BackgroundDonor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells.

To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.Varicella-zoster virus (VZV) infects about 95% of the population, persists throughout life, and may lead to herpes zoster when the virus reactivates. After T-cell-depleted allogeneic stem cell transplantation (TCD alloSCT), reactivation of the virus leads to considerable morbidity (10). Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster. The VZV genome comprises more than 70 unique open reading frames that encode proteins that are coordinately expressed during replication. The product of open reading frame 62, the immediate-early 62 (IE62) protein, is required for the initiation of VZV replication (9) and is expressed at high levels before viral replication has occurred (8). Previous research has demonstrated that IE62-specific T cells were detected after primary VZV infection and in immune subjects (2, 4). In addition, T cells recognizing various other IE proteins and glycoproteins of VZV, as demonstrated by gamma interferon (IFN-␥) production upon stimulation with peptides or lysate derived from these proteins, have been described (1, 6, 13). The VZVspecific memory T cells found in these studies were predominantly CD4 T cells, while no VZV-specific CD8 T cells were demonstrated without prior in vitro expansion, possibly due to the low frequency of VZV-specific CD8 T cells or to the low sensitivity of the screening methods used to detect CD8 T cells by IFN-␥ production upon stimulation. Frey et al. described CD8 epitopes of IE62 detected following in vitro restimulation. However, the HLA restriction and specificity of these T cells were not confirmed (4). Due to the lack of validated VZV-derived immunodominant peptides for major histocompatibility complex (MHC) class I, the analysis of VZV-specific CD8 T-cell responses is hampered (14). To be able to analyze the role of CD8 T cells in VZV reactivation, we therefore set out to identify epitopes for VZV by using VZV-IE62-specific MHC class I peptide complexes.The predictive algorithms BIMAS (11) and SYFPEITHI (12) were used to select potential HLA-A2 binding peptides from the IE62 protein. Peptides with a score of Ն3 (BIMAS) or Ն20 (SYFPEITHI) were considered to have potentially significant binding affinity. The 81 resulting 9-mer peptides were synthesized and tested for binding affinity with the REVEAL MHC-peptide b...

The incidence of Varicella Zoster Virus (VZV) reactivation after T cell depleted allogeneic stem cell transplantation (TCD alloSCT) is 40% the first year after transplantation. CD8 T cells are important in herpes virus reactivations. To analyze the role of CD8 T cells in VZV reactivation, we set out to develop multimeric MHC peptide complexes to be able to identify VZV specific CD8 T cells. Predictive algorithms were used to generate potential immunogenic peptides from Immediate Early (IE) proteins 4, 62 and 63 of VZV that bind in HLA-A2. 127 nonamer peptides were synthesized and tested by a high throughput peptide binding assay (REVEAL5™ assay). Of the peptides that were capable of sufficient binding to the MHC molecules, ProVE™ Pentamers were synthesized. 61 Pentamers were produced and used to screen for HLA-A2 restricted VZV specific T cells in peripheral blood derived from patients with clinical VZV reactivation after TCD alloSCT. In 6 out of 18 HLA-A2 positive patients after VZV reactivation, at a median of 43 days after onset of reactivation, T cells stained positive with the multimeric MHC complex that binds the ALW peptide of the IE62 protein of VZV (IE62-ALW-A2). The percentage of IE62-ALW-A2 positive T cells detected in these 6 patients ranged from 0.02% – 0.13% of CD8 T cells, with a median of 0.04% of CD8 T cells. To confirm the specificity of the Pentamer positive T cells, the IE62-ALW-A2 Pentamer positive T cells were sorted from one patient single cell per well, and expanded. The T cell clones exerted cytolytic activity against HLA-A2 positive EBV-lymphoblastoid cell lines loaded with the IE62-ALW-A2 peptide, whereas unloaded target cells were not lysed. HLA-A2 positive COS cells transfected with the IE62 gene and thus endogenously expressing the IE62 protein were recognized by IE62-ALW-A2 positive T cells as demonstrated by IFNg production. In addition, peripheral blood lymphocytes were stimulated in vitro with the IE62-ALW-A2 peptide in combination with IL-2 and IL-15, and we demonstrated with this procedure that IE62-ALW-A2 positive CD8 T cells were present at a low frequency in another 3 of the 12 IE62-ALW-A2 negative patients. To study whether the immune response against the IE62-ALW-A2 epitope correlated with the course of the clinical infection, we studied the percentage of IE62-ALW-A2 positive T cell during a VZV reactivation in one patient. Six days prior to reactivation, only 0.03% of the CD8 T cells were IE62-ALW-A2 positive. At 42 days after onset of the reactivation, 0.23% of the CD8 T cells were Pentamer positive. After the reactivation resolved, IE-62-ALW-A2 specific CD8 T cells declined to 0.09% at day 49 and 0.03% at day 145 after reactivation. In conclusion, we identified by using a high throughput peptide binding assay and Pentamer production, a new immune dominant epitope of VZV that is recognized by HLA-A2 restricted CD8+ T cells. We demonstrate that IE62-ALW-A2 specific T cells can recognize and lyse target cells that endogenously express the IE62 protein of VZV. In addition, we demonstrate that IE62-ALW-A2 specific T cells expand during the VZV specific immune response. This dominant epitope of VZV in combination with multimeric complexes can be a useful tool to study the immune response to VZV infection and possibly to VZV vaccination.