BACKGROUND:We aimed to investigate if iron deficiency was associated with infection susceptibility in a large cohort of healthy individuals. STUDY DESIGN AND METHODS:The Danish Blood Donor Study is a national ongoing prospective study of blood donors. We included 94,628 donors with 338,290 ferritin measurements from March 2010 to October 2022. We performed sex-stratified multivariable Cox regression to estimate the risk of infection for iron-deficient donors compared with iron-replete donors. Infection was defined as either a filled prescription of antibiotics registered in the Danish National Prescription Registry (NPR), or a hospital contact with infection registered in the Danish National Patient Registry (DNPR). RESULTS:Iron deficiency was associated with an overall increased risk of infection (defined as prescriptions of antibiotics) for women (hazard ratio [HR] 1.08, 95% confidence interval [CI] 1.02-1.15). Subgroup analyses showed an increased risk of respiratory tract infections (HR 1.16, 95% CI 1.05-1.28) and urinary tract infections (HR 1.16, 95% CI 1.04-1.29). Iron deficiency was not associated with overall risk of infection for men (HR 1.02, 95% CI 0.82-1.28). For both men and women, no association was found between iron deficiency and hospital contacts for infections. CONCLUSION:Iron deficiency was associated with an increased risk of infection in female blood donors. However, effect sizes were small, and there was no association between iron deficiency and hospital contacts for infection. Consequently, risk of infection should not be considered an apprehension regarding blood donation. These findings support the role of iron in immune function and monitorization of iron stores in female blood donors.

Supplementary Figure S5. Stimulated cytokine response in patients with or without pre-treatment infections.

Supplementary Figure S1. Innate immune responses at baseline vs 2-3 cycles of BTKi.

Supplementary Table S2. Gating strategies for the immune cell subset populations included in this study.

Supplementary Table S1. Overview over the applied 10-color flow cytometry panel for the two tubes included in this study.

AbstractPurpose: Patients with chronic lymphocytic leukemia (CLL) have increased risk of severe infections. Although adaptive immune dysfunction is well described, clinical tools for identifying patients at risk are lacking, warranting investigation of additional immune components. In contrast to chemotherapy, targeted agents could spare or even improve innate immune function. Therefore, we investigated innate immune phenotypes and function in patients with CLL before and during targeted treatment. Experimental Design: Baseline and consecutive blood samples were collected from patients with CLL treated with acalabrutinib (n = 17) or ibrutinib+venetoclax (n = 18) in clinical trials. Innate immune function was assessed by TruCulture, a whole-blood ligand-stimulation assay quantifying cytokine release in response to standardized stimuli. Innate immune phenotypes were characterized by flow cytometry. As a proxy for infections, we mapped antimicrobial use before and during treatment. Results: At baseline, patients with CLL displayed impaired stimulated cytokine responses to the endotoxin lipopolysaccharide (LPS) along with deactivated monocytes, enrichment of myeloid-derived suppressor cells and metamyelocytes, and elevated (unstimulated) proinflammatory cytokines. Two/three cycles of acalabrutinib or ibrutinib normalized LPS-stimulated responses, in parallel with decreased duration of infections. Innate immune profiles and elevated proinflammatory cytokines further normalized during longer-term acalabrutinib or ibrutinib+venetoclax, paralleled by decreased infection frequency. Conclusions: Innate immune impairment and infection susceptibility in patients with CLL were restored in parallel during targeted therapy. Thus, targeted treatment may reduce the risk of infections in CLL, as currently under investigation in the PreVent-ACaLL phase 2 trial of acalabrutinib+venetoclax for high-risk CLL (NCT03868722).

Supplementary Figure S6. Clinical trajectories with 3 years of follow up.

Supplementary Figure S4. Changes in immune cell activation marker expression during targeted treatment.

Supplementary Table S5. Baseline patient- and disease related characteristics for patients with or without history of infections.

Supplementary Table S4. Baseline patient- and disease related characteristics.

Supplementary Table S3. Supplementary Table S3. Representativeness of study participants.

This document describes how the Nomenclature for Properties and Units (NPU) terminology can be applied to differentiate between cell subpopulations of the hematopoietic lineage. The clusters of differentiation molecules are included in the NPU syntax, together with its correct affiliations to indicate their presence or absence. This allows for identification and isolation of cell populations, subsets, and differentiation stages, which is essential for correct diagnosis and treatment of several malignancies and autoimmune diseases.

Background Recent studies have shown extensive crosstalk between our immune system and the gut microbiome (GM). The host immune system plays a vital role in the maintenance of GM homeostasis by establishing a balance between eliminating invading pathogens and promoting the growth of beneficial microbes, whereas GM can modulate the immune system by cytokine production. As chronic lymphocytic leukemia (CLL) is characterized by a high rate of infectious complications and an altered immune microenvironment, it is warranted to uncover the link between microbiome composition and immune dysregulation in CLL patients. Thus, we investigated the microbiome pattern and potential connections to disease characteristics and phenotypes and distribution within the immune microenvironment in CLL patients requiring treatment. Methods Blood and feces samples were collected from 17 patients with CLL prior to targeted treatment with acalabrutinib or ibrutinib+venetoclax in clinical trials. T-cell maturation subsets, Treg/Th17 subsets, and activation- and exhaustion profiles were assessed in fresh blood samples by an 8-tube, 10 color flow cytometry panel (DuraClone), including one tube containing beads for calculating absolute concentrations. Fecal samples were subjected to shotgun sequencing using Illumina HiSeq and taxonomical profiling was done using an in-house bioinformatics pipeline. The bioinformatics analysis involved microbial species abundances comparison and co-occurrence identification across patient clusters. Electronic health records provided data on clinical baseline characteristics. Case report forms and data on use of antimicrobial drugs will be assessed at a later stage to investigate potential associations with infections or toxicity related adverse events. All patients provided written informed consent and the study was approved by the National Ethics Committee and the Data Protections Agency. Results Unsupervised clustering analyses based on GM composition data revealed two GM clusters, where we found an overlap constituting approximately 40% of the cohort between Binet stage C (7/10 patients) and GM cluster 2 (7/10 patients) (Figure 1A). GM cluster 2 was characterized by differentially abundant species such as Bacteroides fragilis and Eggerthela lenta, demonstrating the two highest fold change and elevated relative abundances (Figure 1B). Closer examination of the taxonomic composition characteristic for the 7 patients revealed a small set of indicator microbiome species, where Clostridium innocuum together with Clostridiales sp., Bacteroides fragilis and Escherichia coli, were the most significantly abundant co-occurring species for CLL patients with Binet C. Preliminary examination of T-cell subset data showed generally lower concentrations of T-cells in patients from GM cluster 2 compared to GM cluster 1, where the 7 patients overlapping with Binet C furthermore had decreased levels of Th17 CD4+ and Tc17 CD8+ cells compared to both the normal reference level and the rest of the cohort (Figure 1C). Conclusion Our preliminary results suggest a link between CLL stage at time of needing treatment, gut microbiome composition, represented by a subset of highly abundant bacterial species, and T-cell distribution with reduced Th17 and Tc17 subsets, to be further investigated. To our knowledge, we are thus among the first to present data connecting the gut microbiome to tumor microenvironmental changes in patients with CLL. Moving forward we will further explore microbiome-microenvironment interactions with focus on the production of short-chain fatty acids (bacterial metabolites which play crucial roles as immunomodulatory molecules) to investigate connections between microbiome, immune phenotypes, and clinical outcomes including infections, treatment-related toxicity, and disease progression, to light on novel avenues for therapeutic interventions in CLL.

Background The immune pathogenesis underlying the diverse clinical course of COVID-19 is poorly understood. Currently, there is an unmet need in daily clinical practice for early biomarkers and improved risk stratification tools to help identify and monitor COVID-19 patients at risk of severe disease. Methods We performed longitudinal assessment of stimulated immune responses in 30 patients hospitalized with COVID-19. We used the TruCulture whole-blood ligand-stimulation assay applying standardized stimuli to activate distinct immune pathways, allowing quantification of cytokine responses. We further characterized immune cell subsets by flow cytometry and used this deep immunophenotyping data to map the course of clinical disease within and between patients. Results Here we demonstrate impairments in innate immune response pathways at time of COVID-19 hospitalization that are associated with the development of severe disease. We show that these impairments are transient in those discharged from hospital, as illustrated by functional and cellular immune reconstitution. Specifically, we identify lower levels of LPS-stimulated IL-1β, and R848-stimulated IL-12 and IL-17A, at hospital admission to be significantly associated with increasing COVID-19 disease severity during hospitalization. Furthermore, we propose a stimulated immune response signature for predicting risk of developing severe or critical COVID-19 disease at time of hospitalization, to validate in larger cohorts. Conclusions We identify early impairments in innate immune responses that are associated with subsequent COVID-19 disease severity. Our findings provide basis for early identification of patients at risk of severe disease which may have significant implications for the early management of patients hospitalized with COVID-19.

Defects in the interleukin-12/interferon-gamma (IFN-γ) pathway and anti-IFN-γ antibodies have been associated with severe nontuberculous mycobacteria (NTM) infections. Consequently, disseminated NTM infections should prompt investigations for immunodeficiency. Herein, we report a case of a treatment refractory and ultimately disseminated and fatal Mycobacterium avium complex infection in a 71-year-old woman of Thai origin. Simultaneously, she had recurrent Salmonella kentucky cultured from stool samples and chronic perianal HSV-2 lesions. Late in the course of disease, anti-IFN-γ autoantibodies were demonstrated. Clinical studies investigating immunomodulating therapy and treatment among patients with anti-IFN-γ autoantibodies are lacking and, in this case, treatment seemed of a more palliative nature.

Background: JC-virus (JCV) associated granular cerebellar neuronopathy (JCV-GCN), which causes severe ataxia, is a rare complication of severe immunodeficiency and caused by a variant of JCV with a VP1-deletion. JCV-GCN has not yet been reported in patients with autoimmune lymphoproliferative syndrome (ALPS-FAS). We report a 34-year old woman with autoimmune lymphoproliferative syndrome (ALPS-FAS) and common variable immunodeficiency (CVID) who developed JCV-GCN which progressed to fatal progressive multifocal leukoencephalopathy ( PML). Methods: Extensive longitudinal virologic, genetic and immunologic characterisation by JCV CSF quantification and sequencing, peripheral blood flowcytometry and exome sequencing. Results : A diagnosis of JCV-GCN was established by findings of severe cerebellar vermis atrophy on MRI and a JCV variant with a VP1-deletion (JCV-VP1) in CSF. During a period of 18 months, at which treatment with high dose immunoglobulin, cidofovir, mirtazapine and high dose fucidic acid were given, cerebellar ataxia was relatively stable. In CSF a mixture of predominantly JCV-VP1 with only low levels of wildtype JCV was found. However, classic PML ensued, with rising JCV- CSF levels and a complete change to JCV-wildtype quasi species without JCV-VP1. Immunological findings were unusual, with disappearance of the original ALPS-FAS phenotype. Trio-exome sequencing identified a heterozygote de novo variant of unknown significance in RAG1. FAS mutations were not detected in the patient or in the parents, suggesting that her previously diagnosed ALPS-FAS-FAS was caused by somatic mosaicism. At the progression to PML, increased expression of T-cell PD-1 was demonstrated, suggesting T-cell exhaustion. Treatment with pembrolizumab, an inhibitor of PD-1, was attempted but followed by rapid and fatal PML progression. Conclusion: JCV-GCN may complicate ALPS and may progress to PML. In JCV-GCN, wild-type and JCV-VP-1 can co-exist in CSF. Quantification and sequencing of JCV in CSF is helpful to characterize and monitor disease and treatment. Progression of disease may be associated with development of T-cell exhaustion, but the benefit PD-1 inhibitor treatment is uncertain.

Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through recognition and phagocytosis of pathogens, and mediation of acute inflammation through secretion of pro-inflammatory cytokines. Furthermore, monocytes constitute an important link between innate and adaptive immunity through antigen presentation to lymphocytes. Patients with chronic lymphocytic leukemia (CLL) have an increased risk of infection, despite often displaying normal neutrophil and monocyte counts. CLL-cells depend on interactions with the immune microenvironment (IME) for proliferation and survival, while inducing changes in surrounding immune cells. However, changes in myeloid cell function in patients with CLL remain sparsely documented. Since the btk-inhibitor ibrutinib impairs B-cell receptor signaling and disrupts CLL-IME interactions (Niemann et al, CCR, 2016), we investigated the impact of ibrutinib and venetoclax on monocyte- and neutrophil phenotype and function in CLL patients. Methods: Nine patients treated with ibrutinib 420 mg daily for 8 weeks followed by addition of venetoclax with an initial 5-week ramp up period were included. Blood samples were taken at baseline, after 8 weeks ibrutinib monotherapy, and after another 8 weeks of ibrutinib and venetoclax combination therapy. Immune phenotype was assessed in whole blood by an 8-tube, 10 color flow cytometry panel with custom designed lyophilized antibodies (Duraclone). Immune function was characterized using TruCulture, a whole blood-ligand stimulation assay applying the toll like receptor (TLR) ligands heat killed candida albicans (TLR 2,4,6), lipopolysaccharide (LPS; TLR4), resiquimod (single-stranded RNA-virus analog, TLR7,8), and Poly:IC (double-stranded RNA-virus analog, TLR3), after which the cytokine response was measured. Informed consent from patients and approval from the ethics committee was obtained. Results: Monocyte and neutrophil counts, as wells as distribution of mature and immature neutrophils, were within normal range at baseline (n=9) and remained unchanged throughout treatment. At baseline, expression of HLA-DR on monocytes was suppressed, but increased significantly upon combination treatment with ibrutinib and venetoclax (p=0.04). HLA-DR expression on neutrophils was high at baseline, remained unchanged upon ibrutinib treatment (n=8), but declined after addition of venetoclax (n=7) (p<0.01). LPS-stimulated TNF-α and IL-6 production was suppressed at baseline, IL-6 levels increased significantly upon ibrutinib monotherapy, and levels of both TNF-α and IL-6 almost normalized upon addition of venetoclax to ibrutinib (p=0.02 for TNF-α, p=0.009 for IL-6). Conclusion: Here we show for the first time that myeloid leukocytes in CLL patients exhibit an altered HLA-DR expression at baseline, which is paralleled by an impaired cytokine response to LPS stimulation. Following treatment with ibrutinib and venetoclax, HLA-DR expression increased on monocytes and decreased on neutrophils, both representing normalization. In parallel, LPS-induced TNF-α and IL-6 production improved, likely reflecting an improved monocytic function, as monocytes respond strongly to whole-blood LPS-stimulation. Increased monocyte HLA-DR expression has previously been linked to improved cytokine response (Belge et al, J Immunol, 2002). The contribution of neutrophils to the improved cytokine response is unclear, since the function of HLA-DR expressing neutrophils in CLL is largely unknown. We propose a model where ibrutinib and venetoclax eradicate CLL-cells, disrupt tumor-IME interactions, and maybe directly impact microenvironmental cell signaling, thereby restoring neutrophil and monocyte function and improving immune function. Further investigation of myeloid function in CLL at baseline and upon different treatment regimens is warranted to guide personalized treatment based on modulation of infection-risk. Disclosures Svanberg: Novo Nordisk Foundation: Research Funding; Abbvie: Other: Travel grant. Kater:Genentech: Research Funding; Roche: Other: Travel funding, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Niemann:Acerta: Consultancy, Research Funding; Roche: Other: Travel grant; Astra Zeneca: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding; CSL Behring: Consultancy; Janssen: Consultancy, Other: Travel grant, Research Funding; Gilead: Other: Travel grant; Sunesis: Consultancy; Abbvie: Consultancy, Other: Travel grant, Research Funding.

Purpose: To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1 alpha (IL-1 alpha), interleuldn-6 (IL-6), interleuldn-10 (IL-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon a (IFN alpha). Because the c-aAbs can be transferred to patients through blood transfusion, the assay was used to assess c-aAb levels in a cohort of patients who were receiving blood transfusions and subsequently presented with or without febrile reactions.Materials and methods: The microsphere-based Luminex platform was used. Recombinant forms of human IL-1 alpha, IL-6, IL-10, GM-CSF, and IFNa were gently coupled to MAG-PLEX beads. Plasma IgG binding was measured with phycoerythrin (PE)-labeled secondary antibodies. Previously confirmed c-aAb positive and negative donor plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay.Results: The assay detected specific and saturable immunoglobulin G (IgG) binding to each of the tested cytokines in previously confirmed c-aAb positive plasmas and in preparations of pooled normal immunoglobulin. Confirmed c-aAb negative plasmas gave no saturable binding. The detection limit of the cytokine autoantibodies was estimated to be between 1 pM and 10 pM. The recovery of confirmed cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients with or without febrile transfusion reactions. The c-aAb level before and after the blood transfusions varied only slightly and in an irregular manner.Conclusion: This assay simultaneously detected up to five different c-aAbs in pooled human IgG and in plasma from individual blood donors, and it was deemed suitable for larger screenings. Based on confirmed antibody binding characteristics and the resultant reactivity in this multiplex assay, a classification of the c-aAb levels was suggested. The screening results of the recipients who received blood transfusions indicate that more studies are needed to clarify the role of antibodies, if any, in transfusion medicine and in high-dose immunoglobulin treatment. (C) 2015 Elsevier B.V. All rights reserved.