Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65

Qi Sun, Karen Pollok, Robert L. Burton, Li Jun Dai, William Britt, David J. Emanuel, Kenneth G. Lucas

Research output: Contribution to journalArticle

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Abstract

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV- transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-dapleted CTL fractions indicated that CD8+ CTL mediated the pp65- specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV- infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

Original languageEnglish (US)
Pages (from-to)3242-3250
Number of pages9
JournalBlood
Volume94
Issue number9
StatePublished - Nov 1 1999
Externally publishedYes

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T-cells
Cytotoxic T-Lymphocytes
Cytomegalovirus
Human Herpesvirus 4
Viruses
Cells
Cell Line
Cytotoxicity
Stem cells
Antigen-Presenting Cells
Immunotherapy
Assays
Transplants
Histocompatibility Antigens Class I
Peptides
Epstein-Barr Virus Infections
Chromium
Vaccinia virus
Cytomegalovirus Infections
Stem Cell Transplantation

ASJC Scopus subject areas

  • Hematology

Cite this

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65. / Sun, Qi; Pollok, Karen; Burton, Robert L.; Dai, Li Jun; Britt, William; Emanuel, David J.; Lucas, Kenneth G.

In: Blood, Vol. 94, No. 9, 01.11.1999, p. 3242-3250.

Research output: Contribution to journalArticle

Sun, Qi ; Pollok, Karen ; Burton, Robert L. ; Dai, Li Jun ; Britt, William ; Emanuel, David J. ; Lucas, Kenneth G. / Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65. In: Blood. 1999 ; Vol. 94, No. 9. pp. 3242-3250.
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abstract = "Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV- transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-dapleted CTL fractions indicated that CD8+ CTL mediated the pp65- specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV- infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.",
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