IL-27 Promotes IL-10 Production by Effector Th1 CD4⁺ T Cells: A Critical Mechanism for Protection from Severe Immunopathology during Malaria Infection

IL-10 production increases during primary infection and CD4⁺ T cells are the major source

The timing of production of a regulatory cytokine such as IL-10 may be crucial for the outcome of P. chabaudi infection. Therefore, to define which cells produced IL-10 during the acute infection, and whether the appearance of IL-10 in these cells coincided with control of pathology and acute infection, we determined the numbers of IL-10⁺ splenic cells throughout the acute phase of P. chabaudi infection in C57BL/6 mice (days 0–30) by FACS analysis (Fig. 1). After the peak of parasitemia (characteristically around day 7), a dramatic increase in the numbers of cells producing IL-10 (gated as shown in Fig. 1A) was evident, reaching a maximum of 2.5 × 10⁶ cells/spleen on day 15 (Fig. 1B). The numbers of IL-10–producing cells rapidly decreased thereafter, but spleens of animals infected for 30 d still contained more IL-10⁺ cells than uninfected mice. The presence of a large number of IL-10–producing cells at this time coincided with the control of pathology, which is usually observed between days 10 and 15 (13).

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FIGURE 1.

CD4⁺ cells are the major producers of IL-10 during acute P. chabaudi infection. C57BL/6 mice were infected i.p. with 10⁵ parasitized RBC, and after different time points, the splenocytes were harvested, stimulated in culture with PMA and ionomycin for 6 h, and IL-10 production was detected using intracellular staining. A, The gating strategy is shown. B, Total numbers of IL-10–producing cells per spleen during infection (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 compared with day 0 [D0]; t test; n = 7–9 per time point). No analysis was performed on day 25. The parasitemia curve is also presented. C, Pie charts represent the distribution of different cell subsets among IL-10⁺ cells during analysis. Bar graph shows total numbers of IL-10⁺ cells per spleen for each cell population on specific time points (non-T non-B cells = CD3⁻CD4⁻CD8⁻CD19⁻NK1.1⁻ cells). Data are representative of three independent experiments. D, Several cell types (CD11b^hi, CD11c^hi, NK1.1⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, and CD19⁺) were sorted on day 7 of infection, and IL-10 transcription was analyzed by quantitative RT-PCR (mean ± SEM, n = 3–5). N.D., not determined.

IL-10 can be produced by cells of both the innate and adaptive immune system (5). We therefore used FACS analysis to determine which splenic cells (CD4⁺, CD8⁺, CD19⁺ B cells, NK cells, NKT cells) were responsible for this production during the acute P. chabaudi infection. Already from day 5, CD4⁺ cells were clearly the biggest IL-10⁺ population, accounting for nearly 50% of total IL-10–secreting cells, as shown in the pie charts (Fig. 1C). On days 15, 20, and 30 postinfection, the CD4⁺ cells accounted for up to 70% of the IL-10⁺ cells with >2 × 10⁶ cells per spleen. The total number of IL-10⁺ CD4⁺ T cells declined as the infection progressed to the chronic stage, but at day 30 of infection, the numbers were still greater than those observed in uninfected mice (0.7 × 10⁶ on day 30 and 0.09 × 10⁶ on day 0; p = 0.0002; t test). Regarding the frequency of IL-10⁺ cells in the spleen during this infection, we observed that when the whole spleen was considered, these frequencies do not change significantly (data not shown); however, we observed an ∼10-fold increase in the frequency of IL-10⁺ cells within the CD4⁺ population (from 0.46 ± 0.15% on day 0 to 4.19 ± 0.08% on day 15; data not shown), which means that the increase in the total numbers of IL-10⁺ CD4⁺ T cells is not only due to the characteristic splenomegaly during this infection. Therefore, these results show CD4⁺ T cells to be the major source of IL-10, with maximum numbers after the peak of parasitemia.

We also found that B cells (CD19⁺) produced IL-10. CD19⁺ cells represented nearly 40% of the IL-10⁺ cell population in naive mice (day 0, Fig. 1C). This proportion decreased after day 10 of infection. Production of IL-10 by non-T non-B cells (CD3⁻CD4⁻CD8⁻CD19⁻NK1.1⁻ cells) was also evident (∼25% of IL-10–producing cells at day 10).

Although myeloid cells (monocytes, macrophages, and DCs) can also produce IL-10 (5, 46), we could not reliably detect this by FACS analysis in these cells. Therefore, we analyzed IL-10 transcription by these cells using quantitative RT-PCR at the peak of parasitemia (day 7), when the highest Ag load is present, and therefore, both myeloid and lymphoid cells are activated. The various different cell populations (CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺, NK1.1⁺, CD11b^high, CD11c^high) were sorted from the spleens of infected C57BL/6 mice. In agreement with the FACS data presented in Fig. 1A–C, a large upregulation of IL-10 transcription was observed in CD4⁺ T cells, whereas other cell types, such as CD11b^hi (43), CD11c^hi, CD19⁺, and CD8⁺ T cells, showed much lower levels of IL-10 mRNA transcripts (Fig. 1D). NK1.1⁺ cells showed no increase in IL-10 transcription compared with naive cells. Thus, despite the relative widespread upregulation of IL-10 transcription among cells of both the innate and adaptive immune response, CD4⁺ T cells expressed the highest level of IL-10 mRNA, confirming them as the major source of IL-10 during P. chabaudi infection.

The majority of IL-10–producing CD4⁺ T cells also produce IFN-γ, and their numbers increase transiently after the peak of parasitemia

Studies have shown that IFN-γ⁺ Th1 cells that coproduce IL-10 have an important function in downregulating the immune response to T. gondii (17) and L. major (16), and in controlling autoimmune responses (47). Therefore, we speculated that IL-10 produced by CD4⁺ T cells in conjunction with IFN-γ during infection in this malaria model also plays an important role. To examine this hypothesis, we analyzed splenocytes for cytokine production by flow cytometry at different time points postinfection. We observed both IL-10⁺ IFN-γ⁺ coproducing cells and IL-10⁺ single-producing cells. Both types of IL-10⁺ cells were present throughout infection, but the greatest number and percentages were observed on day 15 (Fig. 2A, 2B), coinciding with clinical signs of pathology. The frequencies of IL-10–producing, single-positive CD4⁺ T cells were low and relatively constant throughout. In fact, there were ∼6-fold more IL-10⁺ IFN-γ⁺ double-producing CD4⁺ T cells than those producing only IL-10, suggesting an important role for CD4⁺ T cells producing both IL-10 and IFN-γ in the control of immunopathology.

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FIGURE 2.

IFN-γ⁺ CD44^hi CD4⁺ T cells are the main source of IL-10 during acute infection. Spleen cells from C57BL/6 mice were harvested on different days postinfection and stimulated in culture with PMA and ionomycin, which was followed by intracellular staining for cytokines. A, Representative dot plots show gated CD4⁺ T cells. Production of IL-10 and IFN-γ was assessed throughout acute infection and mean percentages are presented. B, Total numbers of IL-10⁺ and IFN-γ⁺ IL-10⁺ CD4⁺ T cells per spleen (mean ± SEM; n = 7–9 per time point; t test). C, MFI for IFN-γ (day 5) and IL-10 (day 15) observed for the different cytokine-producing cell populations. D, Representative histograms show CD44 surface staining and (E) expression of transcription factor T-bet on CD4⁺ T cells from day 15-infected mice. F, CD4⁺ T cells from day 15-infected mice were stained for CD45RB, CD127, ICOS, and CD62L, which was followed by intracellular staining for IFN-γ and IL-10. Representative histograms are presented, and the bar graphs show the MFI for each cell population and the respective marker. Data are representative of three (A–D, F) and two independent experiments (E). Mean ± SEM; n = 6–9; t test; *p < 0.05, **p < 0.01, ***p < 0.001.

The induction of these IL-10⁺ Th1 CD4⁺ T cells has been shown to require high Ag load (48). In Fig. 2A, we also observe this correlation, as double-producing cells are induced after the peak of parasitemia, when the greatest amounts of Ag are available, and later, when parasitemia drops, the frequency of those cells also decreases.

Interestingly, IL-10⁺ IFN-γ⁺ double-producing cells expressed higher levels of IFN-γ than single IFN-γ⁺ cells at the beginning of infection (day 5, mean fluorescence intensity [MFI]: 1072 ± 154.1 and 509.5 ± 48.15, respectively; p = 0.028; t test; Fig. 2C). Similarly, at the time of maximum numbers of double IL-10⁺ IFN-γ⁺ cells (day 15), MFI of IL-10 was greater in these cells compared with the single IL-10 producers (138.5 ± 3.0 and 102.5 ± 2.86, respectively; p = 0.028; t test).

The higher levels of IFN-γ and IL10 in the double-positive cells may indicate that they are highly activated or more differentiated. Indeed, IL-10⁺ IFN-γ⁺ CD4⁺ T cells expressed more CD44 (MFI: 125.2 ± 6.3) than either IFN-γ (66.8 ± 0.9; p = 0.0022; t test) or IL-10 (28.6 ± 0.9; p = 0.005; t test) single-producing CD4⁺ T cells (Fig. 2D). These double producers also expressed the highest levels of the ICOS and the lowest levels of IL-7Rα (CD127) compared with IFN-γ and IL-10 single producers (Fig. 2F), demonstrating an increased activation state.

Critically, the expression of the transcription factor T-bet in IL-10⁺ IFN-γ⁺ Th1 cells was at similar levels to those of IFN-γ⁺ Th1 cells, demonstrating that highly activated effector Th1 cells are the major source of IL-10 during P. chabaudi infection (Fig. 2E).

Mice with Il10 deletion specifically in T cells reproduce the phenotype observed in Il10^−/− mice

As effector Th1 cells were the major source of IL-10 after the peak of infection, the next step was to determine whether IL-10 from T cells was important to control immune-mediated pathology in vivo. We therefore asked whether mice carrying a deletion of Il10 specifically in T cells, CD19⁺ B cells, or macrophages and neutrophils (LysM⁺ cells) would reproduce the phenotype observed in completely deficient Il10^−/− mice.

For these experiments, we used Il10^flox/flox mice (35) crossed to CD4-Cre (35, 36), CD19-Cre (39), or LysM-Cre (37, 38), and the appropriate Il10^flox/floxCre⁻ littermate controls. The mice were infected with 10⁵ P. chabaudi, and survival rates, weight loss, temperature decline, and anemia (RBC/ml) were monitored for 20 d. As shown in Fig. 3A, only those mice unable to secrete IL-10 specifically from T cells (Il10^fl/flCD4Cre⁺ mice) fully reproduced the pathological features of the infection in the Il10^−/− mice, in that they exhibited substantial weight loss, hypothermia, and anemia, and only 30–40% of mice survived the acute infection, despite the fact that in these mice other cells are competent in secreting IL-10. Therefore, not only are T cells the major source of IL-10 during P. chabaudi infection, but they are the critical source of protective IL-10, which promotes protection from severe immunopathology. Importantly, lack of IL-10 in T cells did not affect the duration or magnitude of the low/subpatent chronic P. chabaudi AS infection that follows the acute infection (data not shown).

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FIGURE 3.

Mice with Il10 deletion specifically in T cells reproduce the phenotype observed in Il10^−/− mice. Using the Cre/loxP recombination system, specific deletions of Il10 were performed in T cells, CD19⁺ cells, or monocyte/neutrophil (LysM⁺) cells. These mice are designated as Il10^fl/flCD4Cre⁺, Il10^fl/flCD19Cre⁺, and Il10^fl/flLysMCre⁺ mice, respectively. A, Mice from these lineages were infected i.p. with 10⁵ pRBC. Survival rates and signs of pathology (weight loss, temperature, and RBC counts) were monitored. The graphs for weight loss, temperature, and RBC counts present data from days 10, 9, and 11 postinfection, respectively, when the major differences were observed between the groups (mean ± SEM; n = 10–20; t test). Respective Cre⁻ littermate controls for each mouse line were used. Experiments were performed four times with similar results. B, Plasma cytokines were detected at the peak of parasitemia (day 7; mean ± SEM; n = 7–10; each symbol indicates an individual mouse). Red dotted line represents the cytokine values for naive mice.

Interestingly, mice carrying B cells unable to produce IL-10 (Il10^fl/flCD19Cre⁺) also presented partial signs of pathology, with 15% mortality rate during the infection, suggesting that IL-10–producing B cells may contribute to control of malaria disease. These Il10^fl/flCD19Cre⁺ mice have significantly lower frequencies and total numbers of IL-10⁺ IFN-γ⁺ double-producing and IL-10⁺ single-producing CD4⁺ T cells compared with Il10^fl/flCD19Cre⁻ controls (Supplemental Fig. 2A, 2B), yet comparable numbers of IFN-γ⁺ single-producing CD4⁺ T cells and Foxp3⁺ CD4⁺ T cells (Supplemental Fig. 2B, 2C). Thus, the increase in the severity of immunopathology observed in mice unable to secrete IL-10 specifically from B cells (Il10^fl/flCD19Cre⁺ mice) during P. chabaudi infection may result from their inability to generate sufficient numbers of IL-10–secreting CD4⁺ T cells.

IL-10 from monocytes and neutrophils did not appear to be required for the control of immunopathology, as there was no mortality, and weight loss, anemia, or hypothermia in Il10^fl/flLysMCre⁺ mice was similar to their respective Cre⁻ controls and WT C57BL/6 mice. Similar to infections in Il10^−/− mice (12, 13), the effects of the deletion of Il10 in any of the cell populations could not readily be ascribed to differences in parasitemias because these were similar between the Il10^fl/fl Cre⁺ mice and their respective Cre⁻ controls, and not significantly different from those of WT C57BL/6 (Supplemental Fig. 2D).

Similarly to Il10^−/− mice (12, 13), infected Il10^fl/flCD4Cre⁺ mice exhibited greater amounts of the proinflammatory cytokines IFN-γ, TNF-α, and IP-10 compared with infected WT and Il10^fl/flCre⁻ control mice (Fig. 3B), indicating the more inflammatory nature of the host response to P. chabaudi when IL-10 was lacking in T cells. Taken together, these results demonstrate a major protective role for IL-10 produced by T cells, most likely CD4⁺ T cells, against immune-mediated pathology during an acute blood-stage P. chabaudi infection.

IL-10 from WT CD4⁺ T cells rescues immunodeficient mice from death

Because CD4⁺ T cells were found to be the major producers of IL-10 during a P. chabaudi infection, and T cell-derived IL-10 is important for the regulation of pathology in this infection, we wanted to verify directly that IL-10 from CD4⁺ T cells prevented pathology. Therefore, WT C57BL/6 or Il10^−/− CD4⁺ T cells were adoptively transferred into Rag1^−/− mice, which were then infected with P. chabaudi and the acute-stage pathology monitored. In these experiments, the aim was to observe a direct protective role for CD4-derived IL-10 in the acute stage of infection when immunopathology is most evident. Rag1^−/− mice infected with P. chabaudi normally exhibit severe weight loss, hypothermia, and anemia, and ∼40% die already within the first 15 d of infection (Fig. 4A). As shown in Fig. 4A and 4B, all Rag1^−/− mice that received WT CD4⁺ T cells survived for the duration of the analysis, exhibited significantly less weight loss, less hypothermia, and less anemia than Rag1^−/− mice receiving Il10^−/− CD4⁺ T cells or unreconstituted Rag1^−/− mice, demonstrating that IL-10–sufficient CD4⁺ T cells controlled pathology in this model. By contrast, recipient mice transferred with Il10^−/− CD4⁺ T cells showed equivalent pathology to unreconstituted Rag1^−/− mice and, in fact, required longer to recover.

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FIGURE 4.

IL-10 from CD4⁺ T cells rescues immunodeficient mice from death. On day −1, Rag1^−/− mice received 10⁷ naive CD4⁺ T cells i.v. either from C57BL/6 WT or Il10^−/− mice. Unreconstituted Rag1^−/− mice were used as controls. A, After infection with 10⁵ parasitized RBC i.p. on day 0, all mice were monitored for 15 d and pathology was assessed. Survival rate and weight loss are shown (weight-loss graph: statistical analysis was performed using both transferred groups, but comparing mice transferred with WT CD4⁺ cells and unreconstituted mice showed similar results). B, Additional pathology measurements are presented: temperature, RBC counts, and hemoglobin (HGB). C, Total numbers of parasitized RBC (pRBC) per liter of blood on days 7 and 9 postinfection (mean ± SEM; n = 12–20/group). The experiments were performed three times. D, Percentages and total numbers of Foxp3⁺ CD4⁺ T cells per spleen of recipient Rag1^−/− mice on day 10 postinfection (each symbol indicates an individual mouse). Before infection, Rag1^−/− mice receiving either WT or Il10^−/− CD4⁺ T cells presented similar frequencies of Foxp3⁺ CD4⁺ T cells (∼14%; data not shown). Mean ± SEM; n = 10 per group; *p < 0.05, **p < 0.01, ***p < 0.001; t test (A, C, D); one-way ANOVA (B).

The differences in pathology in Rag1^−/− mice receiving either WT or Il10^−/− CD4⁺ T cells could not be explained by the differences in parasitemia, because they both showed similar numbers of parasitized RBC/l (Fig. 4C). Therefore, these data confirm that IL-10 from CD4⁺ T cells is critical to limit the immunopathology during P. chabaudi infection but is not important for the control of parasitemia.

As it has been demonstrated that transfer of CD4⁺ T cells into lymphopenic hosts, such as Rag1^−/− mice, can result in a preferential expansion of Foxp3⁺ Tregs (49), we assessed the numbers of Foxp3⁺ CD4⁺ T cells in the reconstituted Rag1^−/− mice. Before transfer, purified cells were assessed for the expression of Foxp3, and both WT and Il10^−/− CD4⁺ T cells presented similar frequencies of Foxp3⁺ cells (∼14%; data not shown). Surprisingly, after transfer and infection, Rag1^−/− mice receiving WT CD4⁺ T cells had significantly higher percentages and greater numbers of Foxp3⁺ CD4⁺ T cells per spleen compared with those mice receiving Il10^−/− CD4⁺ T cells (Fig. 4D), suggesting that Il10^−/− Foxp3⁺ CD4⁺ T cells are not able to expand postinfection, or that the effector cells outgrew the regulatory cells. Thus, it is possible that the transferred WT CD4⁺ T cells were better able to ameliorate immunopathology through increased Foxp3⁺ Treg frequencies, which may, in turn, regulate the outcome of disease directly through IL-10 production or via other suppressor mechanisms. Therefore, the next step was to determine whether Foxp3⁺ CD4⁺ Tregs were the critical source of IL-10 for the control of pathology in this model.

Foxp3⁺ Tregs produce IL-10 during infection but effector Th1 cells are the major source

We have shown that CD4⁺ T cells are the critical source of IL-10 for the attenuation of immunopathology in P. chabaudi infection, and that IFN-γ⁺ IL-10⁺ CD4⁺ T cells are the major producers of IL-10 and represent highly activated effector Th1 cells. However, they may not necessarily be the critical source regulating pathology; the single IL-10⁺ CD4⁺ T cell subset may be important and could represent a small population of suppressive Foxp3⁺ Tregs.

To examine whether IL-10–producing Tregs could be a source of protective IL-10 during this P. chabaudi infection, we used mice harboring the EGFP sequence inserted into the Foxp3 gene (Foxp3-EGFP reporter mice) (34) to determine the contribution of Foxp3⁻ and Foxp3⁺ CD4⁺ T cells to protective IL-10 production. It is important to point out that there was a significant increase in the total numbers of Foxp3⁺ Tregs in the spleen postinfection (Fig. 5A), which could be observed already from day 5. Moreover, analysis of IL-10 transcription in sorted EGFP-Foxp3⁺ Tregs confirmed a large upregulation of IL-10 transcription in these cells during infection (Fig. 5B). CD4⁺ Foxp3⁺ and Foxp3⁻ splenic T cells were sorted based on EGFP protein expression, and the two populations were then restimulated in vitro and stained for intracellular cytokines. Fig. 5C clearly shows that Foxp3⁻ effector CD4⁺ T cells are the main source of IL-10 during acute infection, reaching nearly 2 × 10⁶ secreting cells on day 15, compared with only 0.2 × 10⁶ Foxp3⁺ CD4⁺ Tregs producing this cytokine.

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FIGURE 5.

Foxp3⁺ CD4⁺ T cells produce IL-10 during infection, but effector CD4⁺ cells are the major source. CD4⁺ T cells were harvested from Foxp3-EGFP reporter mice on different days postinfection, and Foxp3⁺ cells were sorted based on their expression of EGFP protein. After stimulation with PMA and ionomycin for 6 h, intracellular staining was performed for IL-10 and IFN-γ. A, Total numbers of Foxp3⁺ EGFP cells per spleen during infection with 10⁵ parasitized RBC (mean ± SEM; n = 3–5 per time point; *p < 0.05 compared with naïve mice; t test). B, EGFP-Foxp3⁺ cells were sorted on day 10 of infection, and IL-10 transcription was analyzed using RT-PCR (n = 3). C, Total numbers of Foxp3⁻ and Foxp3⁺ cells producing IL-10 per spleen (n = 3–4 per time point; t test). D, Representative plots show mean percentages of cytokine-producing sorted Foxp3⁻ and Foxp3⁺ cells. Total numbers of Foxp3⁺ and Foxp3⁻ cells, per spleen producing only IL-10 or IL-10 in combination with IFN-γ are presented (mean ± SEM; n = 3–4 per time point; t test). A and B, Data are representative of two independent experiments. C and D, Combined data from two or three experiments for each time point.

The majority of IL-10⁺ Foxp3⁻ effector cells coproduced IFN-γ, whereas the majority of IL-10⁺ Foxp3⁺ cells did not produce IFN-γ, as observed in the representative dot plots (Fig. 5D). The bar graphs showing the total numbers of cytokine-producing cells confirm Foxp3⁻ effector cells as the major source of IL-10 (Fig. 5D). Therefore, although the total number of Foxp3⁺ Tregs increases during infection and IL-10 production is upregulated by these cells, IFN-γ⁺ IL-10⁺ double-producer Foxp3⁻ effector CD4⁺ T cells remain the prevailing source of IL-10. Unexpectedly, our intracellular analysis also revealed a minor population of IFN-γ⁺ single-producing Foxp3⁺ cells (Fig. 5D), which have also been observed in other parasitic infections (50, 51). Their function is currently unknown.

IL-10 from Foxp3⁻ CD4⁺ T effector cells is essential and sufficient to control immune-mediated pathology during acute P. chabaudi infection

It remained to be determined whether IL-10 from Foxp3⁺ Tregs was required to limit immunopathology or whether IL-10 from effector Th1 cells was sufficient. To address this question, we sorted Foxp3⁺ CD4⁺ Tregs from naive C57BL/6 WT mice (CD45.1 or CD45.2) or Il10^−/− mice (CD45.2) based on their high-level expression of CD25, whereas effector cells were sorted as CD25⁻. These 2 populations were cotransferred at a ratio of 7 effector CD25⁻ CD4⁺ T cells (3.5 × 10⁶) to 1 regulatory CD25⁺ CD4⁺ T cell (0.5 × 10⁶) into Rag1^−/− mice 3 wk before infection with P. chabaudi. Before transfer, the sorted cells were assessed for Foxp3 expression to verify that the majority of both WT and Il10^−/− CD25⁺ CD4⁺ T cells were Foxp3⁺ (>86%), and that CD25⁻ CD4⁺ cells were almost entirely Foxp3⁻ (Fig. 6A). These results confirm that Foxp3⁺ Tregs can develop in the absence of IL-10 (52). Using the allotypic markers CD45.1 and CD45.2, we were able to determine that in mice receiving WT effector and WT regulatory CD4⁺ T cells, the percentage of Foxp3⁺ regulatory CD25⁺ cells after reconstitution (day 0) and postinfection (day 10), and their ratio to effector CD25⁻ cells, remained constant (Fig. 6B). However, in mice receiving WT effector and Il10^−/− regulatory CD4⁺ T cells, the percentage of regulatory CD25⁺ cells expressing Foxp3 had decreased after 3 wk of reconstitution (day 0); in addition, their ratio to effector CD25⁻ cells decreased significantly postinfection (day 10; p = 0.05; t test; Fig. 6B).

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FIGURE 6.

IL-10 from Foxp3⁻ CD4⁺ T cells is essential and sufficient to limit pathology. Rag1^−/− mice were reconstituted with naive cells: 0.5 × 10⁶ CD4⁺CD25⁺ and 3.5 × 10⁶ CD4⁺CD25⁻ T cells either from C57BL/6 WT (CD45.1 or CD45.2) or Il10^−/− (CD45.2) mice. After 3 wk of reconstitution, the mice were infected with 10⁵ infected RBC, and analysis of pathology was performed for 15 d. A, Using intranuclear staining, representative histograms show Foxp3 expression in CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells before transfer into immunocompromised mice. B, On days 0 and 10 postinfection, spleens from reconstituted mice were harvested, and the percentages of Foxp3⁺ cells in the CD45.1 and CD45.2 cell populations were assessed, as well as the ratio between them. Dotted lines represent the original frequency of Foxp3⁺ cells in the CD25⁺ population before transfer. C, Postinfection, signs of pathology were detected using several parameters: temperature, weight loss, and RBC count. Data are representative of two independent experiments. Mean ± SEM; n = 3–4 (B); n = 5–7 (C); *p < 0.05, **p < 0.01; t test.

Infected Rag1^−/− mice reconstituted with the mixture of WT effector and WT regulatory CD4⁺ T cells presented equivalent pathology to those reconstituted with total WT CD4⁺ T cells (Fig. 6C), verifying the transfer model. Rag1^−/− mice reconstituted with WT effector and Il10^−/− Tregs recovered better from the pathology, with less hypothermia and lower anemia, compared with those reconstituted with total WT CD4⁺ T cells (Fig. 6C), demonstrating that despite a lower ratio of Foxp3-expressing CD25⁺ cells to effector CD4⁺ T cells, these mice were nevertheless protected from severe disease. Furthermore, these two groups present similar total numbers of effector cells, which is unlikely to be the reason for less severe disease in mice receiving WT effector and Il10^−/− Tregs when compared with those reconstituted with total WT CD4⁺ T cells (53.87 ± 10.48 × 10⁵ and 44.70 ± 7.94 × 10⁵ cells, respectively; p = 0.55). Therefore, IL-10 derived from Foxp3⁺ Tregs is not required for protection from severe immunopathology during P. chabaudi infection. Rag1^−/− mice reconstituted with Il10^−/− effector and WT regulatory CD4⁺ T cells displayed increased hypothermia, weight loss, and anemia compared with control mice receiving total WT CD4⁺ T cells (Fig. 6C), and showed little sign of recovery from the infection. Furthermore, the survival curves clearly confirm the severe immune-mediated pathology in mice receiving Il10^−/− effector and WT regulatory CD4⁺ T cells because they present significantly higher mortality (Supplemental Fig. 3). Regarding the parasite burden, no significant difference in peripheral parasitemia was observed between the groups. These data clearly demonstrate that IL-10 derived from Foxp3⁺ Tregs is not required for protection from severe immunopathology. IL-10 from effector CD4⁺ T cells is the critical source and is sufficient to limit immunopathology during P. chabaudi infection without contributing to an increase in parasite burden.

IL-27, but not IL-21, is necessary to induce optimal IL-10 production by IFN-γ⁺ Th1 cells

IL-27 has recently been implicated in the regulation of IL-10 production by T cells (53), and IL-21 has been shown to amplify the IL-27 signal for IL-10 production by Tr1 cells (54). We evaluated the role of these pathways in inducing IL-10 production by IFN-γ⁺ Th1 cells in this model of malaria. Il21^−/− mice were infected with P. chabaudi, and their cytokine profile was analyzed by intracellular staining at day 15 postinfection. Interestingly, despite having lower total numbers of spleen cells (data not shown), IL-21–deficient mice showed greater frequencies and numbers of IL-10⁺ IFN-γ⁺ double-producing Th1 cells and IL-10⁺ single-producing CD4⁺ T cells compared with WT mice (Fig. 7A), clearly demonstrating that IL-21 is not required for IL-10 production in CD4⁺ T cells during P. chabaudi infection.

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FIGURE 7.

Generation of IL-10⁺ IFN-γ⁺ CD4⁺ T cells is independent of IL-21 but requires IL-27 signaling. A, WT and Il21^−/− mice were injected with 10⁵ P. chabaudi-infected RBC, and 15 d later the splenocytes were harvested for intracellular analysis. Representative plots show IL-10 and IFN-γ production by gated CD4⁺ T cells. Mean percentages of cytokine-producing cells are shown. Total numbers of CD4⁺ T cells per spleen producing only IL-10 or IL-10 in combination with IFN-γ are presented (each dot represents an individual mouse; mean ± SEM; n = 5; t test). Data are representative of three independent experiments. B, After sublethal irradiation, Rag1^−/− mice were reconstituted with 1.7 × 10⁶ total bone marrow cells from WT (CD45.1⁺), Il27ra^−/− (CD45.2⁺) mice, or a mixture of both at a ratio of 50:50, and they were designated as 100% WT, 100% Il27ra^−/−, and 50%:50% WT: Il27ra^−/− chimeras, respectively. Nine weeks after the transplantation, all mice were infected with 10⁵ P. chabaudi, and 8 d later, they were culled for analysis. Representative contour plots show IL-10 and IFN-γ production by gated CD4⁺ T cells. These cells were selected based on their expression of the allotypic markers CD45.1 or CD45.2. The total numbers of IL-10⁺ IFN-γ⁺ double producers, IL-10⁺ single producers, and IFN-γ⁺ single producers per spleen are shown for WT and IL-27ra^−/− cells. C, The graphs for temperature and weight loss represent data from days 8 and 7, respectively, when the major differences were observed between the groups (mean ± SEM; n = 6–11; t test).

To address the role of IL-27 for the production of IL-10 by IFN-γ⁺ Th1 cells, we generated mixed bone marrow chimeras in which WT CD45.1⁺ and Il27ra^−/− CD45.2⁺ (40) bone marrow cells were transferred to sublethally irradiated Rag1^−/− mice at various ratios. As controls, chimeras receiving 100% WT CD45.1⁺ or 100% Il27ra^−/− CD45.2⁺ bone marrow cells were also generated. After 7 wk, the chimerism was determined. We selected those mixed chimeric mice with ∼50% reconstitution with Il27ra^−/− T cells (Supplemental Fig. 4) for enumeration of IL-10⁺ IFN-γ⁺, IL-10⁺, and IFN-γ⁺ CD4⁺ T cells postinfection with P. chabaudi. Pathology was also analyzed. Using the gating strategy shown in Supplemental Fig. 4, we gated the WT and Il27ra^−/− CD4⁺ T cells based on the expression of their allotypic marker. IL-27R–deficient IFN-γ⁺ CD4⁺ T cells were significantly impaired in their ability to produce IL-10, with reduced frequencies and numbers of IL-10⁺ IFN-γ⁺ CD4⁺ T cells in chimeric mice reconstituted with 100% Il27ra^−/− bone marrow compared with 100% WT bone marrow, and in Il27ra^−/−, compared with WT, CD4⁺ T cells that coexisted in mixed chimeras (Fig. 7B). In addition, there was defective induction of IL-10⁺ single-producer CD4⁺ T cells in the Il27ra^−/−, compared with WT, chimeras, whereas the number of IFN-γ⁺ single-producer CD4⁺ T cells per spleen was not affected by the absence of IL-27R (Fig. 7B).

As IL-10⁺ IFN-γ⁺ Th1 cells are necessary to control immune-mediated pathology induced during acute P. chabaudi infection, it would be expected that chimeric mice reconstituted with only Il27ra^−/− bone marrow cells, which generated the lowest number of IL-10⁺ IFN-γ⁺ Th1 cells, would present significantly increased symptoms of pathology, including hypothermia and weight loss, compared with chimeras reconstituted with 100% WT bone marrow cells. This was observed (Fig. 7C). In addition, mixed bone marrow chimeric mice also presented with a partial increase in the severity of pathology (Fig. 7C). In conclusion, our results clearly demonstrate that the induction of IL10⁺ IFN-γ⁺ CD4⁺ T cells, which provide critical protection from immunopathology during P. chabaudi infection, requires signaling through the IL-27 pathway.