The immune system plays a role in the pathogenesis of myelodysplastic syndrome (MDS) but its precise contribution to disease development and control is not fully clarified. T cell activation could ...reflect undesired autoimmune reactions against normal hematopoietic precursor cells as well as effective immune-surveillance against dysplastic clones. We have investigated lymphocyte subsets and activation markers of 42 low risk and intermediate-1 risk MDS patients and compared them to those of intermediate-2 risk, high risk MDS patients, and healthy donors. In low and intermediate-1 risk MDS patients, we have found an activated state of lymphocytes, determined by increased percentages of effector T cells with cytotoxic profile, increased number of clonal expansions, increased frequencies of Wilms' Tumor 1 (WT1) specific lymphocytes and decreased regulatory T cell activation, as compared to healthy donors. Moreover, we demonstrate autologous T cell mediated cytotoxicity against aberrant hematopoietic precursor cells. These findings provide evidence for the existence of immune-surveillance in the pathogenesis of low and intermediate-1 risk MDS patients. Our data are important for adequate evaluation of the role of immune-modulatory drugs in the future and justify the reconsideration of the role of immune-suppressing therapy for low and intermediate-1 risk MDS patients.
Signal regulatory protein-α (SIRPα) is a transmembrane receptor selectively expressed on myeloid and neuronal cells. The broadly expressed cell surface CD47 molecule acts as a major extracellular ...ligand. The SIRPα cytoplasmic tail contains ITIM motifs that upon CD47 binding mediate the recruitment and activation of the cytosolic tyrosine phosphatases SHP-1 and SHP-2. SIRPα signaling negatively regulates many signaling pathways leading to reduced tumor migration, survival and cell transformation. Interestingly, a reduced SIRPα expression has been found in >70% of acute myeloid leukemia (AML) patients, including APL (FAB M3). Since SIRPα-derived signals are involved in the inhibition of myeloid cell growth, reduced SIRPα expression might contribute the proliferative advantage of leukemic cells. We hypothesized that the reduced expression of SIRPα in APL might be the result of aberrant promoter hypermethylation, which could have potential relevance for innovative therapy. The aim of this study was therefore to investigate protein expression of SIRPα in pediatric APL and evaluate the methylation status of the PTPNS1 gene (encoding SIRPα). Immunocytochemical staining was performed on cryo-preserved cytospins using a commercially available antibody (Ab2971, Abcam). The leukemic cell lines THP-1, Kasumi-1 were used as positive controls. Negative controls were performed by omitting the first antibody and the inclusion of the ALL cell line CCRF-CEM. SIRPα protein expression was evaluated by two independent investigators by scoring the intensity of the staining: negative (−), low (+/−) and positive (+). Methylation specific PCR (MSP) was set up in order to study the methylation pattern of the PTPNS1 promoter in the APL cell line NB4 and 10 APL patient samples (containing >80% blasts). Two primer sets (named MSP1 and MSP2, respectively) were used which recognized different regions of the PTPNS1 promoter. SIRPα expression in THP-1 cells was high, while expression in Kasumi-1 cells was low. The lymphoblastic cell line CCRF-CEM did not express SIRPα. These findings were in agreement with results obtained by Western blotting using the same antibody. The APL cell line NB4 stained positive for SIRPα. In the APL patients, SIRPα expression was low (n=7) or absent (n=2) in 9/10 (90%) samples. Expression was high in 1 sample (10%). MSP revealed methylation in one part of the PTPNS1 promoter region. The MSP1 primer set showed both methylated and unmethylated bands in the NB4 cell line as well as in all 10 APL patient samples. No methylation was detected using the MSP2 primer set. In conclusion, we observed no to low expression of SIRPα in the majority of APL patient samples (90%). In addition, we observed methylation of the PTPNS1 promoter in APL patient samples, indicating that aberrant promoter methylation might be involved in the reduced SIRPα expression in pediatric APL. This could have important implications for future alternative treatment options for APL patients directed towards demethylation of the gene and re-expression of this inhibitory signaling protein.
The WHO classification of myeloid disorders contribute to a more refined classification and prognostication of myelodysplastic syndromes (MDS). The considerable differences in clinical behaviour of ...pure refractory anemia (RA) versus refractory cytopenia with multilineage dysplasia (RCMD) with or without ringsideroblasts are of importance in the management of patients with MDS. Flow cytometry may add additional diagnostic criteria to adequately discriminate RA from RCMD (+/− ringsideroblasts; (RS)) and may contribute in identifying Idiopathic Cytopenia of Undetermined Significance. We developed a 4-colour flow-cytometric procedure that comprises all differentiation stages of granulocytic, monocytic and erythroid lineages, instrumental for the recognition of various subpopulations within all three lineages in normal bone marrow samples. In 43 evaluable patients with MDS (RA, RARS, RCMD, RCMD-RS, MDS-U, RAEB-1 and 2), aberrant expression of differentiation antigens were demonstrated in 1 or more lineages. Flow-cytometry identified aberrancies in granulopoiesis and monocytopoiesis in 93% and 74% of the cases, respectively. In the majority of cases abnormal relations between CD13, CD16, CD11b, CD15 and HLA-DR were prominent in the granulopoiesis. In 34% of the cases a striking monocytopenia was detected, whereas in 59% abnormal surface expression of CD14, CD36 and CD33 indicating aberrant differentiation of monocytes. We defined aberrant myeloid blasts by a leukaemia associated phenotype (LAP) according to the definitions used in acute myeloid leukaemia. In 47% of the patients a LAP was detectable by demonstrating co-expression of CD5, CD7, CD19 and CD56 on CD34+ myeloid blasts. In all patients diagnosed as RA/RARS and MDS-U (n=12) according to WHO criteria, additional flow aberrancies were identified including a leukaemia associated phenotype of myeloid blasts in 41% of the cases. Only in 3 out of 28 cases with RCMD/RCMD-RS no erythroid aberrancies were detectable by flow-cytometry. In 9 normal control BM samples, no flow-cytometric abnormalities were present. It is concluded that flow-cytometry in MDS identifies aberrancies in the granulocytic and monocytic lineages and may classify patients with multi-lineage aberrancies not otherwise determined by cytology (WHO). Flow-cytometry may discriminate pure RA or MDS-U from RCMD. Since new drugs are emerging in low-risk MDS, the value of flow-cytometry might be of importance to further refine the classification in MDS. The exact role of these aberrant differentiation patterns on IPSS, clinical behaviour, impact on treatment decisions and as tool in disease monitoring have to be determined in future prospective studies.
In the search for new treatment modalities to eradicate minimal residual disease cells (MRD) in acute myeloid leukemia (AML), immunotherapy provides an attractive option. AML blasts show ...differentiation towards leukemic dendritic cells (DC), providing the unique opportunity to generate DC harbouring the full range of tumour antigens. In a large cohort of AML samples (n=154) AML-DC were generated by two culture methods, i.e. in presence of cytokines GM-CSF, TNF-α, SCF, Flt-3L, IL-3 and IL-4 (n=147) or calcium ionophore (CI) and IL-4 (n=108). Median AML-DC yield, defined by phenotypical DC characteristics, in the cytokine-based cultures was 12% (range:0–70%). Considering cultures yielding ≥10% AML-DC successful, 58% (85/147) of cytokine-based cultures were successful and 61% (66/108) of CI cultures. Overall, functional AML-DC generated with either method was possible in 66% (101/154) of patients. Identification of AML blast populations with DC differentiation capacity is important to select patients eligible for immunisation programs. Interestingly, presence of Flt-3 internal tandem duplication (ITD) was strongly correlated with decreased DC differentiation capacity in both culture methods (cytokine-based culture: p<0.001; CI-culture: p=0.03) suggesting that constitutive activation of tyrosine kinase receptors inhibits differentiation towards DC. In multiparameter regression analysis, powerful predictors for cytokine-based AML-DC culture outcomes were Flt-3 ITD (regression coefficient B:-3.41, p<0.001), CD14 (B:3.28, p<0.001) and TNFα-RI (B:2.82, p<0.001). This regression model predicts 88% of culture outcomes. ROC curves show high sensitivity (95%) and specificity (76%) with an AUC of 0.93 (p<0.001). In 25% of unsuccessful cytokine-based cultures, the CI-based culture method provides an alternative. This percentage increases to 56% if Flt-3 ITD+ AML samples are left out, emphasizing Flt-3 ITD+ blasts' inability to differentiate towards leukemic DC. In conclusion, AML-DC cultures are successful in most patients. Selection of patients is well possible based upon the presence of Flt-3 ITD and the expression of CD14 and TNFα-RI. Based on these results, we are currently entering patients in a phase I/II clinical vaccination trial.
In intermediate-2 (Int-2) and high risk patients with myelodysplastic syndromes (MDS), treatment with azacitidine is associated with hematological improvement and prolonged overall survival (OS) in ...patients who respond to therapy. However, only half of the patients who are treated will benefit from this treatment. It is a major challenge to predict which patients are likely to respond to treatment. The aim of this study was to investigate the predictive value of immunophenotyping for response to treatment with azacitidine of Int-2 and high risk MDS patients.
Bone marrow aspirates were analyzed by flow cytometry in 42 patients with Int-2 and high risk MDS, chronic myelomonocytic leukemia, or low blast count acute myeloid leukemia before treatment and after every third cycle of azacitidine. A flow score was calculated using the flow cytometric scoring system (FCSS).
The presence of myeloid progenitors with an aberrant immunophenotype was significantly associated with lack of response (p = 0.02). A low pretreatment FCSS was associated with significantly better OS compared with a high pretreatment FCSS (p = 0.03). A significant decrease in FCSS was observed in patients with complete response after three cycles azacitidine compared to patients with progressive disease (p = 0.006).
Absence of aberrant myeloid progenitor cells at baseline and/or a decrease in the FCSS during treatment identified Int-2 and high risk MDS patients who are likely to respond to treatment with azacitidine.
Dendritic cell (DC)-based immunotherapy faces new challenges since efficacy of DC vaccines in clinical trials has been inconsistent. Strategies to improve immune responses induced by DC are currently ...being explored. We have recently shown the feasibility of generating fully functional DC from Acute Myeloid Leukemic (AML) blasts, but with varying expression levels of the important costimulatory molecule CD86. To overcome this variability, we developed a novel bispecific diabody (BsDb) simultaneously and agonistically targeting CD40 on AML-DC and CD28 on naïve T cells. Beside optimization of CD28-mediated signaling, the resulting cellular cross-linking was also hypothesized to increase the strength and duration of T cell/AML-DC interactions, thus increasing T cell responsiveness to AML antigens. Indeed the αCD40/αCD28-bispecific diabody provokes increased T cell-DC cluster formation as assessed by light microscopy. Significant increased cluster formation was observed when T cells and AML-DC were cocultured in presence of the BsDb as compared to T cells incubated with a control protein (46%±2 versus 22%±1 respectively, p<0.05). Prior incubation of T cells and/or AML-DC with CD28 or CD40, respectively, completely prevented cluster formation in presence of the BsDb indicating specific binding of the BsDb to CD40 and CD28. The αCD40/αCD28 BsDb significantly increases T cell proliferation induced by AML-DC as compared to the unstimulated cocultures, in a dose dependent manner, as evaluated by mixed lymphocyte reactions (fold increased T cell proliferation of cocultures stimulated with BsDb as compared to unstimulated cocultures:170%±12, p<0.05). In addition, BsDb is capable of DC maturation induction as shown by significant increased mean fluorescence index (MFI) of the maturation markers CD80 (MFI of AML-DC cultured in presence of control protein vs AML-DC cultured in presence of BsDb: 22±5 vs 12±3, p<0.05) and CD83 (4±1 vs 1.5±0.5, p<0.05). In order to determine the effect of aCD40/aCD28-bispecific diabody-mediated cross-linking of AML-derived DC and CD8+ T cells on the induction efficiency of tumor-specific CTL, AML-DC derived from the HLA-A2+ AML cell line MUTZ-3 were pre-incubated with the aCD40/aCD28-bispecific diabody, loaded with the heteroclitic variant of the aa988 epitope of hTERT, and used as stimulator cells in an HLA-A2-matched allogeneic in vitro CTL induction protocol. In total nine parallel bulk cultures, were stimulated twice with peptide-loaded MUTZ-3 DC, either pulsed with control protein or the aCD40/aCD28-bispecific diabody. hTERT988Y-specific CD8+ T cells could be detected in 5/9 individual cultures when stimulated with DC pulsed with the aCD40/aCD28-bispecific diabody, whereas in only 1/9 individual cultures hTERT988Y-specific CD8+ T cells could be detected when stimulated with DC pulsed with the control protein. Thus, priming efficacy of tumor-specific cytotoxic T cells can also be improved by cross-linking AML-DC and T cells with the αCD40/αCD28 diabody. We propose that the αCD40/αCD28-bispecific diabody can serve as a potent therapeutic tool to effectively augment anti-tumor T cell responses elicited by AML-DC.
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.
