Therapeutic effects of PB502 on IPS. (A-D) Lethally irradiated B6.WT mice were injected with BALB/c.Ifng^−/− T cells and BALB/c.WT TCD BM cells. (A-C) Recipients were intraperitoneally injected with PB502 (20 mg/kg) or L-Kyn (100 mg/kg) with or without CH-223191(10 mg/kg) each day from day 3 through day 8. Lungs were harvested on days 14 (A-B) and 21 (C). (A) Representative image of hematoxylin and eosin staining (×200; left) and pathologic scoring (right; n = 5 per group). (B) Levels of cytokine messenger RNA (mRNA; n = 5 per group). (C) Lung cells were stained for CD4, CD44, and Foxp3 and assessed by flow cytometry. Representative dot plots for Foxp3⁺ or CD44^hi cells on gated CD4⁺ T cells (left) and composition of Foxp3⁺CD4⁺, Foxp3⁻CD44^hiCD4⁺, and CD44^int/loCD4⁺ T cells (right; n = 3-5 per group). (D-E) Recipient mice were intraperitoneally injected with PB502 (20 mg/kg) or L-Kyn (100 mg/kg) each day from day 7 through day 12. Lungs were harvested on day 27. (D) Data were analyzed and presented as described in panel C. (E) Isolated CD4⁺ T cells were restimulated with anti-CD3/CD28 for 24 hours, and the concentration of IL-10 and IL-17A was measured in the cell culture supernatants (n = 6 per group). (F) Naïve CD4⁺ T cells isolated from B6 splenocytes or human peripheral blood mononuclear cells were cultured under Treg differentiation conditions (anti-CD3/CD28, IL-2, and TGF-β) with PB502 (1 μM) or L-Kyn (100 μM) for 3 (mouse cells) or 5 days (human cells). Representative dot plots for Foxp3⁺CD4⁺ T cells are presented. (G-H) Naïve CD4⁺ T cells were cultured under Th17 differentiation conditions (anti-CD3/CD28, IL-6, TGF-β, IL-23, anti–IFN-γ, and anti–IL-4 for mouse cells and anti-CD3/CD28, TGF-β, IL-1β, and IL-23 for human cells) with PB502 (1 μM) or L-Kyn (100 μM) for 3 (mouse cells) or 5 days (human cells). (G) Representative dot plots for Foxp3⁺CD4⁺ T cells. (H) Concentrations of IL-10 in cell culture supernatants of mouse cells. (I) Lethally irradiated B6 mice were injected with BALB/c.Ifng^−/− T cells (2 × 10⁶) and BALB/c.WT TCD BM cells (1 × 10⁷). Recipients were intraperitoneally injected with PB502 (20 mg/kg) or L-Kyn (100 mg/kg) each day from day 3 through day 16. Survival rates are presented at the left and clinical GVHD scores at the right. Results are representative of at least 3 independent experiments with similar results. Data represent mean ± standard error of the mean. One- or 2-way analysis of variance (A-H) and Mantel-Cox log-rank test (I) were performed. *P < .05, **P < .01, ***P < .001.