Fig 1.
IL-21 is produced during P. chabaudi infection and required to control chronic infection.
(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.
Fig 2.
IL-21 is produced by CD4+ T cells during P. chabaudi infection.
(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.
Fig 3.
IL-21 is co-expressed with IFN-γ and IL-10 during P. chabaudi infection.
(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.
Fig 4.
Mice bearing T cells deficient in IL-21 fail to control chronic P. chabaudi infection.
Course of a P. chabaudi infection in mixed BM chimeric mice generated as described with the scheme in (A) and detailed in Materials and Methods and S1 Table, (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 S1 Table). 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.
Fig 5.
IL-21-producing Tfh cells are activated during acute P. chabaudi infection.
(A) Flow cytometric analysis of representative naïve (top row) and infected mice (8 days post-infection, bottom row). Gates show frequency of CD3+CD4+CD44high cells expressing CXCR5 and PD-1. (B) Frequency and (C) total numbers of Tfh cells, defined as CD3+CD4+CD44highCXCR5+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+CD44highCXCR5+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.
Fig 6.
Flow cytometry analysis of B cell responses during P. chabaudi infection in WT C57BL/6, Il21-/- and Il21r-/- mice.
(A-D) Analysis of Mature (M) Transitional 1 (T1) and Transitional 2 (T2) on CD19+B220+ gated B cells in the spleen based on the pattern of IgD and IgM expression. (E-G) Analysis of GL-7highCD38low GC cells on CD19+IgD– gated B cells in the spleen. (H-I) Analysis of B220+CD138+ plasmablasts in the spleen. (J-L) Analysis of B220+CD138high plasmablasts and B220–CD138high plasma cells in the BM (1 femur plus 1 tibia per mouse). 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<0.05; # #, P<0.01). The Mann Whitney U test was used to compare with its respective basal level when sets of data of only 2 time points were available (*, P<0.05). Bars represent median values. Data are representative of at least two independent experiments and were pooled from groups of 3–4 mice per time point.
Fig 7.
P. chabaudi-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.
(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) MSP121-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.
Fig 8.
Mice bearing IL-21R-deficient B cells fail controlling chronic P. chabaudi infection and activating P. chabaudi-specific-IgG-responses.
(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 Materials and Methods and S1 Table). (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 S1 Table). 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) MSP121-specific IgG antibodies determined by ELISA 32 days post-infection in different mixed BM chimeric groups (4–5 mice per group, details in S1 Table). (D) Total number of MSP121-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 S1 Table). 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.
Fig 9.
Mice deficient in IL-21 signaling fail to develop immunity to a secondary P. chabaudi infection.
(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 materials and methods, and re-infected with 105 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.