Primary B Lymphocytes Infected with Kaposi's Sarcoma-Associated Herpesvirus Can Be Expanded In Vitro and Are...

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 thein vivocontrol of KSHV-infected B lymphocytes.

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 malignanciesin 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.