Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation

Abstract

Viral infection or reactivation remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. We now show that infusions of single cytotoxic T lymphocyte (CTL) lines (5 x 10(6)-1.35 x 10(8) cells/m(2)) with specificity for 2 commonly detected viruses, Epstein-Barr virus (EBV) and adenovirus, can be safely administered to pediatric transplantation recipients receiving partially human leukocyte antigen-matched and haploidentical stem cell grafts (n = 13), without inducing graft-versus-host disease. The EBV-specific component of the CTLs expanded in vivo and persisted for more than 12 weeks, but the adenovirus-specific component only expanded in vivo in the presence of concomitant adenoviral infection. Nevertheless, adenovirus-specific T cells could be detected for at least 8 weeks in peripheral blood, even in CTL recipients without viral infection, provided the adenovirus-specific component of their circulating lymphocytes was first expanded by exposure to adenoviral antigens ex vivo. After infusion, none of these 13 high-risk recipients developed EBV-associated lymphoproliferative disease, while 2 of the subjects had resolution of their adenoviral disease. Hence, bispecific CTLs containing both EBV- and adenovirus-specific T cells can safely reconstitute an antigen responsive "memory" population of CTLs after human leukocyte antigen-mismatched stem cell transplantation and may provide antiviral activity. This trial was registered at www.clinicaltrials.gov as #NCT00590083.

Figure 1

Immunophenotype of bivirus-specific CTL lines generated for clinical use. Reactivity of CTL lines (n = 20) with antibodies against the T-cell surface antigens CD3, CD4, CD8, and CD56, the monocyte surface antigen CD14, and the B-cell surface antigen CD19. Lines made from haploidentical donors are represented by circles (n = 8), and those made from matched unrelated donors are represented by triangles (n = 12).

Figure 2

Virus-specific activity of CTL as determined by Cr release assay. ⁵¹Cr release at 4 hours after coincubation of CTL lines with autologous nontransduced EBV-LCL (EBV target), autologous EBV-LCL transduced with Ad5f35^null vector, or recipient PHA blasts (allogeneic target). Lines made from haploidentical donors are shown in the top panel (n = 8) and those made from matched unrelated donors are shown in the bottom panel (n = 12). The data are mean percentage lysis (± SEM) of targets by all 20 CTL lines at E/T ratios of 40:1, 20:1, 10:1, and 5:1.

Figure 3

Virus-specific activity of CTL as determined by IFN-γ ELIspot assay. T-cell specificity and cytokine production were assessed using the IFN-γ ELIspot assay by direct stimulation with hexon pepmix (adenovirus), penton pepmix (adenovirus), irradiated EBV-LCL at an E/T ratio of 1:1 (EBV), and an irrelevant pepmix (control). Results are presented as SFCs/10⁵ CTLs. Lines made from haploidentical donors are represented by circles (n = 8), and those made from matched unrelated donors are represented by triangles (n = 12).

Figure 4

In vivo expansion and clinical benefits of EBV-specific T cells. (A) Frequencies of peripheral blood T cells responding to EBV-LCL by IFN-γ secretion before and after CTL infusion in 12 patients. Individual haploidentical recipients (n = 5) are represented by circles, and MUD recipients are represented by triangles (n = 7). Mean values are represented by lines: solid for haploidentical transplant recipients and dashed for MUDs. (B) Increase in the mean frequency of EBV-LCL-responsive peripheral blood T cells in patients 1, 2, and 3 is related to a decrease in EBV-DNA levels.

Figure 5

Effects of adenovirus-specific CTL in vivo. (A) ELIspot analysis of the frequency of peripheral blood T cells responding to irradiated Ad5f35-infected donor PBMCs by IFN-γ secretion before and after CTL infusion in patients without (n = 9; left panel) and with (n = 2; right panel) adenovirus infection at the time of receiving CTL. (B) Imaging studies are shown before and after CTL in a patient (patient 2) with adenovirus pneumonia who had a complete clinical response after T-cell infusion. A chest CT scan and chest radiograph demonstrate bilateral interstitial infiltrates. The follow-up chest radiograph 2 weeks after CTL infusion is reported as normal (C). Marked increase in adenovirus-specific T-cell precursor frequency in patient 2, and marked decrease in adenoviral load in stool from patient 10 after CTL infusion and clearance of virus coincident with a transient increase in adenovirus-specific T cells.

Figure 6

Amplification of adenovirus-specific T cells in vitro. (A) PBMCs from a haploidentical transplant recipient without adenovirus infection were stimulated in vitro with adenovirus antigens to amplify the frequency of adenovirus-reactive T cells and reanalyzed 9 days later by IFN-γ ELIspot. (B) Amplification of adenovirus-specific T cells from a MUD recipient without infection showing an increase in the adenovirus-specific T-cell precursor frequency using IFN-γ ELIspot.