T cells modulate Epstein-Barr virus latency phenotypes during infection of humanized mice.

UNLABELLED: Human B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cells in vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ(-/-) mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferated in vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that the in vitro growth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8(+) cell-depleted mice, and it was absent from CD4(+) cell-depleted mice. These results indicate that CD4(+) T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4(+) T cells contributed to this process through their CD40L expression. IMPORTANCE: At primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4(+) T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.

Cytomegalovirus-seropositive children show inhibition of in vitro EBV infection that is associated with CD8+CD57+ T cell enrichment and IFN-γ.

EBV, a human herpesvirus, is commonly acquired during childhood and persists latently in B cells. EBV seropositivity has been connected to immunomodulatory effects such as altered T and NK cell functional responses as well as protection against early IgE sensitization; however, owing to the asymptomatic presentation during childhood little is known regarding the infection process in children of different ages. In this study, we used mononuclear cells from cord blood and from 2- and 5-y-old EBV-naive children for in vitro EBV infection. We show that the degree of EBV-induced B cell activation and expansion differs between age groups and in particular in relationship to IFN-γ production capacity. EBV infection induced redistribution between B cell subsets with enrichment of IgD(+)CD27(+) cells (commonly referred to as non-switched memory) in infected cord blood cell cultures, and of IgD(-)CD27(+) cells (switched memory) in cell cultures from older children. We also related results to serostatus to CMV, a persistent herpesvirus that can affect differentiation status of T and NK cells. As compared with CMV(-) children, the EBV-induced enrichment of IgD(-)CD27(+) B cells was significantly reduced in infected cell cultures from CMV(+) children. This effect was associated with high levels of IFN-γ and frequencies of highly mature CD8(+)CD57(+) T cells in CMV(+) children. Our results demonstrate that both a child's age and serostatus to CMV will have an impact on EBV-induced B cell activation and expansion, and they point to the ability of viruses with immunomodulatory functions, such as CMV, to affect immune responses within the host system.

Modification of cell differentiation, one of the mechanisms in the surveillance of malignancy.

Most humans carry the potentially life-endangering Epstein-Barr virus (EBV). The immediate danger after infection is imposed by proliferation of the B cells that carry the viral genome. Although a number of different cell types can be infected with EBV, B lymphocytes are exceptionally sensitive; they express a set of virus-encoded proteins, which collaborate with host proteins to induce proliferation. This phenomenon can be demonstrated in vitro with experimentally infected B cells. These viral genes are expressed only in B lymphocytes and are restricted to a defined differentiation stage. This limitation is of high importance for the maintenance of the controlled EBV-carrier state of humans. The emergence of EBV-induced B-cell malignancies is counteracted by highly efficient immunologic mechanisms. Recognition of EBV-transformed immunoblasts in an MHC class I-restricted manner by cytotoxic CD8 T cells and, to a lesser extent, by CD4 T cells, is thought to play the major role. The in vitro experimental results are in accordance with the emergence of EBV(+) B-cell malignancies in immunosuppressive conditions. In this Masters primer, we emphasize that in addition to eliminating B cells that carry the virus genome, the regulatory circuit of the immune response also operates in surveillance, particularly in the early phase of infection. This mechanism involves T-cell-mediated regulation of B-cell differentiation. Because of the strict dependence of the viral growth program on the expression of host cell factors, altering the differentiation state can curb the proliferation of B cells that harbor the viral genome.

The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.