Rapid Generation of Cytomegalovirus Specific T Lymphocytes Using Commercially Available CMV pp65 Peptides.

Adoptive immunotherapy with cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) can result in resolution of viremia and reconstiution of CMV specific immunity following allogeneic stem cell transplantation. Most recipients of allogeneic transplants who develop CMV reactivation are successfully treated with anti-viral agents, however some have persistent viremia or develop CMV disease despite these medications. In these situations the development of CMV specific T cells may be impractical due to time contraints or institutional limitations. We have expanded CMV pp65 specific CTL from 5/6 normal blood donors of diverse HLA backgrounds in 10–15 days using adherent cells (monocytes) as antigen presenting cells, pulsed with a commercially available source of CMV pp65 peptides, which includes 138 CMV pp65 15mers. These pulsed monocytes are incubated at a 1:5 ratio with non-adherent peripheral blood lymphocytes, resulting in a population of CD4 and CD8+ CMV pp65 specific T cells after 2 weekly stimulations. While >80% of these cells were CD3+, 29–78% were CD4+, and 15–55% were CD8+. These CTL have specific cytotoxicity against CMV pp65, and intracellular cytokine analysis showed specific interferon-gamma production in response to CMV pp65 target cells. Table I depicts the phenotype as well as interferon-gamma production and specific cytotoxicity in response to stimulation with B lymphoblastoid cell lines (BLCL) expressing pp65. There was no reactivity to allogeneic targets by cytokine analysis or in chromium release assays. CTL sufficient for a dose of 30–50 × 10⁶ CTL were routinely generated from 40–50 cc of whole blood. Considering the short time frame and relative ease with which these CTL can be expanded, this methodology could be readily adopted in clinical situations of persistent or therapy refractory CMV infections in allogeneic stem cell transplant patients as an adjuvant to anti-viral therapy.