[Specific suppression in regulatory T cells by Foxp3 siRNA contributes to enhance the in vitro anti-tumor immune response in hepatocellular carcinoma patients].
OBJECTIVE
To design the suitable sequence siRNA of Foxp3 to interfere the function of regulatory T cells(Treg), and to evaluate whether the suppression of Treg could enhance the anti-tumor immune response in hepatocellular carcinoma patients or not.


METHODS
Foxp3-specific siRNAs by chemical synthesis were delivered into regulatory T cells. The inhibition efficiencies of Foxp3-specific siRNAs were evaluated by real-time PCR and fluorescently stained for intracellular Foxp3 and analyzed using multiparameter FCM. The Foxp3(+) Treg subpopulation was selectively analyzed for surface expression levels of CD127, CTLA-4 and GITR. The suppression of Treg to CD4(+)CD25(-) T cells or anti-tumor specific CD8(+) T-cell responses induced by tumor specific antigen (NY-ESO-1b) was evaluated by CFSE, Elispot and Pentamer analysis.


RESULTS
A subpopulation of Tregs with reduced levels of Foxp3 mRNA and protein mediated by siRNA was CD127 up-regulation and CTLA-4 or GITR down-regulation compared with those in Foxp3(high) Tregs. Knockdown of intracellular Foxp3 in Treg reduced suppression of Foxp3+ Treg to proliferative capacity of CD4(+)CD25(-) T cells. IFN-gamma released of NY-ESO-1-specific CD8(+) T cells from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected(132+/-55 vs 27+/-11, P<0.05). Frequency of NY-ESO-1b-specific CD8(+) T cells by Pentamer analysis from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected (0.21%+/-0.17% vs 0.57%+/-0.39%, P<0.05).


CONCLUSIONS
Knockdown of intracellular Foxp3 in Treg mediated by siRNAs can inhibit the suppression of Foxp3+ Treg and enhance the anti-tumor immune response in hepatocellular carcinoma patients in vitro.
Zhang
Heng-hui
HH
Peking University People's Hospital, Peking University Hepatology Institute, Beijing 100044, China.
Fei
Ran
R
Xie
Xing-wang
XW
Wang
Li
L
Luo
Hao
H
Wang
Xue-yan
XY
Wei
Lai
L
Chen
Hong-song
HS
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000276:immunology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014162:Transfection
C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D034741:RNA, Small Interfering