ALBERT

Description: During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. The Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17-92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17-92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17-92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL. Funded by: FAPESP, CNPq and CAPES. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4835 The development and functionality of CD4+CD25hi regulatory T cells (Tregs) depends on stable FoxP3 expression, a central regulator of Treg differentiation. It is believed that this is accomplished by regulatory regions in the promoter and 3 evolutionarily conserved noncoding sequences, termed CNS1, CNS2 (or TSDR) and CNS3. The activation of TCR (with anti-CD2/3/28) in CD4+CD25− naïve T cells from PBMCs, in the presence of IL-2, TGF-β and atRA, induces the generation of Foxp3+ induced regulatory T cells (iTreg). While demethylation of 5mC residues in the CNS2 is associated with stable FoxP3 expression in nTregs, the epigenetic events involved in the regulation of FoxP3 in iTregs remains unexplored. Recently, the oxidation of 5-mC, originating hidroxymethylated 5-hmC residues, have been described as a key mechanism of active demethylation, with roles in biological processes, such as regulation of pluripotency and differentiation of hematopoietic stem cells. In contrast to PBMCs, in umbilical cord blood (UCB) T cells are mainly naïve making UCB an attractive source for the development of protocols for generation of iTregs. Here, we evaluated the iTregs generation from UCB naïve T cells. In addition, we compared the expression of FoxP3 on iTregs and on naturally occurring Tregs (nTregs) obtained from PBMCs. Also, we evaluated the methylation pattern of promoter and CNS2 and CNS3 in nTregs, fleshly isolated naïve T cells, activated naïve T cells (Teff), and iTregs. Finally, we evaluated the ability of iTregs, to suppress the proliferation of activated T cells, as compared to nTregs. For this, CD4+CD25-CD45RA+ naïve T cells were immunomagnetically isolated from UCB and activated with anti-human CD2/CD3/CD28 beads (1:2 beads:cell ratio) in the presence of IL-2 (50 U/ml) with (iTregs) or without (Teff) TGF-β (5 ng/ml) and atRA (100 nM) for 5 days. In parallel, PBMCs from 5 individuals were obtained for nTregs phenotypic characterization. CD4+ gated cells from iTregs and from PBMC were analyzed by flow cytometry for FoxP3 expression in the CD25+, CD25hi and CD25−population. nTregs (CD4+CD25+CD127−) were immunomagnetically isolated from PBMCs and CD4+CD25hi and CD4+CD25− populations were FACS-sorted from iTreg to observe the potential in regulate the proliferation of CD3+ T cells (CFSE staining). Finally, methylation pattern analysis of FoxP3 locus, including CNS2 and CNS3, were performed in naïve T cells, nTregs, iTreg and Teff. The mean percentage of FoxP3+ cells in CD4+CD25hi from iTreg was 98.5%, as compared to 82.4% in PBMCs. Interestingly, the percentage of FoxP3+ cells in total CD4+CD25+ was higher in cells from iTreg (97,3%) than on PBMCs (26,8%). Moreover, while the percentage of FoxP3+ cells in the CD4+CD25− population, was very low in PBMCs (2,8%), up to 55% of the cells derived from iTreg were FoxP3+. The immunossupression assay showed that, compared to activated CD3+ T cells cultured alone, nTregs (CD4+CD25+CD127−) decreased the proliferation of CD3+ T cells in 55%, while iTregs (CD4+CD25hi) decreased the proliferation in 46%. Interestingly, the CD4+CD25− population from iTreg (55% of FoxP3+ cells) also decreased the proliferation of CD3+ T cells, but to a lower extent (21%). Additionally, while naïve T cells and Teff presented low level of 5hmC in both segments evaluated of CNS2 (∼1%); upon in vitro induction, iTregs presented 5hmC levels comparable to that of nTregs (5–11% and 5% respectively), in line to FoxP3 expression. Furthermore, CNS3, which was found to be partially demethylated in naïve T cells and nTregs (45 and 50% respectively), presented even higher levels of demethylation upon activation in iTregs and nTregs (77 and 82% respectively). In summary, we show that functional Foxp3+CD4+CD25hi T cells can be generated in vitro from UCB naïve T cells. Additionally, our results indicate that active demethylation of CNS2 occurs in a TGF-β and atRA-dependent manner during iTregs generation. Moreover, the partial demethylation of CNS3 observed in naïve T cells and nTregs, and the increased demethylation promoted by activation (in Teff and iTreg), is consistent with the role of CNS3 as a pioneer element that initiates FoxP3 transcription. Our results contribute to the understanding of the epigenetic mechanisms underlying the differentiation of Tregs and may help in the development of protocols for the generation of functional iTregs for future therapeutic applications. Support: FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

Description: Mesenchymal stem cells (MSC) are multipotent precursors present in the bone marrow (BM), capable of differentiating into osteoblasts, adipocytes, chondrocytes and myoblasts. In addition to BM, MSC can be obtained from a variety of adult and fetal tissues. Additionally pericytes, that form a continuous subendothelial network of the vascular bed, share many properties with MSC, whereas the name stromal cell or fibroblast is often used interchangeably with MSC. How similar are MSCs obtained from different human tissues and what is their relationship with fibroblasts and pericytes? To answer these questions we have examined the expression of a set of 30 genes in 18 different human cell lines cultured in vitro: 7 MSC cultures from different sources (3 adult tissues: bone marrow, saphena vein, adipose tissue, and 4 fetal tissues: artery, liver, umbilical artery and umbilical vein), 2 of fibroblasts, 2 MSC cultures differentiated in vitro into adipocytes or osteoblasts, and one each of pericytes from the human retina, of bone marrow, endothelial cells, liver, brain, skeletal muscle and heart. Genes were selected on the basis of previous results of SAGE for MSC, CD34+ cells and fibroblasts. Cluster analysis using the software Cluster 3.0 UPGMA (average linkage) showed that all the MSC lines formed a very close cluster with pericytes and fibroblasts, separated from other normal human cells. Changes of the expression of highly expressed MSC genes were modest during in vitro differentiation of MSCs into osteoblasts or adipocytes, although the differentiation was accompanied by the increased expression of tissue specific genes. In addition, all the MSC lines had similar immunophenotypic markers and capacity for in vitro differentiation. Similarity of the gene expression profiles of MSC and fibrobasts were further confirmed by clustering of the 1,000 top expressed tags of SAGE libraries for 21 normal human tissues. Despite the similarity, expression of genes related to angiogenesis, especially CXCL6, was higher in MSC from umbilical vein, adult saphena vein and in pericytes than in BM-derived MSC. In conclusion, MSC that can be obtained from a variety of adult and fetal tissues have very similar biological markers, differentiation potential and gene expression profiles. Comparison of these characteristics also shows that MSC, fibroblasts and pericytes are very closely related, representing probably different functional states of the same cell. Finally, differentiaton of MSC into osteoblasts or adipocytes is marked by the expression of tissue specific genes, without dramatical changes of the overall profile.

Description: Background: Cancer testis antigens have become the most extensively studied antigen group in the field of tumor immunology. Aims: This study aims to analyze global expression of 14 CT (cancer/testis) antigens in MM to identify possible prognostic markers and therapeutic targets. Patients and Methods: The expression of MAGEA1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA10, MAGEA12, BAGE1, MAGEC1/CT7, GAGE family, LAGE-1, PRAME, NY-ESO-1, SPA17 and SSX1 was studied by RT-PCR in: 15 normal tissues, one pool of 10 normal bone marrow samples, three normal tonsils and bone marrow aspirates from six normal donors, three monoclonal gammophaties of undetermined significance (MGUS), five solitary plasmacytomas, 39 MM samples (95% advanced stage) and MM cell line U266. CodeLink Human UniSet I Bioarrays 10,000 genes was used for arrays analyses. Results: SPA17 was positive in all normal tissues and was excluded for further analyses. MAGEC1/CT7 was positive in bone marrow aspirates from one MGUS and in one plasmacytoma. U266 cell line was positive for all CT antigens, except SSX1. The frequencies of CT antigens expression in MM patients were: MAGEC1/CT7 = 30/39 (77%); LAGE-1 = 19/39 (49%); MAGEA3/6 = 16/39 (41%); MAGEA2 = 14/39 (36%); GAGE family = 13/39 (33%); NY-ESO-1 = 13/39 (33%); BAGE-1 = 12/39 (28%); MAGEA1 = 10/39 (26%); PRAME = 9/39 (23%); SSX-1 = 10/39 (26%); MAGEA12 = 8/39 (20.5%); MAGEA4 and MAGEA10 = 0%. Cox’s regression model showed that GAGE family positivity and number of positive CT antigens 〉 6 were independent prognostic factors when all patients were analyzed. However, MAGEC1/CT7 expression was the only independent prognostic factor when non-transplanted patients where analyzed. Three samples predominantly positive (〉 6) and three samples predominantly negative (0 or 1) for the 13 analyzed CT antigens were submitted to microarrays analyses. 147 genes were overexpressed in predominantly positive CT antigens samples. Conclusions: Based on our findings, MAGEC1/CT7, MAGEA3/6 and LAGE-1 seem good candidates for immunotherapy, since together they are overexpressed in 85% of our MM cases. Besides, GAGE family expression, number of CT antigens 〉 6 and MAGEC1/CT7 seem to have impact on MM prognosis. Also, the results of arrays analyses corroborate the hypothesis that MM can be separate in two groups: predominantly positive and predominantly negative for CT antigens, meaning that these antigens may have important role for MM biology.

Description: Mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation in vitro and in vivo. One of the ways by which MSCs may modulate immune responses is by the induction of CD4+CD25+FOXP3+ regulatory T cells (Treg), which suppress alloreactivity in vitro and prevent or attenuate GVHD in animal models. TGF-b (TGFB1 gene), one of the factors secreted by MSC, is known to induce the expression of FOXP3 and to drive the generation of Tregs from CD4+CD25− T cells. T cell activation is characterized by the expression of many surface molecules, and CD69 is one of the earliest markers, transiently expressed following activation. CD69 is selectively expressed at sites of chronic inflammation and recent in vivo and in vitro results indicate that this receptor may modulate the inflammatory response, by inducing TGF-b production. Despite the importance of secreted factors, cell to cell contacts promote increased lymphocyte immune modulation. Interestingly, TGF-b is known to induce the expression of b-IG-H3 (TGFBI gene), a secreted extracellular matrix adaptor protein, whose expression is higher on hematopoietic stem cells adherent to MSC. Given the potential role of CD69 as a regulatory molecule, we explored the effects of MSC on the expression of CD69, TGFB1, FOXP3 and TGFBI on co-cultured T-cells. Peripheral blood mononuclear cells (PBMC) from 6 individuals were co-activated by anti-CD3/CD28 beads and cultured either in the presence or in the absence of MSC (5:1) adhered to the bottom of culture wells. IL2 (20U/ml) was added in the 3rd day for full activation. Cultured PBMC were collected 1, 3 and 5 days after activation. Percentage of CD69+ cells and proliferation of activated lymphocytes (cell division tracking by CFSE) were evaluated by flow cytometry on gated CD3+ lymphocytes. Transcripts levels of TGFB1, TGFBI, FOXP3 and IL10 were determined by real time PCR and normalized using ACTB as an endogenous control. Relative expression levels were calculated in comparison with activated PBMC cultivated alone at the 5th day. Proliferation of lymphocytes co-cultured with MSC was significantly inhibited. As expected, activation of lymphocytes cultured alone was evidenced by the expression of CD69 in 5% of cells in the first day (mean percentage), followed by a decrease in the subsequent days (4% and 3%, respectively). Interestingly, lymphocytes co-cultured with MSC displayed a completely different pattern, with a similar initial activation (7%) which was followed by significant increase in the 3rd day (16%), maintained in the 5th day (14%). Furthermore, TGFBI and IL10 were both expressed at significantly higher levels on PBMC co-cultured with MSC, compared to PBMC alone, in all days evaluated. In addition, their transcript levels decreased faster on PBMC cultured alone. In the other hand, TGFB1 and FOXP3 levels in PBMC cultured alone or in the presence of MSC were similarly higher in the 1st day, but decreased till the 5th day, when their levels were slightly, but significantly higher on PBMC co-cultured with MSC, compared to PBMC cultured alone. We demonstrate for the first time that co-culture with MSC causes a sustained increase of the CD69 marker on CD3+ lymphocytes, which is accompanied by increased levels of TGFB1, TGFBI, FOXP3 and IL10 on total PBMC. Our results are in line with the proposed immunoregulatory role of CD69. In addition, higher TGFBI levels on PBMC may increase lymphocyte adherence to MSC, thus favoring immune modulation. This work was supported by FINEP, CNPq and FAPESP.

Description: Introduction: Absence of mutations in IgVH genes or higher number of ZAP70+ cells (as a surrogate marker) in chronic lymphocytic leukemia (CLL) B-cells defines a patient group with a poorer clinical course. These features relate to the role of BCR signalling in the proliferation and survival of CLL B-cells, and establish a link between these markers and the biology of CLL prognostic subgroups. The identification of additional players in this context may help to better understand the molecular basis of this disease and contribute to develop new therapeutic approaches. A search for genes potentially related to BCR signalling, when comparing mutated and unmutated CLL cases using serial analysis of gene expression, revealed a 4-fold increase of CD72 tags in unmutated samples, a specific B cell surface glycoprotein known to transmit both positive and negative signals in BCR signalling. Objective: This finding lead us to explore the potential role of CD72 on BCR signalling in distinct CLL prognostic subgroups, as defined by ZAP70 expression. Methods: Percentage of ZAP70+ and CD72+ cells were evaluated by flow cytometry on gated CD19+CD5+ cells in 25 CLL samples. Positive cases for ZAP70 and CD72 were defined using a cut-off of 35% and 40% positive cells, respectively. Real time PCR was used to quantify the expression levels of 3 genes related to proliferation and survival, RELB, Beta-Catenin (CTNNB1) and AKT1, on 16 CD19+ enriched (purity 〉 90%) CLL samples. Results: Samples were classified as 11 ZAP70+ and 14 ZAP70−. Median percentage of CD72+ cells in ZAP70+ was significantly higher than for ZAP70− cases (82% compared to 39%, respectively, P=0.0029). Furthermore, percentages of CD72 and ZAP70 were positively correlated (r=0.5930 and P=0.0009). Interestingly, ZAP70+ cases were restricted to CD72+ cases (n=11, CD72+ZAP70+ [+/+]), whereas six CD72+ cases were ZAP70− (ZAP70−CD72+ [−/+]). Finally, there were 8 cases CD72−ZAP70− [−/−]. No differences among these 3 groups were observed in regard to laboratory parameters (white blood cells, total lymphocytes, lymphocyte percentage, haemoglobin, haematocrit and platelet number). Despite the reduced number of samples analysed (6 +/+, 6 −/− and 4 −/+), transcripts for RELB (P

Description: Abstract 3065 Poster Board III-2 Deregulations of miRNA expression and function in B-cell acute lymphoblastic leukemia (B-ALL) have been associated with specific recurrent citogenetic abnormalities and clinical outcomes. In contrast, there is few data about miRNAs in T-cell acute lymphoblastic leukemia (T-ALL). We have determined the miRNA expression profile of 48 T-ALL patients' blasts and compared with normal mature T cells. We used the Taqman MicroRNA Assay Human Panel to screen 164 known mature miRNA sequences. Normal CD3+ cells were isolated from peripheral blood of four healthy subjects by immunomagnetic labeling. Total RNA was pooled and reverse transcribed with specific looped RT primers, and expression was evaluated by quantitative real-time PCR (RQ-PCR). Reactions were performed in duplicates and samples with a coefficient of variation greater than 5% were excluded. Furthermore, we considered as differentially expressed those miRNAs with fold change values higher than 10 or lower than 0.1. With this strategy we identified four miRNAs that were hyper-expressed (miR-181a, miR-181b, miR-213 and miR-29b) and three hypo-expressed (miR-150, miR-95, miR-338) in the leukemic pool. In order to confirm our findings, we then performed the analysis of miR-181a, miR-181b and miR-29b expression on 52 individual samples (48 T-ALL and 4 normal T cell samples) using RQ-PCR. Forty-five (93.7%) and 46 (95.8%) of the T-ALL samples presented expression levels of miR-29b and of miRs 181a/181b higher than the maximum detected in the normal samples. The analysis of the predicted targets for these three miRNAs was performed using miRNApath. MAPK signaling was the pathway with the highest number of target genes with 60 genes, of which MAP4K4, FOS, RAP1B, AKT3 and NLK were commonly targeted by all three miRNAs. As deregulation of the MAPK pathway in T-ALL has been previously described, we hypothesized that the hyper-expression of miR-29b, miR-181a and miR181b may be associated with this aberrant MAPK signaling. Disclosures No relevant conflicts of interest to declare.