recombinant mouse il 21 receptor fc  (R&D Systems)

Journal: PLoS Pathogens

Article Title: Neddylation contributes to CD4 + T cell-mediated protective immunity against blood-stage Plasmodium infection

doi: 10.1371/journal.ppat.1007440

Figure Lengend Snippet: (A) Left, RNA-Seq analysis of activated CD44 hi CD4 + T cells from 3 mice at day 5 of P . yoelii 17XNL infection. Heat map showing differential expression of the signature genes related to Th1, Th2, Th17, Tfh and Treg lineages between Uba3 fl/fl and Uba3 ΔT mice. Right, validation of Bcl-6 expression by quantitative RT-PCR for activated CD4 + T cells as described above. (B) Representative counter plots and bar graphs showing the proportions and numbers of Tfh (PD-1 + CXCR5 + ) cells among activated CD4 + CD44 hi T cells in spleens of Uba3 fl/fl and Uba3 ΔT mice at days 0, 7 and 16 p.i. (n = 8–9 per group). (C) Immunoblotting for Bcl-6 in splenic CD4 + T cells from Uba3 fl/fl and Uba3 ΔT mice at days 0, 5, 7 p.i. (n = 4 per group), bar graphs showing densitometry of the bands relative to that of Uba3 fl/fl mice. (D) Intracellular staining of IL-21 in splenic CD4 + T cells from Uba3 fl/fl and Uba3 ΔT mice prior to (day 0) and at day 7 p.i. (n = 6 per group). Data are representative of two or more replicate experiments and are shown as mean±SEM. * p <0.05, *** p <0.01 by Student’s t test.

Article Snippet: IL-21 was detected using recombinant mouse IL-21 receptor/human Fc chimera (R&D Systems) and PE-conjugated anti-human IgG Fc (ebioscience), as previously described [ ].

Techniques: RNA Sequencing Assay, Infection, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Frontiers in Immunology

Article Title: NK1.1 Expression Defines a Population of CD4 + Effector T Cells Displaying Th1 and Tfh Cell Properties That Support Early Antibody Production During Plasmodium yoelii Infection

doi: 10.3389/fimmu.2018.02277

Figure Lengend Snippet: NK1.1 + CD4 + T cells express Tbet and Bcl6 and produce effector cytokines. (A) Representative intracellular staining of NK1.1 − and NK1.1 + CD4 + T cells for the transcription factors Tbet, Bcl6, RORγt, Gata3, and Foxp3. Cells previously gated on live, CD44 hi CD62L lo CD4 + TCRβ + splenocytes. Gates based on FMO controls shown in Supplemental Figure . (B) Frequency of NK1.1 + and NK1.1 − CD4 + T cells expressing Tbet, Bcl6 or, Tbet and Bcl6 on days 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01. (C) Median fluorescence intensity (MFI) for Tbet and Bcl6 in NK1.1 + and NK1.1 − CD4 + T cells on day 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. * p < 0.05. (D) Representative intracellular cytokine staining of NK1.1 − and NK1.1 + CD4 + T cells previously gated on live CD4 + TCRβ + T cells, on day 7 post-infection. Splenocytes were stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A before being stained for IFN-γ, IL-21, and TNFα. (E) The frequency of single, double, or triple cytokine-producing cells at day 7 post-infection. Significance assessed via an unpaired non-parametric Mann-Whitney test. * p < 0.05. Data are representative of three independent experiments (error bars, s.e.m.).

Article Snippet: For IL-21 staining, recombinant mouse IL-21 receptor fused to human Fc (R & D Systems) staining was performed first, followed by secondary anti-human Fc-PE Ab (ThermoFisher Scientific) staining.

Techniques: Staining, Expressing, Transformation Assay, Fluorescence, Infection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A) IL-21 mRNA in spleen cells of P . chabaudi -infected mice measured by real-time quantitative RT-PCR. Parasitemia (B) and total rbc counts (C) were determined in WT C57BL/6 (closed circles), Il21 -/- (open circles) and Il21r -/- (open squares) mice. (D) Individual examples of spleens from Il21r -/- (a) Il21 -/- (b) and WT C57BL/6 (c) mice at day 120 post-infection, and a spleen from an age-matched WT C57BL/6 naïve mouse (d). Bar, 1 cm. (E) Total number of nucleated live splenocytes were determined with a hemocytometer in WT C57BL/6 (black bars), Il21 -/- (open bars) and Il21r -/- (stripped bars) mice. (F) Numbers of Ter119 + and Ter119 – cells in the spleen of WT C57BL/6 (black bars) and Il21r -/- (striped bars) at day 32 post-infection. Data are representative of two or more independent experiments and are obtained in groups of 5–10 mice per time point. Statistical significance was obtained using Mann Whitney U test or Kruskal-Wallis test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Error bars correspond to mean ± SEM.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Infection, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A) Flow cytometry plots showing individual examples of IL-21 expression on mononuclear cells from WT C57BL/6 and Il21 -/- mice at day 8 post-infection (top row). For the gating strategy, singlet cells were first selected, followed by live cells and mononuclear cells. In the bottom row, the IL-21-producing mononuclear cells detected in WT C57BL/6 mice, identified by red dots, were overlaid on the plots corresponding to the different combinations of surface biomarkers. (B) Cumulative data showing the differential combination of expression (+) or absence of expression (–) of each surface marker (indicated in the bottom left) on IL-21-producing mononuclear cells. (C) Flow cytometry plots showing individual examples of IL-21 expression on CD3 + CD4 + T cells at day 8 post-infection. (D) Cumulative data showing the percentage (left) and total numbers (right) of IL-21-producing CD4 + T cells in the spleen of WT C57BL/6 mice at different days post-infection. Data are representative of at least two independent experiments and were obtained in groups of 4–5 mice per time point. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01; ***, P<0.001); or comparing each surface marker combination with every other surface marker combination within each time point (# #, P<0.01). Bars represent median values.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Flow Cytometry, Expressing, Infection, Marker

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A-C) Flow cytometry plots showing individual examples for days 8 and 15 post-infection of different cytokine combinations studied in CD3 + CD4 + T cells from the spleen of WT C57BL/6 mice. (D) IL-21-producing CD4 + T cells (red) overlaid on the plots corresponding to IFN-γ vs IL-10 on gated CD3 + CD4 + T cells. Cumulative data showing the percentage (E) and total numbers (F) of IL-21-producing CD4 + T cells co-expressing IFN-γ, IL-4 and IL-10 in the spleen of WT C57BL/6 mice. The differential combination of expression (+) or absence of expression (–) of each cytokine (indicated in the bottom left) is shown for each subset at different days post-infection. Data are representative of at least two independent experiments and were obtained in groups of 4–6 mice per time point. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point, corresponding to each cytokine combination with its respective basal level (day 0 post-infection). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Bars represent median values.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Flow Cytometry, Infection, Expressing

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: Course of a P . chabaudi infection in mixed BM chimeric mice generated as described with the scheme in (A) and detailed in and , (B) with fully functional B cells and T cells deficient in the Il21 gene ( Il21 -/- T cells, closed squares), and (C) with fully functional B cells and T cells deficient in the Il21r gene ( Il21r -/- T cells, closed triangles) infected with P . chabaudi . As controls, mixed BM chimeric mice with BM from Tcra -/- and Ighm mice were generated ( Il21 +/+ and Il21r +/+ T cells, open squares, details in ). Statistical significance was obtained using Mann Whitney U test. **, P<0.01; ***, P<0.001. The graphs show the mean ± SEM of the parasitemia at different time points in 7–10 mice per group. Data are representative of two independent experiments.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Infection, Generated, Functional Assay, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A) Flow cytometric analysis of representative naïve (top row) and infected mice (8 days post-infection, bottom row). Gates show frequency of CD3 + CD4 + CD44 high cells expressing CXCR5 and PD-1. (B) Frequency and (C) total numbers of Tfh cells, defined as CD3 + CD4 + CD44 high CXCR5 + PD-1 + , in WT C57BL/6, Il21 -/- , Il21r -/- and Ighm mice. (D) Flow cytometric analysis representative of infected WT C57BL/6 mice (8 days post-infection) corresponding to IL-21 intracellular staining on CD4 + T cells (red), overlaid on side scatter light vs CD44 (left) and CXCR5 vs PD-1 (right) from CD3 + CD4 + T cells. Numbers show frequency of IL-21-producing CD4 + T cells with high expression of CD44 (left), and their differential expression of CXCR5 and PD-1 (right). (E) Differential combination of expression (+) or absence of expression (–) of CD44, CXCR5 and PD-1 (bottom left) on IL-21-producing CD3 + CD4 + T cells at different days post-infection in the spleen of WT C57BL/6 mice. (F) Flow cytometric analysis of IFN-γ (green line) on CD3 + CD4 + CD44 high CXCR5 + PD-1 + IL-21 + T cells from the spleen of WT C57BL/6 mice, 8 days post- P . chabaudi infection (representative of 4 mice). (G) Serum IL-6 at day 6 post- P . chabaudi infection. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01; ***, P<0.001), or comparing with the data obtained from the WT C57BL/6 group (#, P<0.05; # #, P<0.01). Bars represent median values. Data are representative of at least two independent experiments and were obtained in groups of 4–7 mice per time point.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Infection, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of P . chabaudi -infected rbc determined by ELISA. Antibody units (AU) were calculated based on the P . chabaudi -specific antibody levels of a hyper-immune standard plasma defined as 1000 U. In the cases where levels of antibodies were below background, arbitrary values of 2 log lower than the mean value observed in WT C57BL/6 mice were set to be able to perform the statistical test. (D) MSP1 21 -specific IgG-producing ASC in BM obtained from one femur and one tibia, and (E) MBC per spleen, determined by ELISPOT 32 days post-infection. Statistical significance was obtained using the Kruskal-Wallis test comparing each time point with its respective basal level (day 0 post-infection) (*, P<0.05; **, P<0.01), or comparing with the data obtained from the WT C57BL/6 group (# #, P<0.01). The Mann Whitney U test was used in the case of IgG subtypes, comparing Il21 -/- vs WT C57BL/6 mice (#, P<0.05). Bars represent median values. Data are representative of at least two independent experiments and were obtained in groups of 3–8 mice per time point.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A) Scheme describing the approach applied to generate mice with fully functional T cells and B cells deficient in the Il21 or the Il21r gene (details in and ). (B) Course of a P . chabaudi infection in mixed BM chimeric mice with B cells deficient in the Il21r gene ( Il21r -/- B cells, closed circles); as controls, mixed BM chimeric mice with BM from Tcra -/- and Ighm mice were generated ( Il21r +/+ B cells, open squares, details in ). Statistical significance was obtained using the Mann Whitney U test (**, P<0.01). The graph shows the mean ± SEM of the parasitemia at different time points in 7–10 mice per group. (C) MSP1 21 -specific IgG antibodies determined by ELISA 32 days post-infection in different mixed BM chimeric groups (4–5 mice per group, details in ). (D) Total number of MSP1 21 -specific IgG MBC in spleens from different mixed BM chimeric groups, determined by ELISPOT on days 120–150 post-infection (4–5 mice per group, details in ). Statistical significance was obtained using the Kruskal-Wallis test comparing the data obtained from the group of Rag2 -/- mice reconstituted with BM from WT C57BL/6 mice (BL/6→ Rag2 -/- . # #, P<0.01). Bars represent median values. Data are representative of two independent experiments.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Functional Assay, Infection, Generated, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: PLoS Pathogens

Article Title: Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

doi: 10.1371/journal.ppat.1004715

Figure Lengend Snippet: (A) Scheme describing the experimental approach. CQ = chloroquine. (B and C) P . chabaudi -infected mice were treated with chloroquine to eliminate parasitemia as described in the , and re-infected with 10 5 P . chabaudi- infected rbc (day 0 post-secondary infection). The graphs show the course of secondary P . chabaudi infection in WT C57BL/6 (black circles), Il21 -/- (red circles) and Il21r -/- (brown circles) mice; course of primary infection in Il21 -/- (gray circles) and Il21r -/- (gray squares) are overlaid. (D and E) Number of Tfh cells per spleen post-primary and secondary infection, respectively. (F and G) Number of IFN-γ + CD4 + T cells per spleen post-primary and secondary infection, respectively. Data are representative of two independent experiments and are obtained in groups of 3–10 mice per time point. Statistical significance was obtained using Mann Whitney U test (**, P<0.01) or Kruskal-Wallis test (#, P<0.05). Error bars correspond to mean ± SEM.

Article Snippet: Briefly, cells were fixed in 2% paraformaldehyde and stored in staining buffer overnight at 4°C, permeabilized in Perm/Wash buffer (BD Pharmingen), and incubated in Perm/Wash with recombinant mouse IL-21 receptor/ human Fc chimera (5mg/mL; R&D Systems).

Techniques: Infection, MANN-WHITNEY