Fig 1.
Schematic overview of the intracellular calcineurin pathway in T cells and amplification of the NFATc1/A isoform [11].
The calcineurin pathway is activated upon antigenic stimulation of the CD3/TCR complex in combination with co-stimulatory signals. This in turn activates the signal molecules phospholipase C-γ (PLC-γ) and inositol 1,4,5-trisphosphate (IP3) causing an influx of calcium and the opening of calcium channels in the membrane to maintain intracellular calcium levels. Upon interaction between calcium and the small calcium-binding protein, calmodulin, the phosphatase calcineurin is activated, which dephosphorylates NFAT. There are 13 phosphorylation sites present on NFAT that are known to be dephosphorylated upon activation. Dephosphorylation causes the translocation of NFAT to the nucleus where it will initiate gene transcription through the interaction with other transcription factors, such as AP-1. The signaling pathway is regulated by other signaling pathways, such as the MAPK pathway (JNK) and NFκB pathway. Once in the nucleus, NFAT will act as transcription factor and regulates the production of cytokines and the amplification of the isoform NFATc1/A in a positive autoregulatory feedback loop. The intracellular signaling pathways can also be activated by using PMA/ionomycin as a stimulus, while calcineurin inhibitors, such as TAC, are known to inhibit the calcineurin pathway.
Fig 2.
Gating strategy for the total NFATc1 expression in T cell subsets.
(A) Gating of CD3+ T cell subsets. Cells were gated for their expression of CD14 and CD3, where after CD3+CD14- T cells were gated for their expression of CD4 or CD8. Within these populations, the expression of CD28 was determined to identify the CD4+CD28+, CD8+CD28+ and CD8+CD28- T cell subsets. B) Example of the total NFATc1 expression in CD3+CD14- cells, either unstimulated or stimulated with PMA/ionomycin. FSC) Forward scatter; SSC) sideward scatter.
Fig 3.
NFATc1 expression (MFI) in the total study population of TAC-treated patients.
Unstimulated (grey) and PMA/ionomycin stimulated (white) blood samples were stained for the expression of NFATc1 in CD3+ (upper left graph), CD4+ (upper middle graph), CD8+ (upper right graph), CD4+CD28+ (lower left graph), CD8+CD28+ (lower middle graph) and CD8+CD28- (lower right graph) T cells. Data are plotted as box and whiskers (Tukey style); n = 23 isotype controls, n = 10 healthy controls, n = 11 TAC patients before transplantation; *) p < 0.05, **) p < 0.01, ***) p < 0.001.
Fig 4.
Immunosuppressive drug therapy effects on NFATc1 amplification in T cell subsets.
(A) NFATc1 amplification in CD4+CD28+, CD8+CD28+ and CD8+CD28- T cells before transplantation (n = 21 kidney transplant patients). (B, C, D) Inhibition of NFATc1 amplification (MFI) after a TAC-(grey) or BELA-(white) based therapy. Delta NFATc1 expression (amplification) was determined at different time points after transplantation and compared to the samples before transplantation in CD4+CD28+ (B), CD8+CD28+ (C) and CD8+CD28- T cells (D). The number of patients that were measured at each time point is shown on the x-axis in parentheses. n = 11 TAC-treated patients and n = 10 BELA-treated patients at day 0. Data are plotted as box and whiskers (Tukey style); *) p < 0.05, **) p < 0.01 compared to day 0.
Fig 5.
Spearman correlations of NFATc1 amplification with TAC or MPA pre-dose concentrations.
(A) NFATc1 amplification inversely correlated with TAC pre-dose concentrations in time. The correlation was only seen in CD4+CD28+ (left graph) and CD8+CD28+ cells (middle graph) and not in CD8+CD28- cells (right graph). (B) MPA pre-dose concentrations were not correlated to NFATc1 amplification levels in CD4+CD28+ (left graph) and CD8+CD28+ cells (middle graph) nor in CD8+CD28- cells (right graph). TAC-treated patients were included for this analysis when blood samples were analyzed for NFATc1 amplification at all time points: before transplantation and day 4, 30, 90, 180 and 360 after transplantation. N = 7; rs, Spearman correlation coefficient.
Fig 6.
Individual drug effects on NFATc1 amplification in T cell subsets.
TAC, at a high concentration of 50 ng/ml, significantly inhibited the expression of NFATc1 in both CD4+CD28+ (upper graph), CD8+CD28+ (lower left graph) and CD8+CD28- cells (lower right graph) compared to the sample without drugs. Data are plotted as mean ± SEM; n = 5; *) p < 0.05.