Inhibition of Fas-mediated apoptosis by antigen: implications for lymphomagenesis

Abstract

Apoptotic deletion of expanded B cell populations is essential in avoidance of autoimmune disease and immune regulation of some B cell malignancies. The role of CD4+ T cells in B cell apoptosis is evident from the high incidence of B cell tumors and autoimmunity in patients with T cell diseases such as the acquired immune deficiency syndrome (AIDS). We have previously demonstrated that in Epstein-Barr Virus (EBV) negative Burkitt's lymphoma (BL), a tumor derived from proliferating centroblasts of the germinal center, the malignant lymphocytes can be induced to express Fas (CD95) by ligation of CD40 at the B cell surface. Upon CD40 engagement, BL cells are sensitized to T-cell derived death signals provided by Fas ligand (FasL, CD95L). HBL-3 is a cell line derived from an AIDS-related BL in which the tumor IgM binds the human erythrocyte "i" antigen. To determine whether Fas-mediated apoptosis of BL cells is reduced in the context of antigen to which the tumor IgM binds, we stimulated HBL-3 cells with CD40 ligand (CD40L, CD154) in the presence and absence of human erythrocytes expressing the "i" antigen, and measured Fas-mediated apoptosis upon exposure to an agonistic anti-Fas antibody. We observed that HBL-3 cells were sensitized to Fas-mediated death by exposure to CD40L. When i+ RBCs were present, Fas-mediated apoptosis in HBL-3 cells was reduced by greater than 30%. In contrast, there was no reduction in Fas-mediated apoptosis in the presence of i- (I+) RBCs. These findings demonstrate that Fas-mediated deletion of BL cells is inhibited upon surface IgM engagement by antigen for which the malignant clone has affinity.

FIGURE 1

Sensitivity of HBL-3 cells to Fas-mediated cytolysis is enhanced by exposure to CD40L. HBL-3 cells were exposed to media, or to the irradiated CD40L− (clone B2.7) or CD40L+ T cell mutants (clone D1.1), and after 48 h of coculture the viable B cells were purified by density centrifugation prior to exposure to an agonistic anti-Fas antibody (clone CH11) at one of three doses. Inhibition of HBL-3 proliferation in response to Fas ligation was determined by reduction of the fluorometric growth indicator alamarBlue.

FIGURE 2

Fas-mediated apoptosis in HBL-3 cells is inhibited in the presence of i+ RBCs but not i− RBCs. HBL-3 cells were exposed to media alone (histograms A,B), or stimulated by coculture with irradiated CD40L+ T cells in the absence (C,D) or presence of human i+ (E) or i− (I+) (F) RBCs at 1 × 10⁴ RBCs/ml. After 48 h, the viable HBL-3 cells were isolated by Ficoll-Hypaque centrifugation, re-aliquoted at 0.5 × 10⁶ cells/ml; and then exposed to media alone (A,C), to an agonistic anti-Fas antibody (B, D–F), or as a control, to an isotype-matched antibody to CD23 (not shown) in the presence or absence of RBCs at 1 × 10⁴ RBCs/ml. After an additional 18 h, the cells were analyzed for DNA content by immunofluorescence flow cytometry. The percent of cells with subdiploid (apoptotic) DNA is indicated for each sample.

FIGURE 3

Chromatin fragmentation is reduced in CD40-stimulated HBL-3 cells exposed to i+ RBCs prior to Fas engagement. HBL-3 cells were exposed to media (A), or to irradiated CD40L+ T cells in the absence (B) or presence of either i−/I+ (C) or i+ RBCs (D). After 48 h, the cells were pulsed with ³H-thymidine for 16 h, washed, and exposed in triplicate to an agonistic anti-Fas antibody or a control antibody, at 200 ng/ml, in the presence or absence of i+ or i− RBCs, as indicated. After an additional 24 h, the cells were harvested and assessed for intact chromatin by the JAM assay. Each panel represents the results for cells of one initial culture circumstance, as indicated. Within each panel, the bars demonstrate the effects of reagents added at the 48 h time point.

FIGURE 4

Model for inhibition of Fas-mediated apoptosis in HBL-3 cells by RBC antigen. In the context of a B lymphocyte binding antigen through IgM at the surface, as in an HBL-3 cell binding to “i” at the RBC surface, an activated CD4+ T cell recognizing the same antigen would provide “help” to that B cell by CD40L, expressed at the T cell surface. Upon CD40 engagement, the germinal center or malignant BL cell is induced to synthesize DNA, differentiate, and express activation antigens including Fas. The activated, Fas-expressing B cell is primed for apoptosis via FasL, which is also expressed at the surface of activated CD4+ T cells. When the Fas receptor is engaged by FasL, apoptosis is inhibited by cross-linking of the antigen receptor (IgM). In HBL-3 lymphoma cells, in which IgM binds “i” Fas-mediated apoptosis of the tumor cells is reduced in the presence of i+ RBCs, but not in the presence of I+ (i−) RBCs.