sCTLA-4 Is Present in Sera from Pediatric ALL Figure 3. A correlation between sCTLA-4 and neoplastic B cells is apparent. The surface immunophenotype of PBMCs from B-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. The correlation between serum sCTLA-4 levels and the percentage of CD3 expressing cells (T lymphocytes), as well the correlation with CD38 (early and late stages of the B and T-cell maturation) and CD10 expression (a diagnostic marker of human acute lymphocytic leukemia) were no significant (Panels A, B and C). Otherwise, there was a significantly positive correlation between the level of sCTLA-4 and the frequency of HLA-DR and CD19 membrane expressing cells (Panels D and E). In addition, a positive correlation between the sCTLA-4 levels and the percentage of CD19+/CD1d+ cells (marker correlated with poor prognosis) was observed (Panel F). doi:10.1371/journal.pone.0044654.g003 membrane of T and B lymphocytes, but also on different tumor cells [8–12]. In addition, alternative splicing of mRNA encoding the CTLA- 4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. There is abundance of literature showing the presence of sCTLA-4 in autoimmune diseases [20–28], and also in allergic asthma [29] and allergy to hymenoptera venom [30]. The release of sCTLA-4 from acute lymphoblastic leukaemia cells may constitute a strategy for immune-surveillance escape. In fact, 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 [23,28,30,31]. Association of type-1 diabetes, Graves’ disease and CD with a point mutation in exon 1 of CTLA-4 (i.e. position 49A/G) leading to a Thr/Ala substitution in the leader peptide has been described in several different populations [16–22]. Moreover, single nucle- otide polymorphisms (SNPs) with a point mutation in exon 1 of CTLA-4 gene have been linked to susceptibility to several autoimmune disease [31]. In fact, higher levels of sCTLA-4 were observed in individuals with +49A/G and CT60A haplotype. Interestingly, although the data in this field of research remain controversial, it has been recently shown that the same SNPs in the CTLA-4 coding region are also associated with susceptibility to human cancer [36], such as cervical squamous cell carcinoma [37], colorectal cancer [38], hepatocellular carcinoma [39], gastric cancer [40] and acute myeloid leukemia [41]. These results suggest that expression of CTLA-4 would be associated with a decreased ability of the immune system to detect and eliminate tumor-associated antigens. Thus, the presence of a more tolerogenic CTLA-4 genotype, for instance related to a more frequent production of sCTLA-4, could represent a potential form of tumor immune evasion. In the present study we demonstrate the presence at significantly elevated levels of a circulating soluble form of CTLA-4 in 70% of B-ALL pediatric patients with active disease. In these series of experiments, data of expression of surface CTLA-4 are not available to us, however we presume that it could be similar to previous results published [11,12]: neoplastic cells from most B- CLLs were positive for all CTLA-4 epitopes at either the surface or the cytoplasm. The elevation of SCTLA4 observed in this series of patients appear to somehow conflict, by a theoretical point of view, with the observation of increased sCTLA-4 serum levels in patients with autoimmune diseases. However, it should be considered that, at least in an in vitro system, sCTLA-4 is able to suppress proliferation of T lympocytes (both in a MLR and in an antigen specific experimental conditions] [23,30]. Finally, it is known that B lymphocytes are able to produce CTLA-4 [11]. In this report, it is shown that sCTLA-4 transcript is expressed also by malignant B cells, at least in ALL paediatric patients. The correlation observed among CD1d expression [7] and the higher levels of sCTLA-4 in B-ALL patients suggest a possible role of this soluble molecule as a marker of progression to malignancy, or as a marker of severity of the neoplastic disease. Unfortunately, we cannot yet definitely support this hypothesis. It should be interesting to follow-up the serum expression of this molecule and, possibly, to correlate its levels to the decreasing of neoplastic cell numbers following therapy. Author Contributions Conceived and designed the experiments: GP RS DS. Performed the experiments: RS CT FF DS. Analyzed the data: RS MB GP DS. Contributed reagents/materials/analysis tools: ML GDR. Wrote the paper: GP MB DS. PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44654