Milano, Italy) according to the manufacturer’s recommendations. The single-stranded cDNA was synthesized using 1 mg of total RNA by reverse transcription by incubating it at 42uC for 1 h with 20 pmol of oligo(dT) (Roche Molecular Biochemicals, Basel, Switzerland), 500 mM dNTPs (Roche Molecular Biochemicals), 30 U of RNase inhibitor (5 Prime 3 Prime, Boulder, CO), and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD) in a total volume of 20 microL. PCR reactions were performed with cDNA and primers designed to amplify the entire coding sequence of CTLA-4:59- ATGGCTTGCCTTGGATTTCAGCGGCACAAGG-39 and 59-TCAATTGATGGGAATAAAATAAGGCTGAAATTGC-39 (predicted size: 672 bp). PCR reaction was as follows: 94uC for 5 min, 30 cycles 94uC for 1 min, 60uC for 1 min and 72uC for 1 min followed by a final extension at 72uC for 5 min. The amplified fragments were separated on 1% agarose gel and visualized by ethidium bromide. As controls, the G3PDH primers were used: 59-ACATCGCTCAGAACACCTATGG-39, and 59- GGGTCTACATGGCAACTGTGAG-39. Statistical Analysis Statistical analysis was performed using GraphPad Prism software 4?0 (GraphPad Software Inc., CA, USA). Because of the skewed nature of some of the data non-parametric tests were used throughout. Associations were calculated using Spearman rank correlations. Differences between unpaired groups were evaluated by the Mann–Whitney U-test. P values ,0.05 were considered significant. Results Plasma sCTLA-4 Levels in Patients with ALL In this study, the presence of circulating sCTLA-4 was evaluated in the sera of all normal donors and patients enrolled in this study (Fig. 1, panel A). Plasma sCTLA-4 levels in 54 normal blood donors ranged from 0.00 ng/ml to 36.06 ng/ml (mean 6 standard deviation, 1.071 ng/ml 65.318 ng/ml). A wider range of levels was observed in paediatric patients with ALL. Among patients with ALL, sCTLA-4 levels ranged from 0.02 ng/ml to 870.8 ng/ml (132.0 ng/ml 6208.7 ng/ml). The majority of patients with B-ALL (42%) had sCTLA-4 levels higher the range observed in normal donors, and, overall, there was a significant difference between the levels detected in normal donors and the levels in either patients with B-ALL (P,0.0001). In addition, we confirmed the presence of sCTLA-4 mRNA in PBMCs isolated from ALL patients. As shown in Fig. 1, panel C, a mRNA form encoding for sCTLA-4 was evident, suggesting that the molecule we measured in ELISA tests was a result of an alternative splicing. Finally, Western-blot of human serum was probed with specific mAb anti-CTLA-4. As shown in Fig. 1, panel D, three different samples resulted positive by ELISA analyses (BBG14, BBG33 and BBG56), were characterized by a band of approximately 23 kDa, which is consistent with previous reports [23,29,31]. Association of sCTLA-4 Levels with Immunophenotype Classification The ALL patients were classified in relation to the membrane and cytoplasmic immunophenotype of peripheral blood mononu- clear 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 statistical difference between the three ALL groups and the control samples (Fig. 1, panel B). The results obtained evaluating serum sCTLA-4 concentrations in the 3 subgroups sCTLA-4 Is Present in Sera from Pediatric ALL demonstrated a different ability of the 3 groups in producing sCTLA-4. As shown in Fig. 1B, serum levels of sCTLA-4 ranged from 0.0 ng/ml to 669.7 ng/ml (97.166169.3 ng/ml) in the common ALL group, from 0.0 to 236.0 (86.176101.1 ng/ml) in pre-B ALL, and from 0.0 to 870.8 (271.46350.3 ng/ml) in pro-B ALL. As resulted from statistical analyses, no correlation was evidenced among the three ALL groups. Otherwise, all of the three groups of patients were significantly different from the donor group (p,0.005). Association of Serum sCTLA-4 Levels with HLA-DR, CD19, CD10, CD3, and CD38 Expression on PBMCs in ALL Patients There was no significant correlation between sCTLA-4 levels and either age (data no shown), hemoglobin levels, or platelet numbers in either patient group (Fig. 2, panels A and B). However, the levels of sCTLA-4 in patients with ALL were correlated with leukocyte counts (Fig. 2, panel C). PBMCs of patients with ALL were stained with different mAb and the percentage of positive cells was compared with the level of sCTLA-4 measured in the corresponding serum. Surprisingly, the correlation between serum sCTLA-4 levels and the percentage of CD3 expressing cells was no significant (r = 0.1, p = 0.54) (Fig. 3, panel A). In fact it is described that T lymphocytes are able to produce the spliced soluble form of CTLA-4. On the other hand, there was no significant correlation also when CD38 expression (a receptor preferentially expressed at both early and late stages of the B and T-cell maturation) and CD10 expression (one of the most significant cell surface marker in the diagnostic of human acute lymphocytic leukemia) were compared to the level of sCTLA-4 (r = 0.0647, p = 0.73, and r = 0.1022, p = 0.53 respec- tively) (Fig. 3, panels B and C respectively). Otherwise, there was a significantly positive correlation between the level of sCTLA-4 and the frequency of HLA-DR and CD19 membrane expressing cells (r = 0.6075, p,0.001 and r = 0.7062 p,0.001, respectively) (Fig. 3, panels D and E). Plasma sCTLA-4 Levels and CD1d We have previously observed that CD1d is expressed in ALL subsets with poor prognosis and that CD1d+ blasts are able to interact with NKT cells via CD1d [7]. For these reasons we analyzed the correlation between these markers. The amount of sCTLA-4 levels in patients with ALL were compared to the percentage of CD19+/CD1d+ cells. As shown in Fig. 3 panel F, there is a positive correlation between these two markers (r = 0.824, p,0.001). Discussion CTLA-4 was first described as an important negative regulator of T-cell activation and proliferation, interacting with B7 molecules on antigen-presenting cells. It is therefore a plausible candidate as a susceptibility gene in diseases with T-cell mediated pathogenesis. Expression and function of CTLA-4 inhibitory receptor have been investigated mainly in T lymphocytes [13–17]. More recently, the expression of membrane CTLA-4 is demon- strated also on cells different from T lymphocytes, such as B cells, monocytes or dendritic cells [9,10] in correlation with the activation state of the cell. Of note, the expression of CTLA-4 at the cellular membrane level is often controversial, mainly due to the low levels of expression combined with poor capability of commercial mAb anti-CTLA-4. Otherwise, the expression of this inhibitory receptor was previously demonstrated not only on the PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44654