CD103-CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin

Abstract

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received considerably less attention. Here, we demonstrate that TLR5 expression by dendritic cells (DCs) allows a 1,000-fold enhancement of T-cell sensitivity to flagellin, and this enhancement did not require the expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T-cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of ovalbumin (OVA)-specific T cells to OVA. In the spleen, the exquisite T-cell sensitivity to flagellin was regulated by CD4-CD8α- DCs and was blocked by a monoclonal antibody to TLR5. In the mesenteric lymph nodes, flagellin-specific T-cell activation was regulated by a population of CD103-CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DC subsets controls the sensitivity of the adaptive immune response to flagellated pathogens.

Figure 1. In vivo expansion of flagellin-specific T cells requires TLR5, but not Myd88

Wild-type, TLR5-deficient, and Myd88-deficient mice were adoptively transferred with 1×10⁶ CD90.1 OT-II or SM1 TCR transgenic T cells and immunized with 100µg OVA plus 1µg flagellin. Three days later clonal expansion of OT-II and SM1 T cells was assessed in the spleen. (A) FACS plots show CD4 and CD90.1 staining of the gating on live cells. (B) Graphs show the mean percentage +/− SEM of OT-II and SM1 T cells, (★★★) denotes a p value <0.0001, (★★) denotes a p value <0.01, and (★) denotes a p value <0.05, as analyzed by two-way ANOVA.

Figure 2. Robust flagellin-specific T cell responses in NLRC4-deficient mice

Wild-type and NLRC4-deficient mice were adoptively transferred with CFSE labeled SM1 T cells and immunized intravenously the following day with 1µg of flagellin and 10µg of LPS. (A) FACS plot show expansion of SM1 T cells (CD4 and CD90.1 positive) in the spleen of wild-type and NLRC4-deficient mice three days after immunization. (B) Bar plots show the percentage of SM1 T cells in the spleen of wild-type and NLRC4-deficient mice three days after immunization. There is no significant difference between immunized groups, as evaluated by unpaired t-test (p<0.05).

Figure 3. TLR5 enhances T cell activation to low doses of flagellin

1×10⁶ CFSE labeled TCR transgenic T cells were adoptively transferred into wild-type and TLR5-deficient mice and the following day were immunized with different doses of ovalbumin (1–100µg) or flagellin (100pg–100µg) plus 10 µg of LPS. Three days later clonal expansion of (A) OT-II or (B–C) SM1 T cells was examined in the spleen. Upper plots show staining of CD4 and CD90.1 to identify transgenic T cells and lower plots show CD11a and CFSE staining after gating on OT-II and SM1 T cells. Each plot is a representative of three mice per group and two independent experiments. (D) Bar plots showing the percentage and total number of OT-II T cells in the spleen of Ova (1–100µg) immunized animals. (E–F) Plots show the percentage and total number (±SEM) of SM1 T cells in the spleen of (E) wild-type and (F) TLR5-deficient mice immunized with flagellin. Graphs combine the results of two independent experiments with three mice per group. Statistical significance was examined using unpaired t-test between the groups immunized with Ova (100µg) or flagellin (1µg) and transfer only. (★★★) denotes a p value <0.0001, (★★) denotes a p value <0.01, and (★) denotes a p value <0.05, as analyzed by unpaired t-test.

Figure 4. TLR5 expression by CD11c+DCs controls the sensitivity of flagellin-specific T cell activation

DCs isolated from C57BL/6 (wild-type) and TLR5-deficient mice were cultured with TCR transgenic (SM1 or OT-II) T cells and various concentrations of antigen (flagellin or OVA). (A) Graph shows increased expression of CD69 on gated (CD4+CD90.1+) OT-II T cells after 16 hours of incubation with wild-type DCs and OVA (1ng–100µg)/ml. (B) Graph shows increased expression of CD69 on (CD4+CD90.1+) SM1 T cells after 16 hours of incubation in the presence of wild-type (solid line) or TLR5 deficient DCs (dotted line) with flagellin (1ng–10µg)/ml or flagellin peptide (6µM). (C) DCs were cultured with TCR transgenic OT-II T cells and various concentrations of OVA (µg/ml), OVA plus flagellin, or OVA plus LPS. Graph shows increased expression of CD69 on gated (CD4+CD90.1+) OT-II T cells after 16 hours of incubation with wild-type DCs. (D) DCs isolated from wild-type, TLR5-deficient, or Myd88-deficient mice were cultured with OT-II T cells and OVA (100µg/ml) or OVA plus flagellin (1ng/ml), or OVA plus LPS (1ng/ml). Graph shows increased expression of CD69 on (CD4+CD90.1+) OT-II T cells after 16 hours of incubation with wild-type, TLR5-deficient, or Myd88-deficient DCs.

Figure 5. DC expression of TLR5, but not Myd88, is required to activate flagellin-specific T cells

DCs from the spleen of wild-type, TLR5-deficient and Myd88-deficient mice were incubated with flagellin and SM1 T cells to examine T cell activation. (A–B) 1×10⁵ DCs were cultured with 1×10⁵ SM1 T cells for 16 (CD69 and CD25) or 48h (CD44 and CD62L) in the presence of 10 ng/ml of flagellin (solid line) or medium alone (shaded area). (A) Plots show cell surface expression of CD69, CD25, CD44, or CD62L after gating on (CD4+CD90.1+) SM1 T cells. (B) Production of IL-2 in culture supernatants was assessed by ELISA 48 hours after incubation with different concentrations of flagellin or medium alone. Graph shows mean IL-2 +/− SEM in tissue culture replicates after stimulation. (C) Prior treatment of DCs with a monoclonal anti-TLR5 antibody (10µg/ml) for 60 min inhibited flagellin-specific activation of SM1 T cells. Plots show CD69 surface staining after gating on CD4+CD90.1 SM1 T cells. All data are representative of three independent experiments.

Figure 6. Splenic DC subsets have differential ability to activate flagellin-specific T cells

CD8α+, CD8α− CD4+CD8α− and CD4−CD8α− splenic CD11c+ DC subsets were purified using DC enrichment kits and were analyzed for their ability to activate flagellin-specific T cells in vitro. DC subsets were incubated with SM1 T cells at a 1:1 ratio (1×10⁵ cells/well) in the presence of 10ng/ml of flagellin or 6µM of flagellin peptide (427–441). (A) Upper and lower panel showed the activation of SM1 T cells in the presence of CD11c+CD8α+ lymphoid or CD11c+CD8α-myeloid DCs as evaluated by CD69 and CD25 expression on gated SM1 T cells, 16h post incubation. (B) CD4+CD8α− and CD4−CD8α− myeloid DC subsets 1×10⁵ were co-cultured with 1×10⁵ SM1 T cells for 16hours in the presence of flagellin and T cell activation analyzed by examining CD69 expression on gated SM1 T cells. Data are representative of two independent experiments completed in triplicate.

Figure 7. TLR5 is required for splenic and mucosal DCs to activate flagellin specific T cells

CD11c+ dendritic cells enriched from (A) spleen, (B) peripheral lymph nodes or (C) mesenteric lymph nodes of wild-type and TLR5-deficient mice. FACS plots (left) show activation of SM1 T cells after 16h of culture with wild-type DCs (black) or TLR5-deficient DCs (grey) in the presence or absence of 10ng/ml of flagellin or flagellin peptide (6µM). T cell activation was analyzed by the expression of CD69 on gated SM1 T cells (CD4+CD90.1+). Graphs (right) show increased expression of CD69 on (CD4+CD90.1+) SM1 T cells after 16 hours of incubation in the presence of wild-type (λ) or TLR5-deficient DCs (σ) with flagellin (1ng–100ng)/ml or flagellin peptide (6µM). Each data point is representative of mean of two independent experiments done in triplicate, (★★★) denotes a p value <0.0001, as analyzed by two-way ANOVA. (D) CD11c+ dendritic cells from mesenteric lymph nodes were incubated with monoclonal TLR5 antibody (10µg/ml) for 60 min and then cultured in a 1:1 ratio with SM1 cells in the presence of flagellin (1ng/ml) and flagellin peptide (6µM). 16 hours later, CD69 expression was measured on T cells by flow cytometry. Data are representative of two independent experiments.

Figure 8. Mesenteric lymph node DC subsets have differential ability to activate flagellin specific T cells

(A) CD103 positive or negative myeloid DCs from mesenteric lymph nodes were enriched from CD11c+ DCs using magnetic beads and cultured with SM1 T cells for 16h in the presence of flagellin (10ng/ml) or flagellin peptide (6µM). FACS plots (left) show CD69 expression on gated SM1 T cells. Bar graphs (right) show dose dependent activation of SM1 T cells cultured with CD103 positive or negative fractions. Each bar shows the mean percentage of CD69+ cells after gating on SM1 T cells. (B) FACS plots show activation of SM1 T cells cultured for 16h with CD11b positive or negative dendritic cells fraction in the presence or absence of flagellin (10ng/ml) or flagellin peptide (6µM). Bar graphs (right) show dose dependent activation of SM1 T cells cultured with CD11b+ or CD11b− MLN DCs. Each bar shows the mean percentage of CD69+ cells after gating on SM1 T cells. (C) CD11b+ dendritic cells pretreated with monoclonal anti-TLR5 antibody were cultured with SM1 T cells and T cell activation was examined 16h later. (★★) denotes a p value <0.01 and ns denotes a p value >0.05 as analyzed by unpaired t-test.