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The Journal of Immunology, 2003, 170: 3187-3194.
Copyright © 2003 by The American Association of Immunologists

CD4+ T Cell-Induced Differentiation of EBV-Transformed Lymphoblastoid Cells Is Associated with Diminished Recognition by EBV-Specific CD8+ Cytotoxic T Cells 1

Aaruni Khanolkar*, Zheng Fu*, L. Joey Underwood*, Kristy L. Bondurant*, Rosemary Rochford{dagger} and Martin J. Cannon2,*

* Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and {dagger} Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4+ T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4+ T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8+ CTL. In contrast, CD4+ T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8+ CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8+ CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8+ T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4+ T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8+ CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4+ T cell contact was prevented, indicated a major role for CD4+ T cell cytokines in the differentiation of LCL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epstein-Barr virus is a ubiquitous human herpesvirus that persists throughout life as a latent infection of resting memory B cells (1). EBV-specific CD8+ CTL play a central role in the maintenance of an asymptomatic carrier state through their ability to recognize viral latent Ags associated with B cell transformation (2, 3). EBV-specific CD8+ CTL responses are inhibited in cyclosporine A (CsA)3-treated transplant patients (4, 5), who are accordingly at high risk of developing EBV-associated posttransplant lymphoproliferative disorders (PTLD) and lymphoma (6, 7, 8). EBV-specific CTL can inhibit the development of EBV-driven human B cell tumors in chimeric SCID/hu mice (9, 10, 11), and EBV-specific CTL immunotherapy has been used for the prevention or treatment of PTLD (12, 13).

In contrast with the acknowledged importance of virus-specific CD8+ T cells in the control of latent EBV infection in healthy individuals, relatively little is known of the CD4+ T cell response to EBV. Virus-specific CD4+ CTL that lyse in vitro-transformed lymphoblastoid cell lines (LCL) have been described (11, 14, 15), and CD4+ T cells can induce Fas-mediated apoptosis in LCL (16). The observation that EBV-specific CD4+ T cells may also use the perforin/granzyme pathway for lysis of LCL strengthens the view that the CD4+ T cell response may be a significant component of virus-specific immunosurveillance (14). This proposal is supported by the recent identification of CD4+ CTL that are specific for EBV-encoded nuclear Ag (EBNA)1, an Ag that is essential for episomal maintenance of EBV in transformed B cells (17).

Although CD4+ CTL may fulfil a protective role against disease, noncytotoxic CD4+ helper T cells may contribute to EBV pathogenesis, through their ability to promote B cell activation, proliferation, and differentiation. Experiments in SCID/hu mice engrafted with human PBL show that the presence of T cells is required for the development of EBV-driven lymphoproliferative disease and tumors (18, 19), suggesting that T cell help for B cell activation is a necessary and critical step for viral reactivation from latency. This idea is reinforced by recent studies (20) showing that CD4+ T cell-mediated activation of latently infected resting B cells induces expression of BZLF1, an immediate-early transactivator of viral lytic cycle replication, followed by de novo infection and rapid outgrowth of EBV-transformed B cells. The observation that T cell-mediated B cell activation induces BZLF1 mRNA expression rather than transformation-associated transcripts such as EBNA2 supports the two-step model of EBV transformation in which viral replication is followed by secondary infection and transformation of bystander B cells (21, 22). Although these findings point to a critical role for CD4+ T cell-mediated B cell activation in viral pathogenesis, the ability of CD4+ T cells to promote B cell differentiation may also influence the viral life cycle. Studies in SCID/hu mice have clearly shown that tumorigenesis is accompanied by plasmacytoid differentiation and production of high levels of human Ig (23, 24, 25). Plasmacytoid differentiation of EBV-driven B cell tumors in SCID mice also results in down-regulation of viral latent gene expression (25). In this report, we show that CD4+ T cells induce differentiation of EBV-transformed B cells from a lymphoblastoid phenotype to an intermediate phenotype with some plasmacytoid features. Furthermore, we show that differentiation is accompanied by resistance of LCL to lysis by EBV-specific CD8+ CTL. These observations suggest that CD4+ T cell interactions with EBV-transformed B cells may have a significant influence on the differentiation state of infected B cells and on patterns of viral gene expression and latency.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Lymphoblastoid cell lines

EBV-transformed LCL were established by infection of PBL with the B95-8 strain of EBV in the presence of 1 µg/ml CsA, and maintained in RPMI 1640 supplemented with 10% FCS, 3 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-ME (RPMI/10), as described (26), except that in some cases the medium was supplemented with 10% human AB (HuAB) serum (Gemini Bio-Products, Calabasas, CA) (RPMI/10Hu) rather than 10% FCS.

EBV-specific CD4+ T cell lines

CD4+ T cell lines (and matching LCL) were derived from two healthy adult individuals (donors 1 and 2) and one heart transplant patient (donor 3), who was receiving CsA (100 mg, oral, twice daily). The blood level of CsA at the time of blood drawing was 167 ng/ml. CD4+ T cells were purified from PBL by positive selection with anti-CD4-coupled Dynabeads (Dynal, Lake Success, NY). PBL (3 x 107) were incubated with 2 x 107 anti-CD4 Dynabeads in 2 ml of PBS plus 2% HuAB serum at 4°C for 1 h. Dynabead-rosetted CD4+ T cells were magnetically separated, washed five times with cold PBS plus 2% HuAB serum, and subsequently recovered by incubation with 60 µl of CD4-Detachabead reagent (Dynal) in 150 µl of RPMI/10Hu. Purified CD4+ T cells (3 x 105/ml) were cocultured with autologous, irradiated (7500 cGy) LCL (2 x 105/ml) in RPMI/10Hu for 9–11 days, after which period the T cells were restimulated with irradiated LCL. Recombinant human IL-2 (50 U/ml; provided by the Biological Response Modifiers Program, National Cancer Institute, Bethesda, MD) was added to the cultures at this time. EBV-specific CD4+ T cells were subsequently maintained by restimulation with irradiated LCL every 14–21 days, with interim 50–70% changes of fresh medium plus IL-2 every 2–4 days. LCL used for CD4+ T cell stimulation were maintained in RPMI/10Hu, to avoid stimulation of CD4+ T cells specific for bovine serum proteins.

The specificity of the CD4+ T cell lines was confirmed in proliferation assays against LCL and EBV-uninfected normal, activated B lymphoblasts (14, 20). Briefly, CD4+ T cells proliferated strongly in response to stimulation with autologous LCL maintained in medium supplemented with FCS or HuAB serum, but failed to recognize HLA class II-mismatched allogenic LCL. In addition, responses against autologous LCL were markedly inhibited (50–80% reduction in stimulation indices) in the presence of blocking anti-HLA class II mAb. CD4+ T cells failed to mount proliferative or cytotoxic responses against autologous normal (i.e., non-EBV-transformed) B lymphoblasts activated with anti-CD40 mAb and IL-4 in the presence of CDw32-transfected L cells (27), as previously reported (20). CDw32 encodes an Fc{gamma}R, which cross-links the anti-CD40 mAb, thereby delivering an activation signal to the B cells. Flow cytometric analysis indicated that the normal B lymphoblasts were phenotypically comparable with LCL. Like LCL, normal B lymphoblasts expressed high levels of CD23 and adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3). Finally, like LCL, the anti-CD40-activated B lymphoblasts expressed high levels of CD86 and HLA-DR, which are critical for costimulatory and Ag-presenting function. Collectively, this analysis shows that the normal B lymphoblasts are fully activated and phenotypically comparable to LCL.

All three CD4+ T cell lines were cytotoxic against LCL, predominantly through the perforin/granzyme pathway (14). Flow cytometric analysis of intracellular IFN-{gamma} and IL-4 expression revealed a distribution of Th1 and Th2 phenotypes, and an intermediate IFN-{gamma}+IL-4+ double-positive subset (14, 20).

EBV-specific CD8+ CTL

EBV-specific CD8+ T cell lines were derived from each donor, as described (26). PBL (2 x 106/ml) were stimulated with autologous, irradiated (7500 cGy) LCL (5 x 104/ml) in RPMI/10Hu. After 9–11 days, T cells (2 x 105/ml) were restimulated with irradiated LCL (2 x 105/ml) in RPMI/10Hu plus 50 U/ml rIL-2. T cell lines were subsequently maintained by restimulation with irradiated LCL every 14–21 days, with interim 50–70% changes of fresh medium plus IL-2 (100 U/ml) every 2–4 days. After three to four restimulation cycles, T cell lines were typically >90% CD8+ by flow cytometric analysis. CD8+ T cells were further isolated (to >98% purity) by positive selection with anti-CD8 Dynabeads (Dynal).

Alloreactive CD8+ CTL

Responder PBL (2 x 106/ml) from an allogenic, HLA class I-mismatched individual were stimulated with irradiated PBL (2 x 105/ml) from donor 1. After 9–11 days, alloreactive T cells (2 x 105/ml) were restimulated with irradiated PBL (2 x 105/ml) from donor 1 in RPMI/10Hu plus 50 U/ml IL-2. T cell lines were subsequently maintained by restimulation with irradiated donor 1 PBL every 14–21 days, with interim 50–70% changes of fresh medium plus IL-2 (100 U/ml) every 2–4 days. After the third restimulation, alloreactive CD8+ T cells were isolated (to >98% purity) by positive selection with anti-CD8 Dynabeads.

Peptide-specific CD8+ CTL

Monocyte-derived dendritic cells (DC) from donor 1 were generated in AIM-V medium (Invitrogen, Grand Island, NY) supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml), as described (28). After 5–6 days, DC maturation was induced by addition of TNF-{alpha} (1000 U/ml), IL-1{beta} (500 U/ml), and PGE2 (1 µM) for a further 2 days. Mature DC were pulsed for 1–2 h at 37°C with 50 µg/ml peptide 170–178 (SLGRWPWQV) from hepsin, a tumor Ag overexpressed in ovarian cancer (29) and prostate cancer (30, 31). Hepsin 170–178 binds HLA A*0201 and is recognized by HLA A*0201-restricted CD8+ CTL. Peptide-loaded DC were washed twice before culture with PBL from donor 1 at a responder:stimulator ratio of 30:1. After 7 days, T cells were collected and restimulated with peptide-pulsed DC. For the second and third DC stimulations, the medium was supplemented with 50–100 U/ml IL-2, and the culture period was extended to 14 days. After the third cycle, CD8+ T cells were purified by positive selection with anti-CD8 Dynabeads. Subsequent passages of CD8+ T cell lines used peptide-loaded autologous PBL as APC.

CD4+ T cell and LCL cocultures

LCL (2 x 106/well) were cocultured with irradiated (3300–3800 cGy) EBV-specific CD4+ T cells (2 x 106/well) in a final volume of 4 ml of RPMI/10 in 12-well Costar tissue culture plates (Corning, Corning, NY). In some experiments, a blocking mAb specific for CD40 ligand (CD40L) was added at 50 µg/ml.

For transwell cocultures, 12-well plates were coated with anti-CD3 mAb (40 µg/ml OKT3 in PBS, 0.75 ml/well, overnight at 4°C). After aspiration of the OKT3 mAb, CD4+ T cells were added (2 x 106/well in 1.5 ml of RPMI/10). Polycarbonate membrane transwells with a 0.4-µm pore size (Corning) were then placed in the wells, and LCL were added to each transwell (5 x 105 cells/transwell in 0.5 ml of RPMI/10). Both conventional cocultures and transwell cocultures were incubated for 4 days at 37°C in 7% CO2.

Phenotypic analysis of LCL

Expression of LCL surface markers was assessed by flow cytometry, as described (25). Residual contamination of cocultured LCL by any surviving CD4+ T cells was tested by staining for CD3-expressing cells. Abs used were FITC-anti-CD23, PE-anti-CD38, PE-anti-CD54, FITC-anti-CD3 (all from Caltag, Burlingame, CA), and PE-anti-CD58 (BD Biosciences, San Jose, CA). The anti-HLA class I (W6/32) mAb was prepared from a hybridoma obtained from the American Type Culture Collection (Manassas, VA). FITC-conjugated goat anti-mouse IgG was from Sigma-Aldrich (St. Louis, MO). Flow cytometric analysis was conducted with a FACScan (BD Biosciences), using LYSIS II software (BD Biosciences) and WinMDI 2.8 software (kindly provided by J. Trotter (The Scripps Research Institute, La Jolla, CA)).

Lymphoproliferation assays

The proliferative capacity of LCL following 4-day coculture with CD4+ T cells was tested by transfer of LCL to 96-well plates (5 x 104 cells/well) and incubation for a further 2, 3, or 4 days in RPMI/10. Proliferation was determined by addition of [3H]thymidine (1 µCi/well) for the final 6 h of each microwell culture. All assays were performed in 6-fold replicate wells.

Cytotoxicity assays

EBV-specific CD8+ CTL activity against normal and cocultured LCL was measured in a standard 51Cr-release assay (26). LCL were labeled with 50 µCi of Na251CrO4 for 1 h and washed three times before incubation in 96-well round-bottom microtiter plates (1 x 104 targets/well) with CD8+ CTL at the indicated effector:target ratios for 6 h at 37°C in 7% CO2. Released 51Cr in the supernatants was measured with a Cobra Auto gamma counter (Packard, Meriden, CT).

RNase protection assays (RPA)

RNA was extracted from control LCL and LCL cocultured with CD4+ T cells by the method of Chomczynski and Sacchi (32). RNA from 106 cells was analyzed using a multiprobe RPA for expression of the EBV latent transcripts latent membrane protein (LMP)1, LMP2A, EBNA1, EBNA2, and EBNA6, and the lytic transcripts BZLF1, BRLF1, early Ag D, and gp350 (33). A riboprobe to measure the housekeeping gene L32 was included in each assay. The RPA was performed exactly as described (34). Protected probe species were resolved by denaturing PAGE and visualized by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differentiation of LCL following coculture with CD4+ T cells

The differentiation state of LCL was judged by the relative expression of CD23 and CD38 (25, 35). CD23 is a marker of mature, activated immunoblasts, the expression of which is up-regulated by viral EBNA2 and LMP1 (36, 37), and CD38 has utility as a marker for plasmacytoid differentiation. In vitro-transformed LCL display a phenotype that resembles proliferating, Ag-stimulated B cell immunoblasts, and are typically CD23highCD38low, whereas plasmacytoid differentiation of EBV-transformed LCL in vivo is accompanied by reduced CD23 expression and increased CD38 expression (25, 35). Following coculture with autologous, irradiated EBV-specific CD4+ T cells for 4 days, LCL from donor 1 showed a marked increase in CD38 expression, but little change in CD23 expression was observed (Fig. 1, A and C). In contrast, CD4+ T cells from donor 3 not only induced an increase in the proportion of CD38+ LCL, but also induced a substantial reduction in CD23 expression. In this instance, a CD23lowCD38high subset constituted the major population of LCL following coculture (Fig. 1, B and D). The increase in CD38high cells cannot be attributed to residual T cell contamination, because irradiation of the CD4+ T cells precluded their survival beyond the first 2 days of coculture. The absence of T cells after 4 days of coculture was confirmed by flow cytometric analysis.



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  		FIGURE 1. EBV-specific CD4+ T cells induce differentiation of EBV-transformed B cells. LCL from donors 1 and 3 were cocultured with autologous irradiated EBV-specific CD4+ T cells for 4 days and assayed for CD23 and CD38 expression by two-color flow cytometry. The phenotypes of control LCL are shown in A (donor 1) and B (donor 3). Cocultured LCL are shown in C (donor 1) and D (donor 3). Cocultures conducted in the presence of 50 µg/ml anti-CD40L blocking mAb are shown in E (donor 1) and F (donor 3).

 
The interaction of B cell CD40 with its ligand on activated T cells plays multiple roles in B cell activation, proliferation, and differentiation (38). To test whether CD40-CD40L interactions were involved in differentiation of LCL, we conducted coculture experiments in the presence of a blocking anti-CD40L mAb at a concentration known to inhibit T cell-mediated activation of resting B cells (20). We found that blockade of CD40 engagement had no effect on the ability of CD4+ T cells to induce differentiation of LCL (Fig. 1, E and F). CD4+ T cell-induced differentiation was accompanied by loss of proliferative capacity (Fig. 2). CD4+ T cell-mediated inhibition of LCL proliferation has also been described by other investigators (16, 39, 40), but growth inhibition has not previously been associated with phenotypic changes reflecting B cell differentiation. Up to a 10-fold increase in Ig secretion by cocultured LCL relative to control LCL was observed over 4 days. However, overall Ig production remained very low, with control LCL producing 180 ng/ml after 2 days and 550 ng/ml after 4 days, whereas cocultured LCL produced 2.2 µg/ml after 2 days and 5 µg/ml after 4 days.



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  		FIGURE 2. CD4+ T cell-induced differentiation of LCL results in reduced proliferative capacity. LCL from donor 1 were cocultured with autologous irradiated EBV-specific CD4+ T cells for 4 days. Control LCL ({blacktriangleup}) and cocultured LCL ({blacksquare}) were then transferred to microwells, and proliferation was recorded by [3H]thymidine uptake after a further 2, 3, and 4 days of culture. Each determination was from 6-fold replicate microwells. Results are presented as mean values from four independent experiments.

 
Cocultured LCL show reduced vulnerability to lysis by EBV-specific CD8+ CTL

LCL cocultured with CD4+ T cells for 4 days were tested for their susceptibility to lysis by autologous, EBV-specific CD8+ CTL. We found that in standard 6-h 51Cr-release assays, cocultured LCL from donors 1 and 3 showed a marked reduction in their sensitivity to lysis (Fig. 3). Essentially the same results were also seen from cocultures with CD4+ T cells and LCL from donor 2 (not shown). Incorporation of blocking anti-CD40L mAb during coculture failed to reverse the loss of sensitivity of LCL to EBV-specific CD8+ CTL (Fig. 3). In contrast, coculture with CD4+ T cells did not alter the sensitivity of LCL to lysis by CD8+ CTL specific for an exogenously loaded peptide Ag in assays in which both control LCL and cocultured LCL were pulsed with the target peptide (Fig. 4A). Furthermore, control LCL and cocultured LCL were equally sensitive to lysis by alloreactive CD8+ CTL (Fig. 4B). These observations suggest that resistance of CD4+ T cell-cocultured LCL to lysis by EBV-specific CD8+ CTL (Figs. 3 and 4C) is related to the Ag specificity of the CTL, and cannot be attributed to an intrinsic, differentiation-related insensitivity to CD8+ T cell-mediated lysis.



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  		FIGURE 3. CD4+ T cell-induced differentiation of LCL is associated with decreased sensitivity to lysis by EBV-specific CD8+ CTL. LCL from donors 1 (A) and 3 (B) were cocultured with irradiated EBV-specific CD4+ T cells for 4 days and tested for sensitivity to lysis by autologous EBV-specific CTL in a 6-h 51Cr-release assay. Targets were control LCL (•), cocultured LCL ({blacktriangleup}), and LCL cocultured with CD4+ T cells and 50 µg/ml anti-CD40L mAb ({blacksquare}).

 


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  		FIGURE 4. CD4+ T cell-induced differentiation of LCL does not alter sensitivity to lysis by alloreactive CD8+ CTL or CTL specific for an externally loaded peptide. LCL from donor 1 were cocultured with irradiated EBV-specific CD4+ T cells for 4 days and tested for susceptibility to lysis by CD8+ CTL specific for an exogenously loaded peptide, SLGRWPWQV (A), HLA class I-mismatched alloreactive CD8+ CTL (B), and EBV-specific autologous CD8+ CTL (C) in a 6-h 51Cr-release assay. Sensitivity to peptide-specific CD8+ CTL was tested against control LCL and cocultured LCL pulsed for 1 h with 50 µg/ml peptide and washed three times before incorporation in the assay. Targets were control LCL ({blacktriangleup}) and cocultured LCL ({blacksquare}).

 
Expression of HLA class I and adhesion molecules following coculture of LCL with CD4+ T cells

The levels of expression of HLA class I molecules and accessory adhesion molecules such as CD54 (ICAM-1) and CD58 (LFA-3) may influence the sensitivity of LCL to lysis by CD8+ CTL. Flow cytometric analysis of control and cocultured LCL revealed no changes in expression of HLA class I or CD54 (Fig. 5). However, expression of CD58 by LCL was reduced following coculture (Fig. 5C). The mean fluorescence intensity for CD58 expression by cocultured LCL was 55, compared with a mean fluorescence intensity of 98 for control LCL.



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  		FIGURE 5. Expression of adhesion molecules and HLA class I following coculture of LCL with CD4+ T cells. LCL from donor 1 were cocultured with autologous irradiated EBV-specific CD4+ T cells for 4 days. Expression of HLA class I (A), CD54 (B), and CD58 (C) by control LCL (light histograms) and cocultured LCL (bold histograms) was determined by flow cytometry.

 
Is EBV-specific CD8+ T cell cytotoxicity dependent on expression of CD58 by LCL?

Previous work has suggested that target cell CD58 adhesion with T cell CD2 plays an important role in the sensitivity of target cells to lysis by CTL (41, 42), including EBV-specific CD8+ CTL (43). As LCL coculture and differentiation are accompanied by modulation of CD58 expression, it is thus possible that the reduced vulnerability to EBV-specific CTL is at least partly attributable to loss of CD58. To address this question, we conducted cytotoxicity assays in which CD58-CD2 adhesion was blocked with anti-CD58 mAb. We found that anti-CD58 mAb strongly inhibited lysis of LCL by both EBV-specific CD8+ CTL (Fig. 6A) and alloreactive CD8+ CTL (Fig. 6B). These results contrasted with cytotoxicity assays against cocultured LCL, which were efficiently killed by alloreactive CD8+ CTL, but showed reduced sensitivity to lysis by EBV-specific CD8+ CTL (Fig. 4). As the control and cocultured LCL were killed with equal efficiency by alloreactive CTL, which require CD58 expression for efficient lysis of targets, we conclude that the differentiation-related reduction of CD58 expression is unlikely to be sufficient to account for the reduced sensitivity of cocultured LCL to lysis by EBV-specific CTL.



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  		FIGURE 6. Lysis of LCL by EBV-specific CD8+ CTL or alloreactive CD8+ CTL is blocked by anti-CD58 mAb. LCL were tested for sensitivity to lysis by EBV-specific CD8+ CTL (A) or alloreactive CD8+ CTL (B) in a 6-h 51Cr-release assay in the presence of 10 µg/ml anti-CD58 mAb ({blacksquare}) or 10 µg/ml isotype control Ab ({blacktriangleup}).

 
CD4+ T cell-induced differentiation of LCL is associated with reduced viral gene expression

We have previously shown that plasmacytoid differentiation of EBV-transformed B cells in the SCID mouse tumor model is associated with loss of viral latent gene expression (25). As viral latent gene products are targets for CD8+ CTL, we asked whether CD4+ T cell-induced differentiation of LCL in vitro might also be associated with changes in viral gene expression. Multiprobe RPA for viral mRNA expression by cocultured LCL revealed, to varying extents, reduced expression of LMP1, LMP2A, EBNA1, EBNA2, and EBNA6 (Fig. 7). In particular, expression of EBNA6, which is a target Ag for EBV-specific CTL from donor 1 (26), was almost totally lost by day 4 of coculture. These observations suggest that reduced viral latent gene expression following CD4+ T cell coculture of LCL may account for the reduced recognition and lysis of cocultured LCL by EBV-specific CD8+ CTL. This conclusion is strengthened by the observation that cocultured LCL retain their sensitivity to lysis by both alloreactive CTL and CTL specific for an exogenously loaded peptide Ag (Fig. 4).



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  		FIGURE 7. Modulation of viral gene expression following coculture of LCL with CD4+ T cells. Expression of viral mRNA by LCL from donor 3 was determined by RPA following coculture with EBV-specific CD4+ T cells for 3 or 4 days (D3 and D4, respectively). Lane 1 represents control LCL, and lane 2 represents cocultured LCL. The probes, with protected sizes in brackets, are LMP1 (329 bp), EBNA2 (296 bp), EBNA1 (205 bp), EBNA6 (178 bp), LMP2A (170 bp) (latent transcripts), BZLF1 (Z, 269 bp), BRLF1 (R, 258 bp) (immediate-early transcripts), early Ag D (182 bp) (early transcript), and gp350 (143 bp) (late transcript). The L32 probe was 76 bp. Note that each probe in the control marker lane migrates more slowly than the protected species: this is due to nonhomologous flanking sequences transcribed along with the probe that are not protected by mRNA.

 
The viral immediate-early genes BZLF1 and BRLF1, the early gene EA-D, and the late gene gp350 were expressed by control LCL, and in each case, coculture of LCL with CD4+ T cells resulted in reduced expression (Fig. 7). CD4+ T cell-induced modulation of viral gene expression is thus not confined to the transformation-associated latent genes. We also noted some loss of expression of the L32 ribosomal mRNA species, suggesting that overall gene expression may be reduced. This finding is consistent with our earlier observation of reduced L32 expression in the plasmacytoid tumor cells recovered from SCID/hu mice (25), and may be a reflection of the loss of proliferative capacity of differentiated cells (Fig. 2 and Ref.25). RNA from precisely the same number of cells was analyzed in each track, as described previously (25), and thus the reduced level of L32 RNA is unlikely to be due to a gel loading error.

Is cell-cell contact required for CD4+ T cell-induced differentiation of LCL?

Although we found that blockade of CD40-CD40L interaction does not abrogate T cell-induced differentiation of LCL, other cell surface receptor-ligand interactions may play a role in this process. To address this question, CD4+ T cells and LCL from donor 1 were cocultured in a transwell system which prevents physical contact but allows passage of soluble factors. Increased expression of CD38 and almost total loss of the CD23highCD38low lymphoblastoid subset was observed following transwell coculture of LCL with anti-CD3-activated CD4+ T cells (Fig. 8, A and B). No phenotypic changes were seen following transwell cocultures conducted in the absence of plate-bound anti-CD3, indicating that differentiation of LCL was dependent on CD4+ T cell activation. LCL from transwell cocultures with activated CD4+ T cells were strongly resistant to lysis by autologous, EBV-specific CD8+ CTL (Fig. 8C). These results are comparable with those derived from standard cocultures (Figs. 1 and 3), suggesting that CD4+ T cell cytokines play a central role in inducing differentiation of LCL, and also in inducing resistance to lysis by EBV-specific CD8+ CTL.



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  		FIGURE 8. CD4+ T cell-derived cytokines induce differentiation of LCL and reduce sensitivity to lysis by EBV-specific CD8+ CTL. Transwell cocultures were used to determine whether differentiation of LCL was induced by cell surface receptor-ligand interactions or by soluble CD4+ T cell-derived cytokines. CD4+ T cells from donor 1 were placed in the lower chamber in the presence of plate-bound anti-CD3 mAb. Autologous LCL were placed in the upper chamber. After 4 days, CD23 and CD38 expression by control LCL (also cultured in transwells, but with no CD4+ T cells in the lower chamber) (A) and cocultured LCL (B) were assayed by two-color flow cytometry. Transwell-cocultured LCL were also tested for lysis by autologous EBV-specific CD8+ CTL in a 6-h 51Cr-release assay (C). Targets were control LCL (•) and cocultured LCL ({blacktriangleup}).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro transformation of B cells with EBV results in the generation of immortalized cell lines with a lymphoblastoid phenotype. LCL express high levels of B cell activation markers such as CD11a, CD23, CD54, and CD58, and high levels of viral latent gene products involved in transformation and continuous proliferation. Many of the transformation-associated viral gene products are targets for EBV-specific CD8+ CTL immunosurveillance in vivo, and thus the CD8+ CTL response is able to maintain an asymptomatic state in latently infected immunocompetent individuals (2, 3). In contrast with the in vitro phenotype of LCL, there is increasing evidence in vivo that EBV-transformed B cells are not fixed at the lymphoblastoid stage of B cell differentiation and retain the capacity to differentiate further to a plasmacytoid or mature plasma cell phenotype. Studies of EBV-associated lymphoproliferative disorders in SCID/hu chimeric mice have clearly demonstrated that tumorigenesis is accompanied by the production of high levels of human Ig, and that the majority of tumor cells bear the morphological and phenotypic characteristics of plasma cells (23, 24, 25). In addition, plasmacytoid differentiation of EBV-transformed B cells in the SCID/hu mouse model has been associated with loss of viral latent gene expression (25) and in some cases a switch to viral lytic cycle gene expression (35). Loss of latent gene expression in association with plasmacytoid differentiation of LCL in vitro has also been reported (44). Clinical observations on the pathology of EBV-associated PTLD provide further evidence for plasmacytoid differentiation of EBV-transformed B cells in vivo (8, 45, 46, 47), and loss of viral latent gene expression in PTLD has been reported (45, 48). Of particular note, expression of viral LMP and EBNA2 was preferentially seen in immunoblastic PTLD, whereas PTLD with the morphological and phenotypic appearance of plasmacytomas did not stain for LMP or EBNA2 (45). These observations provide strong evidence that patterns of viral gene expression are closely tied to the differentiation state of the B cell.

Why is there a discrepancy between the in vitro phenotype of EBV-transformed LCL and the in vivo phenotype of EBV-associated PTLD? Activation, proliferation, and differentiation of normal B cells is heavily influenced by CD4+ helper T cell responses. CD4+ T cells that express CD40L promote activation of resting B cells, and T cell cytokines, notably IL-4, IL-6, and IL-10, play key roles in B cell proliferation and plasma cell differentiation (49, 50, 51). These findings support the proposal that EBV-specific CD4+ T cells may exert a strong influence on the differentiation state of EBV-transformed B cells. To test this hypothesis, we conducted in vitro coculture experiments with LCL and autologous irradiated EBV-specific CD4+ T cells, and found that cocultured LCL displayed an altered phenotype relative to control LCL, as documented by changes in CD23 and CD38 expression, and showed a marked loss of proliferative capacity. Collectively, these features bear close comparison with the plasmacytoid phenotype of EBV-associated B cell tumors in SCID/hu mice (25).

One of the more notable outcomes of CD4+ T cell-induced differentiation of LCL is the reduced sensitivity of cocultured LCL to lysis by virus-specific CD8+ CTL. This observation is again comparable to EBV-associated tumor cells from SCID/hu mice, which, relative to autologous LCL, are also markedly resistant to lysis by EBV-specific CD8+ CTL (52). There are at least three potential mechanisms that could account for evasion of EBV-specific CTL. First, differentiation may endow the B cell with intrinsic resistance to CD8+ T cell cytotoxicity, either through blockade of receptor-mediated or perforin/granzyme-induced apoptotic pathways, or through down-regulation of Ag processing and presentation. Second, differentiation could result in loss of expression of HLA class I and/or adhesion molecules such as CD54 and CD58, both of which may be important for conjugate formation between target cells and CTL. Finally, evasion of lysis may be attributed to changes in viral latent gene expression. To address the first possibility, we pulsed control and cocultured LCL with an exogenous peptide and tested their sensitivity to lysis by autologous peptide-specific CD8+ CTL. The control and cocultured LCL were equally susceptible to lysis by peptide-specific CTL, indicating that CD4+ T cell-induced differentiation does not alter intrinsic sensitivity to CD8+ T cell cytotoxicity. Furthermore, control and cocultured LCL were also equally susceptible to lysis by donor-specific alloreactive CTL, indicating that endogenous Ag processing and presentation were unimpaired. With respect to the second possibility, flow cytometric analysis showed that expression of HLA class I and CD54 by LCL was unchanged following coculture, but reduced CD58 expression was noted, a finding that is consistent with the previously reported differentiation-associated loss of CD58 expression by EBV-driven human B cell tumors in the SCID/hu mouse model (25).

Does CD58 play a role in recognition and lysis of LCL by EBV-specific CTL? Blocking studies with anti-CD58 mAb clearly showed that target CD58 interaction with CD2 on the T cell is required for EBV-specific CTL killing of LCL, suggesting that the differentiation-related loss of CD58 by cocultured LCL may at least partly contribute to evasion of lysis. However, control LCL and cocultured LCL were equally sensitive to lysis by alloreactive CTL, notwithstanding the finding that alloreactive CD8+ T cell cytotoxicity against LCL could be blocked with anti-CD58 mAb. Because reduced CD58 expression by cocultured LCL is not sufficient to abrogate lysis by alloreactive CTL, we infer that reduced CD58 expression is also unlikely to be responsible for evasion of lysis by EBV-specific CTL. These results are consistent with earlier studies indicating that LFA-3 is an important element in recognition and lysis, but is not the sole determinant of LCL sensitivity to CTL (53, 54).

Collectively, these results strongly point to the third possibility, i.e., that differentiation-related evasion of EBV-specific CD8+ T cell cytotoxicity is most likely to be a consequence of changes in viral gene expression. RPA showed that differentiation of cocultured LCL was accompanied by a loss of viral latent gene expression, including EBNA6, a known target Ag for EBV-specific CTL from donor 1 (26).

The reduction in expression of both latent and lytic viral genes contrasts with our earlier observation of a switch to lytic cycle replication in plasmacytoid cells from EBV-driven B cell tumors in SCID/hu mice (35). However, the switch to lytic cycle replication in SCID/hu mice was observed in B cell tumors resulting from infection with wild-type EBV, whereas this study was conducted with LCL transformed with the standard B95-8 laboratory strain, which has limited capacity for lytic cycle replication in human B cells.

Although plasmacytoid differentiation in itself is associated with cell cycle arrest, loss of viral latent gene expression may also make a direct contribution to inhibition of LCL proliferation following coculture with CD4+ T cells. Growth of LCL is driven by the transforming viral genes EBNA2 and LMP1; the expression of both these latent genes is markedly diminished by day 4 of coculture.

Although a cytotoxic CD4+ T cell response may in part result in negative selection of more differentiated LCL which express lower levels of viral target Ags, our results strongly point to an active process in which CD4+ T cell signals drive differentiation and modulation of viral gene expression. Transwell cocultures, in which LCL are physically separated from activated CD4+ T cells (and thus not subject to lysis) confirmed that CD4+ T cell-mediated differentiation of LCL is an active, cytokine-mediated process and not simply the result of cytotoxic selection. The CD4+ T cell cytokines involved in differentiation of LCL have not yet been identified. Leading candidates include IL-6 and IL-10, both of which have also been identified as growth and differentiation factors for EBV-transformed B cells (49, 55, 56, 57, 58). A further consideration is that, although it is tempting to assume that differentiation and loss of viral gene expression are closely linked, the possibility remains that the two phenomena are independent events, mediated by different cytokines. Identification of the specific CD4+ T cell cytokines that drive differentiation and/or modulation of viral gene expression will undoubtedly throw some light on this important question.

In conclusion, we have shown that EBV-specific CD4+ T cell responses can induce phenotypic changes in EBV-transformed LCL. In this context, it is notable that high levels of IL-4, IL-6, and IL-10 have been detected in the serum or tumor tissues of PTLD patients (59, 60, 61, 62, 63), suggesting a Th2 CD4+ T cell response that would favor B cell differentiation and thus account for the plasmacytoid features commonly associated with PTLD. We have also shown that CD4+ T cell-induced differentiation of LCL is associated with modulation of viral gene expression and marked resistance to lysis by EBV-specific CD8+ CTL. CD4+ T cell interactions with EBV-transformed B cells may thus play a role in the pathogenesis of EBV infection.


   Acknowledgments
 
We thank Randy Noelle (Dartmouth Medical School, Lebanon, NH) for kindly providing mAb specific for CD40L.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants CA63931 and CA73556. Back

2 Address correspondence and reprint requests to Dr. Martin J. Cannon, Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205. E-mail address: cannonmartin{at}uams.edu Back

3 Abbreviations used in this paper: CsA, cyclosporine A; LCL, lymphoblastoid cell line; PTLD, posttransplant lymphoproliferative disorder; HuAB, human AB; DC, dendritic cell; RPA, RNase protection assay; CD40L, CD40 ligand; LMP, latent membrane protein; EBNA, EBV-encoded nuclear Ag. Back

Received for publication September 10, 2002. Accepted for publication January 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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