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Purpose of Review Immune checkpoint inhibitors (ICIs) have improved the field of cancer, especially in patients with advanced malignancies. Nevertheless, cardiovascular immune-related adverse events (irAEs) with high mortality and morbidity have been observed, including myocarditis, pericarditis, and vasculitis. To date, only a few clinical risk factors have been described and are currently being investigated. Recent Findings In this review, we address the four most prevailing risk factors for cardiovascular irAEs. ICI combination therapy is a predominant risk factor for developing ICI-mediated myocarditis. Additionally, ICI combined with other anti-cancer treatments (e.g., tyrosine kinase inhibitors, radiation, chemotherapy) seems to increase the risk of developing cardiovascular irAEs. Other risk factors include female sex, pre-existing cardiovascular disease, and specific tumors, on which we will further elaborate in this review. Summary An a priori risk strategy to determine who is at risk to develop these cardiovascular irAEs is needed. Insights into the impact of risk factors are therefore warranted to help clinicians improve care and disease management in these patients.

List of used antibodies, plasmids, gRNA sequences and datasets. Figures showing expression data, gene correlations, cell line clonality, screen replicates, and protein expression in neuroblastoma cells.

Basemean and fold change scores per indivudual gRNA used in the CRISPR screen resulting in a significant TRUE of FALSE hit in the screen.

Neuroblastoma is the second most common tumor in children. The cause of neuroblastoma is thought to lie in aberrant development of embryonic neural crest cells and is accompanied by low MHC-1 expression and suppression of the NF-κB transcription factor, thereby gearing cells toward escape from immunosurveillance. Here, we assess regulation of the MHC-1 gene in neuroblastoma to enhance its immunogenic potential for therapeutic T-cell targeting. A genome-wide CRISPR screen identified N4BP1 and TNIP1 as inhibitory factors of NF-κB-mediated MHC-1 expression in neuroblastoma. Patients with advanced stage neuroblastoma who expressed high levels of TNIP1 and N4BP1 exhibited worse overall survival. Depletion of N4BP1 or TNIP1 increased NF-κB and MHC-1 expression and stimulated recognition by antigen-specific CD8+ T cells. We confirmed that TNIP1 inhibited canonical NF-κB member RelA by preventing activation of the RelA/p50 NF-κB dimer. Furthermore, N4BP1 inhibited both canonical and noncanonical NF-κB through binding of deubiquitinating enzyme CEZANNE, resulting in stabilization of TRAF3 and degradation of NF-κB-inducing kinase NIK. These data suggest that N4BP1/CEZANNE or TNIP1 may be candidate targets for immunotherapy in neuroblastoma tumors and should lift NF-κB suppression, thereby triggering increased peptide/MHC1-mediated tumor reactivity to enhance therapeutic T-cell targeting.Significance:Aberrant regulation of NF-κB and MHC-1 in neuroblastoma tumors provides new targets for immunotherapeutic approaches against neuroblastoma.

Immunostimulatory cytidyl guanosyl (CpG) motifs are of great interest as cancer vaccine adjuvants. They act as potent inducers of Th1 responses, including the activation of cytotoxic CD8+ T lymphocytes (CTL). Whereas animal models have provided clear evidence that CpG enhances antitumor immunity, clinical trials in humans have thus far been less successful. Applying cryosurgery as an instant in situ tumor destruction technique, we now show that timing of CpG administration crucially affects colocalization of antigen and CpG within EEA-1+ and LAMP-1+ compartments within dendritic cells in vivo. Moreover, antigen/CpG colocalization is directly correlated with antigen cross-presentation, the presence of CTL, and protective antitumor immunity. Thus, failure or success of CpG as a vaccine adjuvant may depend on colocalization of antigen/CpG inside DCs and hence on the timing of CpG administration. These data might aid in the design of future immunotherapeutic strategies for cancer patients. [Cancer Res 2008;68(13):5390–6]

Supplementary Figure 1 from In vivo Colocalization of Antigen and CpG within Dendritic Cells Is Associated with the Efficacy of Cancer Immunotherapy

Pediatric cancers are characterized by a relatively low mutational burden and therefore, children are thought to be poor candidates for T cell-engaging immunotherapies. Here, we performed a multidimensional characterization of the tumor immune microenvironment in newly diagnosed children with acute myeloid leukemia (AML) and non-leukemic controls. We identified a subset of pediatric AML patients with remarkably high levels of T cell infiltration and a relatively low abundance of anti-inflammatory macrophages in the bone marrow. In addition, we detected large T cell networks that colocalized with B cells in immune-infiltrated samples, resembling tertiary lymphoid structures as described in solid tumors. Using spatial transcriptomics, we dissected the composition of these structures and revealed unique hotspots of anti-tumor immunity. This work raises the possibility that a subset of pediatric AML patients may benefit from T cell-engaging immunotherapies and encourages further study of these lymphoid structures in the context of immunotherapy in AML.Statement of significanceA subset of pediatric AML patients harbors hotspots of anti-tumor immunity in the tumor microenvironment. The presence of tertiary lymphoid structure-like aggregates in the bone marrow of pediatric AML encourages the evaluation of T cell-engaging immunotherapies in this population, as well as the development of novel antibody-based and cellular therapies.

Background Increasing evidence shows that pediatric atopic dermatitis (AD) differs from adult AD on a biologic level. Broad biomarker profiling across a wide range of ages of pediatric patients with AD is lacking. Objective Our aim was to identify serum biomarker profiles in children with AD aged 0 to 17 years and compare these profiles with those previously found in adults with AD. Methods Luminex multiplex immunoassays were used to measure 145 biomarkers in serum from 240 children with AD (aged 0-17 years). Principal components analysis followed by unsupervised k-means clustering were performed to identify patient clusters. Patients were stratified into age groups (0-4 years, 5-11 years, and 12-17 years) to assess association between age and cluster membership. Results Children aged 0 to 4 years had the highest levels of TH1 cell–skewing markers and lowest levels of TH17 cell–related markers. TH2 cell–related markers did not differ significantly between age groups. Similar to the pattern in adults, cluster analysis identified 4 distinct pediatric patient clusters (TH2 cell/retinol–dominant, skin-homing–dominant, TH1 cell/TH2 cell/TH17 cell/IL-1–dominant, and TH1 cell/IL-1/eosinophil–inferior clusters). Only the TH1 cell/TH2 cell/TH17 cell/IL-1–dominant cluster resembled 1 of the previously identified adult clusters. Although no association with age or age of onset seemed to be found, disease severity was significantly associated with the skin-homing–dominant cluster. Conclusion Four distinct patient clusters based on serum biomarker profiles could be identified in a large cohort of pediatric patients with AD, of which 1 was similar to previously identified adult clusters. The identification of endotypes driven by distinct underlying immunopathologic pathways might be useful to define pediatric patients with AD who are at risk of persistent disease and may necessitate different targeted treatment approaches.

Immune checkpoint inhibitors (ICI) have revolutionized the treatment landscape of advanced malignancies, but come with a diverse spectrum of immune-related adverse events (irAEs). Mechanistic studies can aid the transition from expert-opinion to evidence-based irAE treatment strategies. We aimed to longitudinally characterize peripheral blood T and B cell dynamics in ICI-treated patients by multicolor flow cytometry and serum multiplex immunoassay at baseline, ±3 weeks and ±6 weeks or upon clinically relevant irAEs. We analyzed samples from 44 ICI-treated patients (24 anti-PD-1 monotherapy, 20 combined anti-PD-1/anti-CTLA-4; cICI), of whom 21 developed irAEs, and 10 healthy donors. IrAEs after cICI were characterized by significantly enhanced proliferation of Th1-associated, mainly (CD4+) effector memory T cells, as well as Th17-associated immune responses and germinal center activation (reflected by CXCL13 and IL-21 increases). We observed no changes in CD21lo, memory, class-switched or newly activated B cell subsets. Especially double-positive PD-1+LAG-3+CD8+T cells showed enhanced cytotoxic capacity in patients with irAEs after cICI. Within anti-PD-1 monotherapy, irAEs were associated with modestly enhanced Th1-associated responses reflected by increased serum CXCL9 and CXCL10. In conclusion, ICI-induced toxicity is dominated by enhanced Th1-associated responses, but in cICI we also found evidence for Th17-associated responses and germinal center activation. Together, our data add to the growing body of evidence that irAEs may be driven by newly activated CD4+helper T cells, specifically after cICI. This study also supports tailored irAE treatment, based on ICI regimen, and to deploy specific strategies such as Th17 inhibition especially in cICI-associated irAEs.

Contains fulltext : 292311.pdf (Publisher’s version ) (Open Access) Bacillus Calmette-Guerin (BCG) vaccination has been hypothesized to reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severity, and/or duration via trained immunity induction. Health care workers (HCWs) in nine Dutch hospitals were randomized to BCG or placebo vaccination (1:1) in March and April 2020 and followed for 1 year. They reported daily symptoms, SARS-CoV-2 test results, and health care-seeking behavior via a smartphone application, and they donated blood for SARS-CoV-2 serology at two time points. A total of 1,511 HCWs were randomized and 1,309 analyzed (665 BCG and 644 placebo). Of the 298 infections detected during the trial, 74 were detected by serology only. The SARS-CoV-2 incidence rates were 0.25 and 0.26 per person-year in the BCG and placebo groups, respectively (incidence rate ratio, 0.95; 95% confidence interval, 0.76 to 1.21; P = 0.732). Only three participants required hospitalization for SARS-CoV-2. The proportions of participants with asymptomatic, mild, or moderate infections and the mean infection durations did not differ between randomization groups. In addition, unadjusted and adjusted logistic regression and Cox proportional hazards models showed no differences between BCG and placebo vaccination for any of these outcomes. The percentage of participants with seroconversion (7.8% versus 2.8%; P = 0.006) and mean SARS-CoV-2 anti-S1 antibody concentration (13.1 versus 4.3 IU/mL; P = 0.023) were higher in the BCG than placebo group at 3 months but not at 6 or 12 months postvaccination. BCG vaccination of HCWs did not reduce SARS-CoV-2 infections nor infection duration or severity (ranging from asymptomatic to moderate). In the first 3 months after vaccination, BCG vaccination may enhance SARS-CoV-2 antibody production during SARS-CoV-2 infection. IMPORTANCE While several BCG trials in adults were conducted during the 2019 coronavirus disease epidemic, our data set is the most comprehensive to date, because we included serologically confirmed infections in addition to self-reported positive SARS-CoV-2 test results. We also collected data on symptoms for every day during the 1-year follow-up period, which enabled us to characterize infections in detail. We found that BCG vaccination did not reduce SARS-CoV-2 infections nor infection duration or severity but may have enhanced SARS-CoV-2 antibody production during SARS-CoV-2 infection in the first 3 months after vaccination. These results are in agreement with other BCG trials that reported negative results (but did not use serological endpoints), except for two trials in Greece and India that reported positive results but had few endpoints and included endpoints that were not laboratory confirmed. The enhanced antibody production is in agreement with prior mechanistic studies but did not translate into protection from SARS-CoV-2 infection.

Severe uveitis is a rare complication of interleukin-4 receptor alpha blocking by dupilumab in topic dermatitis (AD) patients. The aim of this study was to describe five moderate-to-severe AD patients who developed uveitis during dupilumab treatment and to compare the proteomic profile of aqueous humor (AqH) of dupilumab-associated uveitis (n=3/5 available samples) with non-infectious uveitis (n=27) and cataract controls (n=11). Included patients were treated at the University Medical Center Utrecht (the Netherlands). Active dupilumab-associated uveitis complicated by serous detachment, cystoid macular edema, or secondary glaucoma developed within a median of 6.0 months (interquartile range 2.3–16.5 months) after starting dupilumab. Uveitis resolved after discontinuation of dupilumab and/or treatment with local or systemic corticosteroids. Proteomic profiling of AqH revealed that the molecular profile of dupilumab-associated uveitis resembled that of non-infectious uveitis. In conclusion, dupilumab-associated uveitis is a severe adverse event of dupilumab therapy, requiring urgent referral to an ophthalmologist.

Mucopolysaccharidoses (MPS) are devastating inherited diseases treated with hematopoietic cell transplantation (HCT). However, disease progression, especially skeletal, still occurs in all patients. Secondary inflammation has been hypothesized to be a cause. To investigate whether systemic inflammation is present in untreated patients and to evaluate the effect of HCT on systemic inflammation, dried blood spots (n = 66) of patients with MPS (n = 33) treated with HCT between 2003 and 2019 were included. Time points consisted of pre-HCT and, for patients with MPS type I (MPS I), also at 1, 3, and 10 years of follow-up. Ninety-two markers of the OLINK inflammation panel were measured and compared with those of age-matched control subjects (n = 31) by using principal component analysis and Wilcoxon rank sum tests with correction. Median age at transplantation was 1.3 years (range, 0.2-4.8 years), and median time of pre-HCT sample to transplantation was 0.1 year. Normal leukocyte enzyme activity levels were achieved in 93% of patients post-HCT. Pretransplant samples showed clear separation of patients and control subjects. Markers that differentiated pre-HCT between control subjects and patients were mainly pro-inflammatory (50%) or related to bone homeostasis and extracellular matrix degradation (33%). After 10 years’ follow-up, only 5 markers (receptor activator of nuclear factor kappa-Β ligand, osteoprotegerin, axis inhibition protein 1 [AXIN1], stem cell factor, and Fms-related tyrosine kinase 3 ligand) remained significantly increased, with a large fold change difference between patients with MPS I and control subjects. In conclusion, systemic inflammation is present in untreated MPS patients and is reduced upon treatment with HCT. Markers related to bone homeostasis remain elevated up to 10 years after HCT and possibly reflect the ongoing skeletal disease, making them potential biomarkers for the evaluation of new therapies.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies.

Background Atopic dermatitis (AD) is a highly heterogeneous disease, both clinically and biologically, whereas patients are still being treated according to a “one-size-fits-all” approach. Stratification of patients into biomarker-based endotypes is important for future development of personalized therapies. Objective Our aim was to confirm previously defined serum biomarker-based patient clusters in a new cohort of patients with AD. Methods A panel of 143 biomarkers was measured by using Luminex technology in serum samples from 146 patients with severe AD (median Eczema Area and Severity Index = 28.3; interquartile range = 25.2-35.3). Principal components analysis followed by unsupervised k-means cluster analysis of the biomarker data was used to identify patient clusters. A prediction model was built on the basis of a previous cohort to predict the 1 of the 4 previously identified clusters to which the patients of our new cohort would belong. Results Cluster analysis identified 4 serum biomarker–based clusters, 3 of which (clusters B, C, and D) were comparable to the previously identified clusters. Cluster A (33.6%) could be distinguished from the other clusters as being a “skin-homing chemokines/IL-1R1–dominant” cluster, whereas cluster B (18.5%) was a “TH1/TH2/TH17-dominant” cluster, cluster C (18.5%) was a “TH2/TH22/PARC-dominant” cluster, and cluster D (29.5%) was a “TH2/eosinophil-inferior” cluster. Additionally, by using a prediction model based on our previous cohort we accurately assigned the new cohort to the 4 previously identified clusters by including only 10 selected serum biomarkers. Conclusion We confirmed that AD is heterogeneous at the immunopathologic level and identified 4 distinct biomarker-based clusters, 3 of which were comparable with previously identified clusters. Cluster membership could be predicted with a model including 10 serum biomarkers.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune dysregulation syndrome characterized by uncontrolled immune cell activation. Timely diagnosis is important, since early treatment can improve survival rates. However, completing all assessments needed to reach ≥5 positive criteria out of the 8 HLH-2004 criteria can be time consuming and may delay timely initiation of treatment. Hence, we applied a data-driven approach to identify a minimal parameter set for early decision-making towards the initiation of HLH-specific treatment. We retrospectively evaluated 165 patients from five Dutch tertiary hospitals with suspected HLH. Sixteen pHLH (median age 0.5 years) and 70 sHLH patients (median age 8.7 years) were identified using the HLH-2004 criteria. Clustering analysis and multi-receiver operator characteristics were used to identify parameters distinctive of HLH. The presence of either increased ferritin, cytopenia in ≥2 lineages, or splenomegaly distinguished HLH from non-HLH cases with a negative predictive value of 100%. A minimal parameter set consisting of 2 major criteria (phagocytosis and splenomegaly) and 3 minor criteria (cytopenia, increased ferritin, and increased triglycerides/low fibrinogen) predicted HLH with 95% (88–99) sensitivity and 94% (86–98) specificity. This finding was replicated in an independent retrospective validation cohort of 109 US patients (n = 109). By dividing a subset of the HLH-2004 criteria into major and minor criteria, this strategy uses the evaluation of less than 5 criteria to quickly identify patients with HLH. When confirmed in a prospective setting, this approach could be of value for timely diagnosis and treatment of HLH. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-021-01005-7.

Background The CLN3 disease spectrum ranges from a childhood‐onset neurodegenerative disorder to a retina‐only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well‐known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity. Methods Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal‐associated membrane protein 1 (LAMP‐1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis. Results Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole‐shaped LAMP‐1 expression, suggesting the use of LAMP‐1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP‐1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in‐depth revealing that intracellular LAMP‐1 expression was most pronounced in T‐cells of classical‐protracted CLN3 disease while it was most pronounced in B‐cells of “retina‐only” CLN3 disease. Conclusion Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.

Optimal fludarabine exposure has been associated with improved treatment outcome in allogeneic hematopoietic cell transplantation, suggesting potential benefit of individualized dosing. A randomized controlled trial (RCT) comparing alternative fludarabine dosing strategies to current practice may be warranted, but should be sufficiently powered for a relevant end point, while still feasible to enroll. To find the optimal design, we simulated RCTs comparing current practice (160 mg/m2 ) to either covariate-based or therapeutic drug monitoring (TDM)-guided dosing with potential outcomes being nonrelapse mortality (NRM), graft failure, or relapse, and ultimately overall survival (covering all three aforementioned outcomes). The inclusion in each treatment arm (n) required to achieve 80% power was calculated for all combinations of end points and dosing comparisons. The trial requiring the lowest n for sufficient power compared TDM-guided dosing to current practice with NRM as primary outcome (n = 70, NRM decreasing from 21% to 5.7%). We conclude that a superiority trial is feasible.

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).

We prospectively studied clofarabine-fludarabine-busulfan (CloFluBu)-conditioning in allogeneic hematopoietic cell therapy (HCT) for lymphoid and myeloid malignancies and hypothesized that CloFluBu provides a less toxic alternative to conventional conditioning regimens, with adequate antileukemic activity. All patients receiving their first HCT, from 2011-2019, were included and received CloFluBu. The primary endpoint was event-free survival (EFS). Secondary endpoints were overall survival (OS), graft-versus-host disease (GvHD)-relapse-free survival (GRFS), treatment-related mortality (TRM), cumulative incidence of relapse (CIR), acute and chronic GvHD (aGvHD and cGvHD), and veno-occlusive disease (VOD). Cox proportional hazard and Fine and Gray competing-risk models were used for data analysis. One hundred fifty-five children were included: 60 acute lymphoid leukemia (ALL), 69 acute myeloid leukemia (AML), and 26 other malignancies (mostly MDS-EB). The median age was 9.7 (0.5 to 18.6) years. Estimated 2-year EFS was 72.0% ± 6.0 in ALL patients, and 62.4% ± 6.0 in AML patients. TRM in the whole cohort was 11.0% ± 2.6, incidence of aGvHD 3 to 4 at 6 months was 12.3% ± 2.7, extensive cGvHD at 2 years was 6.4% ± 2.1. Minimal residual disease-positivity prior to HCT was associated with higher CIR, both in ALL and AML. CloFluBu showed limited toxicity and encouraging EFS. CloFluBu is a potentially less toxic alternative to conventional conditioning regimens. Randomized prospective studies are needed.

Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about heterogeneity and functional specialization of human DC subsets in (local) inflammatory conditions. We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from a local inflammatory site, namely synovial fluid (SF) from patients suffering from a chronic inflammatory condition, Juvenile Idiopathic Arthritis (JIA). cDC1, a relatively small dendritic cell subset in blood, are strongly enriched in SF. SF cDC1 showed a quiescent immune signature without a clear inflammatory profile low expression of PRR, chemokine, and cytokine receptors, and poor induction of T cell proliferation and cytokine production, but selective production of IFNλ upon polyinosinic:polycytidylic acid exposure. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including IL-17. Although the cDC2 and monocytes showed an overlapping transcriptional core profile, there were clear differences in the transcriptional landscape and functional features, indicating that these cell types retain their lineage identity in chronic inflammatory conditions. In conclusion, our findings suggest that at the site of inflammation, there is specific functional programming of human DC subsets, especially cDC2. In contrast, the enriched subset of cDC1 remains relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a more regulatory role.

Immune checkpoint inhibition (ICI) can induce durable responses in patients with advanced malignancies. Three cases of hematological neoplasia following ICI for solid tumors have been reported to date. We present five patients treated at our tertiary referral center between 2017 and 2021 who developed chronic myeloid leukemia (two patients), acute myeloid leukemia, myelodysplastic syndrome and chronic eosinophilic leukemia during or after anti-PD-1-based treatment. Molecular analyses were performed on pre-ICI samples to identify baseline variants in myeloid genes. We hypothesize that PD-1 blockade might accelerate progression to overt myeloid malignancies and discuss potential underlying mechanisms.

COVID-19 vaccinations are recommended for children with cancer but data on their vaccination response is scarce. This study assesses the antibody and T-cell response following a 2- or 3-dose vaccination with BNT162b2 mRNA COVID-19 vaccine in children (5–17 years) with cancer. For the antibody response, participants with a serum concentration of anti-SARS-CoV-2 spike 1 antibodies of >300 binding antibody units per milliliter were classified as good responders. For the T-cell response, categorization was based on spike S1 specific interferon-gamma release with good responders having >200 milli-international units per milliliter. The patients were categorized as being treated with chemo/immunotherapy for less than 6 weeks (Tx < 6 weeks) or more than 6 weeks (Tx > 6 weeks) before the first immunization event. In 46 patients given a 2-dose vaccination series, the percentage of good antibody and good T-cell responders was 39.3% and 73.7% in patients with Tx < 6 weeks and 94.4% and 100% in patients with Tx > 6 weeks, respectively. An additional 3rd vaccination in 16 patients with Tx < 6 weeks, increased the percentage of good antibody responders to 70% with no change in T-cell response. A 3-dose vaccination series effectively boosted antibody levels and is of value for patients undergoing active cancer treatment.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct., The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme of the European Commission (grant LSHB-CT-2006-018708) as 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). TS and LS were supported by a Scientific Grant from the Medical University of Silesia Nr. PCN-1-050/K/0/K.

Severe acute respiratory syndrome coronavirus 2 infection after coronavirus disease 2019 vaccination raises concerns about the emergence of vaccine escape variants. Here we characterize 14 breakthrough infections among 5860 fully vaccinated Dutch health care workers ≥14 days after the final dose of vaccination with either BNT162b2, mRNA-1273, or Ad26.COV2.S. These breakthrough infections presented with regular B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants and high viral loads, despite normal vaccine-induced B- and T-cell immune responses detected by live virus neutralization assays and ELISpot. High-risk exposure settings, such as in households, indicate a potential risk of viral transmission despite full vaccination.

Active immunotherapy approaches for neuroblastoma (NB), a pediatric cancer of the sympathetic nervous system, has met with limited success. Especially challenging is the genetic heterogeneity of NB which makes it difficult to identify factors that consistently indicate the likelihood of an effective immune response and thereby select patients who are most likely to benefit from immunotherapy. Hence, we undertook an unbiased analysis of gene expression signatures from >500 well-annotated primary NBs representing diverse clinical and genetic subtypes to identify of predictors of immune response. Using clustering analysis of bulk transcriptomic signatures from these tumors, we identified a subset of NBs that was notable for the high expression of genes associated with anti-tumor immune response. These “immunogenic” tumors showed a predominance of gene expression signatures derived from malignant cells with primitive neural crest-like or mesenchymal properties, one of the two cell states that shape intratumoral heterogeneity in NB. In contrast, tumors that expressed committed, adrenergic neuron-like signatures were less immunogenic. Single-cell (sc) RNA-seq and immunohistochemistry analysis further confirmed that NBs comprise both adrenergic and mesenchymal tumor cells, and that the presence of mesenchymal cells positively associated with immune cell infiltration into the TME. scRNA-seq also revealed that mesenchymal NB cells were enriched for inflammatory gene signature. Gene expression analysis of isogenic pairs of adrenergic and mesenchymal cells showed that mesenchymal NBs differentially upregulate genes involved in regulating antigen processing and presentation, MHC class I expression, type-I interferon and TLR3 signaling, and NK cell activation. This is achieved through a permissive chromatin landscape at the promoters of these immune regulatory genes that support their high expression in mesenchymal cells. By contrast, in adrenergic cells, tumor-intrinsic immune genes are epigenetically silenced by the PRC2 complex and PRC2 inhibition leads to increased immune cell activation. Remarkably, induction of the mesenchymal state in adrenergic cells through transcriptional reprogramming by PRRX1 or therapy resistance is accompanied by the epigenetic activation of innate and adaptive immune response genes. Functionally, the inherent immunogenicity of mesenchymal cells promotes T cell infiltration by secreting inflammatory cytokines, enables efficient targeting by antigen-specific cytotoxic T and NK cells, and imparts responsiveness to immune checkpoint blockade in a syngeneic NB model. In conclusion, our study uncovers an unappreciated link between immunogenicity and tumor lineage state in NB, and rationalizes future interrogations into (i) avenues through which the vulnerability of mesenchymal cells to immune-mediated targeting could be harnessed clinically and (ii) how perturbation of epigenetically-regulated cell states could be harnessed to promote anti-tumor immune response. Citation Format: Satyaki Sengupta, Sanjukta Das, Angela C. Crespo, Annelisa M. Cornel, Anand G. Patel, Navin R. Mahadevan, Marco Campisi, Alaa K. Ali, Bandana Sharma, Jared H. Rowe, Rogier Versteeg, Rudolf Jaenisch, Stefani Spranger, Rizwan Romee, Brian C. Miller, David A. Barbie, Stefan Nierkens, Michael A. Dyer, Judy Lieberman, Rani E. George. Divergent tumor cell states in neuroblastoma possess distinct immunogenic phenotypes [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A08.

Brain tumors are the leading cause of disease-related death in childhood and strong efforts are required to develop innovative and efficient therapeutic strategies for patients with high-risk disease. Key critical factor of pediatric brain tumors is their molecular heterogeneity; one of the aspects that contributes to such heterogeneity is the intrinsic capacity of the tumor cells to organize, shape and exploit the surrounding brain tumor microenvironment (TME) to sustain tumor growth and malignant progression. TME was proved to play a crucial role in several malignancies, but in pediatric brain malignancies this has not been fully elucidated yet. Here, we aimed at characterizing the TME cell populations and their contributions in ependymoma and medulloblastoma, two of the most common pediatric brain tumor entities. Single-cell transcriptome analysis (n=65 ependymomas; n=39 medulloblastomas) of publicly available tumor datasets, as well as newly generated data of primary tumors and matching patient-derived tumor xenografts (PDX), showed an extensive heterogeneity of TME cell types with distinctive expression signatures. Amongst the identified TME populations, analysis revealed pro-inflammatory and proliferating myeloid cells, tumor-infiltrating lymphocytes, active regulatory T cells and vascular progenitor cells. Comparative analysis between primary and PDX tumors showed that tumor cells stimulated the host microenvironment, which in turn exhibited tumor-associated stromal signatures. Applying a deconvolution method, using our single cell data as reference, on a bulk tumor cohort including PFA ependymomas with different degree of immune infiltration, we observed an enrichment of polarized macrophages and microglia cells in tumor with high infiltration. Next, we identified TME markers involved in tumor-supportive functions, such as immune suppression (TIGIT, FOXP3, BCL2, CD19, ICAM2), pro-inflammatory stimuli (CCL3, CCL4, IL1B, GPNMB), extracellular matrix remodeling (COL4A1) and angiogenic processes (ANGPT2). TME markers with emerging role in ependymoma and medulloblastoma can be considered as possible targets for tailored and more effective anti-tumor therapeutic strategies.

Background The immunotherapeutic drug dinutuximab, which binds to disialoganglioside (GD2) and activates natural killer (NK) cells, is part of the standard regimen in high-risk neuroblastoma (NB) patients. However, dinutuximab only results in tumor reduction in a subset of patients, and survival rates of high-risk neuroblastoma patients are below 60%. Novel immunotherapies are therefore needed. Current in vitro models lack the ability to study novel immunotherapies with high-throughput screening (HTS). We aimed to optimize NB organoid-lymphocyte cocultures for HTS, and possibly personalized testing, of novel antibody-mediated and cellular immunotherapies. Materials and Methods Two patient-derived organoids (691B: GD2+MHC-I- and 691T: GD2-MHC-I+) were transduced with an endogenous luciferase construct to use D-luciferin-induced bioluminescence as readout for cell growth. The growth rate, optimal seeding density and optimal pre-culture time per organoid were determined by density curves, and the number of needed cells was downscaled to facilitate HTS. After pre-culture, the luciferase-transduced organoids were co-cultured with primary PBMCs from healthy donors, PRAME-TCR transduced T cells or CAR-T cells.1 Several effector:target (E:T) ratios and timepoints were tested to identify the optimal window for read-out of dinutuximab-induced antibody-dependent cytotoxicity (ADCC) and T-cell mediated cytotoxicity. The required number of immune cells per ratio was calculated based on the expansion rate of organoid cells after 48 and 72 hours. Results The density screens showed an optimal seeding density of 5000-10.000 organoid cells per well, yielding a high luminescence signal while minimizing the number of cells needed. Already at the lowest E:T ratio (1:3), we observed killing of the MHC-I expressing 691T organoid, likely based on allogeneic recognition of the organoids by T cells. The killing efficacy increased with higher E:T ratios and co-culture time. Pre-culturing of organoids for 72 hours before addition of effector cells resulted in formation of larger 3D spheres, which reduced the killing efficacy for all E:T ratios. ADCC effects of dinutuximab were studied in GD2+MHC-I- 691B organoids. Addition of dinutuximab resulted in 25% increase of killing after 24 hours and reached up to 70% increase after 72 hours for 10:1 and 20:1 E:T ratios. Higher E:T ratios were likely needed because NK cells make up a smaller proportion of PBMCs than T cells. Dinutuximab did not increase killing of the GD2- organoid, confirming specificity of the antibody. T cell mediated killing was almost 100% for MHC-I+691T organoids after 24 hours of culturing with PRAME-TCR transduced T cells and CAR-T cells at a 1:3 E:T ratio, showing the high anti-tumor cytotoxicity of these cells and potential for HTS at very low E:T ratios. Conclusions We have developed a robust in vitro bioluminescence-based organoid/lymphocyte co-culture assay with a low cell input, to facilitate high-throughput screening of novel antibody-based or cellular immunotherapies, possibly combined with chemotherapeutic or targeted compounds. In the future this method may be applied for personalized drug screens. Reference Avital L Amir, et al. PRAME-Specific Allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer. Clin Cancer Res 2011. Disclosure Information F. van den Ham: None. W.M. Kholosy: None. K. Ober: None. A.M. Cornel: None. S. Nierkens: None. J. Anderson: None. J.J. Molenaar: None. J. Wienke: None.

Background Neuroblastoma (NBL) is the most common pediatric solid tumor and responsible for about 15% of all pediatric cancer deaths. The majority of high-risk (HR) patients suffers from relapse after intense therapy regimens, resulting in a 5-year survival rate of only 40%. Even though the potential of immune interference in HR-NBL is shown by the additive effect of anti-GD2 monoclonal antibody therapy to the treatment protocol, long-term follow-up studies reveal that the beneficial effect of immunotherapy diminishes over time. We hypothesize that this is a result of inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL and its microenvironment. One of the most remarkable immunomodulatory strategies of NBL tumors is the absence of MHC-I surface expression, thereby preventing cytotoxic T cell recognition and killing. MHC-I lacking cells are known to be subjected to NK cell mediated cytotoxicity, however, we have shown that NBL is able to evade this by temporary upregulating surface expression of MHC-I, thereby becoming temporarily more prone to T cell mediated cytotoxicity. The aim of this project is to identify pharmacological strategies to enhance adaptive immune activation and therewith immunogenicity of HR-NBL. Materials and Methods FDA-approved drug libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cell lines using high-throughput flow cytometry analyses optimized for adherent NBL cells. The effect of positive hits was subsequently confirmed in a panel of NBL patient-derived tumeroids. Alterations in the transcriptome and translatome upon incubation with compounds of interest were further studied to identify potential additional immunomodulatory effects in NBL. Ultimately, compound treated NBL cell lines and tumeroids were co-cultured with PRAME reactive tumor-specific T cells and healthy-donor NK cells to determine the in vitro effect on T- and NK cell cytotoxicity. Results Drug library screening revealed MHC-I upregulation upon treatment of NBL cell lines and patient-derived tumeroids with multiple histon deacetylase inhibitors (HDACi). Further investigation of immunomodulatory effects of HDACi in NBL revealed enhanced expression of several additional players of the antigen presenting machinery, immunoproteasome expression, and MICA/MICB upregulation in NBL cells. We show that in untreated NBL cells, plasticity of MHC-I expression causes evasion of both NK- and T cell mediated cytotoxicity. Intriguingly, co-culture of NBL cells with tumor-specific T cells and healthy-donor NK cells upon treatment with the HDACi Entinostat resulted in enhanced in vitro T- and NK cell activation and cytotoxicity. Conclusions We show pharmacological upregulation of MHC-I, other antigen presenting machinery players, and the NKG2D ligands MICA/MICB upon HDACi in HR-NBL. Pre-treatment of NBL with HDACi resulted in enhanced in vitro T- and NK cell mediated cytotoxicity, substantiating HDACi as a potential strategy to improve adaptive immune engagement and therewith immunogenicity to aid NBL treatment. This work was supported by the Villa Joep Foundation [IWOV-Actief.51391.180034]. Disclosure Information A.M. Cornel: None. D.A.M.H. van den Beemt: None. E. Dunnebach: None. M.P. van Dierselhuis: None. S. Nierkens: None.

BackgroundThe addition of monoclonal antibody therapy against GD2 to the treatment of high-risk neuroblastoma led to improved responses in patients. Nevertheless, administration of GD2 antibodies against neuroblastoma is associated with therapy-limiting neuropathic pain. This severe pain is evoked at least partially through complement activation on GD2-expressing sensory neurons.MethodsTo reduce pain while maintaining antitumor activity, we have reformatted the approved GD2 antibody ch14.18 into the IgA1 isotype. This novel reformatted IgA is unable to activate the complement system but efficiently activates leukocytes through the FcαRI (CD89).ResultsIgA GD2 did not activate the complement system in vitro nor induced pain in mice. Importantly, neutrophil-mediated killing of neuroblastoma cells is enhanced with IgA in comparison to IgG, resulting in efficient tumoricidal capacity of the antibody in vitro and in vivo.ConclusionsOur results indicate that employing IgA GD2 as a novel isotype has two major benefits: it halts antibody-induced excruciating pain and improves neutrophil-mediated lysis of neuroblastoma. Thus, we postulate that patients with high-risk neuroblastoma would strongly benefit from IgA GD2 therapy.

Simple Summary Immunotherapy may be an attractive treatment option to increase survival, and to reduce treatment-related side effects, for children with acute myeloid leukemia (AML). While immunotherapies have shown successes in many cancer types, the development and subsequent clinical implementation have proven difficult in pediatric AML. To expedite the development of immunotherapy, it will be crucial to understand which pediatric AML patients are likely to respond to immunotherapies. Emerging research in solid malignancies has shown that the number and phenotype of immune cells in the tumor microenvironment is predictive of response to several types of immunotherapies. Such a predictive model may also be applicable for AML and, thus, knowledge on the immune cells infiltrating the bone marrow environment is needed. Here, we discuss the current state of knowledge on these infiltrating immune cells in pediatric AML, as well as ongoing immunotherapy trials, and provide suggestions concerning the way forward. Abstract Immunotherapeutic agents may be an attractive option to further improve outcomes and to reduce treatment-related toxicity for pediatric AML. While improvements in outcome have been observed with immunotherapy in many cancer types, immunotherapy development and implementation into patient care for both adult and pediatric AML has been hampered by an incomplete understanding of the bone marrow environment and a paucity of tumor-specific antigens. Since only a minority of patients respond in most immunotherapy trials across different cancer types, it will be crucial to understand which children with AML are likely to respond to or may benefit from immunotherapies. Immune cell profiling efforts hold promise to answer this question, as illustrated by the development of predictive scores in solid cancers. Such information on the number and phenotype of immune cells during current treatment regimens will be pivotal to generate hypotheses on how and when to intervene with immunotherapy in pediatric AML. In this review, we discuss the current understanding of the number and phenotype of immune cells in the bone marrow in pediatric AML, ongoing immunotherapy trials and how comprehensive immune profiling efforts may pave the way for successful clinical trials (and, ultimately, implementation into patient care).

INTRODUCTION: Dupilumab has recently been approved for the treatment of moderate to severe atopic dermatitis (AD) in adults. Daily practice data on dupilumab treatment are scarce.OBJECTIVE: To study the effect of 16-week treatment with dupilumab on clinical response and serum biomarkers in adult patients with moderate-severe AD in daily practice.METHODS: Data were extracted from the BioDay registry, a prospective multicenter registry. Sixteen-week clinical effectiveness of dupilumab was expressed as number of patients achieving EASI-50 (Eczema Area and Severity Index) or EASI-75, as well as patient-reported outcomes measures (Patient-Oriented Eczema Measure, Dermatology Life Quality Index, Numeric Rating Scale pruritus). Twenty-one biomarkers were measured in patients treated with dupilumab without concomitant use of oral immunosuppressive drugs at five different time points (baseline, 4, 8, 12, and 16 weeks).RESULTS: In total, 138 patients treated with dupilumab in daily practice were included. This cohort consisted of patients with very difficult-to-treat AD, including 84 (61%) patients who failed treatment on ≥2 immunosuppressive drugs. At week 16, the mean percent change in EASI score was 73%. The EASI-50 and EASI-75 were achieved by 114 (86%) and 82 (62%) patients after 16 weeks of treatment. The most reported side effect was conjunctivitis, occurring in 47 (34%) patients. During dupilumab treatment, disease severity-related serum biomarkers (TARC, PARC, periostin, and IL-22), eotaxin-1, and eotaxin-3 significantly decreased.CONCLUSION: Treatment with dupilumab significantly improved disease severity and decreased severity-related serum biomarkers in patients with very difficult-to-treat AD in a daily practice setting.

Fludarabine is the most frequently used agent in conditioning regimens for allogeneic hematopoietic cell transplantation (HCT). Body surface area–based dosing leads to highly variable fludarabine exposure. We studied the relation between fludarabine exposure and clinical outcomes. A retrospective, pharmacokinetic-pharmacodynamic analysis was conducted with data from patients undergoing HCT with fludarabine (160 mg/m2) as part of a myeloablative conditioning (busulfan targeted to an area under the plasma-concentration-time curve [AUC] of 90 mg*h/L) and rabbit antithymocyte globulin (6-10 mg/kg; from day −9/−12) between 2010 and 2016. Fludarabine exposure as AUC was calculated for each patient using a previously published population pharmacokinetic model and related to 2-year event-free survival (EFS) by means of (parametric) time-to-event models. Relapse, nonrelapse mortality (NRM), and graft failure were considered events. One hundred ninety-two patients were included (68 benign and 124 malignant disorders). The optimal fludarabine exposure was determined as an AUC of 20 mg*h/L. In the overexposed group, EFS was lower (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.1-3.5; P = .02), due to higher NRM (HR, 3.4; 95% CI, 1.6-6.9; P < .001) associated with impaired immune reconstitution (HR, 0.43; 95% CI, 0.26-0.70; P < .001). The risks of NRM and graft failure were increased in the underexposed group (HR, 3.3; 95% CI, 1.2-9.4; P = .02; HR, 4.8; 95% CI, 1.2-19; P = .02, respectively). No relationship with relapse was found. Fludarabine exposure is a strong predictor of survival after HCT, stressing the importance of optimum fludarabine dosing. Individualized dosing, based on weight and “renal function” or “therapeutic drug monitoring,” to achieve optimal fludarabine exposure might improve survival.

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for patients with a variety of malignant and non-malignant diseases. Despite its life-saving potential, HCT is associated with significant morbidity and mortality. Reciprocal interactions between hematopoietic stem cells (HSCs) and their surrounding bone marrow (BM) niche regulate HSC function during homeostatic hematopoiesis as well as regeneration. However, current pre-HCT conditioning regimens, which consist of high-dose chemotherapy and/or irradiation, cause substantial short- and long-term toxicity to the BM niche. This damage may negatively affect HSC function, impair hematopoietic regeneration after HCT and predispose to HCT-related morbidity and mortality. In this review, we summarize current knowledge on the cellular composition of the human BM niche after HCT. We describe how pre-HCT conditioning affects the cell types in the niche, including endothelial cells, mesenchymal stromal cells, osteoblasts, adipocytes, and neurons. Finally, we discuss therapeutic strategies to prevent or repair conditioning-induced niche damage, which may promote hematopoietic recovery and improve HCT outcome.

IntroductionInsight into inflammation patterns is needed to understand the pathophysiology of HIV and related cardiovascular disease (CVD). We assessed patterns of inflammation related to HIV infection and CVD risk assessed with carotid intima media thickness (CIMT).MethodsA cross-sectional study was performed in Johannesburg, South Africa, including participants with HIV who were virally suppressed on anti-retroviral therapy (ART) as well as HIV-negative participants who were family members or friends to the HIV-positive participants. Information was collected on CVD risk factors and CIMT. Inflammation was measured with the Olink panel ‘inflammation’, allowing to simultaneously assess 92 inflammation markers. Differences in inflammation patterns between HIV-positive and HIV-negative participants were explored using a principal component analysis (PCA) and ANCOVA. The impact of differentiating immune markers, as identified by ANCOVA, on CIMT was assessed using linear regression while adjusting for classic CVD risk factors.ResultsIn total, 185 HIV-positive and 104 HIV negative participants, 63% females, median age 40.7 years (IQR 35.4 – 47.7) were included. HIV-positive individuals were older (+6 years, p ConclusionHIV positive patients on stable ART and HIV negative controls had similar immune activation patterns. CVD risk in HIV-positive participants was mediated by inflammation markers included in this study.

Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect. CB T cells are readily available in CB banks and show low toxicity, high proliferation rates, and increased anti-leukemic effect upon transfer. However, the combination of anti-tumor gene modification and preservation of advantageous immunological traits of CB T cells represent major challenges for the harmonized production of T cell therapy products. In this manuscript, we optimized a protocol for expansion and lentiviral vector (LV) transduction of CB CD8+ T cells, achieving a transduction efficiency up to 83%. Timing of LV treatment, selection of culture media, and the use of different promoters were optimized in the transduction protocol. LentiBOOST was confirmed as a non-toxic transduction enhancer of CB CD8+ T cells, with minor effects on the proliferation capacity and cell viability of the T cells. Positively, the use of LentiBOOST does not affect the functionality of the cells, in the context of tumor cell recognition. Finally, CB CD8+ T cells were more amenable to LV transduction than peripheral blood (PB) CD8+ T cells and maintained a more naive phenotype. In conclusion, we show an efficient method to genetically modify CB CD8+ T cells using LV, which is especially useful for off-the-shelf adoptive cell therapy products for cancer treatment., Graphical abstract, The manuscript proposes an efficient method to gene modify cord blood-derived CD8+ T cells using lentiviral vectors and a transduction enhancer (LentiBOOST). Future application of these findings can improve the generation of allogeneic T cell therapies for the treatment of cancer.

Simple Summary Neuroblastoma is a type of childhood cancer accounting for approximately 15% of childhood cancer deaths. Despite intensive treatment, including immunotherapy, prognosis of high-risk neuroblastoma is poor. Increasing amounts of research show that the fighting capacity of the immune system is very important for the outcome of neuroblastoma patients. Therefore, we investigated the fighting capacity of immune cells in blood at diagnosis and during the different phases of therapy. In this study, we observed both processes that stimulate and processes that decrease fighting capacity of immune cells in neuroblastoma patients during therapy. Despite this, we show that overall fighting capacity of the immune system of neuroblastoma patients is impaired at diagnosis as well as during therapy. In addition, we observed a lot of variation between patients, which might explain differences in therapy efficacy between patients. This study provides insight for improvement of therapy timing as well as new therapy strategies enhancing immune cell fighting capacity. Abstract Despite intensive treatment, including consolidation immunotherapy (IT), prognosis of high-risk neuroblastoma (HR-NBL) is poor. Immune status of patients over the course of treatment, and thus immunological features potentially explaining therapy efficacy, are largely unknown. In this study, the dynamics of immune cell subsets and their function were explored in 25 HR-NBL patients at diagnosis, during induction chemotherapy, before high-dose chemotherapy, and during IT. The dynamics of immune cells varied largely between patients. IL-2- and GM-CSF-containing IT cycles resulted in significant expansion of effector cells (NK-cells in IL-2 cycles, neutrophils and monocytes in GM-CSF cycles). Nonetheless, the cytotoxic phenotype of NK-cells was majorly disturbed at the start of IT, and both IL-2 and GM-CSF IT cycles induced preferential expansion of suppressive regulatory T-cells. Interestingly, proliferative capacity of purified patient T-cells was impaired at diagnosis as well as during therapy. This study indicates the presence of both immune-enhancing as well as regulatory responses in HR-NBL patients during (immuno)therapy. Especially the double-edged effects observed in IL-2-containing IT cycles are interesting, as this potentially explains the absence of clinical benefit of IL-2 addition to IT cycles. This suggests that there is a need to combine anti-GD2 with more specific immune-enhancing strategies to improve IT outcome in HR-NBL.

Immunologia; Recerca mèdica Inmunología; Investigación médica Immunology; Medical research Omidubicel (nicotinamide-expanded cord blood) is a potential alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-identical donor is lacking. A phase I/II trial with standalone omidubicel HCT showed rapid and robust neutrophil and platelet engraftment. In this study, we evaluated the immune reconstitution (IR) of patients receiving omidubicel grafts during the first 6 months post-transplant, as IR is critical for favorable outcomes of the procedure. Data was collected from the omidubicel phase I-II international, multicenter trial. The primary endpoint was the probability of achieving adequate CD4+ T-cell IR (CD4IR: > 50 × 106/L within 100 days). Secondary endpoints were the recovery of T-cells, natural killer (NK)-cells, B-cells, dendritic cells (DC), and monocytes as determined with multicolor flow cytometry. LOESS-regression curves and cumulative incidence plots were used for data description. Thirty-six omidubicel recipients (median 44; 13–63 years) were included, and IR data was available from 28 recipients. Of these patients, 90% achieved adequate CD4IR. Overall, IR was complete and consisted of T-cell, monocyte, DC, and notably fast NK- and B-cell reconstitution, compared to conventional grafts. Our data show that transplantation of adolescent and adult patients with omidubicel results in full and broad IR, which is comparable with IR after HCT with conventional graft sources.