Cytokine production profile of BL cells during different types of EBV latency. We monitored the production of 42 cytokines by three type I BL cell lines (Akata, Daudi and Mutu) and their virus-negative counterparts. The data are summarized in Figure 1 and color-coded according to cytokine concentration (Fig. 1). There were five cytokines that were common to all type I BL cell lines: IL-10, IP-10/CXCL10, MDC/CCL22, MIP-1α/CCL3 and MIP-1β/CCL4, albeit at varying levels (Table 1). This suggested that these cytokines might be involved in BL pathogenesis and that this cytokine signature might be characteristic of type I EBV latency in BL (Fig. 1). To investigate whether this pattern of cytokine production was specifically induced by EBV, we compared the types and concentrations of cytokines produced by type I BL cells with their corresponding EBV-negative counterparts (Fig. 2). The color-coded scale in Figure 1 was sufficient to represent overall trends but was not suitable for analyzing changes in cytokine concentrations. Thus, a new color-coded scale was applied to analyze the magnitude of the differences in cytokine concentrations between EBV-positive and EBV-negative cells in more detail. The levels of IL-10, MDC/CCL22 and MIP-1α/CCL3 were 11.9-, 185.1- and 2.4-fold higher, respectively (average values for all three cell lines), in type I BL cells than in their EBV-negative counterparts (Fig. 2), suggesting that these three cytokines are upregulated by type I EBV latency and might mediate in part the pathogenic potential of EBV. Other cytokines, such as IP-10/CXCL10 and MIP-1β/CCL4, were upregulated in a cell line-dependent manner, which suggests that their expression does not have to be upregulated by EBV during the evolution of BL (Fig. 2).
Figure 1. Cytokine production profiles of various lymphoid cells. To compare cytokine production levels, the data were color-coded according to the expression level, as indicated in the legend. Data represent the average of two independent experiments for Epstein–Barr virus (EBV)-negative Akata and B lymphoblastoid cell line (B-LCL) donor 4. Two independent oligoclonal pools were assayed for B-LCL donors 1 and 2. For Akata/B95-8, a recombinant B95-8 virus containing the G418-resistance gene inserted into the viral TK gene locus was used. For B-LCL/Akata, a recombinant Akata virus containing the G418-resistance gene and a green fluorescence protein (GFP) expression cassette inserted into the viral TK gene locus was used. For MKN28/Akata, a recombinant Akata virus containing the G418-resistance gene inserted into the viral TK gene locus was used. In type I EBV latency, EBNA1, BARF0, and EBER are expressed. In type III EBV latency, all of the latency-associated EBV genes are expressed, including EBNA1, EBNA2, EBNA3, EBNA-LP, EBER, BARF0, and LMP. BL, Burkitt’s lymphoma; EGF, epidermal growth factor; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; GRO, growth-related oncogene; IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; PDGF, platelet-derived growth factor; RANTES, regulated upon activation, normal T cell expressed and secreted; TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
Download figure to PowerPoint
Table 1. Summary of cytokines characteristic to transformed B cells with distinct EBV latency
|BL signature cytokines||IL-10, IP-10/CXCL10, MIP-1a/CCL3, MIP-1b/CCL4|
|Upregulated in BL cells by type I EBV latency||IL-10, MDC/CL22, MIP-1a/CCL3|
|B-LCL/DLBCL signature cytokines||IL-8/CXCL8, IL-10, IL-13, IP-10/CXCL10, MIP-1a/CCL3, MIP-1b/CCL4, PDGF-AA, RANTES/CCL5|
Figure 2. Induction of cytokine production during type I and type III Epstein–Barr virus (EBV) latency in different B cell lines and an epithelial cell line MKN28. To visualize the magnitude of cytokine induction, the data were color-coded according to fold difference in expression level, as indicated in the legend. Comparisons were between EBV-negative cells and their EBV-positive counterparts. If a cytokine was not detected, the theoretical lower limit of detection, as described by the manufacturer’s overnight incubation protocol, was used to estimate the fold-difference value. BL, Burkitt’s lymphoma; EGF, epidermal growth factor; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; GRO, growth-related oncogene; IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; PDGF, platelet-derived growth factor; RANTES, regulated upon activation, normal T cell expressed and secreted; TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
Download figure to PowerPoint
Somewhat counterintuitively, the production of certain cytokines in EBV-negative BL cells was downregulated by type I EBV latency. The profile of downregulated cytokines was unique to each cell line. In Akata cells, fibroblast growth factor-2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), IL-1α, IL-2, IL-3 and IL-4 were downregulated 2.5- to 3.3-fold (Fig. 2). These cytokines were upregulated during type III EBV latency, which indicates that Akata cells are able to express them. One possible explanation for these results is that EBV expresses a repressor of these cytokines during type I latency in Akata cells, whereas during type III latency, EBV expresses a strong inducer of these cytokines that can counteract the repressor function. In Daudi cells, type I EBV latency resulted in a 3.3-fold decrease in IP-10/CXCL10, whereas in the other two cell lines (Akata and Mutu), IP-10/CXCL10 was upregulated by type I EBV latency (Fig. 2).
Akata and Mutu cells support type III EBV latency. Infection of EBV-negative Akata cells with B95-8 EBV results in type III latency. Mutu cells with type III EBV latency have been isolated in vitro, which was a spontaneous shift from type I–III latency. Type III Akata and Mutu cells produced IL-10, IP-10/CXCL10, MDC/CCL22, MIP-1α/CCL3, MIP-1β/CCL4 and RANTES/CCL5 at high levels, a signature that, with the exception of RANTES/CCL5, overlapped that of BL cells (Fig. 1). Type III Mutu cells were unique in that they also produced high levels of IFN-α2 (Fig. 1). Cytokine production in type III BL cells was more active than in type I cells (Fig. 1). The magnitude of IL-10, MDC/CCL22 and MIP-1α/CCL3 induction during type III EBV latency was 48.6-, 5441.8- and 65.1-fold, respectively (average values for two cell lines), in Akata and Mutu cells (Fig. 2). These results suggest that type III latency-associated viral genes are more potent cytokine inducers than those expressed during type I latency. Although the production levels were modest, the many other following cytokines were upregulated in type III BL cells compared with their EBV-negative counterparts: FGF-2, Flt-3L, fractalkine/CX3CL1, IFNγ, IL-1α, IL-1β, IL-1ra, IL-6, IL-8/CXCL8, IL-10, IL-12(p70), IL-15, IL-17, IP-10/CXCL10, MCP-1/CCL2, MCP-3/CCL7, MDC/CCL22, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, sCD40L, TNFα, TNFβ and vascular endothelial growth factor (VEGF) (Fig. 2).
Previously, we reported that EBV-encoded small RNA (EBER) play a role in the induction of IL-10 in Akata cells and contribute to their malignant phenotype.(22,32) We investigated whether the expression of EBER was responsible for the induction of type I BL cytokines. The expression of EBER in EBV-negative Akata cells resulted in the upregulation of IL-10 production by approximately 2.5-fold (Fig. 1), consistent with a previous report.(22) However, the levels of MDC/CCL22 and MIP-1α/CCL3 were unaffected by EBER (Fig. 1). These results suggest that an as-yet unidentified viral factor is responsible for the upregulation of MDC/CCL22 and MIP-1α/CCL3. Ectopic expression of EBER also failed to downregulate the levels of FGF-2, G-CSF, IL-1β, IL-2, IL-3 and IL-4 in Akata cells, which suggests that EBER do not function as a repressor of these cytokines during type I EBV latency (Fig. 1).
Cytokine signature of the BL-like cell line BJAB. The BL-like cell line BJAB exhibited a similar cytokine profile to BL cells but with some key differences. Both types of cells produced IL-10, IP-10/CXCL10, MDC/CCL22, MIP-1α/CCL3 and MIP-1β/CCL4 (Fig. 1), which was the BL cytokine signature. This is noteworthy because the histology of BL-like lymphoma is similar to BL, even though BL-like lymphoma does not harbor an Ig/c-myc translocation and is negative for EBV, strongly suggesting the role of BL signature cytokines in the pathogenesis. Unlike the BL cell lines, BJAB cells produced high levels of fractalkaine/CX3CL1, RANTES/CCL5, TNFβ and VEGF (Fig. 1). The level of MDC/CCL22 was substantially lower than the BL cell lines. Thus, the BJAB cytokine signature was distinct from the BL cytokine signature. Epstein–Barr virus infection of BJAB cells, which results in type III latency, was associated with the upregulation of IL-4, IL-8/CXCL8, MDC, sIL-2Ra, TNFα and TNFβ, and downregulation of eotaxin/CCL11, FGF-2, Flt-3L, IL-1ra, IL-6, IL-12(p70), IL-13, IP-10/CXCL10, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5 and VEGF (Fig. 2). The levels of MIP-1α/CCL3 and MIP-1β/CCL4 production in EBV-infected BJAB cells were markedly reduced to 0.6% and 2.3%, respectively, relative to the uninfected cells (Fig. 2). This strong repression of cytokine production by EBV was unique to BJAB cells. Similar to BJAB cells, EBV also establishes type III latency in Akata and Mutu cells. Type III EBV latency in Akata and Mutu cells did not result in the downregulation of any cytokines (Fig. 2). These results indicated that the cellular genetic background of BJAB, a BL-like lymphoma, differs from that of BL cells. To our knowledge, this is the first report of differences in cytokine production associated with type III EBV latency in BL and BL-like cells.
Previously, it was reported that MDC/CCL22 levels are upregulated upon EBV infection in BJAB cells, and that LMP-1 induces the expression of IL-6, IL-8/CXCL8, IL-10, IP-10/CXCL10, RANTES/CCL5, TNFα and TNFβ.(12,16,17,33–35) In Akata cells, we reproduced the upregulation of these cytokines by LMP-1, although the color code did not show modest upregulation of IL-6, IL-8/CXCL8, TNFα and TNFβ for their low expression levels (2.9-, 6.9-, 8.3-, and 11.0-fold, respectively; Fig. 1). Our data were consistent with these earlier results in terms of induction of IL-8/CXCL8, MDC/CCL22, TNFα and TNFβ by type III EBV latency in BJAB cells, which express LMP-1. However, unlike previous results, type III EBV latency in BJAB cells was not associated with the upregulation of IL-10 and resulted in the downregulation of IL-6, IP-10/CXCL10 and RANTES/CCL5. One explanation for these seemingly controversial results is that there might be viral genes expressed during type III EBV latency in BJAB cells that can counteract the function of LMP-1. It is worth noting that the analysis of viral gene function using transfection assays might not accurately reflect events that are relevant to viral infection.
Cytokine production profile of B-LCL. There were more cytokines produced by B-LCL than BL cells, and the levels of the cytokines produced were higher than in BL cells (Fig. 1). The cytokine profiles varied among B-LCL from different donors, and the cytokine profiles of independent cultures of B-LCL from the same donor also exhibited some variation (Fig. 1). However, almost all the B-LCL produced the following cytokines: IL-8/CXCL8, IL-10, IL-13, IP-10/CXCL10, MDC/CCL22, MIP-1α/CCL3, MIP-1β/CCL4, PDGF-AA, RANTES/CCL5, TNFα, TNFβ and VEGF (Fig. 1). The cytokine signature of the B-LCL was distinct from the BL and BL-like cell lines in that the levels of certain cytokines, namely MIP-1β/CCL4, PDGF-AA, RANTES/CCL5 and TNFβ, were substantially higher (Fig. 1). There was no evidence of virus strain-specific cytokine signatures in B-LCL, in that the cytokine profiles induced by EBV of B95-8 and Akata origin were similar.
Cytokine production footprints for other cell types. To determine the specificity of the BL and B-LCL cytokine signatures, we investigated the cytokine production profiles of PBMC from a healthy donor and several different T cell lines. Cytokine production by naïve PBMC was active and the following cytokines were produced at high levels: granulocyte–macrophage colony-stimulating factor (GM-CSF), growth-related oncogene (GRO), IL-1ra, IL-8/CXCL8, MCP-1/CCL2, MDC/CCL22, sIL-2Ra and TNFα (Fig. 1). Unlike the B-LCL, PBMC were poor producers of IL-10, MIP-1α/CCL3 and TNFβ (Fig. 1). These results suggest that the cytokine signature of PBMC is distinct from that of BL and B-LCL. We also analyzed three acute T cell lymphoblastic leukemia-derived cell lines (CEM, Jurkat and MOLT-4) and the HTLV-1-transformed MT-4 T cell line. The T cell lines produced only a few cytokines, namely VEGF (by MOLT-4 cells), MIP-1α/CCL3, RANTES/CCL5 and sIL-2Ra (by MT-4 cells) (Fig. 1). Importantly, none of the T cell lines produced detectable levels of IL-10, IL-12 (p40), IL-13, MDC/CCL22 and MIP-1β/CCL4, a cytokine phenotype that could theoretically be used to distinguish between tumor cells of T cell and B cell origin (Fig. 1). All four T cell lines exhibited unique cytokine signatures, which suggests that these cell lines are genetically divergent and that the cytokines produced might play distinct roles in the pathogenesis of T cell malignancies. Additionally, we analyzed an epithelial cell line, MKN28, in which EBV establishes type I latency. According to the comparison between EBV-positive and EBV-negative counterparts (Fig. 2), EBV type I latency in MKN28 cells upregulated expressions of G-CSF, GRO, IL-1α, IL-4, IL-8/CXCL8, IL-10 and IP-10/CXCL10, whereas it downregulated expressions of MDC/CCL22, PDGF-AA/BB, sLI-2Ra and TNFα. This profile was unique to MKN28 cells, as highlighted by the reduction of MDC/CCL22, which was increased in all the BL cell lines upon type I EBV latency. Interestingly, however, the induction of IL-10 was consistently seen among all cell lines with type I EBV latency. Overall, the results indicate that the cytokine signatures of BL and B-LCL are unique and can be differentiated from those of other malignancies.