antigen specific memory th17 cell recall response  (ATCC)

Journal: Immunity

Article Title: Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Figure 1. Immunization with Heat-Killed K. pneumoniae Induces Antigen-Specific Th17 Responses (A) Radial cladogram of IL-17A(A), IL-17D, IL-17F protein families from different organisms including mammal (dark blue), bird (red), fish (sky blue), frog (pink), C. intestinalis (lime), and oyster (green). The following abbreviations are used: Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos Taurus; Rn, Rattus norvegicus; Ss, Sus scrofa; Ec, Equus caballus; Md, Monodelphis domestica; Ol, Oryzias latipes; Sr, Salmo salar; Tr, Takifugu rubripes; Dr, Danio rerio; Gg, Gallus gallus; Ci, Ciona intestinalis; Xt, Xenopus tropicalis; Cg, Crassostrea gigas.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae-43816 (ATCC) Th17 cells but not GD17 cells; cells responded to K. pneumoniae43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1b and IL-23 (Figure S1F), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1b and IL-23. (B) C57BL/6 mice were immunized intranasally with 20 mg heat-killed K. pneumo Naivemice were sacrificed 24 hr after 104 liveK. pneumoniae-43816 (ATCC) (sero intracellular cytokine staining (upper panel).

Techniques:

Journal: Immunity

Article Title: Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Figure 2. Characterization of Immunization-Induced T Cell Responses C57BL/6 mice were immunized intranasally with 20 mg of heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) at day 0 and sacrificed 1, 2, and 4 weeks after lung, spleen, and mediastinal lymph nodes were collected and intracellular staining was performed. Each time point has a group of three mice. (A) Representative flow cytometry plots of IL-17A and IFN-g staining on CD4 gated cells in the spleen. (B) Representative flow cytometry plots of T-bet (upper panel) and Rorgt (lower panel) staining on IL-17A+ (Th17 blue line) and IFN-g+ (Th1 red line) from the spleen. Mean fluorescent intensity of T-bet or Rorgt is shown on each plot.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae-43816 (ATCC) Th17 cells but not GD17 cells; cells responded to K. pneumoniae43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1b and IL-23 (Figure S1F), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1b and IL-23. (B) C57BL/6 mice were immunized intranasally with 20 mg heat-killed K. pneumo Naivemice were sacrificed 24 hr after 104 liveK. pneumoniae-43816 (ATCC) (sero intracellular cytokine staining (upper panel).

Techniques: Staining, Cytometry

Journal: Immunity

Article Title: Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Figure 3. Mucosal Immunity to an Autologous Bacterial Challenge Is Mediated by B Cells and Th17 Cells (A) C57BL/6 mice were immunized intranasally with 20 mg of heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) at day 0 and day 7. Four weeks after the 2nd

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae-43816 (ATCC) Th17 cells but not GD17 cells; cells responded to K. pneumoniae43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1b and IL-23 (Figure S1F), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1b and IL-23. (B) C57BL/6 mice were immunized intranasally with 20 mg heat-killed K. pneumo Naivemice were sacrificed 24 hr after 104 liveK. pneumoniae-43816 (ATCC) (sero intracellular cytokine staining (upper panel).

Techniques:

Journal: Immunity

Article Title: Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: 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 an ELISA plate, and lung homogenate from naive mice (labeled as N) and K. pneumoniae-43816 (ATCC) (serotype K2)-immunized mice (labeled as V) was tested for crossreactivity. K. pneumoniae strain numbers and serotypes are also listed. (B) Mediastinal lymph nodes from immunized C57BL/6 mice were labeled 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 mg heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) at day 0 and day 7.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae-43816 (ATCC) Th17 cells but not GD17 cells; cells responded to K. pneumoniae43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1b and IL-23 (Figure S1F), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1b and IL-23. (B) C57BL/6 mice were immunized intranasally with 20 mg heat-killed K. pneumo Naivemice were sacrificed 24 hr after 104 liveK. pneumoniae-43816 (ATCC) (sero intracellular cytokine staining (upper panel).

Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Cell Culture, Staining

Journal: Immunity

Article Title: Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Figure 5. Cross-Serotype Protection in Heterologous Challenge Is Mediated by Th17 not Th1 (A) C57BL/6 and Ifng/ mice were immunized intranasally with 20 mg heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) at day 0 and day 7. Twenty-eight days after the 2nd immunization, immunized mice and naive unimmunized mice were infected with 104 K. pneumoniae-396 (serotype K1) and sacrificed 24 hr later. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has four to five mice. (B) IL-17F-Thy1.1 reporter mice were immunized with heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) and were sacrificed 1 week later. Lung single cells were prepared and stained with CD4 and Thy1.1. Flow-cytometry-sorted 105 Th17 (CD4+Thy1.1+) per mouse or 105 Th-non17F (CD4+Thy1.1-) per mouse was intravenously transferred to Rag2/Il2rg/ mice. Four weeks after transfer, mice were infected with 104 K. pneumoniae-396 (serotype K1) and sacrificed at 24 hr. Lung burdens were assessed by CFU. Each group has three to four mice. (C) IL-17F-Thy1.1 reporter mice were immunized with heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) and were sacrificed 1 week later. Lung single cells were prepared and stained with CD4 and Thy1.1. flow-cytometry -orted 105 Th17 (CD4+Thy1.1+) per mouse were intravenously transferred to Rag2/ mice. Four weeks after transfer, mice were infected with 104 K. pneumoniae-396 (serotype K1). Control IgG, a-IL17RC or a-IFN-g were given i.t. 12 hr after infection and mice were sacrificed at 24 hr after infection. Lung and spleen burdens were assessed by CFU. Each group has four mice. (D) Il17ra/ mice with B cell depletion (0.1 mg anti-CD20 i.p. weekly) mice were immunized intranasally with 20 mg heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) at day 0 and challenge with 104 K. pneumoniae-396 (serotype K1) on day 28. Control IgG or a-IFN-g were given i.t. 12 hr after infection and mice were sacrificed at 24 hr after infection. Lung bacterial burdens were assessed by CFU. Each group has four to five mice. Data represent the mean ± SD of each group.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae-43816 (ATCC) Th17 cells but not GD17 cells; cells responded to K. pneumoniae43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1b and IL-23 (Figure S1F), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1b and IL-23. (B) C57BL/6 mice were immunized intranasally with 20 mg heat-killed K. pneumo Naivemice were sacrificed 24 hr after 104 liveK. pneumoniae-43816 (ATCC) (sero intracellular cytokine staining (upper panel).

Techniques: Infection, Staining, Flow Cytometry, Cytometry, Control

Journal: Immunity

Article Title: Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

doi: 10.1016/j.immuni.2011.10.018

Figure Lengend Snippet: Figure 6. The Th17 Response to Klebsiella OMPs Is Partially MHC-II Dependent (A) flow cytometry sorted CD4 T cells from K. pneumoniae-43816 (ATCC) (serotype K2)-immunized mice mediastinal lymph nodes were labeled with CFSE and cultured with flow cytometry sorted CD11+ DCs from C57BL/6 or MHC-II KO spleen for 4 days in the presence or absence of 10 ml/ml OMP from K. pneumoniae- 396 (serotype K1) or K. pneumoniae-43816 (ATCC) (serotype K2). Th17 proliferation was measure by intracellular IL-17 staining and CFSE dilution. Data are representative from two independent experiments. (B) IL-1b levels in the culture supernatants were measured by Luminex. (C) C57BL/6 mice were immunized with heat-killed K. pneumoniae-43816 (ATCC) (serotype K2) (labeled as HKKP) or 1 mg LPS or 1 mg LPS plus 10 mg OMPs isolated from K. pneumoniae-43816 (ATCC) (serotype K2). Seven days later, mice were sacrificed and lungs were harvested for intracellular IL-17 and IFN-g analysis on gated CD4+ T cells by flow cytometry. (D) Total Th1 (CD4+IFN-g+) and Th17 (CD4+IL-17+) cells were graphed. (E) Four weeks after immunization, LPS or LPS+OMP-immunized mice were challenged with 104 live K. pneumoniae-396 (serotype K1), bacterial burden was determine by lung and spleen CFU. Data are from two independent experiments with three to five mice in each group. (F) Ighm/ mice were immunized with 10 mg OMP isolated from K. pneumoniae-43816 (ATCC) (serotype K2) and challenged with K. pneumoniae-396 (serotype K1) 4 weeks later. Control IgG or anti-IL17RC was given i.t. 12 hr after infection. Mice were sacrificed at 24 hr, and bacterial burden was determined by lung CFU. Each group has five mice. Data represent the mean ± SD of each group.

Article Snippet: To further support this, equal numbers of sorted GD17 and Th17 cell were cultured with heat-killed K. pneumoniae-43816 (ATCC) Th17 cells but not GD17 cells; cells responded to K. pneumoniae43816 (ATCC) restimulation and produced IL-17 in the presence or absence of neutralizing antibodies against IL-1b and IL-23 (Figure S1F), indicating the antigen-specific memory Th17 cell recall response occurred independently of IL-1b and IL-23. (B) C57BL/6 mice were immunized intranasally with 20 mg heat-killed K. pneumo Naivemice were sacrificed 24 hr after 104 liveK. pneumoniae-43816 (ATCC) (sero intracellular cytokine staining (upper panel).

Techniques: Cytometry, Labeling, Cell Culture, Staining, Luminex, Isolation, Control, Infection