Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis.
Effector-memory T cells expressing Fas (Apo-1/CD95) are switched to an apoptotic program by cross-linking with Fas-ligand (FasL). Consequently, tumors that express FasL can induce apoptosis of infiltrating Fas-positive T lymphocytes and subdue any antitumor host immune response. Since Epstein-Barr virus (EBV)-associated tumors such as Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) express FasL, we determined whether EBV-specific cytotoxic T lymphocytes (EBV-CTLs) could be modified to resist this evasion strategy. We show that long-term down-modulation of Fas can be achieved in EBV-CTLs by transduction with small interfering RNA (siRNA) encoded in a retrovirus. Modified T cells resisted Fas/FasL-mediated apoptosis compared with control cells and showed minimal cleavage of the caspase3 substrate poly(ADP-ribose) polymerase (PARP) protein after Fas engagement. Prolonged Fas stimulation selected a uniformly Fas(low) and FasL resistant population. Removal of responsiveness to this single death signal had no other discernible effects on EBV-CTLs. In particular, it did not lead to their autonomous growth since the modified EBV-CTLs remained polyclonal, and their survival and proliferation retained dependence on antigen-specific stimulation and on the presence of other physiologic growth signals. EBV-CTLs with knocked down Fas should have a selective functional and survival advantage over unmodified EBV-CTLs in the presence of tumors expressing FasL and may be of value for adoptive cellular therapy.
Dotti
Gianpietro
G
Center for Cell and Gene Therapy, Baylor College of Medicine, 6621 Fannin St, MC 3-3320, Houston, TX 77030, USA. gdotti@bcm.tmc.edu
Savoldo
Barbara
B
Pule
Martin
M
Straathof
Karin C
KC
Biagi
Ettore
E
Yvon
Eric
E
Vigouroux
Stephane
S
Brenner
Malcolm K
MK
Rooney
Cliona M
CM
D000818:Animals; D000941:Antigens / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D017252:CD3 Complex / Q000096:biosynthesis; D019556:COS Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002857:Chromium / Q000378:metabolism; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D053222:Fas Ligand Protein; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000378:metabolism; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D019014:fas Receptor / Q000096:biosynthesis / Q000378:metabolism*
D000941:Antigens; D017252:CD3 Complex; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D019014:fas Receptor; D002857:Chromium; D007371:Interferon-gamma; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases