Effects of JES6-1 treatment on the early CD4⁺ T cell response to <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 7 p.i., splenic CD4⁺ T cells were analyzed for CD122, CD25, CD69 and CD44 expression and cell size (FSC). Data show gated CD4⁺ T cells expressing high levels of activation markers and large size (n = 3–4). (B) Non-stimulated (basal) and anti-CD3 mAb stimulated IFN-γ and IL-17 production was evaluated in CD4⁺ T cells from the same groups of mice. (C) Numbers of CD4⁺ T cells per spleen were determined in the same groups of mice. (D) On day 4 p.i., PRBC-stimulated CD4⁺ T cell proliferation was analyzed <i>in vitro</i> in the presence or absence of JES6-1 mAb. Histograms show CFSE fluorescence in gated CD4⁺ T cells. The means ± SD (n = 3–4) of the percentages of replicating (CFSE^LO) cells are shown. In A–D, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with non-stimulated cells. Data are representative of three separate experiments.</p

Effects of JES6-1 treatment on the splenic CD4⁺CD25⁺FoxP3⁺ cell population during <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were infected with 10⁶ PRBC. On day 7 p.i., CD4⁺ T cells were analyzed for CD25 and FoxP3 expression and cell size (FSC). Dot plots represent gated CD4⁺ T cells. Dot plots and histograms show a representative mouse from each group. Numbers inside dot plots refer to means ± SD (n = 4) of cell percentages in each gate. Histograms show gated CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3⁻ cells in relation to CD4⁺CD25⁻FoxP3⁻ cells. Numbers inside histograms refer to means ± SD (n = 4) of large cell percentages. (B) On days 0, 2 and 4 p.i., C57BL/6 mice were treated with JES6-1 mAb. Data represent the means ± SD (n = 4) of CD4⁺CD25⁺FoxP3⁺ cell percentages and numbers per spleen on days 7 and 20 of infection. In A–B, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with cells from non-treated (NT) mice; and #p<0.05 with CD25⁺FoxP3⁺ cells. Data are representative of two separate experiments.</p

Secretion of IL-2 and expression of activation markers by splenic CD4⁺ T cells during <i>P. chabaudi</i> malaria.

<p>(A) IL-2 secretion was analyzed in gated CD4⁺ T cells obtained from C57BL/6 mice on days 3, 5, 7 and 15 p.i. with 10⁶ PRBC. Numbers inside dot plots refer to means ± SD (n = 3–4) of cell percentages in the upper right gate. (B) Cell size (FSC) and expression of CD25 and CD122 were evaluated in gated CD4⁺IL-2⁻ and CD4⁺IL-2⁺ cells of the same groups of mice. (C) On day 7 p.i., CD122, mTGF-β, CD45RB, CTLA-4 and GITR expression was analyzed in gated CD4⁺CD25⁺ cells, subdivided into small and large cells. Numbers inside dot plots refer to means ± SD (n = 3–4) of cell percentages in each gate. The mean fluorescence intensity (MFI) of CD4⁺CD25⁻ cells (controls) stained with mAb to mTGF-ß and GITR was comparable to respective isotopic controls (data not shown). In A–C, there was a significant difference (*p<0.05) between experimental conditions and non-stimulated (NS) cells from non-infected mice. Cells from non-infected mice stimulated with anti-CD3 mAb were used as positive controls. Dot plots and histograms show a representative mouse of each group. Data are representative of three separate experiments.</p

Effects of JES6-1 treatment on splenic CD4⁺ T cell phenotype and proliferative response to PRBC during chronic <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 20 p.i., CD62L and CD45RB expression was evaluated in gated CD4⁺ T cells. Numbers inside dot plots refer to means ± SD (n = 3–4) of cell percentages in each gate. (B) CD69 expression was analyzed in gated CD4⁺ T cells from the same groups of mice. (C) <i>In vitro</i> proliferative response of PRBC-stimulated CD4⁺ cells cultured in the presence or absence of JES6-1 mAb. CD4⁺ T cells were obtained from 30-day infected mice treated <i>in vivo</i> or not with JES6-1 mAb on days 0, 2 and 4 of infection. Histograms show CFSE fluorescence in gated CD4⁺ T cells. The means ± SD (n = 3–4) of the percentages of replicating (CFSE^LO) cells are shown. In A–C, significant differences compared experimental conditions *p<0.05 with cells from non-infected (NI) mice; **p<0.05 with non-treated (NT) mice or cells; and # p<0.05 with non-treated (NT) cells from JES6-1-treated mice. Data are representative of two separate experiments.</p

Effects of JES6-1 treatment on the regulation of acquired immune responses to <i>P. chabaudi</i> malaria.

<p>(A) C57BL/6 mice were treated with JES6-1 mAb on days 0, 2 and 4 p.i. with 10⁶ PRBC. On day 30 p.i., TNF-α and IFN-γ production was evaluated in spleen cell supernatants (mean ± SD, n = 4). (B) Serum titers of parasite-specific IgG2a were determined in the same groups of mice. (C) Parasitemia curves were evaluated in C57BL/6 mice injected with spleen cells from JES6-1-treated mice and non-treated (NT) mice on day 30 p.i. and challenged with 10⁸ PRBC (mean ± SD, n = 4). In A–B, significant differences compared experimental conditions *p<0.05 with non-infected (NI) mice; and **p<0.05 with non-treated (NT) mice. In C, significant differences compared to experimental conditions *p<0.05 with mice transferred with cells from non-treated (NT) mice. Data are representative of two separate experiments.</p

Expression of IL-2R α and ß chains in splenic CD4⁺ T cells during <i>P. chabaudi</i> malaria.

<p>(A) Parasitemia curves and CD4⁺ T cell numbers per spleen were evaluated in C57BL/6 mice infected with 10⁶ PRBC (mean ± SD, n = 4). (B) Basal (non-stimulated) proliferation and IFN-γ production in infected mice. Percentages of replicating (CSFE^LO) CD4⁺ T cells and CD4⁺IFN-γ⁺ cells are shown (mean ± SD, n = 4). (C) On days 4, 7 and 30 p.i., CD25 and CD122 expression was analyzed in gated CD4⁺ T cells. Numbers inside dot plots refer to means ± SD (n = 3–4) of cell percentages in each gate. (D) On day 7 p.i., CD25⁻CD122⁺, CD25⁺CD122⁺, CD25⁻CD122⁻ and CD25⁺CD122⁻ cells in gated CD4⁺ T cells of the same groups of mice were analyzed according to cell size (FSC). Numbers inside histograms refer to means ± SD (n = 3–4) of large cell percentages. In A–D, *p<0.05, compared to non-infected mice (day 0). Dot plots and histograms show a representative mouse of each group. Data are representative of three separate experiments.</p