Increased proportions of circulating PD-1+ CD4+ memory T cells and PD-1+ regulatory T cells associate with good response to prednisone in pulmonary sarcoidosis.

BACKGROUND: The treatment response to corticosteroids in patients with sarcoidosis is highly variable. CD4+ T cells are central in sarcoid pathogenesis and their phenotype in peripheral blood (PB) associates with disease course. We hypothesized that the phenotype of circulating T cells in patients with sarcoidosis may correlate with the response to prednisone treatment. Therefore, we aimed to correlate frequencies and phenotypes of circulating T cells at baseline with the pulmonary function response at 3 and 12 months during prednisone treatment in patients with pulmonary sarcoidosis. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell populations in 22 treatment-naïve patients and 21 healthy controls (HCs). Pulmonary function tests at baseline, 3 and 12 months were used to measure treatment effect. RESULTS: Patients with sarcoidosis showed an absolute forced vital capacity (FVC) increase of 14.2% predicted (± 10.6, p < 0.0001) between baseline and 3 months. Good response to prednisone (defined as absolute FVC increase of ≥ 10% predicted) was observed in 12 patients. CD4+ memory T cells and regulatory T cells from patients with sarcoidosis displayed an aberrant phenotype at baseline, compared to HCs. Good responders at 3 months had significantly increased baseline proportions of PD-1+CD4+ memory T cells and PD-1+ regulatory T cells, compared to poor responders and HCs. Moreover, decreased fractions of CD25+ cells and increased fractions of PD-1+ cells within the CD4+ memory T cell population correlated with ≥ 10% FVC increase at 12 months. During treatment, the aberrantly activated phenotype of memory and regulatory T cells reversed. CONCLUSIONS: Increased proportions of circulating PD-1+CD4+ memory T cells and PD-1+ regulatory T cells and decreased proportions of CD25+CD4+ memory T cells associate with good FVC response to prednisone in pulmonary sarcoidosis, representing promising new blood biomarkers for prednisone efficacy. TRIAL REGISTRATION: NL44805.078.13.

Measuring burden of disease in both asthma and COPD by merging the ACQ and CCQ: less is more?

Symptoms of asthma and COPD often overlap, and both diseases can co-exist in one patient. The asthma control questionnaire (ACQ) and clinical COPD questionnaire (CCQ) were developed to assess disease burden in respectively asthma or COPD. This study explores the possibility of creating a new questionnaire to assess disease burden in all obstructive lung diseases by integrating and reducing questions of the ACQ and CCQ. Data of patients with asthma, COPD and asthma-COPD overlap (ACO) were collected from a primary and secondary care center. Patients completed ACQ and CCQ on the same day. Linear regression tested correlations. Principal Component Analysis (PCA) was used for item reduction. The secondary cohort with asthma and COPD patients was used for initial question selection (development cohort). These results were reproduced in the primary care cohort and secondary cohort of patients with ACO. The development cohort comprised 252 patients with asthma and 96 with COPD. Correlation between ACQ and CCQ in asthma was R = 0.82, and in COPD R = 0.83. PCA determined a selection of 9 questions. Reproduction in primary care data (asthma n = 1110, COPD n = 1041, ACO = 355) and secondary care data of ACO patients (n = 53) resulted in similar correlations and PCA-derived selection of questions. In conclusion, PCA determined a selection of nine questions of the ACQ and CCQ: working title 'the Obstructive Lung Disease Questionnaire'. These results suggest that this pragmatic set of questions might be sufficient to assess disease burden in obstructive lung disease in both primary as secondary care.

Aberrant B cell receptor signaling in circulating naïve and IgA+ memory B cells from newly-diagnosed autoantibody-positive rheumatoid arthritis patients.

OBJECTIVE: Altered B cell receptor (BCR) signaling has been implicated in the pathogenesis of rheumatoid arthritis (RA). Here we aimed to identify signaling aberrations in autoantibody-positive and autoantibody-negative RA patients by performing a comprehensive analysis of the BCR signaling cascade in different B cell subsets. METHODS: We first optimized phosphoflow cytometry for an in-depth analysis of BCR signaling across immunoglobulin isotypes in healthy donors. Subsequently, we compared BCR signaling in circulating B cell subsets from treatment-naïve, newly-diagnosed autoantibody-positive RA and autoantibody-negative RA patients and healthy controls (HCs). RESULTS: We observed subset-specific phosphorylation patterns of the BCR signalosome in circulating B cells from healthy donors. Compared with HCs, autoantibody-positive RA patients displayed enhanced responses to BCR stimulation for multiple signaling proteins, specifically in naïve and IgA+ memory B cells. Whereas in unstimulated healthy donor B cells, the phosphorylation status of individual signaling proteins showed only limited correlation, BCR stimulation enhanced the interconnectivity in phosphorylation within the BCR signalosome. However, this strong interconnectivity within the BCR signalosome in stimulated B cells from HCs was lost in RA, especially in autoantibody-positive RA patients. Finally, we observed strong correlations between SYK and BTK protein expression, and IgA and IgG anti-citrullinated protein antibody concentrations in serum from autoantibody-positive RA patients. CONCLUSION: Collectively, the isotype-specific analysis of multiple key components of the BCR signalosome identified aberrant BCR signaling responses in treatment-naïve autoantibody-positive RA patients, particularly in naïve B cells and IgA+ memory B cells. Our findings support differential involvement of dysregulated BCR signaling in the pathogenesis of autoantibody-positive and autoantibody-negative RA.

Airway epithelial cell response to RSV is mostly impaired in goblet and multiciliated cells in asthma.

BACKGROUND: In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most-but not all-studies. Moreover, the molecular mechanisms causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood. METHODS: Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (n=8) and healthy donors (n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After 3 days, cells were processed for single-cell RNA sequencing. RESULTS: A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed. CONCLUSION: Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.

Aberrant characteristics of peripheral blood innate lymphoid cells in COPD, independent of smoking history.

Background: Distinguishing asthma and COPD can pose challenges in clinical practice. Increased group 1 innate lymphoid cells (ILC1s) have been found in the lungs and peripheral blood of COPD patients, while asthma is associated with elevated levels of ILC2s. However, it is unclear whether the inflammatory characteristics of ILC1s and ILC2s differ between COPD and asthma. This study aims to compare peripheral blood ILC subsets and their expression of inflammatory markers in COPD patients, asthma patients and controls. Methods: The study utilised multi-colour flow cytometry to analyse peripheral blood ILC populations in clinically stable COPD patients (n=38), asthma patients (n=37), and smoking (n=19) and non-smoking (n=16) controls. Results: Proportions of peripheral blood inflammatory CD4+ ILC1s were significantly higher in COPD patients than in asthma. Proportions of CD4- ILC1s were increased in COPD patients compared to asthma patients and smoking controls. Frequencies of CD117- ILC2s were significantly reduced in COPD patients compared with asthma patients. In contrast, the fraction of inflammatory CD45RO+ cells within the CD117- ILC2 population was significantly increased. Principal component analyses showed that combined features of the circulating ILC compartment separated COPD patients from asthma patients and both control groups. Conclusion: Our in-depth characterisation of ILC1 and ILC2 populations in peripheral blood revealed significant differences in their phenotypes between COPD and asthma patients and smoking or non-smoking controls. These findings suggest a role for both ILC subsets in COPD disease pathology, independent of smoking history, and may have implications for patient stratification and therapy development.

FastCAR: fast correction for ambient RNA to facilitate differential gene expression analysis in single-cell RNA-sequencing datasets.

Cell type-specific differential gene expression analyses based on single-cell transcriptome datasets are sensitive to the presence of cell-free mRNA in the droplets containing single cells. This so-called ambient RNA contamination may differ between samples obtained from patients and healthy controls. Current ambient RNA correction methods were not developed specifically for single-cell differential gene expression (sc-DGE) analyses and might therefore not sufficiently correct for ambient RNA-derived signals. Here, we show that ambient RNA levels are highly sample-specific. We found that without ambient RNA correction, sc-DGE analyses erroneously identify transcripts originating from ambient RNA as cell type-specific disease-associated genes. We therefore developed a computationally lean and intuitive correction method, Fast Correction for Ambient RNA (FastCAR), optimized for sc-DGE analysis of scRNA-Seq datasets generated by droplet-based methods including the 10XGenomics Chromium platform. FastCAR uses the profile of transcripts observed in libraries that likely represent empty droplets to determine the level of ambient RNA in each individual sample, and then corrects for these ambient RNA gene expression values. FastCAR can be applied as part of the data pre-processing and QC in sc-DGE workflows comparing scRNA-Seq data in a health versus disease experimental design. We compared FastCAR with two methods previously developed to remove ambient RNA, SoupX and CellBender. All three methods identified additional genes in sc-DGE analyses that were not identified in the absence of ambient RNA correction. However, we show that FastCAR performs better at correcting gene expression values attributed to ambient RNA, resulting in a lower frequency of false-positive observations. Moreover, the use of FastCAR in a sc-DGE workflow increases the cell-type specificity of sc-DGE analyses across disease conditions.

Immunological profiling in long COVID: overall low grade inflammation and T-lymphocyte senescence and increased monocyte activation correlating with increasing fatigue severity.

Background: Many patients with SARS-CoV-2 infection develop long COVID with fatigue as one of the most disabling symptoms. We performed clinical and immune profiling of fatigued and non-fatigued long COVID patients and age- and sex-matched healthy controls (HCs). Methods: Long COVID symptoms were assessed using patient-reported outcome measures, including the fatigue assessment scale (FAS, scores ≥22 denote fatigue), and followed up to one year after hospital discharge. We assessed inflammation-related genes in circulating monocytes, serum levels of inflammation-regulating cytokines, and leukocyte and lymphocyte subsets, including major monocyte subsets and senescent T-lymphocytes, at 3-6 months post-discharge. Results: We included 37 fatigued and 36 non-fatigued long COVID patients and 42 HCs. Fatigued long COVID patients represented a more severe clinical profile than non-fatigued patients, with many concurrent symptoms (median 9 [IQR 5.0-10.0] vs 3 [1.0-5.0] symptoms, p<0.001), and signs of cognitive failure (41%) and depression (>24%). Immune abnormalities that were found in the entire group of long COVID patients were low grade inflammation (increased inflammatory gene expression in monocytes, increased serum pro-inflammatory cytokines) and signs of T-lymphocyte senescence (increased exhausted CD8+ TEMRA-lymphocytes). Immune profiles did not significantly differ between fatigued and non-fatigued long COVID groups. However, the severity of fatigue (total FAS score) significantly correlated with increases of intermediate and non-classical monocytes, upregulated gene levels of CCL2, CCL7, and SERPINB2 in monocytes, increases in serum Galectin-9, and higher CD8+ T-lymphocyte counts. Conclusion: Long COVID with fatigue is associated with many concurrent and persistent symptoms lasting up to one year after hospitalization. Increased fatigue severity associated with stronger signs of monocyte activation in long COVID patients and potentially point in the direction of monocyte-endothelial interaction. These abnormalities were present against a background of immune abnormalities common to the entire group of long COVID patients.

Circulating T cells in sarcoidosis have an aberrantly activated phenotype that correlates with disease outcome.

RATIONALE: Disease course in sarcoidosis is highly variable. Bronchoalveolar lavage fluid and mediastinal lymph nodes show accumulation of activated T cells with a T-helper (Th)17.1 signature, which correlates with non-resolving sarcoidosis. We hypothesize that the peripheral blood (PB) T cell phenotype may correlate with outcome. OBJECTIVES: To compare frequencies, phenotypes and function of circulating T cell populations in sarcoidosis patients with healthy controls (HCs) and correlate these parameters with outcome. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell subsets in treatment-naïve patients and HCs. The disease course was determined after 2-year follow-up. Cytokine production was measured after T cell stimulation in vitro. MEASUREMENTS AND MAIN RESULTS: We observed significant differences between patients and HCs in several T cell populations, including CD8+ and CD4+ T cells, Th1/Th17 subsets, CD4+ T memory stem cells, regulatory T cells (Tregs) and γδ T cells. Decreased frequencies of CD4+ T cells and increased frequencies of Tregs and CD8+ γδ T cells correlated with worse outcome. Naïve CD4+ T cells displayed an activated phenotype with increased CD25 expression in patients with active chronic disease at 2-year follow-up. A distinctive Treg phenotype with increased expression of CD25, CTLA4, CD69, PD-1 and CD95 correlated with chronic sarcoidosis. Upon stimulation, both naïve and memory T cells displayed a different cytokine profile in sarcoidosis compared to HCs. CONCLUSIONS: Circulating T cell subpopulations of sarcoidosis patients display phenotypic abnormalities that correlate with disease outcome, supporting a critical role of aberrant T cell activation in sarcoidosis pathogenesis.

Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK.

Introduction: Cytotoxic CD8+ T cell (CTL) exhaustion is a dysfunctional state of T cells triggered by persistent antigen stimulation, with the characteristics of increased inhibitory receptors, impaired cytokine production and a distinct transcriptional profile. Evidence from immune checkpoint blockade therapy supports that reversing T cell exhaustion is a promising strategy in cancer treatment. Ibrutinib, is a potent inhibitor of BTK, which has been approved for the treatment of chronic lymphocytic leukemia. Previous studies have reported improved function of T cells in ibrutinib long-term treated patients but the mechanism remains unclear. We investigated whether ibrutinib directly acts on CD8+ T cells and reinvigorates exhausted CTLs. Methods: We used an established in vitro CTL exhaustion system to examine whether ibrutinib can directly ameliorate T cell exhaustion. Changes in inhibitory receptors, transcription factors, cytokine production and killing capacity of ibrutinib-treated exhausted CTLs were detected by flow cytometry. RNA-seq was performed to study transcriptional changes in these cells. Btk deficient mice were used to confirm that the effect of ibrutinib was independent of BTK expression. Results: We found that ibrutinib reduced exhaustion-related features of CTLs in an in vitro CTL exhaustion system. These changes included decreased inhibitory receptor expression, enhanced cytokine production, and downregulation of the transcription factor TOX with upregulation of TCF1. RNA-seq further confirmed that ibrutinib directly reduced the exhaustion-related transcriptional profile of these cells. Importantly, using btk deficient mice we showed the effect of ibrutinib was independent of BTK expression, and therefore mediated by one of its other targets. Discussion: Our study demonstrates that ibrutinib directly ameliorates CTL exhaustion, and provides evidence for its synergistic use with cancer immunotherapy.

Overcoming immune checkpoint blockade resistance in solid tumors with intermittent ITK inhibition.

Cytotoxic CD8 + T cell (CTL) exhaustion is driven by chronic antigen stimulation. Reversing CTL exhaustion with immune checkpoint blockade (ICB) has provided clinical benefits in different types of cancer. We, therefore, investigated whether modulating chronic antigen stimulation and T-cell receptor (TCR) signaling with an IL2-inducible T-cell kinase (ITK) inhibitor, could confer ICB responsiveness to ICB resistant solid tumors. In vivo intermittent treatment of 3 ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) with ITK inhibitor significantly improved ICB therapy. ITK inhibition directly reinvigorate exhausted CTL in vitro as it enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX. Our study demonstrates that intermittent ITK inhibition can be used to directly ameliorate CTL exhaustion and enhance immunotherapies even in solid tumors that are ICB resistant.

Type-2 CD8+ T-cell formation relies on interleukin-33 and is linked to asthma exacerbations.

CD4+ T helper 2 (Th2) cells and group 2 innate lymphoid cells are considered the main producers of type-2 cytokines that fuel chronic airway inflammation in allergic asthma. However, CD8+ cytotoxic T (Tc) cells - critical for anti-viral defense - can also produce type-2 cytokines (referred to as 'Tc2' cells). The role of Tc cells in asthma and virus-induced disease exacerbations remains poorly understood, including which micro-environmental signals and cell types promote Tc2 cell formation. Here we show increased circulating Tc2 cell abundance in severe asthma patients, reaching peak levels during exacerbations and likely emerging from canonical IFNγ+ Tc cells through plasticity. Tc2 cell abundance is associated with increased disease burden, higher exacerbations rates and steroid insensitivity. Mouse models of asthma recapitulate the human disease by showing extensive type-2 skewing of lung Tc cells, which is controlled by conventional type-1 dendritic cells and IFNγ. Importantly, we demonstrate that the alarmin interleukin-33 (IL-33) critically promotes type-2 cytokine production by lung Tc cells in experimental allergic airway inflammation. Our data identify Tc cells as major producers of type-2 cytokines in severe asthma and during exacerbations that are remarkably sensitive to alterations in their inflammatory tissue micro-environment, with IL-33 emerging as an important regulator of Tc2 formation.

Inflammatory and tolerogenic myeloid cells determine outcome following human allergen challenge.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.

3D chromatin reprogramming primes human memory TH2 cells for rapid recall and pathogenic dysfunction.

Memory T cells provide long-lasting defense responses through their ability to rapidly reactivate, but how they efficiently "recall" an inflammatory transcriptional program remains unclear. Here, we show that human CD4+ memory T helper 2 (TH2) cells carry a chromatin landscape synergistically reprogrammed at both one-dimensional (1D) and 3D levels to accommodate recall responses, which is absent in naive T cells. In memory TH2 cells, recall genes were epigenetically primed through the maintenance of transcription-permissive chromatin at distal (super)enhancers organized in long-range 3D chromatin hubs. Precise transcriptional control of key recall genes occurred inside dedicated topologically associating domains ("memory TADs"), in which activation-associated promoter-enhancer interactions were preformed and exploited by AP-1 transcription factors to promote rapid transcriptional induction. Resting memory TH2 cells from patients with asthma showed premature activation of primed recall circuits, linking aberrant transcriptional control of recall responses to chronic inflammation. Together, our results implicate stable multiscale reprogramming of chromatin organization as a key mechanism underlying immunological memory and dysfunction in T cells.

Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation.

B-cell acute lymphoblastic leukemia (B-ALL) reflects the malignant counterpart of developing B cells in the bone marrow (BM). Despite tremendous progress in B-ALL treatment, the overall survival of adults at diagnosis and patients at all ages after relapse remains poor. Galectin-1 (GAL1) expressed by BM supportive niches delivers proliferation signals to normal pre-B cells through interaction with the pre-B cell receptor (pre-BCR). Here, we asked whether GAL1 gives non-cell autonomous signals to pre-BCR+ pre-B ALL, in addition to cell-autonomous signals linked to genetic alterations. In syngeneic and patient-derived xenograft (PDX) murine models, murine and human pre-B ALL development is influenced by GAL1 produced by BM niches through pre-BCR-dependent signals, similarly to normal pre-B cells. Furthermore, targeting pre-BCR signaling together with cell-autonomous oncogenic pathways in pre-B ALL PDX improved treatment response. Our results show that non-cell autonomous signals transmitted by BM niches represent promising targets to improve B-ALL patient survival.

Decoding the genetic and epigenetic basis of asthma.

Asthma is a complex and heterogeneous chronic inflammatory disease of the airways. Alongside environmental factors, asthma susceptibility is strongly influenced by genetics. Given its high prevalence and our incomplete understanding of the mechanisms underlying disease susceptibility, asthma is frequently studied in genome-wide association studies (GWAS), which have identified thousands of genetic variants associated with asthma development. Virtually all these genetic variants reside in non-coding genomic regions, which has obscured the functional impact of asthma-associated variants and their translation into disease-relevant mechanisms. Recent advances in genomics technology and epigenetics now offer methods to link genetic variants to gene regulatory elements embedded within non-coding regions, which have started to unravel the molecular mechanisms underlying the complex (epi)genetics of asthma. Here, we provide an integrated overview of (epi)genetic variants associated with asthma, focusing on efforts to link these disease associations to biological insight into asthma pathophysiology using state-of-the-art genomics methodology. Finally, we provide a perspective as to how decoding the genetic and epigenetic basis of asthma has the potential to transform clinical management of asthma and to predict the risk of asthma development.

B Cell Signaling and Activation in Autoimmunity.

Autoreactive B cells play a key role in the initiation or aggravation of many systemic and tissue-specific autoimmune disorders [...].