Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A) FACS profile of spleen and liver mononuclear cells in WT, Il17rb −/− and Il15 L117P mice on a B6 background. Numbers are percentage of gated cells. α-GalCer/CD1d dimer + i NKT cells and α-GalCer/CD1d dimer − NK1.1 + NK cells were slightly decreased in Il17rb −/− mice and markedly reduced in Il15 L117P mice. (B) IL-17RB expression in spleen and liver i NKT cells of WT B6, Il17rb −/− and Il15 L117P mice. Shaded profiles in the histograms indicate the background staining with isotype matched control mAb. (C, D) In vitro cytokine production by spleen i NKT cells from Il17rb −/− and Il15 L117P mice (C) and by liver i NKT cells from Il17rb −/− mice (D). Sorted i NKT cells (5×10 4 /100 µL) from spleen and liver of WT B6 and Il17rb −/− mice were co-cultured with BM-DCs (5×10 3 /100 µL) for 48 h in the presence of the indicated doses of α-GalCer. The Il17rb −/− i NKT cells produced IFN-γ at levels equivalent to WT, while T H 2 and T H 17 cytokine production, except for IL-4, were severely impaired. (E) i NKT cell-dependent cytokine production in WT B6 and Il17rb −/− mice in vivo. α-GalCer (2 µg) was i.v. injected and the levels of cytokines in serum were analyzed at the indicated time points. The serum IFN-γ levels were similar in both mice, whereas production of T H 2 and T H 17 cytokines, except for IL-4, was significantly reduced in the Il17rb −/− mice. Cytokines were measured by ELISA or a cytometric bead array system at the indicated time points. Data are mean ± SDs from three mice and repeated three times with similar results.

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Expressing, Staining, In Vitro, Cell Culture, Produced, In Vivo, Injection, Enzyme-linked Immunosorbent Assay

Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A, B) FACS profiles of thymus (A) and enriched thymic i NKT cells (B) in WT, Il17rb −/− and Il15 L117P mice on a B6 background. (A) α-GalCer/CD1d dimer + i NKT cells were slightly decreased in Il17rb −/− mice and markedly reduced in Il15 L117P mice. (B) There was a loss of the NK1.1 − population in Il17rb −/− thymic i NKT cells, while Il15 L117P thymic i NKT cells showed impairment of the NK1.1 + population. (C) IL-17RB and CD122 expression by thymic i NKT cell populations of B6 mice. IL-17RB and CD122 expression in CD4 − and CD4 + of CD44 lo NK1.1 − (Stage 1), CD44 lo NK1.1 + (Stage 2), and CD44 hi NK1.1 + (Stage 3) populations were analyzed. IL-17RB expression was observed in Stages 1/2, while CD122 expression was in the Stage 3 cells. (D, E) Profiles of thymic i NKT cells in B6 and Il15 L117P mice showing expression of IL-17RB and CD4 (D) and further divided into CD44 and NK1.1 subpopulations (E). The percentage of IL-17RB + i NKT cells was increased due to the loss of expansion of IL17RB − i NKT cells in Il15 L117P mice. CD4 − and CD4 + , IL-17RB + i NKT cells were almost all Stage 1 and Stage 2 in both WT B6 and Il15 L117P mice. On the other hand, the majority of CD4 − and CD4 + , IL-17RB − i NKT cells were Stage 3 in both WT B6 and Il15 L117P mice. Loss of expansion of CD4 − and CD4 + , IL-17RB − i NKT cells was also observed in Il15 L117P mice. (F, G) Percentage (F) and cell number (G) of the total i NKT cells and the four subtypes (i.e. IL-17RB +/− and CD4 +/− ) in B6, Il17rb −/− and Il15 L117P mice based on their CD44 and NK1.1 expression patterns. The number of CD4 − and CD4 + , IL-17RB − i NKT cells was significantly decreased especially in Stage 3 in Il15 L117P mice compared to WT B6 mice. By contrast, CD4 − and CD4 + , IL-17RB + i NKT cells in Il15 L117P mice were present in numbers comparable to WT. Results are representative of those from three independent experiments. (H, I) Development of i NKT subtypes in Stages 1 and 2. Stage 1 and 2 cells in the four i NKT subtypes (i.e. IL-17RB +/− and CD4 +/− ) from WT B6 mice were sorted and cocultured with dGuo treated 15 dpc FT lobes from Jα18 −/− mice (1,000 cells/well). 10 d after culture, cells were recovered and analyzed the surface expression pattern in CD44 versus NK1.1 (H) and CD4 versus IL-17RB (I). IL-17RB − precursors gave rise through Stage 2 to Stage 3 cells with IL-17RB − , while IL-17RB + subtypes gave rise to Stage 2 cells with IL-17RB + . Results are representative of those from three independent experiments.

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Expressing

Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A, B, D, H) Quantitative PCR analysis of thymic i NKT subtypes. Thymic i NKT cells further divided into four subtypes based on the expression of CD4 and IL-17RB (red, CD4 − IL-17RB + ; orange, CD4 + IL-17RB + ; blue, CD4 − IL-17RB − ; green, CD4 + IL-17RB − ). One representative out of three experiments is shown (mean ± SEM). (A) The purity of the sorted cells was confirmed by the relative Il17rb and Cd4 mRNA expression levels in the respective subtypes. Il2rb ( = Cd122 ) expression was restricted to CD4 − and CD4 + , IL-17RB − i NKT cells. (B) Expression of T H 1/T H 2/T H 17 related genes. T H 1 related: Ifng , Tbx21 and Stat4 , T H 2 related; Il4 and Gata3 , and T H 17 related: Il17a , Il22 and Rorc transcripts were analyzed. (D) Expression of cytokine receptor genes. Receptor for IL-12, IL-23, and IL-25 were analyzed. The component chains of the various receptors are IL-12 receptor: IL-12Rβ2/IL-12Rβ1; IL-23 receptor: IL-23R/IL-12Rβ1; IL-25 receptor: IL-17RB/IL-17RA. (H) Expression of chemokine receptor genes. Ccr4 , Ccr6 , Ccr7 , Cxcr3 , and Cxcr6 . (C, E, F, G) In vitro cytokine production by thymic i NKT cell subtypes (red, CD4 − IL-17RB + ; orange, CD4 + IL-17RB + ; blue, CD4 − IL-17RB − ; green, CD4 + IL-17RB − ). Sorted thymic i NKT subtypes (5×10 4 cells/100 µL) were co-cultured with BM-DCs (5×10 3 /100 µL) for 48 h in the presence of α-GalCer (100 ng/µL) (C), IL-12 (10 ng/µL) (E), IL-23 (10 ng/µL) (F), or IL-25 (10 ng/µL) (G). Levels of IFN-γ, IL-4, IL-9, IL-10, IL-13, IL-17A, and IL-22 were analyzed. The data are representative of three independent experiments (mean ± SEM).

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Real-time Polymerase Chain Reaction, Expressing, In Vitro, Cell Culture

Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A–C) FACS profile of peripheral i NKT cells in B6 mice. α-GalCer/CD1d dimer + TCRβ + i NKT cells (A) and i NKT subtypes based on the expression of CD44 and NK1.1 (B) or CD4 and IL-17RB (C) in spleen, liver, BM, lung, inguinal LN, and mesenteric LN from B6 or Il17rb −/− mice. Numbers indicate percentage of total mononuclear cells (A) and i NKT cells (B, C). (D) Number of cells of each i NKT subtype based on the expression of IL-17RB and CD4 in thymus and periphery of B6 and BALB/c mice. Cell numbers were calculated based on the results from , , and S8B. IL-17RB + i NKT cells were mainly localized in spleen, lung, inguinal LN, and mesenteric LN, whereas hardly any were observed in liver and BM. One representative experiment of three is shown.

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Expressing

Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A) Global gene expression profiles in i NKT subtypes in the thymus and spleen. Tree view representation of clustering analysis among the four i NKT subtypes in thymus and spleen from B6 and BALB/c. The values represent coefficients between the indicated panels. r 2 >0.95 in red, 0.85< r 2 <0.95 in orange, and r 2 <0.85 in blue. One representative experiment of three is shown. (B) Plasticity and stability of i NKT subtypes. The four i NKT cell subtypes in the thymus were sorted and each subtype (5×10 5 ) was i.v. transferred into independent Jα18 −/− mice ( n = 3). 10 d after transfer, α-GalCer/CD1d dimer + TCRβ + cells in spleen were analyzed by FACS for the expression of IL-17RB and CD4. Representative data from three experiments are shown. (C–F) In vitro cytokine production by splenic i NKT cell subtypes (red, CD4 − IL-17RB + ; orange, CD4 + IL-17RB + ; blue, CD4 − IL-17RB − ; green, CD4 + IL-17RB − ). Sorted splenic i NKT subtypes (5×10 4 cells/100 µL) were co-cultured with BM-DCs (5×10 3 /100 µL) for 48 h in the presence of α-GalCer (100 ng/µL) (C), IL-12 (10 ng/µL) (D), IL-23 (10 ng/µL) (E), and IL-25 (10 ng/µL) (F). Levels of IFN-γ, IL-4, IL-9, IL-10, IL-13, IL-17A, and IL-22 in the supernatants were analyzed by ELISA or CBA. Data are mean ± SD of triplicate wells. One representative experiment of three is shown.

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Expressing, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A, B) Quantitative analysis of Rorc (A) and E4bp4 (B) in i NKT cell subtypes after cytokine treatment. Sorted i NKT cell subtypes (5×10 4 /100 µL) from thymus (left) or spleen (right) were co-cultured with BM-DCs (5×10 3 /100 µL) in the presence or absence of IL-23 (10 ng/ml) or IL-25 (10 ng/ml) for 24 h. The i NKT cell subtypes were then sorted again and analyzed for expression of the indicated genes by quantitative real-time PCR. The data are representative of three independent experiments (mean ± SEM). (C, D) Cytokine production by CD4 + IL-17RB + i NKT cells in response to IL-25. Sorted CD4 + IL-17RB + i NKT cells (5×10 4 /100 µL) from thymus (C) or spleen (D) of B6 or E4bp4 −/− mice were co-cultured with BM-DCs (5×10 3 /100 µL) in the presence of IL-25 (10 ng/ml) for 48 h and then the levels of the indicated cytokines in the tissue culture media were analyzed. i NKT cells from B6 were compared to those from E4bp4 −/− mice. ** p <0.01 calculated by t test. The data are representative of three independent experiments (mean ± SD).

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Development and Function of Invariant Natural Killer T Cells Producing T H 2- and T H 17-Cytokines

doi: 10.1371/journal.pbio.1001255

Figure Lengend Snippet: (A) Schematic showing the protocol for RSV-induced AHR. Mice were i.n. administered with RSV (10 6 pfu) or PBS alone as a control 4 times at 10-day intervals. Mice were i.p. immunized with rec Gs/alum (50 µg/2 mg) 4 d after first RSV infections. Three days after the last RSV administration, mice were exposed i.n. to rec Gs and were measured 1 d later. (B) Development of RSV-induced AHR in BALB/c, but not in Jα18 −/− or Il17rb −/− mice. Changes in R L are depicted. The RSV-infected, rec Gs immunized, BALB/c mice had a greatly increased AHR compared to the other three groups. Results are expressed as the mean ± SEM. * p <0.05 and ** p <0.01. (C, D) Total and differential cell counts (C) and cytokines (D) in BAL fluid. BAL fluid was collected 24 h after challenge of the mice depicted in (B) with intranasal rec Gs. Results are expressed as the mean ± SEM. * p <0.05 and ** p <0.01. (E) Histological examination of lung tissues by H&E and PAS staining. RSV infected, rec Gs immunized, BALB/c, Jα18 −/− or IL17rb −/− , mice were compared with control BALB/c mice (rec Gs alone). Bars indicate 100 µm. (F) AHR development after cell transfer of spleen IL-17RB + i NKT cells into Jα18 −/− mice. Indicated cell numbers of sorted IL-17RB + , IL-17RB − i NKT cells or total i NKT cells from spleen, or PBS control, were i.v. transferred into rec-Gs/alum-sensitized Jα18 −/− mice 24 h before RSV treatment (on the day 9, 19, and 29), and then challenged with rec Gs (24 h) and measurement of lung resistance (48 h). Each group of IL-17RB + i NKT cell-transferred mice was compared to other three groups. * p <0.05, ** p <0.01 calculated by Kruskal Wallis test. The results represent one out of four experiments with five mice in each group.

Article Snippet: Biotinylated anti-mouse IL-17RB (B5F6) was generated previously and detected by staining with PE or PE-Cy7 Avidin (BD Biosciences).

Techniques: Infection, Staining