as B lymphocytes [9], monocytes and monocyte-derived dendritic cells [10]. In addition, the surface expression of CTLA-4 in neoplastic cell populations from different leukemia subtypes was demonstrated by Pistillo et al. [11]. These authors suggest a possibile physiopathological role of this receptor: the expression of CTLA-4 in leukemias (as well as in a number of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma) might indicate the ability to interact with the CD80/CD86 ligands on antigen-presenting cells, and to transduce a relevant immunosuppressive signal [11,12]. A native soluble form of CTLA-4 (sCTLA-4), deriving from lack of transmembrane sequence, has been described [22]. High concentration of sCTLA-4 were observed in sera of patients with autoimmune thyroid diseases [22,23], as well as in patients with other autoimmune diseases, such as type-1 diabetes [24], diffuse cutaneous systemic sclerosis [25], systemic lupus erythematosus [26], myasthenia gravis [27] and celiac disease [28]. In addition, raised plasma levels of sCTLA-4 were observed in patients with allergic asthma [29] and allergy to hymenoptera venom, but not in allergic rhinitis [30]. Soluble CTLA-4 may have important immunoregulatory functions. The effect of sCTLA-4 binding to CD80/CD86 molecules might depend on the activation state of the cells involved, interfering with T cell costimulation and with T cell responses. Thus, sCTLA4 might act indirectly both as inhibitor or as enhancer of the immune response [22,29,31]. In addition, elevated levels of soluble CD80 [32,33], and CD86 in some leukemia patients have been demonstrated, and elevated sCD86 levels have been reported as a marker of poor prognosis in acute myeloid leukemia [34,35]. However, the mechanisms for the production of sCTLA-4, sCD28, sCD80 and sCD86, and their correlation with hemato- logical malignancy activity have not been well elucidated. As previously mentioned, leukemic B-ALL cells have been proved to be able to express CTLA-4 on their surface [11]. Pistillo et al. evidentiated an apparent different expression of CTLA-4 on the membrane of leukemic B cells depending on the mAb utilized. Flow cytometric analysis carried out with a panel of anti–CTLA-4 human single chain fragment of variable domain antibodies, all containing leukemic cells at high frequency (greater than 75%) in peripheral blood or bone marrow aspirate samples. On the other hand, when commercially available mAb were used, the percent- age of CTLA-4 expression on leukemic B cell surface decrease to 20% (vs. 90% in cytoplasm) [11]. The release of a soluble form of CTLA-4 provides a potentially powerful means by which APC and/or malignant cells may modulate the co-stimulatory signals normally delivered by the membrane form of CTLA-4. In an attempt to further evaluate the immunopathological roles of T-cell costimulatory molecules and to search for potential surrogate markers in ALL, we investigated the plasma concentration of sCTLA-4 in ALL pediatric patients, compared with normal healthy subjects and their relationship with specific membrane-expressed markers. Materials and Methods Patients The study population included 80 children with ALL of B-cell precursor origin. ALL cases were part of a bank of cryo-preserved samples collected between 1995 and 2008. Samples were analyzed on the basis of the availability of frozen sera and, therefore, are not representative of a consecutive series of patients. Clinical data included age, sex, white blood cell count at diagnosis, assessment sCTLA-4 Is Present in Sera from Pediatric ALL of hepatomegaly, splenomegaly, and BM and central nervous system (CNS) involvement [7]. Written informed consent was obtained from parents, all samples were obtained following the ethical guidelines of the most recent Declaration of Helsinki (Edinburg 2000) and approved by the Ethics Committee of Ospedale Bambino Gesu` , Italy. Diagnosis and categorization resulted from morphologic analysis of BM aspirates, immunophe- notyping, and cytogenetic and molecular genetic analysis of PB and/or BM samples [7]. Immunophenotyping included analysis with a standard panel of monoclonal antibodies (mAb) to CD19, CD10, CD34, cytoplasmic and surface Ig. Cases were classified as pro-B ALL (n = 8), common ALL (n = 54), pre-B ALL (n = 18). No mature B-ALL cases were observed in this series. Classification of ALL according to age allowed identification of six infant ALL (,1 year) and 74 non-infant ALL cases. Fourty-five age matched normal serum samples were obtained (age range from 2 to 6), after parents’ written informed consent, from children undergoing diagnostic laboratory procedures, which did not reveal hematological abnormalities neither autoimmune disorders. In addition, it is well known that sCTLA-4 levels are virtually undetectable in normal controls [22,23,28–30]. Other- wise, this report shows for the first a description of normal values in a pediatric group. Antibodies and Immunostaining For ALL cell staining the following mAb were used: CD1d- phycoerythrin (PE), CD10-PE, CD10-fluorescein isothiocyanate (FITC), CD38-FITC, CD19-peridinin chlorophyll protein-cyanine dye 5.5. (PerCP-Cy5.5), CD34-FITC, CD3-FITC, CD19-FITC, CD80-FITC, CD86-FITC, CD11a-FITC and fluorochrome- conjugated isotype-matched control mAbs (Becton Dickinson Italia, Milan, Italy). B-CLL cells (26105) were incubated for 20 min with mAb at 4uC. Cells were then washed and resuspended in 300 mixro liters cold PBS before flow cytometric analysis (FACScalibur, Becton Dickinson), using the CellQuest software (Becton Dickinson). ELISA Specific ELISA kits were used for measuring serum sCTLA-4 levels (Bender), according to the manufacturer’s protocol. Each sample was diluted 1:10 and tested in triplicate. Deviation between triplicates was ,10% for any reported value. The lowest sensitivity threshold is 0.1 pg/ml. The analytical response was linear approximately between 0.162 and 1.200 of absorbance values (corresponding to 0.1– 50 ng/ml) as assessed by serial dilution test using a strongly positive serum (data not shown). Western blotting. Western blotting was used to detect serum sCTLA-4. Proteins were separated by 10–20% gradient PAGE in a discontinuous buffer system on a Mini-Protean system (Bio-Rad, Segrate, Milano, Italy). The separated components were electro- blotted onto PVDF membranes. The blots were washed with 0.15 M NaCl, 0.05 M Tris (TBS), pH 7.5, with 0.3% Tween 20 and reacted with a 1:100 dilution of the anti-CTLA-4 mAb (clone 14D3, IgG2a, eBiosciences, San Diego, CA, USA) for 1 h at room temperature, washed, and then reacted with reporter antibody (HRP-conjugated anti-mouse IgG) for 1 h. The blots were then developed by the use of a commercially available chemilumine- sence detection kit (BMB, Indianapolis, IN) according to the manufacturers instructions. mRNA analysis by RT-PCR. B lymphocytes were separated from peripheral blood mononuclear cells, and purified in vitro using the MACS CD20 isolation kit (Miltenyi Biotech, Auburn, CA USA). Total RNA was extracted using RNAse Mini kit (Qiagen, PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44654