Human epicardial fat has a beige profile and contains higher type 2 innate lymphoid cells than subcutaneous fat.

OBJECTIVE: Epicardial adipose tissue (EAT) is a visceral fat that has been associated with coronary artery disease and atrial fibrillation. Previous work has revealed that EAT exhibits beige features. METHODS: First, a new pan-genomic microarray analysis was performed on previously collected paired human EAT and thoracic subcutaneous AT (thSAT) from the EPICAR study (n = 31) to decipher a specific immune signature and its link with browning genes. Then, adaptive (T and B cells) and innate lymphoid cell (ILC1, ILC2, and ILC3) immunophenotyping assay panels, including CD127, CD117, and prostaglandin D2 receptor 2, were performed on prospectively collected paired human multiorgan donors (n = 18; INTERFACE study). RESULTS: In the EPICAR study, a positive correlation between the T helper cell subtype Th2 immune pathway and browning genes was found in EAT versus thSAT (r = 0.82; p < 0.0001). In the INTERFACE study, this correlation was also observed (r = 0.31; p = 0.017), and a preponderance of CD4+T cells, CD8+T cells, and a few B cells was observed in all ATs (p < 0.0001). An increase in ILCs was observed in visceral AT (VAT) (i.e., EAT + VAT; 30 ± 5 ILCs per gram of AT) compared with subcutaneous counterparts (i.e., thSAT + abdominal SAT; 8 ± 2 ILCs per gram of AT; p = 0.001), with ILC1 being the most frequent (ILC1 > ILC3 > ILC2). Numbers of ILCs per gram of AT correlated with several Th2 or browning genes (IL-13, TNF receptor superfamily member 9 [TNFRSF9], and alkaline phosphatase, biomineralization associated [ALPL]). Interestingly, a specific increase in EAT-ILC2 compared with other ATs was observed, including a significant proportion expressing CD69 and/or CD25 activation markers (97.9% ± 1.2%; p < 0.0001). Finally, more natural killer cells were observed in EAT + VAT than in thSAT + abdominal SAT (p = 0.01). Exclusion of patients with coronary artery disease in the EPICAR and INTERFACE studies did not modify the main findings. Gene expression phenotyping confirmed specific upregulation of Th2 pathway and browning genes (IL-33 and uncoupling protein 1 [UCP-1]) in EAT. CONCLUSIONS: This is the first study, to our knowledge, to provide a comparison between innate and adaptive lymphoid cells in human EAT. Further studies are ongoing to decipher whether these cells could be involved in EAT beiging.

Effect of Prior Treatment With Fingolimod on Early and Late Response to Rituximab/Ocrelizumab in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Real-life studies noted that the risk of disease activity in multiple sclerosis (MS) after switching to rituximab (RTX) or ocrelizumab (OCR) may be unequal depending on prior disease-modifying therapy (DMT), with a higher risk associated with fingolimod (FING). METHODS: We performed a retrospective analysis of a structured prospective data collection including all consecutive patients with relapsing MS who were prescribed RTX/OCR in the MS center of Marseille. Cox proportional hazards models were applied to clinical and MRI outcomes. RESULTS: We included 321 patients with a median (interquartile range [IQR]) follow-up of 3.5 years (1.5-5) after RTX/OCR initiation. At the first RTX/OCR infusion, the mean (SD) age of patients was 37 (10) years, and the median (IQR) disease duration was 8 years (3-15): 68 patients did not receive treatment before RTX/OCR and 108 switched from FING, 47 from low efficacy therapy, and 98 from natalizumab. For statistical analysis, the group "FING" was divided into "short-FING" and "long-FING" groups according to the median value of the group's washout period (27 days). On Cox proportional hazards analysis, for only the "long-FING" group, the risk of relapse within the first 6 months of RTX/OCR was increased as compared with patients without previous DMT (hazard ratio [HR]: 8.78; 95% CI 1.72-44.86; p < 0.01). Previous DMT and washout period duration of FING had no effect on B-cell levels at 6 months. Beyond the first 6 months of RTX/OCR, age <40 years was associated with increased risk of relapse (HR: 3.93; 95% CI 1.30-11.89; p = 0.01), male sex with increased risk of new T2 lesions (HR: 2.26; 95% CI 1.08-4.74; p = 0.03), and EDSS ≥2 with increased risk of disability accumulation (HR: 3.01; 95% CI 1.34-6.74; p < 0.01). Previous DMT had no effect on the effectiveness of RTX/OCR beyond 6 months after initiation. DISCUSSION: For patients switching from FING to RTX/OCR, the risk of disease reactivation within the first 6 months of treatment was increased as compared with patients with other DMT or no previous DMT only when the washout period exceeded 26 days. Neither FING nor other previous DMT reduced the effectiveness of RTX/OCR beyond the first 6 months of treatment.

Clinical, biological, electrophysiological and therapeutic profile of patients with anti-MAG neuropathy according to MYD88L265P and CXCR4 mutations and underlying haemopathy.

INTRODUCTION: Anti-MAG neuropathies are associated with an IgM monoclonal gammopathy of undetermined significance (MGUS) or with a malignant haemopathy. Our objective was to determine whether the presence of a haemopathy or somatic mutations of MYD88 and CXCR4 genes influences disease presentation and response to rituximab (RTX). METHODS: We included 79 patients (mean age 74 years, disease duration 9.68 years) who had a bone marrow aspiration with morphologic and immunophenotypic analysis. MYD88L265P and CXCR4 mutations were analysed in peripheral B cells. Information collected included: inflammatory neuropathy cause and treatment sensory sum score (ISS), MRC testing, overall neuropathy limitation scale (ONLS), Rash-built Overall Disability Score (RODS), ataxia score, anti-MAG titres, peak IgM dosage, neurofilament light chain levels, motor and sensory amplitudes, motor unit index (MUNIX) and motor unit size index (MUSIX) sum scores. Efficacy of RTX was evaluated at 12 months in 26 patients. RESULTS: Malignant haematological disorders were discovered in 17 patients (22%): 13 Waldenstrom macroglobulinemia, 3 marginal zone lymphoma and one mantle cell lymphoma. MYD88L265P mutation was detected in 29/60 (48%) patients and CXCR4 in 1 single patient. Disease severity, biological and electrophysiological data and response to RTX were comparable in patients with MGUS/lymphoma and patients with/without MYD88L265P mutation. ISS was lower and MUSIX higher in patients improved by RTX. CONCLUSIONS: MYD88L265P mutation and underlying haemopathies are not predictive of a more severe disease. However, in cases of resistant and progressive neuropathy, they provide an opportunity to prescribe newly available drugs such as Bruton tyrosine kinase inhibitors.

Efficacy of Rituximab Outlasts B-Cell Repopulation in Multiple Sclerosis: Time to Rethink Dosing?

BACKGROUND AND OBJECTIVES: Patients with multiple sclerosis (PwMS) receiving extended dosing of rituximab (RTX) have exhibited no return of disease activity, which suggests that maintenance of deep depletion of circulating B cells is not necessary to maintain the efficacy of RTX in MS. METHODS: This was a prospective monocentric observational study including all consecutive PwMS who started or continued RTX after 2019, when the medical staff decided to extend the dosing interval up to 24 months for all patients. Circulating B-cell subsets were monitored regularly and systematically in case of relapse. The first extended interval was analyzed. RESULTS: We included 236 PwMS (81% with relapsing-remitting MS; mean [SD] age 43 [12] years; median [range] EDSS score 4 [0-8]; mean relapse rate during the year before RTX start 1.09 [0.99]; 41.5% with MRI activity). The median number of RTX infusions before extension was 4 (1-13). At the time of the analysis, the median delay in dosing was 17 months (8-39); the median proportion of circulating CD19+ B cells was 7% (0-25) of total lymphocytes and that of CD27+ memory B cells was 4% (0-16) of total B cells. The mean annual relapse rate did not differ before and after the extension: 0.03 (0.5) and 0.04 (0.15) (p = 0.51). Similarly, annual relapse rates did not differ before and after extension in patients with EDSS score ≤3 (n = 79) or disease duration ≤5 years (n = 71) at RTX onset. During the "extended dosing" period, MRI demonstrated no lesion accrual in 228 of the 236 patients (97%). Five patients experienced clinical relapse, which was confirmed by MRI. In these patients, the level of B-cell subset reconstitution at the time of the relapse did not differ from that for patients with the same extension window. DISCUSSION: The efficacy of RTX outlasted substantial reconstitution of circulating B cells in PwMS, which suggests that renewal of the immune system underlies the prolonged effect of RTX in MS. These findings suggest that extended interval dosing of RTX that leads to a significant reconstitution of circulating B cells is safe in PwMS, could reduce the risk of infection, and could improve vaccine efficacy.

Avdoralimab (Anti-C5aR1 mAb) Versus Placebo in Patients With Severe COVID-19: Results From a Randomized Controlled Trial (FOR COVID Elimination [FORCE]).

OBJECTIVES: Severe COVID-19 is associated with exaggerated complement activation. We assessed the efficacy and safety of avdoralimab (an anti-C5aR1 mAb) in severe COVID-19. DESIGN: FOR COVID Elimination (FORCE) was a double-blind, placebo-controlled study. SETTING: Twelve clinical sites in France (ICU and general hospitals). PATIENTS: Patients receiving greater than or equal to 5 L oxygen/min to maintain Sp o2 greater than 93% (World Health Organization scale ≥ 5). Patients received conventional oxygen therapy or high-flow oxygen (HFO)/noninvasive ventilation (NIV) in cohort 1; HFO, NIV, or invasive mechanical ventilation (IMV) in cohort 2; and IMV in cohort 3. INTERVENTIONS: Patients were randomly assigned, in a 1:1 ratio, to receive avdoralimab or placebo. The primary outcome was clinical status on the World Health Organization ordinal scale at days 14 and 28 for cohorts 1 and 3, and the number of ventilator-free days at day 28 (VFD28) for cohort 2. MEASUREMENTS AND MAIN RESULTS: We randomized 207 patients: 99 in cohort 1, 49 in cohort 2, and 59 in cohort 3. During hospitalization, 95% of patients received glucocorticoids. Avdoralimab did not improve World Health Organization clinical scale score on days 14 and 28 (between-group difference on day 28 of -0.26 (95% CI, -1.2 to 0.7; p = 0.7) in cohort 1 and -0.28 (95% CI, -1.8 to 1.2; p = 0.6) in cohort 3). Avdoralimab did not improve VFD28 in cohort 2 (between-group difference of -6.3 (95% CI, -13.2 to 0.7; p = 0.96) or secondary outcomes in any cohort. No subgroup of interest was identified. CONCLUSIONS: In this randomized trial in hospitalized patients with severe COVID-19 pneumonia, avdoralimab did not significantly improve clinical status at days 14 and 28 (funded by Innate Pharma, ClinicalTrials.gov number, NCT04371367).

Innate lymphoid cell recovery and occurrence of GvHD after hematopoietic stem cell transplantation.

Lymphocytes are essential for microbial immunity, tumor surveillance, and tissue homeostasis. However, the in vivo development and function of helper-like innate lymphoid cells (ILCs) in humans remain much less well understood than those of T, B, and NK cells. We monitored hematopoietic stem cell transplantation (HSCT) to determine the kinetics of ILC development in both children and adults. It was found that, unlike NK cells, helper-like ILCs recovered slowly, mirroring the pattern observed for T cells, with normalization achieved at 1 year. The type of graft and the proportion of CD34+ cells in the graft did not significantly affect ILC reconstitution. As HSCT is often complicated by acute or chronic graft-versus-host disease (GVHD), the potential role of ILC subsets in maintaining tissue integrity in these conditions was also analyzed. It was found that GVHD was associated with lower levels of activated and gut-homing NKp44+ ILCP, consistent with a non-redundant role of this ILC subset in preventing this life-threatening disorder in lymphopenic conditions.

Discrimination of COVID-19 From Inflammation-Induced Cytokine Storm Syndromes Using Disease-Related Blood Biomarkers.

OBJECTIVE: Infection with the novel coronavirus SARS-CoV-2 triggers severe illness with high mortality in a subgroup of patients. Such a critical course of COVID-19 is thought to be associated with the development of cytokine storm, a condition seen in macrophage activation syndrome (MAS) and secondary hemophagocytic lymphohistiocytosis (HLH). However, specific data demonstrating a clear association of cytokine storm with severe COVID-19 are still lacking. The aim of this study was to directly address whether immune activation in COVID-19 does indeed mimic the conditions found in these classic cytokine storm syndromes. METHODS: Levels of 22 biomarkers were quantified in serum samples from patients with COVID-19 (n = 30 patients, n = 83 longitudinal samples in total), patients with secondary HLH/MAS (n = 50), and healthy controls (n = 9). Measurements were performed using bead array assays and single-marker enzyme-linked immunosorbent assay. Serum biomarker levels were assessed for correlations with disease outcome. RESULTS: In patients with secondary HLH/MAS, we observed pronounced activation of the interleukin-18 (IL-18)-interferon-γ axis, increased serum levels of IL-1 receptor antagonist, intercellular adhesion molecule 1, and IL-8, and strongly reduced levels of soluble Fas ligand in the course of SARS-CoV-2 infection. These observations appeared to discriminate immune dysregulation in critical COVID-19 from the well-recognized characteristics of other cytokine storm syndromes. CONCLUSION: Serum biomarker profiles clearly separate COVID-19 from MAS or secondary HLH in terms of distinguishing the severe systemic hyperinflammation that occurs following SARS-CoV-2 infection. These findings could be useful in determining the efficacy of drugs targeting key molecules and pathways specifically associated with systemic cytokine storm conditions in the treatment of COVID-19.

Granulocyte Macrophage Colony-Stimulating Factor-Specific Autoantibodies and Cerebral Nocardia With Pulmonary Alveolar Proteinosis.

In this study, we report the history of a 40-year-old man with a primary cerebral abscess caused by Nocardia abscessus that led to the discovery of autoimmune pulmonary alveolar lipoproteinosis (anti-granulocyte-macrophage colony-stimulating factor [GM-CSF] autoantibodies). Anti-GM-CSF autoantibodies promote immunodeficiency and should be monitored to prevent opportunistic and disseminated infections and to diagnose asymptomatic pulmonary alveolar lipoproteinosis.

Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia.

Natural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis.

Coronavirus disease 2019 (COVID-19) is a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in a pandemic1. The C5a complement factor and its receptor C5aR1 (also known as CD88) have a key role in the initiation and maintenance of several inflammatory responses by recruiting and activating neutrophils and monocytes1. Here we provide a longitudinal analysis of immune responses, including phenotypic analyses of immune cells and assessments of the soluble factors that are present in the blood and bronchoalveolar lavage fluid of patients at various stages of COVID-19 severity, including those who were paucisymptomatic or had pneumonia or acute respiratory distress syndrome. The levels of soluble C5a were increased in proportion to the severity of COVID-19 and high expression levels of C5aR1 receptors were found in blood and pulmonary myeloid cells, which supports a role for the C5a-C5aR1 axis in the pathophysiology of acute respiratory distress syndrome. Anti-C5aR1 therapeutic monoclonal antibodies prevented the C5a-mediated recruitment and activation of human myeloid cells, and inhibited acute lung injury in human C5aR1 knock-in mice. These results suggest that blockade of the C5a-C5aR1 axis could be used to limit the infiltration of myeloid cells in damaged organs and prevent the excessive lung inflammation and endothelialitis that are associated with acute respiratory distress syndrome in patients with COVID-19.

Functional and genetic testing in adults with HLH reveals an inflammatory profile rather than a cytotoxicity defect.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls [DCs]) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.

Pathophysiology of IgG4-related disease: A T follicular helper cells disease?

IgG4-related disease is a chronic inflammatory disease characterized by clinical, biological and pathological unifying findings. Because these criteria are not always all together available in patients and because biological and pathological markers are not totally specific, the diagnosis should be retained after exclusion of mimickers. Since the individualization of IgG4-RD, several studies have allowed to better characterize immunological abnormalities associated with this particular condition. B and T cell oligoclonal activation is associated with T helper 2 cytokine production leading to IgG4 production and profibrotic cytokine release. A central role for T follicular helper 2 cells is suggested from recent findings. We summarize here recent advances in understanding of immune abnormalities in IgG4-related disease.

Imbalance of Circulating Innate Lymphoid Cell Subpopulations in Patients With Septic Shock.

Background: Septic shock, a major cause of death in critical care, is the clinical translation of a cytokine storm in response to infection. It can be complicated by sepsis-induced immunosuppression, exemplified by blood lymphopenia, an excess of circulating Treg lymphocytes, and decreased HLA-DR expression on circulating monocytes. Such immunosuppression is associated with secondary infections, and higher mortality. The effect of these biological modifications on circulating innate lymphoid cells (ILCs) has been little studied. Methods: We prospectively enrolled patients with septic shock (Sepsis-3 definition) in the intensive care unit (ICU) of Timone CHU Hospital. ICU controls (trauma, cardiac arrest, neurological dysfunction) were recruited at the same time (NCT03297203). We performed immunophenotyping of adaptive lymphocytes (CD3+ T cells, CD19+ B cells, CD4+CD25+FoxP3+ Treg lymphocytes), ILCs (CD3-CD56+ NK cells and helper ILCs - ILC1, ILC2, and ILC3), and monocytes by flow cytometry on fresh blood samples collected between 24 and 72 h after admission. Results: We investigated adaptive and innate circulating lymphoid cells in the peripheral blood of 18 patients in septic shock, 15 ICU controls, and 30 healthy subjects. As expected, the peripheral blood lymphocytes of all ICU patients showed lymphopenia, which was not specific to sepsis, whereas those of the healthy volunteers did not. Circulating CD3+ T cells and CD3-CD56+ NK cells were mainly concerned. There was a tendency toward fewer Treg lymphocytes and lower HLA-DR expression on monocytes in ICU patients with sepsis. Although the ILC1 count was higher in septic patients than healthy subjects, ILC2, and ILC3 counts were lower in both ICU groups. However, ILC3s within the total ILCs were overrepresented in patients with septic shock. The depression of immune responses has been correlated with the occurrence of secondary infections. We did not find any differences in ILC distribution according to this criterion. Conclusion: All ICU patients exhibit lymphopenia, regardless of the nature (septic or sterile) of the initial medical condition. Specific distribution of circulating ILCs, with an excess of ILC1, and a lack of ILC3, may characterize septic shock during the first 3 days of the disease.

High-Dimensional Single-Cell Analysis Identifies Organ-Specific Signatures and Conserved NK Cell Subsets in Humans and Mice.

Natural killer (NK) cells are innate lymphoid cells (ILCs) involved in antimicrobial and antitumoral responses. Several NK cell subsets have been reported in humans and mice, but their heterogeneity across organs and species remains poorly characterized. We assessed the diversity of human and mouse NK cells by single-cell RNA sequencing on thousands of individual cells isolated from spleen and blood. Unbiased transcriptional clustering revealed two distinct signatures differentiating between splenic and blood NK cells. This analysis at single-cell resolution identified three subpopulations in mouse spleen and four in human spleen, and two subsets each in mouse and human blood. A comparison of transcriptomic profiles within and between species highlighted the similarity of the two major subsets, NK1 and NK2, across organs and species. This unbiased approach provides insight into the biology of NK cells and establishes a rationale for the translation of mouse studies to human physiology and disease.