Generation of Cytomegalovirus pp65-Specific T Cells from Seronegative Donors.

Cytomegalovirus (CMV) can cause significant disease in allogeneic stem cell transplant (SCT) recipients. Adoptive transfer of donor derived CMV-specific cytotoxic T lymphocytes (CTL) is currently used for prophylaxis and treatment of CMV infections in allogeneic transplant recipients, but this is only available for patients whose donors are CMV seropositive. We developed a method to prime and expand CMVpp65-specific T cells from the peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. Monocyte-derived dendritic cells isolated from four CMV seronegative donors were pulsed with CMVpp65 peptides for two hours prior to T cell stimulation. CMVpp65 peptides consisted of 15 mers with an 11 amino acid overlap. T cells obtained from CMV seronegative donors were primed every seven days for three weeks using CMVpp65 peptide pulsed dendritic cells. Interleukin-7 and interleukin-12 were initially added to the T cell cultures to promote CTL differentiation. After ten days, interleukin-2 together with interleukin-7 and interleukin-12 were added at three day intervals. Intracellular cytokine staining (ICS) and ⁵¹Cr-release assays were used to measure the activity and specificity of CMVpp65 primed CMV seronegative donor T cells seven to ten days after the last stimulation. A four color ICS analysis revealed that CMVpp65-specific IFN-γ-producing CD4+ (Th1) T cells were expanded from two of the four seronegative donors; one of these two donors had CMVpp65-specific killing as measured by ⁵¹Cr-release assay, the other displayed non-specific killing. One of the other two donors exhibited CMVpp65 killing but lacked IFN-γ production. PBMC isolated from a CMV seronegative donor three weeks post CMV vaccination also exhibited CMV-specific killing after only one week in vitro stimulation, with no CMV specificity pre-vaccination. In conclusion CMV-specific effector cells can be expanded from some CMV seronegative donors using CMVpp65 peptide pulsed dendritic cells, interleukin-2, interleukin-7, and interlukin-12. These cells may have the potential to be used in adoptive cell therapy to prevent or treat CMV infections in SCT recipients from CMV seronegative donors. An alternative strategy for patients with persistent CMV infection may be to use CTL derived from a vaccinated donor. Analysis of CMVpp65-specific T cells from additional seronegative donors is in progress to further optimize ex vivo expansion.