Figures and data
Early PD-1 upregulation in activating T cells.
(A, B) PD-1 upregulation after antigenic stimulation of CD4+ T cells. Purified CD4+ CD62L+ T cells from DO11.10 TCR-transgenic mouse were stimulated with OVA323-339 peptide (1 μg/ml) in the presence of A20 cells for indicated duration of time. Numbers represent percentages of PD-1+ within CD4+ cells (A) and mean fluorescence intensity of PD-1 (B). Data represent average ± SEM of triplicate samples. (C) PD-1 upregulation on DO11.10 T cells by different concentrations of OVA peptide. Anti-PD-L1 mAb or isotype control (5 μg/ml) was added at the beginning of co-culture. (D) T cell proliferation after stimulation. CFSE-labeled DO11.10 CD4+ CD62L+ T cells were stimulated as in (C). (E) PD-1-dependent inhibition of cytokine production from DO11.10 T cells. Cytokine levels in the culture supernatant were determined after 3 days of stimulation with OVA323-339 (0.2 μg/ml). Data represent average ± SEM of triplicate (B, C) or quadruplicate (E) samples. *p < 0.05, ***p < 0.001; Student’s t-test.
PD-L1 blockade unleashes the development of IL-4-producing Th2 cells.
(A) Development of effector cells expressing T-bet or GATA3. Purified CD4+ CD62L+ DO11.10 T cells were stimulated with OVA323-339 (1 μg/ml) in a Th1 or Th2-skewing condition. Intracellular staining for T-bet and GATA3 expression was performed after 1 and 4 days. (B, C) PD-1-dependent regulation of Th1 and Th2 cell differentiation. Naïve DO11.10 T cells were stimulated in the neutral condition (B) or Th1/Th2-skewing conditions (C) for 3 days using PD-L1-deficient or PD-L1-expressing parental A20 cells as APC. Subsequently, cytokine-producing activities were evaluated by stimulating activated T cells (1.5 x 105 cells) with immobilized anti-CD3 mAb for 24 h. (D-J) Naïve DO11.10 T cells were stimulated for 3 days using parental A20 cells as APC in the presence or absence of anti-PD-L1 mAb. T cell stimulation was conducted in the neutral (D-F) or skewing condition for Th1 (G, H) and Th2 cells (I, J). Activated T cells were restimulated in the same condition for 2 more days. Intracellular staining for cytokines was performed on day 5. Percentages of IFN-γ- and IL-4- positive events in CD4+ cells (D, E, G-J) and IL-4+ event / IFN-γ+ event ratios in individual samples (F) are shown. Data represent average ± SEM of triplicate (B, C) or quadruplicate (E, F, H, J) samples. *p < 0.05, ***p < 0.001; Student’s t-test.
PD-1 agonist antibody strongly inhibits the induction of cytokine-producing Th2 cells.
hPD-1-tranduced DO11.10 T cells were stimulated with OVA323-339 in Th1 or Th2- skewing condition using PD-L1-deficient A20 cells as APC. (A) hPD-1 expression in DO11.10 T cells after the transduction of wild-type hPD-1 or YFYF mutant. (B, C) The effects of anti-hPD-1 agonist mAb, HM266, on hPD-1 WT-transduced DO11.10 T cells in Th1 (B) or Th2-skewing condition (C). The concentrations of HM266 or isotype control were 5 μg/ml. Intracellular cytokine staining was performed on day 5. (D-G) The involvement of PD-1 signaling in Th2 inhibition by HM266. DO11.10 T cells expressing hPD-1 WT or hPD-1 YFYF were stimulated with OVA323-339 (1 μg/ml) in Th1 or Th2-skewing condition. Representative plots of intracellular cytokine staining on day 5 (D, E) and proportions of IFN-γ- (F) and IL-4-producing cells (G) within CD4+ cells. Data represent average ± SEM of duplicate samples.
J116 inhibits the induction of IL-4-producing Th2 cells.
(A) The immunosuppressive activity of anti-hPD-1 mAb J116. The PD-1 agonistic activity was determined by the inhibition of IL-2 production from DO11.10 T cell hybridoma. OVA323-339 peptide (2 μg/ml) was added to stimulate hPD-1-transduced DO11.10 cells in the presence of IIA1.6 cells that express murine FcγRIIB but lack PD-L1. (B-G) DO11.10 T cells were transduced with wild-type hPD-1 or YFYF mutant and skewed into Th1 or Th2 cells in the presence of J116 or mouse IgG1 isotype control (5 μg/ml). The concentration of OVA323-339 was 1 μg/ml. Intracellular cytokine staining was performed on day 5. Representative plots of cytokines staining (B, C) and percentages of IFN-γ- (D) and IL-4-producing cells (E) in CD4+ T cells. The same number of T cells were restimulated with anti-CD3 mAb on day 5, and IFN-γ- (F) and IL-4 levels (G) after 24 h were determined in the culture supernatant. Data represent average ± SEM of triplicate (A, F, G) or duplicate (D, E) samples. **p < 0.01; Student’s t-test.
Immune deviation by PD-1 agonist antibody in a Th2-prone condition.
hPD-1- tranduced DO11.10 T cells were stimulated with OVA323-339 (1 μg/ml) in the presence of HDM-stimulated BMDC supernatant using PD-L1-deficient A20 cells as APC. The concentrations of HM266 or isotype control were 5 μg/ml. (A, B) Representative plots of IFN-γ- (A) and IL-4-producing CD4+ cells (B) on day 5. DO11.10 T cells received transduction of either wild-type hPD-1 or YFYF mutant. (C, D) Proportions of IFN-γ- (C) and IL-4-producing cells (D) within CD4+ cells. (E) IL-4+ event / IFN-γ+ event ratios in individual samples. Data represent average ± SEM of duplicate samples.
Spontaneous PD-1 upregulation in T cells sufficiently suppress functional differentiation into Th2 cells when stimulated with PD-1 agonist antibody.
CD4+ CD62L+ cells from hPD-1-KI DO11.10 mice were stimulated with OVA323-339-pulsed PD-L1-deficient A20 cells. (A, B) Early PD-1 upregulation in the stimulated hPD-1-KI DO11.10 cells. OVA323-339 concentration was 1 μg/ml. Numbers indicate percentages of PD-1+ in CD4+ cells and mean fluorescence intensity of PD-1 (B). (C, D) The inhibition of Th2 cell induction by HM266, anti-hPD-1 agonist mAb. hPD-1-KI DO11.10 cells were stimulated with the indicated concentrations of OVA323-339 for 3 days. HM266 or control mouse IgG1 was added at 5 μg/ml. The activated CD4+ T cells were restimulated with immobilized anti-CD3 mAb for 6 h. IFN-γ-producing cells in Th1-skewing condition (C) and IL-4-producing cells in Th2-skewing condition (D) were evaluated by subsequent intracellular cytokine staining. (E-G) Cytokine-producing activities of activated CD4+ T cells. IFN-γ (E), IL-4 (F) and IL-13 (G) levels in the culture supernatant were evaluated after restimulation of activated T cells (2 x 105 cells) on day 3 with immobilized anti-CD3 mAb for 24 h. Data represent average ± SEM of triplicate samples. *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t-test.
PD-1 agonist antibody ameliorates allergic inflammation.
(A) The induction of allergic asthma in HDM-sensitized hPD-1 knock-in mice. Treatment with anti-hPD-1 agonist mAb, HM266, was done in two different schedules as indicated. (B-D) Infiltrated cell numbers in the BAL. Number of eosinophils (C) and CD4+ T cells (D) were calculated from flow cytometric analysis. (E) PAS staining of the lung sections. Scale bars represent 100 μm. (F-H) Intracellular cytokine staining of CD4+ T cells from the lung tissue (F, G) and BAL fluid (H, I). Panels indicate percentages in CD4+ T cells (F-H) and the numbers of cytokine-producing CD4+ T cells. (J, K) Plasma levels of total IgE (J) and HDM-specific IgE levels (K) on day 7. Data represent average ± SEM of vehicle control (n = 3), HDM + ctrl Ab (n = 6), HDM + preventive HM266 (n = 6) and HDM + therapeutic HM266 (n = 4) groups. *p < 0.05, **p < 0.01, ***p < 0.001; Tukey-Kramer test.
PD-1 expression on CD4+ T cells from allergic hPD-1 knock-in mice.
(A) HDM extract induced lung allergy was induced on hPD-1 knock-in mice as indicated. Mice was euthanized 4 h after the final intranasal challenge. (B) BAL cell numbers. Numbers of eosinophils and CD4+ T cells were calculated from flow cytometry analysis. (C) Representative FACS plots of CD69, TIGIT, and hPD-1 expression on CD4+ T cells collected from BAL or lung cells. (D) Percentages of CD69+, TIGIT+, and hPD-1+ cells in CD4+ T cells. Data represent average ± SEM of 6 mice. **p < 0.01, ***p < 0.001; Student’s t-test.
