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

<div><p>Interleukin-21 signaling is important for germinal center B-cell responses, isotype switching and generation of memory B cells. However, a role for IL-21 in antibody-mediated protection against pathogens has not been demonstrated. Here we show that IL-21 is produced by T follicular helper cells and co-expressed with IFN-γ during an erythrocytic-stage malaria infection of <i>Plasmodium chabaudi</i> in mice. Mice deficient either in IL-21 or the IL-21 receptor fail to resolve the chronic phase of <i>P</i>. <i>chabaudi</i> infection and <i>P</i>. <i>yoelii</i> infection resulting in sustained high parasitemias, and are not immune to re-infection. This is associated with abrogated <i>P</i>. <i>chabaudi</i>-specific IgG responses, including memory B cells. Mixed bone marrow chimeric mice, with T cells carrying a targeted disruption of the <i>Il21</i> gene, or B cells with a targeted disruption of the <i>Il21r</i> gene, demonstrate that IL-21 from T cells signaling through the IL-21 receptor on B cells is necessary to control chronic <i>P</i>. <i>chabaudi</i> infection. Our data uncover a mechanism by which CD4+ T cells and B cells control parasitemia during chronic erythrocytic-stage malaria through a single gene, <i>Il21</i>, and demonstrate the importance of this cytokine in the control of pathogens by humoral immune responses. These data are highly pertinent for designing malaria vaccines requiring long-lasting protective B-cell responses.</p></div

<i>P</i>. <i>chabaudi</i>-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.

<p>(A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of <i>P</i>. <i>chabaudi</i>-infected rbc determined by ELISA. Antibody units (AU) were calculated based on the <i>P</i>. <i>chabaudi</i>-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 <i>Il21</i>^<i>-/-</i> 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.</p

IL-21 is produced during <i>P</i>. <i>chabaudi</i> infection and required to control chronic infection.

<p>(A) IL-21 mRNA in spleen cells of <i>P</i>. <i>chabaudi</i>-infected mice measured by real-time quantitative RT-PCR. Parasitemia (B) and total rbc counts (C) were determined in WT C57BL/6 (closed circles), <i>Il21</i>^<i>-/-</i> (open circles) and <i>Il21r</i>^<i>-/-</i> (open squares) mice. (D) Individual examples of spleens from <i>Il21r</i>^<i>-/-</i> (a) <i>Il21</i>^<i>-/-</i> (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), <i>Il21</i>^<i>-/-</i> (open bars) and <i>Il21r</i>^<i>-/-</i> (stripped bars) mice. (F) Numbers of Ter119⁺ and Ter119^– cells in the spleen of WT C57BL/6 (black bars) and <i>Il21r</i>^<i>-/-</i> (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.</p

IL-21 is co-expressed with IFN-γ and IL-10 during <i>P</i>. <i>chabaudi</i> infection.

<p>(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.</p

Mice deficient in IL-21 signaling fail to develop immunity to a secondary <i>P</i>. <i>chabaudi</i> infection.

<p>(A) Scheme describing the experimental approach. CQ = chloroquine. (B and C) <i>P</i>. <i>chabaudi</i>-infected mice were treated with chloroquine to eliminate parasitemia as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004715#sec009" target="_blank">materials and methods</a>, and re-infected with 10⁵<i>P</i>. <i>chabaudi-</i>infected rbc (day 0 post-secondary infection). The graphs show the course of secondary <i>P</i>. <i>chabaudi</i> infection in WT C57BL/6 (black circles), <i>Il21</i>^<i>-/-</i> (red circles) and <i>Il21r</i>^<i>-/-</i> (brown circles) mice; course of primary infection in <i>Il21</i>^<i>-/-</i> (gray circles) and <i>Il21r</i>^<i>-/-</i> (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.</p

<i>P</i>. <i>chabaudi</i>-specific IgG B-cell responses are abrogated in the absence of IL-21 signaling.

<p>(A) IgG, (B) IgG subtypes (day 32 post-infection) and (C) IgM antibodies specific for a lysate of <i>P</i>. <i>chabaudi</i>-infected rbc determined by ELISA. Antibody units (AU) were calculated based on the <i>P</i>. <i>chabaudi</i>-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 <i>Il21</i>^<i>-/-</i> 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.</p

Mice deficient in IL-21 signaling fail to develop immunity to a secondary <i>P</i>. <i>chabaudi</i> infection.

<p>(A) Scheme describing the experimental approach. CQ = chloroquine. (B and C) <i>P</i>. <i>chabaudi</i>-infected mice were treated with chloroquine to eliminate parasitemia as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004715#sec009" target="_blank">materials and methods</a>, and re-infected with 10⁵<i>P</i>. <i>chabaudi-</i>infected rbc (day 0 post-secondary infection). The graphs show the course of secondary <i>P</i>. <i>chabaudi</i> infection in WT C57BL/6 (black circles), <i>Il21</i>^<i>-/-</i> (red circles) and <i>Il21r</i>^<i>-/-</i> (brown circles) mice; course of primary infection in <i>Il21</i>^<i>-/-</i> (gray circles) and <i>Il21r</i>^<i>-/-</i> (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.</p

IL-21-producing Tfh cells are activated during acute <i>P</i>. <i>chabaudi</i> infection.

<p>(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^highCXCR5⁺PD-1⁺, in WT C57BL/6, <i>Il21</i>^<i>-/-</i>, <i>Il21r</i>^<i>-/-</i> and <i>Ighm</i> 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^highCXCR5⁺PD-1⁺IL-21⁺ T cells from the spleen of WT C57BL/6 mice, 8 days post-<i>P</i>. <i>chabaudi</i> infection (representative of 4 mice). (G) Serum IL-6 at day 6 post-<i>P</i>. <i>chabaudi</i> 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.</p

IL-21 is produced by CD4⁺ T cells during <i>P</i>. <i>chabaudi</i> infection.

<p>(A) Flow cytometry plots showing individual examples of IL-21 expression on mononuclear cells from WT C57BL/6 and <i>Il21</i>^<i>-/-</i> 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.</p

Flow cytometry analysis of B cell responses during <i>P</i>. <i>chabaudi</i> infection in WT C57BL/6, <i>Il21</i>^<i>-/-</i> and <i>Il21r</i>^<i>-/-</i> mice.

<p>(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-7^highCD38^low 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⁺CD138^high plasmablasts and B220^–CD138^high 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.</p