Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells

Abstract

In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Figure 1

CCL20 expression in HL cell lines and primary tumors. A: Q-PCR demonstrates the up-regulation of CCL20 mRNA in EBV-positive L591 HL cells, compared with EBV-negative L591-SD3 cells (top) and in KM-H2 cells infected with a recombinant EBV, compared with EBV-negative KM-H2 control cells. These differences were statistically significant (P = 0.004 for L591 and P = 0.0002 for KM-H2-EBV). All Q-PCR data are presented as the mean of three replicates. For comparability across all Q-PCR assays RQ values for CCL20 in L428 HL cells (data not shown) were normalized to a value of 1. Asterisk denotes P value of <0.05. B: ELISA shows increased CCL20 protein in the supernatant of EBV-positive L591 and KM-H2 cells compared to their EBV-negative counterparts. C: Left: Q-PCR analysis of CCL20 expression in purified GC B cells (GCB I, GCB II), EBV-negative primary HL tumors (light gray bars) and EBV-positive primary HL tumors (dark gray bars). CCL20 expression was higher in HL tissue than in GC B cells; and generally in EBV-positive tumors than in EBV-negative tumors. Right: GCOS signal for CCL20 determined from the microarray analysis of HRS cells microdissected from five EBV-positive primary NS tumors (HLB, HLE, HLG, HLI, HLJ) (dark gray bars) compared to that from three EBV-negative tumors (HLM, HLL, HLH) (white bars), purified GC B cells (GC#1 to GC#3) (light gray bars), and microdissected GC cells (GC1) (black bar).

Figure 2

CCL20 protein expression in primary HL. A: CCL20 expression in normal tonsil. GC B cells show very low or undetectable levels of CCL20 protein. In contrast, rare small lymphocytes (white arrows) scattered throughout the GC and within the mantle zone (MZ) were positive. B and C: Strong staining in HRS cells (black arrows) of primary EBV-positive HL. D: Absence of CCL20 expression in HRS cell (black arrow) of EBV-negative HL; small lymphocytes in the background are positive (white arrow).

Figure 3

Up-regulation of CCL20 in HL cells is mediated by the EBV-encoded EBNA1. A: Q-PCR analysis of CCL20 mRNA expression in EBV-negative KM-H2 cells (top) and L428 cells (bottom) 48 hours after transient transfection with either EBNA1 expression vector or control empty vector. In both cell lines the ectopic expression of EBNA1 resulted in the up-regulation of CCL20 mRNA. These differences were statistically significant (P = 0.013 for KM-H2 and P = 0.009 for L428). The RQ value for CCL20 in the vector-only control was set at 1. B: RT-PCR confirms the expression of EBNA1 in the KM-H2 cells (top) and L428 cells (bottom) 48 hours after transfection with EBNA1 expression vector. EBV-positive L591 cells were used as a positive control for EBNA1 expression.

Figure 4

Enhanced migration of PBMCs toward conditioned medium from EBV-positive L591 HL cells. A: Increased migration of PBMCs to conditioned medium from EBV-positive L591 cells compared with that to conditioned medium from EBV-negative L591-SD3 cells or RPMI plus 10% FCS alone (P < 0.0000 and P < 0.0000, respectively). This enhanced migration could be inhibited by the preincubation of L591-conditioned medium with anti-CCL20 antibody (left) but remains unaffected in the presence of an isotype control antibody shown here as a separate experiment (P = 0.0034 compared to either RPMI or medium from L591-SD3 cells, right). B: Increased PBMC migration was also observed to conditioned medium from EBV-positive KM-H2 cells (P < 0.0003 compared to EBV-negative KM-H2 medium). This could be inhibited by pretreatment with anti-CCL20 antibody.

Figure 5

Increased migration of regulatory T cells toward conditioned medium from EBV-positive L591 HL cells. A: Flow cytometric analysis reveals a significant increase in the proportion of CD4⁺ T cells expressing FOXP3 within the population of cells that migrated toward conditioned medium from EBV-positive L591 cells (n = 6, P = 0.026). B: Flow cytometry shows that the proportion of CD4⁺ cells expressing FOXP3 was higher in those PBMCs that migrated to conditioned medium from L591 EBV-positive cells (black bars) compared to those that migrated to conditioned medium from L591-SD3 EBV-negative cells (white bars). This increased migration could be abolished by addition of anti-CCL20 antibody (gray bars). Shown here are results for three separate donors.

Figure 6

Increased migration of regulatory T cells toward conditioned medium from EBNA1-expressing L428 cells. A: ELISA reveals increased CCL20 in the supernatant of EBNA1-expressing L428 cells. B: Increased PBMC migration was observed to conditioned medium from EBNA1-expressing L428 cells. This could be inhibited by pretreatment with anti-CCL20 antibody. C: Flow cytometry shows that the proportion of CD4⁺ cells expressing FOXP3 was higher in those PBMCs that migrated to conditioned medium from EBNA1-expressing L428 cells (black bars) compared to those that migrated to conditioned medium from EBNA1-negative control cells (L428-GFP, white bars). This increased migration could be abolished by addition of anti-CCL20 antibody (dark gray bars).