Generation of EBV-specific CD4+ cytotoxic T cells from virus naive individuals.
Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9-11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4(+) (71 +/- 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.
Savoldo
Barbara
B
Center for Cell and Gene Therapy and Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
Cubbage
Michael L
ML
Durett
April G
AG
Goss
John
J
Huls
M Helen
MH
Liu
Zhensheng
Z
Teresita
Lopez
L
Gee
Adrian P
AP
Ling
Paul D
PD
Brenner
Malcolm K
MK
Heslop
Helen E
HE
Rooney
Cliona M
CM
D000328:Adult; D015704:CD4 Antigens / Q000096:biosynthesis*; D002353:Carrier State / Q000276:immunology / Q000821:virology; D002461:Cell Line, Transformed; D002648:Child; D002675:Child, Preschool; D017077:Culture Media, Conditioned; D003601:Cytotoxicity Tests, Immunologic / Q000592:standards / Q000706:statistics & numerical data; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology / Q000821:virology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D020031:Epstein-Barr Virus Infections / Q000276:immunology / Q000821:virology; D005260:Female; D005312:Fetal Blood / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D007223:Infant; D008213:Lymphocyte Activation; D008297:Male; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis; D015203:Reproducibility of Results; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism / Q000821:virology
D015704:CD4 Antigens; D017077:Culture Media, Conditioned; D018984:Epitopes, T-Lymphocyte; D015375:Receptors, Interleukin-2