Figure 1.
IFNγ+IL-10+ T-bet+ CD4+ T cells expand during chronic L. donovani infection.
(A and B) C57BL/6 mice were infected i.v. with 3×107 Leishmania donovani amastigotes, and at the indicated times post infection (p.i.), splenomegaly (A) and tissue parasite burden (B) were determined. (C and D) Frequency (C) and total number (D) of IL-10 and IFNγ producing splenocytes in naïve and day 28 infected mice after PMA and Ionomycin stimulation. (E and F) Frequency (E) and total number (F) of IL-10 and IFNγ producing splenocytes in naïve and day 28 infected mice after antigen stimulation. (G and H) T-bet, CD127 and Foxp3 expression (H) by different cytokine producing populations of CD3ε+CD4+ T cells from d28 infected mice (G). Data are from n = 4 mice per group and representative of 2–3 independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001 for infected vs. naïve mice.
Figure 2.
Costimulatory molecule expression on cDCs during chronic infection.
(A) Splenic cDCs were gated as MHCIIhiCD11chi cells (top panel) and further gated into the three major subsets based on CD4 and CD8α expression (bottom panel). (B) Surface expression of CD40, CD80, CD86 and PD-L1 on the entire cDC population. (C–F) Surface expression of CD40 (C), CD80 (D), CD86 (E) and PD-L1 (F) by each cDC subset. Data are expressed as mean fold change ± SEM of the MFI on cDCs from infected mice compared to naïve mice (n = 4 mice per group). Data is representative of 3 experiments.
Figure 3.
CD11chi splenic cDCs acquire a regulatory cytokine profile.
(A) Sorting strategy and representative sort purities of unfractionated cDCs (top left panel) and each sorted cDC subset. (B) IL-12p70 secretion by CD11chiMHCIIhi cDCs from naïve and/or day 28 infected mice, in the presence or absence of LPS. (C and D) Secretion of IL-12/23p40 by cDC subsets in the absence (B) or presence (C) of LPS. (E and F) Spontaneous IL-27p28 secretion by unfractionated cDCs (E) and sorted cDC subsets (F). (G and H) IL-10 secretion by unfractionated cDCs (G) and sorted (H) cDC subsets.
Figure 4.
Autocrine IL-10 but not IL-27p28 regulates IL-12 and IL-10 production by cDCs.
(A) IL-12p70 and (B) IL-10 secretion was determined in cultures of cDCs sorted from d28 infected mice cultured with or without LPS and in the presence or absence of neutralizing mAbs to IL-10R or IL-27p28. Data are representative of 3 experiments. * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 5.
Deletion of CD11c+ cells in DTx treated (CD11c-cre×Rosa26iDTR)F1 mice.
(A) Experimental protocol: Infected (CD11c-cre×Rosa26iDTR)F1 mice received 4 ng/g DTx i.p. at 48 hour intervals from d21 until day 28 p.i. (B and C) Impact of CD11c+ cell ablation on cells expressing CD11c and MHCII, shown by representative dot plot (B) and by extent of depletion CD11cloMHCII−, CD11cloMHCIIhi and CD11chiMHCIIhi (cDCs) relative to untreated mice (C). (D–F) Impact of DTx treatment on CD11chi cDC subsets, shown by representative dot plot (D), as total number of cells in DTx treated (black bars) vs. control (open bars) mice (E) and by extent of depletion (F). Data are from 2 pooled experiments (n = 8 mice). *** = p<0.001 for DTx vs. PBS treated groups.
Figure 6.
Deletion of CD11c+ cells reduces pathology and enhances resistance to L. donovani.
Infected (CD11c-cre×Rosa26iDTR)F1 mice were treated as shown in Figure 6A and at d28 p.i. mice were assessed for splenomegaly (A), spleen parasite burden (B) and spontaneous NO production by adherent splenocytes (C). Data are pooled from two independent experiments (n = 8 mice). * = p<0.05, other p values as indicated, for DTx vs. PBS treated animals.
Figure 7.
CD11c+ cells regulate IL-10 production by Th1 cells.
Splenic T cells from the mice used in Figure 7 were analysed for cytokine production (A–C) Frequency of antigen-specific CD4+ T cells producing IFNγ (A), IL-10 (B) or both (C) in mice with or without DTx treatment. (D–F) Total number of antigen-specific CD4+ T cells producing IFNγ (D), IL-10 (E) or both (F) in mice with and without DTx treatment. Data are pooled from two independent experiments (n = 8 mice). * = p<0.05, other p values as indicated, for DTx vs. PBS treated animals.
Figure 8.
Removal of CD11c+ cells affects splenomegaly and NO production even in the absence of neutrophils.
DTx-treated (CD11c-cre×Rosa26iDTR)F1 mice were administered neutrophil depleting mAb 1A8 or control mAb (2A3) every 48 h from d21 post infection. Mice were analyzed at day 28 post infection. (A–C) Splenomegaly and NO production (B) and spleen parasite burden (C) at d28 post infection in neutrophil depleted and neutrophil replete DTx-treated mice. Data are shown as mean ± SEM (n = 3 or 4 mice per group).
Figure 9.
CD11chi cDCs are required for the expansion/maintenance of IFNγ+IL-10+ CD4+ T cells.
(A) 12 h after initiation of DTx treatment, (CD11c-cre×Rosa26iDTR)F1 mice were reconstituted i.v. with CD11chiMHCIIhi cDCs or CD11clo/int cells obtained from day 21-infected congenic B6J.CD45.1 donors. (B) Purity of transferred cells. (C–E) Splenomegaly (C), spleen parasite burden (D), and spontaneous NO production (E) in recipient mice. (F and G) The frequency (F) and number (G) of antigen-specific CD4+ T cells producing IFNγ and IL-10 in recipient mice. Data are pooled from two independent experiments (n = 7–10 mice). * = p<0.05, ** = p<0.01, *** = p<0.001 for indicated comparisons between groups.