Figure 4. Loss of Toll-like receptor signaling results in reduced B-1a responses to both phosphatidylcholine and the microbiota.

(A) Frequency of live B-1a cells in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice in the peritoneal cavity (PerC) and (B) spleen, as measured by flow cytometry. B-1a cells defined as CD19⁺CD23^–CD43⁺CD5⁺. (C) Serum IgM titers in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice, as measured by ELISA. (D) Percentage of SYBR⁺μMT^-/- fecal bacteria bound by serum IgM from WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice, as measured by flow cytometry. (E) Representative flow cytometry plot and quantification (F-G) of percentage of B-1a cells in the (F) peritoneal cavity (PerC) and (G) spleen bound by fluorescein-labeled phosphatidylcholine liposomes in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice. (H) Schematic showing adoptive transfer of CD19⁺ pooled bone marrow and spleen cells (B cells) from either C57BL/6 (WT) (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) into B cell deficient μMT^-/- recipeint mice at 2 days of age and sacrificed at 10 weeks of age. (I-J) Frequency of live B-1a cells in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) B cell recipient mice in the (J) peritoneal cavity (PerC) and (J) spleen, as measured by flow cytometry. (K) Serum IgM titers in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) B cell recipient mice, as measured by ELISA. (L) Percentage of SYBR⁺ fecal bacteria bound by serum IgM from WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) B cell recipient mice, as measured by flow cytometry. Error bars indicate the mean (± SEM). *p<0.05, **p<0.01, and ***p<0.001 (unpaired two-tailed Student's t-test). Each data point represents an individual mouse. Data are pooled from at least three independent experiments (A-G; I-L).

Figure 4—figure supplement 1. TLR expression on B cell subsets using TLR reporter mice.

(A–E) Expression of TLR2, TLR4, TLR5, TLR7, and TLR9 on peritoneal cavity B-2 cells (CD19⁺CD23⁺) and B-1a cells (CD19⁺CD23^–CD43⁺CD5⁺) using Tlr2^IRES-GFP, Tlr4^IRES-YFP, Tlr5^{IRES-TdTomato}, Tlr7^{IRES-TdTomato}, Tlr9^IRES-GFP reporter mice, respectively, or C57BL/6 mice as controls. Each data point represents an individual mouse. Error bars indicate the mean (± SEM). *p<0.05, **p<0.01, and ***p<0.001 (unpaired two-tailed Student's t-test). Data are representative of at least two experiments.

Figure 4—figure supplement 2. Loss of Toll-like receptor signaling results in reduced B-1a responses to both phosphatidylcholine and the microbiota in co-housed mice.

WT (black) and Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice were cohoused for 4 weeks post weaning to normalize microbiota between mice. (A-B) Percentage of B-1a cells bound by fluorescein-labeled phosphatidylcholine liposomes (PtC+) in the (A) peritoneal cavity (PerC) and (B) spleen in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice, as measured by flow cytometry. B-1a cells defined as CD19⁺CD23^–CD43⁺CD5⁺. (C) Serum IgM titers in 7 wk old WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice, as measured by ELISA. (D) Percentage of SYBR⁺ fecal bacteria bound by serum IgM from WT (black) or Tlr2^-/-Tlr4^-/-Unc93b1^3d/3d (blue) mice, as measured by flow cytometry. Error bars indicate the mean (± SEM). *p<0.05, **p<0.01, and ***p<0.001 (unpaired two-tailed Student's t-test). Each data point represents an individual mouse. Data are pooled from four independent experiments.