Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers

Abstract

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA-peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 x 10(3)/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy.

Figure 1.

Selection of CMV-specific CTLs from blood samples using large-scale magnetic separation. (A): HLA–peptide tetramer staining of donor PBMCs before selection and in the positive and negative fractions after selection. (B) Cytotoxicity assay of positively selected cells performed directly after selection or after 8 d of culture on autologous peptide-loaded cells. ♦, 0.5 μM peptide; □, DMSO control. (C) Growth characteristics of cells in the positive fraction reveal a 30-fold expansion after 8 d. Cells were expanded with autologous lymphoblastoid cells and allogeneic feeder cells. (D) Positively selected CMV-specific CTLs lyse autologous and HLA-matched fibroblasts, which have been infected with CMV. CTLs were cultured for 8 d and subsequently were tested on fibroblasts that were infected with CMV. The E:T ratio in all cases was 2:1. □, Mock-infected fibroblasts; ▪, CMV-infected fibroblasts. All results are representative of at least three independent experiments.

Figure 2.

CMV-specific immune reconstitution and CMV viremia after adoptive transfer of CMV-specific CTLs. The vertical bar indicates the timing of adoptive transfer and the absolute number of CMV-specific CTLs administered. Data from patients 1, 2 and 3 (A–C) are shown. ▪, Absolute count of tetramer⁺ T cells; ▴, viral load. Persistence of the infused T cells was assessed in patient 2 by PCR detection of the clonotypic TCR CDR3 sequence of the infused cells in PBMC samples that were taken from the patient. FACS analysis of donor PBMCs demonstrated that 80% of the CMV-specific CTLs expressed the TCRBV11 chain. TCRBV11-specific PCR was performed on positively selected donor CMV-specific CTLs, and the PCR products were cloned and sequenced to determine the dominant TCR clonotype. A single TCR CDR3 sequence, YLCARFDGRAGETQYFGPG, comprised 90% of all TCRBV11 sequences. A clonotype-specific primer (5′-gtttgacgggagggccggtga–3′) was synthesized and used with a TCRBC primer for clonotype-specific PCR which was performed on patient PBMCs that were isolated before infusion and at days 5, 28, 44, 66, and 100 after infusion.

Figure 3.

CMV-specific immune reconstitution and CMV viremia after adoptive transfer of CMV-specific CTLs in patients. The vertical bar indicates the timing of adoptive transfer and the absolute number of CMV-specific CTLs that was administered. Data from patients 7, 8, and 9 (A–C) is shown. ▪, Absolute count of tetramer⁺ T cells or expressed as percentage of CD8 T cells; ▴, viral load. Persistence of the infused T cells was assessed in patient 7 by PCR detection of the clonotypic TCR CDR3 sequence of the infused cells in PBMC samples that were taken from the patient (Fig. 2); >90% of tetramer-binding cells expressed TCRBV3. A single TCR CDR3 sequence, SGEQGMDEQYFGPGTRLTVT, made up all TCRBV3 sequences. A clonotype-specific primer (5′-gggaacagggaatggacgag–3′) was used for clonotype-specific PCR that was performed on patient PBMCs that were isolated before infusion and at days 1, 5, 7, 12, and 15 after infusion (A).