Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist

Figures & data

Figure 1. Dysregulated T cell function induced by CD28SA stimulation. (A) Human CD4⁺ T_EMs were stimulated for 1 to 4 d with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3 and NIB1412); control category included cells without any treatment (Control). Cells were harvested at indicated time points and stained with fluorochrome-conjugated anti-CD4 and anti-TCR antibodies followed by flow cytometric analysis. Population of CD4⁺TCR⁺ cells are shown in the upper right quadrant as percentages of total T cells. Results are representative of four independent experiments. (B) Human CD4⁺ T_EMs cells were stimulated with the indicated concentrations of plate-bound antibodies and proliferation was measured three days post-activation by ³H-labeled thymidine incorporation. The vertical axis represents mean cpm ± SD from triplicate wells. The data are representative of four independent experiments, (*P < 0.05, ***P < 0.001; unpaired t test). (C) Cell surface staining of human CD4⁺ T_EMs stimulated for 4 d with 5 μg/ml of plate-bound anti-CD3 mAb and/or 10 μg/ml of NIB1412. The percentages of the CD4⁺ CD95⁺ cells are shown in the upper right quadrant. Results are representative of three independent experiments (D) Human CD4⁺ T_EMs were stimulated for 1 to 6 d, fixed with ice cold 70% ethanol, stained with propidium iodide and cells in S-phase were quantified by flow cytometry. Results are representative of at least four independent experiments. (E) IL-2 concentrations in the supernatants from human CD4⁺ T_EMs stimulated for 24, 48 and 72 h were determined by enzyme-linked immunosorbent assay (ELISA). The IL-2 titers from four independent experiments (mean ± SD of replicate samples) are expressed as picograms per mL on a log₁₀ scale. (*p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA).

Figure 2. Enhanced cell surface expression of LFA-1 and CCR5 on CD28SA-activated CD4⁺ effector memory T cells. Human CD4⁺ T_EMs were stimulated for 1 to 4 d with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3&NIB1412); control category included cells without any treatment (Control). Cells were harvested at indicated time points and stained with fluorochrome-conjugated anti-CD4 and anti-LFA (A) or anti-CD4 and anti-CCR5 (B) antibodies followed by flow cytometric analysis. (A) Population of CD4⁺LFA⁺ cells are shown in the upper right quadrant as percentages of total T cells. The cells are shown as percentages of total T cells in the upper right quadrant. (B) Population of CD4⁺CCR5⁺ cells are shown in the upper right quadrant as percentages of total T cells. Results are representative of four independent experiments.

Figure 3. Adhesion and transmigration of CD28SA-activated CD4⁺ effector memory T cells. Human CD4⁺ T_EMs were stimulated with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3 and NIB1412); control category included cells without any treatment (Control). Cells were harvested at 48 h and labeled with CellTracker™ Green CMFDA. (A) Immunofluorescent microscopy of HDMEC monolayer with adherent CMFDA-labeled T_EMs initially stimulated with the indicated antibodies and stained for F-actin and DNA using fluorescently labeled phalloidin (red) and DAPI (blue), respectively. (Scale bar = 20 microns). Phase optics was adjusted to emphasize adherent cells. (B) CMFDA-labeled T_EMs were added to confluent HDMECs and incubated for 30 min. Cells were washed and the remaining adherent cells visualized under fluorescence microscopy. Vertical axis represents the number of adherent T_EMs per field (**p < 0.01; ***p < 0.001, unpaired t test). Results from four independent donors are represented as means ± SD (C) Transmigration of stimulated CD4⁺ T_EMs across HDMEC layer on transwell inserts. Transmigrated cells were quantified by fluorescence and the results are presented as the mean (± SD) number of transmigrated cells from 3 replicate wells per condition. Results are representative of three independent experiments (***p < 0.001; unpaired t test).

Figure 4. Failure of CD4⁺ effector memory T cells to upregulate cell surface PD-1 after CD28SA-activation. Human CD4⁺ T_EMs were stimulated for 1 to 4 d with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3 and NIB1412); control category included cells without any treatment (Control). (A) Cells were harvested at indicated time points and stained with fluorochrome-conjugated anti-CD4 and anti-PD-1 antibodies followed by flow cytometric analysis. (B) Cells harvested at day 3 were stained for intracellular PD-1 following fixation/permeabilization treatment. Population of CD4⁺ PD-1⁺ cells are shown in the upper right quadrant as percentages of total T cells. Results are representative of four independent experiments.

Figure 5. Absence of PD-1 mediated regulation of T cell function in CD28SA- stimulated T cells. (A) Schematic of the protocol used to investigate the functional significance of PD-1 pathway on NIB1412-activated CD4⁺ T cells. (B) Human PBMCs were stimulated for 48 h with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3 and NIB1412); control category included cells without any treatment (Control). Cells were then re-stimulated with anti-CD3 only (CD3,1 μg/ml) or with anti-CD3 and 10 μg/ml of rPD-L1 (CD3 and rPD-L1). Cells were harvested and stained with fluorochrome-conjugated anti-CD4 antibody, fixed, permeabilized, stained with fluorochrome-conjugated anti-IFNγ antibody and analyzed by flow cytometry. The CD4⁺ population is shown in light gray and the CD4⁻ population in dark gray. The percentages of the CD4⁺ IFNγ⁺ cells are shown in the upper right quadrant. Results are representative of four independent experiments.

Figure 6. Effects of PD-1 engagement on phospho PTEN and casein kinase 2 levels in CD4⁺ effector memory T cells stimulated by anti-CD3 and CD28SA. Human CD4⁺ T_EMs were stimulated with anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); rPD-L1 (rPD-L1, 10μg/ml); with anti-CD3 mAb and rPD-L1 (CD3 + rPD-L1) or with NIB1412 and rPD-L1 (NIB1412 + rPD-L1). At the end of 72 h, the cells were lysed and extracted protein separated by SDS-PAGE. pPTEN – S380/T382/383 (A) and CK2 (B) protein levels were assessed by immunoblotting and β-actin was used as loading control. All blots are representative of four independent experiments. pPTEN and CK2 levels were quantified by densitometry, normalized to β-actin and expressed as a percentage of pPTEN or CK2 levels in untreated cells. Data are represented as mean ± SEM of four independent experiments. Statistical analysis was performed using unpaired t test (*p < 0.05, **p < 0.01). Phospho PTEN – pPTEN; Casein Kinase 2 – CK2