Increased Number of IL-17 Producing CD4+ T Cells in Low Risk Myelodysplastic Syndrome (MDS)

Abstract Immunological responses are increasingly recognized as important in the initiation and progression of MDS. Indeed, autoimmune diseases commonly occur in association with low risk MDS. Different studies, including our own, have shown that Foxp3+ Tregs play a role in the inhibition of tumour immune surveillance in high risk MDS; however, less clear are the potential factors which are inducing inflammatory response in low risk MDS that may also predispose to autoimmunity. CD4+IL 17 producing T cells (Th17) are considered as important pro-inflammatory T cells and contribute to the pathogenesis of a number of autoimmune diseases as well as some malignancies. The aim of this study was to investigate the potential role of Th17 cells in low risk MDS. We show for the first time that Th17 cells are markedly increased in low risk MDS in comparison to high risk disease and healthy age matched controls. We also describe an inverse relationship between numbers of Th17 cells and naturally occurring Tregs in MDS. 88 patients with newly diagnosed MDS, and 15 age matched healthy donors, were recruited. Analysis of Th17 cells was performed in a subset of 43 patients. WHO classifications were as follows: 19% had RA, 33% had RCMD, 32% had RAEB I & II and 16% had 5q- syndrome. A clear difference was observed in the median percentage and absolute number of Th17 cells between patients with low risk (based on IPSS) compared to high risk MDS (p<0.01). In low risk, but not high risk MDS, CD4+ cells had greater median number of committed Th17 cells than healthy donors (p<0.01). The absolute number of polyclonal Foxp3+ Tregs inversely correlated with Th17 cells, with higher Tregs in the high risk MDS when compared to low risk MDS or healthy donors (p<0.001), consistent with our previous report. Analysis according to IPSS, BM blast percentage or WHO classification revealed the same patterns of Th17 and Tregs expression, with the higher risk phenotypes being associated with significantly higher Treg number and lower risk phenotypes with greater Th17 cells. This finding was specific for Th17 as there was no significant difference in the number of circulating Th1 and Th2 cells between MDS subtypes. Co-culture of Tregs from patients with MDS with their own T effectors lead to suppression of supernatant IFNγ levels. Importantly, Tregs did not have an inhibitory effect on IL17 production and indicates that reduced Th17 number in the presence of high Tregs does not indicate active suppression of the Th17 phenotype. To investigate differences in serum cytokines between low and high risk MDS and healthy donors, serum samples from patients were analyzed by Luminex for multiple cytokines. The cytokines IL12(p<0.01) IL7(p<0.005) IFNγ(p<0.01) and RANTES (p<0.005) were significantly higher in patients with low risk compared to high risk MDS and healthy controls. The inhibitory cytokine IL10 and soluble IL2 receptor were present at higher levels in the serum of patients with high risk MDS (p<0.01 and p<0.005 respectively). To confirm that the peripheral blood cytokines are a true reflection of cytokine concentration in the bone marrow, the concentration of 30 different cytokines was measured by Luminex in 10 matched bone marrow and peripheral blood samples from these patients and there was no significant difference between these two sets of samples. In addition, the level of apoptosis, assessed by TUNEL assay, was also higher in the bone marrow of low risk patients. In conclusion, the “unfavourable” Th17 to Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and bone marrow apoptosis. These findings may help identify subtype of low risk MDS patients likely to respond to immunosuppressive therapy.

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Expansion of Polyclonal CD4+CD25high Foxp3+ Regulatory T-Cells in High Risk Myelodysplastic Syndromes (MDS).

Blood ◽

10.1182/blood.v108.11.2641.2641 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2641-2641

Author(s):

Shahram Y. Kordasti ◽

Wendy Ingram ◽

Janet Hayden ◽

David Darling ◽

Linda Barber ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

High Risk ◽

Regulatory T Cells ◽

Autoimmune Diseases ◽

Immunosuppressive Therapy ◽

Absolute Number ◽

Low Risk ◽

Refractory Anaemia ◽

Bone Marrow Blasts

Abstract Foxp3+ regulatory T-cells(Tregs) play a central role in maintaining tolerance. A reduction in Tregs activity is a key feature of autoimmune diseases, whereas their expansion in malignant diseases leads to suppression of the host anti-tumour responses. Expansion of oligoclonal cytotoxic T cells, inhibition of MDS CD34+ progenitors proliferation and response to immunosuppressive therapy suggest that autoimmunity contributes to the pathophysiology of low risk MDS. However, this is not generally a feature of high risk MDS. We hypothesise that in high risk MDS patients an increased number of Tregs may suppress immune responses against the dysplastic clone. In early MDS, these cells may be reduced and or be associated with coexistent autoimmune diseases, an uncommon but recognised association of low risk MDS. We therefore studied CD4+ CD25high Foxp3+ and CD8+ CD25+Foxp3+ Tregs in peripheral blood of MDS patients and examined correlation with bone marrow blasts, cytogenetic Status, IPSS score and progression to AML. Clonality of CD4+CD25+ Tregs was assessed by TCR spectratype analysis of CDR3 size distribution and by CDR3 sequence analysis. 52 patients with MDS (30 male, 22 female) with a mean age of 62 years (range 40 to 82 years) were studied. According to WHO classification, 5/52 (10%) had a diagnosis of 5q− syndrome, 9/52(17%) refractory anaemia (RA), 18/52 (35%) refractory cytopenia with multilineage dysplasia (RCMD), 16/52 (31%) refractory anaemia with excess blasts (RAEB) and 4/52 (7%) patients MDS/MPD (one with JAK2 V617F Mutation). Cytogenetic study shows normal pattern in 57%, 5q- in 22%, stable cytogenetic in 10% and complex findings in 11%. All samples were taken at diagnosis prior to any treatment. In addition 5 samples were analyzed pre and post 5-Azacytidine therapy. The absolute number of CD4+CD25highFoxp3+ regulatory T cells in 5q- syndrome was 0.5±0.28×107/l, RA0.6±0.56×107/l, RCMD1.42±0.97×107/l, RAEB2.8±2.2×107/l and MDS/MPD 2.9±2×107/l. In cases with <5% bone marrow blasts (RA, 5q− & RCMD)absolute number and percentage of Tregs was significantly lower than those with ≥5% BM blasts (p=0.001). The mean number was also significantly lower at 0.73±0.57×107/l in low risk cases(IPSS 0) compared with 2±1.5×107/l in intermediate and high risk groups (p=0.008). CD8+ Tregs were not significantly different between the subtypes of MDS and between low and high risk IPSS subgroups. Tregs number did not differ significantly between various cytogenetic subgroups. The spectratype of CD4+CD25+ TCR amplicons, showed a polyclonal pattern and the overall complexity of Vβ subfamilies was not different between low risk and high risk MDS, suggesting that the expanded Tregs in high risk MDS are not clonal and likely to arise by peripheral expansion rather than an antigen-driven response. CD4+ CD25high Foxp3+ Tregs in five patients were studied pre and post 5-Azacytidine. The numbers were significantly decreased after treatment in the one patient who responded to treatment (p=0.001), whereas Treg numbers were unchanged or increased in non responsive cases. In 5 RCMD cases with concomitant autoimmune diseases the percentage of Tregs was lower than other patients within the same subgroup of MDS, however, this did not achieve statistical significance. The findings indicate Tregs are altered in MDS and may be important in the pathophysiology of MDS. Monitoring of Tregs numbers can be a useful indicator for disease progression and response to immunosuppressive therapy.

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Increased Expression of Circulating Mir-22 in the Peripheral Blood of Patients with Myelodysplastic Syndromes Was Associated with Poorer Prognosis

Blood ◽

10.1182/blood.v126.23.2854.2854 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2854-2854

Author(s):

Yuesheng Meng ◽

Fanli Hua ◽

Ying Li ◽

Song Gao

Keyword(s):

Bone Marrow ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Risk Group ◽

High Risk Group ◽

Low Risk ◽

Expression Levels ◽

Healthy Donors ◽

Chi Square Analysis

Abstract The molecular pathogenesis of myelodysplastic syndromes (MDS) has not been completely elucidated. Deregulation of expression of some microRNA has been implicated in hematological disorders including MDS. An oncogenic role for miR-22 was recently suggested in MDS in which the expression of miR-22 was increased. However, there were corroborations showing that it could be a tumor suppressor in acute myeloid leukemia. In this study, we examined the expression levels of circulating miR-22 in the plasma of patients with MDS and evaluated its significance in clinical context. The diagnosis of MDS was made according to the WHO classifications and subtypes included 8 cases of RA, 14 cases of RCMD and 6 cases of RARS/RCMD-RS, 10 cases RAEB1 and 7 cases RAEB2. Twenty cases of healthy donors were enrolled as normal control. Written informed consent for sample collection was obtained from all subjects enrolled. EDTA-anticoagulated peripheral blood samples were collected and centrifuged. Circulating microRNAs were purified from the plasma with miRNeasy serum kit (Qiagen). Expression of miR-22 was reverse- transcripted and measured with stem-loop real time quantitative polymerase chain reaction assay. First of all, we proved that the expression of miR-22 was detectable in the plasma of patients with MDS as well as of healthy donors. Our result showed that the expression levels of circulating miR-22 in MDS patients were higher than that in healthy donors (medians 1.360 vs 1.000, P<0.05), among which 13 patients (37.1%) showed marked increase of miR-22 expression (>2 times). Due to the high heterogeneity of MDS and relatively smaller sample size, the subtypes were grouped into two major categories based on IPSS: the low-risk group (including RA, RCMD and RARS/RCMD-RS, totally 18 cases) and the high-risk group (RAEB1 and RAEB2, 17 cases in total). Relative levels of circulating miR-22 were higher in the high-risk group than in the low-risk group (medians= 1.081 vs 2.317, P<0.05). A follow-up study revealed an association of the expression levels of miR-22 with prognosis. Patients with increased expression of miR-22 had poorer clinical response (Chi-square analysis, P<0.05) and lower overall survival rate (Kaplan-Meier analysis, P<0.05). We compared the levels of miR-22 in the plasma with that of mononuclear cells of bone marrow in some MDS patients and found that the relative levels of miR-22 in the plasma and bone marrow cells were not directly co-related (P>0.05), indicating it could be an independent prognostic factor. In summary, this study confirmed an oncogenic presence of miR-22 in MDS. Given the fact that there is a lack of blastic cells in the bone marrow of patients with low-risk MDS but their plasma or serum is easy to obtain, the detection of circulating miR-22 may be of value as a molecular marker in the prognosis of MDS. Disclosures No relevant conflicts of interest to declare.

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Th17 Cells Are Increased in the Bone Marrow but Not the Peripheral Blood in Multiple Myeloma and Demonstrate Defective Functionality and Aberrant Phenotypes

Blood ◽

10.1182/blood.v124.21.3420.3420 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3420-3420

Author(s):

Christopher Parrish ◽

Gina B Scott ◽

Graham Cook ◽

Gordon Cook

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

Cell Lines ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Th17 Cells ◽

Mononuclear Cells ◽

Healthy Donors ◽

One Way Anova

Abstract Introduction Immune dysfunction in multiple myeloma (MM) is now a well accepted, if not yet completely understood, phenomenon contributing to both an increased incidence of infection and suboptimal responses to immunotherapies. The recently described Th17 subset of T helper cells is expanded in a range of cancers, and may contribute either to the evolution or control of tumours, but its role in myeloma pathogenesis remains unclear. We therefore investigated the presence, phenotype and function of this novel immune subset in a cohort of MM patients and healthy donors. Methods Peripheral blood (PB) samples were taken from healthy donors (HC, n=27) and MM patients (n=31); mononuclear cells (PBMC) were isolated by density centrifugation. Bone marrow aspirate (BM) samples were taken from MM patients (n=7) and HC (n=3); mononuclear cells were isolated by red cell lysis (BioLegend). For phenotyping, cells were treated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A and assessed by flow cytometry. Where stated, cells were stimulated with candidal wall mannoprotein MP65 (Peptivator, Miltenyi Biotec). For coculture experiments, CD4+T cells were isolated from PBMC using magnetic bead separation (MACS, Miltenyi Biotec) and cultured with human myeloma cell lines (HMCLs: U266B, JIM3, KMS11) with or without human bone marrow stromal cell lines (BMSC: HS-5, HS-27). Statistical analysis was performed using GraphPad Prism v6. Results As noted in previous studies, patients with myeloma have a marked CD4+ T-cell lymphopenia. Within the CD4+ population, MM patients also had a reduced frequency of IL-17+ cells (Th17) compared to HC: 0.41% (95% CI 0.27-0.56) vs. 0.81% (95% CI 0.47-1.15), p=0.0241 (student’s t-test). Using the mean fluorescence intensity for IL-17 as a surrogate indicator of cytokine production, IL-17 production in Th17 from MM patients was significantly reduced compared to HC (p=0.0195). Advancing disease stage correlated significantly with a reduction in PB Th17 frequency (p<0.0001, one-way ANOVA), with those with relapsed disease having the lowest levels (0.10%). In contrast, Th17 cells were expanded in the BM of MM patients compared to HC: 0.9% (95% CI 0.22-1.58) vs. 0.2% (95% CI -0.1198-0.5192), p=0.15). Th17 frequency was not significantly related to serum paraprotein concentration, prior lines of therapy, previous exposure to immunomodulatory novel agents (thalidomide, lenalidomide, bortezomib) or high-dose therapy, frequency of residual normal plasma cells in BM, serum creatinine, or high risk cytogenetic or molecular markers (del13 by FISH or IGHrearrangement). Th17 frequency was, however, lower in patients with IgG isotype disease compared to IgA (p=0.036) or light chain disease (p=0.0264). PB Th17 cells from myeloma patients showed a marked reduction in expression of CD161 compared with HC: 25.5% vs. 54.2%; this was again a stage-related phenomena (p=0.0019, one-way ANOVA). CCR6 expression on Th17s was also reduced in MM compared to controls in a stage-related manner (p=0.0036). Furthermore, PB Th17 cells in MM had impaired capacity to produce IL-17 in response to stimulation with a fungal antigen: 7% responded cf. 21% in HC (p=NS). To determine what, if any, effect MM tumour cells exerted on Th17 homeostasis, we employed a coculture model. CD4+ T cells from HC were cultured with mitomycin C-treated HMCLs for 7 days. Presence of HMCLs resulted in an expanded Th17 population compared to control cultures of CD4+ alone: 2.4% (95% CI: 0.13-1.30) vs..0.7% (95% CI: -0.20-5.07), p=0.09. The expansion of Th17 cells was augmented when BMSC were included (p=0.0476). We observed higher CD161 expression on coculture-derived Th17s when stromal cells were present. Conclusions Multiple abnormalities are apparent in Th17 immunity in MM: their frequency is decreased in PB and increased in BM, in a stage-related manner suggestive of either a homing defect or a tumour microenvironmental expansion. Our in vitro co-culture data suggest tumour-derived Th17 expansion may be responsible. The Th17 phenotype in MM is aberrant, with the relative absence of key lineage markers and attenuated functional responses to fungal antigens. Since IL-17 has been reported to be a growth factor for MM cells, these abnormalities may contribute both to the propagation of the malignant clone and the impaired anti-infective immunity seen in this disease. Disclosures No relevant conflicts of interest to declare.

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Expansion of CD141int Dendritic Cells in Myelodysplastic Syndrome (MDS)

Blood ◽

10.1182/blood.v120.21.3856.3856 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3856-3856

Author(s):

Nathalie Kerkhoff ◽

Shahram Kordasti ◽

Thomas Seidl ◽

Arjan A. Van de Loosdrecht ◽

Ghulam J. Mufti

Keyword(s):

Bone Marrow ◽

T Cells ◽

Dendritic Cells ◽

High Risk ◽

Peripheral Blood ◽

Immune Dysregulation ◽

Dependent Manner ◽

Hla Dr ◽

Healthy Donors ◽

Exact Function

Abstract Abstract 3856 Introduction Immunological responses play an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). Several studies have confirmed that immune dysregulation in MDS may play a critical role in the initiation and progression of the dysplastic clone. We have already shown the importance of expanded Tregs in high risk MDS and its reverse correlation with the number of Th17 cells in low risk disease. However, the potential role of dendritic cells (DCs) in this immune dysregulation and in the expansion of Tregs in MDS is not fully understood. DCs are professional antigen presenting cells (APCs) and potent stimulators of T cells through (cross-)presentation of antigens via MHC class I and II molecules to CD8+ cytotoxic T lymphocytes (CTL) and CD4+ T helper (Th) cells, respectively. DCs are also important in the development of specific anti-tumor T-cell responses. Some subtypes of DCs also play an important role in the expansion of regulatory T cells (Tregs) and induce immune-suppression and editing. There are 3 main subpopulations of DCs in human: plasmacytoid DCs (CD303+ cells) and two types of myeloid DCs (CD1c+ and CD141+ cells). The aim of this study was to investigate the frequency of different DC subsets in the bone marrow and peripheral blood of MDS patients. Patients and methods Twelve peripheral blood and 26 bone marrow samples from MDS patients and 11 peripheral blood and 4 bone marrow samples from healthy donors (HDs) were studied. Three different DC subsets were investigated by flow-cytometry: plasmacytoid DC (Lineage−, HLA-DR+, CD303+), myeloid DC 1 (Lineage−, HLA-DR+, CD1c+) and myeloid DC 2 (Lineage−, HLA-DR+, CD141hi). Results Decreased frequency of DCs in peripheral blood compared to BM Frequencies of all DC subsets were significantly lower in patients' peripheral blood compared to patients' bone marrow (CD1c+ DC 2.56×102 v 7.02×102 and 0.06% v 0.33%, p=0.001; CD141+ DC 4.18×103 v 6.04×103 and 0.44% v 2.44%, p=0.005; CD303+ DC 0.06×102 v 1.07×102 and 0.01% v 0.13%, p=0.045). DC subsets The percentage of the CD1c+ DC subset was significantly reduced in the peripheral blood of MDS patients compared to HD (1.46×103 v 0.26×103, 0.18% v 0.05%, p=0.001) (figure 1). Interestingly, we have noticed an additional subset of DCs, which was not described before. The definition of this subset was based on intermediate expression of CD141 compared to previously described myeloid DCs. The CD141int subset was the only DC subset that was increased in peripheral blood of MDS patients compared to HD (0.41×102 v 2.30×102, 0.01% v 0.13%, p=0.019). Both CD1c+ and CD141int DC subsets were increased in the bone marrow of MDS patients compared to healthy donors. However, these differences were not statistically significant. Discussion Although the decreased number of circulating DCs is reported in MDS, our data suggest that this decrease is more profound in the peripheral blood compared to BM. We have also shown that this decrease is mainly in CD1c+ myeloid DCs. In this study, we describe two subsets of CD141 DCs, CD141hi and CD141int. The CD141int subpopulation is the only DC subset that is increased in MDS patients compared to HD. Our data suggest that DCs from peripheral blood may migrate to the bone marrow in response to an aberrant BM microenvironment in MDS. Nevertheless, the pattern of decrease in DC numbers is not uniform and CD141int DCs are in fact increased. It is already known that CD141+ DCs, play an important role in the induction of Tregs in an IL-10 dependent manner. We have also reported an increased number of Tregs in high-risk MDS, which correlates, with higher risk of progression toward AML. Our data suggest that CD141+ DCs may play a role in the expansion of Tregs in MDS. However, the exact function of this subset of DCs in MDS and their effect on CD4+ T-cells polarization needs to be investigated in more details. Disclosures: No relevant conflicts of interest to declare.

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Regulatory T Cells Fail to Suppress Fast Homeostatic Proliferation In Vitro

Life ◽

10.3390/life11030245 ◽

2021 ◽

Vol 11 (3) ◽

pp. 245

Author(s):

Daniil Shevyrev ◽

Valeriy Tereshchenko ◽

Elena Blinova ◽

Nadezda Knauer ◽

Ekaterina Pashkina ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Autoimmune Diseases ◽

Peripheral Blood ◽

Treg Cells ◽

Homeostatic Proliferation ◽

Suppressive Activity ◽

Healthy Donors

Homeostatic proliferation (HP) is a physiological process that reconstitutes the T cell pool after lymphopenia involving Interleukin-7 and 15 (IL-7 and IL-15), which are the key cytokines regulating the process. However, there is no evidence that these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the functional activity of Tregs stimulated by HP cytokines from patients with rheumatoid arthritis as compared with that of those from healthy donors. Since T cell receptor (TCR) signal strength determines the intensity of HP, we imitated slow HP using IL-7 or IL-15 and fast HP using a combination of IL-7 or IL-15 with anti-CD3 antibodies, cultivating Treg cells with peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio. We used peripheral blood from 14 patients with rheumatoid arthritis and 18 healthy volunteers. We also used anti-CD3 and anti-CD3 + IL-2 stimulation as controls. The suppressive activity of Treg cells was evaluated in each case by the inhibition of the proliferation of CD4+ and CD8+ cells. The phenotype and proliferation of purified CD3+CD4+CD25+CD127lo cells were assessed by flow cytometry. The suppressive activity of the total pool of Tregs did not differ between the rheumatoid arthritis and healthy donors; however, it significantly decreased in conditions close to fast HP when the influence of HP cytokines was accompanied by anti-CD3 stimulation. The Treg proliferation caused by HP cytokines was lower in the rheumatoid arthritis (RA) patients than in the healthy individuals. The revealed decrease in Treg suppressive activity could impact the TCR landscape during lymphopenia and lead to the proliferation of potentially self-reactive T cell clones that are able to receive relatively strong TCR signals. This may be another explanation as to why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease in Treg proliferation under IL-7 and IL-15 exposure can lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis in the case of lymphopenia.

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WT1-Peptide Vaccination Shows High Immunogenicity and Clinical Activity in Patients with Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v106.11.406.406 ◽

2005 ◽

Vol 106 (11) ◽

pp. 406-406 ◽

Cited By ~ 3

Author(s):

Keilholz Ulrich ◽

Carmen Scheibenbogen ◽

Anne Letsch ◽

Anne Marie Asemissen ◽

Wolf Karsten Hofmann ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

High Risk ◽

Clinical Outcome ◽

Peripheral Blood ◽

Great Promise ◽

Clinical Activity ◽

Peptide Vaccination ◽

Before And After ◽

Disease Stabilization

Abstract BACKGROUND: The transcription factor Wilms tumor protein 1 (WT1) holds great promise for immunotherapy of leukemia. WT1 is strongly expressed in the majority of leukemic blasts, is essential for blast proliferation, and is spontaneously immunogenic. METHODS: In the present phase II trial, 12 HLA-A2+ patients with AML without curative treatment option, were vaccinated with WT1.126–134 peptide mixed with adjuvant KLH as T-helper protein and GM-CSF 4 times bi-weekly, then monthly. RESULTS: Patients characteristics, immune responses and clinical outcome are shown in table 1. Patient characteristics, immunologic response, and clinical outcome Pat FAB/caryotype previous chemotherapy disease status at study onset no. of vaccinations clinical outcome WT1Tetr+ T cells in PB after vaccination WT1Tetr+ T cells in BM after vaccination *PB, peripheral blood; BM, bone marrow; MDS, myelodysplastic syndrome; MPD, myeloproliferative disease. 1 M4 yes 2.PR 15 CR 12 months 0.49% 0.87% 2 M2 11q23 yes 1.CR 18 cCR 30+ months 0.43% 0.91% 3 M2 no PD 4 SD 3 months 0.42% 0.80% 4 M6 yes PD 4 PD neg. neg. 5 M2 yes 1.PR 6 PD 0.37% 0.51% 6 M1 yes 2. PR 9 PD 0.43% 0.40% 7 M2 yes 2.PR 9 PD 2.00% 1.36% 8 M7 yes PD 4 PD neg. neg. 9 M5b yes 2.CR 12 cCR 8+ months 0.44% 0.33% 10 sAML from MDS no PD 12 SD 8 months 0.23% 0.13% 11 sAML from MPS no PD 12 SD 9+ months 0.22% 0.53% 12 M4 no PD 8 SD 3 months 1.11% 1.35% WT1-specific T cells could be detected in 3 patients before vaccination. An induction or enhancement of a T cell response against WT1 was observed in 10 of 12 patients after 2 – 6 vaccinations ranging from 0.22 to 2.00% (median 0.43%) in peripheral blood and from 0.33 to 1.36% (median 0.80%) in bone marrow as analysed by tetramer and cytokine staining. At study onset 6 patients had progressive AML (PD) with 40 – 90% marrow blasts, 4 patients partial remission (PR) following chemotherapy and two patients complete remission (CR) at high risk for relapse. Four of the 6 patients with progressive AML had disease stabilization for 3, 3, 8 and 9 months, which is ongoing in the latter patient. Disease stabilization was accompanied by a decrease/normalization of peripheral blasts in two patients and a >50% decrease in RBC transfusion requirements in a patient with AML evolved from MDS. One patient with PR at study onset had an early relapse and then achieved CR for 12 months (patient 1). Both patients vaccinated in CR are in continuous hematological CR (cCR) for 8+ and 30+ months (patient 2 and 9). The remaining 5 patients had PD after 4 – 9 vaccinations. Bone marrow WT1 RNA levels as molecular disease marker paralleled the clinical course as they decreased 1 – 2 logs in the 3 patients with CR or cCR after 6 vaccinations (Fig. 1A), stabilized or decreased in all 4 patients with SD (Fig. 1B), and increased 1 – 2 logs in 4 of the 5 patients with PD (Fig. 1B). No significant toxic effects were observed. CONCLUSION: WT1 peptide vaccination can efficiently induce a specific immune response and has clinical activity in the absence of significant toxicity. These results warrant further studies of WT1 vaccination in AML patients at high risk for relapse. Fig. 1 WT1 levels in bone marrow before and after 6 vaccinations in patients with CR or cCR (A), SD (B) or PD (C) after vaccination. Fig. 1. WT1 levels in bone marrow before and after 6 vaccinations in patients with CR or cCR (A), SD (B) or PD (C) after vaccination.

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Stathmin 1 Is Involved In The Highly Proliferative Phenotype Of High-Risk Myelodysplastic Syndromes and Acute Leukemia Cells

Blood ◽

10.1182/blood.v122.21.861.861 ◽

2013 ◽

Vol 122 (21) ◽

pp. 861-861

Author(s):

João Agostinho Machado-Neto ◽

Paula de Melo Campos ◽

Patricia Favaro ◽

Mariana Lazarini ◽

Irene Lorand-Metze ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Low Risk ◽

Leukemia Cells ◽

Healthy Donors ◽

Stathmin 1 ◽

Marrow Cells

Abstract Introduction : Stathmin 1, also known as Oncoprotein 18 (OP18) or Leukemia-associated phosphoprotein p18 (LAP18), is an important cytoplasmic microtubule-destabilizing protein that plays a critical role in the process of mitosis, proliferation and accurate chromosome segregation through regulation of microtubule dynamics. High levels of Stathmin 1 have been reported in solid tumors and have been associated with poor prognosis in various types of cancers. The identification of overactive proteins in leukemia cells, compared to normal hematopoietic cells, as well as understanding the molecular and cellular basis of the disease may provide new therapeutic opportunities. Aims: To evaluate Stathmin 1 expression in proliferating and non-proliferating hematopoietic cells, in bone marrow cells from healthy donors and from patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In addition, we evaluated the effect of Stathmin 1 silencing on proliferation and apoptosis in the U937 acute myeloid leukemia cell line. Materials and Methods: A panel of human leukemia cell lines that included myeloid (K562, KU812, NB4, HL60, P39, HEL, U937, KG1 and THP1) and lymphoid cells (Jurkat, MOLT4, Daudi, Raji, Namalwa and Karpas 422) in exponential growth was used. Peripheral blood lymphocytes (PBL) were induced, or not, to proliferate upon PHA stimulation for 72 hours. A total of 30 healthy donors and 117 patients at diagnosis (MDS=52 [low-risk=36, high-risk=16], AML=49, and ALL=16) were included in the study. Stathmin 1 gene and protein expression was evaluated by qPCR and Western blot. Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector and cell growth was examined by MTT assay, clonogenicity by colony formation and apoptosis by AnnexinV/PI. Appropriate statistical analyses were performed; results are expressed as median (minimum- maximum). Results: A higher expression of Stathmin 1 was observed in all leukemia cell lines, when compared with normal non-proliferating hematopoietic cells. We also observed a marked increase in Stathmin 1 expression in PBL induced to proliferate with PHA after 72 hours. Stathmin 1 transcripts were significantly increased in total bone marrow cells from patients with AML (2.01 [0.35-8.88]; p=.0009) and ALL (2.94 [1.16-10.82]; p=.0004), compared with healthy donors (1.01 [0.38-4.08]). No difference in Stathmin 1 expression was observed between healthy donors and MDS patients. When the MDS group was stratified by the WHO classification into low and high-risk MDS, Stathmin 1 expression was significantly higher in the high-risk, when compared with low-risk MDS (1.62 [0.42–3.28] vs. 1.13 [0.36–2.61], p=.03). Similar results were found in isolated CD34+ bone marrow cells, Stathmin 1 transcripts were significantly increased in CD34+ AML cells compared with CD34+ normal cells, and in high-risk compared with low-risk MDS (all p≤.02). Interestingly, 3 out of 5 MDS patients showed a significant increase in Stathmin 1 transcripts after disease progression. Also, a significant positive correlation was observed between percentage of bone marrow blasts and Stathmin 1 expression in MDS patients (p=.03; r=.31). In U937 leukemia cells, Stathmin 1 silencing significantly reduced cell proliferation (p=.02) and clonal growth (p<.0001), but did not modulate apoptosis. Conclusions: Stathmin 1 is overexpressed in high-risk MDS and acute leukemia cells, and is upregulated during MDS progression, suggesting that Stathmin 1 plays a role in the highly proliferative phenotype. Our study adds new insights to the role of Stathmin 1 in leukemogenesis. Future studies are necessary to validate whether Stathmin 1 is a predictive marker for MDS progression, and to determinate whether Stathmin 1 is a “driver” or a “passenger” during malignant transformation. Disclosures: No relevant conflicts of interest to declare.

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Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.3464.3464 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3464-3464

Author(s):

Yang Song ◽

Yu-tong Wang ◽

Xiao-jun Huang ◽

Yuan Kong

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Endothelial Cells ◽

Th17 Cells ◽

Immune Thrombocytopenia ◽

Perivascular Cells ◽

Platelet Production ◽

Cd34 Cells ◽

Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

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Enrichment of functional CD8 memory T cells specific for MUC1 in bone marrow of patients with multiple myeloma

Blood ◽

10.1182/blood-2004-01-0366 ◽

2005 ◽

Vol 105 (5) ◽

pp. 2132-2134 ◽

Cited By ~ 86

Author(s):

Carmen Choi ◽

Mathias Witzens ◽

Marianna Bucur ◽

Markus Feuerer ◽

Nora Sommerfeldt ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Hematologic Malignancies ◽

Myeloma Cell ◽

Interferon Γ ◽

Multiple Myeloma Cell ◽

Healthy Donors

AbstractMultiple myeloma (MM) is one of the most common hematologic malignancies. Despite extensive therapeutical approaches, cures remain rare exceptions. An important issue for future immunologic treatments is the characterization of appropriate tumor-associated antigens. Recently, a highly glycosylated mucin MUC1 was detected on a majority of multiple myeloma cell lines. We analyzed bone marrow and peripheral blood of 68 patients with HLA-A2–positive myeloma for the presence and functional activity of CD8 T cells specific for the MUC1-derived peptide LLLLTVLTV. Forty-four percent of the patients with MM contained elevated frequencies of MUC1-specific CD8 T cells in freshly isolated samples from peripheral blood (PB) or bone marrow (BM) compared with corresponding samples from healthy donors. BM-residing T cells possessed a higher functional capacity upon specific reactivation than PB-derived T cells with regard to interferon γ (IFN-γ) secretion, perforin production, and cytotoxicity.

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GDF11 Increased in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v126.23.5224.5224 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5224-5224

Author(s):

Yu Han ◽

Huaquan Wang ◽

Zonghong Shao

Keyword(s):

Bone Marrow ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Risk Group ◽

Low Risk ◽

Mean Corpuscular Hemoglobin ◽

Corpuscular Hemoglobin ◽

Normal Controls

Abstract Objective To analyze the concentration of growth differentiation factor 11(GDF11) in peripheral blood of patients with myelodysplastic syndrome (MDS), so as to evaluate the relationships between these changes and erythropoiesis functions and to explore the role of GDF11 in the pathogenesis of MDS. Methods The concentration of GDF 11 in peripheral blood was detected by enzyme-linked immuno sorbent assay in 44 MDS patients and 10 normal controls from September 2014 to June 2015 at our hospital. The percentage of nucleated erythrocyte (CD235a) in bone marrow was detected by flow cytometry. The correlation between these changes and erythropoiesis functions, including red blood cell count, hemoglobin, reticulocyte (RET%), hematokrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC) and late erythroblast in bone marrow were evaluated. Results (1)The concentration of GDF11(128.67±47.62)in high-risk MDS patients was significantly higher than that of low-risk MDS patients (65.96±36.55,p<0.01)and higher than that of normal controls (29.76±10.10,p<0.01); The concentration of GDF11 in low-risk MDS patients was significantly higher than that of normal controls (p<0.05). (2) The expression of CD235a in high-risk group(38.49±5.42)was not different with that in low-risk group(42.64±7.36, p>0.05). (3)In high-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET%, RBC, MCHC, Hct in peripheral blood and late erythroblast, CD235a+ cells in bone marrow(r=-0.437,r=-0.428,r=-0.444,r=-0.553,r=-0.661,r=-0.436,r=-0.52,all p<0.05),and the expression of GDF11 was positively correlated with MCV(r=0.52, p <0.05),but it was not correlated with MCH (p >0.05).(4) In low-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET% (r=-0.491Ar=-0.606,both p<0.05),it was not correlated with RBC, MCHC, MCV, MCH, Hct, late erythroblast and CD235a+ cells (all p>0.05). Conclusion GDF11 increased in patients with MDS and it was negatively correlated with late erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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