Clinical-grade preparation of human natural regulatory T-cells encoding the thymidine kinase suicide gene as a safety gene.
BACKGROUND
Human CD4+CD25+FOXP3+ natural regulatory T-cells (nTreg) have a great therapeutic potential for the induction of tolerance in allo-transplanted patients or for the control of severe auto-immune diseases. However, clinical-grade production of nTreg remains difficult to achieve because of the absence of a truly specific surface marker and of their low frequency that implies a need for their ex vivo expansion. Furthermore, safety issues should be taken into consideration due to the risk of either uncontrolled nTreg-induced immunosuppression or uncontrolled proliferation of autoreactive contaminating T-cells particularly in an auto-immune context.


METHODS
We compared different clinical-grade conditions for immuno-magnetic selection and ex vivo expansion of nTreg. For safety, expanded cells were genetically modified with retroviral vectors co-expressing human CD90 and HSV1 thymidine kinase. The CD90 surface marker and thymidine kinase allow for selection and elimination of transduced cells by ganciclovir, respectively.


RESULTS
We showed that (i) nTreg could be enriched in a one step using CD25 microbeads, were functionally suppressive and mainly FOXP3+; (ii) using anti-CD28- and anti-CD3-coated beads, interleukin-2 and rapamycin, nTreg were expanded 150-200-fold after 3 weeks. Under these clinical-grade conditions, they remained suppressive, and no major alteration of the TCR repertoire was observed; (iii) after efficient retroviral transduction and CD90 selection, nTreg maintained their suppressive activity; (iv) transduced nTreg could be eliminated by ganciclovir upon activation.


CONCLUSIONS
The efficient procedure reported here for the preparation of nTreg, whose safety has been ensured, is now applicable for further clinical trials.
Guillot-Delost
Maude
M
CNRS UMR 7087 & Université Pierre et Marie Curie-Paris6, 75013 Paris, France.
Cheraï
Mustapha
M
Hamel
Yamina
Y
Rosenzwajg
Michelle
M
Baillou
Claude
C
Simonin
Ghislaine
G
Leclercq
Virginie
V
Mariotti-Ferrandiz
Maria Encarnita
ME
Six
Adrien
A
Bon-Durand
Véronique
V
Maury
Sébastien
S
Salomon
Benoit L
BL
Cohen
José L
JL
Klatzmann
David
D
Lemoine
François M
FM
D000911:Antibodies, Monoclonal / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D016923:Cell Death / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D051858:Forkhead Transcription Factors / Q000378:metabolism; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D018189:Immunomagnetic Separation; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D008863:Microspheres; D012190:Retroviridae / Q000235:genetics*; D020123:Sirolimus / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology*; D018800:Thy-1 Antigens / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D013997:Time Factors; D014161:Transduction, Genetic
D000911:Antibodies, Monoclonal; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D018800:Thy-1 Antigens; D013937:Thymidine Kinase; D015774:Ganciclovir; D020123:Sirolimus