Azidothymidine Sensitizes Primary Effusion Lymphoma Cells to Kaposi Sarcoma-Associated Herpesvirus-Specific CD4+ T Cell Control and Inhibits vIRF3 Function.

Samantha J Williamson,Michael Stürzl,Andrew D Hislop,Shereen Sabbah,Samantha M Nicol,Dirk P Dittmer

doi:10.1371/journal.ppat.1006042

Samantha J Williamson, Michael Stürzl + Show 4 more

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https://doi.org/10.1371/journal.ppat.1006042

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Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with the development of Kaposi sarcoma and the B lymphocyte disorders primary effusion lymphoma (PEL) and multi-centric Castleman disease. T cell immunity limits KSHV infection and disease, however the virus employs multiple mechanisms to inhibit efficient control by these effectors. Thus KSHV-specific CD4+ T cells poorly recognize most PEL cells and even where they can, they are unable to kill them. To make KSHV-infected cells more sensitive to T cell control we treated PEL cells with the thymidine analogue azidothymidine (AZT), which sensitizes PEL lines to Fas-ligand and TRAIL challenge; effector mechanisms which T cells use. PELs co-cultured with KSHV-specific CD4+ T cells in the absence of AZT showed no control of PEL outgrowth. However in the presence of AZT PEL outgrowth was controlled in an MHC-restricted manner. To investigate how AZT sensitizes PELs to immune control we first examined BJAB cells transduced with individual KSHV-latent genes for their ability to resist apoptosis mediated by stimuli delivered through Fas and TRAIL receptors. This showed that in addition to the previously described vFLIP protein, expression of vIRF3 also inhibited apoptosis delivered by these stimuli. Importantly vIRF3 mediated protection from these apoptotic stimuli was inhibited in the presence of AZT as was a second vIRF3 associated phenotype, the downregulation of surface MHC class II. Although both vFLIP and vIRF3 are expressed in PELs, we propose that inhibiting vIRF3 function with AZT may be sufficient to restore T cell control of these tumor cells.

Highlights

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human γ-herpesvirus which infects endothelial cells and establishes a latent infection in B lymphocytes. It is associated with the endothelial cell malignancy Kaposi sarcoma (KS) and the B lymphocyte disorders primary effusion lymphoma (PEL) and multi-centric Castleman disease (MCD)[1]
Culturing PELs in the thymidine analogue azidothymidine (AZT) induces sensitivity to apoptosis mediated by Fas-ligand and TNF-related apoptosis inducing ligand (TRAIL) [19,20] and as these can be expressed by T cells, we asked whether AZT would sensitize PELs to control by KSHV-specific CD4+ T cells
We initially determined whether a panel of established KSHV-specific CD4+ T cell clones [16] (Table 1) expressed Fas-ligand and TRAIL transcripts by qRT-PCR analysis after stimulation with autologous target cells sensitized with their cognate peptide-epitope

Summary

Introduction

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human γ-herpesvirus which infects endothelial cells and establishes a latent infection in B lymphocytes. Proteins expressed within KSHV malignancies include the genome maintenance protein LANA, a cyclin D homologue vCyclin, an NF-κB activator with pro-survival function vFLIP, and the Kaposin proteins of which the best characterized, Kaposin B, functions to stabilize cytokine mRNAs (for review see Schulz and Cesarman[5]) Some of these genes show intrinsic features which likely minimize exposure to CD8+ T cells by restricting synthesis of their encoded protein, reducing the supply of defective ribosomal products (DRiPs) that are thought to be the source of CD8+ T cell peptide-epitopes[6]. Infected B cells express the ubiquitin ligases K3 and K5, which induce endocytosis of surface MHC class I and co-stimulatory molecules such as ICAM and CD86[13,14] These multiple layers of immune evasion mechanisms represent a challenge for T cell mediated control of infected cells

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