Figure 1.
Protective immunity to N. brasiliensis re-infection is IL-13 and IL-4Rα dependent.
IL-4Rα−/− and IL-4Rα−/lox mice were infected for 5 or 7 days post-secondary N. brasiliensis infection (A). Intestinal worm burdens were then quantified (B). Pulmonary mucus production was established by PAS staining (C). Serum Antibody titers of N. brasiliensis specific IgG1 were determined by ELISA (D). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B-cell populations (E). IL-4−/−, IL-13−/− and IL-4Rα−/lox mice were infected for 5 days post-secondary N. brasiliensis infection and intestinal worm burdens were then quantified (F). Pulmonary mucus production was established by PAS staining (G). Serum Antibody titers of N. brasiliensis specific IgG1 were determined by ELISA (H). Data is representative of 3–4 independent experiments. n = 4–6 mice per group.
Figure 2.
B cell IL-4Rα expression is required for optimal immunity to N. brasiliensis re-infection.
MB1CreIL-4Rα−/lox and IL-4Rα−/lox mice were infected for 5 or 7 days post-secondary N. brasiliensis infection and intestinal worm burdens were then quantified (A). Pulmonary mucus production was established by PAS staining (B). Serum Antibody titers of N. brasiliensis IgG1 were determined by ELISA (C). Mediatstinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B cell populations (D). B cells were isolated from N. brasiliensis infected BALB/c, MBcreIL-4Rα−/lox and IL-13−/− and transferred into naïve BALB/c mice (E). Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were then established at day 5 post infection (F & G). The results shown represent 2–4 independent experiments. n = 4–7 mice per group.
Figure 3.
Transfer of N. brasiliensis experienced B cells enhances immunity to N. brasiliensis independently of endogenous B cell populations.
N. brasiliensis infected μMT and BALB/c mice were re-infected with 500xL3 larvae and at day 5 post-secondary infection, the intestinal worm burdens was quantified (A). The possible role for IL-4Rα expressing B cells in boosting immunity independently of endogenous B cells was determined by transfer of B cells isolated from N. brasiliensis infected IL-4Rα−/lox (WT B cells) or MB1Cre IL-4Rα−/lox (IL-4Rα−/− B cells) into naïve μMT mice. These mice were then infected with 500xL3 N. brasiliensis and worm burdens quantified at day 5 post infection (B). Mediastinal lymph node CD3+CD4+ T cell populations IL-13 responses (C) were established by FACS staining. Results shown represent 2 independent experiments. n = 4–7 mice per group.
Figure 4.
B cell MHCII antigen presentation mediates optimal immunity to N. brasiliensis.
Surface expression of CD28 and TCR on CD4+ T cells and CD86 and MHCII on B cells in naive (A) and N. brasiliensis re-infected (B) IL-4Rα−/lox mice and MB1CreIL-4Rα−/lox mice was established by FACS analysis. Histograms: filled gray: isotype control, thin line: IL-4Rα−/lox, thick black line: MB1CreIL-4Rα−/lox. Contributions by MHCII dependent antigen presentation were demonstrated by isolating WT or MHCII−/− B cells from naive or infected mice then adoptively transferring into naive C57BL/6 mice (C). Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were established at day 5 post infection (D). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B cell populations (E). MHC dependent antigen presentation was confirmed by isolating WT and BALB/b B cells from naive or infected mice adoptively transferring into naive BALB/c mice. Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were established at day 5 post infection (F). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B cell populations (G). Data is representative of 2 independent experiments. n = 4–6 mice per group.
Figure 5.
Rapid IL-Rα dependent B cell mediated protection against N. brasiliensis occurs in the lung.
MBcreIL-4Rα−/lox and IL-4Rα−/lox mice were infected for 1 day with N. brasiliensis before spleen B cells were isolated and transferred into naive wild type mice (As in Figure 4C). These were infected with 500xL3 N. brasiliensis and intestinal worm burdens were quantified at day 5 post infection (A). Lung CD3+CD4+ and CD3+CD4+CD44hi T cell populations were analysed by FACS staining (B). Lung CD4+ T cell (C) and mediastinal lymph node CD4+ T cell and B220+ B cell population (D) IL-13 responses were established by intracellular FACS staining. Data is representative of 2 independent experiments. n = 4–6 mice per group.
Figure 6.
B cell MyD88 expression dependent protection against N. brasiliensis infection.
B cells were isolated from WT or MyD88−/− mice 24 hours post N. brasiliensis infection and adoptively transferred into naive WT mice (As in Figure 4C). 24 hours later these mice were infected with 500xL3 N. brasiliensis and subsequently killed 5 days post infection and worm burdens were counted (A). The mediastinal lymph node CD4+ T-cell IL-13 response was established (B). B cells were isolated from naive C57/BL6, MyD88−/− or MHCII−/− mice and pulsed with N. brasiliensis antigen overnight. These were then washed and transferred into naive C57/BL6 mice 24 h prior to infection (C). D5 PI intestinal worm counts are shown (D & F). The mediastinal lymph node CD4+ T-cell IL-13 response was established (E). Data is representative of 2 experiments, n = 5–7 mice per group.