CD4+ T cells classically recognize antigens that are endocytosed and processed in lysosomes for presentation on major histocompatibility complex (MHC) class II molecules. Here, endogenous Epstein-Barr virus nuclear antigen 1 (EBNA1) was found to gain access to this pathway by autophagy. On inhibition of lysosomal acidification, EBNA1, the dominant CD4+ T cell antigen of latent Epstein-Barr virus infection, slowly accumulated in cytosolic autophagosomes. In addition, inhibition of autophagy decreased recognition by EBNA1-specific CD4+ T cell clones. Thus, lysosomal processing after autophagy may contribute to MHC class II-restricted surveillance of long-lived endogenous antigens including nuclear proteins relevant to disease.

IntroductionHodgkin disease (HD) is characterized by a disruption of the lymph node architecture with low numbers of Hodgkin and Reed-Sternberg (H-RS) cells surrounded by an abundance of reactive nonmalignant cells. 1,2 This histologic pattern of HD may be due to the production and release of cytokines from H-RS cells that are involved in the growth of the lymphoma cells and the interactions with bystander cells. [3][4][5] Recently it has been shown that HD cell lines and primary H-RS cells express interleukin-6 (IL-6) and the ␣-chain of the IL-6 receptor (IL-6R; gp80). 3,4,6 IL-6 is synthesized by a variety of cells on stimulation and acts on a wide range of different target cells to regulate cell growth, differentiation, or gene expression. 7,8 IL-6 may act as an autocrine or paracrine growth factor for multiple myeloma, Epstein Barr virus (EBV)-immortalized B cells, and perhaps for H-RS cells, although this has not been proven yet for HD. 3,7,9-13 IL-6 exerts its action via a cell surface receptor complex consisting of at least 2 subunits: the IL-6R gp80 and the signal transducer gp130. 8,14 It has been shown that gp80 is not expressed on normal resting B cells and most Burkitt lymphoma (BL) cell lines 15 ; gp130 is shared by a number of cytokines as a signal transducing receptor component that does not bind IL-6 by itself. 7,8,16 The binding of IL-6 to gp80 leads to an association and dimerization of gp130, followed by the rapid activation of tyrosine kinases of the Janus family (Jak) and a subsequent activation of transcription factors of the signal transducers and activators of transcription (STAT) family. [17][18][19] STAT proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to a number of cytokines. STATs become phosphorylated and translocate as homodimers and heterodimers to the nucleus, where they bind to defined DNA elements within the promoter region of target genes and activate their transcription. [19][20][21] The activation by IL-6 is mainly associated with the tyrosine phosphorylation of STAT3. 19,22 Besides this extracellular mechanism of STAT activation by cytokines evidence also suggests that intracellular events may influence the activation state of STAT proteins and render them independent of extracellular stimuli. Constitutive STAT activation has been reported by several authors for acute leukemias, multiple myeloma, or breast cancer. 13,[23][24][25][26][27][28][29] To further explore the putative role of IL-6 in HD, the expression of gp130 and the influence on the activation of STAT proteins in HD cell lines was analyzed. Neutralizing monoclonal antibodies against IL-6, gp80, gp130, or both receptor subunits did not affect the proliferation of the HD cells or the constitutive activation of STAT molecules. However, the tyrosine kinase inhibitor AG490 inhibited both the constitutive activation of STAT3 and the growth of Hodgkin cell lines in vitro. Materials and methods Cell linesThe HD cell lines L1236, L428, L540, HDLM2, Dev, a...

Since the discovery in 1964 of the Epstein-Barr virus (EBV) in African Burkitt lymphoma, this virus has been associated with a remarkably diverse range of cancer types. Because EBV persists in the B cells of the asymptomatic host, it can easily be envisaged how it contributes to the development of B-cell lymphomas. However, EBV is also found in other cancers, including T-cell/natural killer cell lymphomas and several epithelial malignancies. Explaining the aetiological role of EBV is challenging, partly because the virus probably contributes differently to each tumour and partly because the available disease models cannot adequately recapitulate the subtle variations in the virus-host balance that exist between the different EBV-associated cancers. A further challenge is to identify the co-factors involved; because most persistently infected individuals will never develop an EBV-associated cancer, the virus cannot be working alone. This article will review what is known about the contribution of EBV to lymphoma development.

Although the latent membrane protein-1 (LMP1) of the Epstein-Barr virus (EBV) is believed to be important for the transformation of germinal centre (GC) B cells, the precise contribution of this viral oncogene to lymphoma development is poorly understood. In this study, we used a non-viral vector-based method to express LMP1 in primary human GC B cells. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkin's lymphoma cell lines. Strikingly, LMP1 down-regulated the expression of B-cell-specific genes including B-cell receptor components such as CD79A, CD79B, CD19, CD20, CD22, and BLNK. LMP1 also induced the expression of ID2, a negative regulator of B-cell differentiation. Our data suggest that in EBV-positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed-Sternberg cells, including the loss of B-cell identity.

There is now evidence for both increased and decreased activity of the enzymes controlling the methylation of lysine 27 on histone 3 (H3K27) in cancer. One of these enzymes, KDM6B formally known as JMJD3, a histone demethylase, which removes the trimethyl mark from H3K27, is required for the lineage commitment and terminal differentiation of neural stem cells and of keratinocytes. Our results suggest that KDM6B may also have a role in antigen-driven B-cell differentiation. KDM6B expression increases in B-cell subsets with increasing stage of differentiation, and gene expression profiling shows that KDM6B transcriptional targets in germinal centre B (GC B) cells are significantly enriched for those differentially expressed during memory and plasma cell differentiation. Our results also suggest that aberrant expression of KDM6B may contribute to the pathogenesis of Hodgkin's Lymphoma (HL), an Epstein-Barr virus (EBV) associated malignancy. KDM6B is over-expressed in primary HL and induced by the EBV oncogene, latent membrane protein (LMP1) in GC B cells, the presumptive progenitors of HL. Consistent with these observations, we found that KDM6B transcriptional targets in GC B cells are enriched for genes differentially expressed in HL, and that KDM6B depletion can restore the tri-methylation of H3K27 on these genes.

An important pathogenic event in EpsteinBarr virus (EBV)- IntroductionThe Epstein-Barr virus (EBV) is a ␥-herpesvirus that infects the majority of the world's adult population. In most persons, the virus is carried life-long as an asymptomatic infection where it establishes persistence by latently infecting memory B lymphocytes. However, in a minority of persons, EBV can contribute to the development of one of several B-cell malignancies, including Burkitt lymphoma (BL). 1 In vitro infection of normal B cells with EBV gives rise to lymphoblastoid cell lines (LCLs) in which there is expression of a limited subset of latent virus genes that include the Epstein-Barr nuclear antigens (2, 3A, 3B, 3C, and LP) and the latent membrane proteins (LMP-1, and LMP-2). 2 In contrast, most EBV-associated lymphomas show a more restricted pattern of latency; for example, in the majority of EBV ϩ BL, Epstein-Barr nuclear antigen-1 is the only viral protein expressed. 3,4 As well as maintaining latency in B lymphocytes, the virus can also induce its replicative cycle in these cells. Thus, at any one time, a small proportion of cells in an LCL may spontaneously enter the lytic cycle or be induced to do so by treatment with chemical agents, such as phorbol esters, or by ligation of surface immunoglobulin (Ig). 5 The replicative cycle of EBV is induced by expression of the immediate-early gene, BZLF1, which alone is sufficient to activate downstream lytic genes and complete viral replication in a permissive cell type. 6,7 A number of studies suggest that EBV replicates in terminally differentiated plasma cells. [8][9][10][11][12] These findings are supported by in vitro studies, which show that the BZLF1 promoter is active in memory cells only after they have been differentiated into plasma cells. 12 The intimate association between terminal differentiation and EBV replication in B cells suggests that the switch from latency to the lytic cycle is controlled by factors that normally regulate plasma cell differentiation. We speculated that the absence of such factors could be important in the pathogenesis of EBV-associated lymphomas because virus replication would otherwise result in tumor cell death.We have focused on BLIMP1, a transcription factor encoded by the PRDM1 gene, which orchestrates plasma cell differentiation by repressing genetic programs associated with the germinal center (GC) stages, while at the same time activating those programs associated with plasma cell functions. 13,14 For example, the BLIMP1-mediated silencing of MYC, BCL6, and PAX5 expression has been shown to be required for plasma cell differentiation. [15][16][17][18][19] BLIMP1 also Submitted September 16, 2010; accepted February 21, 2011. Prepublished online as Blood First Edition paper, March 16, 2011; DOI 10.1182 DOI 10. /blood-2010 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Th...

Although Epstein-Barr virus (EBV) usually establishes an asymptomatic lifelong infection, it is also implicated in the development of germinal center (GC) B-cell-derived malignancies, including Hodgkin's lymphoma (HL). Following primary infection, EBV remains latent in the memory B-cell population, where host-driven methylation of viral DNA contributes to the repression of viral gene expression. However, it is still unclear how EBV harnesses the cell's methylation machinery in B cells, how this contributes to viral persistence, and what impact this has on the methylation of cellular genes. We show that EBV infection of GC B cells is followed by upregulation of the DNA methyltransferase DNMT3A and downregulation of DNMT3B and DNMT1. We show that the EBV latent membrane protein 1 (LMP1) oncogene downregulates DNMT1 and that DNMT3A binds to the viral promoter Wp. Genome-wide promoter arrays performed with these cells showed that EBVassociated methylation changes in cellular genes were not randomly distributed across the genome but clustered at chromosomal locations, consistent with an instructive pattern of methylation, and were in part determined by promoter CpG content. Both DNMT3B and DNMT1 were downregulated and DNMT3A was upregulated in HL cell lines, recapitulating the pattern of expression observed following EBV infection of GC B cells. We also found, by using gene expression profiling, that genes differentially expressed following EBV infection of GC B cells were significantly enriched for those reported to be differentially expressed in HL. These observations suggest that EBV-infected GC B cells are a useful model for studying virus-associated changes contributing to the pathogenesis of HL.

Infection of B cells with Epstein-Barr Virus (EBV) induces interleukin-10 (IL-10) production, which may contribute to transformation. IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. However, the mechanism of IL-10 induction is still unclear. Here we demonstrate that IL-10 was specifically induced by the EBV-latent membrane protein 1 (LMP1) in Burkitt's lymphoma (BL) cell lines BL2 and BL41. In two T cell lines (Jurkat, MOLT3), two NHL cell lines (U266, MHH-PREB1), or three Hodgkin's disease (HD) cell lines (L428, L540, and KMH2), LMP1 did not induce IL-10 expression. In contrast, LMP1 activated CD40 or CD54 (ICAM1) expression in the analyzed cell lines. LMP1 derivatives lacking the C-terminal activation regions (CTAR), by deletion of the amino acids between 187 and 351 (Delta CTAR1) or 232 and 386 (Delta CTAR2), alone, or together induced IL-10 at very low amounts compared to wild-type LMP1. Inhibition of LMP1-mediated NF kappa B activation by constitutive repressive I kappa B-alpha only marginally impaired IL-10 expression in BL2 cells, while SB2035080 at 5 microM (a specific p38/SAPK2 inhibitor) led to reduced IL-10 expression. Our findings confirm the role of LMP1 in transactivation of cellular genes possibly important for tumor immunoescape but show that more than one signaling pathway is involved in this activation and suggests the necessity of a defined conformation of CTARs to activate IL-10 involving p38/SAPK2.