Figure 1.

Upregulation of CTLA-4 induced by cAMP in the absence of T cell activation. CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells were purified by immunomagnetic cell sorting, (A) shows the gate strategy and purity of cells after magnetic separation. Purified cells were cultured in the presence of medium alone (medium), CT (1 μg/ml) or FSK (10 μM) for 24 h and then stained with anti-CD4 mAb on the membrane, fixed, permeabilized and stained with anti-CTLA-4. Cytofluorimetric analysis was performed on CD4⁺-gated population. Graphs show the mean of the upregulation of CTLA-4 of five independent donors and the *indicates that the differences between CT or FSK treated as compared with untreated cells are significant (p < 0.05), the variations bars correspond to the SEM (B). CTLA-4 mRNA was evaluated in CD4⁺CD25⁻ and CD4⁺CD25⁺ T lymphocytes purified by FACSvantage cell sorting after staining with anti-CD25 mAb. Cells were cultured in the presence or absence of CT and the mRNA was analyzed after 4 h. RNA integrity and cDNA synthesis were verified by amplifying β2-microglobulin cDNA. An irrelevant cDNA from human tumor cell line (THP-1) has been included in the amplification (lane 6) of (C). The graph (D) represents CTLA-4 mRNA quantification of the two mRNA transcripts (672 and 550 bp fragments), both normalized to those of β2-microglobulin. One of two experiments performed is shown.