sCTLA-4 Is Present in Sera from Pediatric ALL Figure 1. A sCTLA-4 is found in serum of B-ALL patients. Panel A, The concentration of sCTLA-4 was evaluated by ELISA on sera collected from B-ALL patients (n = 64), and a healthy donor group as a control (n = 45). Results are expressed as nanogram per milliliter. Each sample was diluted 1:10 and tested in triplicate. Samples higher to the detection limit of the test (50 ng/ml) were diluted appropriately and tested again. Deviation between triplicates was ,10% for any reported value. Arrow marks 23 kDa species. Panel B, The ALL patients were classified in relation to the membrane immunophenotype of peripheral blood mononuclear cells (PBMCs) in three groups: pro-B ALL (n = 8), common ALL (n = 41), pre-B ALL (n = 15). The analysis of sera sCTLA-4 showed a statistically difference between the three ALL groups and the control samples. No significant differences were observed among the three ALL groups. Panel C, Reverse transcription–PCR was performed in order to verify the presence of the spliced variant of CTLA-4 on PBMCs from a healthy donor (a), on freshly isolated T lymphocytes (b), and from three representative B-ALL patients (1 to 4; sera of patients from 1 to 3 resulted positive for sCTLA-4 ELISA, 4 was negative). The amplification by PCR of the human CTLA-4 coding sequence revealed the constitutive expression by PBMCs from a normal donor of two transcripts whose sizes are: 650 and 550 bp. Otherwise, B-ALL patients preferentially express the light transcript. Arrow marks a 550 bp sCTLA-4 fragment. Panel D, Immunoprecipitation of sCTLA-4 was performed on sera from a representative group of B-ALL patients. 1, 2 and 3 sera were resulted positive during ELISA analysis (showing a mRNA band of 550 bp); 4 was negative (these results were in line with those obtained by PCR). doi:10.1371/journal.pone.0044654.g001 Figure 2. Analyses of correlation between sCTLA-4 and some clinical characteristics in B-ALL patients. As specified in the text, the only significant correlation was between the sCTLA-4 levels and the leukocyte counts (Panel C). No significant correlation was observed between sCTLA-4 levels and either hemoglobin levels (Panel A), or platelet numbers (Panel B). doi:10.1371/journal.pone.0044654.g002 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44654