(B) Anti-OVA antibody IgG1. c57BL/6 and mice mice, and further exhibited the role of TLR2 in the adjuvanticity of TFPR1 in vivo. In addition, we obtained a novel combined adjuvant, TFCAl, based on TFPR1, which can augment antibody and cellular immune responses in mice with different genetic backgrounds, suggesting its promise for vaccine development in the future. value of less than 0.05 was considered statistically significant. 3. Results 3.1. TFPR1 Functions as an Effective Adjuvant for the Model Antigen OVA in C57BL/6 Mice In our previous studies, it was found that the recombinant protein TFPR1 could augment effective immune responses in BALB/c mice [21]. Here, we evaluated the adjuvanticity of TFPR1 in C57BL/6 mice. As in the BALB/c mice, TFPR1 augmented both humoral and intracellular immune responses with a Th1-bised type. C57BL/6 mice immunized with OVA plus TFPR1 experienced higher titers of anti-OVA IgG antibody ( 0.001 vs. OVA) (Physique 1A), Th2-associated subclass IgG1 antibody ( 0.001 vs. OVA) (Physique 1B) and Th1-associated subclass IgG2a antibody ( 0.001 vs. OVA) (Physique 1C). Unlike the BALB/c mice, C57BL/6 mice immunized with OVA plus TFPR1 generated significantly high numbers of IFN–secreting cells ( 0.01 vs. OVA) (Physique 1D). By contrast, alum induced a Th2-biased antibody responses (Physique 1C) and greater amounts of IL-4 (Physique 1E). These results showed that TFPR1 has comparable adjuvanticity for OVA in C57BL/6 mice and BALB/c mice [31], though it induces higher levels of cellular immune responses. Open in a separate window Physique 1 Anti-OVA antibodies and cellular immune responses in C57BL/6 mice immunized with TFPR1 and OVA. C57BL/6 mice were immunized thrice with TFPR1 and OVA, alum and OVA, OVA alone or PBS intramuscularly. On the second week after the final immunization, anti-OVA antibodies were detected by ELISA and cellular immune responses were measured by ELISPOT. (A) Anti-OVA antibody IgG. (B) Anti-OVA antibody IgG1. (C) Anti-OVA antibody IgG2a. (D) Specific IFN-. (E) Specific IL-4. Notice: * stands for 0.05, ** 0.01, and *** 0.001. 3.2. TFPR1 Activates Splenocytes from BALB/c Mice or C57BL/6 Mice to Produce Cytokines with Different Profiles We next compared cytokine profile differences between C57BL/6 and BALB/c mice in vitro by stimulating mouse splenocytes with TFPR1, and a TLR2/1 agonist Pam3CSK4 was used as a positive control. As shown in Physique 2, TFPR1 stimulated splenocytes from C57BL/6 Stevioside Hydrate to secrete significantly higher levels of IFN- ( 0.001) (Physique 2A) and IL-6 ( 0.01) (Physique 2B) but reduced production of IL-8 ( 0.001) Stevioside Hydrate (Physique 2C) and IL-10 ( 0.05) (Figure 2D) compared to splenocytes from BALB/c mice; the positive control, Pam3CSK4, activated splenocytes to generate comparable but Cspg2 different cytokine patterns from TFPR1 in two strains of mice; IFN- was at lower concentrations in the supernatant from C57BL/6 splenocytes than it was in the supernatant from BALB/c spleen cells (Physique 2A). In the mean time, cytokine Stevioside Hydrate profiles were observed to be different between the two kinds of mice in vivo; higher levels Stevioside Hydrate of IFN- ( 0.01) (Physique S1A), IL-6 ( 0.001) (Physique S1B), IL-8 ( 0.01) (Physique S1C) and IL-10 ( 0.001) (Physique S1D) were measured in the sera of C57BL/6 mice than those of BALB/c mice. Taken together, TFPR1 is able to trigger immune cells to produce proinflammatory and regulatory cytokines in both C57BL/6 and BALB/c mice, but with different patterns, which may explain the difference in the immune responses elicited by TFPR1. Open in a separate windows Physique 2 Detection of cytokines secreted by splenocytes from BALB/c and C57BL/6 mice. Splenocytes either from BALB/c or C57BL/6 mice were stimulated with TFPR1 or Pam3CSK4 for 24 h, and cytokines in culture supernatants were measured using ELISA packages. (A) IFN-. (B) IL-6. (C) IL-8. (D) IL-10. Notice: * stands for 0.05, ** 0.01, and *** 0.001. n.s.: no significance. 3.3. TFPR1 Activates TLR2-KO Mice to Generate Lower Levels of Cytokines than Those.