Volume 44, Issue 3, Pages 672-682 (March 2016) Interleukin-27-Producing CD4+ T Cells Regulate Protective Immunity during Malaria Parasite Infection  Daisuke Kimura, Mana Miyakoda, Kazumi Kimura, Kiri Honma, Hiromitsu Hara, Hiroki Yoshida, Katsuyuki Yui  Immunity  Volume 44, Issue 3, Pages 672-682 (March 2016) DOI: 10.1016/j.immuni.2016.02.011 Copyright © 2016 Elsevier Inc. Terms and Conditions

Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 CD4+ T Cells Inhibit IL-2 Production via Soluble Mediator(s) during Malaria Infection (A) IL-2 production and IL-2 mRNA expression by CD4+ T cells from uninfected (○) and P. berghei-infected (•) mice after stimulation with anti-TCR mAb. Data are representative of four experiments. (B) IL-2 production by CD4+ T cells from uninfected mice and those from infected mice mixed at the indicated ratios and stimulated with anti-TCR mAb. Data are representative of six experiments. (C) IL-2 production by anti-TCR-stimulated CD4+ T cells in the presence of the culture supernatant of CD4+ T cells from uninfected (○) and infected (•) mice. Data are representative of four experiments. (D) Mice were adoptively transferred with CFSE-labeled OT-II cells, left uninfected (○) or infected (•) with P. berghei. Three days later, mice were left untreated or treated with OVA and/or an IL-2-expression plasmid. After an additional 4 days, CFSE profiles of OT-II cells were analyzed. Numbers indicate the proportion of cells that divided more than once out of the total number of OT-II cells. The right panel shows a summary (five mice per group). Error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.001 by two-tailed unpaired t test. Results of comparisons are shown versus time 0 (A), 1:0 (B), or versus dose 0 (C). Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Conventional Malaria-Specific CD4+ T Cells Inhibit IL-2 Production (A) Foxp3(hCD2), CD49d, and CD11a expression in CD4+ T cells from uninfected and infected Foxp3hCD2 knockin mice (left panel). Histograms show PD-1 and LAG-3 expression (red) and their isotype controls (gray). (B) IFN-γ (upper panel) and IL-2 (lower panel) production by sorted CD4+ T cells from uninfected C57BL/6 (open bar) and infected Foxp3hCD2 knockin (closed bar) mice and their indicated subpopulations after culture in the presence of dendritic cells and malaria antigen (left) or on anti-TCR-coated plates (right). (C) IL-2 production by CD4+ T cells from uninfected C57BL/6 mice and sorted CD4+ T cell subpopulations from infected Foxp3hCD2 knockin mice co-cultured on anti-TCR-coated plates at the indicated ratios. Data are representative of four experiments. (D and E) Proliferation of CFSE-labeled CD4+ T cells from uninfected B6.SJL mice co-cultured with sorted CD4+ T cell subpopulations from infected Foxp3hCD2 knockin mice (D) or cultured in the lower chamber of the transwell plate during co-culture with sorted CD4+ T cell subpopulations from infected Foxp3hCD2-knockin mice in the upper chamber (E). Ratios of CD4+ T cells that divided more than once were expressed as the mean ratio of the control (responder CD4+ T cells alone). Data are representative of three experiments (D). Error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.001 by two-way ANOVA followed by a Bonferroni’s test. ns, not significant. Results of comparisons are shown versus Foxp3−CD11loCD49dloCD4+ T cells (C) or versus whole CD4+ T cells (D and E). Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Malaria-Specific CD4+ T Cells Produce IL-27 Real-time PCR (A, D, and G) and cytokine production (B, C, E, and F) of sorted CD4+ T cells (A–C), their subpopulations (D–F) and CD11b+/CD11c+ cells (G) from uninfected (open bar) and P. berghei-infected (closed bar) Foxp3hCD2 knockin mice. Cells were cultured on anti-TCR-coated plates (B and E) or in the presence of dendritic cells and malaria antigen (C and F). Error bars indicate SEMs. ∗∗p < 0.001 by two-tailed unpaired t test (A–C) and two-way ANOVA followed by a Bonferroni for post hoc comparison (D–G). ns, not significant (p > 0.05). Data are representative of three experiments. Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 CD4+ T Cells, which Produce a Complex of p28 and EBI3, IL-27, Are Distinct from Those Producing IFN-γ or IL-10 (A) Supernatant was collected after culture of CD4+ T cells from P. berghei-infected mice on anti-TCR mAb-coated plates, and was immunoprecipitated without (−) or with anti-p28 mAb (anti-p28), control mouse IgG, anti-EBI3 mAb (anti-EBI3), or control rat IgG. After the treatment, the levels of p28/EBI3 heterodimer, p28, and EBI3 that remained in the supernatant were determined by ELISA. CD4+ T cells from uninfected mice were cultured in the absence (SUP −) and presence (SUP +) of the remained supernatant at a final concentration of 25%, and the levels of IL-2 production was determined by ELISA (right). (B) CD4+ T cells from mice uninfected (○) or infected (•) with P. berghei were stimulated with anti-TCR mAb-coated plate and PMA/ionomycin, surface stained for CD3/CD4, fixed/permeabiized, stained for p28/IFN-γ, p28/IL-10, IL-10/IFN-γ, and analyzed by flow cytometry (left). Numbers in each quadrant represent percentages of cells. The right panel shows a summary (3–5 mice per group). Error bars indicate SEMs. ns, not significant (p > 0.05). ∗p < 0.05, ∗∗p < 0.001 by two-tailed unpaired t test. Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Requirement of p28 and EBI3 for the Production of IL-27 by CD4+ T Cells (A and B) Production of cytokines by CD4+ T cells from uninfected (open bar) and infected (closed bar) mice was determined after culture on anti-TCR-coated plates (A) or in the presence of dendritic cells and malaria antigen (B). (C) Intracellular cytokine staining of CD4+ T cells from C57BL/6, Il27−/−, and Ebi3−/− mice. Numbers represent percentages of cells. Data are representative of three experiments. Right panel shows a summary of the results. Error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.001 by two-way ANOVA followed by a Bonferroni’s test. ns, not significant. Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Malaria-Specific CD4+ T Cells Inhibit T Cell Responses in an IL-27-Dependent Manner (A) IL-2 production by responder CD4+ T cells from uninfected C57BL/6 mice, and sorted CD11ahiCD49dhiCD4+ T cells from infected C57BL/6, Il27−/−, and Ebi3−/−mice co-cultured at the indicated ratios on anti-TCR-coated plates. (B) Proliferation of CFSE-labeled CD4+ T cells from uninfected B6.SJL mice cultured in the lower chamber of transwell plates with sorted CD4+ T cells from infected C57BL/6, Il27−/−, or Ebi3−/− mice in the upper chamber at the indicated ratios. Antigen-presenting cells and anti-CD3 mAbs were added to both chambers. Proportions of CD4+ T cells that divided more than once were expressed as a ratio of the control (responder CD4+ T cells alone). Data are representative of two experiments. (C) IL-2 production by CD4+ T cells from uninfected C57BL/6 or Il27ra−/− mice and sorted CD4+ T cell subpopulations from infected Foxp3hCD2 knockin mice co-cultured as in (A). (D) Proliferation of CFSE-labeled CD4+ T cells from uninfected C57BL/6 or Il27ra−/− mice co-cultured with sorted CD4+ T cell subpopulations from infected Foxp3hCD2-knock-in mice as in (B). (E) CFSE profiles of labeled OT-II cells adoptively transferred to C57BL/6, Il27−/−, or Ebi3−/− mice, uninfected (○) or infected (•) with P. berghei and immunized with OVA. Numbers indicate the proportion of OT-II cells that divided more than once. The right panel shows a summary. Error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.001 by two-way ANOVA followed by a Bonferroni’s test (A and B) and by two-tailed unpaired t test (C). ns, not significant. Results of comparisons are shown for C57BL/6 versus Il27−/− (A), (B), and Il27ra−/− CD11ahiCD49dhi versus C57BL/6 CD11ahiCD49dhi (C and D). Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 IL-27-Producing CD4+ T Cells Regulate Protective Immune Responses against P. berghei Tcrb−/− mice were adoptively transferred with CD4+ T cells from B6.SJL, Il27−/−, Ebi3−/−, or Il12a−/− mice, and were uninfected (open bar) or infected (closed bar) with P. berghei. (A) Experimental design. (B) IL-2 production by CD4+ T cells cultured on anti-TCR-coated plates. Data are representative of five experiments. (C) Serum levels of IFN-γ 6 days after infection. (D) IFN-γ production by CD4+ T cells cultured with crude malaria antigens and dendritic cells. Data are representative of two experiments. (E) The proportions of CD11ahiCD49dhiCD4+ T cells of the total number of CD4+ T cells for mice uninfected (○) and infected (•) with P. berghei. (F) Parasitemia, weight loss, and survival in mice (11 or 12 mice in each group). Data are summary of three experiments. ∗p < 0.05, ∗∗p < 0.001 by two-way ANOVA followed by a Bonferroni’s test (B–E) or by two-tailed unpaired t test (F left and middle panel, comparison of the indicated time points), and log-rank (Mantel-Cox) test (F right panel, comparison of the curves). ns, not significant. Results of comparisons are shown for uninfected versus infected (B) and infected Tcrb−/− mice transferred with knockout versus B6.SJL CD4+ T cells (C–F). p values of parasitemia and weight loss were unavailable after day 10 due to the death of control mice (F). Error bars indicate SEMs (A–E) or SD (F). Immunity 2016 44, 672-682DOI: (10.1016/j.immuni.2016.02.011) Copyright © 2016 Elsevier Inc. Terms and Conditions