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. Author manuscript; available in PMC: 2012 Dec 23.
Published in final edited form as: Immunity. 2011 Dec 23;35(6):997–1009. doi: 10.1016/j.immuni.2011.10.018

Figure 4.

Figure 4

Cross-serotype protection with heterologous bacterial challenge requires Th17 cells, not B-cells. (A) Various stains of heat-killed K. pneumoniae were coated on a ELISA plate and lung homogenate from naïve mice (labelled as N) and K. Pneumoniae-43816 (ATCC) (serotype K2) immunized mice (labelled as V) were tested for cross reactivity. K. Pneumoniae strain numbers and serotypes are also listed. (B) Mediastinal lymph nodes from immunized C57BL/6 mice were labelled with CFSE and cultured with different serotypes of heat-killed K. Pneumoniae, E. coli, S. aureus and S. pneumoniae for 3 days. Proliferation of IL-17+ cells was analyzed by intracellular IL-17 staining and CFSE dilution. C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 10 days after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 K. Pneumoniae-303 (serotype K16) (C), K. Pneumoniae-396 (serotype K1) (D) or K. Pneumoniae-NDM1+(serotype unknown) (E) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each groups has 4–5 mice. (F)Ighm−/− mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. Four weeks after the 2nd immunization, immunized Ighm−/− mice and naïve unimmunized Ighm−/− mice were infected with 104 live K. pneumoanie-396 (serotype K1) and sacrificed 24h after. A group of immunized mice also received i.t. neutralizing antibody against IL-17A right before infection. Bacterial burden were determined by lung CFU (left panel) and spleen CFU (right panel). Th17 responses in the lung were analyzed by RT-PCR (G). Data are from two independent experiments with 3–4 mice each group. (H) IL-17F-Thy1.1 reporter mice were immunized with heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) and were sacrificed one week after. Lung single cells were prepared and stained with CD4 and Thy1.1. flow cytometry sorted 105 Th17 (CD4+Thy1.1+) per mouse or 105 B cells (CD19+CD4− Thy1.1−) per mouse were intravenously transferred to Rag2−/− mice. Four week after transfer, mice were infected with 104 K. pneumoanie-396 (serotype K1) and sacrificed at 24h. Lung burdens were assessed by CFU. Each group has an N=5.