Rapid, Large-Scale Generation of Highly Pure Cytomegalovirus-Specific Cytotoxic T Cells for Adoptive Immunotherapy

Abstract

Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific cytotoxic T cell (CTL) clones can restore immunity in allogeneic stem cell transplant recipients, providing protection against CMV disease. Current methods for selecting and expanding CMV-specific T cell clones are technically difficult, making adoptive T cell therapy impractical for routine clinical use. In this study, we describe a method for ex vivo generation and expansion of high-purity CMV-specific CTL using peptide-pulsed dendritic cells as antigen-presenting cells. Generation of CMV-specific CTL in numbers sufficient for clinical use in the time span of 4 weeks was accomplished in 6 of 8 CMV-seropositive donors. Examination of pp65 specificity by HLA/peptide tetramer staining demonstrated that a purity of greater than 95% peptide-specific cells could be obtained after two weekly stimulations and retained after further expansion for 3-4 weeks. Median expansion of total cell number was greater than 500-fold and expansion of peptide-specific CTL by tetramer staining was greater than 1.7 x 10(5)-fold. Four weeks after initiating CTL culture, we were able to generate greater than 10(9) total cells that specifically lysed target cells loaded with CMV peptide and cells infected with CMV. This simple and rapid method for generating high-purity CMV-specific CTL for adoptive immunotherapy is currently being examined for routine clinical use for allogeneic stem cell transplantation.

The impact of cytomegalovirus serostatus of donor and recipient before hematopoietic stem cell transplantation in the era of antiviral prophylaxis and preemptive therapy

“…41,42 There have been major laboratory improvements in T-cell therapy, which may make this therapy more readily available in the near future. [41][42][43][44][45][46] The use of cytokines such as interleukin 7 (IL-7) may also prove useful. 47 One potential obstacle for widespread use of immune-based strategies may be the use of systemic steroids.…”

The Early Kinetics of Cytomegalovirus-Specific CD8+T-Cell Responses Are Not Affected by Antigen Load or the Absence of Perforin or Gamma Interferon

“…The finding that improved expansion of mCMV-specific CD8 ϩ T cells is achieved in the absence of IFN-␥ without a major defect in contraction may aid in the development of improved vaccine therapies. Strategies to improve the generation of hCMV-specific T cells ex vivo are being assessed (7,10,34,35), and a better understanding of the factors that control the kinetics of these responses will aid the design of such protocols.…”

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

“…To generate CMV-specific CTLs for functional assays, peptide-loaded autologous dendritic cells (DCs) were first generated as previously described [4,28]. Briefly, PBMCs were incubated for 2 h at 37°C in X-VIVO15 medium.…”

“…CTLs play an important role in controlling viral infection and eliminating cells with malignant potential. In clinical trials involving the adoptive transfer of antigen-specific CTLs, CTL doses of 10 7 -10 9 cells per kilogram of body mass are required to achieve efficacy [4,5]. Thus, there has been considerable interest in developing an in vitro system to expand human CTLs for use in the implementation of adoptive immune therapies.…”