Figure 4. APANs promote CD4⁺ T cell proliferation and IFN-γ release.

(A and B) Spleen tissue sections from WT septic mice (20-hour CLP) were stained with anti-Ly6G, CXCR4, CD86, and CD4 Abs. Representative images of immunohistochemical stains of spleen are shown. Original magnification, ×20 (A); ×200 (B). Scale bar: 50 μm (A); 5 μm (B). (C–E) WT mouse BMDNs (1 × 10⁷) were stimulated with eCIRP (1 μg/mL/10⁶ cells) for 6 hours. FACS-purified APANs, nAPANs, and naive splenic F4/80⁺ macrophage as antigen-presenting cell (APC) controls (1 × 10⁵) were cocultured with splenic CD4⁺ T cells (1 × 10⁵) isolated from naive OT-II transgenic mice. After 72 hours, the cells were stained with anti-CD4, and CD25 Ab, and CSFE dye. The proliferation of (C and D) total CD4⁺ T cells or (C and E) CD4⁺CD25⁺ T cells was assessed. (C) Representative flow cytometry gating of the CD4⁺ T cell proliferation. (D and E) Frequency of total CD4⁺ T cell or CD4⁺CD25⁺ T cell proliferation. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by 1-way ANOVA and SNK test. n = 6/group. *P < 0.05 vs. CD4⁺ T cells only, ^#P < 0.05 vs. nAPAN⁺CD4⁺ T cells. (F and G) APANs, nAPANs, or APCs (1 × 10⁵) were cocultured with OT-II transgenic mouse splenic CD4⁺ T cells (1 × 10⁵), and, 24 hours later, IFN-γ, IL-4, and IL-17 culture supernatant levels were determined by ELISA. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by ANOVA and SNK test. n = 6. *P < 0.05 vs. nAPAN, ^#P < 0.05 vs. APC. (H) APANs, nAPANs, or APCs (1 × 10⁵) were cocultured with OT-II mouse splenic CD4⁺ T cells (1 × 10⁵) in the presence of IgG or IL-12– neutralizing Ab. At 24 hours later, IFN-γ culture supernatant levels were determined by ELISA. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by ANOVA and SNK test. n = 6–12/group. *P < 0.05 vs. nAPAN, ^#P < 0.05 vs. APC, ^†P < 0.05 vs. APAN+IgG.