[Effect of RelB-silenced BMDC pulsed with Talpha146~162 on immunoreaction of T cells primed with TAChR].
OBJECTIVE
To investigate whether RelB-silenced bone marrow-derived dendritic cells (BMDC) pulsed with torpedo acetylcholine receptor (TAChR) immuno-dominant peptide Talpha146~162 can induce tolerance in T cells primed with TAChR.


METHODS
Recombinant lentivirus that produced RelB siRNA and control lentivirus were prepared and used to infect BMDCs. The infected BMDCs were stimulated with LPS,and the resulting cells were designated as DC-siRelB or DC-control, respectively. The mRNA and protein expression of RelB were examined by quantitative real-time PCR and Western blot. Cell surface markers of DC were evaluated by flow cytometry. IL-12 in the supernatant was detected by ELISA. Mice were randomly divided into 6 groups: A1, A2, A3,K1, K2, and K3. On day 0, group A1, A2, and A3 were primed with TAChR in CFA and group K1, K2 and K3 were primed with KLH+CFA. On day 7, group A2 and K2 were injected with Talpha146~162 pulsed DC-siRelB, group A3 and K3 were injected with Talpha146~162 pulsed DC-control, while A1 and K1 group received PBS at the same time. On day 14, lymphocyte proliferative response of the 4 groups were measured.


RESULTS
Recombinant lentivirus including RelBshRNA genes was successfully constructed. RelB siRNA knocked down RelB expression in BMDCs obviously. Compared with DC-control, DC-siRelB expressed a significantly lower level of CD80, CD86, and MHC class II on their surface, producing lower level of IL-12. Compared with group A1 and A3, lymphocyte proliferative response to TAChR of A2 group was suppressed significantly (P<0.05). No different lymphocyte proliferative responses to KLH and ConA were seen in group A1, A2 and A3 (P>0.05). No different lymphocyte proliferative responses were seen in group K1, K2 and K3 (P>0.05).


CONCLUSIONS
Lentiviral-mediated RelB-silenced BMDCs are maturation resistant and can induce antigen-specific tolerance in TAChR primed C57BL/6 mice,which provides a basis for further study of their therapeutic potential in myasthenia gravis.
Department of Neurology, Xiangya Hosptial, Central South University, Changsha 410008, China.
Yong
Y
Zhang
Huan
H
Yang
Bo
B
Xiao
Tefei
T
Lu
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology; D005260:Female; D007108:Immune Tolerance; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*; D051841:Transcription Factor RelB / Q000235:genetics* / Q000378:metabolism
D010446:Peptide Fragments; D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C497594:Relb protein, mouse; C041736:Torpedo acetylcholine receptor, alpha-subunit 1-20; D051841:Transcription Factor RelB