Functional CD4(+) and CD8(+) T-cell responses induced by autologous mitomycin C treated Epstein-Barr virus transformed lymphoblastoid cell lines.
Epstein-Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4(+) and CD8(+) T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4(+) and (granzyme B positive) CD8(+) T-cells rosetting with McLCL. Granzymes A and B, IFN-gamma, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4(+) and CD8(+) T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.
Department of Pathology, University Hospital Vrije Universiteit, 1007 MB Amsterdam, The Netherlands.
P
P
Meij
E
E
Bloemena
N
N
Palmen
A
A
Brink
M B
MB
Vervoort
C J
CJ
Meijer
J M
JM
Middeldorp
D019264:Adoptive Transfer / Q000379:methods; D017951:Antigen Presentation / Q000187:drug effects / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D015925:Cryopreservation / Q000379:methods; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D053804:Granzymes; D004854:Herpesvirus 4, Human / Q000276:immunology* / Q000502:physiology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D016685:Mitomycin / Q000494:pharmacology*; D010641:Phenotype; D012397:Rosette Formation; D012697:Serine Endopeptidases / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D016685:Mitomycin; D007371:Interferon-gamma; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human