 21.3 58.8 10.5 51.5 41.6 59.4 11.8 28.3 8.6 12.3 88.2 IM123.2 (2) GLC 1.5 80.5 6.5 92.5 8.6 31.2 18.0 48.7 25.2 26.4 10.7 12.5 20.3 TLD 4.8 74.7 20.5 84.6 10.7 36.1 48.0 52.8 14.8 17.4 5.5 7.7 68.9 ↵ a Numbers refer to the percentages of tetramer-positive cells with the relevant phenotype or (following peptide stimulation) functional markers. NT, not tested. Underlined values identify key differences between GLC- and TLD-specific cell phenotypes.
↵ b Data for PD-1 staining of tetramer-positive cells are expressed as MFI ratios.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Table S1 (% tetramer+ cells in CD8+ T cells with time post-IM.)
PDF file, 17K.
- Supplemental file 1 -
Table S1 (% tetramer+ cells in CD8+ T cells with time post-IM.)
