Epstein-barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients

Abstract

When Epstein-Barr virus (EBV) infects B cells in vitro, the result is a proliferating lymphoblast that expresses at least nine latent proteins. It is generally believed that these cells are rigorously controlled in vivo by cytotoxic T cells. Consistent with this, the latently infected cells in the peripheral blood of healthy carriers are not lymphoblasts. Rather, they are resting memory B cells that are probably not subject to direct immunosurveillance by cytotoxic T lymphocytes (CTLs). When patients become immunosuppressed, the viral load increases in the peripheral blood. The expansion of proliferating lymphoblasts due to the suppressed CTL response is believed to account for this increase and is considered to be a major risk factor for posttransplant lymphoproliferative disease (PTLD) and AIDS-associated B cell lymphoma. Here we show that there is an increase in the numbers of latently infected cells in the peripheral blood of immunosuppressed patients. However, the cells are not proliferating lymphoblasts. They are all latently infected, resting, memory B cells-the same population of infected cells found in the blood of healthy carriers. These results are discussed in the context of a model for EBV persistence that explains why PTLD is usually limited to the lymph nodes.

Figure 3

Frequencies of virus-infected cells in healthy control and immunosuppressed populations. (A) The graphs show the distribution in the frequencies for each population plotted as their ln values. The resulting curve, for healthy donors, approximates a normal distribution with a mean frequency of 50 infected cells per 10⁷ B cells. The immunosuppressed patients fall into two groups with means of 36 and 1,600, respectively. (B) FACS^® analysis of representative individuals from the healthy controls and from each group of immunosuppressed patients. Note that the level of IgD⁺ B cells in the representative high frequency patient (6.4%) is within the normal range compared with the healthy control (7.2%), whereas it is highly elevated in the low frequency patient (24.7%). In comparison, the memory cells (IgD⁻CD20⁺) are within normal range for both patients (1.4 and 2.1 vs. 1.9% in the control).

Figure 1

The cell surface phenotype of EBV-infected cells in the blood of immunosuppressed individuals. Peripheral lymphocytes were fractionated using mAbs and the MACS^® separation technique. For CD19 and sIg, whole PBMCs were fractionated for the specific marker. For CD23 and IgD, the cells were first selected for expression of the desired marker and the remaining marker-negative B cells were then selected using the pan-B cell marker CD19. The cells were serially diluted, and replicates of each dilution were subjected to DNA PCR for the W repeat of the viral DNA using a technique that will detect a single viral genome in 10⁶ uninfected cells (reference 4). The PCR products were detected by Southern blotting with a specific probe for the W repeat. The position of each lane containing a sample is indicated at the top. Two negative and one positive control were performed for each dilution of cells (not shown). For full experimental details, see Materials and Methods. The frequency of virus-infected cells was calculated using Poisson statistics, and the results are summarized in Table . The cell surface phenotype of the cells tested, the number of cells tested per sample, and the fraction of samples positive at each cell number are given.

Figure 2

Gardella gel analysis of peripheral B cells from immunosuppressed individuals. The gel resolves the faster running linear genomes from slower running episomal genomes. Each sample lane was excised and cut into a series of slices. DNA was extracted from each slice and subjected to EBV-specific DNA PCR. The PCR products from each gel slice were detected by Southern blotting. The position of each lane is indicated at the top. The location of the top and bottom slices of the Gardella gels and the expected migration point of linear and episomal forms of the genome are indicated. The expected location of the PCR product is shown by an arrow. For full experimental details, see Materials and Methods. (A) Analysis of peripheral blood B cells from a healthy carrier as a positive control for the migration of episomes. It has been shown previously that only the episomal form of the virus is detected in these samples. (B) Analysis of whole EBV virions as a positive control for the migration of the linear form of the genome. (C) Analysis of peripheral blood B cells from an immunosuppressed individual who only carries latently infected cells. (D) Analysis of peripheral blood B cells from an immunosuppressed individual who has both the latent (episomal) and lytic (linear) forms of the viral genome.

Figure 4

RT-PCR analysis for the expression of LMP-2a mRNA in infected B cells from immunosuppressed individuals. CD19⁺ B cells were purified by the MACS^® system. RNA was extracted using Trizol, and RT-PCR was performed for LMP-2a. (A) cDNA from a sample containing an estimated 20 infected cells from patient 25. (B) As in A, but spiked with five cells from the EBV-immortalized lymphoblastoid cell line IB4. (C) cDNA from a sample containing an estimated 1,700 infected cells from patient 6. (D) cDNA from 5 IB4 cells in 2 × 10⁵ EBV⁻ BJAB cells. (E) cDNA from 1 IB4 cell in 2 × 10⁵ EBV⁻ BJAB cells. (F) cDNA from 2 × 10⁵ EBV⁻ BJAB cells.