High-dose radiation therapy is a widely used clinical strategy to inhibit tumor growth. However, the rapid generation of excessive reactive oxygen species (ROS) triggers the formation of neutrophil extracellular traps (NETs), which capture free tumor cells in the bloodstream, promoting metastasis. In this study, we developed a hybrid nanoparticle composed of DNase I and gold (DNase I@Au) to enhance radiotherapy efficacy while mitigating metastasis by precisely eliminating NETs. The DNase I@Au nanoparticles, administered via aerosol inhalation, are efficiently delivered to lung tumor tissue, improving radiosensitization and reducing tumor size. Crucially, the nanoparticles could gradually release DNase I, effectively degrading ROS-induced NETs and preventing the interaction of free malignant cells with tumor sites or vasculature, thereby inhibiting metastasis. Therefore, we provide an enzyme and sensitizer co-loaded strategy that offers a promising approach to improve the therapeutic outcome of radiotherapy and reduce the risk of lung cancer metastasis under ROS stimulation.

Radiographic axial spondyloarthritis (r-axSpA) is a chronic rheumatic disease in which innate immune cells and T cells are thought to play a major role. However, recent studies also hint at B cell involvement. Here, we performed an in-depth analysis on alterations within the B-cell compartment from r-axSpA patients.

We performed immune gene expression profiling on total peripheral blood B cells from 8 r-axSpA patients and 8 healthy controls (HCs). Next, we explored B cell subset distribution and B-cell receptor (BCR) signaling responses in circulating B cells from 28 r-axSpA patients and 15 HCs, by measuring spleen tyrosine kinase, phosphoinositide 3-kinase and extracellular signal regulated kinase 1/2 phosphorylation upon α-Ig stimulation using phosphoflow cytometry.

Immune gene expression profiling indicated an elevated pathway score for BCR signaling in total B cells from r-axSpA patients compared with HCs. Flow cytometric analysis revealed an increase in frequency of both total and double-negative 2 (DN2) B cells in r-axSpA patients compared with HCs. In r-axSpA patients, DN2 B cells displayed an isotype shift towards IgA. Remarkably, where DN2 B cells from HCs were hyporesponsive, these cells displayed significant proximal BCR signaling responses in r-axSpA patients. Enhanced BCR signaling responses were also observed in the transitional and naïve B cell population from r-axSpA patients compared with HCs. The enhanced BCR signaling responses in DN2 B cells correlated with clinical disease parameters.

In r-axSpA patients, circulating DN2 B cells are expanded and, together with transitional and naïve B cells, display significantly enhanced BCR signaling responses upon stimulation. Together, our data suggest B cell involvement in the pathogenesis of r-axSpA.

As a heterogeneous B cell subset, age-associated B cells (ABCs) exhibit distinct transcription profiles, extrafollicular differentiation processes, and multiple functions in autoimmunity. TLR7 and TLR9 signals, along with IFN-γ and IL-21 stimulation, are both essential for ABC differentiation, which is also regulated by chemokine receptors including CXCR3 and CCR2 and integrins including CD11b and CD11c. Given their functions in antigen uptake and presentation, autoantibody and proinflammatory cytokine secretion, and T helper cell activation, ABCs display potential in the prognosis, diagnosis, and therapy for autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, multiple sclerosis, neuromyelitis optica spectrum disorders, and ankylosing spondylitis. Specifically targeting ABCs by inhibiting T-bet and CD11c and activating CD11b and ARA2 represents potential therapeutic strategies for SLE and RA. Although single-cell sequencing technologies have recently revealed the heterogeneous characteristics of ABCs, further investigations to explore and validate ABC-target therapies are still warranted.

The posttranslational modification of cellular macromolecules by glycosylation is considered to contribute to disease pathogenesis in autoimmune and inflammatory conditions. In a subgroup of patients with common variable immunodeficiency (CVID), the occurrence of such complications is associated with an expansion of naïve-like CD21low B cells during a chronic type 1 immune activation. The glycosylation pattern of B cells in CVID patients has not been addressed to date.

The objective of this study was to examine the surface glycome of B cells in patients with CVID and associated immune dysregulation.

We performed surface lectin staining on B cells from peripheral blood and tonsils, both ex vivo and after in vitro stimulation. Additionally, we examined the expression of glycosylation-related genes by RNAseq in naïve-like CD21low B cells ex vivo, as well as in naïve CD21pos B cells from healthy controls after in vitro stimulation.

Unlike CD21pos B cells, naïve-like CD21low B cells from CVID patients and CD21low B cells from healthy controls exhibited a unique glycosylation pattern with high levels of α2,6 sialic acids and fucose. This hypersialylation and hyperfucosylation were particularly induced by activation with anti-IgM and interferon-γ (IFN-γ). Transcriptome analysis suggested that naïve-like CD21low B cells possess a comprehensively reorganised glycosylation machinery, with anti-IgM/IFN-γ having the potential to initiate these changes in vitro.

CD21low B cells are hypersialylated and hyperfucosylated. This may implicate altered lectin-ligand interactions on the cell surface potentially affecting the CD21low B-cell function. These glycome changes appear to be driven by the prominent type I immune response in complicated CVID patients. A better understanding of how altered glycosylation influences immune cell function could lead to new therapeutic strategies.

B-cells play a critical role in the formation of immune responses against pathogens by acting as antigen-presenting cells, by modulating immune responses, and by generating immune memory and antibody responses. Here, we studied B-cell subset distributions between regions with higher and lower microbial exposure, i.e. by comparing peripheral blood B-cells from people living in Indonesia or Ghana to those from healthy Dutch residents using a 36-marker mass cytometry panel. By applying an unbiased multidimensional approach, we observed differences in the balance between the naïve and memory compartments, with higher CD11c+ and double negative (DN-IgDnegCD27neg) memory (M)B-cells in individuals from rural tropical areas, and conversely lower naïve B-cells compared to residents from an area with less pathogen exposure. Furthermore, characterization of total B-cell populations, CD11c+, DN, and Breg cells showed the emergence of specific memory clusters in individuals living in rural tropical areas. Some of these differences were more pronounced in children compared to adults and suggest that a higher microbial exposure accelerates memory B-cell formation, which "normalizes" with age.

Auto-immune and inflammatory diseases are heterogenous in their clinical manifestations and prognosis, even among individuals presenting with the same pathology. Understanding the immunological alterations involved in their pathogenesis provides valuable insights in different clinical phenotypes and treatment responses. Immunophenotyping could lead to significant improvements in diagnosis, monitoring, initial treatment decisions and follow-up in autoimmune and inflammatory diseases. Mass cytometry provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, and therefore holds immense potential to evaluate complex cellular systems and for high-dimensional single-cell analysis. The high dimensionality of mass cytometry provides better coverage of immune populations dynamics, with sufficient power to identify rare cell types compared to flow cytometry. In this comprehensive review, we explore how mass cytometry findings contributed in the past decade to a deeper understanding of the cellular actors involved in systemic auto-immune and auto-inflammatory diseases with their respective therapeutic and prognostic impact. We also delve into the bioinformatical approaches applied to mass cytometry to analyze the high volumes of data generated, as well as the impact of the use of complementary single cell RNA sequencing, and their spatial modalities. Our analysis highlights the fact that mass cytometry captures major information on cell populations providing insights on the complex pathogenesis of autoimmune diseases. Future research designs could include mass cytometry findings in association to other -omics to stratify patients in adequate therapeutic arms and provide advancements in personalized therapies in the field of auto-immune and inflammatory diseases.

The use of immunosuppressive treatment is required to prevent rejection events, even a long time after kidney transplantation despite rare recipients achieving long-term graft stability without the need for immunosuppressive treatment, called operationally tolerant patients (TOLs). We comprehensively investigate the immune system of long-term IS recipients (LTTs) and TOLs to highlight their shared and unique immune features. Blood immune cell phenotyping was performed by spectral cytometry. Samples from 34 individuals were analyzed, including 6 LTTs, 8 TOLs, 10 stable patients at 1 year posttransplantation (STAs), and 10 healthy volunteers. B cells differed between LTTs and TOLs with a decreased total B-cell frequency and the acquisition of a memory phenotype in LTTs whereas a naive phenotype is maintained in TOLs. The frequencies of IgD-CD27- B cells and CD11c+ memory B cells are increased in LTTs, with an exhausted phenotype, evoked by a significant decrease in CD25 expression. These CD11c+ B cells display an exhausted phenotype similar to those found in several chronic immune diseases in which they have been shown to participate in their pathophysiology, suggesting active chronic inflammation in LTTs. Altogether, these data indicate that precautions should be taken to minimize IS use.

B cells play a major role in the development and maintenance of systemic lupus erythematosus (SLE). Double negative (DN) B cells defined by the lack of surface expression of IgD and CD27 have attracted recent interest for their sensitivity to Toll-like receptor 7 (TLR7) ligands and their potential role in the production of autoantibodies. Here we aimed at investigating the possible association of DN B cells and their subsets with SLE disease activity specifically in female patients, in which TLR7 gene has been reported to escape X chromosome inactivation.

Peripheral blood mononuclear cells were purified from woman participating to the clinically well-characterized Swiss SLE Cohort Study (SSCS). PBMC from age-matched healthy females were used as controls. PBMC were stained for cell surface markers, intracellular Tbet and analyzed by multicolor cytofluorimetry. Single nucleotide TLR7 polymorphisms were assessed by polymerase chain reaction.

The median SLE disease activity index of the 86 females was 2, IQR [0-6], all but 8 were under chronic SLE treatment. B cells co-expressing CD11c and Tbet were increased, the mean fluorescence intensity (MFI) of CD19 was considerably reduced and we observed a large increase in CD11c + CXCR5-and CD11c-CXCR5-concomitantly with a reduction of CD11c-CXCR5+ B cells in SLE compared to 40 healthy donors (HD). When focusing on the DN B cell subset, we found a reduction of DN1 (CD11c-CXCR5+) and an increase of DN2 (CD11c + CXCR5-) and most impressively of DN3 (CD11c-CXCR5-) cells. The DN subset, particularly DN3, showed the lowest level of CD19 expression. Both DN1 and DN3 percentages as well as the CD19 MFI of DN cells were associated with SLE disease activity. The use of glucocorticoids, immunosuppressants, and antimalarials impacted differentially on the frequencies of DN B cell subsets. CD19 MFI in B cells and the percentage of DN3 were the strongest biomarkers of disease activity. The TLR7 snp3858384 G allele was associated with increased percentages of B cells and CD19+CD11c-CXCR5+ and decreased CD19+CD11c-CXCR5-.

DN3 B cells are strongly associated with SLE clinical activity pointing to their potential involvement in disease pathogenesis, and CD19 expression level performs accurately as disease activity biomarker.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose expression regulates immune cell differentiation. Single-cell transcriptomic profiling was used to ascertain the heterogeneity of AHR expression in human B cell subpopulations. We identified a unique population of B cells marked by expression of AHR, CD9, and myeloid genes such as CD14 and CXCL8. Results were confirmed directly in human PBMCs and purified B cells at the protein level. TLR9 signaling induced CD14, CD9, and IL-8 protein expression in CD19+ B cells. CD14-expressing CD9+ B cells also highly expressed AHR and atypical B cell markers such as CD11c and TBET. In patients with active lupus disease, CD14+ and CD9+ B cells are dysregulated, with loss of CD9+ B cells strongly predicting disease severity and demonstrating the relevance of CD9+ B cells in systemic lupus erythematosus and autoimmune disease.

B cells play a key role in humoral immune responses by producing antibodies. Although there are numerous research on memory B cells definition markers and cytokines on B cell development, different studies have yielded contradictory conclusions due to species studied, the different cells and stimulating agents used. In the current study, we conducted a detailed characterization of B cells in human CBMCs, PBMCs and tonsil, including expression of Igs, activation and memory markers. Furthermore, we found that considerable amounts of IgA and IgG were expressed by CD27- B cells. These "Atypical" memory B cells corresponded to approximately 50% of IgG+ and IgA+B cells in blood, this proportion even reached 90% in tonsil. In addition, we investigated the effect of IL-21 and TGF-β1 on the membrane-bound form and secreted form of Igs using PBMCs and purified blood B cells. There were actual differences between the effect of cytokines on Igs secretion and surface expression. Our study will be helpful to advance the knowledge and understanding of humoral memory.

Lupus is characterized by the autoantibodies against nuclear Ags, underscoring the importance of identifying the B cell subsets driving autoimmunity. Our research focused on the mitochondrial activity and CXCR4 expression in CD11c+ B cells from lupus patients after ex vivo stimulation with a TLR9 agonist, CpG-oligodeoxyribonucleotide (ODN). We also evaluated the response of CD11c+ B cells in ODN-injected mice. Post-ex vivo ODN stimulation, we observed an increase in the proportion of CD11chi cells, with elevated mitochondrial activity and CXCR4 expression in CD11c+ B cells from lupus patients. In vivo experiments showed similar patterns, with TLR9 stimulation enhancing mitochondrial and CXCR4 activities in CD11chi B cells, leading to the generation of anti-dsDNA plasmablasts. The CXCR4 inhibitor AMD3100 and the mitochondrial complex I inhibitor IM156 significantly reduced the proportion of CD11c+ B cells and autoreactive plasmablasts. These results underscore the pivotal roles of mitochondria and CXCR4 in the production of autoreactive plasmablasts.

Despite advances in understanding systemic lupus erythematosus (SLE), many challenges remain in unraveling the precise mechanisms behind the disease's development and progression. Recent evidence has questioned the role of programmed cell death protein 1 (PD-1) in suppressing autoreactive CD4+ T cells during autoimmune responses. Research has investigated the potential impacts of PD-1 on various CD4+ T-cell subpopulations, including T follicular helper (Tfh) cells, circulating Tfh (cTfh) cells, and T peripheral helper (Tph) cells, all of which exhibit substantial PD-1 expression and are closely related to several autoimmune disorders, including SLE. This review highlights the complex role of PD-1 in autoimmunity and emphasizes the imperative for further research to elucidate its functions during autoreactive T-cell responses. Additionally, we address the potential of PD-1 and its ligands as possible therapeutic targets in SLE.

Germline pathogenic variants in BRCA1 and BRCA2 (gpath(BRCA1/2)) represent genetic susceptibility for hereditary breast and ovarian cancer syndrome. Tumor-immune interactions are key contributors to breast cancer pathogenesis. Although earlier studies confirmed pro-tumorigenic immunological alterations in breast cancer patients, data are lacking in healthy carriers of gpath(BRCA1/2). Peripheral blood mononuclear cells of 66 women with or without germline predisposition or breast cancer were studied with a mass cytometry panel that identified 4 immune subpopulations of altered frequencies between healthy controls and healthy gpath(BRCA1) carriers, while no difference was observed in healthy gpath(BRCA2) carriers compared to controls. Moreover, 3 (one IgD-CD27+CD95+ B cell subpopulation and two CD45RA-CCR7+CD38+ CD4+ T cell subpopulations) out of these 4 subpopulations were also elevated in triple-negative breast cancer patients compared to controls. Our results reveal an activated peripheral immune phenotype in healthy carriers of gpath(BRCA1) that needs to be further elucidated to be leveraged in risk-reducing strategies.

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive, heterogeneous non-Hodgkin lymphoma resulting from malignant proliferation of cytotoxic natural killer (NK) or T cells. Previous studies demonstrated variable expression of CD38 on NKTCL tumors. Daratumumab, a human IgGκ monoclonal antibody targeting CD38 with a direct on-tumor and immunomodulatory mechanism of action, was hypothesized to be a novel therapeutic option for patients with relapsed or refractory (R/R) NKTCL. In the phase 2 NKT2001 study (ClinicalTrials.gov Identifier: NCT02927925) assessing the safety and efficacy of daratumumab, a suboptimal overall response rate was seen in R/R NKTCL patients. One patient, whose tumors did not express CD38, responded to treatment, suggesting that the immunomodulatory activities of daratumumab may be sufficient to confer clinical benefit. To understand the suboptimal response rate and short duration of response, we investigated the immune profile of NKTCL patients from NKT2001 in the context of daratumumab anti-tumor activity. Tumor tissue and whole blood were, respectively, analyzed for CD38 expression and patient immune landscapes, which were assessed via cytometry by time-of-flight (CyTOF), multiparameter flow cytometry (MPFC), clonal sequencing, and plasma Epstein-Barr virus (EBV)-DNA level measurements. Changes observed in the immune profiles of NKTCL patients from NKT2001, including differences in B and T cell populations between responders and nonresponders, suggest that modulation of the immune environment is crucial for daratumumab anti-tumor activities in NKTCL. In conclusion, these findings highlight that the clinical benefit of daratumumab in NKTCL may be enriched by B/T cell-related biomarkers.

Systemic autoimmune diseases (SADs) are a significant burden on the healthcare system. Understanding the complexity of the peripheral immunophenotype in SADs may facilitate the differential diagnosis and identification of potential therapeutic targets.

Single-cell mass cytometric immunophenotyping was performed on peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and therapy-naive patients with rheumatoid arthritis (RA), progressive systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). Immunophenotyping was performed on 15,387,165 CD45+ live single cells from 52 participants (13 cases/group), using an antibody panel to detect 34 markers.

Using the t-SNE (t-distributed stochastic neighbor embedding) algorithm, the following 17 main immune cell types were determined: CD4+/CD57- T cells, CD4+/CD57+ T cells, CD8+/CD161- T cells, CD8+/CD161+/CD28+ T cells, CD8dim T cells, CD3+/CD4-/CD8- T cells, TCRγ/δ T cells, CD4+ NKT cells, CD8+ NKT cells, classic NK cells, CD56dim/CD98dim cells, B cells, plasmablasts, monocytes, CD11cdim/CD172dim cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs). Seven of the 17 main cell types exhibited statistically significant frequencies in the investigated groups. The expression levels of the 34 markers in the main populations were compared between HCs and SADs. In summary, 59 scatter plots showed significant differences in the expression intensities between at least two groups. Next, each immune cell population was divided into subpopulations (metaclusters) using the FlowSOM (self-organizing map) algorithm. Finally, 121 metaclusters (MCs) of the 10 main immune cell populations were found to have significant differences to classify diseases. The single-cell T-cell heterogeneity represented 64MCs based on the expression of 34 markers, and the frequency of 23 MCs differed significantly between at least twoconditions. The CD3- non-T-cell compartment contained 57 MCs with 17 MCs differentiating at least two investigated groups. In summary, we are the first to demonstrate the complexity of the immunophenotype of 34 markers over 15 million single cells in HCs vs. therapy-naive patients with RA, SSc, and SLE. Disease specific population frequencies or expression patterns of peripheral immune cells provide a single-cell data resource to the scientific community.

Neuromyelitis optica (NMO) is an autoimmune astrocytopathy mediated by anti-AQP4 antibody-producing B cells. Recently, a B-cell subset highly expressing CD11c and T-bet, originally identified as age-associated B cells, has been shown to be involved in the pathogenesis of various autoimmune diseases. The objective of this study was to determine the relationship between the frequency of CD11chigh B cells per CD19+ B cells in the peripheral blood of patients with NMO and the clinical profiles including the brain volume.

In this observational study, 45 patients with anti-AQP4 antibody-positive NMO in remission and 30 healthy control subjects (HCs) were enrolled. Freshly isolated peripheral blood mononuclear cells were analyzed for immune cell phenotypes. The frequency of CD11chigh B cells per CD19+ B cells was assessed by flow cytometry and was evaluated in association with the clinical profiles of patients. Brain MRI data from 26 patients were included in the study for the analysis on the correlation between CD11chigh B-cell frequency and brain atrophy.

We found that the frequency of CD11chigh B cells in CD19+ B cells was significantly increased in patients with NMO compared with HCs. The expansion of CD11chigh B cells significantly correlated with EDSS, past relapse numbers, and disease duration. In addition, a higher frequency of CD11chigh B cells negatively correlated with total brain, white matter, and gray matter volumes and positively correlated with T2/FLAIR high lesion volumes. When the past clinical relapse episodes of patients with or without the expansion of CD11chigh B cells were compared, relapses in the brain occurred more frequently in patients with CD11chigh B-cell expansion. CD11chigh B cells had distinct features including expression of chemokine receptors associated with migration into peripheral inflammatory tissues and antigen presentation. CD11chigh B-cell frequency was positively correlated with T peripheral helper-1 (Tph-1) cell frequency.

Even during the relapse-free period, CD11chigh B cells could expand in the long disease context, possibly through the interaction with Tph-1 cells. The increased frequency of CD11chigh B cells associated with brain atrophy and disease severity, indicating that this cell population could be involved in chronic neuroinflammation in NMO.

Neurologic immune-related adverse events (irAE-n) are rare but severe toxicities of immune checkpoint inhibitor (ICI) treatment. To overcome diagnostic and therapeutic challenges, a better mechanistic understanding of irAE-n is paramount.

In this observational cohort study, we collected serum and peripheral blood samples from 34 consecutive cancer patients with irAE-n (during acute illness) and 49 cancer control patients without irAE-n (pre- and on-ICI treatment, n = 44 without high-grade irAEs, n = 5 with high-grade nonneurologic irAEs). Patients received either anti-programmed cell death protein (PD)-1 or anti-PD ligand-1 monotherapy or anti-PD-1/anti-cytotoxic T-lymphocyte-associated protein-4 combination therapy. Most common cancers were melanoma, lung cancer, and hepatocellular carcinoma. Peripheral blood immune profiling was performed using 48-marker single-cell mass cytometry and a multiplex cytokine assay.

During acute illness, patients with irAE-n presented higher frequencies of cluster of differentiation (CD)8+ effector memory type (EM-)1 and central memory (CM) T cells compared to controls without irAEs. Multiorgan immunotoxicities (neurologic + nonneurologic) were associated with higher CD8+ EM1 T cell counts. While there were no B cell changes in the overall cohort, we detected a marked decrease of IgD- CD11c+ CD21low and IgD- CD24+ CD21high B cells in a subgroup of patients with autoantibody-positive irAE-n. We further identified signatures indicative of enhanced chemotaxis and inflammation in irAE-n patients and discovered C-X-C motif chemokine ligand (CXCL)10 as a promising marker to diagnose high-grade immunotoxicities such as irAE-n.

We demonstrate profound and partly subgroup-specific immune cell dysregulation in irAE-n patients, which may guide future biomarker development and targeted treatment approaches.

Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell-cell communication, signaling, and migration. The four members of the β2 integrin subfamily are composed of an alternative α (CD11a-d) subunit, which determines the specific receptor properties, and a constant β (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on β2 integrins that are specifically expressed by leukocytes. The pathophysiological role of β2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of β2 integrins in vivo employed mice with a constitutive knockout of all β2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of β2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of β2 integrins by our group has enabled the dissection of cell-specific roles of β2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate β2 integrin activity for therapeutic applications.

Improved characterization of relevant pathogenic pathways in systemic lupus erythematosus (SLE) has been further delineated over the last decades. This led to the development of targeted treatments including belimumab and anifrolumab, which recently became available in clinics. Therapeutic targets in SLE encompass interferon (IFN) signaling, B-T costimulation including immune checkpoints, and increasing modalities of B lineage targeting, such as chimeric antigen receptor (CAR) T cells directed against CD19 or sequential anti-B cell targeting. Patient profiling based on characterization of underlying molecular abnormalities, often performed through comprehensive omics analyses, has recently been shown to better predict patients' treatment responses and also holds promise to unravel key molecular mechanisms driving SLE. SLE carries two key signatures, namely the IFN and B lineage/plasma cell signatures. Recent advances in SLE treatments clearly indicate that targeting innate and adaptive immunity is successful in such a complex autoimmune disease. Although those signatures may interact at the molecular level and provide the basis for the first selective treatments in SLE, it remains to be clarified whether these distinct treatments show different treatment responses among certain patient subsets. In fact, notwithstanding the remarkable amount of novel clues for innovative SLE treatment, harmonization of big data within tailored treatment strategies will be instrumental to better understand and treat this challenging autoimmune disorder. This review will provide an overview of recent improvements in SLE pathogenesis, related insights by analyses of big data and machine learning as well as technical improvements in conducting clinical trials with the ultimate goal that translational research results in improved patient outcomes.

Tonsillar cancer is caused by high-risk human papillomavirus (HPV), tobacco smoking, and alcohol abuse. Aspects of the patient's immune response to this disease have arisen as prognostic factors and treatment targets, reflecting differences in the type and protein expression profile of immune cells. Because tonsillar cancers are heterogenous lesions such data need to be spatially resolved.

In this study, we aim to explore inter-patient and intra-tumoral sources of variation in tonsillar cancer using immunofluorescence and targeted spatial proteomics to interrogate a cohort of 105 patients. Furthermore, we assess prognostic factors and elucidate molecular targets. We have used CD8, CD11c, and Pan-cytokeratin (PanCK) to quantify and locate immune cells driving antigen-specific cellular immunity. Guided by immunofluorescence information, we selected 355 CD8+, CD11c+, or PanCK+ areas inside and outside (i.e., stroma) cancer-cell islets, to quantify 43 immune-related proteins using digital spatial profiling.

Quantitative analysis of immunofluorescence in combination with clinical data revealed that the abundance of total CD8+ cells and CD8+ cells infiltrating cancer-cell islets, respectively, were associated with higher 5-year disease-free survival and overall survival, independently of HPV-status and clinical stage. Comparison of CD8+ cells inside and outside cancer-cell islets revealed an upregulation of effector CD8+ T-cell and immune checkpoint molecules in the former. Among these, the expression of PD-L1 by CD8+ T-cells was associated with lower all-cause mortality in a univariate proportional hazards model. Similarly, a comparison of tumor boundary and stroma CD11c+ cells showed upregulation of both co-stimulatory and immune checkpoint molecules with proximity to tumor cell islets.

Our findings highlight the relevance of analyzing aspects of tumor micro-architecture in the search of prognostic markers and molecular targets for tonsillar cancer. The abundance of intra-tumoral CD8+ T-cells can be considered a positive predictive marker for tonsillar cancer, while the significance of PD-L1 expression by intra-tumoral CD8+ T-cells warrants further evaluation. Location-based differences in CD8+ and CD11c+ cells suggest an immune cell-altering effect on the tumor microenvironment, and grant new insight into which cells that can be targeted by novel therapeutic agents.

The defect of B cell self-tolerance and the continuous antigen presentation by T cells (TCs) mediated by autoreactive B cells (BCs) play a key role in the occurrence and development of systemic lupus erythematosus (SLE). PD-1/PD-L1 signaling axis negatively regulates the immune response of TCs after activation and maintains immune tolerance. However, the effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B/CD4+TCs in the peripheral blood of patients with SLE has not been studied in detail.

PD-1/PD-L1 and Ki-67 levels in peripheral blood (PB) of 50 SLE patients and 41 healthy controls (HCs) were detected through flow cytometry, and then the expression of PD-1+/-cells and PD-L1+/-cells Ki-67 was further analyzed. CD19+B/CD4+TCs were separated for cell culture and the supernatant was collected to determine proliferation and differentiation of TCs. IL-10 and IFN-γ secretion in the supernatant was also determined using ELISA.

The PD-1, PD-L1, and Ki-67 levels on CD19+B/CD4+TCs in patients with SLE were higher than HCs. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ cells was higher than that of PD-L1- cells, and the proliferative activity of PD-1+ cells was higher than that of PD-1- cells. In the system co-culturing CD19+B/CD4+TCs from HCs/SLE patients, activated BCs promoted TCs proliferation and PD-L1 expression among TCs. Addition of anti-PD-L1 to co-culture system restored the proliferation of TCs, and inhibited IL-10/IFN-γ level. The addition of anti-PD-L1 to co-culture system also restored Tfh and downregulated Treg in HCs.

Axis of PD-1/PD-L1 on CD19+B/CD4+TCs in PB of SLE patients is abnormal, and cell proliferation is abnormal. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ and PD-1+ cells compared with PD-L1- and PD-1- cells in SLE patients, respectively. CD19+B/CD4+TCs in SLE patients can interact through PD-1/PD-L1.

SjD (Sjögren's Disease) and SLE (Systemic Lupus Erythematosus) are similar diseases. There is extensive overlap between the two in terms of both clinical features and pathobiologic mechanisms. Shared genetic risk is a potential explanation of this overlap. In this study, we evaluated whether these diseases share causal genetic risk factors. We compared the causal genetic risk for SLE and SjD using three complementary approaches. First, we examined the published GWAS results for these two diseases by analyzing the predicted causal gene protein-protein interaction networks of both diseases. Since this method does not account for overlapping risk intervals, we examined whether such intervals also overlap. Third, we used two-sample Mendelian randomization (two sample MR) using GWAS summary statistics to determine whether risk variants for SLE are causal for SjD and vice versa. We found that both the putative causal genes and the genomic risk intervals for SLE and SjD overlap 28- and 130-times more than expected by chance (p < 1.1 × 10-24 and p < 1.1 × 10-41, respectively). Further, two sample MR analysis confirmed that alone or in aggregate, SLE is likely causal for SjD and vice versa. [SjD variants predicting SLE: OR = 2.56; 95% CI (1.98-3.30); p < 1.4 × 10-13, inverse-variance weighted; SLE variants predicting SjD: OR = 1.36; 95% CI (1.26-1.47); p < 1.6 × 10-11, inverse-variance weighted]. Notably, some variants have disparate impact in terms of effect size across disease states. Overlapping causal genetic risk factors were found for both diseases using complementary approaches. These observations support the hypothesis that shared genetic factors drive the clinical and pathobiologic overlap between these diseases. Our study has implications for both differential diagnosis and future genetic studies of these two conditions.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

CD11c+Tbet+ B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c+Tbet+ B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity.

We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c+ B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c- counterparts.

Dynamics of the response of the 3 CD11c+ B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c+ B-cell subsets were functionally characterized by optimized in vitro cultures.

In contrast to a durable expansion of antigen-specific CD11c- memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c+Tbet+ B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c- counterparts.

Overall, CD11c+Tbet+ B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype.

Histidine decarboxylase (HDC), a histamine synthase, is expressed in various tissues and is induced by proinflammatory cytokines such as TNFα. As they age, C57BL/6 mice show auto-antibody deposition and lymphocyte infiltration into various tissues, including salivary glands. However, the mechanism underlying cell infiltration and the change in HDC expression in salivary glands with aging remain unclear. Thus, we aimed to elucidate the relationship between histamine and inflammaging.

We investigated the change in histology and HDC expression in the major salivary glands (parotid, submandibular, and sublingual) of 6-week- and 9-month-old wild-type mice. We also determined the histological changes, cytokine expression, and anti-aging factor Klotho in the salivary glands of 9-month-old wild-type and HDC-deficient (HDC-KO) mice.

Cell infiltration was observed in the submandibular gland of 9-month-old wild-type mice. Although most cells infiltrating the submandibular glands were CD3-positive and B220-positive lymphocytes, CD11c-positive and F4/80-positive monocyte lineages were also detected. HDC, TNFα, and IL-1β mRNA expression increased in the submandibular gland of 9-month-old wild-type mice. The expression of PPARγ, an anti-inflammatory protein, declined in 9-month-old wild-type mice, and Klotho expression increased in 9-month-old HDC-KO mice. Immunohistochemistry showed that Klotho-positive cells disappeared in the submandibular gland of 9-month-old wild-type mice, while Klotho was detected in all salivary glands in HDC-KO mice of the same age.

Our findings demonstrate the multifunctionality of histamine and can aid in the development of novel therapeutic methods for inflammatory diseases such as Sjogren's syndrome and age-related dysfunctions.

The past few years have provided important insights into the genetic architecture of systemic autoimmunity through aggregation of findings from genome-wide association studies (GWAS) and whole-exome or whole-genome sequencing studies. In the prototypic systemic autoimmune disease systemic lupus erythematosus (SLE), monogenic disease accounts for a small fraction of cases but has been instrumental in the elucidation of disease mechanisms. Defects in the clearance or digestion of extracellular or intracellular DNA or RNA lead to increased sensing of nucleic acids, which can break B cell tolerance and induce the production of type I interferons leading to tissue damage. Current data suggest that multiple GWAS SLE risk alleles act in concert with rare functional variants to promote SLE development. Moreover, introduction of orthologous variant alleles into mice has revealed that pathogenic X-linked dominant and recessive SLE can be caused by novel variants in TLR7 and SAT1, respectively. Such bespoke models of disease help to unravel pathogenic pathways and can be used to test targeted therapies. Cell type-specific expression data revealed that most GWAS SLE risk genes are highly expressed in age-associated B cells (ABCs), which supports the view that ABCs produce lupus autoantibodies and contribute to end-organ damage by persisting in inflamed tissues, including the kidneys. ABCs have thus emerged as key targets of promising precision therapeutics.

B cell activating factor (BAFF) has an important role in normal B cell development. The aberrant expression of BAFF is related with the autoimmune diseases development like Systemic Lupus Erythematosus (SLE) for promoting self-reactive B cells survival. BAFF functions are exerted through its receptors BAFF-R (BR3), transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) that are reported to have differential expression on B cells in SLE. Recently, atypical B cells that express CD11c have been associated with SLE because they are prone to develop into antibody-secreting cells, however the relationship with BAFF remains unclear. This study aims to analyze the BAFF system expression on CXCR5- CD11c+ atypical B cell subsets double negative 2 (DN2), activated naïve (aNAV), switched memory (SWM) and unswitched memory (USM) B cells.

Forty-five SLE patients and 15 healthy subjects (HS) were included. Flow cytometry was used to evaluate the expression of the receptors in the B cell subpopulations. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the soluble levels of BAFF (sBAFF) and IL-21.

We found increased frequency of CXCR5- CD11c+ atypical B cell subpopulations DN2, aNAV, SWM and USM B cells in SLE patients compared to HS. SLE patients had increased expression of membrane BAFF (mBAFF) and BCMA receptor in classic B cell subsets (DN, NAV, SWM and USM). Also, the CXCR5+ CD11c- DN1, resting naïve (rNAV), SWM and USM B cell subsets showed higher mBAFF expression in SLE. CXCR5- CD11c+ atypical B cell subpopulations DN2, SWM and USM B cells showed strong correlations with the expression of BAFF receptors. The atypical B cells DN2 in SLE showed significant decreased expression of TACI, which correlated with higher IL-21 levels. Also, lower expression of TACI in atypical B cell DN2 was associated with high disease activity.

These results suggest a participation of the BAFF system in CXCR5- CD11c+ atypical B cell subsets in SLE patients. Decreased TACI expression on atypical B cells DN2 correlated with high disease activity in SLE patients supporting the immunoregulatory role of TACI in autoimmunity.

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination.

Autoreactive B cells and interferons are central players in systemic lupus erythematosus (SLE) pathogenesis. The partial success of drugs targeting these pathways, however, supports heterogeneity in upstream mechanisms contributing to disease pathogenesis. In this review, we focus on recent insights from genetic and immune monitoring studies of patients that are refining our understanding of these basic mechanisms. Among them, novel mutations in genes affecting intrinsic B cell activation or clearance of interferogenic nucleic acids have been described. Mitochondria have emerged as relevant inducers and/or amplifiers of SLE pathogenesis through a variety of mechanisms that include disruption of organelle integrity or compartmentalization, defective metabolism, and failure of quality control measures. These result in extra- or intracellular release of interferogenic nucleic acids as well as in innate and/or adaptive immune cell activation. A variety of classic and novel SLE autoantibody specificities have been found to recapitulate genetic alterations associated with monogenic lupus or to trigger interferogenic amplification loops. Finally, atypical B cells and novel extrafollicular T helper cell subsets have been proposed to contribute to the generation of SLE autoantibodies. Overall, these novel insights provide opportunities to deepen the immunophenotypic surveillance of patients and open the door to patient stratification and personalized, rational approaches to therapy.

Graves' disease (GD) is a thyroid-specific autoimmune disease with a high prevalence worldwide. The disease is primarily mediated by B cells, which produce autoantibodies against the thyroid-stimulating hormone receptor (TSHR), chronically stimulating it and leading to high levels of thyroid hormones in the body. Interest in characterizing the immune response in GD has motivated many phenotyping studies. The immunophenotype of the cells involved and the interplay between them and their secreted factors are crucial to understanding disease progression and future treatment options. T cell populations are markedly distinct, including increased levels of Th17 and follicular helper T cells (Tfh), while Treg cells appear to be impaired. Some B cells subsets are autoreactive, and anti-TSHR antibodies are the key disease-causing outcome of this interplay. Though some consensus across phenotyping studies will be discussed here, there are also complexities that are yet to be resolved. A better understanding of the immunophenotype of Graves' disease can lead to improved treatment strategies and novel drug targets.

To combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape that characterises the clusters.

Targeted whole blood gene expression and serum cytokines were determined in patients with cSLE, preselected on disease activity state (at diagnosis, Low Lupus Disease Activity State (LLDAS), flare). Unsupervised hierarchical clustering, agnostic to disease characteristics, was used to identify clusters with distinct biological phenotypes. Disease activity was scored by clinical SELENA-SLEDAI (Safety of Estrogens in Systemic Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index). High-dimensional 40-colour flow cytometry was used to identify immune cell subsets.

Three unique clusters were identified, each characterised by a set of differentially expressed genes and cytokines, and by disease activity state: cluster 1 contained primarily patients in LLDAS, cluster 2 contained mainly treatment-naïve patients at diagnosis and cluster 3 contained a mixed group of patients, namely in LLDAS, at diagnosis and disease flare. The biological phenotypes did not reflect previous organ system involvement and over time, patients could move from one cluster to another. Healthy controls clustered together in cluster 1. Specific immune cell subsets, including CD11c+ B cells, conventional dendritic cells, plasmablasts and early effector CD4+ T cells, differed between the clusters.

Using a targeted multiomic approach, we clustered patients into distinct biological phenotypes that are related to disease activity state but not to organ system involvement. This supports a new concept where choice of treatment and tapering strategies are not solely based on clinical phenotype but includes measuring novel biological parameters.

The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist. Historical pandemics include smallpox and influenza, with efficacious therapeutics utilized to reduce overall disease burden through effectively targeting a competent host immune system response. The immune system is composed of primary/secondary lymphoid structures with initially eight types of immune cell types, and many other subtypes, traversing cell membranes utilizing cell signaling cascades that contribute towards clearance of pathogenic proteins. Other proteins discussed include cluster of differentiation (CD) markers, major histocompatibility complexes (MHC), pleiotropic interleukins (IL), and chemokines (CXC). The historical concepts of host immunity are the innate and adaptive immune systems. The adaptive immune system is represented by T cells, B cells, and antibodies. The innate immune system is represented by macrophages, neutrophils, dendritic cells, and the complement system. Other viruses can affect and regulate cell cycle progression for example, in cancers that include human papillomavirus (HPV: cervical carcinoma), Epstein-Barr virus (EBV: lymphoma), Hepatitis B and C (HB/HC: hepatocellular carcinoma) and human T cell Leukemia Virus-1 (T cell leukemia). Bacterial infections also increase the risk of developing cancer (e.g., Helicobacter pylori). Viral and bacterial factors can cause both morbidity and mortality alongside being transmitted within clinical and community settings through affecting a host immune response. Therefore, it is appropriate to contextualize advances in single cell sequencing in conjunction with other laboratory techniques allowing insights into immune cell characterization. These developments offer improved clarity and understanding that overlap with autoimmune conditions that could be affected by innate B cells (B1+ or marginal zone cells) or adaptive T cell responses to SARS-CoV-2 infection and other pathologies. Thus, this review starts with an introduction into host respiratory infection before examining invaluable cellular messenger proteins and then individual immune cell markers.