Cotransfection of DC with TLR4 and MART-1 RNA induces MART-1-specific responses.
BACKGROUND
Cotransfection of dendritic cells (DC) with MART-1 and constitutively active TLR4 (caTLR4) RNA enhances the maturation of DC.


METHODS
Immature DC were cotransfected with RNA constructs encoding MART-1 and caTLR4, and CTL responses were analyzed.


RESULTS
Cotransfection of DC with MART-1 + caTLR4 enhanced the expression of CD80 and CD83 surface markers and increased the secretion of cytokines IL-6, IL-12, and TNFalpha. Neither the native nor the A27L-modified MART-1 RNA could induce significant DC maturation or cytokine secretion. More importantly, DC cotransfected with caTLR4 + MART-1 RNA induced MART-1-specific CTL responses of a higher magnitude than DC transfected with either the native or A27L MART-1 RNA. When the MART-1 RNA-transfected DC were treated with DC-maturing cytokines, the induced CTL were less frequent and less lytic than those induced with MART-1 + caTLR4. A 2- to 100-fold increase in MART-1 tetramer+ cells and 2- to 10-fold increases in IFNgamma secretion and cytotoxicity were seen in CTL induced with MART-1 + caTLR4 compared to CTL induced with either MART-1 or A27L RNA. CTL induced with the mixed RNA displayed high percentages of CD8+ cells coexpressing CD45RA, CD56, and 2B4 antigens. Transfection with caTLR4 alone induced DC maturation, but did not induce lytic CTL, suggesting that CTL responses were induced solely by MART-1 epitopes.


CONCLUSIONS
caTLR4 increases the CTL-inducing capacity of DC generating a lytic response specific for the accompanying antigen. These results demonstrate the possibility of enhancing the immunogenicity of the native MART-1 and other RNA derived from weakly immunogenic tumors in DC-based immunotherapy.
Abdel-Wahab
Zeinab
Z
Department of Surgery, Duke University, Durham, NC 27710, USA.
Cisco
Robin
R
Dannull
Jens
J
Ueno
Tomio
T
Abdel-Wahab
Omar
O
Kalady
Matthew F
MF
Onaitis
Mark W
MW
Tyler
Douglas S
DS
Pruitt
Scott K
SK
D000951:Antigens, Neoplasm / Q000235:genetics; D016827:CD8 Antigens / Q000378:metabolism; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D007371:Interferon-gamma / Q000378:metabolism; D058965:MART-1 Antigen; D008562:Membrane Glycoproteins / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D012313:RNA / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection / Q000379:methods*
D000951:Antigens, Neoplasm; D016827:CD8 Antigens; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D012313:RNA; D007371:Interferon-gamma