Figure 1.
T cell activation alters mRNA expression of specific ion channel subunits.
A, B, Bar diagram summarizing n-fold increase or decrease in intensity of hybridization signals obtained by probing oligonucleotide-based arrays with Cy3 and Cy5 labeled cDNA derived from mRNA isolated from non-activated and PHA-L activated PBMCs, respectively. Gray bars – custom-made array (n = 14) (Tables S1, S2); black bars – Affimetrix array (human U133A 2.0; n = 6). Error bars are SEM. Channel subunit genes are indicated on the left. Nomenclature is from http://www.ncbi.nlm.nih.gov/omim. Brackets - selectivity of corresponding ion channel. cat – cation; n.p. – not present in array; n.d. – not determined. CD25 (IL2RA) served as control. C, Bar diagram summarizing qPCR results for changes in mRNA expression of purified CD4+ and CD8+ T cells activated with anti-CD3/CD28 antibody-coated beads (n = 5–8). D, qPCR analysis of P2RX5 mRNA expression in activated CD4+ T cells in absence (▴) or presence (□) of cycloheximide (n = 3). Error bars are SEM.
Figure 2.
CD4+ T cell activation increases P2RX5 protein expression.
A, Western blot of non-activated (CD4+/−) and activated (CD4+/+) CD4+ T cell lysates and of P2RX5 cDNA transfected (HEK293/+) or mock-transfected (HEK293/m) HEK293 cell lysates. Blots were stained with anti-P2RX5 antibody. Staining with anti-GAPDH antibody (arrow) served as loading control. B, Western blot of protein biotinylated at the cell surface of activated CD4+ T cells (+) and of controls (−). Protein was fractionated with streptavidin-agarose. B – streptavidin-agarose bound fraction; UB – unbound protein fraction. Western blots were stained with antibodies against P2RX5, GAPDH, and CD3ε as indicated by arrows.
Figure 3.
P2RX5 protein is redistributed upon T cell stimulation.
T cells were incubated with either streptavidin beads for control (A, C) or anti-CD3/CD28 antibody-coated streptavidin beads (B, D) for activation. A, B, immunostaining pattern obtained with anti-P2RX5 antibodies (green). C, D, overlayed double fluorescence images obtained with anti-talin (green) and with anti-P2RX5 antibodies (red), pictures represent magnifications of overviews depicted in Fig. S1D. Scale bars – 10 µm.
Figure 4.
P2RX5 knock-down affects CD4+ T cell polarity and IL-10 secretion.
A, Western blot analysis of P2RX5, talin, and LFA-1 expression in activated CD4+ T cells transfected with siRNA as indicated. GAPDH served as loading control. B, C, CD4+ T cells were transfected with indicated siRNA followed by activation for 4 or 24 h. Cells were stained with anti-talin antibodies. Scale bar – 10 µm. D, Bar diagram illustrating relative number of polarized CD4+ T cells seen with talin or LFA-1 immunostains. E, Bar diagram illustrating interleukin production of activated non-transfected (grey bars), control siRNA (white bars) or P2RX5-siRNA transfected (black bars) CD4+ T cells (n = 3 donors). Error bars are SEM, n.s. – non significant; * - p<0.05, ** p<0.01 (Student's T-test).
Figure 5.
Expression of P2RX5 by human T cells is highly dynamic and activation-dependent.
A, Stimulation of human CD4+ TCCs resulted in a significant increase in frequencies of cells expressing P2RX5 on the cell surface. B, Notably, surface expression levels were also upregulated. C, P2RX5 expression was considerably lower on the surface of HEK293 cells. D, Summary of significantly increased frequencies of surface P2RX5-expressing TCCs upon stimulation. Cell surface expression of P2RX5 was also upregulated on CD4+ TCCs. Bars represent mean values ±SEM. Statistical analysis was performed by paired t-test, **p<0.01. E, Sequencing of two CD4+ TCCs cultivated under stimulated (+) or unstimulated (−) conditions revealed that human Th1 and Th1/2 cells were expressing a transcript variant of P2RX5 lacking exon 10.