Figure 6

CD11b⁺Gr1^int cells induced by lipopolysaccharide (LPS) administration suppress T-helper 2 (Th2) cell responses. Th2 cells were generated in vitro using CD4⁺ T cells from DO11.10 mice by incubation under Th2-skewing conditions for 6 days. Conventional dendritic cells (cDCs) and CD11b⁺Gr1^int cells were isolated from the lungs of LPS-treated mice. cDCs and CD11b⁺Gr1^int cells alone (each at 1 × 10⁵ cells per well or at fivefold more numbers of CD11b⁺Gr1^int cells) or in combination as shown were cultured with Th2-polarized DO11.10 CD4⁺ T cells (1 × 10⁶ cells per well) and ovalbumin (OVA) peptide (5 μg ml⁻¹). (a) After cell-surface staining for CD4, intracellular cytokine staining for IL-5 was performed on cocultured cells after 36 h. Cells were incubated with Golgi Stop (BD Biosciences) for the last 4 h of culture, were fixed with CytoFix/CytoPerm (BD Biosciences), permeabilized with Perm/Wash buffer (BD Biosciences), and labeled with anti-interleukin-5 (IL-5) monoclonal antibody (mAb). For analysis by flow cytometry, gating on CD4-positive cells revealed a population with lower CD4 expression and blast-like forward scatter (FSC) vs. side scatter (SSC) properties (not shown), consistent with activated T cells. In each dot plot, the number represents the proportion of IL-5-producing cells (indicated by the box) among activated CD4-positive cells. (b) Cells were stimulated with IL-2 (50 U ml⁻¹) for 15 min and transducer and activator of transcription 5 (STAT5) phosphorylation determined by intracellular staining. (c) Nuclear extracts were analyzed by immunoblotting with antibodies against GATA binding protein 3 (GATA-3) and β-actin. (d) Reversal of suppression of Th2 (IL-5 and IL-13) cytokine production by arginase I (Arg1) inhibitor and anti-IL-10. Culture supernatants were analyzed by multiplex cytokine assay. Data shown are mean±s.d. *P<0.05, **P<0.01, ***P<0.001 with respect to cDC/CD11b⁺Gr1^int (1:5 ratio).

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