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Objectives: Systemic sclerosis (SSc) is characterized by multiple clinical manifestations. Vasculopathy is a main disease hallmark and ranges in severity from an exacerbated Raynaud phenomenon to pulmonary arterial hypertension (PAH). The potential involvement of the immune system in SSc-associated vascular abnormalities is not clear. Here, we set out to study SSc-related immune parameters and determine whether and which peripheral T cell subsets associate with vascular severity in SSc patients., Methods: Peripheral blood and clinical data were collected from 30 SSc patients, 5 patients with idiopathic PAH and 15 age and sex-matched healthy donors (HD). In this cross-sectional cohort, SSc patients with PAH (n = 15) were matched for their age, sex and medication with SSc patients with no signs of PAH (n = 15). Lymphocyte subsets were quantified by multi-colour flow cytometry., Results: SSc patients exhibited elevated percentages of T peripheral helper cells (Tph), CD4+GZMB+ T cells and decreased levels of Th1 cells compared with HD. Increased presence of both CD4+ and CD8+ exhausted-like (CD28-) T cells, characterized by raised cytokine and cytotoxic signature, was also observed in SSc compared with HD blood. Furthermore, IL-4 expressing CD4+CD8+ T cells were significantly increased in SSc peripheral blood. Interestingly, the presence of PAH in SSc was accompanied by a distinct T helper profile, characterized by raised percentages of Th17 and Tph cells., Conclusion: SSc patients with severe vasculopathy (presence of PAH) exhibited a distinct T cell profile, suggesting a potential role of auto-immune inflammation in SSc vascular complications., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Aims/hypothesis: There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity., Methods: Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects., Results: Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications., Conclusions: Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 He, Wang, van Heck, van Cranenbroek, van Rijssen, Stienstra, Netea, Joosten, Tack and Koenen.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Introduction: Immunological non-responders (INR) are people living with HIV (PLHIV) who fail to fully restore CD4+ T-cell counts despite complete viral suppression with antiretroviral therapy (ART). INR are at higher risk for non-HIV related morbidity and mortality. Previous research suggest persistent qualitative defects., Methods: The 2000HIV study (clinical trials NTC03994835) enrolled 1895 PLHIV, divided in a discovery and validation cohort. PLHIV with CD4 T-cell count <350 cells/mm 3 after ≥2 years of suppressive ART were defined as INR and were compared to immunological responders (IR) with CD4 T-cell count >500 cells/mm 3 . Logistic and rank based regression were used to analyze clinical data, extensive innate and adaptive immunophenotyping, and ex vivo monocyte and lymphocyte cytokine production after stimulation with various stimuli., Results: The discovery cohort consisted of 62 INR and 1224 IR, the validation cohort of 26 INR and 243 IR. INR were older, had more advanced HIV disease before starting ART and had more frequently a history of non-AIDS related malignancy. INR had lower absolute CD4+ T-cell numbers in all subsets. Activated (HLA-DR+, CD38+) and exhausted (PD1+) subpopulations were proportionally increased in CD4 T-cells. Monocyte and granulocyte immunophenotypes were comparable. INR lymphocytes produced less IL-22, IFN-γ, IL-10 and IL-17 to stimuli. In contrast, monocyte cytokine production did not differ. The proportions of CD4+CD38+HLA-DR+ and CD4+PD1+ subpopulations showed an inversed correlation to lymphocyte cytokine production., Conclusions: INR compared to IR have hyperactivated and exhausted CD4+ T-cells in combination with lymphocyte functional impairment, while innate immune responses were comparable. Our data provide a rationale to consider the use of anti-PD1 therapy in INR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vos, Navas, Meeder, Blaauw, Groenendijk, van Eekeren, Otten, Vadaq, Matzaraki, van Cranenbroek, Brinkman, van Lunzen, Joosten, Netea, Blok, van der Ven, Koenen and Stalenhoef.)

Objectives: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined., Methods: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment., Results: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7 + cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7 + skin cells and stabilised disease manifestations in a severely affected SSc patient., Conclusion: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells., Competing Interests: Competing interests: YvO is employee of Philikos and owns stocks in Philikos. The remaining authors have no conflicts of interest to report., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Burns are often accompanied by a dysfunctional immune response, which can lead to systemic inflammation, shock, and excessive scarring. The objective of this study was to provide insight into inflammatory pathways associated with burn-related complications. Because detailed information on the various inflammatory mediators is scattered over individual studies, we systematically reviewed animal experimental data for all reported inflammatory mediators. Meta-analyses of 352 studies revealed a strong increase in cytokines, chemokines, and growth factors, particularly 19 mediators in blood and 12 in burn tissue. Temporal kinetics showed long-lasting surges of proinflammatory cytokines in blood and burn tissue. Significant time-dependent effects were seen for IL-1β, IL-6, TGF-β1, and CCL2. The response of anti-inflammatory mediators was limited. Burn technique had a profound impact on systemic response levels. Large burn size and scalds further increased systemic, but not local inflammation. Animal characteristics greatly affected inflammation, for example, IL-1β, IL-6, and TNF-α levels were highest in young, male rats. Time-dependent effects and dissimilarities in response demonstrate the importance of appropriate study design. Collectively, this review presents a general overview of the burn-induced immune response exposing inflammatory pathways that could be targeted through immunotherapy for burn patients and provides guidance for experimental set-ups to advance burn research., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment., Competing Interests: Declaration of interests M.G.N. is a scientific founder and member of the scientific advisory board of TTxD, Lemba, and Biotrip. L.A.B.J. is a scientific founder of TTxD and Lemba. C.B. is a cofounder and scientific advisor of Myllia Biotechnology and Neurolentech., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacillus Calmette-Guèrin - vaccination induces not only protection in infants and young children against severe forms of tuberculosis, but also against non-tuberculosis related all-cause mortality. To delineate different factors influencing mycobacterial growth control, here we first investigate the effects of BCG-vaccination in healthy Dutch adults. About a quarter of individuals already control BCG-growth prior to vaccination, whereas a quarter of the vaccinees acquires the capacity to control BCG upon vaccination. This leaves half of the population incapable to control BCG-growth. Single cell RNA sequencing identifies multiple processes associated with mycobacterial growth control. These data suggest (i) that already controllers employ different mechanisms to control BCG-growth than acquired controllers, and (ii) that half of the individuals fail to develop measurable growth control irrespective of BCG-vaccination. These results shed important new light on the variable immune responses to mycobacteria in humans and may impact on improved vaccination against tuberculosis and other diseases., (© 2024. The Author(s).)

Background: Endogenous steroid hormones have significant effects on inflammatory and immune processes, but the immunological activities of steroidogenesis precursors remain largely unexplored., Methods: We conducted a systematic approach to examine the association between steroid hormones profile and immune traits in a cohort of 534 healthy volunteers. Serum concentrations of steroid hormones and their precursors (cortisol, progesterone, testosterone, androstenedione, 11-deoxycortisol and 17-OH progesterone) were determined by liquid chromatography-tandem mass spectrometry. Immune traits were evaluated by quantifying cellular composition of the circulating immune system and ex vivo cytokine responses elicited by major human pathogens and microbial ligands. An independent cohort of 321 individuals was used for validation, followed by in vitro validation experiments., Findings: We observed a positive association between 11-deoxycortisol and lymphoid cellular subsets numbers and function (especially IL-17 response). The association with lymphoid cellularity was validated in an independent validation cohort. In vitro experiments showed that, as compared to androstenedione and 17-OH progesterone, 11-deoxycortisol promoted T cell proliferation and Candida-induced Th17 polarization at physiologically relevant concentrations. Functionally, 11-deoxycortisol-treated T cells displayed a more activated phenotype (PD-L1 high CD25 high CD62L low CD127 low ) in response to CD3/CD28 co-stimulation, and downregulated expression of T-bet nuclear transcription factor., Interpretation: Our findings suggest a positive association between 11-deoxycortisol and T-cell function under physiological conditions. Further investigation is needed to explore the potential mechanisms and clinical implications., Funding: Found in acknowledgements., Competing Interests: Declaration of interests The authors declare that there is no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab., (Copyright © 2023 Massachusetts Medical Society.)

Introduction: Thermal injury often leads to prolonged and excessive inflammation, which hinders the recovery of patients. There is a notable absence of suitable animal-free models for investigating the inflammatory processes following burn injuries, thereby impeding the development of more effective therapies to improve burn wound healing in patients., Methods: In this study, we established a human full skin equivalent (FSE) burn wound model and incorporated human peripheral blood-derived monocytes and T cells., Results: Upon infiltration into the FSEs, the monocytes differentiated into macrophages within a span of 7 days. Burn-injured FSEs exhibited macrophages with increased expression of HLA-DR + and elevated production of IL-8 (CXCL8), in comparison to uninjured FSEs. Among the T cells that actively migrated into the FSEs, the majority were CD4 + and CD25 + . These T cells demonstrated augmented expression of markers associated with regulatory T cell, Th1, or Th17 activity, which coincided with significant heightened cytokine production, including IFN-γ, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), and TGF-β1. Burn injury did not impact the studied effector T cell subsets or cytokine levels., Discussion: Collectively, this study represents a significant advancement in the development of an immunocompetent human skin model, specifically tailored for investigating burn-induced innate or adaptive immune reactions at the site of burn injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mulder, Vlig, Elgersma, Rozemeijer, Mastenbroek, Middelkoop, Joosten, Koenen and Boekema.)

A substantial part of critically ill patients suffer from sepsis-induced immunosuppression. Reversal of immunosuppression through PD-1 checkpoint inhibition has been proposed as a treatment strategy to overcome immunosuppression in these patients. The PD-1 inhibitor nivolumab, currently used in treatment of cancer, has been evaluated in phase I/II studies in patients with sepsis, demonstrating tolerability and signs of clinical efficacy. No proper dose finding was performed in these studies and, after a single high dose of 480 mg or 960 mg nivolumab, PD-1 inhibition persisted beyond 90 days in the majority of cases. As the duration of sepsis is ~7-10 days, prolonged PD-1 inhibition may unnecessarily induce longer-term immune-related side effects. Based on previously published pharmacokinetic and pharmacodynamic data of nivolumab, a thorough in silico dose finding study for nivolumab in critically ill patients was performed. We found that volume of distribution and clearance of nivolumab were not higher in patients with sepsis compared to the cancer population for which nivolumab is currently approved and showed profound variability. We found that with a single dose of 20 mg nivolumab, the PD-1 receptor occupancy is predicted to stay above the 90% threshold for a median of 23 days (90% prediction interval of 7-78 days). We propose to investigate this dose in critically ill patients as a potential safe and cost-effective pharmacotherapeutic intervention to treat sepsis-induced immunosuppression., (© 2023 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The systemic and local immune response in burn patients is often extreme and derailed. As excessive inflammation can damage healthy tissues and slow down the healing process, modulation of inflammatory responses could limit complications and improve recovery. Due to its complexity, more detailed information on the immune effects of thermal injury is needed to improve patient outcomes. We therefore characterized and quantified subsets of immune cells and mediators present in human burn wound tissue (eschar), sampled at various time points. This study shows that after burn injury, the number of immune cells were persistently increased, unlike the normal wound healing process. There was an immediate, strong increase in neutrophils and a moderate increase in monocytes/macrophages and lymphocytes, especially in the second and third week post burn. The percentage of classical (CD14 high CD16 - ) monocytes/macrophages demonstrated a steady decrease over time, whereas the proportion of intermediate (CD14 high CD16 + ) monocytes/macrophages slowly increased. The absolute numbers of T cells, NK cells and B cells increased up to week 3, while the fraction of γδ T cells was increased only in week 1. Secretome profiling revealed high levels of chemokines and an overall pro-inflammatory cytokine milieu in burn tissue. The local burn immune response shows similarities to the systemic immune reaction, but differs in neutrophil maturity and lymphocyte composition. Altogether, the neutrophil surges, high levels of pro-inflammatory cytokines and limited immunosuppression might be key factors that prolong the inflammation phase and delay the wound healing process in burns., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mulder, Vlig, Fasse, Stoop, Pijpe, van Zuijlen, Joosten, Boekema and Koenen.)

Immune mediated inflammatory diseases (IMIDs) are a heterogeneous group of debilitating, multifactorial and unrelated conditions featured by a dysregulated immune response leading to destructive chronic inflammation. The immune dysregulation can affect various organ systems: gut (e.g., inflammatory bowel disease), joints (e.g., rheumatoid arthritis), skin (e.g., psoriasis, atopic dermatitis), resulting in significant morbidity, reduced quality of life, increased risk for comorbidities, and premature death. As there are no reliable disease progression and therapy response biomarkers currently available, it is very hard to predict how the disease will develop and which treatments will be effective in a given patient. In addition, a considerable proportion of patients do not respond sufficiently to the treatment. ImmUniverse is a large collaborative consortium of 27 partners funded by the Innovative Medicine Initiative (IMI), which is sponsored by the European Union (Horizon 2020) and in-kind contributions of participating pharmaceutical companies within the European Federation of Pharmaceutical Industries and Associations (EFPIA). ImmUniverse aims to advance our understanding of the molecular mechanisms underlying two immune-mediated diseases, ulcerative colitis (UC) and atopic dermatitis (AD), by pursuing an integrative multi-omics approach. As a consequence of the heterogeneity among IMIDs patients, a comprehensive, evidence-based identification of novel biomarkers is necessary to enable appropriate patient stratification that would account for the inter-individual differences in disease severity, drug efficacy, side effects or prognosis. This would guide clinicians in the management of patients and represent a major step towards personalized medicine. ImmUniverse will combine the existing and novel advanced technologies, including multi-omics, to characterize both the tissue microenvironment and blood. This comprehensive, systems biology-oriented approach will allow for identification and validation of tissue and circulating biomarker signatures as well as mechanistic principles, which will provide information about disease severity and future disease progression. This truly makes the ImmUniverse Consortium an unparalleled approach., Competing Interests: GB was employed full time by the company GSK at the time of writing; RJB was employed full time by the company Eli Lilly and Company; DZ was employed full time by the company Pfizer Limited; RH was employed full time by the company Novartis Pharma AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vetrano, Bouma, Benschop, Birngruber, Costanzo, D’Haens, Frasca, Hillenbrand, Iversen, Johansen, Kaser, Koenen, Noehammer, Peyrin-Biroulet, Raes, Ricotti, Rosenstiel, Satagopam, Schreiber, Vermeire, Wollenberg, Weidinger, Ziemek, Danese and ImmUniverse Consortium.)

Biologics that block the T helper (Th) 17 pathway are very effective in the treatment of psoriasis and other inflammatory diseases. However, IL-17 is also crucial for antifungal host defense, and clinical trial data suggest an increase in the incidence of Candida infections during IL-17 inhibitor (IL-17i) therapy. We investigated the innate and adaptive immune responses of patients with psoriasis with a history of skin and/or mucosal candidiasis during IL-17i or IL-12/23 inhibitor therapy, comparing those responses with those of healthy controls. Patients with psoriasis with IL-17i showed significantly lower CD4+Th1-like (CCR6 ‒ CXCR3 + CCR4 ‒ ) and Th1 Th17-like (CD4 + CCR6 + CXCR3 + CCR4 ‒ ) cell percentages. Patient cells stimulated with Candida albicans produced significantly lower IL-6 in the IL-12/23 inhibitor group and IL-1β in the IL-17i group, whereas the release of TNF-α and ROS was similar between patients and controls. IFN-γ and IL-10 production in response to several stimuli after 7 days was particularly decreased in patients receiving IL-17i therapy. Finally, after stimulation with the polarizing cytokines IL-1β and IL-23, the Th17 cytokine response was significantly lower in the IL-17i patient group. These innate and adaptive immune response defects can diminish antifungal host immune response and thereby increase susceptibility to candidiasis in patients treated with IL-17i or IL-12/23 inhibitor., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Because burn injuries are often followed by a derailed immune response and excessive inflammation, a thorough understanding of the occurring reactions is key to preventing secondary complications. This systematic review, which includes 247 animal studies, shows the postburn response of 14 different immune cell types involved in immediate and long-term effects in both wound tissue and circulation. Peripheral blood neutrophil and monocyte numbers increased directly after burns, whereas thrombocyte numbers increased near the end of the first week. However, lymphocyte numbers were decreased for at least 2 weeks. In burn wound tissue, neutrophil and macrophage numbers accumulated during the first 3 weeks. Burns also altered cellular functions because we found an increased migratory potential of leukocytes, impaired antibacterial activity of neutrophils, and enhanced inflammatory mediator production by macrophages. Neutrophil surges were positively associated with burn size and were highest in rats. Altogether, this comprehensive overview of the temporal immune cell dynamics shows that unlike normal wound healing, burn injury induces a long-lasting inflammatory response. It provides a fundamental research basis to improve experimental set-ups, burn care, and outcomes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI)., Methods: This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES., Results: A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes ( NLRP3 and RELA ) and in potentially novel candidate genes, including PSMB10 , DDX1 , KMT2C, and FBXW11 . The FBXW11 canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1β production in primary immune cells extracted from the patient with autoinflammatory disease., Conclusions: Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease., Funding: This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences., Competing Interests: AH, AS, JS, HK, EZ, SH, ES, EH, EL, EJ, GS, Sd, Sv, Ev, SK, MN, RS, AH, Cv No competing interests declared, Fv Reviewing editor, eLife, (© 2022, Hebert et al.)

Systemic chronic inflammation and immune dysfunction are recognized as drivers of the development of non-AIDS related comorbidities (NARCs) in people living with HIV (PLHIV). In order to lower the risk of NARCs, it is critical to elucidate what is the contribution of alterations in the composition and function of circulating immune cells to NARCs-related pathogenesis. Findings from previous immunophenotyping studies in PLHIV are highly heterogeneous and it is not fully understood to what extent phenotypic changes on immune cells play a role in the dysregulated inflammatory response observed. In this study, three flow cytometry panels were designed and standardized to phenotypically and functionally identify the main circulating immune cell subsets in PLHIV. To reduce variability, up to 10 markers out of the approximately 20 markers in each panel were used in a custom dry format DURA Innovations (LUCID product line). Intra-assay precision tests performed for the selected cell subsets showed that the three panels had a %CV below 18% for percent of positive cells and the MFI (mean fluorescent intensity) of lineage markers. Our reported pipeline for immunophenotypic analysis facilitated the discrimination of 1153 cell populations, providing an integrated overview of circulating innate and adaptative immune cells as well as the cells' functional status in terms of activation, exhaustion, and maturation. When combined with unsupervised computational techniques, this standardized immunophenotyping approach may support the discovery of novel phenotypes with clinical relevance in NARCs and demonstrate future utility in other immune-mediated diseases., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

CCR5 is the main HIV co-receptor. We aimed to (1) compare CCR5 expression on immune cells between people living with HIV (PLHIV) using combination antiretroviral therapy (cART) and HIV-uninfected controls, (2) relate CCR5 expression to viral reservoir size and (3) assess determinants of CCR5 expression. This cross-sectional study included 209 PLHIV and 323 controls. Percentages of CCR5+ cells (%) and CCR5 mean fluorescence intensity assessed by flow cytometry in monocytes and lymphocyte subsets were correlated to host factors, HIV-1 cell-associated (CA)-RNA and CA-DNA, plasma inflammation markers and metabolites. Metabolic pathways were identified. PLHIV displayed higher percentages of CCR5+ monocytes and several CD8+ T cell subsets, but lower percentages of CCR5+ naive CD4+ T cells and regulatory T cells (Tregs). HIV-1 CA-DNA and CA-RNA correlated positively with percentages of CCR5+ lymphocytes. Metabolome analysis revealed three pathways involved in energy metabolism associated with percentage of CCR5+ CD8+ T cells in PLHIV. Our results indicate that CCR5 is differently expressed on various circulating immune cells in PLHIV. Hence, cell-trafficking of CD8+ T cells and Tregs may be altered in PLHIV. Associations between energy pathways and percentage of CCR5+ CD8+ T cells in PLHIV suggest higher energy demand of these cells in PLHIV., (© 2022. The Author(s).)

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of C. albicans by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Haas, de Jonge, Koenen, Joosten, Janssen, de Boer, Hendriks, Lefeber and Cambi.)

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Kilic, Li, Bulut, Gupta, Zhang, Qi, Peng, Tsay, Soon, Mekonnen, Ferreira, van der Made, van Cranenbroek, Koenen, Simonetti, Diavatopoulos, de Jonge, Müller, Schaal, Ostermann, Cornberg, Eiz-Vesper, van de Veerdonk, van Crevel, Joosten, Domínguez-Andrés, Xu, Netea and Li.)

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Background: Neutrophil accumulation in the skin is a hallmark of psoriasis. Novel insights on neutrophil phenotypic and functional heterogeneity raise the question to what extent these cells contribute to the sustained inflammatory skin reaction., Objective: We sought to examine the phenotype and functional properties of neutrophils in blood and skin of patients with psoriasis, and the effect of TNF-α and p40(IL-12/IL-23) antibody therapy on circulating neutrophils., Methods: Thirty-two patients with psoriasis were enrolled in an observational study performed in 2 university hospitals. We evaluated neutrophil phenotype and function using in vitro (co)culture stimulation assays, flow cytometry, multiplex immunohistochemistry, and multispectral imaging of patient-derived blood and skin samples., Results: Cluster of differentiation (CD)10 pos and CD10 neg neutrophils were increased in peripheral blood of patients with psoriasis. In CD10 neg neutrophils, different maturation stages were observed, including a subset resembling aged neutrophils that was 3 times more abundant than in healthy individuals. These aged neutrophils displayed suboptimal canonical neutrophil functions and induced IL-17 and IFN-γ production by T cells in vitro, mediated by neutrophil extracellular trap formation. Also, mature and aged neutrophils were present in psoriatic skin and were found in the vicinity of T cells. Upon antibody therapy, numbers of these cells in circulation decreased., Conclusions: Patients with psoriasis reveal a unique neutrophil profile in circulation, and 2 distinct neutrophil subsets are present in psoriatic skin. Targeted biological treatment may aid in the containment of sustained neutrophil-mediated inflammation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors., Result: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target., Conclusion: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.

Twenty to thirty percent of psoriasis (Pso) patients will develop psoriatic arthritis (PsA). Detection of Pso patients that are (at risk for) developing PsA is essential to prevent structural damage. We conducted a systematic search of five bibliographic databases, up to May 2020. We searched for studies assessing markers (clinical, laboratory, genetic) associated with the development or presence of PsA in Pso patients. Study selection and quality assessment of the included studies was performed, followed by a qualitative best evidence synthesis to determine the level of evidence for a marker and its association with concomitant/developing PsA in Pso. Overall, 259 possible markers were identified in 119 studies that met the inclusion criteria. Laboratory markers related to inflammation and bone metabolism reached a strong level of evidence for the association (not prediction) of PsA in Pso. Only CXCL10 showed strong evidence for a positive predictive value for PsA in Pso. The importance of timely detecting PsA in a Pso population, and finding more (bio)markers contributing to early detection, remains high.

The endocrine and the immune systems interact by sharing receptors for hormones and cytokines, cross-control and feedback mechanisms. To date, no comprehensive study has assessed the impact of thyroid hormones on immune homeostasis. By studying immune phenotype (cell populations, antibody concentrations, circulating cytokines, adipokines and acute-phase proteins, monocyte-platelet interactions and cytokine production capacity) in two large independent cohorts of healthy volunteers of Western European descent from the Human Functional Genomics Project (500FG and 300BCG cohorts), we identified a crucial role of the thyroid hormone thyroxin (T4) and thyroid-stimulating hormone (TSH) on the homeostasis of lymphocyte populations. TSH concentrations were strongly associated with multiple populations of both effector and regulatory T cells, whereas B-cell populations were significantly associated with free T4 (fT4). In contrast, fT4 and TSH had little impact on myeloid cell populations and cytokine production capacity. Mendelian randomization further supported the role of fT4 for lymphocyte homeostasis. Subsequently, using a genomics approach, we identified genetic variants that influence both fT4 and TSH concentrations and immune responses, and gene set enrichment pathway analysis showed enrichment of fT4-affected gene expression in B-cell function pathways, including the CD40 pathway, further supporting the importance of fT4 in the regulation of B-cell function. In conclusion, we show that thyroid function controls the homeostasis of the lymphoid cell compartment. These findings improve our understanding of the immune responses and open the door for exploring and understanding the role of thyroid hormones in the lymphocyte function during disease., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Long-term changes in the immune system of successfully treated people living with HIV (PLHIV) remain incompletely understood. In this study, we assessed 108 white blood cell (WBC) populations in a cohort of 211 PLHIV on stable antiretroviral therapy and in 56 HIV-uninfected controls using flow cytometry. We show that marked differences exist in T cell maturation and differentiation between PLHIV and HIV-uninfected controls: PLHIV had reduced percentages of CD4+ T cells and naïve T cells and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells, together with increased Th17/regulatory T cell (Treg) ratios. PLHIV also exhibited altered B cell maturation with reduced percentages of memory B cells and increased numbers of plasmablasts. Determinants of the T and B cell composition in PLHIV included host factors (age, sex, and smoking), markers of the HIV reservoir, and CMV serostatus. Moreover, higher circulating Th17 percentages were associated with higher plasma concentrations of interleukin (IL) 6, soluble CD14, the gut homing chemokine CCL20, and intestinal fatty acid binding protein (IFABP). The changes in circulating lymphocytes translated into functional changes with reduced interferon (IFN)- γ responses of peripheral blood mononuclear cells to stimulation with  Candida albicans  and  Mycobacterium tuberculosis. In conclusion, this comprehensive analysis confirms the importance of persistent abnormalities in the number and function of circulating immune cells in PLHIV on stable treatment., Competing Interests: This study was supported by an AIDS-fonds (#P-29001) Netherlands and an ERC Advanced Grant (#833247). LV was supported by FWO (grant 1.8.020.09.N.00) and Collen-Francqui Research Professor Mandate. SR received a strategic basic research fund of the Research Foundation – Flanders (FWO, 1S32916N). TS and JL were employed by ViiV Healthcare. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van de Wijer, van der Heijden, Horst, Jaeger, Trypsteen, Rutsaert, van Cranenbroek, van Rijssen, Joosten, Joosten, Vandekerckhove, Schoofs, van Lunzen, Netea, Koenen, van der Ven and de Mast.)

Background: The mTOR inhibitor everolimus used in cancer has immune-modulating effects, potentially contributing to an antitumor response but also leading to pulmonary toxicity. We studied the association of immunological cell subsets with antitumor response and pulmonary toxicity in breast cancer patients treated with everolimus plus exemestane., Methods: In this exploratory analysis, peripheral blood mononuclear cells (PBMCs) were collected at baseline and 14, 35, 60, and 90 days after start of treatment, and at the moment of pulmonary toxicity. The percentage and absolute number of T-cells, B-cells, NK-cells, monocytes and numerous subtypes were measured in peripheral blood using flow cytometric analysis and were compared using a (paired) t-test., Results: From 20 patients, a total of 89 samples were collected. At baseline, responders versus non-responders had 0.86% versus 0.32% CD4+ effector cells (CD45RA+CD27-) (p = 0.1266) and non-response could be predicted with 0.71 sensitivity and 0.82 specificity. Patients who developed pulmonary toxicity compared to patients without pulmonary toxicity had relatively more NKT-cells at baseline (6.0% versus 1.3%, p = 0.0068, 59 k versus 12 k * 10 9 /l, p = 0.0081) and at the moment of toxicity (5.2% versus 1.2%, p = 0.0106 and 47 k versus 16 k * 10 9 /l, p = 0.0466). Baseline percentage NKT cells predicted pulmonary toxicity with 0.78 sensitivity and 1.0 specificity., Conclusions: Our results suggest that baseline CD4+ effector cells may be predictive of antitumor responses and baseline NKT cells may be predictive of pulmonary toxicity. These results warrant further validation., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Severe burn injury causes local and systemic immune responses that can persist up to months, and can lead to systemic inflammatory response syndrome, organ damage and long-term sequalae such as hypertrophic scarring. To prevent these pathological conditions, a better understanding of the underlying mechanisms is essential. In this longitudinal study, we analyzed the temporal peripheral blood immune profile of 20 burn wound patients admitted to the intensive care by flow cytometry and secretome profiling, and compared this to data from 20 healthy subjects. The patient cohort showed signs of systemic inflammation and persistently high levels of pro-inflammatory soluble mediators, such as IL-6, IL-8, MCP-1, MIP-1β, and MIP-3α, were measured. Using both unsupervised and supervised flow cytometry techniques, we observed a continuous release of neutrophils and monocytes into the blood for at least 39 days. Increased numbers of immature neutrophils were present in peripheral blood in the first three weeks after injury (0.1-2.8 × 10 6 /ml after burn vs. 5 × 10 3 /ml in healthy controls). Total lymphocyte numbers did not increase, but numbers of effector T cells as well as regulatory T cells were increased from the second week onward. Within the CD4 + T cell population, elevated numbers of CCR4 + CCR6 - and CCR4 + CCR6 + cells were found. Altogether, these data reveal that severe burn injury induced a persistent innate inflammatory response, including a release of immature neutrophils, and shifts in the T cell composition toward an overall more pro-inflammatory phenotype, thereby continuing systemic inflammation and increasing the risk of secondary complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mulder, Vlig, Boekema, Stoop, Pijpe, van Zuijlen, de Jong, van Cranenbroek, Joosten, Koenen and Ulrich.)

Inflammatory disorders like diabetes, systemic lupus erythematodes, inflammatory lung diseases, rheumatoid arthritis and multiple sclerosis, but also rejection of transplanted organs and GvHD, form a major burden of disease. Current classes of immune suppressive drugs to treat these disorders are never curative and side effects are common. Therefore there is a need for new drugs with improved and more targeted modes of action. Potential candidates are the DNA methyl transferase inhibitor 5-azacytidine (Aza) and its derivative 5-aza 2'deoxycitidine (DAC). Aza and DAC have been tested in several pre-clinical in vivo studies. In order to obtain an overview of disorders for which Aza and/or DAC can be a potential treatment, and to find out where information is lacking, we systematically reviewed pre-clinical animal studies assessing Aza or DAC as a potential therapy for distinct inflammatory disorders. Also, study quality and risk of bias was systematically assessed. In the 35 identified studies, we show that both Aza and DAC do not only seem to be able to alleviate a number of inflammatory disorders, but also prevent solid organ rejection and GvHD in in vivo pre-clinical animal models. Aza/DAC are known to upregulate FOXP3, a master transcription factor for Treg, in vitro. Seventeen studies described the effect on Treg, of which 16 studies showed an increase in Treg. Increasing Treg therefore seems to be a common mechanism in preventing inflammatory disorders by Aza/DAC. We also found, however, that many essential methodological details were poorly reported leading to an unclear risk of bias. Therefore, reported effects might be an overestimation of the true effect.

Introduction: Transdermal analysis patches (TAPs) noninvasively measure soluble proteins in the stratum corneum. Ultimately, such local protein profiles could benefit the search for biomarkers to improve personalized treatment in psoriasis. This study aimed to explore the patient friendliness and protein detection by TAP in pediatric psoriasis in daily clinical practice., Methods: In this observational study, TAPs measuring CXC chemokine ligand (CXCL)-1/2, CC chemokine ligand (CCL)-27, interleukin (IL)-1RA, IL-23, IL-1α, IL-8, IL-4, IL-22, IL-17A, vascular endothelial growth factor (VEGF), human beta-defensin (hBD)-2, hBD-1, and kallikrein-related peptidase (KLK)-5 were applied on lesional, peri-lesional, and non-lesional skin sites of psoriasis patients aged >5 to <18 years. Discomfort during TAP removal as an indicator for patient friendliness was assessed by visual analogue scale (VAS; range 0-10)., Results: Thirty-two patients (median age 14.0 years) were included, of which 19 were treated with solely topical agents and 13 with systemic treatment. The median VAS of discomfort during TAP removal was 1.0 (interquartile range 1.0). Significantly higher levels in lesional versus non-lesional skin were found for IL-1RA, VEGF, CXCL-1/2, hBD-2, and IL-8, whereas lower levels were found for IL-1α. Skin surface proteins were measured in both treatment groups, with significant higher lesional levels of KLK-5, IL-1RA, hBD-2, IL-1α, IL-23, and CCL-27 in the systemic treatment group., Conclusion: The TAP platform holds the potential for patient-friendly and noninvasive monitoring of skin-derived proteins in pediatric psoriasis patients in daily clinical practice., (© 2021 The Author(s). Published by S. Karger AG, Basel.)

The application of stem cell-derived secretome in regenerative therapies offers the key advantage that instead of the stem cells, only their effective paracrine compounds are in vivo delivered. Ideally, the secretome can be steered by the culture conditions of the stem cells. So far, most studies use stem cells cultured on stiff plastic substrates, not representative of their native 3D environment. In this study, cells are cultured inside synthetic polyisocyanide (PIC)-based hydrogels, which are minimal, tailorable, and highly reproducible biomimetic matrices. Secretome analysis of human adipose-derived stem cells (multiplex, ELISA) displays that matrix manipulation is a powerful tool to direct the secretome composition. As an example, cells in nonadherent PIC gels secrete increased levels of IL-10 and the conditioned media from 3D culture accelerate wound closure. In all, our PIC-based approach opens the door to dedicated matrix design to engineer the secretome for custom applications.

Background: Expression quantitative trait loci (eQTL) studies are used to interpret the function of disease-associated genetic risk factors. To date, most eQTL analyses have been conducted in bulk tissues, such as whole blood and tissue biopsies, which are likely to mask the cell type-context of the eQTL regulatory effects. Although this context can be investigated by generating transcriptional profiles from purified cell subpopulations, current methods to do this are labor-intensive and expensive. We introduce a new method, Decon2, as a framework for estimating cell proportions using expression profiles from bulk blood samples (Decon-cell) followed by deconvolution of cell type eQTLs (Decon-eQTL)., Results: The estimated cell proportions from Decon-cell agree with experimental measurements across cohorts (R ≥ 0.77). Using Decon-cell, we could predict the proportions of 34 circulating cell types for 3194 samples from a population-based cohort. Next, we identified 16,362 whole-blood eQTLs and deconvoluted cell type interaction (CTi) eQTLs using the predicted cell proportions from Decon-cell. CTi eQTLs show excellent allelic directional concordance with eQTL (≥ 96-100%) and chromatin mark QTL (≥87-92%) studies that used either purified cell subpopulations or single-cell RNA-seq, outperforming the conventional interaction effect., Conclusions: Decon2 provides a method to detect cell type interaction effects from bulk blood eQTLs that is useful for pinpointing the most relevant cell type for a given complex disease. Decon2 is available as an R package and Java application (https://github.com/molgenis/systemsgenetics/tree/master/Decon2) and as a web tool (www.molgenis.org/deconvolution).

Background: Recent clinical trials using regulatory T cells (Treg) support the therapeutic potential of Treg-based therapy in transplantation and autoinflammatory diseases. Despite these clinical successes, the effect of Treg on inflamed tissues, as well as their impact on immune effector function in vivo , is poorly understood. Therefore, we here evaluated the effect of human Treg injection on cutaneous inflammatory processes in vivo using a humanized mouse model of human skin inflammation (huPBL-SCID-huSkin)., Methods: SCID beige mice were transplanted with human skin followed by intraperitoneal (IP) injection of 20-40 × 10 6 allogeneic human PBMCs. This typically results in human skin inflammation as indicated by epidermal thickening (hyperkeratosis) and changes in dermal inflammatory markers such as the antimicrobial peptide hBD2 and epidermal barrier cytokeratins K10 and K16, as well as T cell infiltration in the dermis. Ex vivo -expanded human Treg were infused intraperitoneally. Human cutaneous inflammation and systemic immune responses were analysed by immunohistochemistry and flow cytometry., Results: We confirmed that human Treg injection inhibits skin inflammation and the influx of effector T cells. As a novel finding, we demonstrate that human Treg injection led to a reduction of IL-17-secreting cells while promoting a relative increase in immunosuppressive FOXP3+ Treg in the human skin, indicating active immune regulation in controlling the local proinflammatory response. Consistent with the local control (skin), systemically (splenocytes), we observed that Treg injection led to lower frequencies of IFN γ and IL-17A-expressing human T cells, while a trend towards enrichment of FOXP3+ Treg was observed., Conclusion: Taken together, we demonstrate that inhibition of skin inflammation by Treg infusion, next to a reduction of infiltrating effector T cells, is mediated by restoring both the local and systemic balance between cytokine-producing effector T cells and immunoregulatory T cells. This work furthers our understanding of Treg-based immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships and do not have any conflict of interest., (Copyright © 2020 S. Landman et al.)

Systemic sclerosis (SSc) is an autoimmune disease which is characterized by vasculopathy, tissue fibrosis and activation of the innate and adaptive immune system. Clinical features of the disease consists of skin thickening and internal organ involvement. Due to the heterogeneous nature of the disease it is difficult to predict disease progression and complications. Despite the discovery of novel autoantibodies associated with SSc, there is an unmet need for biomarkers for diagnosis, disease progression and response to treatment. To date, the use of single (surrogate) biomarkers for these purposes has been unsuccessful. Combining multiple biomarkers in to predictive panels or ultimately algorithms could be more precise. Given the limited therapeutic options and poor prognosis of many SSc patients, a better understanding of the immune-pathofysiological profiles might aid to an adjusted therapeutic approach. Therefore, we set out to explore immunological fingerprints in various clinically defined forms of SSc. We used multilayer profiling to identify unique immune profiles underlying distinct autoantibody signatures. These immune profiles could fill the unmet need for prognosis and response to therapy in SSc. Here, we present 3 pathophysiological fingerprints in SSc based on the expression of circulating antibodies, vascular markers and immunomodulatory mediators., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Maintenance of regulatory T cells CD4 + CD25 high FOXP3 + (Treg) stability is vital for proper Treg function and controlling the immune equilibrium. Treg cells are heterogeneous and can reveal plasticity, exemplified by their potential to express IL-17A. TNFα-TNFR2 signaling controls IL-17A expression in conventional T cells via the anti-inflammatory ubiquitin-editing and kinase activity regulating enzyme TNFAIP3/ A20 (tumor necrosis factor-alpha-induced protein 3). To obtain a molecular understanding of TNFα signaling on IL-17 expression in the human effector ( eff Treg, CD25 high CD45RA - ) Treg subset, we here studied the kinome activity regulation by TNFα signaling. Using FACS-sorted naïve ( naïve Treg, CD25 high CD45RA + ) and eff Treg subsets, we demonstrated a reciprocal relationship between TNFα and IL-17A expression; eff Treg (TNFα low /IL-17A high ) and naïve Treg (TNFα high /IL-17A low ). In eff Treg, TNFα-TNFR2 signaling prevented IL-17A expression, whereas inhibition of TNFα signaling by clinically applied anti-TNF antibodies led to increased IL-17A expression. Inhibition of TNFα signaling led to reduced TNFAIP3 expression, which, by using siRNA inhibition of TNFAIP3 , appeared causally linked to increased IL-17A expression in eff Treg. Kinome activity screening of CD3/CD28-activated eff Treg revealed that anti-TNF-mediated neutralization led to increased kinase activity. STRING association analysis revealed that the TNF suppression eff Treg kinase activity network was strongly associated with kinases involved in TCR, JAK, MAPK, and PKC pathway signaling. Small-molecule-based inhibition of TCR and JAK pathways prevented the IL-17 expression in eff Treg. Together, these findings stress the importance of TNF-TNFR2 in regulating the kinase architecture of antigen-activated eff Treg and controlling IL-17 expression of the human Treg. These findings might be relevant for optimizing anti-TNF-based therapy and may aid in preventing Treg plasticity in case of Treg-based cell therapy., (Copyright © 2020 Urbano, He, Heeswijk, Filho, Tijssen, Smeets, Joosten and Koenen.)

Regulatory T cells (Treg) are well-known for their immune regulatory potential and are essential for maintaining immune homeostasis. The rationale of Treg-based immunotherapy for treating autoimmunity and transplant rejection is to tip the immune balance of effector T cell-mediated immune activation and Treg-mediated immune inhibition in favor of Treg cells, either through endogenous Treg expansion strategies or adoptive transfer of ex vivo expanded Treg. Compelling evidence indicates that Treg show properties of phenotypic heterogeneity and instability, which has caused considerable debate in the field regarding their correct use. Consequently, for further optimization of Treg-based immunotherapy, it is vital to further our understanding of Treg proliferative, migratory, and suppressive behavior. It is increasingly appreciated that the functional profile of immune cells is highly dependent on their metabolic state. Although the metabolic profiles of effector T cells are progressively understood, little is known on Treg in this respect. The objective of this review is to outline the current knowledge of human Treg metabolic profiles associated with the regulation of Treg functionality. As such information on human Treg is still limited, where information was lacking, we included insightful findings from mouse studies. To assess the available evidence on metabolic pathways involved in Treg functionality, PubMed, and Embase were searched for articles in English indexed before April 28th, 2019 using "regulatory T lymphocyte," "cell metabolism," "cell proliferation," "migration," "suppressor function," and related search terms. Removal of duplicates and search of the references was performed manually. We discerned that while glycolysis fuels the biosynthetic and bioenergetic needs necessary for proliferation and migration of human Treg, suppressive capacity is mainly maintained by oxidative metabolism. Based on the knowledge of metabolic differences between Treg and non-Treg cells, we additionally discuss and propose ways of how human Treg metabolism could be exploited for the betterment of tolerance-inducing therapies., (Copyright © 2019 Kempkes, Joosten, Koenen and He.)

Sepsis is the cause of more than 5.3 million deaths per year, and novel immunotherapeutic strategies are highly warranted. Human models that mirror sepsis immunology are instrumental to this aim. The response to endotoxin in humans during the first 24 h captures many hallmarks of the inflammatory response observed in sepsis. However, the long-term immunologic effects of human experimental endotoxemia have been sparsely studied and could be determinant for the use of this model in sepsis therapy research. In the present work, we studied the immune-composition of healthy subjects challenged with endotoxin (1 ng/kg) 4 h, 2 days, and 20 days post administration by flow cytometry to study the effects on innate and adaptive immune system, and compared it with the immune-composition in patients during the first 9 days after onset of septic shock. We found several differences and similarities between these groups. Experimental endotoxemia resulted in an increase in absolute numbers of intermediate monocytes, which also displayed lower human leucocyte antigen expression 20 days post endotoxin. These changes differed with those observed in septic shock patients. Another long-term effect of experimental endotoxemia was elevated numbers of effector CD8 cells and an increased percentage of proliferating and cytokine expressing CD8 cells, and these phenomena were also present in sepsis patients. In conclusion, despite considerable differences, experimental endotoxemia captures several long-term aspects of sepsis immunology, specifically the behavior of CD8 T cells, which may eventually aid the development of new therapies for sepsis patients.

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A 3 receptor (A 3 R)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A 3 R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A 3 R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A 3 R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A 3 Rs., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Objective: Platelet hyperreactivity and increased platelet-monocyte aggregation (PMA) are associated with increased cardiovascular risk and inflammation. In a previous cross-sectional study, individuals using a raltegravir (RAL)-based regimen were found to have reduced platelet reactivity and PMA compared with other antiretroviral regimens. Our aim was to investigate whether switching from a nonintegrase inhibitor regimen to a RAL-based regimen reduces platelet reactivity or PMA., Design: An investigator initiated, single-centre, prospective randomized, open-label, blinded endpoint trial., Methods: Forty HIV-infected adults using a nonintegrase inhibitor containing regimen with undetectable viral load were randomized to either continue their regimen or switch to a RAL-based regimen for 10 weeks, continuing the same backbone. The primary outcome was the change in platelet reactivity at week 10, which was determined as the expression of the platelet activation marker P-selectin and binding of fibrinogen before and after ex-vivo stimulation with different platelet agonists. Secondary outcomes included PMA, plasma markers of platelet activation and markers of inflammation and immune cell activation., Results: Twenty-one participants were enrolled in the continuation group and 19 in the RAL group. Baseline characteristics were comparable between groups. There were no differences in the change in platelet reactivity to either platelet agonist at week 10, nor in plasma markers of platelet activation. PMA, C-reactive protein, T-cell activation (CD38HLA-DR) and monocyte (CD14CD16) subsets., Conclusion: Switching a nonintegrase inhibitor containing regimen to a RAL-based regimen does not reduce platelet reactivity, platelet-leukocyte aggregation, inflammation and immune activation in virologically suppressed HIV-infected individuals., Clinical Trial Number: NCT02383355.

Background: Anti-TNF inhibitors successfully improve the quality of life of patients with inflammatory disease. Unfortunately, not all patients respond to anti-TNF therapy, and some patients show paradoxical immune side effects, which are poorly understood. Surprisingly, anti-TNF agents were shown to promote IL-17A production with as yet unknown clinical implications., Objective: We sought to investigate the molecular mechanism underlying anti-TNF-driven IL-17A expression and the clinical implications of this phenomenon., Methods: Fluorescence-activated cell sorting, RNA sequencing, quantitative real-time PCR, Western blotting, small interfering RNA interference, and kinase inhibitors were used to study the molecular mechanisms in isolated human CD4 + T cells from healthy donors. The clinical implication was studied in blood samples of patients with inflammatory bowel disease (IBD) receiving anti-TNF therapy., Results: Here we show that anti-TNF treatment results in inhibition of the anti-inflammatory molecule TNF-α-induced protein 3 (TNFAIP3)/A20 in memory CD4 + T cells. We found an inverse relationship between TNFAIP3/A20 expression levels and IL-17A production. Inhibition of TNFAIP3/A20 promotes kinase activity of p38 mitogen-activated protein kinase and protein kinase C, which drives IL-17A expression. Regulation of TNFAIP3/A20 expression and cognate IL-17A production in T cells are specifically mediated through TNF receptor 2 signaling. Ex vivo, in patients with IBD treated with anti-TNF, we found further evidence for an inverse relationship between TNFAIP3/A20 expression levels and IL-17A-producing T cells., Conclusion: Anti-TNF treatment interferes in the TNFAIP3/A20-mediated anti-inflammatory feedback loop in CD4 + T cells and promotes kinase activity. This puts TNFAIP3/A20, combined with IL-17A expression, on the map as a potential tool for predicting therapy responsiveness or side effects of anti-TNF therapy. Moreover, it provides novel targets related to TNFAIP3/A20 activity for superior therapeutic regimens in patients with IBD., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

In a Dutch consanguineous family with recessively inherited nonsyndromic hearing impairment (HI), homozygosity mapping combined with whole-exome sequencing revealed a MPZL2 homozygous truncating variant, c.72del (p.Ile24Metfs ∗ 22). By screening a cohort of phenotype-matched subjects and a cohort of HI subjects in whom WES had been performed previously, we identified two additional families with biallelic truncating variants of MPZL2. Affected individuals demonstrated symmetric, progressive, mild to moderate sensorineural HI. Onset of HI was in the first decade, and high-frequency hearing was more severely affected. There was no vestibular involvement. MPZL2 encodes myelin protein zero-like 2, an adhesion molecule that mediates epithelial cell-cell interactions in several (developing) tissues. Involvement of MPZL2 in hearing was confirmed by audiometric evaluation of Mpzl2-mutant mice. These displayed early-onset progressive sensorineural HI that was more pronounced in the high frequencies. Histological analysis of adult mutant mice demonstrated an altered organization of outer hair cells and supporting cells and degeneration of the organ of Corti. In addition, we observed mild degeneration of spiral ganglion neurons, and this degeneration was most pronounced at the cochlear base. Although MPZL2 is known to function in cell adhesion in several tissues, no phenotypes other than HI were found to be associated with MPZL2 defects. This indicates that MPZL2 has a unique function in the inner ear. The present study suggests that deleterious variants of Mplz2/MPZL2 affect adhesion of the inner-ear epithelium and result in loss of structural integrity of the organ of Corti and progressive degeneration of hair cells, supporting cells, and spiral ganglion neurons., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Recent regulatory T cell (Treg) based clinical trials support their therapeutic potential in transplantation and auto-inflammatory diseases. However, large numbers of Treg are needed to accomplish therapeutic efficacy. Local injection at the site of inflammation (targeted delivery) may lower the numbers needed for therapy. We evaluated if local delivery of low numbers of human Treg by intradermal injection was able to prevent skin inflammation, using the humanized mouse huPBL-SCID-huSkin allograft model. A dose of only 1 × 10 5 freshly isolated, non expanded Treg injected intradermally in close proximity to the transplanted human skin prevented inflammation of the grafted tissue induced by 4 × 10 7 IP injected human allogeneic PBMCs, (ratio Treg:PBMC = 1:400), as indicated by the inhibition of epidermal thickening, sustained Keratin-10 expression, the absence of Keratin-16 up regulation and prevention of human CD3+ T cell influx. A concomitant reduction of human T cells was observed in lymph nodes and spleen of the mice. Injection of Treg at the contralateral side was also shown to inhibit skin inflammation, suggesting that the inflammatory response was regulated both locally and systemically. In conclusion, local application of Treg may be an attractive way to suppress inflammation in vivo without the need for prior ex vivo expansion.

The immune response to pathogens varies substantially among people. Whereas both genetic and nongenetic factors contribute to interperson variation, their relative contributions and potential predictive power have remained largely unknown. By systematically correlating host factors in 534 healthy volunteers, including baseline immunological parameters and molecular profiles (genome, metabolome and gut microbiome), with cytokine production after stimulation with 20 pathogens, we identified distinct patterns of co-regulation. Among the 91 different cytokine-stimulus pairs, 11 categories of host factors together explained up to 67% of interindividual variation in cytokine production induced by stimulation. A computational model based on genetic data predicted the genetic component of stimulus-induced cytokine production (correlation 0.28-0.89), and nongenetic factors influenced cytokine production as well.

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2'-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4 + T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4 + CD25 high FOXP3 + Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFN γ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFN γ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation in vitro , in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro . Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα-TNFR2 underlying the maintenance of Treg stability in vitro . Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα-TNFR2 loop plays an important role in endorsing Treg stability.

Effective immunity requires a complex network of cellular and humoral components that interact with each other and are influenced by different environmental and host factors. We used a systems biology approach to comprehensively assess the impact of environmental and genetic factors on immune cell populations in peripheral blood, including associations with immunoglobulin concentrations, from ∼500 healthy volunteers from the Human Functional Genomics Project. Genetic heritability estimation showed that variations in T cell numbers are more strongly driven by genetic factors, while B cell counts are more environmentally influenced. Quantitative trait loci (QTL) mapping identified eight independent genomic loci associated with leukocyte count variation, including four associations with T and B cell subtypes. The QTLs identified were enriched among genome-wide association study (GWAS) SNPs reported to increase susceptibility to immune-mediated diseases. Our systems approach provides insights into cellular and humoral immune trait variability in humans., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)