The 3' CCACCA sequence of tRNAAla(UGC) is the motif that is important in inducing Th1-like immune response, and this motif can be recognized by Toll-like receptor 3

Abstract

In this article, the immunogenicity of tRNA and the recognition of tRNA by Toll-like receptors (TLRs) are analyzed. Analyses of the effects of different tRNA(Ala)(UGC) fragments (tRNA(Ala)1-76 [corresponding to positions 1 through 76], tRNA(Ala)26-76, tRNA(Ala)40-76, tRNA(Ala)62-76, tRNA(Ala)1-70, tRNA(Ala)26-70, tRNA(Ala)40-70, and tRNA(Ala)62-70) on the immune responses of hepatitis B surface antigen (HBsAg) were performed with BALB/c mice. Results show that tRNA(Ala)1-76, tRNA(Ala)26-76, tRNA(Ala)40-76, and tRNA(Ala)62-76 adjuvants not only induced stronger T helper (Th) 1 immune responses but also cytotoxic-T-lymphocyte (CTL) responses relative to tRNA(Ala)1-70, tRNA(Ala)26-70, tRNA(Ala)40-70, and tRNA(Ala)62-70 adjuvants in HBsAg immunization. A deletion of the D loop (tRNA(Ala)26-76), anticodon loop (tRNA(Ala)40-76), or TpsiC (tRNA(Ala)62-76) loop of tRNA(Ala)(UGC) does not significantly decrease the adjuvant characteristic of tRNA(Ala)(UGC). However a deletion of the 3'-end CCACCA sequence (tRNA(Ala)1-70, tRNA(Ala)26-70, tRNA(Ala)40-70, and tRNA(Ala)62-70) of tRNA(Ala)(UGC) significantly decreased the adjuvant characteristic in Th1 and CTL immune responses. Moreover, the recognitions of different tRNA(Ala)(UGC) fragments by TLR3, TLR7, TLR8, and TLR9 were analyzed. Results show that a deletion of the 3' CCACCA sequence of tRNA(Ala)(UGC) significantly decreased the recognition by TLR3. We concluded that the 3' CCACCA sequence of tRNA(Ala)(UGC) is the important motif to induce Th1 and CTL responses and this motif can be effectively recognized by TLR3.

FIG. 1.

RNA sequences of different tRNA^Ala(UGC) fragments. The 5′ cap structure sequence (GGGAGA) is not listed in the figure.

FIG. 2.

The anti-HBsAg antibodies (total IgG) after immunization with HBsAg and different adjuvants in BALB/c mice. The sera were collected at weeks 0, 2, 4, 6, and 8, and the antibody titers were determined. Data shown are representative of three independent experiments and are expressed as mean ± SD.

FIG. 3.

The anti-HBsAg subtype IgG1 and IgG2a responses after immunization with HBsAg and different adjuvants at week 8. Data shown are representative of three independent experiments and are expressed as means ± SD.

FIG. 4.

Cytokine responses of T-lymphocyte-enriched splenocytes separated from BALB/c mice after immunization with HBsAg and different adjuvants. Data shown are representative of three independent experiments and are expressed as means ± SD.

FIG. 5.

Lymphoproliferative response of T-lymphocyte-enriched splenocytes separated from BALB/c mice after immunization with HBsAg and different adjuvants. Data shown are representative of three independent experiments and are expressed as means ± SD.

FIG. 6.

The CTL response after immunization with HBsAg and different adjuvants. Lytic activities of effector cells against target cells were assessed. The effector cell-to-target cell ratios (E:T) were set at 12.5:1, 25:1, and 50:1. Data shown are representative of three independent experiments and are expressed as means ± SD.

FIG. 7.

In vitro NF-κB-driven luciferase reporter assays of human TLR3, TLR7, TLR8, and TLR9 activation by different fragments of tRNA^Ala(UGC). These are expressed as activation (n-fold) (luciferase activity upon stimulation divided by luciferase activity of the nonstimulation). Data shown are representative of three independent experiments and are expressed as means ± SD.