Graft versus leukemia response without graft-versus-host disease elicited by adoptively transferred multivirus-specific T-cells

Abstract

A 12-year-old boy with refractory acute lymphoblastic leukemia received a haploidentical transplant from his mother. As prophylaxis for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus, he received ex vivo expanded virus-specific donor T cells 3.5 months after transplant. Four weeks later leukemic blasts bearing the E2A deletion, identified by fluorescent in situ hybridization (FISH), appeared transiently in the blood followed by a FISH-negative hematological remission, which was sustained until a testicular relapse 3.5 months later. Clearance of the circulating leukemic cells coincided with a marked increase in circulating virus-specific T cells. The virus-specific cytotoxic T-cell (CTL) line showed strong polyfunctional reactivity with the patient's leukemic cells but not phytohemagglutinin (PHA) blasts, suggesting that virus-specific CTL lines may have clinically significant antileukemia activity.

Figure 1

Rapid reconstitution of antiviral immunity. The patient received a single dose of donor-derived trivirus-specific T cells 3.5 months following haploidentical transplant from the same donor. Peripheral blood samples were obtained pre- and post-cytotoxic T-cell line (CTL) infusion. Patient peripheral blood mononuclear cells obtained pre versus post infusion were incubated with Epstein-Barr virus-LCL, cytomegalovirus (CMV) pp65 pepmix and Adeno hexon pepmix, and interferon-γ responding T-cells quantified using ELISpot analysis (reported as spot-forming cells per 1 × 10⁵ cells).

Figure 2

Virus- and viral epitope specificity of the cytotoxic T-cell line (CTL). (a) Virus specificity was assessed in an ELISpot assay in which the virus-specific CTLs were challenged with the lymphoblastic cell lines (LCLs) from donor, or peptide libraries covering the open reading frames of cytomegalovirus (CMV) pp65, CMV IE1, Epstein-Barr virus (EBV) LMP2, or adenovirus hexon antigen. (b) Cytotoxic activity of the CTL line was evaluated in a ⁵¹Cr-release assay against viral peptide-pulsed phytohemagglutinin (PHA) blasts. The trivirus-specific CTL were incubated with ⁵¹Cr-loaded, peptide library (CMV pp65 or adenovirus hexon) pulsed or unpulsed PHA blasts, adenovirus hexon peptide library-pulsed PHA blasts or CMV pp65 peptide library-loaded PHA blasts, EBV-LCL from the patient or donor for 4 hours at 37 °C, after which supernatants were harvested to determine lysis. To determine whether the CTL line exhibited reactivity against healthy cells (i.e., alloreactivity), unpulsed PHA blasts derived from the patient were also used as targets. (c) Epitope mapping of the CTL indicated reactivity against known CD8 restricted epitopes for EBV and adenovirus as well as broad reactivity with known CD8⁺ epitopes as well as multiple unidentified epitopes in the pp65 antigen.

Figure 3

Recognition of patient leukemic cells by the virus-specific donor T cell line. The trivirus-specific T cell line was labeled with carboxyfluorescein diacetate, succinimidyl ester and incubated with patient phytohemagglutinin (PHA) blasts, patient bone marrow, containing 85% leukemia blasts, or with cytomegalovirus (CMV)-pulsed Epstein-Barr virus (EBV)-LCL (cognate Ag) for 6 hours in the presence of cytokine secretion inhibitors at 37 °C, 5% CO2, and subsequently examined for the production of granulocyte-macrophage colony stimulating factor, interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α within CD4⁺ (a) and CD8⁺ (b) T cells by intracellular cytokine detection as outlined in the Materials and Methods section. Indicated in each plot is the percentage of the respective subset producing cytokine in response to each separate stimulation. The data show a strong reactivity of donor CD4⁺ T cells with patient leukemic marrow but not with patient PHA blasts, while CD8⁺ T cells show only marginal antileukemia cross-reactivity. (c) Reactivity against known leukemia-associated tumor-associated antigens was tested in an IFN-γ ELISPOT assay using overlapping peptides for tumor-associated antigens. Cells alone indicate cells without any antigen, and actin represents an irrelevant mixture of overlapping peptides. Error bars indicate standard deviation from the mean.