Multifaceted effects of synthetic TLR2 ligand and Legionella pneumophilia on Treg-mediated suppression of T cell activation

Contains fulltext : 97133.pdf (publisher's version ) (Open Access)BACKGROUND: Regulatory T cells (Treg) play a crucial role in maintaining immune homeostasis and self-tolerance. The immune suppressive effects of Tregs should however be limited in case effective immunity is required against pathogens or cancer cells. We previously found that the Toll-like receptor 2 (TLR2) agonist, Pam3CysSK4, directly stimulated Tregs to expand and temporarily abrogate their suppressive capabilities. In this study, we evaluate the effect of Pam3CysSK4 and Legionella pneumophila, a natural TLR2 containing infectious agent, on effector T (Teff) cells and dendritic cells (DCs) individually and in co-cultures with Tregs. RESULTS: TLR2 agonists can directly provide a co-stimulatory signal inducing enhanced proliferation and cytokine production of naive CD4+ Teff cells. With respect to cytokine production, DCs appear to be most sensitive to low amounts of TLR agonists. Using wild type and TLR2-deficient cells in Treg suppression assays, we accordingly show that all cells (e.g. Treg, Teff cells and DCs) contributed to overcome Treg-mediated suppression of Teff cell proliferation. Furthermore, while TLR2-stimulated Tregs readily lost their ability to suppress Teff cell proliferation, cytokine production by Teff cells was still suppressed. Similar results were obtained upon stimulation with TLR2 ligand containing bacteria, Legionella pneumophila. CONCLUSIONS: These findings indicate that both synthetic and natural TLR2 agonists affect DCs, Teff cells and Treg directly, resulting in multi-modal modulation of Treg-mediated suppression of Teff cells. Moreover, Treg-mediated suppression of Teff cell proliferation is functionally distinct from suppression of cytokine secretion

CTLA-4 Signaling Regulates the Intensity of Hypersensitivity Responses to Food Antigens, but is Not Decisive in the Induction of Sensitization

Although food allergy has emerged as a major health problem, the mechanisms that are decisive in the development of sensitization to dietary Ag remain largely unknown. CTLA-4 signaling negatively regulates immune activation, and may play a crucial role in preventing induction and/or progression of sensitization to food Ag. To elucidate the role of CTLA-4 signaling in responses to food allergens, a murine model of peanut allergy was used. During oral exposure to peanut protein extract (PPE) together with the mucosal adjuvant cholera toxin (CT), which induces peanut allergy, CTLA-4 ligation was prevented using a CTLA-4 mAb. Additionally, the effect of inhibition of the CTLA-4 pathway on oral exposure to PPE in the absence of CT, which leads to unresponsiveness to peanut Ag, was explored. During sensitization, anti-CTLA-4 treatment considerably enhanced IgE responses to PPE and the peanut allergens, Ara h 1, Ara h 3, and Ara h 6, resulting in elevated mast cell degranulation upon an oral challenge. Remarkably, antagonizing CTLA-4 during exposure to PPE in the absence of CT resulted in significant induction of Th2 cytokines and an elevation in total serum IgE levels, but failed to induce allergen-specific IgE responses and mast cell degranulation upon a PPE challenge. These results indicate that CTLA-4 signaling is not the crucial factor in preventing sensitization to food allergens, but plays a pivotal role in regulating the intensity of a food allergic sensitization response. Furthermore, these data indicate that a profoundly Th2-biased cytokine environment is insufficient to induce allergic responses against dietary Ag

rapid and robust cd4 and cd8 t nk b cell dendritic cell and monocyte reconstitution after nicotinamide expanded cord blood transplantation

Introduction Nicotinamide-expanded cord blood (NiCord) is a potential alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-id donor is lacking. A phase 1/2 trial with standalone NiCord HCT showed rapid neutrophil- (11 days) and platelet engraftment (34 days). We previously reported that successful CD4+ immune reconstitution (IR) is crucial for infectious and relapse control associated with favorable survival (JACI 2017) and is a better predictor for event-free survival than neutrophil reconstitution. We performed unique in-depth immune monitoring to evaluate and compare IR after NiCord and conventional HCT. Methods In this phase1/2 international multicenter trial, we compared IR after NiCord HCT to cohorts of adolescent and young adult (AYA) patients receiving either unmanipulated cord blood transplantation (unCBT) or T-repleted-unrelated bone marrow transplantation (BMT). All patients received HCT for a hematologic malignancy with myeloablative conditioning without serotherapy. Immune monitoring was performed (harmonized sampling, handling and analyses) in a central lab. The primary endpoint was probability of achieving CD4+ IR (>50*106/L within 100 days). Secondary endpoints were IR of B-cells, CD4+ and CD8+ T-cells, natural killer (NK)-cells, monocytes, and dendritic cells (DC) 7-365 days after HCT. In addition, TREC analyses were performed on CD3+ MACs-sorted cells. Linear-mixed effects modelling in LOESS-regression curves and two-sided log-rank test for univariate comparisons in cumulative incidence plots were used. Results 27 NiCord recipients (median 41.5; 13.4-61.7yrs) were included. NiCord cell dose consisted of median 6.4*106 CD34+/kg, and 2.3*106 CD3+T-cells/kg of the co-infused negative fraction (following CD133+ selection). Of these patients, 91% achieved successful CD4+ IR, which was comparable (p=0.76, Figure 1) to the 27 unCBT (median 15.4; 12.2-22.1 yrs) and 20 BMT (median 14.3; 12.1-19.7 yrs) recipients included in this study. We observed similar reconstitution of T-cells (p=0.15), monocytes (p=0.94), conventional DCs (p=0.41), and plasmacytoid DCs (p=0.52). Interestingly, reconstitution of NK-cells (p Conclusions In-depth immune monitoring reveals fast and full IR after NiCord HCT in adult patients, which is equal or even faster to IR after unCBT or BMT, despite the younger age of the AYA cohorts (expected to reconstitute faster). This may be explained by the higher stem cell dose and higher proliferative capacity of the NiCord-expanded product. Optimal comparison of IR in NiCord vs. unCBT in a randomized phase 3 trial is underway

Route of Administration of the TLR9 Agonist CpG Critically Determines the Efficacy of Cancer Immunotherapy in Mice

Contains fulltext : 81648.pdf (publisher's version ) (Open Access)BACKGROUND: The TLR9 agonist CpG is increasingly applied in preclinical and clinical studies as a therapeutic modality to enhance tumor immunity. The clinical application of CpG appears, however, less successful than would be predicted from animal studies. One reason might be the different administration routes applied in most mouse studies and clinical trials. We studied whether the efficacy of CpG as an adjuvant in cancer immunotherapy is dependent on the route of CpG administration, in particular when the tumor is destructed in situ. METHODOLOGY/PRINCIPAL FINDINGS: In situ tumor destruction techniques are minimally invasive therapeutic alternatives for the treatment of (nonresectable) solid tumors. In contrast to surgical resection, tumor destruction leads to the induction of weak but tumor-specific immunity that can be enhanced by coapplication of CpG. As in situ tumor destruction by cryosurgery creates an instant local release of antigens, we applied this model to study the efficacy of CpG to enhance antitumor immunity when administrated via different routes: peritumoral, intravenous, and subcutaneous but distant from the tumor. We show that peritumoral administration is superior in the activation of dendritic cells, induction of tumor-specific CTL, and long-lasting tumor protection. Although the intravenous and subcutaneous (at distant site) exposures are commonly used in clinical trials, they only provided partial protection or even failed to enhance antitumor responses as induced by cryosurgery alone. CONCLUSIONS/SIGNIFICANCE: CpG administration greatly enhances the efficacy of in situ tumor destruction techniques, provided that CpG is administered in close proximity of the released antigens. Hence, this study helps to provide directions to fully benefit from CpG as immune stimulant in a clinical setting

Case Report:Immune dysregulation associated with long-lasting regression of a (pre)leukemic clone

Regression of leukemia in the absence of disease-modifying therapy remains poorly understood, although immunological mechanisms are thought to play a role. Here, we present a unique case of a 17-year-old boy with immune dysregulation and long-lasting regression of a (pre)leukemic clone in the absence of disease-modifying therapy. Using molecular and immunological analyses, we identified bone marrow features associated with disease control and loss thereof. In addition, our case reveals that detection of certain fusion genes with hardly any blasts in the bone marrow may be indicative of an accompanying oncogenic fusion gene, with implications for disease surveillance- and management in future patients.</p

Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis

INTRODUCTION: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. MATERIALS AND METHODS: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. RESULTS: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. CONCLUSION: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals

Quality assessment of a large multi-center flow cytometric dataset of acute myeloid leukemia patients—A EuroFlow study

Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis.The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as a Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). S.M. was supported by Acción Estratégica en Salud (AES) (Grant PI21_01115) and the grant of CIBERONC of the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain and FONDOS FEDER (no. CB16/12/00400)

Early and Long-Term Effects of Dupilumab Treatment on Circulating T-Cell Functions in Patients with Moderate-to-Severe Atopic Dermatitis

Dupilumab, a mAb targeting IL-4 receptor alpha (IL-4Rα), markedly improves disease severity in patients with atopic dermatitis. However, the effect of IL-4Rα blockade on dynamics of circulating skin-homing T cells, which are crucial players in the pathologic mechanism of atopic dermatitis, has not been studied yet. In addition, it remains unknown whether dupilumab treatment induces long-lasting T- and B-cell polarization. Therefore, we studied the short- and long-term effects of dupilumab treatment on IL-4Rα expression and T-cell cytokine production within total and skin-homing (cutaneous lymphocyte antigen+/CCR4+) subpopulations in patients with moderate-to-severe atopic dermatitis. Dupilumab treatment completely blocked IL-4Rα expression and signal transducer and activator of transcription 6 phosphorylation in CD19+ B cells and CD4+ T cells within 2 hours of administration and through week 52. Although no change in the proportion of skin-homing T-cell subsets was found, dupilumab treatment significantly decreased the percentage of proliferating (Ki67+) and T helper type 2 and T helper type 22 cytokine-producing skin-homing CD4+ T cells at week 4. No evidence of general T helper type cell skewing following a year of dupilumab treatment was found. In summary, dupilumab treatment rapidly and stably inhibited IL-4Rα, which was accompanied by a strong early functional immunological effect specifically on skin-homing T cells without affecting overall T helper type cell skewing in the long term

Targeted Proteomics Reveals Inflammatory Pathways that Classify Immune Dysregulation in Common Variable Immunodeficiency

Patients with common variable immunodeficiency (CVID) can develop immune dysregulation complications such as autoimmunity, lymphoproliferation, enteritis, and malignancy, which cause significant morbidity and mortality. We aimed to (i) assess the potential of serum proteomics in stratifying patients with immune dysregulation using two independent cohorts and (ii) identify cytokine and chemokine signaling pathways that underlie immune dysregulation in CVID. A panel of 180 markers was measured in two multicenter CVID cohorts using Olink Protein Extension Assay technology. A classification algorithm was trained to distinguish CVID with immune dysregulation (CVIDid, n = 14) from CVID with infections only (CVIDio, n = 16) in the training cohort, and validated on a second testing cohort (CVIDid n = 23, CVIDio n = 24). Differential expression in both cohorts was used to determine relevant signaling pathways. An elastic net classifier using MILR1, LILRB4, IL10, IL12RB1, and CD83 could discriminate between CVIDid and CVIDio patients with a sensitivity of 0.83, specificity of 0.75, and area under the curve of 0.73 in an independent testing cohort. Activated pathways (fold change > 1.5, FDR-adjusted p < 0.05) in CVIDid included Th1 and Th17-associated signaling, as well as IL10 and other immune regulatory markers (LAG3, TNFRSF9, CD83). Targeted serum proteomics provided an accurate and reproducible tool to discriminate between patients with CVIDid and CVIDio. Cytokine profiles provided insight into activation of Th1 and Th17 pathways and indicate a possible role for chronic inflammation and exhaustion in immune dysregulation. These findings serve as a first step towards the development of biomarkers for immune dysregulation in CVID