Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation

Abstract

Adoptive immunotherapy with antigen-specific cytotoxic T lymphocytes (CTLs) has proven effective in restoring cellular immunity to cytomegalovirus (CMV) and preventing viral reactivation after allogeneic stem cell transplantation (SCT). In an effort to develop a cost-effective, relatively rapid method of CMV CTL expansion, we investigated the use of a pool of overlapping CMV peptides. Because the possibility exists of vaccinating CMV-seronegative donors, and these individuals may have T cell responses predominantly against IE-1, commercially available peptide mixes for pp65 as well as IE-1 were used to stimulate CTLs from 10 seropositive donors. Of these 10 donors, 4 responded to pp65 only, 1 did not respond to either pp65 or IE-1, 4 responded to both pp65 and IE-1, and 1 responded to IE-1 only. These CMV- specific T cells included a mixture of CD4(+) and CD8(+) effectors, and specific cytotoxicity correlated with interferon-gamma production. The costs associated with a 28-day maintenance course of intravenous ganciclovir, cidofovir, foscarnet, and valganciclovir, as well as the preparation and shipping a single dose of CTLs, were determined. The price of generating CMV CTLs using this method was comparable to or less expensive than a 28-day maintenance course for these agents, not including the costs associated with drug administration, supportive care, and the treatment of drug-related complications. Considering the relative ease, low cost, and the fact that CTL administration can result in CMV-specific immune reconstitution, this option should be considered for patients with CMV reactivation or for prophylaxis in patients at high risk for infection.

Figure 1. Cytotoxicity of CTL

Peripheral blood monocytes were pulsed with pooled CMV pp65 and IE-1 peptides and incubated with peripheral blood lymphocytes from 10 healthy CMV sero-positive donors. Targets include autologous B cell blasts (BB), BB pulsed with pp65 and IE-1 overlapping peptides, and allogeneic BB.

Figure 2. Cytotoxicity of CMV CTL

Peripheral blood lymphocytes were stimulated with autologous monocytes pulsed with pp65 and IE-1 overlapping peptides. Specific cytotoxicity was measured using B cell blasts (BB) infected with either vaccinia encoding pp65 or IE-1 (vvp65, vvIE1), as well as BB pulsed with either peptide mix. Autologous and allogeneic BB were used as negative controls.

Figure 3. IFN-γ production of T cells

Peripheral blood monocytes were pulsed with pooled CMV pp65 peptides and IE1 peptides and incubated with nonadherent peripheral blood lymphocytes. IFN-γ producing T cells specific for these CMV peptides were analyzed at day 10 of culture by intracellular staining. A). CD8+ T cell response to CMV pooled peptides. B). CD4+ T cell response to the CMV peptides.