A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants

Abstract

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.

Figure 1. Specific CD8+ proliferation induced by BLS-OVA_257–264.

C57BL/6J mice received CFSE-labeled OT-I CD8+ cells and were then immunized s.c. with BLS-OVA_257–264 (BLS-OVAp), OVA in IFA, OVA_257–264 (OVAp), OVAp in IFA, BLS or PBS (control). After either 20 h or 5 days, draining lymph nodes were removed, and the CFSE label was analyzed by FACS. The intensity of CFSE fluorescence in CD8+ cells (gated on CFSE+ cells) as an indicator of cell proliferation is shown. Histograms depict representative results for BLS-OVAp and control at 20 h (A) or 5 days (C). Bars represent CFSE mean fluorescence intensity (MFI) + SD for all groups (n = 10) at 20 h (B) or 5 days (D). **p<0.0001 or *p<0.01 compared to control. Data of three independent experiments have been pooled.

Figure 2. Early activation of specific cells induced by BLS-OVA_257–264.

C57BL/6J mice received CFSE-labeled OT-I CD8+ cells and were then immunized s.c. with BLS-OVA_257–264 (BLS-OVAp), OVA in IFA, OVAp, OVAp in IFA, BLS or PBS (control). At 20 h, draining (inguinal) lymph nodes were removed, and CD69 fluorescence was analyzed by FACS. A: Histograms show CD69 expression in CFSE+ cells. B: Percentage of CFSE+ cells expressing CD69. Bars represent the fold increase of the mean % + SD (n = 9). **p<0.001 and *p<0.05 compared to control. Data of two independent experiments have been pooled.

Figure 3. Changes induced by BLS-OVA_257–264 in the phenotype of CD8+-specific cells.

C57BL/6J mice received CFSE-labeled OT-I CD8+ cells and were then immunized s.c. with BLS-OVAp, OVA in IFA, OVAp, OVAp in IFA, BLS or PBS (control). After 5 days, inguinal lymph nodes were removed, and the expression of CD44 and CD127 was analyzed by FACS. A: Dot plots show CD44 and CFSE expression in lymph node cells. B: Bars represent the percentage+SD of CFSE+ cells expressing high levels of CD44+ (n = 10); *p<0.001 compared to control. C: Bars represent the MFI+SD of CD127 in CD44^high CFSE+ cells (n = 10); *p<0.05 compared to control. Pooled data of three experiments are shown.

Figure 4. Cross presentation in TLR4-defficient mice.

DCs from C57BL/10ScNJ (TLR4-defficient) or C57BL/10J (wild type) mice were incubated with BLS-OVA_257–264 (BLS-OVAp), OVA or PBS. CD8+ cells from OT-I mice were stained with CFSE and added to the DCs. At 20 h, non-adherent cells were removed and analyzed by FACS. A: Bars represent the mean percentage of specific CD8+ cells that proliferated (gated on CFSE+cells)+SD (n = 6). B: Mean percentage of CD8+ cells expressing CD69+SD (n = 6). *p<0.05 compared to PBS. Data from two independent experiments have been pooled.

Figure 5. Specific cytotoxicity induced by BLS-OVA_257–264.

A: Representative overlayed histograms of CFSE^high and CFSE^low populations within CFSE+ cells from inguinal lymph nodes of C57BL/6J mice immunized with BLS-OVA_257–264 (BLS-OVAp) in PBS, BLS-OVAp in AlOH or PBS (control). B: Bars represent the mean percentage+SD of specific cytotoxicity in lymph nodes of mice immunized with BLS-OVAp in AlOH, OVA in AlOH, BLS-OVAp in PBS, OVA in PBS, OVAp in PBS, BLS in PBS or with PBS (n = 12). *p<0.05 compared to control. Data from two independent experiments have been pooled (6 mice per group).

Figure 6. IFN-γ induced by BLS.

A: C57BL/10J or C57BL/10ScNJ were immunized with BLS in the right hind footpad. At 48 h total RNA from draining lymph nodes was obtained. The expression of IFN-γ was determined by RT-PCR. Data are expressed as the fold increase of mRNA in draining versus non-draining lymph nodes (n = 6). B: C57BL/10J or C57BL/10ScNJ mice were immunized with BLS or PBS. Splenocytes were re-stimulated with BLS in vitro. IFN-γ in the supernatants were measured by ELISA. ND: Not detectable. Bars represent means+SDs (n = 6). *p<0.05 compared to control. Data of two independent experiments have been pooled.

Figure 7. BLS-OVA_257–264 induces a CTL response via TLR4.

A: Representative histograms of CFSE^high and CFSE^low populations within CFSE+ cells from inguinal lymph nodes of C57BL/10J or C57BL/10ScNJ mice immunized with BLS-OVA_257–264 (BLS-OVAp) or OVA in IFA. The percentage of specific lysis is shown. B: Bars represent the mean percentage+SD of specific cytotoxicity in lymph nodes of C57BL/10J or C57BL/10ScNJ mice immunized with BLS-OVAp in PBS, OVA in IFA or with PBS (n = 12). *p<0.05 compared to control. Data from two independent experiments have been pooled (6 mice per group).