Article Figures & Data
Figures
- FIG. 1.
Schematic drawing of a model for EBV persistence. Virions enter the mucosal surface of the nasopharynx through saliva. The virus enters the lymphoid tissue of Waldeyer's ring, where it infects resting naive B cells and drives them to become proliferating lymphoblasts through expression of nine latent proteins (the growth program) (46). These blasts can then differentiate into resting memory cells by a process analogous to the germinal center reaction by using the default program (46). Once in resting memory cells, all viral protein expression ceases (the latency program) (16, 46). Latently infected memory cells circulate in the periphery and return to the lymphoid tissue, where at some unknown rate, they differentiate into plasma cells and release infectious virus to initiate a new round of infection (Laichalk and Thorley-Lawson, submitted). Each stage of the cycle is subject to immunosurveillance (CTL against infected cells and antibody against virions), with the exception of the memory cells, which are invisible because they express no viral proteins (16). In the absence of an immune response, the cycle will amplify until the memory B compartment is filled with latently infected cells. Once the immune response is activated, the cycle is dramatically reduced or perhaps even completely blocked and the reservoir of latently infected memory cells decays.
- FIG. 2.
EBV-infected cells in the periphery of AIM patients are CD27 positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
- FIG. 3.
EBV-infected cells in the periphery of AIM patients are IgD negative. PBMCs were stained for expression of CD20, a pan-B-cell marker, and IgD (upper panel). The naive (IgD positive) and memory (IgD negative) B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
- FIG. 4.
Cell cycle stage of EBV-infected cells in the blood of AIM patients. B cells from an EBV-transformed cell line (A) or from the blood of AIM patients (B) were stained with the vital DNA dye Hoechst 33342. The AIM B cells were then separated into the G0/G1 and S/G2/M fractions by FACS, and the frequency of infected cells in each population was measured by limiting dilution DNA PCR (C).
- FIG. 5.
B cells, replicating EBV, are rare in the blood of AIM patients. PBMCs (A) or purified B cells (B) were isolated, and the frequency of virus-infected cells expressing the BZLF1 or EBER1 genes was measured by performing a limiting dilution RT-PCR assay. Serial dilutions of cells were prepared, and multiple samples of each dilution were tested for expression of each RNA by RT-PCR. BZLF1 is the gene that initiates viral replication, and EBER1 is believed to be widely expressed in EBV-infected cells. The number of infected cells tested was predetermined by limiting dilution DNA PCR. (C) A control experiment with an EBV-positive cell line demonstrating that the technique can detect a single infected cell expressing BZLF1.
- FIG. 6.
Comparison of frequencies of EBV-infected B cells in blood of AIM patients, immunosuppressed allograft patients, and healthy carriers. The AIM data (n = 20) (open bars) are from the patients listed in Table 4, the data from the immunosuppressed allograft patients (n = 25) (filled bars) have been published previously (2). The healthy carriers (n = 46) (hatched bar) are a new cohort, but similar data have been published previously by our laboratory (22, 29, 33) and are not detailed here. The frequencies of infected cells in this figure are expressed as the natural logs of the number of infected cells in 107 total B cells, not memory B cells.
- FIG. 7.
Detection of high levels of EBV-infected memory B cells in blood of AIM patients. IgD-negative memory B cells were isolated as described in the legend to Fig. 3. Serial dilutions of the cells were then prepared (e.g., 0.75 means that, on average, 7.5 of 10 replicates will have 1 cell), and multiple replicates of each dilution were tested for the presence of EBV either by DNA PCR and Southern blotting for the viral DNA (A) (patient 1 in Table 4) or by real-time PCR for EBER1 RNA (B) (patient 2 in Table 4).
- FIG. 8.
Time course of resolution of EBV infection in blood of AIM patients. The frequencies of infected cells are expressed as the natural logs of the number of infected cells per 107 memory B cells. The dashed horizontal line represents the upper limit, and unbroken line represents the mean of the number of infected memory B cells detected in healthy carriers.
Tables
- TABLE 1.
Phenotype of EBV-infected cells in blood of AIM patients
Patient no. Marker Frequency/105 cellsa Enriched Depleted 1 CD27 250 5.0 2 CD27 170 2.0 3 IgD 7.4 2,500 4 IgD 15 1,400 5 IgD 120 12,000 6 IgD 1.1 9,800 7 Ig 51 <0.7 8 Ig 4,500 170 9 CD5 1.1 220 10 CD5 100 770 ↵ a Enriched, marker-positive population; Depleted, marker-negative population.
- TABLE 2.
Cell cycle distribution of latently infected cells
Patient no. Cell cycle stage Frequency of infected cells/106 cells % of B cells % of infected B cellsa 1 G0/G1 14,000 97 95 S/G2/M 26,000 2.8 5 2 G0/G1 900 98 97 S/G2/M 1,400 2.2 3.4 3 G0/G1 14,000 91 90 S/G2/M 16,000 9 10 ↵ a The percentage of infected B cells was calculated from the data in columns 3 and 4, e.g., for patient 1, for every 106 B cells; 97% are in G0/G1 and the frequency of infected cells is 14,000/106 cells. This is equal to 0.97 × 106 G0/G1 B cells with 13,800 infected cells, and 2.8% are in S/G2/M and the frequency of infected cells is 26,000/106 cells. This is equal to 0.028 × 106 S/G2/M B cells with 730 infected cells. The total number of infected cells is 13,800 plus 730, which equals 14,530 cells. The percentage of cells in G0/G1 is 13,800 × 100/14,350, which equals 95%, and the percentage of cells in S/G2/M is 730 × 100/14,350, which equals 5%.
- TABLE 3.
Percentage of EBV-infected cells in blood of AIM patients replicating EBV
Patient no. % Memory B cells infected % Infected cells expressing BZLF1 1 17 0.003 2 5 0.021 3 4.8 <0.001 4 0.27 <0.01 5 0.13 <0.01 - TABLE 4.
Frequency of infected memory cells and total B cells in blood of AIM patients
Patient no. % of memory B cells % of total B cells 1 46 7.6 2 36 6.2 3 25 4.0 4 17 3.7 5 12 2.4 6 9.1 2.4 7 5.6 2.1 8 4.8 1.3 9 3.6 1.2 10 3.3 0.83 11 2.5 0.63 12 1.4 0.43 13 1.3 0.33 14 0.83 0.15 15 0.56 0.078 16 0.25 0.070 17 0.22 0.053 18 0.18 0.032 19 0.10 0.026 20 0.06 0.007
