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. Author manuscript; available in PMC: 2020 Mar 15.
Published in final edited form as: J Immunol. 2019 Feb 4;202(6):1755–1766. doi: 10.4049/jimmunol.1801229

Figure 2. Blockade of ICOSL does not decrease IL-10 production during chronic T. gondii infection, but leads to expanded T cell populations in the brain.

Figure 2.

(A) ICOS expression on T cells and (B) ICOSL expression on infiltrating myeloid cells isolated from the brains of chronically infected C57BL/6 mice using flow cytometry. Immune cell populations were gated as described in Figure 1C. (C) IL-10 production was assessed using chronically infected control or α-ICOSL-treated IL-10-eGFP reporter (Tiger) mice. The frequency of IL-10-GFP+ CD4 effector and Treg cells isolated from the brain is shown (n=3–4 per group, data is pooled from two independent experiments and analyzed using randomized block ANOVA). (D) The relative expression of IL-10 mRNA in chronically infected whole brains from C57BL/6 mice treated with control or α-ICOSL blocking antibody. Relative expression was normalized to the control (IgG-treated) group (n=4 per group, data is pooled from two independent experiments and analyzed using randomized block ANOVA). (E-N) Chronically infected C57BL/6 mice were treated with an α-ICOSL blocking antibody or control rat IgG. (E) Total T cell numbers isolated from the brain were analyzed by flow cytometry (n=3–4 per group, representative data is pooled from 5 independent experiments and analyzed by randomized block ANOVA). (F-G) Representative brain sections stained for CD3 (green) from control (F) or α-ICOSL-treated (G) mice. (H) Parasite-specific CD4+ effector T cells were identified by flow cytometry using an MHCII-peptide tetramer. (I) IFNγ production from T cells isolated from the brains of control and α-ICOSL-treated mice was measured following ex vivo restimulation (n=3–5 per group, data is pooled from five independent experiments and analyzed using randomized block ANOVA). (J) qRT-PCR was done using mRNA isolated from whole brains of chronically infected mice after α-ICOSL blockade. Relative expression was normalized to the control (IgG-treated) group (n=3–5 per group, data is pooled from two independent experiments and analyzed using randomized block ANOVA). (K) DC and infiltrating macrophage numbers isolated from the brains of chronically infected control or α-ICOSL-treated mice was determined by flow cytometry (n=3–5 per group, data is pooled from five independent experiments and analyzed using randomized block ANOVA). (L) Representative flow plots of the neutrophil population and (M) total numbers of neutrophils isolated from the brain. Number in plots indicates the mean frequency ± standard error (n=3–4 per group, data is pooled from three independent experiments and analyzed using randomized block ANOVA). (N) Total cyst numbers from the brains of chronically infected control and α-ICOSL-treated mice enumerated by light microscopy (n=4–5 per group, data is pooled from three independent experiments and analyzed using randomized block ANOVA). * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001 for all panels.