Relationship between immunoglobulin production and immortalization by Epstein Barr virus

After infection with Epstein Barr virus (EBV), human B lymphocytes actively secrete immunoglobulin (Ig) and are immortalized to become long-term cell lines. In these studies, we investigated the relationship between these virally induced processes utilizing limiting dilution culture techniques, and asked whether all B cells stimulated by EBV to secrete Ig are also immortalized. The activation of B cells by EBV resulting in Ig production and immortalization involved a single precursor cell, required live viral particles, and was independent of immunity to EBV by the lymphocyte donor. However, the precursor frequency of B cells activated to secrete Ig (mean 4.7%) was higher than the precursor frequency of B cells activated to long-term in vitro growth (mean 2.1%). When examined at a single cell level, it appeared that although the vast majority of the immortalized B cells also secrete Ig, only approximately 50% of the B cell precursors induced by EBV to secrete Ig go on to form long-term cell lines. In addition, although immortalized B cell clones producing all major classes of Ig were detected, IgM-committed precursors were more likely to become immortal than were precursors committed to IgG or IgA production. In contrast to these findings in B cells freshly infected with EBV, Ig production was almost always associated with evidence of long-term growth when B cells from previously established EBV-induced B cell lines were tested in identical limiting dilution cultures. Thus, after infection with EBV, human B cells can either become transiently activated to proliferate and to secrete Ig, or become transformed into long-term cell lines most of which produce Ig.     

Heterogeneity of EBV-transformable human B lymphocyte populations

Although most human B cells express receptors for Epstein Barr virus (EBV), few (usually less than 1%) are readily transformed into B lymphoblastoid cell lines after exposure to EBV. Transformable cells previously have been found to be mostly resting B lymphocytes. We recently developed a limiting dilution culture system which permits the growth of EBV-transformed B lymphocytes with high efficiency. Because in this system up to over 30% of peripheral blood- or tonsil-derived B cells respond to EBV, we re-examined the properties of EBV-transformable cells. Frequencies of transformable lymphocytes were determined by Poisson analysis. EBV-susceptible B cells committed to IgM, IgG, or IgA secretion were found to occur in the range of 3 to 27, 0.1 to 6, and 0.1 to 5 per 100 B cells, respectively. Under our culture conditions, a major proportion of the IgM-committed cells derived from large lymphocytes which appeared to have entered the cell cycle. This population contains most of the EBV-responsive cells detected and, therefore, most of the additional cells responding in our culture system. In contrast, precursors of IgG- or IgA-producing lymphoblast lines were small cells with DNA contents typical for the G0/G1 phases of the cell cycle. Anti-immunoglobulin antibodies were used in panning experiments to separate B cell subpopulations which expressed different immunoglobulin isotypes on their surface. In limiting dilution cultures of these purified B lymphocytes subsets, it was found that virtually all precursors of IgM-producing cell lines expressed surface IgM (sIgM) before their infection and transformation by EBV. The "cloning efficiency" of positively selected, large sIgM+ cells approached 100%. In contrast, sIgG or sIgA were found only on cells committed to the production of IgG or IgA, respectively. The expression of sIgD was examined by using sequential panning procedures. Virtually all IgM-committed lymphocytes and a subset of cells committed to IgA secretion were found among the sIgD+ cells. The majority of cells committed to IgA production and all IgG-committed cells were found in the sIgD- B cell population. Our findings indicate that the EBV-susceptible B cell subset of normal lymphocytes is heterogeneous with respect to cell size, cell cycle, sIg determinants, and efficiency of transformation. On the basis of our findings and previous reports, we propose a model in which transformability is a B cell-inherent property. Factors unrelated to the virus but present in our culture system appear responsible for the enhanced vulnerability to viral transformation in some cells which entered into the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epstein-Barr virus induces an oxidative stress during the early stages of infection in B lymphocytes, epithelial, and lymphoblastoid cell lines

The study investigates the direct effect of Epstein-Barr virus infection on the oxidative profile of in vitro cultivated human cells. For this purpose, a panel of human EBV target cells presenting heterogeneity in their cellular and culture types (epithelial cells or lymphocytes; primary culture or continuous cell culture) was selected. These cells are purified human B lymphocytes, DG75, 293, and HepG2 cell lines. The oxidative stress was evaluated during the early stages of infection (2, 12, and 24 h) by measuring malondialdehyde, the end product of the lipid peroxidation, as well as the activities of two antioxidant enzymes: catalase and superoxide dismutase. The obtained results were compared with those of the untreated cells and the K562 cell line which has no interaction with EBV. The incubation of the different target cells with EBV induced an oxidative stress in the purified B lymphocytes, DG75, and 293, but not in HepG2 and K562. This oxidative stress was highlighted by an increase in MDA level (P < 0.05), which began 2 h after the addition of the virus and persisted after 12 and 24 h. Simultaneously, a decrease in catalase and superoxide dismutase activities was observed (P < 0.05), suggesting an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species (ROS). The efficiency of EBV infection, assessed by viral DNA PCR amplification, was confirmed in 293 and DG75 but not in HepG2, which was in total concordance with their oxidative profiles. In conclusion, the EBV infection of B and epithelial cells leads to the establishment of an oxidative stress which can play a key role during the viral transformation.     

An Epstein-Barr virus that expresses only the first 231 LMP1 amino acids efficiently initiates primary B-lymphocyte growth transformation

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.     

Factor(s) required by EBV transformed lymphocytes to grow under limiting dilution conditions

IL-6 was demonstrated to promote growth of EBV transformed lymphocytes. However IL-6 was ineffective at promoting growth of EBV transformed lymphocytes cultured at the single cell level under limiting dilution conditions. On the contrary, HECS, which is known to contain IL-6, supported very efficiently the growth of 1–2 EBV transformed cells. When IL-6 was removed from HECS, by using specific antibodies, no reduction in HECS activity was observed, indicating that probably more than one growth factor is required to support the growth of EBV transformed cells cultured at very low cell numbers in the absence of feeder cells.Abbreviations EBV Epstein Barr virus - FCS Foetal calf serum - HECS Human endothelial culture supernatant - IL-6 Interleukin 6 - PBL Peripheral blood lymphocytes - PBS Phosphate buffered saline     

A growth factor different from IL-6 promotes the growth of Epstein-Barr virus-transformed cells at low cell density

Interleukin-6 (IL-6) was recently described to support the growth of Epstein-Barr (EBV) transformed lymphocytes. However no effect was reported on cloning of EBV transformed lymphocytes under limiting dilution conditions. In this paper we demonstrate that the Supernatant of Human Endothelial Cells (HECS) contains a factor(s) different from IL-6 which induces high cloning efficiency of EBV transformed cell lines, cultured under limiting dilution conditions. Furthermore HECS is superior to IL-6 in all the passages of EBV lymphocytes transformation.     

Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells

We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation.     

Primary CD4+ T-cell responses provide both helper and cytotoxic functions during Epstein-Barr virus infection and transformation of fetal cord blood B cells

Most humans carry Epstein-Barr virus (EBV) in circulating memory B cells as a latent infection that is controlled by an immune response. When infected by EBV, B lymphocytes in fetal cord blood are readily transformed to lymphoblastoid cell lines (LCL). It is frequently assumed that this high efficiency of transformation is due to the absence of a primary immune response. However, cord blood lymphocytes stimulated with autologous LCL yield CD4+ T cells that can completely inhibit the growth of LCL by a major histocompatibility complex-restricted cytotoxic mechanism mediated by granulysin and granzyme B. Because EBV-transformed B cells maintain the phenotype of antigen-activated B-cell blasts, they can potentially receive inhibitory or helper functions from CD4+ T cells. To assess these functions, the effect of EBV-specific CD4+ T cells on the efficiency of virus transformation of autologous B cells was assayed. Paradoxically, although the cytotoxic CD4+ T-cell lines reduced EBV B-cell transformation at a high effector/target ratio of 10:1, they caused a twofold increase in B-cell transformation at the lower effector/target ratio of 1:1. Th1-polarized CD4+ T cells were more effective at inhibiting B-cell transformation, but Th2-polarized cell lines had reduced cytotoxic activity, were unable to inhibit LCL growth, and caused a 10-fold increase in transformation efficiency. Tonsil lymphoid follicles lacked NK cells and CD8+ T cells but contained CD4+ T cells. We propose that CD4+ T cells provide helper or cytotoxic functions to EBV-transformed B cells and that the balance of these functions within tonsil compartments is critical in establishing asymptomatic primary EBV infection and maintaining a stable lifelong latent infection.     

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.     

Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells.

In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.     

The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues.

Recombinant Epstein-Barr viruses (EBVs) were made with mutated latent membrane protein 1 (LMP1) genes that express only the LMP1 amino-terminal cytoplasmic and six transmembrane domains (MS187) or these domains and the first 44 amino acids of the 200-residue LMP1 carboxy-terminal domain (MS231). After infection of primary B lymphocytes with virus stocks having small numbers of recombinant virus and large numbers of P3HR-1 EBV which is transformation defective but wild type (WT) for LMP1, all lymphoblastoid cell lines (LCLs) that had MS187 or MS231 LMP1 also had WT LMP1 provided by the coinfecting P3HR-1 EBV. Lytic virus infection was induced in these coinfected LCLs, and primary B lymphocytes were infected. In over 200 second-generation LCLs, MS187 LMP1 was never present without WT LMP1. Screening of over 600 LCLs infected with virus from MS231 recombinant virus-infected LCLs identified two LCLs which were infected with an MS231 recombinant without WT LMP1. The MS231 recombinant virus could growth transform primary B lymphocytes when cells were grown on fibroblast feeders. Even after 6 months on fibroblast feeder layers, cells transformed by the MS231 recombinant virus died when transferred to medium without fibroblast feeder cells. These data indicate that the LMP1 carboxy terminus is essential for WT growth-transforming activity. The first 44 amino acids of the carboxy-terminal cytoplasmic domain probably include an essential effector of cell growth transformation, while a deletion of the rest of LMP1 can be complemented by growth on fibroblast feeder layers. LMP1 residues 232 to 386 therefore provide a growth factor-like effect for the transformation of B lymphocytes. This effect may be indicative of the broader role of LMP1 in cell growth transformation.     

High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.     

Epstein-Barr virus recombinants from BC-1 and BC-2 can immortalize human primary B lymphocytes with different levels of efficiency and in the absence of coinfection by Kaposi's sarcoma-associated herpesvirus

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.     

Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors.

Since Epstein-Barr virus (EBV) infection of Burkitt's lymphoma (BL) cells in vitro reproduces many of the activation effects of EBV infection of primary B lymphocytes, mRNAs induced in BL cells have been cloned and identified by subtractive hybridization. Nine genes encode RNAs which are 4- to > 100-fold more abundant after EBV infection. Two of these, the genes for CD21 and vimentin, were previously known to be induced by EBV infection. Five others, the genes for cathepsin H, annexin VI (p68), serglycin proteoglycan core protein, CD44, and the myristylated alanine-rich protein kinase C substrate (MARCKS), are genes which were not previously known to be induced by EBV infection. Two novel genes, EBV-induced genes 1 and 2 (EBI 1 and EBI 2, respectively) can be predicted from their cDNA sequences to encode G protein-coupled peptide receptors. EBI 1 is expressed exclusively in B- and T-lymphocyte cell lines and in lymphoid tissues and is highly homologous to the interleukin 8 receptors. EBI 2 is most closely related to the thrombin receptor. EBI 2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. EBI 2 is also expressed at lower levels in a promyelocytic and a histiocytic cell line and in pulmonary tissue. These predicted G protein-coupled peptide receptors are more likely to be mediators of EBV effects on B lymphocytes or of normal lymphocyte functions than are genes previously known to be up-regulated by EBV infection.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

Interferon production potentials of various human lymphoblastoid cell lines

A number of human lymphoblastoid cells were examined concerning their ability to produce spontaneously liberated and virus-induced interferon (IFN). It was found that, in addition to B cells, various T and nonT-nonB lymphoblastoid cells responded well to Sendai virus infection to form IFN, the characterization of which has been recently reported (20). One B lymphoblastoid cell line from an infectious mononucleosis (IM) patient produced a large amount of IFN-alpha and might become an alternative source of IFN production. Among 68 cell lines examined, 35 cell lines liberated 10 U/ml or more of IFN spontaneously in culture fluid. The presence of Epstein-Barr virus (EBV) genome or its activation appears to have no correlation with the spontaneous liberation of IFN. Spontaneously produced IFN from three cell lines was characterized as IFN-alpha. Comparatively higher amounts of IFN were produced in cells from IM patients than those from Burkitt's lymphoma cases or healthy adults. Spontaneously produced IFN was detected more easily in cells transformed by EBV alone than in those transformed by EBV and a tumor promoter, TPA.     

Epstein Barr virus binding induces internalization of the C3d receptor: a novel immunotoxin delivery system

Epstein Barr virus (EBV) infection of human B lymphocytes is initiated by selective binding of the virus to the C3d receptor (EBV/C3d receptor) on the cell surface and results in polyclonal proliferation of infected cells. In these studies we examined the fate of the EBV/C3d receptor during viral infection by using an immunotoxin made from a monoclonal antibody (HB5) reactive with the receptor and the potent toxin, gelonin. Binding of the HB5-gelonin conjugate to the EBV/C3d receptor before EBV infection (at concentrations as low as 10(-11) M) significantly inhibited the subsequent polyclonal proliferation of virus-infected B lymphocytes. HB5 antibody and gelonin alone did not inhibit proliferation. Because internalization of gelonin-antibody conjugates is required to cause cytotoxicity, these results indicate that infection of B lymphocytes with EBV selectively induced endocytosis of the EBV/C3d receptor with concomitant internalization of the immunotoxin. Proliferation of B lymphocytes that were activated by prior infection with EBV, or activated by cross-linking of their surface immunoglobulin molecules, was not inhibited by the antibody-toxin conjugate even at concentrations as high as 10(-7) M. Also, the growth of B lymphoblastoid cell lines cultured in the presence or absence of infectious EBV was not inhibited by HB5-gelonin. Thus, our results suggest that the EBV/C3d receptor is internalized only during the infection of normal B lymphocytes by EBV, with co-internalization of immunotoxin, and indicate that internalization of the EBV/C3d receptor-immunotoxin complex does not occur simply as a consequence of activation and proliferation of B lymphocytes. The use of a ligand to induce endocytosis of its receptor offers a new strategy for the selective delivery of immunotoxins to cells and may be more generally applicable.