ABSTRACT

We have developed an efficient and rapid method for detection of EBV-specific CD8⁺ T cells. Peripheral blood mononuclear cells (PBMCs) are stimulated with autologous lymphoblastoid cell lines (LCLs) for 5 hours and intracellular accumulation of interferon-γ (IFN-γ) is assessed by multiparameter fluorescence-activated cell sorter (FACS) analysis. The proportion of EBV-specific CD8⁺ T cells ranged around 1% in PBMCs of healthy long-term EBV carriers. Class I restriction of IFN- γ production was confirmed using an anti-class I monoclonal antibody (mAb).

We have applied the technique to study CD8⁺ T cell responses in EBV-associated diseases and demonstrated 34-54% of HLA-DR⁺/ CD8⁺ and 34-60% of CD45RO⁺/ CD8⁺ T cells in PBMCs of febrile patients suffering from infectious mononucleosis (IM) to be EBV-specific using FACS analysis. Decline of CD8⁺ T cell sub-populations, namely HLA-DR⁺, CD45RO⁺ and EBV-specific T cells, paralleled the drop in EBV genome load. These data indicate that antigen-driven expansion of CD8⁺ T cells and subsequent contraction with antigen decline in vivo in human is effective for clearing virus-infected cells with minimal disturbance of the homeostasis of the immune system.

The EBV-associated lymphoproliferative disorder (LPD) constitutes a serious complication after allogeneic bone marrow transplantation (BMT). Dynamics of EBV-specific cytotoxic T lymphocytes (CTLs), which are important in controlling EBV, during LPD were here fully elucidated. FACS analysis for IFN- γ production revealed that more than 70% of CD8⁺ T cells in PBMCs of a patient suffering with LPD on day 47 after BMT, were EBV-specific. The patient`s lymphocytes were directly cytotoxic to donor-derived LCLs, thus being blocked by an anti-class I antibody. Counts of EBV-specific CD8⁺ T cell declined in parallel with the EBV genome load and full recovery from LPD was obtained with relaxation of immuno- suppressive drugs. The results illustrate longitudinal dynamics of EBV-specific CTLs during post-transplant LPD as well as the advantages of FACS analysis for decision making of treatment. 

In conclusion, the sensitive and rapid nature of flow cytometric assays for EBV-specific CD8⁺ T cell frequency endows significant advantages for evaluation of EBV-specific CD8⁺ T cell responses in PBMCs of patients with EBV-related disease.