Mycophenolic Acid Inhibits Natural Killer Cell Proliferation and Cytotoxic Function: A Possible Disadvantage of Including Mycophenolate Mofetil in the Graft-Versus-Host Disease Prophylaxis Regimen

Effect of immunosuppressants on the proliferation of NK cells induced by IL-2 and IL-15. (A) NK cells isolated from healthy individuals were cultured in the presence of IL-2 (100 U/mL) and IL-15 (10 ng/mL) with a vehicle control (EtOH 0.1%, MPA 10 μg/mL, CSP 1000 ng/mL, TAC 20 ng/mL, or MTX 100 ng/mL) for 7 days. The vertical axis represents the fold expansion (mean ± SD; n = 6) of NK cells calculated by dividing the number of cells after culture by that before culture. An asterisk indicates values showing significantly less degree of proliferation (P <.05) than the vehicle controls. (B) CFSE contents in CD3^-CD56⁺ cells were determined using flow cytometry at days 1, 5, and 9 of culture. The figure shows a representative set of histograms from a healthy individual. The results from 5 other individuals showed similar results. (C) NK cells were cultured in the presence of IL-2 (100 U/mL) and IL-15 (10 ng/mL) with MPA (1-100 μg/mL) for 7 days. The vertical axis represents the fold expansion (mean ± SD; n = 4) of NK cells calculated by dividing the number of cells after culture by that before culture. An asterisk indicates values showing significantly less proliferation (P <.05) compared with the vehicle controls.

Changes in the NK cell phenotype after a 1-week culture with different immunosuppressants. NK cells were cultured in the presence of IL-2 (100 U/mL) and IL-15 (10 ng/mL) with different immunosuppressants or a vehicle for 7 days, and the percentages of CD56^dimCD16⁺ and CD56^higCD16^- cells before and after culture were determined. (A) Scattergrams of a representative donor from 4 different ones. The analyses of other individuals produced similar results. (B) The percentages of CD56^highCD16^- NK cells in the total CD3^-CD56⁺ cells after culture in the presence of various immunosuppressants. The y-axis represents the mean percentage ± SD of CD56^highCD16^- NK cells calculated from 4 different experiments. An asterisk indicates a value showing a significant difference in the CD56^highCD16^- NK cell percentage compared with the vehicle control.

Changes in the expression of NK cell–activating receptors after culture with immunosuppressants. The changes in the expression levels of NKp30, NKp44, NKp46, and NKG2D in NK cells from 5 different individuals after a 1-week culture with immunosuppressants are shown. The vertical axis represents relative changes in the geometric MFI (gMFI) that is calculated by (gMFI of the receptor in each immunosuppressant -gMFI of the receptor in control)/gMFI of the receptor in control) × 100. An asterisk indicates a significant difference compared with the vehicle control. “–” denotes the mean of the relative change.

Cytotoxicity of cultured NK cells against leukemia cell lines. (A) Cytotoxicities by NK cells against K562 and Daudi cells at a 1:1 E:T ratio after the 1-week culture in the presence of different immunosuppressants were compared. The mean ± SD cytotoxicity from experiments using 3 different donors is shown. An asterisk indicates a significant difference compared with the control. (B) Cytotoxicities by NK cells against K562 and Daudi cells at a 1:1 E:T ratio after the 1-week culture in the presence of different concentration of MPA were compared. The mean ± SD cytotoxicities of experiments using NK cells from 3 different donors are shown.

Effect of guanosine on the MPA-induced NK cell inhibition. NK cells were cultured in the presence of 10 μg/mL MPA with or without 100 μM guanosin for 1 week, and the effects of guanosine on MPA-induced NK cell growth inhibition (A) and cytotoxicity inhibition (B) were assessed. The vertical axis in A and B represent the mean ± SD of the fold expansion calculated by dividing the cell number after culture by that before culture and of the cytotoxicity by cultured NK cells against K562 cells at a 1:1 E/T ratio determined from 4 different donors. An asterisk indicates a significant difference (P <.05) compared with vehicle controls.

Effect of MPA on p27^Kip1 expression by T cells and NK cells. T cells and NK cells were harvested at the indicated time points after culture in the presence of IL-2 (100 U/mL) with or without MPA (10 μg/mL), and p27^Kip1 protein in the T cell and NK cell lysate was detected by Western blot analysis with anti-p27^Kip1–specific monoclonal antibody. The figure shows representative results from 5 experiments.