Donor Lymphocytes Stimulated with CMV pp65 Gene Modified Dendritic Cells for Prophylaxis of CMV Following Allogeneic Haemopoietic Stem Cell Transplantation.

Abstract

Background: Cytomegalovirus (CMV) reactivation post-allogeneic haemopoietic stem cell transplant (HSCT) causes morbidity and mortality. Pharmacological therapy is limited by bone marrow suppression that can result in opportunistic infection. Adoptive transfer of ex-vivo generated CMV-specific T-cells has the potential to rapidly restore immunity, prevent CMV infection and circumvent the need for pharmacotherapy.Methods: We have recruited 18 patients into a trial of donor-derived CMV-specific T-cells generated using dendritic cells transduced with a fiber modified adenoviral vector encoding the immunodominant CMV matrix protein pp65. Thus far, 8 patients have received T-cells prophylactically starting at day 28 post-HSCT. Recipients were monitored for adverse reactions, CMV reactivation by polymerase chain reaction (PCR) and CMV-specific immune reconstitution.Results: 18 cultures have been completed with an 8.5–30 fold increase in total cell number (mean 2.1 × 108, range 0.8–5.1 × 108). Products consisted of T-cells (median 96.5%) displaying an effector memory phenotype (CD45RO+, CD62L−) and a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. All cultures exhibited negligible alloreactivity against recipient derived targets (0–3.6% specific lysis at E:T ratio of 20:1) but strong killing of CMV pp65 peptide pulsed targets (43–79% specific lysis at E:T 20:1). Killing was also observed against targets labeled with adenoviral antigens (2.7–12.5% lysis at E:T 20:1) demonstrating the bi-virus specificity of these cultures. In HLA-B7 and HLA-A2 donors, percentages of tetramer binding T-cells accounted for 3.2 to 40% of cells. Enrichment of cells recognizing known HLA-A24, B8 and B35 epitopes was not observed despite these cultures exhibiting strong cytotoxic activity against pp65 pulsed targets. Reasons for non-infusion include early death post-transplant (3 patients), failure of cultures to meet release criteria (2 patients), severe graft versus host disease (1 patient) and refusal (1 patient). 3 patients are currently awaiting infusion. Patients infused have been followed from 21 to 189 days post infusion. In 5 patients we have demonstrated rapid, polyepitope, CMV pp65-specific immune reconstitution using tetramer and ELISPOT analysis. CMV reactivation by PCR has occurred in 3 patients but none have progressed to CMV disease, suggesting the functional capacity of the cells to control viral reactivation. One patient received pharmacotherapy with valaciclovir while no other patients have required anti-viral antibiotics. There have been no infusion related adverse reactions. Graft versus host disease, non-CMV infections and other adverse events have not exceeded expected rates.Conclusions: Prophylactic adoptive transfer of CMV-specific T-cells is safe, hastens CMV-specific immune reconstitution and may reduce the need for anti-CMV pharmacotherapy in allogeneic HSCT recipients. Our data indicate the potential of specific cellular therapy to control opportunistic infections in severely immunosuppressed patients post-HSCT.