Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to ...increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients' survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4(+) T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4(+) T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4(+) T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3(+)/interleukin-17(+) cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4(+) T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.
Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients ...awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study).
Healthy controls and age- and sex-matched hemodialysis patients without recent illness/autoimmune disease on established, complication-free hemodialysis for a minimum of 6 months were recruited. Circulating regulatory T cells were studied by flow cytometry to compare the regulatory T cell subpopulations. Regulatory T cells from members of each group were compared for suppressive function and plasticity (IL-17-producing capacity) before and after in vitro expansion with and without Rapamycin, using standard assays.
Both groups had similar total regulatory T cells and subpopulations I and III. In each subpopulation, regulatory T cells expressed similar levels of the function-associated markers CD27, CD39, HLA-DR, and FOXP3. Hemodialysis regulatory T cells were less suppressive, expanded poorly compared with healthy control regulatory T cells, and produced IL-17 in the absence of Rapamycin. However, Rapamycin efficiently expanded hemodialysis regulatory T cells to a functional and stable cell product.
Rapamycin-based expansion protocols should enable clinical trials of cell-based immunotherapy for the induction of tolerance to renal allografts using hemodialysis regulatory T cells.
Summary
Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in ...association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3+ CD4+ IL‐17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS (P < 0·01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4+CD25high FoxP3+ regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease (P < 0·005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis (P < 0·01). Tregs from MDS patients suppressed interferon‐γ (IFN‐γ) secretion by effector CD4+ T cells but had no effect on interleukin (IL)‐17 production. In addition, the serum levels of IL‐7, IL‐12, RANTES and IFN‐γ are significantly elevated in low risk MDS, while inhibitory factors, such as IL‐10 and soluble IL‐2 receptor, are significantly higher in high risk disease. The ‘unfavourable’ Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.
Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell‐based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL‐17 ...under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL‐17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL‐17 in vitro when activated in the presence of IL‐1β, but not IL‐6. “IL‐17 potential” is restricted to population III (CD4+CD25hiCD127loCD45RA−) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL‐17 induction. Importantly, we find that IL‐17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL‐17. Finally, we show that CD161+ population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL‐17‐producing Treg‐cell population at these sites. As IL‐17 production from this Treg‐cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.
T
reg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell‐based clinical therapy. However, human
T
reg cells are “plastic”, and are able to produce
IL
...‐17 under inflammatory conditions. Here, we identify and characterize the human
T
reg subpopulation that can be induced to produce
IL
‐17 and identify its mechanisms. We confirm that a subpopulation of human
T
reg cells produces
IL
‐17 in vitro when activated in the presence of
IL
‐1β, but not
IL
‐6. “
IL
‐17 potential” is restricted to population III (
CD
4
+
CD
25
hi
CD
127
lo
CD
45
RA
−
) Treg cells expressing the natural killer cell marker
CD
161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of
T
reg cells and retain their suppressive function following
IL
‐17 induction. Importantly, we find that
IL
‐17 production is
STAT
3 dependent, with
T
reg cells from patients with
STAT
3 mutations unable to make
IL
‐17. Finally, we show that
CD
161
+
population III
T
reg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant
IL
‐17‐producing
T
reg‐cell population at these sites. As
IL
‐17 production from this
T
reg‐cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.
Sweet's syndrome (SS) is an acute febrile neutrophilic dermatosis. It has been associated with malignant disease, especially acute myeloid leukaemia (AML) and drugs, particularly granulocyte colony ...stimulating factor (GCSF). No cause is found in the rest and is labeled idiopathic. We describe 31 patients with a readily diagnosed form of SS, which we believe represent an autoimmune phenomenon secondary to the myelodysplastic syndrome (MDS).
A retrospective study was conducted to identify patients with SS with underlying diagnosed or occult haematological disorders over a 7 year period. The skin histology was reviewed independently by histopathologist and additionally frequency of CD4+ and CD8+ T cells, B cells and NK cells were investigated in 6 patients with chronic relapsing SS in comparison with 4 healthy age matched donors.
We identified 31 patients with SS in a cohort of 744 patients with MDS and 215 with AML seen between 2004-2011. The median age was 58 yrs (37-82 yrs), with male female ratio of 1.2;1 (male 17, female 14). Of these, 74% (N=23) were associated with myelodysplastic syndrome, 13 %( N=4) with AML, 6% (n=2) with chronic myeloid leukemia, 3% (n=1) with acute lymphoblastic leukaemia and 3% (n=1) with polycythaemia rubra vera.
We grouped the patients into those with a chronic relapsing/remitting type of skin eruption (n=15) and the second group consisting of patients with a single episode of classical SS (n=16).
Patients presenting with this chronic relapsing remitting form of SS (n=15) were not generally known to have MDS at the time of their initial skin eruption. The median time from diagnosis of SS to diagnosis of MDS was 17 months (1.4 years) (range 0-157 months). The WHO subtypes of MDS were RCMD (N= 13), RAEB-1 (N=1) and MDS/MPN-U (N=2).All except two patients (trisomy 8 and del 11q) had normal bone marrow karyotype. The IPSS risk groups were; Low (N=10), Int-1 (n=5).Transfusion dependency was subsequently seen in 6 of 15 patients. Progression to high-risk occurred in two patients (RAEB 1), whilst none had leukaemic transformation.
The clinical features of the chronic form were identical to those described by Sweet ; raised, tender plaques which were red and urticated. Some of the lesions had mamillated (“nipple like”) elevation on the surface of these plaques. They were found to be scattered on the torso and limbs, neck and face. Larger more nodular plum coloured lesions may also be found.
All 15 patients had constitutional symptoms including fever and sweats at the time of skin eruptions. Arthralgia was seen in a majority of patients (n=12).Additionally, other associated autoimmune conditions or dermatological conditions seen included seronegative rheumatoid arthritis (n=1), relapsing polychondritis (n=1), pyoderma gangrenosum (n=1) and Behcets disease (n-1).
Compared to MDS without SS (n=711), patients with SS and MDS were on an average 8 years younger, low/int-1 risk, less likely to receive MDS therapy and had lower propensity to leukaemic transformation (all p<0.001).
The frequency of γδ Tcells (3.6% ± 1.66% v 2.2% ± 0.6%, p=0.51), effector CD4+ T cells (12.6% ± 5.7 v 4.5% ± 1.8%, p=0.2) and resting Tregs (11.1 ±2.5 v 5.5% ± 0.6%, p=0.4) in patients were higher compared to healthy aged match donors.
The chronic relapsing remitting of SS was recalcitrant to treatment. Most patients had to be maintained on a higher doses of prednisolone (>15-20mg) to prevent recurrent episodes. The response to immunosuppressive therapy (IST) was variable with median of 4 ISTs (range 0-12) per patient. The treatment associated with complete resolution of the skin eruptions with no relapses were 5-azacitidine in four patients, infliximab in one patient and one with methotrexate but other agents were disappointing. Corticosteroids were effective in all patients; however doses of prednisolone below 15mgs resulted invariably in relapse of SS.
The cause in 16 patients could be attributed either to administration of GCSF or after chemotherapy. The eruption was brief and disappeared spontaneously or following withdrawal of GCSF
We describe a chronic debilitating episodic clinically distinctive skin eruption with features of Sweet's syndrome but not always definitive histopathology often associated with immunological abnormalities affecting other systems (especially rheumatological) universally related to underlying occult ‘lower risk' MDS.
No relevant conflicts of interest to declare.
Foxp3+ regulatory T cells (Tregs) play a central role in maintaining immune tolerance. A reduction in the function of Tregs is a key feature of autoimmune diseases, whereas their expansion in ...malignant diseases leads to the suppression of host antitumor responses. We analyzed the absolute number of CD4+ and CD8+ Tregs in the peripheral blood of 52 patients with myelodysplastic syndrome (MDS) and show a significant correlation between increased number of CD4+ Tregs and MDS subgroups with 5% or more bone marrow blasts (P < .001), high International Prognostic Scoring System (IPSS) score (P < .001), and disease progression (P < .001), whereas no correlation between CD8+ Tregs and prognostic variables was observed. The CD4+ Tregs showed a polyclonal spectratype, and the percentage of the naive subset was significantly higher in the high-risk patients compared with low-risk or healthy age-matched donors (P = .032). Our data suggest that CD4+ Treg expansion is a feature of high-risk MDS and progression to aggressive subtypes of the disease.
The myeloproliferative neoplasms (MPN), in particular myelofibrosis, are associated with elevated levels of inflammatory cytokines and constitutional symptoms. Treatment with JAK inhibitors (JAKi) ...have lead to marked improvement in symptoms and splenomegaly. Signaling through the JAK pathway is critical for T cell development and differentiation. However the baseline immune signature remains largely undescribed in MPN as does the effect of JAK inhibition on the immune subsets in this disease.
The % and absolute number of CD4+ T cell subsets (TH1, TH2 and TH17 and Foxp3+ T regulatory cells) in peripheral blood (PB) were investigated by flow cytometry. T cells were stimulated and stained intracellularly for IFNg, IL-4, IL-17 & TNFα. Tregs were defined as CD4+ CD25highCD27+FOXp3+. The serum level of 30 cytokines was also measured by Luminex. Patients received either ruxolitinib (n=21) or SAR302503 (n=13) as JAKi.
We analysed 50 MPN patients (30 Myelofibrosis, 15 Polycythemia Vera, 5 Essential Thrombocythemia) and 14 healthy donors (HD). 34 patients were treated with JAKi and sequential PB samples were obtained at 1, 3, 6 and 12 month intervals (median follow up 6 months). Tregs are significantly lower in MPN patients compared to HD and drop further following treatment (p<0.0001 and p=0.0049 respectively). There was no difference at baseline in the T effector subsets between the groups including TH1, TH2 and TH17 secreting cells but there was a significant increase in TH17 following JAKi therapy (fig 1a). JAKi resulted in a significant decrease (p=0.03) in CD4 T cells secreting pro-inflammatory cytokines at 3 months follow up although this was less evident at 6 months follow up and occurred irrespective of disease response to treatment. This silencing was confirmed by both intracellular staining and luminex assay of supernatants including a significant decrease in Interleukin-2 receptor (IL-2r) p=0.0007, Interferon gamma induced protein (IP-10) p=0.0006, monokine induced by gamma interferon (MIG) p=0.0008 and hepatocyte growth factor (HGF) p=0.0009.
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This finding was reproduced in-vitro in healthy peripheral blood mononuclear cells (PBMCs). PBMCs were treated with the JAKi ruxolitinib (100-300NM) in the presence or absence of plate bound anti-CD3/28 stimulation and cultured for 5 days. Tregs were reduced in number and there was a considerable increase in the percentage of “cytokine negative” or “silent” T effector cells by FACS analysis compared to untreated or vehicle treated cells (median of 42 % of CD4 to 91% of CD4) (fig 1b). This finding was reproduced by Luminex cytokine assay of supernatants. Western blot demonstrated a reduction in pSTAT3 in ruxolitinib treated cells. To assess the effect of JAKi on Treg function, healthy isolated Tregs were treated with ruxolitinib and co cultured with CFSE labeled autologous T effector cells. Short term JAKi treated Tregs were unable to suppress the proliferation of T effector cells compared to Tregs treated with vehicle alone. Similarly, proliferation rate and function of Tregs was reduced following 4 weeks expansion in the presence of ruxolitinib compared to expanded Tregs in the presence of ATRA and rapamycin as a control.
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Tregs are significantly lower in MPN patients compared to healthy controls in keeping with the inflammatory environment of MPN and decrease further with JAKi. Surprisingly, T effector numbers were not significantly different to healthy controls at baseline and TH1 and TH2 subsets did not change with therapy. However, secretion of proinflammatory cytokines from these cells was blocked with JAKi both invivo and invitro resulting in a functional silencing of T effectors. Interestingly, TH17 subsets increase with treatment possibly representing a polarization from a Treg phenotype to a TH17 phenotype, suggesting the re-establishment of immune-surveillance against the malignant clone. Further investigation is required to confirm the hypothesis that these expanded TH17 cells originate from Tregs or previously “silenced” CD4 T cells.
Harrison:Novartis: Honoraria; Sanofi : Honoraria.
Abstract 4718
Improved experimental therapies are needed for Multiple Myeloma (MM). Despite major progress in treatment and initial induction of remission, myeloma remains an incurable disease. ...Although immunotherapy and, in particular, the employment of NK cells offers an approach of interest for the treatment of Multiple Myeloma (MM), recent studies have shown that myeloma cells utilise a number different mechanisms to impair NK and T cell functions. Important amongst these mechanisms is the reduced expression of CD80 in the sub-populations of PBMC isolated from myeloma patients.
We have previously demonstrated CD80/IL-2 mediated stimulation of NK and T cells isolated from AML patients (as measured by proliferation, cytokine release and target cell specific cytolytic activity). In the present study we have examined the ability of genetically modified MM cells engineered to express CD80 and IL-2 to stimulate NK cell functions. These studies confirm the ability of MM cells to suppress NK cell functions in healthy PBMC and show that in contrast to the unmodified MM cells, the CD80/IL-2 expressing MM cells are able to stimulate a moderate increase in NK and T cell numbers and a significant increase in the fraction of NK cells with activatory receptors (NKp44, NKp30, NKp46) and activation markers (CD69) on the cell surface of both NK and T cells. More importantly for potential therapeutic applications the stimulated NK cells show increased cytolytic activity against the unmodified MM cells.
This data suggest that CD80/IL-2 MM cells may be able to overcome the immune suppressive functions of unmodified MM cells and to stimulate NK, and T cell mediated responses against the unmodified MM cells. Therefore CD80/IL-2 expressing MM cells may provide a suitable cellular vaccine for NK cell stimulation and possibly the induction of broader ranging immunological responses against multiple myeloma cells.
No relevant conflicts of interest to declare.
Immune derangements and altered T cell hemostasis play an important role in the pathogenesis of Myelodysplastic syndromes (MDS) contributing to the increased clone susceptibility to accelerated ...apoptosis. In addition, escape of immune surveillance may be a mechanism of MDS disease progression. The association between MDS and autoimmune diseases (AID) is well described. Small case series reported distinct clinical features and outcome for MDS patients with AID. We report here the largest cohort examining the prevalence of AID among MDS patients, compare characteristics and outcome of MDS patients with and without AID.
We identified all confirmed MDS cases through the Moffitt Cancer Center (MCC) MDS database and King's College Hospital (KCH). Therapy related MDS (t-MDS) cases were excluded. All charts were reviewed for documented past or active AID and its treatment. Patients were divided into 2 groups, those with de novo MDS associated with AID (AIDA-MDS) and those with no documented AID (non AIDA-MDS). Baseline characteristics were compared between the two groups. Chi square test was used for comparison of categorical variables and t-test for continuous variables. Kaplan-Meier estimates were used for overall survival (OS).
At time of this analysis 1408 pts were included, 1044 cases from MCC and 364 at KCH. We identified 391 MDS patients with AID (28%). The median duration of follow up was 74 months (mo) (95% CI 69-78)
Hypothyroidism was the most common AID identified, accounting for 44% (n=171) of cases with AID (12% among all MDS cases in this analysis). Other AID with ≥5% prevalence included ITP 12% (n=46), rheumatoid arthritis 10% (n= 41), gout 9% (n=36), and psoriasis 7% (n=28). To confirm the observed rate of hypothyroidism among our cohort, we explored the rate among MDS pts in the SEER registry where 45% of those registered as MDS had one or more hypothyroid claims (ICD code 244.9) among Medicare beneficiaries (2000-2005). (The prevalence of subclinical hypothyroidism is about 5% of women and in 3% of men with higher prevalence in elderly general population)
Baseline characteristics comparing AIDA-MDS (n=391) and non-AIDA-MDS (n=1017) (summarized in Table-1) were similar except AID were more common in females, RA or RCMDWHO subtype and pts were less RBC transfusion dependent.
Table 1Baseline characteristicsAIDA-MDS n=391Non AIDA MDS n= 1017P valueAge> 60293 (75%)739 (73%)0.4GenderFemale172 (44%)305 (30%)<0.005WHORA53 (14%)95 (9%)0.02RCMD131 (34%)299 (29%)RARS32 (8%)90 (9%)Del 5q12 (3%)45 (4%)RAEB I60(15%)193 (19%)RAEB II56 (14%)169 (17%)AML8 (2%)36 (4%)CMML8 (2%)24 (2%)MDS U13 (3%)10 (1%)MDS/MPN -U18 (5%)50 (5%)unknown06 (1%)IPSSLow85 (23%)183 (20%)0.25Int-1186 (50%)464 (50%)Int-283 (22%)204(22%)High22 (5%)82 (8%)R-IPSSVery low68 (19%)128 (14%)0.1Low127 (35%)306(34%)Intermediate77 (21%)188 (21%)High51 (14%)142 (16%)Very High41 (11%)135 (15%)Hypocellular marrowMCC only/Yes31 (10%)70 (10%)0.5Marrow FibrosisMCC only/Grade 2-340 (13%)95 (13%)0.7PNH clonedetected6 (2%)14 (1%)0.8LGL cloneMCC only / >50024 (8%)41 (6%)0.053T cell clonalityMCC only / + Beta or gamma28/71 (39%)55/112 (40%)0.5KarytotypeFavorable260 (66%)640 (65%)0.4Intermediate67 (17%)152 (15%)Poor59 (15%)183 (19%)missing5 (1%)15 (2%)Trisomy 8Yes44 (12%)89 (9%)0.3RBC transfusionDependence256 (66%)729 (72%)0.016HMA (MCC)Azacitidine168 (54%)344 (47%)0.8Decitabine38 (12%)119 (16%)0.1
Median OS was 60 mo (95% CI 50-70) for AIDA-MDS compared to 45 mo (95% 40-49) for those with no AIDA-MDS (log rank test, p = 0.006). By multivariate analysis adjusting for revised IPSS and age >60, AID was a statistically significant independent factor for OS (HR 0.78 (95% CI 0.66-0.92) (p=0.004). The rate of AML transformation was 23% (n=89) among AIDA-MDS compared to 30% (n=301) in non AIDA-MDS (p=0.006). There were no observed differences in response to treatment including azacitidine or Lenalidomide among evaluable patients for response.
AID are commonly associated with MDS, accounting for 28% of patients in our large cohort. Hypothyroidism was the most prevalent AID (12%) with similar high observation among Medicare MDS beneficiaries. AID was significantly more common in women, and associated with more RA/RCMD WHO subtypes with significantly reduced risk of AML transformation and death.
No relevant conflicts of interest to declare.
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