IL-10 is a pleiotropic cytokine expressed during malaria, a disease characterized by short-lived, parasite-specific Ab responses. The role of IL-10 in regulating B cell responses during malaria is not known. In this study we report that IL-10 is essential for anti-Plasmodium humoral immunity. We identify that germinal center (GC) B cell reactions, isotype-switched Ab responses, parasite control, and host survival require B cell-intrinsic IL-10 signaling. IL-10 also indirectly supports humoral immunity by suppressing excessive IFN-γ, which induces T-bet expression in B cells. Genetic ablation of either IFN-γ signaling or T-bet expression in B cells substantially enhanced GC B cell responses and anti-Plasmodium Ab production. Together, our data show that B cell-intrinsic IL-10 enhances whereas B cell-intrinsic IFN-γ and T-bet suppress GC B cell responses and anti-Plasmodium humoral immunity. These data identify critical immunoregulatory circuits in B cells that may be targeted to promote long-lived humoral immunity and resistance to malaria.

Plasmodium parasites are responsible for more than 200 million infections and nearly 500,000 malaria deaths each year (1). Control and clearance of Plasmodium parasites depends on parasite-specific secreted Ab responses (2) and resistance against severe malaria correlates with the acquisition of humoral immunity (3). However, anti-Plasmodium Ab responses are short lived (4) and dominated by extrafollicular, IgM-secreting B cell reactions (5), leaving individuals susceptible to repeated infections (6). The reasons for the short-lived nature of anti-Plasmodium Ab responses remains a major knowledge gap.

IL-10 is induced following experimental and clinical malaria (7, 8) and is linked to suppression of Th1 cell–mediated immunity and parasite control during experimental Plasmodium infection (8). However, in experimental models IL-10 is also essential for limiting severe malarial disease, immunopathology, and host survival (8, 9). Despite these reports, it is not known whether IL-10 promotes or limits humoral immunity or whether it acts directly or indirectly to modulate B cell function and protective humoral immunity during malaria.

Given the pleiotropic activities of IL-10 and the complexity of its regulation during Plasmodium infection, we investigated the role of IL-10 signaling in the regulation of humoral immunity during experimental Plasmodium yoelii infection of mice, which mimics critical aspects of Plasmodium falciparum pathogenesis and immunity. Utilizing biochemical and genetic approaches, we identified an essential and multifaceted role for IL-10 in anti-Plasmodium humoral immunity. Our data show that B cell-intrinsic IL-10 signaling is required for effective germinal center (GC) B cell and parasite-specific Ab responses, as well as parasite control and host survival. Moreover, IL-10 indirectly promotes humoral immunity by limiting excessive IFN-γ. B cell-intrinsic IFN-γ signaling promotes T-bet expression in GC B cells, and genetic deletion of either the IFN-γ receptor or T-bet in B cells promotes GC-derived Plasmodium-specific Ab responses. Together, our data show that malaria-associated IL-10 enhances whereas IFN-γ and T-bet directly limits anti-Plasmodium B cell activity, humoral immunity, and parasite clearance. Our study provides critical insight into the molecular mechanisms regulating anti-Plasmodium Ab responses. Our study also highlights the importance of how understanding the individual and combined activities of IL-10 and IFN-γ may contribute to the short-lived nature of anti-Plasmodium humoral immunity.

C57BL/6J WT, Il10−/−, Il10Rβ−/−, μMT, Tbx21−/−, and Ifnγr1−/− (8 wk old) mice were purchased from the Jackson Laboratories. The University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee approved all experiments. P. yoelii (clone 17XNL, obtained from MR4; ATCC) was routinely passaged through mosquitos. Infections were initiated by a serial transfer of 106P. yoelii parasite-infected RBCs i.v. Parasitemia was measured by detecting RNA/DNA content in Ter119+ RBCs via flow cytometry. For IL-10R blockade studies, wild-type (WT) mice were injected i.p. with 200 μg of α-IL-10R (1B1.3A; Bioxcell) or rat γ Ig (rIgG) every 3 d starting on day 1 postinfection (p.i.) For studies neutralizing IFN-γ, Il10−/− mice were injected i.p. with 500 μg of α-IFN-γ (XMG1.2; Bioxcell) or rIgG every 2 d beginning 1 d prior to infection.

For 1:1 chimeras, WT recipient (CD45.1) mice were irradiated with 6.5 and 5.5 Gy, separated by 12 h. Bone marrow from WT (CD45.1) and Il10Rβ−/− (CD45.2) or Ifnγr1−/− (CD45.2) mice were mixed 1:1 and 107 cells were injected i.v. Chimerism was assessed at 5 wk and mice were infected with P. yoelii at 6 wk post reconstitution. For BWT, BIL10R−/−, BT-bet−/−, and BIFNgR−/− chimeras, WT (CD45.2) mice were irradiated with 6.5 and 5.5 Gy, separated by 12 h. CD45.1 bone marrow from WT, Il10Rβ−/−, Ifnγr1−/−, or Tbx21−/− and μMT (CD45.1/2) were mixed 1:9 and 107 cells were injected i.v. Chimerism was assessed at 6 wk and mice were infected with P. yoelii at 8 wk. For B cell–specific deletion of specified genes, chimerism for all mice was >80%. All chimeric mice were maintained on oral sulfamethoxazole for 2 wk after irradiation.

Mouse splenocytes were subjected to RBC lysis and stained with fluorescently labeled Abs obtained from Tonbo, eBioscience, BD Biosciences, and BioLegend. Anti-mouse Abs included CD19 (clone 1D3), T and B cell activation marker (clone GL7), Fas (Jo2), CD45.1 (clone A20), CD45.2 (clone 104), T-bet (clone 4B10), and Bcl-6 (clone K112-91). Transcription factor staining was done using Foxp3/transcription factor staining buffer (Tonbo). Data were acquired with a Stratedigm S1200Exi flow cytometer and analyzed using FlowJo software (TreeStar).

Plates (Nunc) were coated with 18 μg/ml P. yoelii lysate and blocked with 2.5% BSA + 5% goat serum. Serum samples were diluted and Abs were detected by HRP-conjugated secondary Abs. End-point titers were extrapolated from a sigmoidal 4PL (where X is the log concentration) standard curve after developing with SureBlue Reserve TMB Kit (KBL). The threshold for end-point titers is the mean + 4 SD of naive mouse sera.

Statistical analyses and end-point titers were performed using GraphPad Prism 6 software (GraphPad). Specific tests of statistical significance are detailed in the figure legends.

IL-10 is a potent immunosuppressive cytokine that both constrains T cell immunity and protects mice from severe malarial disease during experimental Plasmodium infection (8, 9). In vitro, IL-10 promotes human B cell survival, class-switching, and Ab production (1012). In vivo, IL-10 is reported to limit GC reactions and humoral immunity during influenza infection (13) and following ultraviolet irradiation (14). To test the relevance of IL-10 in regulating parasite control, mortality, and humoral immunity during Plasmodium infection, we administered an IL-10R blocking Ab to mice every 3 d starting on day 1 p.i. IL-10R blockade resulted in mortality in ∼50% of mice between days 7 and 11 p.i. (Fig. 1A), consistent with severe lethal immunopathology observed in Plasmodium-infected Il10−/− mice (8, 9). Although blockade of IL-10 signaling modestly improved parasite control through day 10 p.i., parasite control was lost after the second week of infection in surviving mice (Fig. 1B). Consistent with loss of parasite control, serum titers of parasite-specific IgG1 and IgG2b were reduced 15- and 22-fold, respectively, in α-IL-10R–treated mice (Fig. 1C). Notably, IL-10R blockade did not alter the levels of parasite-specific IgM (Fig. 1C), supporting that the secretion of parasite-specific IgM is not sufficient for parasite control. In line with reduced secretion of parasite-specific IgG1 and IgG2b, the frequency and number of GC B cells were reduced ≥10-fold in α-IL-10R–treated mice, compared with control treated mice (Fig. 1D). Consistent with α-IL-10R treatment, P. yoelii–infected Il10−/− mice also exhibited dramatic reductions in GC B cells and isotype-switched secreted Ab compared with infected WT mice (not depicted). Together, these data show that IL-10 signaling is critical for supporting GC B cell reactions and GC-derived Ab responses. The absence of GC B cells and lack of GC-derived parasite-specific secreted IgG in α-IL-10R–treated mice further coincides with the loss of parasite control after the second week of the infection. These data are consistent with clinical studies showing severe malaria is associated with elevated proinflammatory cytokine levels (15), reductions in serum IL-10 levels (16), and reduced P. falciparum–specific Ab responses (17). These reports and our data collectively support that IL-10 both limits severe malaria and critically sustains anti-Plasmodium humoral immunity.

FIGURE 1.

IL-10 is essential for anti-Plasmodium humoral immunity and parasite clearance. Mice were infected with 1 × 106P. yoelii parasitized RBCs and administered 200 μg of α-IL-10R (n = 22) or rIgG (n = 16) every 3 d starting on day 1 p.i. Survival data (A) are pooled from four independent experiments. (B) Parasitemia (% RBCs infected) in α-IL-10R– and rIgG–treated mice surviving through day 18 p.i. Data are pooled from three independent experiments. (C) P. yoelii parasite lysate-specific IgM, IgG1, and IgG2b end-point titers on day 18 p.i. Data are pooled from three independent experiments. (D) Representative dot plot (left) and summary data (right) depicting the proportion and total number of GC B cells (CD19+GL7+Fas+) on day 18 p.i. Data are pooled from three independent experiments. Data in (B)–(D) are mean ± SEM. Data in (A) were analyzed by log-rank Mantel–Cox test. Data in (B) were analyzed using multiple t tests and correcting for multiple comparisons using the Holm–Sidak method. Data in (C) and (D) were analyzed using unpaired, nonparametric Mann–Whitney tests. *p ≤ 0.05, ****p < 0.0001. L.O.D., limit of detection.

FIGURE 1.

IL-10 is essential for anti-Plasmodium humoral immunity and parasite clearance. Mice were infected with 1 × 106P. yoelii parasitized RBCs and administered 200 μg of α-IL-10R (n = 22) or rIgG (n = 16) every 3 d starting on day 1 p.i. Survival data (A) are pooled from four independent experiments. (B) Parasitemia (% RBCs infected) in α-IL-10R– and rIgG–treated mice surviving through day 18 p.i. Data are pooled from three independent experiments. (C) P. yoelii parasite lysate-specific IgM, IgG1, and IgG2b end-point titers on day 18 p.i. Data are pooled from three independent experiments. (D) Representative dot plot (left) and summary data (right) depicting the proportion and total number of GC B cells (CD19+GL7+Fas+) on day 18 p.i. Data are pooled from three independent experiments. Data in (B)–(D) are mean ± SEM. Data in (A) were analyzed by log-rank Mantel–Cox test. Data in (B) were analyzed using multiple t tests and correcting for multiple comparisons using the Holm–Sidak method. Data in (C) and (D) were analyzed using unpaired, nonparametric Mann–Whitney tests. *p ≤ 0.05, ****p < 0.0001. L.O.D., limit of detection.

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IL-10R is expressed on a broad array of hematopoietic cells (18). Thus, we next assessed whether IL-10 acts directly on B cells to promote GC B cell responses. To do this we generated mixed bone marrow chimeric mice containing WT and Il10rβ−/− compartments at a 1:1 ratio, which allowed us to dissect the role of IL-10 signaling on B cells in the same infected host. We observed clear reductions in GC B cell differentiation among Il10rβ−/− B cells compared with WT B cells (Fig. 2A), showing that B cell–intrinsic IL-10 signaling plays a direct role in promoting GC B cell responses. To explore the physiological relevance of B cell–intrinsic IL-10R signaling on parasite control and secretion of GC-derived Ab responses, we generated mixed bone marrow chimeric mice in which IL-10Rβ–deficiency was restricted to B cells (BIL10R−/−). Strikingly, relative to BWT mice, BIL10R−/− mice were unable to control parasitemia after the second week of infection (Fig. 2B), succumbing to infection similar to α-IL-10R–treated mice (Fig. 1B). Consistent with the loss of parasite control, BIL10R−/− mice exhibited 5- to 6-fold reductions in parasite-specific secreted IgG1, and IgG2b (Fig. 2C), which was further linked to nearly 6-fold numerical reductions in GC B cells (Fig. 2D). Importantly, total numbers of CD19+ B cells were not significantly different between BWT mice and BIL10R−/− mice (Supplemental Fig. 1), supporting that IL-10 signaling regulates GC B cell responses, not B cell survival. Together, these data show that B cell–intrinsic IL-10 signaling critically supports GC B cell differentiation and promotes GC-derived Ab responses. Moreover, the loss of IL-10R signaling in B cells phenocopies the dysregulated humoral immune response observed in P. yoelii–infected α-IL-10R–treated mice (Fig. 1C). Thus, B cell–intrinsic IL-10 signaling is essential for the generation of GC B cell and Ab responses, parasite control, and host survival during experimental malaria.

FIGURE 2.

B cell-intrinsic IL-10 signaling is essential for anti-Plasmodium humoral immunity. (A) Representative dot plots and summary data showing the proportion of GC (CD19+GL7+) B cells among WT and Il10rβ−/− B cells in Plasmodium infected WT:Il10rβ−/− (1:1) chimeric mice on day 10 p.i. connect proportions of GL7+ GC B cells among WT and Il10rβ−/− cells in the same mouse. Data are representative of three independent experiments. (BD) Mixed bone marrow chimeric mice in which B cells were either WT (BWT) or deficient in expression of Il10rβ−/− (BIL-10R−/−) were infected with P. yoelii. Parasite burdens (B), parasite-specific secreted Ab responses (C), representative dot plots and total number of GC B cells [CD19+GL7+Fas+, (D)]. Data in (B) are pooled from two independent experiments. Data in (C) and (D) are from day 21 p.i. and represent two independent experiments. Data in (B)–(D) are mean ± SEM. Data in (A) were analyzed using paired, nonparametric Wilcoxon matched-pairs signed rank test. Data in (B) were analyzed using multiple t tests and correcting for multiple comparisons using the Holm–Sidak method. Data in (C) and (D) were analyzed using unpaired, nonparametric Mann–Whitney tests. **p ≤ 0.01.

FIGURE 2.

B cell-intrinsic IL-10 signaling is essential for anti-Plasmodium humoral immunity. (A) Representative dot plots and summary data showing the proportion of GC (CD19+GL7+) B cells among WT and Il10rβ−/− B cells in Plasmodium infected WT:Il10rβ−/− (1:1) chimeric mice on day 10 p.i. connect proportions of GL7+ GC B cells among WT and Il10rβ−/− cells in the same mouse. Data are representative of three independent experiments. (BD) Mixed bone marrow chimeric mice in which B cells were either WT (BWT) or deficient in expression of Il10rβ−/− (BIL-10R−/−) were infected with P. yoelii. Parasite burdens (B), parasite-specific secreted Ab responses (C), representative dot plots and total number of GC B cells [CD19+GL7+Fas+, (D)]. Data in (B) are pooled from two independent experiments. Data in (C) and (D) are from day 21 p.i. and represent two independent experiments. Data in (B)–(D) are mean ± SEM. Data in (A) were analyzed using paired, nonparametric Wilcoxon matched-pairs signed rank test. Data in (B) were analyzed using multiple t tests and correcting for multiple comparisons using the Holm–Sidak method. Data in (C) and (D) were analyzed using unpaired, nonparametric Mann–Whitney tests. **p ≤ 0.01.

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Bcl-6 is an essential transcriptional regulator of GC B cell differentiation, maintenance, class-switch recombination, and somatic hypermutation (19). T-bet, the master transcriptional regulator of T helper 1 cells, can also be expressed by GC B cells and several reports highlight that T-bet promotes class switching and plasma cell differentiation (20, 21). By contrast, Plasmodium infection of T-bet deficient (Tbx21−/−) mice is associated with enhanced parasite control and larger GC-derived anti-Plasmodium Ab responses (22), supporting that T-bet limits GC responses and anti-malarial humoral immunity. To dissect if IL-10 regulates the transcription factor profile of GC B cells, we first evaluated Bcl-6 and T-bet expression in GC B cells recovered from Plasmodium-infected WT and Il10−/− mice. The proportion of T-bet+ GC B cells (Fig. 3A) and ratio of T-bet+ to Bcl-6+ GC B cells (Fig. 3B) were elevated 2-fold in Il10−/− mice, compared with WT mice, supporting that GC B cells in Il10−/− mice preferentially express T-bet. By extension, Bcl-6+ GC B cells outnumbered T-bet+ GC B cells 2-fold in WT mice (Fig. 3A, 3B), indicating that GC B cells in WT mice preferentially express Bcl-6. We identified that significantly larger proportions of Il10Rβ−/− GC B cells express T-bet+ compared with WT GC B cells in 1:1 chimeric mice (Fig. 3C), consistent with a B cell-intrinsic role for IL-10 signaling in regulating the transcription factor profile of parasite-specific GC B cells. Notably, there were no differences in the proportion of WT or Il10Rβ−/− GC B cells expressing Bcl-6 (not depicted), supporting that IL-10 directly limits expression of T-bet, but not Bcl-6.

FIGURE 3.

IL-10 limits and IFN-γ promotes T-bet expression in GC B cells. (A and B) WT and Il10−/− mice were infected with P. yoelii. (A) Representative dot plots and summary data depicting the proportion of GC B cells (CD19+GL7+Fas+) expressing Bcl-6 and T-bet on day 10 p.i. (B) Ratio of T-bet+ GC B cells to Bcl-6+ GC B cells. Data are pooled from three independent experiments. (C) Proportion of T-bet+ GC B cells (CD19+GL7+) in WT:Il10rβ−/− chimeric mice on day 10 p.i. Lines connect proportions of T-bet+ GC B cells among WT and Il10rβ−/− cells in the same mouse. Data are representative of two independent experiments. (D and E) WT and Il10−/− mice were infected with P. yoelii. Il10−/− mice were treated with either 500 μg of rIgG or α-IFN-γ every 2 d starting on day-1. (D) Representative dot plots and summary data showing the proportion GC B cells (CD19+GL7+Fas+) expressing Bcl-6 and T-bet on day 10 p.i. (E) Ratio of T-bet+ GC B cells to Bcl-6+ GC B cells in mice from each experimental group. Data are pooled from two independent experiments. (FH) WT:Ifnγr1−/− mixed bone marrow chimeric mice were infected with P. yoelii. Representative dot plots and summary data showing the proportion GC B cells (CD19+GL7+) (F), Bcl-6+ GC B cells (G), and T-bet+ GC B cells (H) among WT and Ifnγr1−/− cells recovered on day 10 p.i. Lines connect proportions in the same mice. Data are representative of two independent experiments. In (C), (G), and (H), gray histograms represent isotype control staining. Data in (A), (B), (D), and (E) are mean ± SEM. Data in (A) and (B) were analyzed using unpaired, nonparametric Mann–Whitney tests. Data in (C) and (F)–(H) were analyzed using paired, nonparametric Wilcoxon matched-pairs signed rank tests. Data in (D) and (E) were analyzed using unpaired, nonparametric Kruskal–Wallis test correcting for multiple comparisons via Dunn’s tests.

FIGURE 3.

IL-10 limits and IFN-γ promotes T-bet expression in GC B cells. (A and B) WT and Il10−/− mice were infected with P. yoelii. (A) Representative dot plots and summary data depicting the proportion of GC B cells (CD19+GL7+Fas+) expressing Bcl-6 and T-bet on day 10 p.i. (B) Ratio of T-bet+ GC B cells to Bcl-6+ GC B cells. Data are pooled from three independent experiments. (C) Proportion of T-bet+ GC B cells (CD19+GL7+) in WT:Il10rβ−/− chimeric mice on day 10 p.i. Lines connect proportions of T-bet+ GC B cells among WT and Il10rβ−/− cells in the same mouse. Data are representative of two independent experiments. (D and E) WT and Il10−/− mice were infected with P. yoelii. Il10−/− mice were treated with either 500 μg of rIgG or α-IFN-γ every 2 d starting on day-1. (D) Representative dot plots and summary data showing the proportion GC B cells (CD19+GL7+Fas+) expressing Bcl-6 and T-bet on day 10 p.i. (E) Ratio of T-bet+ GC B cells to Bcl-6+ GC B cells in mice from each experimental group. Data are pooled from two independent experiments. (FH) WT:Ifnγr1−/− mixed bone marrow chimeric mice were infected with P. yoelii. Representative dot plots and summary data showing the proportion GC B cells (CD19+GL7+) (F), Bcl-6+ GC B cells (G), and T-bet+ GC B cells (H) among WT and Ifnγr1−/− cells recovered on day 10 p.i. Lines connect proportions in the same mice. Data are representative of two independent experiments. In (C), (G), and (H), gray histograms represent isotype control staining. Data in (A), (B), (D), and (E) are mean ± SEM. Data in (A) and (B) were analyzed using unpaired, nonparametric Mann–Whitney tests. Data in (C) and (F)–(H) were analyzed using paired, nonparametric Wilcoxon matched-pairs signed rank tests. Data in (D) and (E) were analyzed using unpaired, nonparametric Kruskal–Wallis test correcting for multiple comparisons via Dunn’s tests.

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IL-10 is a potent suppressor of IFN-γ production (23). Furthermore, T-bet expression can be regulated by IFN-γ in B cells (24). Thus, we predicted that the transcription factor profiles of GC B cells are additionally regulated by IFN-γ during Plasmodium infection. In support of this, neutralization of IFN-γ in Plasmodium-infected Il10−/− mice reestablished GC B cell profiles that match those observed in GC B cells recovered from WT mice, with Bcl-6+ GC B cells outnumbering T-bet+ GC B cells 2-fold (Fig. 3D, 3E). To test whether B cell–intrinsic IFN-γ signaling limits GC B cell responses and/or induces T-bet expression in GC B cells, we generated and infected WT:Ifnγr1−/− (1:1) bone marrow chimeric mice with P. yoelii. Ifnγr1−/− B cells were more likely to differentiate into GC B cells (Fig. 3F). Moreover, larger proportions of Ifnγr1−/− GC B cells expressed Bcl-6 (Fig. 3G), whereas reduced proportions expressed T-bet (Fig. 3H). These data show that IFN-γ acts directly on B cells to regulate their relative expression of Bcl-6 and T-bet. Collectively, these data demonstrate that B cell–intrinsic IL-10 signaling limits the formation of T-bet+ GC B cells, whereas B cell–intrinsic IFN-γ signaling promotes the development of T-bet+ GC B cells. The data depicted in this study mechanistically link the deleterious effects of excessive IFN-γ (25) to B cell–intrinsic, direct alterations of the GC B cell transcription factor profile with preferential induction of T-bet and loss of Bcl-6 expression.

Because IFN-γ limited GC B cell reactions in a B cell-intrinsic manner, we hypothesized that B cell–intrinsic IFN-γ signaling would by extension constrain GC-derived, parasite-specific secreted Ab responses. To test this, we generated mixed bone marrow chimeric mice in which B cells specifically lacked Ifnγr1 (BIFNgR−/−). In support of our hypothesis, Plasmodium-infected BIFNgR−/− mice exhibited 1.5- and 2.6-fold increases in parasite-specific secreted IgG1 and IgG2b, respectively, relative to BWT mice (Fig. 4A). Thus, IFN-γ acts directly on B cells to limit GC B cell responses (Fig. 3F) and class-switched, parasite-specific IgG1 and IgG2b Ab responses during experimental malaria. Because B cell–intrinsic IFN-γ signaling potently stimulated T-bet expression (Fig. 3H), we additionally tested whether T-bet is mechanistically linked to suppressing GC B cell responses and subsequent secretion of Plasmodium-specific Ab. To do this, we generated mixed bone marrow chimeric mice in which T-bet deficiency was restricted to the B cell compartment (BT-bet−/−). Plasmodium-infected BT-bet−/− mice exhibited a ∼3.5-fold increase in the total number of GC B cells, relative to Plasmodium-infected BWT mice (Fig. 4B). The numerical enhancement of GC B cell responses in BT-bet−/− mice was additionally linked to a 4.1-fold increase in IgG1 titers and a 3.6-fold increase in IgG2b titers, relative to parasite-specific Ab titers in BWT mice (Fig. 4C). These data show that B cell–intrinsic expression of T-bet limits the magnitude of the Plasmodium infection–induced GC B cell responses, resulting in less parasite-specific secreted Ab. Of note, relative to BWT mice, neither BIFNgR−/− nor BT-bet−/− mice exhibited enhanced parasite control (not depicted). This could be due to each experimental group exceeding a threshold of secreted Ab necessary to resolve the infection or potential deleterious impacts on B–T communication and/or parasite-specific effector CD4 T cell function. This latter possibility is unlikely as GC-Tfh responses are also elevated in the BIFNgR−/− and BT-bet−/− chimeras (not depicted). Nevertheless, these data conclusively show that B cell–intrinsic IFN-γ signaling and T-bet expression limit anti-Plasmodium humoral immunity.

FIGURE 4.

IFN-γ signaling and T-bet expression in B cells limits humoral immunity. (A) Parasite-specific secreted Ab endpoint titers in Plasmodium-infected mixed bone marrow chimeric mice in which B cells were either WT (BWT) or deficient in expression of IFN-γ receptor 1 (BIFNgR−/−). Titers were determined on day 21 p.i. Data are pooled from two independent experiments. (B) Representative dot plots and summary data showing the proportion and total number of CD19+ GC B cells (CD19+GL7+Fas+) on day 21 p.i. in Plasmodium-infected mixed bone marrow chimeric mice in which B cells were either WT (BWT) or deficient in expression of T-bet (BT-bet−/−). (C) Parasite lysate specific total IgG, IgG1, and IgG2b end-point titers on day 21 p.i. in BWT and BT-bet−/− chimeric mice. Data are pooled from two independent experiments. Data in (A)–(C) were analyzed using unpaired, nonparametric Mann–Whitney tests. Data in (A) and (C) are mean ± SEM.

FIGURE 4.

IFN-γ signaling and T-bet expression in B cells limits humoral immunity. (A) Parasite-specific secreted Ab endpoint titers in Plasmodium-infected mixed bone marrow chimeric mice in which B cells were either WT (BWT) or deficient in expression of IFN-γ receptor 1 (BIFNgR−/−). Titers were determined on day 21 p.i. Data are pooled from two independent experiments. (B) Representative dot plots and summary data showing the proportion and total number of CD19+ GC B cells (CD19+GL7+Fas+) on day 21 p.i. in Plasmodium-infected mixed bone marrow chimeric mice in which B cells were either WT (BWT) or deficient in expression of T-bet (BT-bet−/−). (C) Parasite lysate specific total IgG, IgG1, and IgG2b end-point titers on day 21 p.i. in BWT and BT-bet−/− chimeric mice. Data are pooled from two independent experiments. Data in (A)–(C) were analyzed using unpaired, nonparametric Mann–Whitney tests. Data in (A) and (C) are mean ± SEM.

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In this study we show that B cell–intrinsic T-bet expression impairs GC B cell differentiation and limits GC B cell derived Ab responses during experimental malaria. These data are in contrast to recent reports supporting that T-bet promotes GC B cell responses in models of acute and chronic viral infection and spontaneous autoimmunity (20, 24). We performed comparative studies and identified that LCMV clone 13-infected BT-bet−/− mice exhibited a 2-fold reduction in the magnitude of the GC B cell response and a 2.4-fold reduction in LCMV-specific IgG on day 21 p.i. (Supplemental Fig. 2), confirming the importance of B cell–intrinsic T-bet expression in promoting humoral immunity during chronic viral infection. Although the precise reasons for the differential role of T-bet in B cells responding to chronic viral and parasitic infections are not currently clear, they may be linked to greater perturbations of pro- and anti-inflammatory networks during malaria. Indeed, IFN-γ is potently induced during Plasmodium infection (26), which may be further linked to sustained expression of T-bet in parasite-specific lymphocyte subsets. Moreover, when T-bet is in excess in CD4 T cells, it can directly form a complex with Bcl-6, which prevents Bcl-6 from repressing its target promoters, including Blimp-1 (27). Although T-bet/Bcl-6 complexes have not been reported in B cells, our data identify that T-bet is inversely correlated to Bcl-6 expression in GC B cells. Reduced Bcl-6 expression or function may limit B cell class-switching and affinity maturation, resulting in the preferential expansion of IgM-expressing Ab secreting cells. In support of this notion, recent work from our laboratory has identified that B cell-intrinsic IFN-γ signaling and T-bet expression drive the expansion of short-lived plasmablasts that play no appreciable role in parasite control (J.J. Guthmiller, R.L. Pope, A.C. Graham, R. A. Zander, A. Chitrakar, R.K. Tweten, J. Li, and N.S. Butler, submitted for publication). Thus, B cell–intrinsic IFN-γ signaling and T-bet expression directly regulate fate determination of parasite-specific B cells during Plasmodium infection.

Our study also shows that IL-10 and IFN-γ signaling events differentially regulate the expression of T-bet in GC B cells. Thus, we propose a model in which IL-10 regulates anti-Plasmodium humoral immunity through two distinct mechanisms. First, B cell–intrinsic IL-10 signaling directly promotes GC B cell responses via limiting B cell expression of T-bet. Second, IL-10 suppresses local and systemic IFN-γ, which induces T-bet expression in GC B cells and limits their number and function. As a composite our data reveal critical mechanisms of B cell regulation during malaria that may contribute to the suboptimal and short-lived nature of anti-Plasmodium humoral immunity. Our findings may also be relevant for developing new interventions to improve anti-Plasmodium humoral immunity following infection or vaccination.

We thank John Harty, Mark Lang, and Lauren Zenewicz for critical feedback and the University of Oklahoma Health Sciences Center Flow Cytometry Laboratory for technical assistance.

This work was supported by grants from the American Heart Association (16PRE27660002 to J.J.G.), the National Institutes of Health/National Institute of Allergy and Infectious Diseases (T32AI007633 to R.A.Z.; R01AI125446 and R01AI127481 to N.S.B.), and the National Institutes of Health/National Institute of General Medical Sciences under Grant 8P20GM103447.

The online version of this article contains supplemental material.

Abbreviations used in this article:

GC

germinal center

p.i.

postinfection

rIgG

rat γ Ig

WT

wild type.

1
World Health Organization
.
2015
.
World Malaria Report 2015
.
World Health Organization
,
Geneva, Switzerland
.
2
Cohen
S.
,
McGREGOR
I. A.
,
Carrington
S.
.
1961
.
Gamma-globulin and acquired immunity to human malaria.
Nature
192
:
733
737
.
3
Crompton
P. D.
,
Kayala
M. A.
,
Traore
B.
,
Kayentao
K.
,
Ongoiba
A.
,
Weiss
G. E.
,
Molina
D. M.
,
Burk
C. R.
,
Waisberg
M.
,
Jasinskas
A.
, et al
.
2010
.
A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.
Proc. Natl. Acad. Sci. USA
107
:
6958
6963
.
4
Kinyanjui
S. M.
,
Conway
D. J.
,
Lanar
D. E.
,
Marsh
K.
.
2007
.
IgG antibody responses to Plasmodium falciparum merozoite antigens in Kenyan children have a short half-life.
Malar. J.
6
:
82
.
5
Leoratti
F. M.
,
Durlacher
R. R.
,
Lacerda
M. V.
,
Alecrim
M. G.
,
Ferreira
A. W.
,
Sanchez
M. C.
,
Moraes
S. L.
.
2008
.
Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria.
Malar. J.
7
:
186
.
6
Tran
T. M.
,
Li
S.
,
Doumbo
S.
,
Doumtabe
D.
,
Huang
C. Y.
,
Dia
S.
,
Bathily
A.
,
Sangala
J.
,
Kone
Y.
,
Traore
A.
, et al
.
2013
.
An intensive longitudinal cohort study of Malian children and adults reveals no evidence of acquired immunity to Plasmodium falciparum infection.
Clin. Infect. Dis.
57
:
40
47
.
7
Jagannathan
P.
,
Eccles-James
I.
,
Bowen
K.
,
Nankya
F.
,
Auma
A.
,
Wamala
S.
,
Ebusu
C.
,
Muhindo
M. K.
,
Arinaitwe
E.
,
Briggs
J.
, et al
.
2014
.
IFNγ/IL-10 co-producing cells dominate the CD4 response to malaria in highly exposed children.
PLoS Pathog.
10
:
e1003864
.
8
Couper
K. N.
,
Blount
D. G.
,
Wilson
M. S.
,
Hafalla
J. C.
,
Belkaid
Y.
,
Kamanaka
M.
,
Flavell
R. A.
,
de Souza
J. B.
,
Riley
E. M.
.
2008
.
IL-10 from CD4CD25Foxp3CD127 adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection.
PLoS Pathog.
4
:
e1000004
.
9
Li
C.
,
Corraliza
I.
,
Langhorne
J.
.
1999
.
A defect in interleukin-10 leads to enhanced malarial disease in Plasmodium chabaudi chabaudi infection in mice.
Infect. Immun.
67
:
4435
4442
.
10
Brière
F.
,
Servet-Delprat
C.
,
Bridon
J. M.
,
Saint-Remy
J. M.
,
Banchereau
J.
.
1994
.
Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3.
J. Exp. Med.
179
:
757
762
.
11
Rousset
F.
,
Garcia
E.
,
Defrance
T.
,
Péronne
C.
,
Vezzio
N.
,
Hsu
D. H.
,
Kastelein
R.
,
Moore
K. W.
,
Banchereau
J.
.
1992
.
Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.
Proc. Natl. Acad. Sci. USA
89
:
1890
1893
.
12
Levy
Y.
,
Brouet
J. C.
.
1994
.
Interleukin-10 prevents spontaneous death of germinal center B cells by induction of the bcl-2 protein.
J. Clin. Invest.
93
:
424
428
.
13
Sun
K.
,
Torres
L.
,
Metzger
D. W.
.
2010
.
A detrimental effect of interleukin-10 on protective pulmonary humoral immunity during primary influenza A virus infection.
J. Virol.
84
:
5007
5014
.
14
Chacón-Salinas
R.
,
Limón-Flores
A. Y.
,
Chávez-Blanco
A. D.
,
Gonzalez-Estrada
A.
,
Ullrich
S. E.
.
2011
.
Mast cell-derived IL-10 suppresses germinal center formation by affecting T follicular helper cell function.
J. Immunol.
186
:
25
31
.
15
Clark
I. A.
,
Budd
A. C.
,
Alleva
L. M.
,
Cowden
W. B.
.
2006
.
Human malarial disease: a consequence of inflammatory cytokine release.
Malar. J.
5
:
85
.
16
Mahanta
A.
,
Kar
S. K.
,
Kakati
S.
,
Baruah
S.
.
2015
.
Heightened inflammation in severe malaria is associated with decreased IL-10 expression levels and neutrophils.
Innate Immun.
21
:
546
552
.
17
Rovira-Vallbona
E.
,
Moncunill
G.
,
Bassat
Q.
,
Aguilar
R.
,
Machevo
S.
,
Puyol
L.
,
Quintó
L.
,
Menéndez
C.
,
Chitnis
C. E.
,
Alonso
P. L.
, et al
.
2012
.
Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study.
Malar. J.
11
:
181
.
18
Moore
K. W.
,
de Waal Malefyt
R.
,
Coffman
R. L.
,
O’Garra
A.
.
2001
.
Interleukin-10 and the interleukin-10 receptor.
Annu. Rev. Immunol.
19
:
683
765
.
19
Fukuda
T.
,
Yoshida
T.
,
Okada
S.
,
Hatano
M.
,
Miki
T.
,
Ishibashi
K.
,
Okabe
S.
,
Koseki
H.
,
Hirosawa
S.
,
Taniguchi
M.
, et al
.
1997
.
Disruption of the Bcl6 gene results in an impaired germinal center formation.
J. Exp. Med.
186
:
439
448
.
20
Barnett
B. E.
,
Staupe
R. P.
,
Odorizzi
P. M.
,
Palko
O.
,
Tomov
V. T.
,
Mahan
A. E.
,
Gunn
B.
,
Chen
D.
,
Paley
M. A.
,
Alter
G.
, et al
.
2016
.
Cutting edge: B cell-intrinsic T-bet expression is required to control chronic viral infection.
J. Immunol.
197
:
1017
1022
.
21
Wang
N. S.
,
McHeyzer-Williams
L. J.
,
Okitsu
S. L.
,
Burris
T. P.
,
Reiner
S. L.
,
McHeyzer-Williams
M. G.
.
2012
.
Divergent transcriptional programming of class-specific B cell memory by T-bet and RORα.
Nat. Immunol.
13
:
604
611
.
22
Oakley
M. S.
,
Sahu
B. R.
,
Lotspeich-Cole
L.
,
Majam
V.
,
Thao Pham
P.
,
Sengupta Banerjee
A.
,
Kozakai
Y.
,
Morris
S. L.
,
Kumar
S.
.
2014
.
T-bet modulates the antibody response and immune protection during murine malaria.
Eur. J. Immunol.
44
:
2680
2691
.
23
D’Andrea
A.
,
Aste-Amezaga
M.
,
Valiante
N. M.
,
Ma
X.
,
Kubin
M.
,
Trinchieri
G.
.
1993
.
Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells.
J. Exp. Med.
178
:
1041
1048
.
24
Domeier
P. P.
,
Chodisetti
S. B.
,
Soni
C.
,
Schell
S. L.
,
Elias
M. J.
,
Wong
E. B.
,
Cooper
T. K.
,
Kitamura
D.
,
Rahman
Z. S.
.
2016
.
IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity.
J. Exp. Med.
213
:
715
732
.
25
Zander
R. A.
,
Obeng-Adjei
N.
,
Guthmiller
J. J.
,
Kulu
D. I.
,
Li
J.
,
Ongoiba
A.
,
Traore
B.
,
Crompton
P. D.
,
Butler
N. S.
.
2015
.
PD-1 co-inhibitory and OX40 Co-stimulatory crosstalk regulates helper T cell differentiation and anti-Plasmodium humoral immunity.
Cell Host Microbe
17
:
628
641
.
26
Rhee
M. S.
,
Akanmori
B. D.
,
Waterfall
M.
,
Riley
E. M.
.
2001
.
Changes in cytokine production associated with acquired immunity to Plasmodium falciparum malaria.
Clin. Exp. Immunol.
126
:
503
510
.
27
Oestreich
K. J.
,
Mohn
S. E.
,
Weinmann
A. S.
.
2012
.
Molecular mechanisms that control the expression and activity of Bcl-6 in TH1 cells to regulate flexibility with a TFH-like gene profile.
Nat. Immunol.
13
:
405
411
.

The authors have no financial conflicts of interest.

Supplementary data