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Summary Background Refractory coeliac disease type 2 is a rare subtype of coeliac disease with high mortality rates; interleukin 15 (IL-15) is strongly implicated in its pathophysiology. This trial aimed to investigate the effects of AMG 714, an anti-IL-15 monoclonal antibody, on the activity and symptoms of refractory coeliac disease type 2. Methods This was a randomised, double-blind, placebo-controlled, phase 2a study of adults with a confirmed diagnosis of refractory coeliac disease type 2. Patients were randomly assigned at a 2:1 ratio to receive seven intravenous doses over 10 weeks of AMG 714 (8 mg/kg) or matching placebo. Biopsy samples were obtained at baseline and week 12 for cellular analysis and histology. The change in the proportion of aberrant intraepithelial lymphocytes from baseline to week 12 with respect to all intraepithelial lymphocytes was the primary endpoint and was quantified using flow cytometry. Secondary endpoints were the change in aberrant intraepithelial lymphocytes with respect to intestinal epithelial cells; intestinal histological scores (villous height-to-crypt depth ratio; VHCD); intraepithelial lymphocyte counts; Marsh score; and patient-reported symptom measures, including the Bristol stool form scale (BSFS) and gastrointestinal symptom rating scale (GSRS). Main analyses were done in the per-protocol population of patients who received their assigned treatment, provided evaluable biopsy samples, and did not have major protocol deviations; only patients with non-atypical disease were included in the analyses of aberrant intraepithelial lymphocytes, including the primary analysis. Safety was assessed in all patients who received at least one dose of study drug. This study is registered at ClinicalTrials.gov (NCT02633020) and EudraCT (2015-004063-36). Findings From April 13, 2016, to Jan 19, 2017, 28 patients were enrolled and randomly assigned to AMG 714 (n=19) and placebo (n=9). Six patients were not included in the primary analysis because of protocol deviation (one in the AMG 714 group), insufficient biopsy samples (one in the AMG 714 group), and atypical intraepithelial lymphocytes (three in the AMG 714 group and one in the placebo group). At 12 weeks, the least square mean difference between AMG 714 and placebo in the relative change from baseline in aberrant intraepithelial lymphocyte percentage was −4·85% (90% CI −30·26 to 20·56; p=0·75). The difference between the AMG 714 and placebo groups in aberrant intraepithelial lymphocytes with respect to epithelial cells at 12 weeks was −38·22% (90% CI −95·73 to 19·29; nominal p=0·18); the difference in change in Marsh score from baseline was 0·09% (95% CI −1·60–1·90; nominal p=0·92); the difference in VHCD ratio was 10·67% (95% CI −38·97 to 60·31; nominal p=0·66); and the difference in change in total intraepithelial lymphocyte count was −12·73% (95% CI −77·57–52·12); nominal p=0·69). Regarding symptoms, the proportion of patients with diarrhoea per the BSFS score decreased from ten (53%) of 19 at baseline to seven (37%) of 19 at week 12 in the AMG 714 group and increased from two (22%) of nine at baseline to four (44%) of nine at week 12 in the placebo group (nominal p=0·0008); and the difference between the groups in change in GSRS score was −0·14 (SE 0·19; nominal p=0·48). Eight (89%) patients in the placebo group and 17 (89%) in the AMG 714 group had treatment-emergent adverse events, including one (11%) patient in the placebo group and five (26%) in the AMG 714 group who had serious adverse events. The most common adverse event in the AMG 714 group was nasopharyngitis (eight [42%] patients vs one [11%] in the placebo group). Interpretation In patients with refractory coeliac disease type 2 who were treated with AMG 714 or placebo for 10 weeks, there was no difference between the groups in terms of the primary endpoint of aberrant intraepithelial lymphocyte reduction from baseline. Effects on symptoms and other endpoints suggest that further research of AMG 714 may be warranted in patients with refractory coeliac disease type 2. Funding Celimmune and Amgen.

Goals: To validate cut-off values of CD 3+ T-cell receptor gammadelta chain (TCRγδ + ) intraepithelial lymphocyte (IEL) in the (differential) diagnosis of celiac disease (CD). Background: CD is characterized by an increase in gamma-delta IEL (CD 3+ TCRγδ + IEL). Study: Percentages were determined by flow cytometric analysis of IELs from small bowel biopsies in 213 CD and 13 potential CD (PCD) patients and in total 112 controls. A cut-off value for percentages of CD 3+ TCRγδ + IEL to differentiate active CD and controls was obtained from a receiver operating characteristic curve and implemented in controls and PCD patients. Results: Percentage of CD 3+ TCRγδ + IEL was significantly increased in the majority of CD patients, irrespective of the presence of villous atrophy. A cut-off value of 14% for CD 3+ TCRγδ + IEL resulted in 66.3% sensitivity and 96.6% specificity for CD diagnosis (area under the curve, 88.6%). Conclusions: A percentage of ≥14% CD 3+ TCRγδ + IEL has a high specificity for CD diagnosis and can be of diagnostic help in cases where diagnosis is not straightforward.

Natural killer (NK) cells are a population of innate immune cells known to play a pivotal role against tumor spread. In multiple murine models, it was shown that physical exercise had the potential to increase NK cell antitumor activity through their mobilization and tissue redistribution in an interleukin (IL)‐6 and epinephrine‐dependent manner. The translation of this finding to patients is unclear. In this randomized pilot trial, we analyzed blood samples of patients with resectable breast or colon cancer who were randomized into an evidence‐based moderate‐high intensity resistance and aerobic exercise intervention (n = 8) or a control group (n = 6) during the first 9–12 weeks of (neo)adjuvant chemotherapy. In this pilot, we did not solely focus on statistical significance, but also explored whether average between‐group differences reached 10%. NK cell degranulation was preserved in the exercise group whereas it decreased in the control group resulting in a between‐group difference of 11.4% CD107a+ degranulated NK cells (95%CI = 0.57;22.3, p = 0.04) in the presence and 13.8% (95%CI = −2.5;30.0, p = 0.09) in the absence of an anti‐epidermal growth factor receptor monoclonal antibody (EGFR‐mAb). In line, the between‐group difference of tumor cell lysis was 7.4% (95%CI = −9.1;23.9, p = 0.34), and 13.7% (95%CI = −10.1;37.5, p = 0.23) in favor of the exercise group in the presence or absence of EGFR mAb, respectively. Current explorative analyses showed that exercise during (neo)adjuvant chemotherapy may benefit NK cell activity. Future studies with a larger sample size are needed to confirm this finding and to establish its clinical potential. Trial registration: Dutch trial register number NTR4105., We analyzed blood samples of patients with resectable breast or colon cancer who were randomized into an evidence‐based exercise intervention (n = 8) or control group (n = 6) during the first 9–12 weeks of (neo)adjuvant chemotherapy. NK cell degranulation was preserved during exercise and, although not statistically significant, cytotoxicity levels were 7%–14% higher compared to controls suggesting that physical exercise during (neo)adjuvant chemotherapy might benefit NK cell activity.

Our understanding of the coronavirus disease-19 (COVID-19) immune response is almost exclusively derived from studies that examined blood. To gain insight in the pulmonary immune response we analysed BALF samples and paired blood samples from 17 severe COVID-19 patients. Macrophages and T cells were the most abundant cells in BALF. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells and expressed higher levels of the exhaustion marker PD-1 than in peripheral blood. Prolonged ICU stay associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. In conclusion, the bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood.SummaryThe bronchoalveolar immune response in severe COVID-19 strongly differs from the peripheral blood immune profile. Fatal COVID-19 associated with T cell activation blood, but not in BALF.

It has been suggested that gluten may play a role in equine inflammatory small bowel disease (ISBD). Previous work showed an association between equine gluten-sensitive enteropathy and IgA antibodies to tissue transglutaminase (TGA) in serum. The purpose of this study is to investigate the prevalence of IgA antibodies to TGA in a group of healthy non–gluten-free sport ponies and to present a case of tentative gluten-sensitive enteropathy and dermatitis in a horse. Blood samples were obtained from 40 healthy jumping ponies. The ponies comprised 12 mares, 8 stallions, and 20 geldings with an average age of 9.0 ± 3.8 years (±SD; range 3–19 years). Sera were tested for IgA antibodies against human recombinant TGA. Significance (P < .05) of the correlation between TGA titer and age in these ponies was assessed using Pearson test (two tailed). In addition, to further illustrate tentative equine gluten-sensitive enteropathy and dermatitis, the clinical course in an 11-year-old Dutch Warmblood sport horse gelding has been described. The average TGA titer was 21.4 ± 13.6 AU/mL (range 2–65 AU/mL). There was a significant (P = .013) correlation (r = 0.389) between age and TGA titer in ponies. One of the 40 ponies (2.5%) showed an elevated TGA titer. An elevated TGA titer decreased after a gluten-free ration for 3 months in an 11-year-old Warmblood gelding with a tentative diagnosis of ISBD associated with full remission of the generalized skin reaction. To our best knowledge, this is the first study assessing TGA antibodies in sera from healthy non–gluten-free ponies and showing a correlation with age. The presented case could be the first one of a horse with a tentative diagnosis of gluten-sensitive enteropathy combined with dermatitis. Given the reported findings, this study warrants further investigations into gluten-sensitive enteropathy and dermatitis in individual horses affected with ISBD.

Background: Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets. Objective: To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT. Methods: Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1β) in the absence or presence of NiSO4. The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSElow/neg CD3+CD4+ T-lymphocytes upon nickel stimulation compared to the percentage of CFSElow/neg CD3+CD4+ T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test. Results: Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4+ cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails. Conclusions: Here, we describe an optimized and highly accurate flow cytometric LPT which comprises of CFSE-labelled cells cultured in autologous serum (not heat inactivated) and without the presence of T cell skewing cytokines. Clinical relevance: The sensitivity and specificity of LPT is enhanced, compared to HI HPS, when autologous serum without skewing cytokines is used.

ObjectiveWe aimed to determine the predictive capacity and diagnostic yield of a 10-fold increase in serum IgA antitissue transglutaminase (tTG) antibody levels for detecting small intestinal injury diagnostic of coeliac disease (CD) in adult patients.DesignThe study comprised three adult cohorts. Cohort 1: 740 patients assessed in the specialist CD clinic at a UK centre; cohort 2: 532 patients with low suspicion for CD referred for upper GI endoscopy at a UK centre; cohort 3: 145 patients with raised tTG titres from multiple international sites. Marsh 3 histology was used as a reference standard against which we determined the performance characteristics of an IgA tTG titre of ≥10×ULN for a diagnosis of CD.ResultsCohort 1: the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for IgA tTG levels of ≥10×ULN at identifying individuals with Marsh 3 lesions were 54.0%, 90.0%, 98.7% and 12.5%, respectively. Cohort 2: the sensitivity, specificity, PPV and NPV for IgA tTG levels of ≥10×ULN at identifying individuals with Marsh 3 lesions were 50.0%, 100.0%, 100.0% and 98.3%, respectively. Cohort 3: the sensitivity, specificity, PPV and NPV for IgA tTG levels of ≥10×ULN at identifying individuals with Marsh 3 lesions were 30.0%, 83.0%, 95.2% and 9.5%, respectively.ConclusionOur results show that IgA tTG titres of ≥10×ULN have a strong predictive value at identifying adults with intestinal changes diagnostic of CD. This study supports the use of a no-biopsy approach for the diagnosis of adult CD.

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of connective tissue diseases, collectively known as myositis. Diagnosis of IIM is challenging while timely recognition of an IIM is of utter importance considering treatment options and otherwise irreversible (severe) long-term clinical complications. With the EULAR/ACR classification criteria (2017) considerable advancement has been made in the diagnostic workup of IIM. While these criteria take into account clinical parameters as well as presence of one autoantibody, anti-Jo-1, several autoantibodies are associated with IIM and are currently evaluated to be incorporated into classification criteria. As individual antibodies occur at low frequency, the development of line blots allowing multiplex antibody analysis has improved laboratory diagnostics for IIM. The Euroline myositis line-blot assay (Euroimmun) allows screening and semi-quantitative measurement for 15 autoantibodies, i.e. myositis specific antibodies (MSA) to SRP, EJ, OJ, Mi-2α, Mi-2β, TIF1-γ, MDA5, NXP2, SAE1, PL-12, PL-7, Jo-1 and myositis associated antibodies (MAA) to Ku, PM/Scl-75 and PM/Scl-100. To evaluate the clinical significance of detection and levels of these autoantibodies in the Netherlands, a retrospective analysis of all Dutch requests for extended myositis screening within a 1 year period was performed. A total of 187 IIM patients and 632 non-IIM patients were included. We conclude that frequencies of MSA and MAA observed in IIM patients in a routine diagnostic setting are comparable to cohort-based studies. Weak positive antibody levels show less diagnostic accuracy compared to positive antibody levels, except for anti-NXP2. Known associations between antibodies and skin involvement (anti-MDA5, anti-TIF1-γ), lung involvement (anti-Jo-1), and malignancy (anti-TIF1-γ) were confirmed in our IIM study population. The availability of multiplex antibody analyses will facilitate inclusion of additional autoantibodies in clinical myositis guidelines and help to accelerate diagnosing IMM with rare but specific antibodies., Highlights • Similar frequency of MAA and MSA in diagnostic setting compared to cohort studies. • Positive levels of MSA/MAA show better diagnostic accuracy compared to weak positive levels. • Anti-NXP2 differentiates already at weak positive level between IIM and non-IIM patients. • Anti-TIF1-γ is associated with malignancy and anti-MDA5 and TIF1-γ with skin disease. • Anti-Jo-1 is associated with lung disease.

Background Titanium is being increasingly used. Although it is considered to be a non-allergenic material, allergic reactions to it have been reported. Titanium dioxide has been found to be an unreliable patch test material. Few studies to date have profiled titanium allergy, and it therefore remains difficult to distinguish its manifestations. Objectives To evaluate alternatives for titanium dioxide as a patch test preparation, and to profile titanium reactions and manifestations. Methods A retrospective chart review was conducted with 458 patients who underwent patch testing with at least 1 of 5 different titanium salts. Results At least 1 positive result was noted in 5.7% of the patients. The frequency of positive results for the tested salts ranged from 0.9% to 7.9%. Titanium(IV) oxalate hydrate had the highest yield and titanium dioxide the lowest. Erythema, dermatitis and local swelling were the most common objective complaints. In 16 (61.5%) patients, the test result had partial or full clinical relevance. Conclusions No titanium-specific risk factors and clinical picture could be identified. Titanium dioxide is not adequately sensitive for identifying titanium allergy. The titanium salts seem to be possible superior patch test preparations, but appear to be unsuitable if used singly. The patient's medical history and clinical picture remain crucial in the diagnostic work-up.

Objectives. This study aimed to assess the impact of celiac disease (CD) on oral health and xerostomia. Study Design. Members of the Dutch Celiac Society (n = 5522) were invited to complete an online questionnaire based on the Oral Health Impact Profile 14 (OHIP-14) and Xerostomia Inventory (XI). Acquaintances and partners of the CD respondents served as the comparison group. In total, data of 740 patients with CD and 270 comparison participants were evaluated. Results. The median age of the responding patients with CD (55 years) was similar to the median age in the comparison group (53 years). Oral health problems, including aphthous stomatitis, painful mouth, and gingival problems, were more frequently reported by patients with CD. Mean OHIP-14 score (4.9 vs 2.6; P Conclusions. This study showed that oral health problems are more commonly experienced in adult patients with CD than in the comparison group. Collaboration between dentists and gastroenterologists is recommended to increase detection of undiagnosed CD.

Aim: Celiac disease (CD) is strongly associated with HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8. Up to 99.7% of all CD patients are positive for either one or two of these genetic markers, demonstrating a high negative predictive value. This has led to the development of diagnostic kits that, instead of providing a full HLA-DQ typing, detect only these three HLA-DQ types. Our aim was to compare three different kits for their performance, utilization, and costs. Because 0.4-3.6% of all CD patients test positive for HLA-DQ7 and negative for the aforementioned types, information provided by the kits regarding DQ7 alpha and beta chains was evaluated as well. Materials and Methods: Fifty DNA samples previously typed with the SSCP method were analyzed using three commercial kits. Results and Discussion: All kits report hetero- or homozygosity for HLA-DQ2.5. The XeliGen kit directly detects HLA-DQ7, but is relatively expensive. The MLPA kit is the least expensive in terms of reagents and may indirectly detect HLA-DQ7. The CeliaSCAN kit is easy to use and provides indirect information about HLA-DQ7.5. Conclusion: All kits correctly identify the CD risk genes. The resources of the laboratory and the intended use should determine the preference for any of the HLA-DQ typing kits herein described.

Immune-mediated enteropathies are caused by excessive reactions of the intestinal immune system towards non-pathogenic molecules. Enteropathy leads to malabsorption-related symptoms and include (severe) chronic diarrhea, weight loss and vitamin deficiencies. Parenteral feeding and immunosuppressive therapy are needed in severe cases. Celiac disease has long been recognized as the most common immune-mediated enteropathy in adults, but the spectrum of immune-mediated enteropathies has been expanding. Histological and clinical features are sometimes shared among these enteropathies, and therefore it may be challenging to differentiate between them. Here, we provide an overview of immune-mediated enteropathies focused on clinical presentation, establishing diagnosis, immunopathogenesis, and treatment options.

Aminobisphosphonates (NBP) are used for treatment of metastatic bone disease. Frequently, patients undergoing NBP-treatment experience side-effects, known as acute phase response (APR), resulting from cytokine production by Vγ9Vδ2-T cells. As opposed to NBP, statins reduce intracellular phosphoantigen levels and prevent NBP-induced Vγ9Vδ2-T cell activation in vitro. We conducted a pilot study in patients with (bone-)metastasized malignancies receiving NBP-treatment and evaluated the phenotype and function of circulating Vγ9Vδ2-T cells in vivo and the effects of statins on Vγ9Vδ2-T cell responses and the associated APR. We observed reduced expression of perforin, granzyme B and HLA-DR on Vγ9Vδ2-T cells in patients treated with NBP and statins. However, statins could not prevent NBP-induced changes in circulating Vγ9Vδ2-T cell numbers or production of IFNγ and TNFα. Consistent with this, simvastatin could not prevent the occurrence of APR upon NBP-infusion. These observations call for the exploration of alternative strategies to prevent collateral APR upon NBP treatment.

Background: Refractory coeliac disease type II (RCDII) frequently transforms into an enteropathy-associated T-cell lymphoma (EATL) and therefore requires intensive treatment. Current evaluated treatment strategies for RCDII include cladribine (2-CdA) and autologous stem cell transplantation (auSCT). Objective: The purpose of this study was to evaluate long-term survival and define clear prognostic criteria for EATL development comparing two treatment strategies. Methods: A total of 45 patients were retrospectively analysed. All patients received 2-CdA, after which they were either closely monitored (monotherapy, n = 30) or a step-up approach was used including auSCT (step-up therapy, n = 15). Results: Ten patients (22%) ultimately developed EATL; nine of these had received monotherapy. Absence of histological remission after monotherapy was associated with EATL development (p = 0.010). Overall, 20 patients (44%) died with a median survival of 84 months. Overall survival (OS) within the monotherapy group was significantly worse in those without histological remission compared to those with complete histological remission(p = 0.030). The monotherapy group who achieved complete histological remission showed comparable EATL occurrence and OS as compared to the step-up therapy group (p = 0.80 and p = 0.14 respectively). Conclusion: Histological response is an accurate parameter to evaluate the effect of 2-CdA therapy and this parameter should be leading in the decisions whether or not to perform a step-up treatment approach in RCDII.

Background & Aims: In most cases celiac disease (CD) is successfully treated with a gluten-free diet (GFD). However, some patients become refractory to the GFD. Refractory CD (RCD) patients have an increased risk for developing enteropathy associated T-cell lymphoma and early diagnosis is therefore of importance. Currently, RCD diagnosis relies on endoscopy and adequate serological markers are lacking. Antibodies against glycoprotein-2 (GP2A) were described in Crohn's disease (CrD) and active CD but not in ulcerative colitis (UC), suggesting this is a specific marker for small intestinal lesions.Methods: Sera obtained from patients visiting our outpatient clinic for routine serological tests for diagnosis and/or follow-up of inflammatory bowel disease (n=78), active CD (n=45), GFD (n=34) and RCD (n=15) were analysed for GP2A titres.Results: Increased GP2A-IgA levels in CrD and active CD as compared to controls (p

Department of Oral and Maxillofacial Surgery/Oral Pathology, Academic Centre for Dentistry(ACTA), Amsterdam, The NetherlandsBACKGROUND: The immune system plays an impor-tant role in tumour immune surveillance. Head andneck squamous cell carcinoma patients are oftenimmune compromised.OBJECTIVE: To chart the baseline levels of T-cellsubpopulation frequencies in patients with cancer priorto treatment.SUBJECTS AND METHODS: Blood samples of patientswere taken at the time of diagnosis, analysed with flow-cytometry and compared with blood samples of healthydonors.RESULTS: Compared to healthy donors, a significantshift from naive to effector memory T cells wasobserved. This effect was most prominent in stage IIpatients. A similar shift from naive to effector memoryT cells was noted in patients with oropharynx or larynxsquamous cell carcinomas. Furthermore, the percent-age of effector memory and effector T cells was higherin the group of patients with human papillomavirus-positive oropharyngeal squamous cell carcinomas,compared with patients with human papillomavirus-neg-ative tumours, suggestive of virus-induced T-cell activa-tion.CONCLUSION: Here, we provide a simple and easilyimplementable tool to document T lymphocyte subsetsin the peripheral blood of head and neck cancerpatients, which might be useful for prognosis and/ortherapy response prediction.Oral Diseases (2012) doi:10.1111/odi.12037Keywords: head and neck cancer; T cell subpopulations; HPV;peripheral blood lymphocytes; prognostic factor

This review aims at summarizing the present knowledge on the clinical consequences of concomitant coeliac disease (CD) in adult patients with type 1 diabetes mellitus (T1DM). The cause of the increased prevalence of CD in T1DM patients is a combination of genetic and environmental factors. Current screening guidelines for CD in adult T1DM patients are not uniform. Based on the current evidence of effects of CD on bone mineral density, diabetic complications, quality of life, morbidity and mortality in patients with T1DM, we advise periodic screening for CD in adult T1DM patients to prevent delay in CD diagnosis and subsequent CD and/or T1DM related complications.

Treatment of chronic myeloid leukemia with tyrosine kinase inhibitors, such as imatinib mesylate, dasatinib and nilotinib, results in high rates of cytogenetic and molecular responses. However, in many patients, minimal residual disease is detected by molecular techniques. Since chronic myeloid leukemia cells are particularly good targets for immune surveillance mechanisms, we explored active specific immunotherapy using leukemia lysate-loaded dendritic cells in vitro. Our data show the potency of dendritic cell-based vaccination strategies for the induction of T cell-mediated responses to eradicate minimal residual disease.

Introduction: A small proportion of coeliac disease (CD) patients become refractory (RCD) to a gluten-free diet (GFD) showing uncontrolled immune-mediated enteropathy. Some of these patients exhibit a high risk to develop enteropathy-associated T-cell lymphoma (EATL). Aim: The aim of the study was to find whether a lack of circulating homeostatic T cells, such as Treg, Tγδ+ or iNKT cells would be associated with the development of RCD or EATL. Patients and methods: We investigated in a total of 137 CD patients [28 untreated, 80 responsive to GFD and 29 RCD (14 type I and 15 type II)] and 73 age-matched healthy volunteers the frequency of Treg, Tγδ+ and iNKT lymphocytes by flow cytometric analysis of peripheral blood. Results: Circulating Tγδ+ cell and Treg frequencies in RCD were comparable to those in healthy controls. However, RCD patients had significantly reduced numbers of circulating iNKT cells, as compared to age-matched patients responding to a GFD. This reduction was similar in RCD patients with and without aberrant intraepithelial lymphocytes and in patients with EATL. Circulating iNKT cell numbers were not reduced in untreated coeliac patients. GFD treated patients had a significantly increased proportion of CD4+ iNKT cells. Conclusion: Follow-up studies are necessary to determine whether CD4+ iNKT cells control the immune response against gluten and their absence contributes to the progression to RCD and EATL.

The huHMFG-1 (AS1402) antibody is a humanised IgG1 directed against MUC1 and is currently in clinical trials for the treatment of breast carcinoma. Adenocarcinomas over-express and shed MUC1, and high MUC1 serum levels are associated with progressive disease. Here, we have investigated the effects of MUC1 present in sera from breast and ovarian cancer patients and that of NK cells on in vitro huHMFG-1-mediated ADCC, performed with and without the addition of various cytokines. Screening for patients with high levels of NK cells bearing the FcγRIIIa-158V polymorphism, adjusting the dosage to circulating levels of MUC1 and co-administration of NK cell activating cytokines may increase the efficacy of huHMFG-1 treatment.

Purpose Evading antitumor immune responses is an important aspect of the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Invariant CD1d-restricted natural killer T (iNKT) cells play an allegedly pivotal role in such responses via transactivation of immune effector cells. It has been reported that iNKT cells are reduced in peripheral blood of cancer patients compared with healthy controls. Here, we investigated whether the extent of this deficiency affected disease outcome in HNSCC patients. Patients and Methods In a prospective study, circulating iNKT cell numbers were evaluated in 47 patients before radiotherapy. Patients were stratified in three groups based on iNKT cell levels, and clinical data were obtained during a median follow-up period of 31 months. Results A small, compared with an intermediate or large, circulating iNKT cell fraction was significantly associated with decreased 3-year overall survival rate (39% v 75% and 92%, respectively), disease-specific survival rate (43% v 87% and 92%, respectively), and locoregional control rate (31% v 74% and 92%, respectively) in HNSCC patients. Cox regression revealed that the iNKT cell level, as well as clinical T stage, was an independent prognostic parameter even after correction for the confounding effect of age. Conclusion A severe circulating iNKT cell deficiency was related to poor clinical outcome in HNSCC patients, suggesting their critical contribution to antitumor immune responses. Furthermore, screening for iNKT cell levels may be useful for determining which patients can benefit from immunotherapeutic adjuvant therapies aimed at reconstitution of the circulating iNKT cell pool.

Early recognition followed by treatment of rheumatoid arthritis (RA) helps to maintain joint integrity and functional capacity,1 suggesting it may be beneficial to intervene in patients with arthralgia before RA develops. Anticitrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are established predictive markers,2 ,3 but only 20–40% of ACPA and/or RF positive patients with arthralgia develop RA within 2 years.4 Recently, we have demonstrated that the type I interferon (IFN) signature correctly identifies 52% of patients with arthralgia who will develop RA within 2 years.5 ,6 Our previous study suggested that a B cell related gene signature was associated with protection against arthritis development,6 and could aid in the prediction of arthritis development. We therefore studied the clinical value of the B cell signature, comprising CD19, CD20, CD79α and CD79β, for the prediction of arthritis development in an independent cohort of 115 ACPA and/or RF positive patients with arthralgia followed for median 22 months5 and explored the phenotypical nature of this B cell signature. In total, 44 patients (38%) developed arthritis (defined as one or more swollen joints) within 2 years. Of these, 4 patients had undifferentiated arthritis and 40 patients fulfilled the 2010 American College of Rheumatology (ACR)/ European League Against Rheumatism (EULAR) criteria for RA. Patients were stratified into B cellhigh …

Objectives: Enteropathy associated T-cell lymphoma (EATL) is a rare non-Hodgkin lymphoma that may complicate coeliac disease and typically occurs in patients with refractoriness to the gluten-free diet. The majority of these patients harbour a clonal expansion of intraepithelial lymphocytes (IELs) with an aberrant phenotype in the small intestine which are thus considered as the 'precursor' lymphoma cells. We describe a 51-year-old female patient with refractory coeliac disease (RCD) who developed an EATL with manifestations in the proximal small intestine and in a mesenteric lymph node that did not evolve from regular type 'aberrant' αβ-T-cells but rather from a clonal expansion of γδ-T-cells. Methods: Duodenal biopsies and lymphoma tissue from a patient with refractory coeliac disease whom developed an EATL were extensively studied by immunophenotypical, T-cell receptor immunogenetic and chromosomal analysis. Results: Flow cytometric analysis of duodenal IELs revealed an unusual large clonal expansion of CD30 negative γδ-T-cells in a patient with RCD. When the patient clinically deteriorated 18 months later, a substantial part (30%) of this cell population did express CD30. In addition, identical immunogenetic aberrancies had developed in a prehepatic lymph node. Conclusions: We here report on a case of extraintestinal EATL that originated from a clonal γδ-IEL population rather than from aberrant IEL. This EATL displayed a distinctive pattern of immunophenotypical, T-cell receptor immunogenetic and chromosomal aberrancies as compared to classical EATL, defining this lymphoma as a novel variant of EATL.

Different strategies have been developed to identify those refractory celiac disease (RCD) patients who are at risk to develop an enteropathy associated T-cell lymphoma (EATL). Flow cytometric analysis of intra-epithelial lymphocytes (IEL) with an aberrant phenotype is considered the golden standard but is not widely available. Immunohistochemistry (IHC) and T-cell receptor (TCR) rearrangement studies are commonly available but may lack sensitivity and specificity. Here, we compared the three different methods in the workup of patients suspected for RCD. Duodenal biopsies from control patient (n = 5), RCD patients with moderately increased aberrant IEL populations (20–50 %: n = 14), and RCD patients with high numbers of aberrant IEL (>50 %: n = 5) as determined by flow cytometry were analysed by IHC and TCR-γ chain rearrangement analysis. Three pathologists scored the slides independently. Sensitivity of IHC and TCR-γ rearrangement analysis in RCD patients with high numbers of aberrant IELs was 100 and 71 %, respectively. RCD patients with aberrant cells between 25 and 50 % however, were missed by IHC and TCR in 50 and 57 % of cases, respectively. In addition, inter-rater reliability analysis of the IHC scoring revealed coder-pair Kappa coefficients between 0.28 and 0.85. Immunohistochemistry and to a lesser extent TCR-γ clonality analysis are sensitive in identifying patients with high numbers of aberrant IEL populations, yet miss half of RCD patients with moderately increased numbers. In addition, IHC has a high inter-observer variability. Therefore, patients suspected for RCD should undergo flow cytometric analysis of the duodenum.

Aims: Vaccination with acute myeloid leukemia (AML)-derived dendritic cells (DCs) is a promising immunotherapeutic approach to prevent relapse of AML. However, in clinical practice AML-derived DC culture is unfeasible in 40% of cases. Here, we demonstrate that AML cells can be expanded in vitro prior to differentiation with cocktails of cytokines with known myeloid growth-promoting effects. Results: Nine out of 13 initially CD14- samples gain de novo CD14 (>10%) expression (69% increment; p = 0.01) after in vitro expansion. These expanded CD14+ leukemic cells displayed a high probability (six out of six initially CD14- samples tested) to differentiate into DCs upon culture with GM-CSF, TNF-α and IL-4. Conclusion: Induction of CD14 on initially CD14- AML cells potentially increases the number of patients eligible for DC-based immunotherapy.

Harvesting the potential of the immune system in order to eradicate (residual) acute myeloid leukemia (AML) cells is the long pursued goal of immunotherapy in AML. Strategies using apoptotic tumor cell vaccines have been explored for many years, without significant clinical improvements. In recent years insight has been gained into the mechanisms activating and interfering with tumor-directed immunity. With the arrival of novel immune-modulating agents allowing for the interference with regulatory molecules and interaction with immune-propelling mechanisms, new doors are opening for increasing vaccination efficacy. Combined with advances in the design of apoptotic tumor-based vaccines, we are on the verge of creating an effective AML vaccine strategy, offering a much needed novel therapeutic option for this devastating disease.

Active immunotherapy may prevent the relapse of acute myeloid leukemia (AML) by inducing leukemia-specific T cells. Here, we investigated whether Wilms' tumor 1 (WT1) and preferentially expressed antigen in melanoma (PRAME)-specific T cells could be induced upon the priming of healthy donor- and AML patient-derived T cells with HLA-A2-matched, peptide-loaded allogeneic dendritic cells. AML-reactive, tetramer (Tm)-binding and interferon-producing, cytotoxic T lymphocytes specific for PRAME could readily be isolated from healthy individuals and maintained in culture. In this setting, priming efficacy was significantly higher for PRAME than for WT1. The priming of T cells from patient-derived material proved to be near-to-impossible: No leukemia-associated antigen (LAA)-specific T cell could be primed in 4 patients that had recently achieved a complete response (CR), and in only 1 out of 3 patients exhibiting a sustained CR we did observe WT1-specific T cells, though with a low frequency. These findings suggest that the functionality and/or repertoire of T cells differ in healthy subjects and AML patients in CR, and may have repercussions for the implementation of active vaccination approaches against AML.

IgG reactivity against the immunodominant region aa6-35 of Human Papillomavirus (HPV) type-16 E7 was determined in a peptide-based ELISA in a cohort study of women with initial mild to moderate cervical dyskaryosis. On the basis of HPV DNA patterns, as determined by PCR in cervical smears prior to IgG testing, HPV-16-positive patients were grouped as having either a cleared, a fluctuating, or a persistent HPV-16 infection. In a cross-sectional study at the start of serological follow-up, positive IgG reactivities were found more often in the total group of HPV-16-positive patients (20.0%) than in patients consistently typed as HPV-negative over a period of at least 12 months prior to testing (3.1%, p < 0.04). The highest proportion of positive responders was found in patients with a cleared HPV-16 infection (29.4%). Also, IgG reactivities found in HPV-16 clearance patients were significantly higher than in patients with a persistent infection (p < 0.008). In a subsequent longitudinal study over a period of up to 27 months, consistently positive reactivities were observed in patients with cleared viral infections who showed seroreactivity in the cross-sectional study, while mostly negative reactivities were found in patients with viral persistence. HPV-16 E7-specific IgG subclass responses were determined in a selection of 19 CIN and 11 HPV-16-positive cervical carcinoma (CeCa) patients with positive E7-specific IgG responses. IgG2 was predominant in the CIN patients, suggesting the presence of IFNgamma (Th1) at the site of HPV infection. In the CeCa patients IgG1 and IgG2 were produced equally, possibly indicating a rise in Th2 cytokines. Our data suggest that HPV-16 E7 IgG reactivity in a subset of CIN patients with viral clearance may result from successful Th1 responses.

Background and objectives A subgroup of rheumatoid arthritis (RA) patients displays elevated type I IFN response gene (IRG) expression in peripheral blood, which has shown clinical relevance in relation to disease onset and therapy response. Identification of the cell type(s) contributing to this IFN signature could provide insight into its functional consequences and pathologic role in RA. This study aimed to investigate the contribution of the major peripheral leukocyte subsets to the IFN signature in RA. Methods Blood was collected from 26 early RA patients and lysed directly or separated into mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs). Using flow cytometry, PBMCs were sorted into CD4 + T cells, CD8 + T cells, CD19 + B cells and CD14 + monocytes. mRNA expression levels of three IRGs (RSAD2, IFI44L and MX1), as well as type I IFN receptors IFNAR1 and IFNAR2, were determined in blood and cell subsets by qPCR. IRG expression was averaged to calculate an IFN score for each sample. Results Patients were designated “IFN high ” (n = 8) and “IFN low ” (n = 18) based on the IFN score cutoff in peripheral blood from healthy controls. As expected, IFN scores were significantly higher in all cell subsets from IFN high patients compared to IFN low patients. This difference was remarkably large for the PMN fraction (mean 25-fold, p Both IFNAR1 and IFNAR2 expression was highest in the PMN fraction compared to the other subsets, suggesting increased sensitivity of PMNs to type I IFNs. Concordantly, we observed IFNAR1 and IFNAR2 upregulation compared to healthy controls selectively in RA PMNs (p ≤ 0.0077) but not in the PBMCs. Conclusions PMNs are the main contributors to the whole blood type I IFN signature in RA patients, which seems due to increased sensitivity to type I IFN signalling. Considering the well-established role of neutrophils in the pathology of RA, this further supports a pathologic role of type I IFN activity in the disease.

For over a century, research has sought ways to boost the immune system of cancer patients in order to eradicate tumors that arose after escaping presumptive immunosurveillance. With increasing knowledge of the immune system, immunotherapeutic strategies to break tolerance to the tumor evolved from largely nonspecific to more specific and increasingly potent forms of cancer vaccination. Overall however, immunotherapeutic strategies for the treatment of cancer have had limited clinical success and an urgent need exists, therefore, to introduce more effective, knowledge-based therapeutic approaches. Invariant natural killer T (iNKT) cells constitute an evolutionary conserved T lymphocyte lineage with dominant immunoregulatory and antitumor effector cell properties. iNKT specifically recognize the glycolipid α-galactosylceramide (α-GalCer/KRN7000) in the context of the CD1d antigen-presenting molecule resulting in their activation. Activated iNKT have been shown to promote the development of a long-lasting Th1-biased proinflammatory antitumor immune response in a variety of murine tumor-metastasis models of liver, lung and lymph nodes, including colon and lung carcinoma, lymphoma, sarcoma, and melanoma, suggesting broad clinical applicability. Here, we will provide an overview of the preclinical data of α-GalCer that formed the basis for subsequent clinical trials in advanced cancer patients, review these clinical trials, focusing on our own experience with α-GalCer, and discuss future perspectives.

Introduction Immune response plays an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). It has been shown that a consistent immunological signature in MDS comprises an increase in Th17 cells in low risk MDS and an expansion of Tregs in high risk subtypes. The presence of high Treg numbers in lower risk stages is an independent prognostic value and is associated with poor survival. Whereas the immune response in low risk MDS is pro-inflammatory, high risk MDS is characterized by immunosuppression which could contribute into the expansion of the dysplastic clone and malignant transformation. The exact role of dendritic cells (DC) in inducing this immune dysfunction is yet to be understood. The aim of this comparative study was therefore to investigate the frequency and function of different DC subsets in the bone marrow (BM) and peripheral blood (PB) of MDS patients compared to healthy donors (HD). Patients and methods The enumeration of DC subsets was performed in 110 MDS and in 19 HD bone marrow samples. In peripheral blood 34 MDS and 18 HD samples were investigated. DC were identified by the expression of HLA-DR and the lack of the lineage markers CD14 and CD19. They were further subdivided into CD303+ plasmacytoid DC (pDC), and the CD11c+ myeloid subsets CD1c+ myeloid DC 1 (mDC1), CD141hi myeloid DC 2 (mDC2) and CD16+ 6-Sulfo LacNac (Slan) DC. Furthermore, their ability to upregulate co-stimulatory molecules was assessed by flow cytometry and the secretion of cytokines in response to TLR ligands was analyzed using Cytometric Bead Array (CBA). Their capacity to induce allogeneic T cell proliferation was evaluated in a mixed leukocyte reaction (MLR). Results The frequencies of pDC, mDC1 and mDC2 but not of SlanDC were significantly lower in patients' BM compared to HDs' BM (pDC 0.33% vs 0.72%, p=0.01; mDC1 0.30% vs 0.73%, p Conclusion Our data clearly point to both a numeric and functional impairment of myeloid DC subsets in BM as well as in PB of patients with MDS. A further decline of DC frequencies in high risk MDS may support the therapeutic targeting of DC in the BM microenvironment of these patients to redress immune dysfunction and possibly prevent further progression to acute myeloid leukemia. Extensive research is needed to further delineate the differences in DC function and immune composition in MDS risk categories. Disclosures Mufti: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The myelodysplastic syndromes (MDS) constitute a group of heterogeneous clonal haemopoietic stem cell disorders, characterized by ineffective and dysplastic haematopoiesis with varying degrees of peripheral cytopenia. Low-risk MDS is characterized by increased apoptosis in the bone marrow (BM) with autoimmune characteristics whereas the advanced or high-risk stages involve immune evasion and secondary DNA damage, giving cells growth potential to progress into acute myeloid leukaemia (AML). Nevertheless, the causes of MDS remain poorly defined and it is not clear how the disease progresses from an early stage to advanced MDS and AML. Although there are clear indications for a role of the immune system, the exact mechanism by which the immune response contributes to the progression is not yet clear. New insights into the pathophysiology of MDS with regard to the immune system will be instrumental for the development of novel patient-oriented therapies. This review is focused on the role of immune responses in MDS and the implications for the development of novel immune therapies.

Therapeutic vaccination with dendritic cells (DCs) is recognized as an important experimental therapy for the treatment of minimal residual disease in acute myeloid leukemia. Many sources of leukemia-associated antigens and different methods for antigen loading of DCs have been used in an attempt to optimize anti-tumor responses. For instance, monocyte-derived DCs have been loaded with apoptotic whole-cell suspensions, necrotic cell lysates, tumor-associated peptides, eluted peptides and cellular DNA or RNA. Furthermore, monocyte-derived DCs can be chemically or electrically fused with leukemic blasts, and DCs have been cultured out of leukemic blasts. However, it remains a challenge in cancer immunotherapy to identify which of these methods is the most optimal for antigen loading and activation of DCs. This review discusses recent advances in DC research and the application of this knowledge towards new strategies for antigen loading of DCs in the treatment of minimal residual disease in acute myeloid leukemia.

Item does not contain fulltext CD1d restricted, alpha-galactosylceramide (alphaGC) responsive invariant (i)NKT cells positively regulate immune responses. Both intravenous and intradermal administered alphaGC are known to activate iNKT cells. iNKT cells become unresponsive to a second intravenous alphaGC injection, whereas no data are available regarding potential anergy upon intradermal administration. Here, comparative analysis of two intradermal versus two intravenous injections in mice demonstrated that iNKT cell anergy was prevented by intradermal injection and when combined with a vaccine, superior tumor protection afforded by intradermally administered alphaGC. Moreover, human skin dendritic cells (DC) took up intradermally injected alphaGC and activated iNKT cells upon migration, while iNKT cells in human skin-draining lymph nodes expanded in response to alphaGC presented either by exogenously added DC or by CD1d positive antigen presenting cells in the lymph nodes. In conclusion, glycolipids such as alphaGC may greatly improve the efficacy of skin immunization strategies, targeting cutaneous and lymph node DC. 01 september 2010

Introduction Flow cytometry is a validated, easy applicable methodology for the determination of intraepithelial lymphocytes (IEL) phenotype in celiac disease (CD). An increase in Gamma-Delta IEL (CD3+TCRγδ+) is a characteristic finding in CD patients yet cut-off values for CD3+TCRγδ+ IEL in CD have not been determined. A validated cut-off value might be helpful in the establishing the diagnosis of CD, especially in those patients with minimal histological abnormalities (i.e., Marsh I) or individuals with positive serology in the absence of histological abnormalities. Methods Flow cytometric immunophenotyping was performed on IELs isolated from fresh small bowel biopsy specimens obtained from histology and serology proven CD patients (n=219) (active CD: n=92, CD in remission: n=127), healthy controls without CD (n=89) and suspected CD (n=14), the latter being defined as Marsh III and/or positive serology. Thirty-one additional patients with non-celiac villous atrophy were included as a disease control group. Follow-up data were available from thirteen CD patients. Percentages of CD3+TCRγδ+ IEL from CD patients and healthy controls were used to calculate a cut-off value for CD3+TCR γδ+ IEL in suspected CD patients using a receiver operating characteristic (ROC) curve. Results A significantly higher percentage of CD3+TCRγδ+ IEL was found in biopsy and serology proven CD patients (median 19.0%, range 1-58%) compared to healthy controls (median 6.0%, range 1-15%) (p 14% CD3+TCRγδ+ IEL might aid in a diagnosis of CD. Furthermore, Flow cytometric immunophenotyping might be helpful in cases of diagnostic doubt on CD in patients compliant to a GFD without the need for a gluten-challenge.

The generation and loading of dendritic cells (DC) ex-vivo for tumor vaccination purposes is laborious and costly. Direct intradermal (i.d.) administration of tumor-associated antigens could be an attractive alternative approach, provided that efficient uptake and cross-presentation by appropriately activated skin DCs can be achieved. Here, we compare the efficiency of i.d. delivery of relatively small apoptotic blebs (diameter ∼0.1–1 μm) derived from MART-1 transduced acute myeloid leukemia (AML) HL60 cells, to that of larger apoptotic cell remnants (ACR; 2–10 μm) in a physiologically highly relevant human skin explant model. Injection of either fluorescently-labelled ACRs or blebs alone did not affect the number or distribution of migrated DC subsets from skin biopsies after 48 hours, but resulted in a general up-regulation of the co-stimulatory molecules CD83 and CD86 on skin DCs that had ingested apoptotic material. We have previously shown that i.d. administration of GM-CSF and IL-4 resulted in preferential migration of a mature and highly T cell-stimulatory CD11hiCD1a+CD14− dermal DC subset. Here, we found that co-injection of GM-CSF and IL-4 together with either ACRs or blebs resulted in uptake efficiencies within this dermal DC subset of 7.6% (±6.1%) and 19.1% (±15.9%), respectively, thus revealing a significantly higher uptake frequency of blebs (P < 0.02). Intradermal delivery of tumor-derived blebs did not affect the T-cell priming and TH-skewing abilities of migratory skin DC. Nevertheless, in contrast to i.d. administration of ACR, the injection of blebs lead to effective cross-presentation of MART-1 to specific CD8+ effector T cells. We conclude that apoptotic bleb-based vaccines delivered through the skin may offer an attractive, and broadly applicable, cancer immunotherapy.

Background Infection with cancer-linked human papillomavirus (HPV) types such as HPV type 16 (HPV16) is the most important risk factor in the development of cervical cancer. It has been shown that immunoglobulin G (IgG) antibody responses against HPV16 virus-like particles (VLPs) are specifically associated with genital HPV16 infection. Purpose The aim of this study was to determine the temporal relationships between the presence of HPV16 VLP-specific IgGs, HPV16 infection patterns, and the course of premalignant cervical disease. Methods Plasma samples from 133 women who had been diagnosed originally with mild to moderate cervical dyskaryosis and enrolled in a prospective non-intervention cohort study conducted in Amsterdam, The Netherlands, from 1991 through 1996 were analyzed for the presence of HPV16 VLP-specific IgGs by use of an enzyme-linked immunosorbent assay. A detailed analysis was performed on 43 women with different HPV16 infection patterns during a follow-up period of 10-34 months. Progression or regression of cervical intraepithelial neoplasia (CIN) lesions was monitored by cytologic and colposcopic testing at intervals of 3-4 months. HPV typing in cervical smears was performed by use of a polymerase chain reaction-based assay. Statistical analysis of the serologic data was performed by use of the Mann-Whitney U test or 2 x 2 table analyses. Results The presence of HPV16 VLP-specific IgGs in the plasma of the patients was found to be associated with the presence of HPV16 DNA in the cervical smear. Significantly higher proportions of patients with persistent HPV16 infections (i.e., who were polymerase chain reaction positive in three to 11 consecutive tests) than of patients with cleared HPV16 infections were found to be positive for the presence of HPV16 VLP-specific IgGs (18 [69.2%] of 26 versus nine [28.1%] of 32, respectively; P = .003). HPV16 VLP-specific IgGs were consistently detected in all women (n = 11) who were persistently HPV16 DNA positive during follow-up and whose disease ultimately progressed to CIN III (histologically diagnosed severe dysplasia or carcinoma in situ). Conclusion HPV16 VLP-specific IgG responses are present in the plasma of a majority of patients with persistent HPV16 infections and histologically confirmed high-grade lesions but only in a smaller subset of patients with cleared HPV16 infections and either normal cervical histology or low-grade CIN lesions. Implications These results suggest that HPV16 VLP-specific antibodies are not responsible for the clearance of virally induced CIN lesions but that they might, in patients with persistent HPV16 infections, be indicative of an increased cervical cancer risk.

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.

Abstract 2193 Introduction. Vaccination with acute myeloid leukemia derived dendritic cells (AML-DC) is a promising immunotherapeutic tool to prevent relapse of AML due to presence of residual AML cells. However, in the clinical setting AML-DC vaccination is hampered in 40% of cases where AML-DC cannot be cultured from diagnosis material or where numbers of AML-cells were too low to obtain sufficient numbers of AML-DC. Here we explore whether expansion prior to differentiation is feasible for those cases where vaccine preparation is hampered due to low numbers of cells. Material & Methods. After obtaining informed consent, peripheral blood or bone marrow mononuclear cells from 51 AML patients were isolated by density centrifugation using Ficoll-Paque. AML blasts were resuspended in culture medium supplemented with different cocktails of cytokines with known myeloid growth-promoting effects (i.e. GM-CSF, G-CSF, SCF, FLT3-L, IL-3). Cells were passaged every 3–4 days when expansion exceeded more than 2-fold. After 1–2 months the capacity the expanded cells to differentiate into DC was tested by culture with IL-4, TNFα, GM-CSF or the calcium ionophore A23187 and IL-4. The cells were analyzed for progenitor (CD34, CD14) and DC (CD1a, DC-SIGN, CD40, CD80, CD86) markers by flow cytometry pre- and post differentiation. Results. Cells from 51 patients were able to proliferate to varying numbers of passages (mean 16, range 1–66). Of all cases, 76% (39/51) could be kept in culture for at least two months, thus leading to an expansion factor of at least 256. Overall, low expression of TNFαRI and high expression of TNFαRII was associated with an ongoing proliferative capacity. No correlation was found with various FAB subtypes. Expansion was achieved by most of the tested cocktails but we identified a slight advantage for the combination of GM-CSF, FLT3L, IL-3 and SCF. Upon culture and expansion, marker expression changed: CD34 expression decreased in 7 out of 9 evaluated patients, whereas CD14 expression increased significantly (p= 0.04) in 8 out of 13 evaluated patients (from no expression on blasts cells to 51% (range 19–89%)). High expression levels of CD14 correlated with the ability to subsequently differentiate into DC, with the preferred GM-CSF, IL-4 and TNFα.differentiation cocktail. In 7 out of 13 expanded cultures DC could be cultured. Conclusion. DC progenitors were successfully expanded from AML blood and bone marrow samples, enabling the generation of sufficient numbers of AML-DC for vaccination purposes. Expanded cells gained CD14 expression, which is a positive predictor for DC-differentiation [Mohty et al. Leukemia 2002; Houtenbos et al. Leukemia 2003]. Thus, more AML patients will become eligible for DC vaccination upon progenitor expansion. Disclosures: No relevant conflicts of interest to declare.