Lipid Motif of a Bacterial Antigen Mediates Immune Responses via TLR2 Signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2−/− DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively; our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen

Direct conjugation of lipid motif on P6 is important for induction of high titers of anti-P6 antibodies and cytokine secretion by antigen-secretion T cells.

<p>WT mice were immunized i.p. with 40 µg of native P6 (▪), non-lipidated rP6 (•), or non-lipidated rP6 plus Pam3Cys (open circle) emulsified in CFA, IFA, and PBS. (<b>A</b>) Anti-P6 Ig levels were measured in pre-immune and post-immune sera by ELISA. (<b>B–D</b>) Frequency of cytokine secreting T cells in spleens from the same animals after 16 weeks were measured by ELISPOT. Splenocytes were assayed directly <i>ex vivo</i> and after 3 day restimulation with BMDCs pulsed with native P6. Plates were developed and spots enumerated microscopically. *p<0.05 2way ANOVA with Bonferroni post-test comparison.</p

Expression of TLR2 on APCs mediates responses to lipoprotein P6.

<p>WT mice were immunized s.c. with 40 µg of native P6 emulsified in CFA and IFA one week later. (<b>A</b>) Proliferation of CD3⁺ cells isolated from draining lymph nodes was measured following 4 day co-culture with syngeneic irradiated WT (black) and TLR2^−/− (gray) BMDCs pulsed with 0.06 µg/ml native P6 or non-lipidated rP6. Thymidine was added to the wells for the last 16 hrs of incubation. (<b>B</b>) BMDCs from WT (black bar) and TLR2^−/− (gray bar) mice were incubated for 1 hr with the indicated stimuli and Dextran-FITC simultaneously (formalin-killed NTHI, f.k.). Cells were harvested and stained with anti-CD11c PE and acquired by two-color flow cytometry (FITC vs PE) to determine endocytic uptake of Dextran-FITC. Results are expressed as percent change in FITC MFI from media control. *p<0.01 1way ANOVA with Bonferroni post-test comparison of WT to TLR2^−/−.</p

Lipid motif on P6 augments T cell proliferation and cytokine production.

<p>WT mice were immunized s.c. with 40 µg of native P6 emulsified in CFA and IFA one week later. (<b>A</b>) Proliferation of CD3⁺ cells isolated from draining lymph nodes was measured following 4 day co-culture with syngeneic irradiated BMDCs pulsed with 0.25, 0.12, and 0.06 µg/ml native P6 (▪) or non-lipidated rP6 (•). Media alone control (▴) was performed for background proliferation of T cells. Thymidine was added to the wells for the last 16 hrs of incubation. (<b>B</b>) Splenocytes from the same animals were co-cultured overnight with 0.06 µg/ml antigen pulsed irradiated BMDCs in ELISPOT plates coated with anti-cytokine mAb. Plates were developed and spots enumerated microscopically. *p<0.01 1way ANOVA with Bonferroni post-test comparison of native P6 to non-lipidated rP6.</p

TLR2 expression is important for antibody and recall cytokine responses against P6.

<p>WT (black bar) and TLR2^−/− (gray bar) mice were immunized i.p. with 40 µg of native P6 emulsified in CFA, IFA, and PBS. (<b>A</b>) Anti-P6 Ig levels were measured in pre-immune and post-immune sera by ELISA. (<b>B–D</b>) Frequency of cytokine secreting T cells in spleens from the same animals after 16 weeks were measured by ELISPOT. Splenocytes were assayed directly <i>ex vivo</i> and after 3 day restimulation with BMDCs pulsed with native P6. Plates were developed and spots enumerated microscopically. *p<0.05 2way ANOVA with Bonferroni post-test comparison of WT and TLR2^−/−.</p

TLR2 expression on APC mediates upregulation of CD40 in response to lipoprotein P6.

<p>BMDCs from WT (top row) and TLR2^−/− (bottom row) mice were incubated overnight with the indicated stimuli. Cells were harvested and stained with anti-CD11c and anti-CD40 and acquired by two-color flow cytometry (FITC vs PE) to determine CD40 expression patterns. Percent of cells expressing CD40 over isotype are provided in each plot. *p<0.05 2way ANOVA with Bonferroni post-test comparison of native P6 between WT and TLR2^−/−. ^†p<0.05 2way ANOVA with Bonferroni post-test comparison of native P6 and non-lipidated rP6 in WT.</p