P2X7 receptor drives Th1 cell differentiation and controls the follicular helper T cell population to protect against <i>Plasmodium chabaudi</i> malaria

<div><p>A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to <i>Plasmodium</i> infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage <i>Plasmodium chabaudi</i> malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during <i>P</i>. <i>chabaudi</i> infection and thus in disease outcome.</p></div

Splenic B6 or <i>P2rx7</i>^-/- CD4 cell co-transfer and protection against <i>Pc</i> infection.

<p>(A-D) Naïve CD4⁺ cells from B6 (CD45.1) and <i>P2rx7</i>^-/- (CD45.2) female mice were co-transferred into <i>Cd4</i>^-/- female mice that were then infected with 1 × 10⁶ <i>Pc</i>-iRBCs. Splenic CD4⁺ cells were analyzed at 30 days p.i. Non-infected mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 5) of one representative experiment out of three. Significant differences were for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) A schematic illustration of the experimental protocol is shown. (B) Dot plot shows CD45.1⁺CD4⁺ and CD45.1^-CD4⁺ cells at 30 days p.i. CD45.1⁺CD4⁺ and CD45.1^-CD4⁺ cell numbers per spleen are shown in the column bar graph. (C) Contour plots show PD1 and CD39 expression in CD4⁺ cells. PD1^hiCD39^loCD4⁺ and PD1^loCD39^hiCD4⁺ cell percentages are shown in the column bar graphs. (D) Histograms show T-bet and Bcl6 expression in PD1^hiCD39^loCD45.1⁺CD4⁺, PD1^loCD39^hiCD45.1⁺CD4⁺, PD1^hiCD39^loCD45.1^-CD4⁺ and PD1^loCD39^hiCD45.1^-CD4⁺cells. FMO controls are shown in the histograms. The MFIs of T-bet and Bcl6 expression are shown in the column bar graphs. (E-F) CD4⁺ cells from B6 and <i>P2rx7</i>^-/- female mice at 20 days p.i. were transferred into <i>Cd4</i>^-/- female mice that were infected with 1 × 10⁵ <i>Pc</i>-iRBCs. <i>Cd4</i>^-/- mice transferred with naïve B6 cells were used as controls. The data were expressed as means ± SD (<i>n</i> = 4–6) of one representative experiment out of three. Significant differences were observed for the (*) mice transferred with B6 cells at 0 and 20 days p.i. and (**) mice transferred with B6 and <i>P2rx7</i>^-/- cells at 20 days p.i. with <i>p</i> < 0.05, using the Mann Whitney U test. (E) A schematic illustration of the experimental protocol is shown. (F) Parasitemia curves are shown.</p

<i>Ex vivo</i> analysis of iRBC uptake by splenic DCs soon after <i>Pc</i> infection.

<p><b>(A-C)</b> B6 mice were i.v. infected with 1 × 10⁸ purified mature CTV-<i>Pc</i> iRBCs (flow cytometry) or GFP-<i>Pc</i> iRBCs (immunofluorescence). Spleens were analyzed after 15 min and in non-infected controls. <b>(A)</b> A representative immunofluorescence image (10x magnification) shows the spleen of an infected B6 mouse. The staining of MOMA-1⁺ metallophilic macrophages and CD19⁺ B cells delineates the RP/MZ from the white pulp (WP). The lower panel details a merged GFP⁺CD11c⁺ cell in the RP. <b>(B)</b> Percentage of CD11c pixels colocalized with GFP pixels and percentages of GFP pixel distribution in the splenic RP/MZ and WP were obtained from immunofluorescence images (means ± SD). <b>(C)</b> Representative contour plots obtained by flow cytometry show CTV staining in the CD11c⁺ cells of naïve mice (-) and recently infected mice. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed, while excluding T cells, B cells, NK cells and RBCs. Data in contour plots show the percentages of CTV⁺ cells in the CD11c⁺ cell population. The numbers of total and CTV⁺CD11c⁺ cells per spleen in recently infected mice were calculated from the data obtained in contour plots (means ± SD). <b>(D-F)</b> B6.CD11c-YFP mice were i.v. infected with 1 × 10⁸ purified mature CTV-<i>Pc</i> iRBCs. Spleens were analyzed by flow cytometry after 15 min and in non-infected controls. <b>(D)</b> Representative contour plots show CTV staining in the CD11c⁺YFP⁺ and F4/80⁺YFP⁺ cells. Data show the percentages of CTV⁺ cells in each population. <b>(E)</b> The numbers of total and CTV⁺ CD11c⁺YFP⁺ and F4/80⁺YFP⁺ cells per spleen were calculated from the data obtained in <b>D</b> (means ± SD). <b>(F)</b> The relative proportions of CD11c⁺ and F4/80⁺ cells in the CTV⁺YFP⁺ cell population were calculated from the data obtained in <b>E</b> (means ± SD). In <b>A</b>, the scale bars correspond to 50 µm. In <b>B</b>, data were calculated using FIJI software. In <b>B</b> and <b>E</b>, significant differences (p < 0.05) between the indicated groups are designated by *. In <b>A-F</b>, one representative experiment out of three (n = 3-4) is shown.</p

Splenic Tfh cell responses in infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-G) B6 and <i>P2rx7</i>^-/- female mice were analyzed at 7,14, 20, 30, 50, 100 and 130 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 3–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) Hematoxilyn-eosin stained sections show splenic follicular hyperplasia in <i>P2rx7</i>^-/- mice (40x magnification; bar scales correspond to 200 μm). (^#) The mean areas of lymphoid follicles are shown. (B) Confocal immunofluorescence images (100x magnification; bar scales correspond to 400 μm) of splenic sections are shown. Tissue slices were stained for CD19 (green), CD4 (red) and GL7 (blue). (C) CD19⁺ and Fas⁺GL7⁺CD19⁺ cell numbers per spleen were determined by flow cytometry. (D) Contour plots show PD1, ICOS and Bcl6 <i>versus</i> CXCR5 expression in CD4⁺ cells. PD1⁺CXCR5⁺CD4⁺, ICOS⁺CXCR5⁺CD4⁺ and Bcl6⁺CXCR5⁺CD4⁺ cell numbers per spleen are shown in the column bar graphs. (E) PD1⁺CXCR5⁺CD4⁺ cell numbers per spleen were determined by flow cytometry. (F) IL-21 concentrations were determined by ELISA in the supernatants of splenocytes stimulated or not with iRBCs (splenocyte/3 iRBCs). (G) Anti-parasite IgM and IgG2c serum concentrations were determined by ELISA.</p

Effects of DC depletion on acute <i>Pc</i> malaria.

<p><b>(A-F)</b> B6 and B6.CD11c-DTR mice were treated with either DTx to deplete CD11c⁺ cells or PBS as a control. The mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs 24 h later. <b>(A)</b> Representative contour plots obtained 24 h after treatment by flow cytometry confirm the efficiency of DTx-induced depletion of splenic CD11c⁺I-A⁺ cells in B6.CD11c-DTR mice. Data show the percentages of CD11c⁺I-A⁺ cells in the splenocyte population. <b>(B)</b> Parasitemia curves are shown (means ± SD). <b>(C)</b> Variations in body weight relative to day 0 are shown (means ± SD). <b>(D)</b> Survival curves are shown. <b>(E)</b> Data show the numbers of CD3⁺CD4⁺ T cells per spleen at days 0 and 4 p.i. (means ± SD). <b>(F)</b> Data show the percentages of proliferating CFSE^lowCD4⁺ T cells and IFN-γ concentrations in the supernatants of spleen cell cultures stimulated for 72 h with iRBCs (means ± SD). In <b>B-D</b>, significant differences (p < 0.05) between the indicated groups are designated by *. In <b>E</b> and <b>F</b>, significant differences (p < 0.05) between all other groups are designated by *. In <b>A-F</b>, one representative experiment out of three (n = 5) is shown.</p

Parasitemia and clinical parameters in B6 and <i>P2rx7</i>^-/- mice infected with <i>Pc</i> and P. <i>yoelii</i> 17NL parasites.

<p>(A-C) B6 and <i>P2rx7</i>^-/- mice were infected with 1 × 10⁶ <i>Pc</i>-iRBCs. The data were expressed as means ± SD (<i>n</i> = 4–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using (A and C) the Mann Whitney U test or (B) the long-rank test. (A) Parasitemia curves are shown. (B) Survival curves are shown. (C) Hemoglobin serum (Hb) concentration, body weight and body temperature were monitored daily. Variation in body weight relative to day 0 is shown. (D) B6 and <i>P2rx7</i>^-/- mice were infected with 1 × 10⁵ <i>P</i>. <i>yoelii</i> 17XNL-iRBCs. The data were expressed as means ± SD (<i>n</i> = 4–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using the Mann Whitney U test. Parasitemia curves are shown.</p

Splenic CD4 T cell responses in acutely infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-G) B6 and <i>P2rx7</i>^-/- mice were analyzed at 4, 5 and 7 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). Female mice were used in the experiments with the exception that females and males were compared. The data were expressed as means ± SD (<i>n</i> = 3–4) of one representative experiment out of three. Significant differences were observed for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) EB-stained CD4⁺ blood cell percentages were determined by flow cytometry, before and after iRBC rupture. The blood samples were collected at 9 a.m. (1.3 ± 0.3% iRBCs at 4 days p.i. and 7.6 ± 0.3% iRBCs at 5 days p.i.; >95% trophozoites and schizonts) and 2 p.m. (7.3 ± 1.2% iRBCs at 4 days p.i. and 15.0 ± 3.7% iRBCs, at 5 days p.i.; >95% ring forms). (B) Splenocytes were stimulated or not with 25–500 μM ATP. EB-stained CD4⁺ cell percentages were determined by flow cytometry. Horizontal gray bar represents the confidence interval obtained with <i>P2rx7</i>^-/- cells. (C) CFSE-stained CD4⁺ cells and APCs (splenocytes from naïve nude mice), at a 1:1 ratio, were stimulated or not with iRBCs (1 splenocyte/ 4 iRBCs). Histograms show CFSE-stained CD4⁺ cells. CFSE^loCD4⁺ cell percentages are shown in the column bar graph. (D) Splenocytes were stimulated or not with iRBCs (1 splenocyte/ 3 iRBCs). Contour plots show intracellular IFNγ- and IL-10-stained CD4⁺ cells. IFNγ⁺IL-10^- and IFNγ⁺IL-10⁺ cell percentages in CD4⁺ cells are shown in the column bar graphs. (E) CD4⁺ cell numbers per spleen were determined by flow cytometry. (F) IFNγ⁺CD4⁺ and IL-10⁺CD4⁺ cell numbers per spleen were determined by flow cytometry. (G) IgM-, IgG2c- and IgG1-secreting cell numbers per spleen were determined by ELISPOT assay.</p

<i>In vivo</i> and <i>ex vivo</i> analysis of iRBC uptake by splenic DCs during crisis.

<p><b>(A-D)</b> B6.CD11-YFP mice were i.p. infected with 1 × 10⁶ mCherry-<i>Pc</i> iRBCs. Spleens were analyzed by CIVM after eight days, at a time of day when mature parasite stages predominated. <b>(A)</b> A snapshot shows the subcapsular RP. <b>(B)</b> CIVM 3D animation shows the presence of few mCherry-<i>Pc</i> iRBCs (red) attached to YFP⁺ cells (green). <b>(C)</b> Percentage of mCherry⁺ cells in the YFP⁺ cell population is shown (mean ± SEM). <b>(D)</b> YFP⁺ cell volume and sphericity are shown. Black, red and blue dots are from three different experiments. Horizontal lines represent mean values and SEM. <b>(E</b> and <b>F)</b> B6 mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs. At eight days p.i., half of the B6 mice were re-infected i.v. with 1 × 10⁸ purified mature CTV-<i>Pc</i> iRBCs. Spleens were analyzed by flow cytometry after 15 min. <b>(E)</b> Representative contour plots show CTV staining in the CD11c⁺ cells of <i>Pc</i>-infected mice that were re-infected or not with CTV-<i>Pc</i> iRBCs. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed. Data show the percentages of CTV⁺ cells in the CD11c⁺ cell population. <b>(F)</b> The numbers of total and CTV⁺CD11c⁺ cells per spleen were calculated from the data obtained in <b>E</b> (means ± SD). <b>(G</b> and <b>H)</b> B6 mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs or GFP-<i>Pc</i> iRBCs. Spleens were analyzed after eight days, at a time of day when mature parasite stages predominated. <b>(G)</b> Representative contour plots obtained by flow cytometry show GFP staining in CD11c⁺ cells of mice that were infected with <i>Pc</i> iRBCs or GFP-<i>Pc</i> iRBC. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed. Data show the percentages of GFP⁺ cells in the CD11c⁺ cell population. <b>(H)</b> Numbers of total and GFP⁺CD11c⁺ cells per spleen were calculated from the data obtained in <b>G</b>. In <b>A</b> and <b>B</b>, the scale bars correspond to 50 µm and 30 µm, respectively. One representative experiment out of three (n = 2) is shown. In <b>C</b> and <b>D</b>, data were obtained using Imaris software. Data from three experiments (n = 2) are shown. In <b>E-H</b>, one representative experiment out of three (n = 5) is shown.</p

Analysis of the interactions between splenic DCs and CD4⁺ T cells after <i>Pc</i> infection.

<p><b>(A)</b> B6.CD11c-YFP mice were i.p. infected with 1 × 10⁶ mCherry-<i>Pc</i> iRBCs. Spleens were analyzed by CIVM after 2 h and 12 h and in non-infected controls. Speed and displacement of YFP⁺ cells are shown. Black, red and blue dots are from three different experiments. Horizontal lines represent mean values and SEM. <b>(B</b> and <b>C)</b> B6 mice were i.p. infected with 1 × 10⁶ mCherry-<i>Pc</i> iRBCs. Spleens were analyzed after 2 h or 24 h and in non-infected controls. <b>(B)</b> Representative immunofluorescence images (10x magnification) show CD11c⁺ cells and CD4⁺ cells in DAPI-stained tissue sections. <b>(C)</b> Percentages of CD11c pixels colocalized with GFP pixels in the immunofluorescence images are shown (means ± SD). <b>(D</b> and <b>E)</b> B6.CD11c-YFP mice were adoptively transferred with 5 × 10⁶ CFP⁺CD4⁺ T cells and i.p. infected with 1 × 10⁶ mCherry-<i>Pc</i> iRBCs. Spleens were analyzed by CIVM after 24 h and in non-infected controls. <b>(D)</b> Snapshots show the subcapsular RP with CFP⁺CD4⁺ cell tracking. <b>(E)</b> Speed and arrest coefficients of CFP⁺CD4⁺ cells are shown. Black, red and blue dots are from three different experiments. Horizontal lines represent mean values and SEM. In <b>B</b> and <b>D</b>, the scale bars correspond to 100 and 50 µm, respectively. In <b>A</b>, <b>C</b> and <b>E</b>, significant differences (p < 0.05) between the indicated groups are designated by *. In <b>A</b> and <b>E</b>, data were calculated using Imaris software. Data from three experiments (n = 2) are shown. In <b>C</b>, data were calculated from eight images (two images per mouse) using FIJI software. In <b>B</b> and <b>C</b>, one representative experiment out of three (n = 4) is shown. In <b>D</b>, one representative experiment out of three (n = 2) is shown.</p

Splenic Th1 cell responses in chronically infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-F) B6 and <i>P2rx7</i>^-/- female mice were analyzed at 20, 30 and 50 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 3–5) of one representative experiment out of three. Significant differences were observed for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) Splenocytes were stimulated or not with 25–500 μM ATP. EB-stained CD4⁺ cell percentages were determined by flow cytometry. Horizontal gray bar represents the confidence interval obtained with <i>P2rx7</i>^-/- cells. (B) CD4⁺ cell numbers per spleen were determined by flow cytometry. (C) Splenocytes were stimulated or not with iRBCs (1 splenocyte/ 3 iRBCs). IFNγ⁺IL-10^-, IFNγ⁺IL-10⁺ and IFNγ^-IL-10⁺ cell percentages in CD4⁺ cells were determined by flow cytometry. (D) IFNγ and IL-10 concentrations were determined by ELISA in the supernatants of splenocytes described in E, which were stimulated or not with 100–300 μM ATP. (E) Histograms show T-bet expression in CD4⁺ cells. FMO controls are shown in the histograms. The MFIs of T-bet expression are shown in the scatter plots. (F) Contour plots show T-bet and P2X7 expression in CD4⁺ cells. T-bet⁺P2X7⁺ cell percentages in CD4⁺ cells are shown in the column bar graph.</p