Figure 3. TRIF-dependent detection of bacterial viability promotes Tfh cell differentiation.

Wild-type (WT) and Trif^−/− mice were vaccinated intraperitoneally as indicated with either 5×10⁷ live ThyA⁻ EC, heat-killed ThyA⁻ EC (HKEC), or HKEC+RNA(30μg).

(A) Quantitative RT-PCR for Bcl6, Il21 and Tb×21 transcripts in total CD4⁺ T cells on day 5 after vaccination. Fold increase over naïve mice is shown.

(B) Flow cytometry dot plots gated on CD4⁺ T cells showing expression for CD25 and CXCR5 (upper panels), and ICOS and PD-1 within gated CXCR5⁺ T cells (lower panel).

(C) Quantitative RT-PCR for Bcl6, Il21 and Tbx21 transcripts in sorted CD44⁺CD4⁺ T cells either CXCR5⁻ (left contour plot) or CXCR5⁺ (right contour plot). Data represent relative expression to β-actin.

(D, E) Flow cytometry dot plots (D) and percentages (E) of CXCR5⁺ICOS⁺CD4⁺ T cells.

(F, G) Flow cytometry dot plots (F) and percentages (G) of CXCR5⁺PD-1⁺CD4⁺ T cells.

(H) Flow cytometry dot plots for CXCR5 and Bcl6 gated on CD4⁺ T cells.

(I) Flow cytometry of CD44⁺CD25⁻CD4⁺ T cells. Gray dots represent the total CD4⁺ T cell population; black dots represent the CXCR5⁺ICOS⁺CD4⁺ T cell population.

(J) Percentages of total CD44⁺CD4⁺ T cells.

(B–J) Data for CD4⁺ T cells in spleens of indicated genotypes before and 5 days post vaccination. Each symbol represents an individual mouse in scatter plots.

NS, not significant (P > 0.05); *, P<0.05, **, P≤0.01and ***, P≤0.001 (two-tailed unpaired t test). Data are mean±s.e.m. Numbers adjacent to outlined areas indicate percent of cells in gates. Data are representative of at least 3 independent experiments. Mouse numbers are in (E), (G) and (J) WT (naive, n=11; +EC, n=13; +HKEC, n=15; +HKEC+RNA, n=9) and Trif^−/− (naive, n=8; +EC, n=9; +HKEC, n=9; +HKEC+RNA, n=7).

See also Figure S3.