Primary B lymphocytes infected with KSHV can be expanded in vitro and are recognized by LANA-specific CD4+ T cells

Kaposi's sarcoma-associated herpesvirus (KSHV) has tropism for B lymphocytes, in which it establishes latency, and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease; however, few models of B lymphocyte infection exist to study immune recognition of such cells. Here, we developed a model of B lymphocyte infection with KSHV in which infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and then challenged with KSHV-specific CD4(+) T cells. The infected cells expressed viral proteins found in PELs, namely, LANA and viral IRF3 (vIRF3), albeit at lower levels, with similar patterns of gene expression for the major latency, viral interleukin 6 (vIL-6), and vIRF3 transcripts. Despite low-level expression of open reading frame 50 (ORF50), transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface-expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM heavy chains with Igλ light chains, recapitulating the features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of major histocompatibility complex (MHC) class II-matched CD4(+) T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8(+) T cells, we suggest that CD4(+) T cells are potentially important effectors for the in vivo control of KSHV-infected B lymphocytes. IMPORTANCE KSHV establishes a latent reservoir within B lymphocytes, but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines, which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes that recapitulates features seen in PEL and MCD by gene expression and cell phenotype analysis, allowing the study of T cell recognition of these cells. Challenge of KSHV-infected B cells with CD4(+) T cells specific for LANA, a protein expressed in all KSHV-infected cells and malignancies in vivo, showed that these effectors could efficiently recognize such targets. Given that the virus expresses immune evasion genes or uses proteins with intrinsic properties, such as LANA, that minimize epitope recognition by CD8(+) T cells, CD4(+) T cell immunity to KSHV may be important for maintaining the virus-host balance

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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

T cell immunity to Kaposi’s sarcoma-associated herpesvirus latent proteins

T cell immunity is important for the control of Kaposi’s sarcoma-associated herpesvirus (KSHV) disease, yet little is known about KSHV-specific immunity in healthy donors. Screening PBMCs from such donors by ELISpot analysis identified weak responses to the KSHV latent antigens; antigens expressed in the virus associated pathologies. We generated T cell clones to the latent proteins LANA and vFLIP and determined whether they recognised target cells. CD8+ clones poorly recognised targets expressing vFLIP or LANA, through mechanisms which reduce target protein synthesis: vFLIP used rare codons in the mRNA encoding this protein, while deleting the acidic repeat of LANA increased its recognition. We then examined whether LANA-specific CD4+ T cells recognised B cells expressing or fed LANA protein. These were recognised, however most KSHV-infected cell lines, in the form of primary effusion lymphoma (PEL) lines, were not. PELs express vIRF3 which inhibits promoter function of the HLA class II transactivator CIITA. Expressing CIITA from a different promoter restored CD4+ T cell recognition of PELs. This study suggests CD8 recognition of the latent antigens tested is inefficient due to the innate properties of the targets but that CD4 T cells can effectively recognise targets if the immune evasion mechanisms are bypassed

Cell death induced in BJAB cells expressing control, vFLIP or vIRF3 transgenes in the presence or absence of 10 μg/ml AZT after challenge with anti-Fas antibody or TRAIL.

<p>Averaged results of at least seven assays where control, vFLIP or vIRF3 transduced cells were grown either with or without 10 μg/ml AZT, transgene expression induced for 24 hours and cells challenged with either anti-Fas antibody or TRAIL and after 48 hours the percentage of dead cells binding Annexin with or without PI staining was then assessed.</p

Surface expression of MHC class II on PELs treated with AZT.

<p>BCBL-1, JSC-1 or VG-1 PEL cells were cultured in either normal medium (No AZT) or medium supplemented with 10 μg/ml AZT for at least 10 days, then stained with MHC class II-specific antibodies or an isotype control and analyzed by flow cytometry. Data is representative of three assays.</p

Characteristics of KSHV-specific CD4+ T cells.

<p>Characteristics of KSHV-specific CD4+ T cells.</p

Outgrowth assays of PEL cells challenged with KSHV-specific T cells in the presence or absence of AZT.

<p>(A). VG-1 PELs which had been treated with 10 μg/ml AZT for at least one week were either pre-sensitized with LAP-peptide or not and seeded in triplicate cultures at doubling dilutions from 10⁴ cells per well to 1 250 cells per well. To these, 10⁴ MHC-matched LAP-specific CD4+ T cells or mismatched CD4+ T cell clones were added and, where indicated, AZT added to a final concentration of 10 μg/ml. Cultures were allowed to grow for 10 days and cell pellets then visualized by microscopy. Images show representative cultures of triplicate wells. (B). Flow cytometric phenotyping of cell populations present at day 10. Cells from the wells highlighted with the black outline in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006042#ppat.1006042.g001" target="_blank">Fig 1A</a> were stained for expression of CD138 or CD4 and analyzed by flow cytometry.</p

Summaries of PEL outgrowth assays after challenge with KSHV-specific CD4+ T cells in the presence or absence of 10 μg/ml AZT.

<p>Cell outgrowth at day 10 was scored after confirming cell identity by flow cytometry for the indicated PEL and T cell combinations. Data is expressed as the minimum number of PELs seeded which successfully outgrew the T cells and the dashed line represents the number of AZT treated PELs seeded in the absence of T cells to achieve outgrowth. Results shown are averages from at least two assays with error bars representing the standard error of the mean. Black arrowheads indicate greater than 10⁴ PELs were required to outgrow the T cells.</p

Titration of AZT inhibition of vIRF3 function in BJAB cells.

<p>vIRF3 and control transduced BJAB cells were cultured in different concentrations of AZT and then transgene expression induced after which the cells were challenged with anti-Fas antibody and the percentage dead cells determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006042#ppat.1006042.g005" target="_blank">Fig 5</a>. Results are averages of three independent assays with error bars representing the standard error of the mean.</p