ALBERT

Description: Recently, we have established a 5-parameter flow cytometry (FCM)-based score allowing for a precise prediction a deletion (5q) in therapy naïve MDS patients. The aim of this prospective study was to test, whether this FCM-based profiling is at least equal to the cytogenetics/FISH-based del(5q) detection for monitoring response to treatment. Overall, 229 FCM investigations were performed in 57 patients with MDS and del(5q) (IPSS-R very low/low: n=24, int: n119, high/very high n=22) including 39 patients with isolated del(5q) or one additional cytogenetic abnormality. The majority of analyses were performed in patients receiving lenalidomide or azacitidine (n=28 and n=21 patients), or in patients receiving chemotherapy and/or allogeneic transplantation (n=3), or growth factors (n=5). The del(5q)-FCM-score includes the following 5 parameters: myeloid progenitors (myPC 〉 2%) = 3 points, CD45 MFI ratio (lymphocytes: myPC ≤ 7.0) = 10, SSC ratio (granulocytes : lymphocytes 〈 6.0) = 2, CD71 (≤ 20%) on granulocytes = 1.5, and female gender = 1.5; a score ≥ 15.0 indicates the presence of del(5q). A standardized FCM (lyse-stain-wash) and cytogenetics/FISH were performed according to ELN guidelines at the TU of Dresden, VUMC of Amsterdam, UH of Bristol, and UH of Guadalajara. Before therapy, in 61 cytogenetic/FISH analyses in 40 MDS patients a del(5q) was detectable. In 53/561 (87%) FCM measurements, performed in parallel, a del(5q) could be predicted using the del(5q)-FCM-score. Monitoring response to treatment, a cytogenetic complete response (CCR) with the disappearance of del(5q) was accompanied by the disappearance of the typical del(5q)-FCM-profile in all of the 50 (19 patients) FCM measurements (sensitivity=100%). Otherwise, in patients without CCR the presence of del(5q) could be predicted by FCM in 96/118 measurements (specificity=81%). Aiming at a still higher specificity, in the following analyses we included only patients who presented with a typical del(5q)-FCM-score before therapy (33 patients; 54 measurements; median score=16.5) resulting in the above described sensitivity of 100% (32 measurements; median score=13.0) as well as a remarkably high specificity of 97% (59/61 measurements; median score=16.5) for response prediction. Next, we compared cytogenetics/FISH and del(5q)-FCM-score with further methods for response monitoring as cytomorphology, two well established diagnostic FCM scores: FCSS (flow cytometry scoring system; Wells et al. 2003) and the diagnostic score (Ogata et al. 2009), as well as the assessment of hematological improvement (HI). Thus, cytomorphology and FCSS, analyzing dyspoiesis of multiple cell lineages, showed a CR or disappearance of a MDS typical FCSS in less than the half of all investigations being in cytogenetic CR (sensitivity: 41% and 38%), but those two methods revealed a high specificity (98% and 97%). On the other side, the analysis of HI was high sensitive (91%) but not specific (49%). The use of the diagnostic Ogata score, considering almost only abnormalities of the myeloid progenitors, ended up with a slightly lower sensitivity (91%) and specificity (89%) as cytogenetics/FISH and the del(5q)-FCM-score. Finally, response monitoring using the del(5q) FCM-score allowed for a better separation of overall survival compared to cytogenetics/FISH (p=0.039 vs. p=0.1725). Flow cytometry-based detection of a del(5q)-specific immunophenotype is feasible and can serve as a rapid tool for response monitoring during treatment with disease-modifying drugs. At the moment, we test whether the better separation of the patients' survival curves applying the FCM-score holds true in a larger patient cohort to be analyzed within the ELNet iMDS-Flow working group. Disclosures Platzbecker: TEVA: Honoraria, Research Funding.

Description: Discriminating between cytopenia(s) due to myelodysplastic syndromes (MDS) and due to other (non-clonal) causes can be challenging, especially when dysplasia as assessed by cytomorphology is minimal, and when other MDS-specific features (such as ring sideroblasts or cytogenetic aberrations) are absent. Current recommendations for diagnosing MDS endorse flow cytometry (FC) as a valuable and informative diagnostic tool. Most FC protocols focus on analyzing the progenitor cells and the maturing myelomonocytic lineage. However, one of the most frequently observed symptoms in MDS is anemia, which is often associated with erythrodysplasia. Therefore, flow cytometric features of nucleated erythroid cells may complement current validated FC tools. The international, multicenter study within the European LeukemiaNet MDS-FC working group (ELNet-IMDS-Flow) reported herein focused on defining those erythroid parameters that enable discrimination of dyserythropoiesis associated with MDS from erythropoiesis in non-clonal cytopenias. This analysis was based on ELNet iMDS-flow guidelines for studying nucleated erythroid cells and their expression of CD117, CD71, CD36, CD235a and CD105. [Westers et al., Leukemia 2012] Nineteen centers (members of the ELNet-iMDS-flow) collected FC data on the erythroid lineage in mainly low grade MDS cases and pathological and normal controls. Bone marrow aspirates were taken after informed consent in accordance with the Declaration of Helsinki and local ethics committee approval. Data from a learning cohort were compared among MDS patients and controls; the results were validated in a separate cohort. The learning cohort comprised 685 cases and the validation cohort 352 cases; in total 191 normal controls, 443 pathological controls, and 403 MDS cases were included. The data revealed that the analysis of the expression pattern of CD71 and that of CD36 on erythroid cells in combination with the percentage of CD117+ erythroid progenitors provides the best discrimination between MDS and non-clonal cytopenia. The selected markers were used to build an FC erythroid dysplasia score which displayed a sensitivity of 59% (95% CI: 49-68%) and a specificity of 84% (95% CI: 77-89%). Of note, not every MDS case shows signs of erythrodysplasia by cytomorphology whereas some non-clonal conditions do. Evaluation of the results in the validation cohort displayed a specificity of 77% (95% CI: 29-50%) and a sensitivity of 39% (95% CI: 66-85%) for separating pathologic controls and MDS cases based on FC erythroid dysplasia. Most “FC-dysplastic” cases in the pathological control group involved reactive conditions and cytopenia associated with infections. The majority of the “FC-dysplastic” controls demonstrated abnormal CD71 expression, which argues against the application of single aberrancies to indicate dysplasia. Considering only the presence of multiple erythroid aberrancies as erythroid dysplasia by FC increased the specificity to 96% and 95% in the learning and validation cohorts, respectively; however, at the cost of a markedly reduced sensitivity (37% and 21%, respectively). Ultimately, analysis of the erythroid and myeloid lineages should be combined to increase both sensitivity and specificity. In summary, the defined erythroid marker combination may aid the diagnostic work-up of cytopenic cases with suspected MDS, particularly in combination with flow cytometric evaluation of the progenitor cells and maturing myelomonocytic lineage. This will be implemented in an upcoming multicenter data collection exercise within ELNet iMDS-flow. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Loosdrecht:Celgene: Consultancy.

Description: It is generally accepted that myelodysplastic syndromes (MDS) most often originate in a multipotent, myelorestricted progenitor population, although primary transformation may occur at the hematopoietic stem cell level. MDS can be classified into low risk and high risk with evolution to acute myeloid leukemia (AML) predominantly in the latter cases. In AML, survival of leukemia-initiating cells, often referred to as leukemic stem cells, after chemotherapy is thought to lead to minimal residual disease and relapse. Hence, in de novo AML a larger size of the stem cell compartment is predictive for poor survival. [Van Rhenen et al.,Clin Cancer Res 2005,11] The monoclonal antibody against the cell surface antigen C-type lectin-like molecule-1, CLL-1, together with lineage infidelity markers enables discrimination of normal and malignant stem cells. [Van Rhenen et al.,Blood 2007,110; Van Rhenen et al.,Leukemia 2007,21] It could be hypothesized that CLL-1 and aberrant marker expression on MDS stem cells together with size of the stem cell compartment may predict leukemic evolution. Therefore, stem cells, defined as CD45dimCD34+CD38−, were analyzed for expression of CLL-1 and aberrant lineage markers in bone marrow samples from 88 MDS patients classified by WHO as 16 RA w/o RS, 42 RCMD w/o RS, 3 MDS-U, 5 hypoplastic MDS, 6 MDS/MPD and CMML, 15 RAEB-1 and 2, 20 AML patients with a known MDS history and 26 healthy controls. Analysis of the CD34+CD38− frequency in all MDS patients and normal controls revealed no significant differences (median 0.0061% vs. 0.0074%, respectively), whereas the frequency of CD34+CD38− cells was 17-fold higher in high risk MDS (RAEB-1 and 2, median: 0.076%) as compared to low risk MDS (median: 0.0046%, p20) could be tested in 11/15 high risk RAEB-1 and 2 cases and in 16/73 of the remaining low risk MDS cases. In these cases, median CLL-1 expression on the CD34+CD38− cells was 1.6% (range 0–50) in low risk and 2.0% (range 0–27) in high risk MDS. Median CLL-1 expression on stem cells was 0.0% (range 0–4.7) in normal controls. Nevertheless, expression of lineage infidelity markers, such as CD5, CD7 and CD56, on CD34+CD38− stem cells in MDS strongly suggests that a considerable part of these stem cells is malignant (median 35% in 7/16 patients tested). Our data show that CLL-1 is virtually absent on stem cells in MDS. Remarkably, median CLL-1 expression on stem cells in AML cases that evolved from MDS (7%, range 0–53, n=9) was manifold lower than in de novo AML (median 45% when excluding non de novo AML [Van Rhenen et al.,Blood 2007,110], p=0.034). Detailed analysis of CLL-1 expression in AML had already revealed that CLL-1 expression increases with differentiation (CD34− 〉 CD34+CD38− 〉 CD34+CD38+). [Bakker et al.,Cancer Res 2004,64;Van Rhenen et al.,Blood 2007,110] Thus, our data suggest that the CD34+CD38− cells in high risk MDS and AML with antecedent MDS are more immature than in most de novo AML, which might explain poor prognosis of AML cases with MDS history. To conclude, our data indicate that CLL-1 is a specific marker of de novo AML, while CLL-1-negative AML may have been evolved from a MDS pre-phase that is further characterized by an increasing size of the stem cell compartment upon progression towards AML.

Description: Purpose: This randomized phase II study (HOVON89) in patients with low/int-1 risk MDS refractory or unlikely to respond to erythropoietin and granulocyte-colony stimulating factor (EPO/G-CSF) assessed efficacy and safety of lenalidomide without (Arm A) or with EPO+/-G-CSF (Arm B) in case of no erythroid response after 4 cycles. Patients and methods: In total 200 patients were randomly 1:1 assigned to either Arm A or Arm B. All patients were treated with lenalidomide (10 mg/day/day 1-21) for a minimum of 6 months in arm A and 12 months in arm B or until loss of response or disease progression. Patients in arm B without hematological improvement-erythroid (HI-E) after 4 cycles received EPO (30,000 IU/wk). In those patients who did not show HI-E after 6 months, EPO was increased to 60,000 IE/wk. G-CSF (3x 300-480 µg/wk) was added if no HI-E was reached at 8 month. The current pre-final evaluation was based on the first180 patients and included 85% non-del5q MDS and15% patients with isolated del5q. The median age was 71years (range 38-89). No differences were observed between both arms regarding sex (55% male), WHO PS, WHO diagnostic subgroup and IPSS, baseline Hb, WBC, platelets, endogenous erythropoietin level, pretreatment with EPO+/-G-CSF (67% of the patients were pretreated) and pre-study transfusions. Patients had received a median of 13 (range 0-72) units of RBC and 4 (range 0-13) within 8 weeks for prior study entry. Results: Adverse events were consistent with the known safety profile of lenalidomide/EPO/G-CSF. HI-E according to IWG criteria was achieved in 38% and 41% of the patients for arm A and B, respectively (p = 0.46). HI-E was significantly lower in non-del5q versus del5q patients (33% vs 78%, respectively). Time-to-HI-E was 3.1 months (median; range 1.6-12.3) for both arms with a median duration of 10 months (range 1 - 76). The median PFS was 14.4 vs 15.4 months in arms A and B (p=0.43). OS was 51.1 and 37.7 months for arm A and B (p=0.09). At 2 years 17% of patients had progressed to AML (no differences between arms). The median FU of patients still alive is 31 months. PFS and OS was significantly longer in those who achieved HI-E, (median 13 vs 19 months, p=0.02 for PFS and median 31 vs 63 months for OS, p

Description: Myelodysplastic syndromes (MDS) are a group of clonal disorders of the bone marrow characterized by peripheral cytopenias. Standard treatment in low- and intermediate-I–risk MDS is supportive therapy consisting of regular transfusions and growth factors, that is, erythropoietin (Epo) and granulocyte-colony-stimulating factor (G-CSF). Because flow cytometric analysis of MDS bone marrow samples can identify clinically relevant subgroups regarding transfusion dependency and disease progression, we addressed the question whether flow cytometry (FCM) was instrumental in predicting response. In 46 patients with low- and intermediate-I–risk MDS that were treated with Epo/G-CSF, low Epo level and low transfusion need were associated with response to Epo/G-CSF. Interestingly, aberrant phenotype of myeloblasts identified nonresponders among patients with the greatest response probability according to the predictive model of Hellström-Lindberg et al. Moreover, aberrant FCM of myeloblasts acted as a significant biomarker for treatment failure in multivariate analysis. A new predictive model based on the basis FCM combined with previously validated Epo levels is proposed defining 3 subgroups with 94%, 17%, and 11% response probability. In conclusion, FCM may add significantly to well-known predictive parameters in selecting MDS patients eligible for Epo/G-CSF treatment. This is of relevance regarding prevention of treatment failure.

Description: Chronic myeloid leukemia (CML) is characterized by the presence of the Philidelphia chromosome which encodes for the fusion protein bcr-abl, a constitutively active tyrosine kinase. Imatinib mesylate (Gleevec, Novartis) is an inhibitor of the kinase activity of bcr-abl and therefore a treatment modality for CML. High rates of cytogenetic responses are found, although in many patients minimal residual disease (MRD) is detected by molecular techniques. Immunotherapy using leukemic dendritic cell (DC) based vaccination might be a feasible approach to eradicate MRD. In a pilot study on CML-DC-based vaccination in advanced CML DTH responses towards CML cells were achieved. (Ossenkoppele et al., Leukemia, 2003, 17, 1424). However, little is known about the effects of Imatinib mesylate on bcr-abl positive CML-DC during vaccination. In this study we investigated effects of Imatinib on culture of CML-DC, their viability, T cell stimulating and migratory capacity in vitro in 17 patients. Imatinib hardly affected the immunophenotypical profile of CML-DC. Expression of CD40, CD80, CD83, HLA-DR and chemokine receptors (CCR5, CCR7, CXCR4) remained stable during overnight exposure. Only the fluorescence intensity of CD86 was significantly higher (1–10μM, p=0.043) and that of CD54 significantly decreased in the highest Imatinib concentration tested (p=0.043). Exposure of Imatinib-free cultured CML-DC to Imatinib significantly reduced the recovery of viable cells and hence the DC yield in a dose-dependent manner (5.3–26% reduction after 20h. for 1–10μM, p=0.043). Migration of mature CML-DC towards CCL19 (MIP3β) was significantly improved in the presence of 1 and 5μM Imatinib which implies increased ability to reach the lymph nodes (p=0.043, mean 27, 36 and 34% migration for 0, 1 and 5μM Imatinib, respectively). Imatinib did not affect T cell stimulating capacity of CML-DC, except at concentrations higher than 3μM (p=0.028). No significant changes were observed for Imatinib exposure of CD34+ DC isolated from normal subjects. In conclusion, Imatinib at low concentrations maintains the immunogenecity of CML-DC. Since Imatinib plasma levels in CML patients are 1.5 and 3.0μM upon 400 and 800mg daily, respectively, our data justify continuation of Imatinib treatment during CML-DC-based vaccination regimens.

Description: Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p

Description: Abstract 3283 Whereas in 80% of the patients suffering from acute myeloid leukemia (AML) complete remission is achieved, the 5-year survival rate is only 30% - 40% due to relapse of the disease. In order to increase overall survival of AML patients, novel or additional therapies are required. Due to their ability to ingest, process and subsequently present antigens to (naïve) lymphocytes in an immunogenic context, dendritic cells (DC) can initiate antigen-specific immunity. Therefore, DC have become a major subject of interest as mediators of anti-tumor immunity in a minimal residual disease setting in AML. Since only few AML-associated antigens (LAA) have been characterized to date, the use of apoptotic tumor blast cells as a source of LAA could be instrumental, due to the presence of both known and unknown LAA. However, the immunogenicity of apoptotic cells remains questionable; e.g. apoptotic tumor cells may not activate DC adequately, but rather impair their function. Previously, it was shown in mice that apoptotic blebs, which detach from the main apoptotic cell during apoptosis, induce DC maturation, whereas the apoptotic cell remnant (ACR) does not. Here, we analyzed monocyte-derived DC (MoDC) function in vitro, after co-culture with either human AML-derived ACR or blebs. Apoptosis of CFSE-labeled HL60 AML cells was induced by Mitoxantrone and ACR and blebs were isolated by differential centrifugation. The apoptotic fractions were subsequently added to allogeneic MoDC without the addition of a maturation stimulus (immature), or with the addition of the cytokine cocktail IL-1β, IL-6, PGE2, and TNFα, supplemented with TLR 7/8 ligand R848, 1 hour after initiation of the co-culture (maturing). Blebs were preferentially ingested compared to ACR by both immature and maturing MoDC (75% vs. 45%, and 75% vs. 40% CFSE positive MoDC (p =〈 0.0001), respectively). Analysis of the capacity of mature MoDC to migrate towards the lymph node-homing chemokine CCL19, in a transwell migration system, demonstrated that MoDC migration was diminished by approximately 50% compared to unloaded mature MoDC, irrespective of the ingestion of blebs or ACR. However, the percentage of CFSE-positive MoDC which were able to migrate towards CCL19 was significantly higher when MoDC had ingested blebs, compared to ACR (18% vs. 7% (p = 0.05), respectively). Although the ingestion of blebs or ACR does not lead to significant changes in phenotypic maturation, CD40 ligation of immature MoDC that had taken up blebs, resulted in the secretion of twice the amount of the pro-inflammatory cytokines IL-6 and IL-12p70 as compared to MoDC that had ingested ACR (p = 0.05). In conclusion, we show that blebs derived from human AML cells are preferentially ingested by both immature and maturing MoDC and that a higher percentage of these MoDC are able to migrate towards CCL19, compared to MoDC that have ingested ACR. Furthermore, compared to ACR, ingestion of blebs by MoDC leads to an increased production of the pro-inflammatory cytokines IL-6 and IL-12. Additional functional analyses are underway to determine whether ingestion of blebs also leads to a more efficient antigen presentation and subsequent anti-leukemic T cell activation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1860 Immune editing is recognized as being important in the pathogenesis of myelodysplastic syndrome (MDS). In low risk MDS, inappropriate immune reactions are thought to contribute to the initiation and progression of the disease. IL-17 producing CD4+ T-helper cells have been associated with various autoimmune diseases and are found in increased frequencies in low-risk MDS. These increased Th17 frequencies are accompanied by a reduced FoxP3+ T-regulatory (Treg) frequency. On the other hand, in high-risk MDS, the elevated number of bone marrow blasts was associated with increased numbers of Treg. These increased Treg may inhibit immune responses directed against the dysplastic blasts and as such permit progression of the disease. The demethylating agents ‘5-aza-2’-deoxycytidine and ‘5-azacitidine (Aza) have shown significant clinical benefits in the treatment of MDS. Although aberrant DNA methylation patterns appear to play a role in the pathogenesis of MDS, clinical responses to DNA demethylating agents could not be related to changes in DNA methylation patterns. Cytokine production and FoxP3 expression are also regulated by epigenetic mechanisms, and FoxP3 expression is increased upon in vitro treatment with Aza. Since cytokines and FoxP3+ Treg may play a role in the pathogenesis of MDS, we set out to investigate the effects of Aza (Vidaza®) treatment on CD4+ T-helper subset frequencies in high-risk MDS patients. Nine patients treated with 75 mg/m2/day subcutaneously for 7 consecutive days in a 28 day cycle were included in this study. Peripheral blood samples were drawn before, 15 days after start of treatment and at the end of the third cycle. There was no change in absolute or relative CD3+ and CD3+CD4+ T-cell numbers during treatment. The proportion of Treg was slightly but not significantly increased (paired student's T-test, p=0.08) on day 15 (11.9 % of CD4+) compared to levels before treatment (9.6 % of CD4+) while numbers dropped to pretreatment levels after the third cycle(9.6 % of CD4+). Cytokine producing cells were enumerated after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin in the presence of Brefeldin A. No significant changes in IFNγ (23.8%, 22.5% and 17.9% of CD4+ cells pretreatment, 15d post treatment and after the third cycle respectively) TNFα (38.3, 35.9 and 39.3% of CD4+ cells) or IL-4 (0.6, 1.0 and 0.6% of CD4+ cells) producing cells were observed. However, the proportion of IL-17 producing cells progressively decreased during treatment (1.1, 0.7 and 0.6% of CD4+ cells; ANOVA, p=0.006). In conclusion, Aza may modulate the immune response and in particular reduce IL-17 responses. This effect of Aza may provide a rationale for expanding the group of patients eligible for Aza treatment with low risk MDS patients, because low risk MDS has been associated with elevated, potential pathogenic, Th17 cells. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 4026 Aberrant DNA methylation and other epigenetic changes play a role in the development of myelodysplastic syndromes (MDS). Epigenetic drugs such as DNA methyltransferase inhibitors are therefore increasingly employed in MDS treatment regimens. Recent studies show that gene methylation processes also regulate T-cell function. Here we analyzed the in vitro effects of the DNA methyltransferase inhibitor ‘5-azacitidine (Aza) on CD4+ T-cell activation. We confirmed the previously described inhibition of proliferation and increased expression of FoxP3, the regulatory T-cell (Treg) marker, by anti-CD3 stimulated T-cells in the presence of 1mM Aza. Here we have sorted CD4+ T-cells isolated form healthy donor peripheral blood into CD25neg resting, CD25dim recently activated and CD25hi Treg cells. Aza facilitated the induction of CD25hiFoxP3+ T-cells from CD25neg (4.7% of vehicle treated cells versus 17.3% of Aza treated cells p=0.0007, n=9) and to a lesser extend from CD25dim (1.2% versus 8.6%, p=0.0015, n=7) CD4+ T-cells, while Aza had no effect on FoxP3 expression in CD25hi sorted cells, FoxP3 expression remained high. In addition, cytokine producing T-cells were enumerated after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin in the presence of Brefeldin A. Aza treatment increased the number of IFNγ producing cells in the total CD4+ population (19.1% versus 40.8%; p