Article Figures & Data
Figures
- FIGURE 1.
PD-1 and PD-L are expressed by human CD4+ T cells upon activation. A, CD4+ T cells were activated with protein-coated microspheres as described in Materials and Methods. At the indicated times, cells were harvested, washed once, and stained with either anti-huPD-1, anti-huPD-L1, anti-CD25, or anti-CD69 Abs. Control Ab isotype (thin line) was included for each staining. Cell populations were analyzed by flow cytometry using a FACScan. A representative experiment is shown (n = 3). B, RNA was prepared from unstimulated or 72 h anti-CD3 activated cells. PCR analysis was performed as described in Materials and Methods. Control indicates PCR performed in the absence of RNA. All RNA samples were also assessed for expression of GAPDH, a housekeeping gene.
- FIGURE 2.
Anti-PD-1 Ab can deliver a negative signal to human CD4+ T cells. A, Purified CD4+ T cells (105 cells/well) were activated with microspheres coated with a fixed concentration of anti-CD3 Ab (1 μg/107 microspheres) and increasing concentrations of PD-L1.Fc or PD1–17 Ab. Activation in the absence of PD-1 engagement was determined using microspheres containing anti-CD3 Ab (1 μg) and murine IgG (4 μg/107 microspheres). For comparison purposes, values are represented as the percentage of anti-CD3 Ab response, where 100% represent cpm obtained when cells were activated with anti-CD3/murine IgG-coated microspheres. A representative experiment is shown (n = 3). B, Cells (105 cells/well) were activated with anti-CD3, anti-CD3/PD-L1.Fc, and anti-CD3/anti-PD1–17 microspheres in the presence of increasing concentrations of soluble anti-CD28 Ab. Two sets of anti-CD3 microspheres are shown, one for each of the corresponding isotypes (muIgG2a for PD-L1.Fc and huIgG1 for anti-PD1–17). Proliferation was measured at 72 h. Similar results were obtained with multiple donors (n = 3). C, Cells (105 cells/well) were activated with anti-CD3, anti-CD3/PD-L1.Fc (CIS), and anti-CD3 + PD-L1.Fc (TRANS) microspheres. CIS microspheres contained both anti-CD3 and PD-L1.Fc on the same bead, TRANS microspheres consist of two beads, one containing anti-CD3, and one containing PD-L1.Fc. To maintain equal bead to cell ratios in CIS and TRANS conditions, microspheres coated with muIgG control were added to CIS cultures. In all cases, control murine Ig was added to achieve a total protein concentration of 5 μg/107 microspheres. Proliferation was measured at 72 h. Similar results were obtained with multiple donors (n = 6).
- FIGURE 3.
Soluble anti-PD-1 Abs enhance T-T cell responses. CD4+ T cell were preactivated for 72 h with anti-CD3/anti-CD28-coated microspheres, harvested, and restimulated with increasing concentrations of PHA in the presence of IL-2 (10 ng/ml) and anti-PD-1 Ab (20 μg/ml) (A) or increasing concentrations of anti-PD-1 Ab in the presence of 0.1% PHA + 10 ng/ml IL-2 (B). Proliferation was measured at 72 h (n = 3).
- FIGURE 4.
ICOS costimulation can be prevented by PD-1 engagement. A, CD4+ T cells were activated with microspheres coated with anti-CD3/control Ig (1 μg/4 μg/107 microspheres), anti-CD3/PD-L1.Fc/control Ig, anti-CD3/ICOS-L.Fc/control Ig, anti-CD3/B7-2.Fc/control Ig, anti-CD3/PD-L1.Fc/ICOS-L.Fc, or anti-CD3/PD-L1.Fc/B7-2.Fc (1 μg/2 μg/2 μg/107 microspheres). Microsphere composition is indicated in the chart below x-axis. Proliferation was measured at 72 h. Statistical analysis indicated that CD3/PD-L (p = 0.0386), CD3/ICOS-L (p = 0.0393), CD3/B7 (p = 0.0019), and CD3/B7/PD-L (p = 0.0099) responses differed significantly from CD3 responses; in contrast, no statistical significance was observed between CD3/ICOS-L/PD-L (p = 0.9836) and CD3 responses. B, Supernatants were harvested at 24 h and IL-2 production analyzed by ELISA (sensitivity 20 pg/ml). Inset, IL-2 transcript relative expression under stimulation conditions. Values are normalized for GAPDH expression (n = 2). C, CD4+ T cells were activated as in A in the presence of a neutralizing murine anti-human IL-2 Ab (20 μg/ml) or control murine IgG. Microsphere composition and presence of neutralizing IL-2 Ab is indicated in the figure below the x-axis. Statistical analysis indicated that CD3/PD-L (p = 0.0209), CD3/ICOS-L (p = 0.0045) responses differed significantly from CD3 responses; no statistical significance was observed between CD3/ICOS-L/PD-L (p = 0.0759) and CD3 responses. CD3/ICOS-L/PD-L plus anti-IL-2 Ab (p = 0.0419) responses differed significantly from CD3/ICOS-L/PD-L responses. Proliferation was measured at 72 h. Summaries of results obtained with four to eight donors are shown. Horizontal lines represent mean value for each condition.
- FIGURE 5.
IL-2, IL-7, and IL-15 but not IL-4 or IL-21 rescue PD-1-mediated inhibition. CD4+ T cells were activated with microspheres coated with anti-CD3 (•) or anti-CD3/PD-L1.Fc (○) in the presence of increasing concentrations of IL-2 (A), IL-7 (B), IL-15 (C), IL-4 (D), and IL-21 (E). Proliferation was measured at 72 h. A representative experiment is shown (n = 6).
- FIGURE 6.
IL-21 activation of primary human CD4+ T cells does not result in STAT5 phosphorylation. CD4+ T cells were activated with anti-CD3 Ab-coated microspheres for 5 and 24 h followed by addition of 100 ng/ml IL-2 or IL-21 for 15 min. Cell lysates were immunoblotted and analyzed for total and phosphorylated STAT1 (A), STAT3 (B), and STAT5 (C) as described in Materials and Methods (n = 3).
Tables
- Table I.
Treatment of T cells with IL-2, IL-7, and IL-15 but not IL-4 and IL-21, leads to up-regulation of IL-2Rα chaina
Media IL-2 IL-4 IL-7 IL-15 IL-21 Isotype Anti-CD25 Isotype Anti-CD25 Isotype Anti-CD25 Isotype Anti-CD25 Isotype Anti-CD25 Isotype Anti-CD25 Anti-CD3 3.44 96.75 5.35 736.86 3.5 94.12 5.31 622.07 4.69 488.4 4.16 100.12 Anti-CD3/PD-L1.Fc 3.12 46.38 4.49 307.03 4.05 51.41 4.18 246.99 4.46 272 4.34 51.42 -
a CD4+ T cells were activated with anti-CD3 Ab-coated microspheres in the presence of optimal concentrations of the indicated cytokines. At 72 h, cells were harvested and stained for IL-2Rα (CD25) expression as described in Materials and Methods (n = 2). Mean fluorescence values are shown, bold numbers indicate increases 4-fold above media control.
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