CD4+ T-cell effectors inhibit Epstein-Barr virus-induced B-cell proliferation

Abstract

In immunodeficient hosts, Epstein-Barr virus (EBV) often induces extensive B-cell lymphoproliferative disease and lymphoma. Without effective in vitro immune surveillance, B cells infected by the virus readily form immortalized cell lines. In the regression assay, memory T cells inhibit the formation of foci of EBV-transformed B cells that follows recent in vitro infection by EBV. No one has yet addressed which T cell regulates the early proliferative phase of B cells newly infected by EBV. Using new quantitative methods, we analyzed T-cell surveillance of EBV-mediated B-cell proliferation. We found that CD4+ T cells play a significant role in limiting proliferation of newly infected, activated CD23+ B cells. In the absence of T cells, EBV-infected CD23+ B cells divided rapidly during the first 3 weeks after infection. Removal of CD4+ but not CD8+ T cells also abrogated immune control. Purified CD4+ T cells eliminated outgrowth when added to EBV-infected B cells. Thus, unlike the killing of EBV-infected lymphoblastoid cell lines, in which CD8+ cytolytic T cells play an essential role, prevention of early-phase EBV-induced B-cell proliferation requires CD4+ effector T cells.

FIG. 1

Effects of FK506 on the kinetics of proliferation of uninfected and EBV-infected PBMCs as measured by incorporation of [³H]dT. PBMCs from EBV-seropositive donors (A and C) or EBV-seronegative donors (B and D) were infected with EBV (shaded bars and triangles, EBV⁺) or a control inoculum (solid bars and circles, EBV⁻) in the presence or absence of FK506. (A and B) Seventeen days after infection, [³H]dT was added to the PBMC cultures; the incorporated radioactivity was measured 24 h later. Results represent the mean ± standard error of the mean (SEM) of triplicate samples for each experimental condition. (C and D) Solid lines indicate addition of FK506; dashed lines represent PBMC cultures without FK506. [³H]dT incorporation was determined at intervals from 2 to 15 days after infection.

FIG. 2

Composition and proliferation of PBMC cultures infected with EBV in the absence or presence of FK506. (A and B) PBMCs from an EBV-seropositive donor were infected with EBV. The numbers of CD3⁺ (black), CD19⁺ (hatched), and CD23⁺ (white) cells were determined by FACS four times after infection with EBV in the absence (A) or presence (B) of FK506. (C and D) PBMCs from an EBV-seropositive donor were incubated with CFSE dye and then infected with EBV in either the absence (C) or presence (D) of FK506. Living cells were analyzed for CD3, CD19, and CD23 expression and the intensity of CFSE fluorescence 3 and 17.5 days after infection. Progressive rounds of cell division are reflected in sequential halvings of fluorescence intensity and a shift to the left in the profile of CFSE staining.

FIG. 3

Effect of initial cell density on the proportion of CD23⁺ B cells in PBMC cultures infected with EBV in the presence and absence of FK506. FACS analysis of expression of CD19 (vertical axis) and CD23 (horizontal axis) in PBMC cultures initiated at starting densities varying between 10⁶ and 2 × 10⁵ cells/ml. All cultures were derived from the same EBV-seropositive individual, simultaneously infected with EBV, and analyzed 16 days after infection.

FIG. 4

Effect of depletion of T cells on the proportion of CD23⁺ B cells in EBV-infected cultures. At the indicated times, cultures of EBV-infected cells originally drawn from EBV-seropositive donors were harvested and analyzed by FACS for the fraction of CD23⁺ B cells. (A) Effect of depletion of CD3⁺ cells. Cultures depleted of CD3-expressing cells prior to infection were initiated at 2 × 10⁵ cells/ml and compared with cultures of PBMC initiated at 10⁶ cells/ml. Both groups of cells were cultured in the presence (solid lines) and absence (dashed lines) of FK506. Following CD3⁺ cell depletion, cultures from donor 1 contained fewer than 3.3% CD3⁺ cells at 5 days after infection, and cultures from donor 2 contained fewer than 0.8% CD3⁺ cells. (B) Effect of depletion of CD4⁺ and CD8⁺ cells. PBMC cultures were initiated at 10⁶ cells/ml; cultures depleted of CD4⁺ cells were initiated at 4 × 10⁵ cells/ml; and cultures depleted of CD8⁺ cells were initiated at 8 × 10⁵ /mL. CD4-depleted and CD8-depleted cultures from donor 3 contained fewer than 1.4% CD4⁺ cells and fewer than 0.7% CD8⁺ cells, respectively. CD4-depleted and CD8-depleted cultures from donor 4 contained fewer than 4.2% CD4⁺ cells and fewer than 0.5% CD8⁺ cells, respectively, measured 3 days after infection. (C) Effect of depletion of different T-cell subpopulations in an experiment utilizing PBMCs from one seropositive donor. All cultures were simultaneously infected with EBV and initiated at the same starting cell density of 2 × 10⁶ cells/ml. Cultures depleted of CD3-, CD4-, and CD8-expressing cells contained less than 0.9% CD3⁺ cells, less than 0.5% CD4⁺ cells, and less than 1.4% CD8⁺ cells respectively, at 8.5 days after infection. Triangles, PBMCs; diamonds, CD3-depleted cells; squares, CD4-depleted cells; and X's, CD8-depleted cells.

FIG. 5

Effect of depletion of T cells on the proportion of CD23⁺ B cells in EBV-infected cultures initiated at different cell densities. PBMC cultures (solid bars) are compared with CD3-depleted (hatched bars), CD4-depleted (stippled bars), and CD8-depleted (white bars) cultures, all derived from the same EBV-seropositive donor. Cultures were infected with EBV and initiated at the indicated cell densities. Cultures depleted of CD3-, CD4-, and CD8-expressing cells contained contaminating residual populations of less than 1.1% CD3⁺ cells, less than 0.6% CD4⁺ cells, and less than 0.8% CD8⁺ cells, respectively, at the time of initiation. Results represent cells analyzed 18 days after infection. ■, PBMCs; ▨, CD3 depleted; ░⃞, CD4 depleted; □, CD8 depleted.

FIG. 6

Effect of depletion of different T-cell subsets on the proportion of dividing CD23⁺ cells. Mixed PBMC cultures or cultures that had been drawn from an EBV-seropositive donors and depleted of CD3-, CD4-, or CD8-expressing cells were incubated with CFSE and infected with EBV. At 8.5 and 12.5 days after infection, CD23⁺ B cells in the cultures were selectively gated and analyzed for CFSE fluorescence intensity (horizontal axis). Cultures depleted of CD3-, CD4-, and CD8-expressing cells contained contaminating residual populations of less than 0.9% CD3⁺ cells, less than 0.5% CD4⁺ cells, and less than 1.4% CD8⁺ cells, respectively, at 8.5 days after infection. Percentages in the upper left corner of each proliferation profile represent the percentage of CD23⁺ B cells in that culture which have undergone proliferation. Percentages in the upper right corner of each profile represent the percentage that have not yet undergone proliferation.

FIG. 7

Effect of addition of T-cell subpopulations on the number of CD23⁺ B cells. (A) EBV-infected cells were either PBMCs or CD3-depleted PBMCs at 2 × 10⁶ cells/ml. (B) EBV-infected target cells were CD3-depleted PBMCs (white hatched bar) seeded at 2 × 10⁵ cells/ml to which were added increasing numbers of purified, positively selected CD3- (dark hatched bars) or CD4-expressing (white bars) T cells. There were fewer than 0.4% contaminating CD19⁺ B cells in the purified CD3⁺ population and less than 0.3% CD8⁺ and 0.2% CD19⁺ cells in the purified CD4⁺ population. The cultures were analyzed for the number of CD23⁺ B cells 17.5 days after infection. Cells in all cultures were initially derived from the same EBV-seropositive donor.