Dithiocarbamates and viral IL-10 collaborate in the immortalization and evasion of immune response in EBV-infected human B lymphocytes

Abstract

Epstein-Barr virus (EBV) is implicated in the development of a number of human malignancies including several subtypes of non-Hodgkin lymphoma (NHL) [G. Pallesen, S.J. Hamilton-Dutoit, X. Zhou, The association of Epstein-Barr virus (EBV) with T cell lymphoproliferations and Hodgkin's disease: two new developments in the EBV Field, Adv. Cancer Res. 62 (1993) 179-239]. Lymphoproliferative disease and NHL occurring in severely immunosuppressed individuals almost always involve EBV and have been extensively studied and modeled in vitro. EBV has also been causally associated with some cases of NHL occurring in otherwise immunocompetent individuals. However, a direct role for EBV in the pathogenesis of neoplasms developing in the presence of an otherwise competent immune system has not been established. We investigated potential interactions between dithiocarbamates (DTC), an important class of thiono-sulfur compounds, and EBV leading to immortalization of human B lymphocytes and evasion of cell-mediated immune response in culture. Primary lymphocyte cultures employing wild-type and recombinant EBV mutants were used to assess the respective roles of DTC and viral genes in lymphocyte transformation and survival. Pretreatment of EBV-infected human B lymphocytes with DTC directly enhanced transformation in the absence of T cells (5 nM) and independently increased survival of transformed cells in the presence of competent autologous T cells (10 nM). Both DTC-induced transformation and immortalization of EBV-infected B lymphocytes were dependent on the expression of viral IL-10. These results provide a biological basis for studying collaborations between chemical and virus that alter lymphocyte biology, and provide a rationale for further molecular epidemiology studies to better understand the potential influence of these interactions on the development of NHL and perhaps other viral-associated malignancies.

Figure 1

Transformation and immortalization of EBV-infected B lymphocytes treated with DMDTC. Human Ficoll-purified PBMC (A) or isolated B lymphocytes (B) were infected with B95-8 EBV supernatants, treated with DMDTC or buffer, cultured for 4 weeks and colonies enumerated. Cells from resulting colonies were then subcultured under limiting dilution conditions for another 4 weeks and cloning efficiency expressed as the percent of immortalized colonies (%) for PBMC (C) or purified B lymphocytes (D). Results represent the average number of colonies per treatment group from triplicate cultures +/− SEM. Asterisks indicate significant difference from buffer-treated cultures using Student t-test (p ≤ 0.05). Data is representative of 1 out of 6 independent experiments performed using PBMC, and 1 out of 8 independent experiments using isolated B lymphocytes. Cells for each experiment were obtained from different donors (n=14).

Figure 2

Kinetics of gene expression in EBV-infected primary B lymphocytes. B cells were harvested at intervals as indicated (hours post-infection) and analyzed by flow cytometry or RT-PCR techniques. (A) Flow cytometric analysis of 2 × 10⁶ EBV-infected B cells treated with PBS (A1) or 100 nM DMDTC (A2) using antibodies against: EBNA2, BZLF-1 and VCA (viral capsid antigen p120). Values represent Mean + S.E.M. (n=4). (B) RT-PCR analysis of RNA extracts from 1 × 10⁶ EBV-infected B cells treated with PBS (B1) or 100 nM DMDTC (B2). Representative agarose gel (n=6) of PCR products for EBNA2, LMP1 and BCRF1 is shown. Primers for the specific transcripts and PCR conditions are indicated in Materials and Methods.

Figure 3

The role of vIL-10 in DMDTC enhanced B cell transformation. (A) The number of vIL-10 secreting cells was determined at 24, 48 and 72 hr in EBV- infected or uninfected B lymphocytes treated with PBS or 100 nM DMDTC. Asterisks indicate significant difference between DMDTC-treated and untreated EBV-infected cultures using Student t-test (p < 0.05). (B) Transformation of EBV-infected and/or DMDTC-pretreated B lymphocytes was evaluated in the presence and absence of anti-vIL10 antibodies. Colonies were scored after 4 weeks in culture. Asterisks indicate significant difference with and without addition of neutralizing anti-IL-10 antibodies using Student t-test (p < 0.05). (C) Effect of DMDTC treatment on purified B lymphocytes co-cultivated with irradiated lymphoblastoid cell lines containing BCRF1-deleted or WT EBV recombinant strains. Colonies were scored after 6 weeks in culture. Error bars represent +/− SEM (n=3). Asterisks indicate significant difference from buffer-treated cultures using Student t-test (p < 0.05).

Figure 4

Influence of DMDTC pretreatment on EBV-infected B lymphocyte immortalization in the presence of T cells. EBV-infected purified B cells were cultured for one week at a density of 500 cells per well in the presence of PBS or 100 nM DMDTC. Cells were washed, varying numbers of autologous purified T cells added, cultured for an additional 4–6 weeks and scored for colonies. Symbols indicate the number of transformants per well from duplicate experiments. The ratio of B:T cells: 500:0 (1), 500:500 (1:1), 500:1000 (1:2), 500:2000 (1:4), 500:4000 (1:8).

Figure 5

Exogenous IL-2 partially restores T lymphocyte response against DMDTC-treated/EBV-infected B lymphocytes. Ficoll-purified PBMC were infected with EBV, treated with 100 nM DMDTC, cultured for 14 days and then supplemented with PBS or 200 U rhIL-2. (A) vIL-10-producing B cells. Asterisks indicate significant difference compared with DMDTC pretreated cells without IL-2 supplementation using Student t test (p < 0.01). Circle indicates significant difference compared with EBV infected cultures without DMDTC pretreatment or IL-2 supplementation using Student t test (p < 0.01); and (B) IL-2-producing T cells were enumerated by ELISPOT at 15, 17, 21 and 28 days post-infection. Data are expressed as the mean +/− SEM for secreting cells (n=4). Asterisks indicate significant difference compared with identical cultures without IL-2 supplementation using Student t test (p < 0.01).