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The performed experiments on the immune evasive functions of BCRF1/vIL-10 pointed to an involvement in cytokine orchestration and the prevention of NK cell-mediated killing of infected cells. The elimination of ΔBCRF1- or double k.o.-infected B cells by NK cells was found to be enhanced. At low effector/target rates CD4+ T cells improved NK cell-mediated killing in ΔBCRF1-infected samples. BCRF1 deficiency was associated with a more pronounced Th1 cytokine response that presumably accounted for the boost of NK cell activity when CD4+ T cells were present.

1.3.1 Endogenous BCRF1 expression does not influence specific T cell recognition in vitro The observation that BCRF1 deficiency did not influence MHC I levels or T cell activity (figures 2.4A, 2.5 and 2.6) contrasted with experiments on vIL-10 effects of previous studies (Zeidler et al., 1997; Bejarano and Masucci, 1998; Salek-Ardakani et al., 2002). Presumably, levels of vIL-10 differed significantly between these studies. Levels of different cytokines, and those of IL-10 in particular, are known to be critical for function (Moore et al., 2001). Inappropriate timing and dosage may even evoke opposing effects (Mocellin et al., 2004).

In general, the ad-hoc recognition of targets by activated T cells is independent of secondary signals in case of a strong TCR trigger (Akdis and Blaser, 2001). The conditions to generate and raise T cell clones enrich exactly those T cells with high TCR affinity to EBV targets (Rickinson and Moss, 1997). This experimental setting might have simply masked the direct impact of secreted vIL-10 on T cells. Nevertheless, physiological vIL-10 levels apparently did not affect MHC-I levels and antigen presentation.

1.3.2 vIL-10 and Th1 cytokines

In experiments with PBMC derived effector cells ex vivo, BCRF1 deletion resulted in an immune phenotype. One of the assays revealed a dampening effect of vIL-10 on the Th1 cytokine response. This observation is in line with the known feature of IL-10 to promote Th2 responses and to impair Th1 cytokine secretion (Moore et al., 2001). Interestingly, production of human IL-10 did not complement BCRF1 deficiency and did not rescue the phenotype. In contrast, IL-10 reached highest levels when cells had been infected with virus that lacked BCRF1, but still did not prevent high Th1 levels (figure 2.7). Apparently, vIL-10 impairs Th1 cytokine production with a higher specificity than human IL-10, which at first glance contrasts the described low affinity of vIL-10 to the IL-10R (Liu et al., 1997). However, a number of reports have associated high IL-10 levels with effector activation (Mocellin et al.,

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2004). The suboptimal binding of vIL-10 probably induces an intermediate signaling of that might rather induce anergy and prevent Th1 cytokine shedding.

CD4+ T cells represent the major source of Th1 and Th2 cytokines, but other cells can also secrete these factors. Dedicated effects of BCRF1 on CD4+ T cells were however observed in experiments that investigated the supportive role of CD4+ T cells in NK cell mediated killing of infected B cells. This observation strongly contributed to the model that vIL-10 indirectly hampers the induction of cellular immune responses via the inhibition of Th1 cytokine synthesis by CD4+ T cells.

Taken together, vIL-10 appears to be of defined inhibitory function for Th1 cytokine responses and might fulfill this task to a larger extent than human IL-10. Hypothetically, the IL10-R induces a weak signaling cascade in case of low affinity binding of viral IL-10 and a strong signal upon high affinity binding of human IL-10. Whether this is indeed the case and why CD4+ T cells react on vIL-10 in a particularly sensitive way remains to be elucidated.

1.3.3 vIL-10 impairs NK cells

NK cells can be a source of IL-10 and have regulatory potential on dendritic cells (Vivier and Ugolini, 2009). However, I observed that NK cells can also be inhibited themselves by vIL-10, representing a remarkable advantage for EBV and presumably also for other IL-10 encoding viruses (Rode et al., 1993; Fleming et al., 1997; Kotenko et al., 2000). For human IL-10 in contrast, several publications postulate stimulating effects on NK cell activity (Petersson et al., 1998; Lauw et al., 2000; Parato et al., 2002). The reason that NK cells react differently to hIL-10 and vIL-10 remains elusive and mechanistic reasons are unclear. Generally speaking, the artificial administration of high concentrations of IL-10 probably leads to different outcomes than cellular secretion.

Interestingly, CD8+ T cells were shown to recruit cytokine receptors to the immunological synapse upon target recognition, which was shown by fluorescent labeling and time lapse analysis (Maldonado et al., 2004; Maldonado et al., 2009). Hence, sensitivity to cytokines can be regulated by the recipient cell and might also be realized by NK cells. The visualization of such processes at the immunological synapse is technically demanding, but would probably bear interesting insight on the general behavior of NK cells during target recognition.

Patients with X-linked lymphoproliferative disease (XLD), a rare disease with abrogated NK and NKT cell development (Hislop et al., 2010), suffer from symptoms highly similar to infectious mononucleosis in case of EBV infection. This observation represents the link that NK cells importantly contribute to the control of an EBV infection in vivo. The EBV microRNA BART2-5’ targets the NK cell activating ligand MICB which implies NK cell

mediated immunity (Nachmani et al., 2009). My experiments could contribute the aspect that synthesis of EBV-encoded vIL-10 reduces NK cell killing of infected B cells in vitro. To which extent this observation differs for NK cell subsets that are comprised in CD56+ cells (Cooper et al., 2001; Godfrey et al., 2004) could not be derived from this data, but remains to be addressed in future investigations.

1.3.4 vIL-10 and hIL-10 – homologues with different posttranscriptional regulation

Viral IL-10 has apparently acquired distinct features that distinguish it from human IL-10. In addition, expression analyses revealed different transcript levels: Human IL-10 messengers are abundant in primary B cells and not affected by EBV infection. BCRF1 transcripts, in contrast, were barely detectable and relative levels per se suggested a minor contribution compared to hIL-10.

Regulation of human IL-10 has been intensively studied and occurs on both transcriptional and post-transcriptional level (Mosser and Zhang, 2008). No comprehensive data exist for BCRF1. The prediction of promoter binding sites 800 bp upstream of the BCRF1 ORF by the PROMO algorithm identified binding sites for many of those transcription factors that also regulate hIL-10 expression (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi? dirDB=TF_8.3, Messeguer et al., 2002). Apparently, also the cis-acting promoter elements of hIL-10 have been integrated in the viral genome within gene uptake (figure 4.1A). The same regulatory elements can hence drive both hIL-10 and BCRF1 expression. Moreover, four BZLF1 binding sites were identified for hIL-10 and presumably also exist for BCRF1, despite they differ in relative distance to the start site of gene transcription (Mahot et al., 2003; Bergbauer et al., 2010). Thus, both genes appear to be also similarly regulated by the viral transactivator BZLF1.

Nevertheless, significant differences in transcript prevalence were detected in this work. In general, cytokine transcripts are highly regulated also at the post-transcriptional level. Tristetraprolin (TTP) represents an important regulator of transcript half-life, as it targets messengers for accelerated decay by binding to an AU-rich element (ARE) (Carballo et al., 1998; Blackshear, 2002). TTP also affects hIL-10 due to its six AUUUA pentamer motifs in the 3’ untranslated region (UTR) (Stoecklin et al., 2008). TTP is induced upon EBV-infection and is of presumable importance for this method of transcript regulation during infection (own observations).

Human IL-10 mRNA is targeted by microRNA hsa-miR106a, which is expressed in all cells of lymphoid origin (Sharma et al., 2009). In total, the 5’UTR of 60bp and the 3’UTR of

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1032bp flank the 537bp of coding sequence in the transcript of human IL-10, rendering it highly prone to additional post-transcriptional regulation mechanisms.

In sharp contrast, EBV genes generally have short UTRs - presumably driven by the evolutionary pressure to reduce the risk of unintended cellular regulation to a minimum. Accordingly, BCRF1 transcripts comprise a 5’UTR of 44bp and a 3’UTR of just 73 bp. The entire transcript lacks both ARE motifs and a consensus sequence for hsa-miR106a.

Hence, the post-transcriptional regulation of hIL-10 and BCRF1 differs fundamentally (figure 4.1B). The low abundance of BCRF1 transcripts might be compensated by a high transcript longevity resulting in levels of vIL-10 protein that are obviously sufficient to cause the distinct phenotypes.

Figure 4.1: Transcriptional and post-transcriptional regulation of hIL-10 and BCRF1. A. Known promoter binding sites for hIL-10 (Mahot et al., 2003; Mosser and Zhang, 2008) and predicted binding sites for BCRF1 (symbols with dotted outlines, predicition by the PROMO algorithm, Messeguer et al., 2002, and Bergbauer et al., 2010). Black arrow: transcriptional start, red arrow: translational start, the scale indicates the approximate distance in nucleotides relative to the translational start.

B. Transcript structure of hIL-10 and BCRF1, boxes indicate translated sequence with black bars representing exon-exon junctions. Known sites of posttranscriptional regulation are indicated (Stoecklin et al., 2008; Sharma et al., 2009). ARE, AU rich element.