Enhanced antigen processing of flagellin fusion proteins promotes the antigen-specific CD8+ T cell response independently of TLR5 and MyD88

Abstract

Flagellin is a highly effective adjuvant for CD4(+) T cell and humoral immune responses. However, there is conflicting data in the literature regarding the ability of flagellin to promote a CD8(+) T cell response. In this article, we report that immunization of wild-type, TLR5(-/-), and MyD88(-/-) adoptive transfer recipient mice revealed the ability of flagellin fusion proteins to promote OVA-specific CD8(+) T cell proliferation independent of TLR5 or MyD88 expression by the recipient animal. Wild-type and TLR5(-/-) APCs were able to stimulate high levels of OVA-specific CD8(+) T cell proliferation in vitro in response to a flagellin fusion protein containing full-length OVA or the SIINFEKL epitope and 10 flanking amino acids (OVAe), but not to OVA and flagellin added as separate proteins. This effect was independent of the conserved regions of flagellin and occurred in response to OVAe alone. Comparison of IFN-γ production by CD8(+) effector cells revealed higher levels of SIINFEKL peptide-MHC I complexes on the surface of APCs that had been pulsed with OVAe-flagellin fusion proteins than on cells pulsed with OVA. Inhibition of the proteasome significantly reduced Ag-specific proliferation in response to OVAe fusion proteins. In summary, our data are consistent with the conclusion that flagellin-OVA fusion proteins induce an epitope-specific CD8(+) T cell response by facilitating Ag processing and not through stimulatory signaling via TLR5 and MyD88. Our findings raise the possibility that flagellin might be an efficient Ag carrier for Ags that are poorly processed in their native state.

FIGURE 1

Flagellin–OVA fusion protein stimulates in vivo OVA-specific CD8⁺ T cell proliferation independent of a requirement for TLR5 or MyD88. C57BL/6 or TLR5^−/− mice received 1 × 10⁶ CFSE-labeled OT-I cells and were immunized the following day with 10⁻¹⁰ moles of OVA, OVA + flagellin, or OVAe-flagellin. Three days later, CFSE dilution (A) and OT-I cell number (B) in the draining popliteal LN was analyzed by flow cytometry. Plots are gated on CD8⁺CD90.1⁺ events. Data are representative of three mice per group. *p < 0.05 compared with the OVA group.

FIGURE 2

Design of flagellin full-length and HV region fusion proteins. OVA_247-274 and OVA_313-349 were fused to N terminus full-length flagellin or the HV region of flagellin from S. enteritidis (residues 180–419) or P. aeruginosa (residues 181–398).

FIGURE 3

The enhanced proliferative response to the SIINFEKL epitope in fusion proteins is not dependent on the conserved regions of flagellin or on TLR5 expression by APCs. Total LN cell suspensions were generated from the s.c. LNs of wild-type and TLR5^−/− mice and mixed with CD8-enriched, CFSE-labeled OT-I cells at a ratio of 9:1. Cells were stimulated with OVA, OVAe, OVAe–flagellin_Se, OVAe–flagellin_Pa, OVAe–HV_Se, OVAe–HV_Pa, or flagellin–OVA and incubated for 3 d at 37°C before harvest. CFSE dilution (A), OT-I cell number (B), and the % divided of CFSE-labeled OT-I cells (C) were determined by flow cytometry. Data are representative of triplicate cultures from two independent experiments.

FIGURE 4

OT-I effector cells produce IFN-γ in response to incubation with TLR5^−/−CD11c⁺ cells pulsed with OVAe, OVAe fusion, or flagellin–OVA fusion proteins, but not with OVA. CD11c⁺ cells were enriched from the LNs of TLR5^−/− mice and pulsed overnight with 10⁻⁷ M immunogen. The next day, CD11c⁺ cells were washed and mixed with OT-I effector cells incubated for 5 h with brefeldin. Cells were then stained to determine intracellular production of IFN-γ by flow cytometry. Data are representative of triplicate cultures from two independent experiments.

FIGURE 5

Inhibition of proteasome function significantly reduces CD8⁺ T cell proliferation in response to stimulation with OVAe–flagellin fusion protein. Total LN cell suspension was generated from the s.c. LNs of TLR5^−/− mice and treated with chloroquine (50 μM) or lactacystin (20 μM) for 2 h before the addition of Ag for 6 h. Cells were then washed and incubated overnight in chloroquine (5 μM) or lactacystin (2 μM). The following day, cells were washed and mixed with CD8-enriched OT-I cells. Cultures were harvested 3 d later, and the number of OT-I cells recovered from each condition was determined by bead-based flow cytometric counting. Data are representative of triplicate cultures from two independent experiments. *p < 0.0001.