Mass Cytometry Research Articles

Aging is the leading risk factor of chronic diseases including cardiovascular diseases and metabolic diseases. Emerging evidence suggests cellular senescence, one of the pillars of aging, plays a causal role in development of these diseases. While we previously reported that senescent cell elimination delays or prevents progression of several aging phenotypes in aged mice, the responsible types of senescent cells contributing to diseases in vivo are not yet fully elucidated. We made high fat diet-induced obesity model (60% fat diet, HFD) and examine the cell type of senescent cells. Single cell mass cytometry analysis using adipose tissue revealed that senescent endothelial cells expressed the DNA damage marker γH2AX and inflammatory factors such as TNFα and IL-6 (Figure 1). To test the hypothesis that senescent endothelial cells are a major contributor to chronic diseases, we developed Tie2-Cre; p16 Ink4a - LOX-ATTAC mice, which allows specific elimination of p16 Ink4a positive senescent endothelial cells upon administration of AP20187 (which only affects Tie2 -expressing p16 Ink4a positive cells carrying the conditionally-expressed ATTAC construct) (Figure 2). We found that targeted removal of these cells alleviated HFD-induced metabolic dysfunction with decrease of inflammatory cytokines (Figure 3). In contrast, transplanting senescent endothelial cells into lean mice caused adipose tissue inflammation and metabolic dysfunction. We found the inflammatory factors from senescent endothelial cells induced inflammation in preadipocytes and expand inflammation and targeting senescent endothelial cells may cut the expanding inflammation. Consistent with these findings, natural flavonoid fisetin, which we previously identified that can induce apoptosis selectively to senescent endothelial cells among other senescent cell types, reduced senescent cell amount in adipose tissue and improved glucose metabolism. Moreover, we confirmed the effect of fisetin against senescent endothelial cells in freshly isolated human omental adipose tissue from obese individuals. The fisetin-treated explants exhibited a significant reduction in both SA-β-gal-positive adipocytes and endothelial cells, as well as fewer p16-positive cells, indicating that fisetin induced apoptosis in senescent endothelial cells. Collectively, these data suggest that elimination of p16 Ink4a positive senescent endothelial cells may be a viable therapeutic strategy for alleviation of metabolic diseases.

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. In this study, we report the generation of a spatial multiomic atlas of PDAC tumors and tumor-adjacent lymph nodes from patients treated with combination neoadjuvant immunotherapies. Using machine learning-enabled hematoxylin and eosin image classification models, imaging mass cytometry, and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within and adjacent to TLS spanning distinct spatial niches and pathologic responses. Unsupervised learning identified TLS-specific spatial gene expression signatures that are significantly associated with improved survival in patients with PDAC. We identified spatial features of pathologic immune responses, including intratumoral TLS-associated B-cell maturation colocalizing with IgG dissemination and extracellular matrix remodeling. Our findings offer insights into the cellular and molecular landscape of TLS in PDACs during immunotherapy treatment.

IFN-γ- GranzymeB + Natural killer cells are induced by IV BCG vaccination and associated with protection against tuberculosis in rhesus macaques.

Engineering unnatural amino acids in peptide linkers enables cathepsin-selective antibody-drug conjugates for HER2-positive breast cancer.

Background: Protein arginine deiminase 4 (PAD4) has emerged as a promising therapeutic target for acute promyelocytic leukemia (APL) because of its role in epigenetic regulation and leukemogenesis. All-trans retinoic acid, a standard differentiation agent in APL therapy, has been shown to upregulate PAD4 expression during leukemic cell maturation. Interestingly, first-generation PAD4 inhibitors also promote differentiation, but simultaneously trigger compensatory PAD4 overexpression, underscoring the unresolved complexity of PAD4 modulation in leukemia therapy. Methods: In this study, we employed mass cytometry and transcriptomic–proteomic integrated analysis to investigate the underlying mechanisms of YW3-56, a dual-function PAD4 inhibitor against protein expression and enzymatic function, in NB4 leukemia cells. Functional validation was conducted using Western blot and metabolic assays. Results: Mass cytometry analysis revealed that YW3-56 reduced leukemia stemness (CD44/CD133), while enhancing myeloid differentiation (CD11b/CD14) and immunogenic activation (CD80/CD86). Multiomics analysis revealed a YW3-56-induced metabolic shift characterized by downregulation of glycolytic enzymes and upregulation of the tricarboxylic acid cycle and pentose phosphate pathway components, indicating a reversal of the Warburg effect. Mechanistically, this metabolic reprogramming was driven by reduced AKT expression and phosphorylation at Thr308, impaired GLUT1 expression and membrane localization, and decreased glucose uptake, which collectively promoted the differentiation of NB4 cells. Additionally, YW3-56 suppressed the downstream mTOR pathway, inducing caspase-3/PARP-mediated apoptosis and inhibiting cell proliferation. Conclusions: Our study demonstrated that YW3-56 exerts multimodal antileukemic effects in APL by simultaneously targeting PAD4-mediated epigenetic regulation, AKT-driven metabolic reprogramming and cellular differentiation, highlighting PAD4-AKT signaling as a promising target for APL combination therapy.

Imaging mass cytometry (IMC) is a powerful multiplexed imaging technology used to investigate cell phenotypes and spatial organization of tissue in health and disease. The spatial resolution of IMC is presently at 1 µm, enabling the resolution of single cells and large subcellular compartments but not submicrometer intracellular structures. Here we report a method to improve the resolution of IMC so that it approaches that of light microscopy. High-resolution IMC (HR-IMC) uses an oversampling approach coupled with point-spread function-based deconvolution to achieve a resolution below 350 nm. We demonstrate the performance of HR-IMC in resolving subcellular structures, such as nuclear foci and mitochondrial networks previously undetectable with IMC, and applied it to visualize chemotherapy-induced perturbation of patient-derived ovarian cancer cells. HR-IMC extends highly multiplex IMC analyses into the subcellular regime, enabling analysis of cell biological features and characteristics of disease.

Intracranial aneurysms (IAs) are pathologic dilatations of cerebral arteries with the potential for rupture and subarachnoid hemorrhage (SAH). While inflammation is implicated in IA pathogenesis, the precise immune regulatory mechanisms underlying IA stability, growth, and rupture remain poorly defined. We aimed to characterize the immune landscape of IAs using a noninvasive endoluminal biopsy method coupled with high-dimensional single-cell phenotyping to identify immune signatures associated with IA states. This prospective observational cohort study was conducted at Yale New Haven Hospital between July 2022 and August 2024. Patients undergoing endovascular treatment for ruptured or unruptured IAs were eligible for inclusion. Endoluminal biopsy samples were obtained using detachable coils and microcatheter tips exposed to the IA lumen during treatment. High-dimensional single-cell phenotyping was performed using mass cytometry by time-of-flight (cyTOF) with a 30-marker panel. Cells were clustered using unsupervised analysis, and immune profiles were compared across IA states. Associations with clinical variables were assessed using Fisher exact tests. Fifteen patients were included (mean age 52.20 ± 12.42 years; 53.33% female). Smoking history included current (26.67%), former (33.33%), and never (40.00%) smokers. Hypertension was present in 8 patients (53.34%), with 4 controlled and 4 poorly controlled. Sample viability was high (mean 93.28% ± 5.11%). All 6 unstable aneurysms (4 ruptured, 2 unruptured but growing) clustered within cyTOF immune group 1 while 6 of 9 stable or undocumented aneurysms clustered in group 2 (p = 0.01; odds ratio = ∞, 95% CI 1.97-∞). Group 1 exhibited neutrophil predominance and upregulation of CD38, HLA-DR, and CCR7. No significant associations were observed between immune group and rupture status, hypertension, smoking, or laterality. This study demonstrates that noninvasive endoluminal biopsy combined with high-dimensional single-cell immune phenotyping can identify distinct immune signatures in IAs. A shift toward neutrophil-dominant inflammation was associated with aneurysm growth and rupture. Limitations include the cross-sectional design, which limits causal inference. These findings may support future development of immune-based biomarkers and targeted immunomodulatory therapies to stabilize IAs and prevent rupture.

Regulated cell death is integral to sculpting the developing brain, yet the relative contributions of extrinsic apoptosis and necroptosis remain unclear. Here, we leverage single-cell mass cytometry (CyTOF) to characterize the cellular landscape of the mouse telencephalon in wild-type (WT), RIPK3 knockout (RIPK3 KO), and RIPK3/Caspase-8 double knockout (DKO) mice. Strikingly, combined deletion of RIPK3 and Caspase-8 leads to a 12.6% increase in total cell count, challenging the prevailing notion that intrinsic apoptosis exclusively governs developmental cell elimination. Detailed subpopulation analysis reveals that DKO mice display selective enrichment of Tbr2⁺ intermediate progenitors and endothelial cells, underscoring distinct, cell type-specific roles for extrinsic apoptotic and necroptotic pathways. These findings provide a revised framework for understanding the coordinated regulation of cell number during telencephalic development and suggest potential mechanistic links to neurodevelopmental disorders characterized by aberrant cell death.

Abstract Background and Aims Peritoneal dialysis (PD) fluids are designed to remove excess water and uremic toxins along an osmotic gradient created by high glucose concentrations (1,360–3,860 mg/dL). Multiple PD fluid exchanges per day add up to a glucose exposure of 50% of the recommended daily energy intake for a healthy diet by intraperitoneal administration alone. Albeit this glucose exposure has been associated with adverse metabolic outcomes in end-stage kidney disease (ESKD) patients on PD, mechanistic studies remain elusive. The aim of this study was to explore the systemic effects of PD induced glucose exposure and the underlying mechanisms leading to adverse clinical outcomes. Method In a murine animal experiment, PD was conducted for 7 weeks via an implanted PD catheter in healthy mice and mice with chronic kidney disease (CKD, via 5/6 nephrectomy). PD and CKD+PD groups were compared to healthy control (HC) and CKD mice otherwise housed and treated with identical conditions. Glucose absorption after intraperitoneal administration of PD fluid was assessed using dynamic small animal positron emission tomography (µPET) scans with subsequent computed tomography (CT) imaging and a glucose analog radiotracer (10 MBq 2-[18F]fluoro-2-deoxy-D-glucose, [18F]FDG). Systemic effects of PD induced glucose exposure were investigated with a combination of multiplexed single-cell imaging mass cytometry, untargeted tissue proteomics, spatial transcriptomics, and enzyme histochemistry of hepatic tissue, routine serum chemistry and targeted serum metabolomics, gut microbiome analysis with 16S rRNA sequencing, and immunofluorescence staining of the gut and gut-vascular barrier. Findings of the experimental animal models were then translated to a previously published cohort of patients on automated PD. In this clinical cohort relationships between peritoneal glucose loads and systemic effects were assessed using linear mixed-effects models. Results During PD treatment, intraperitoneally administered glucose was rapidly taken up by the liver, evident in dynamic µPET scans. Chronic PD treatment (independent of CKD effects) induced hepatic metabolic inflammation, with activation of acute phase response signaling (FDR P < 0.05), the liver X receptor/retinoid X receptor pathway (FDR P < 0.05), significant reduction of hepatic tissue resident macrophages (F4/80+CD11b-, P = 0.016), and glucose-to-cholesterol shunting with activation of fatty acid metabolism resulting in significantly elevated serum cholesterol levels (P = 0.00001) despite an absence of hepatic steatosis or glycogen storage. Hepatic tissue resident macrophage depletion was further associated with increased oxidative stress with pentose phosphate pathway cycling and lipopolysaccharide deposition in (peri) portal areas, without increased gut permeability or gut dysbiosis, but elevated serum bilirubin (P = 0.001). In a clinical cohort of n = 61 automated PD patients intraperitoneal glucose loads were significantly associated with serum gamma-glutamyl transferase levels as marker of hepatic oxidative stress (P = 0.007) and systemic inflammation (P = 0.000007). Conclusion This is the first study to report hepatic oxidative stress and metabolic inflammation induced by PD. The systemic effects of this hepatic glucotoxicity become further evident in stimulation of systemic inflammation in patients on PD. This suggests translatability of the identified pathomechanisms that may contribute to increased cardiometabolic risk in clinical PD. These findings reframe the current understanding of PD and its associated comorbidity burden and may allow to identify novel therapeutic approaches to improve clinical outcomes of ESKD patients on PD.

Abstract Background and Aims Non-invasive monitoring and early detection of rejection are of high priority in kidney transplantation. In our previous work, we established urine flow cytometry (FC) as tool for non-invasive kidney transplant monitoring and rejection detection. We hypothesized that an even deeper phenotyping of urinary cells improves the diagnostic yield of urinary T cells. Therefore, we established Cytometry by time of flight (CyTOF) analysis of urine cells to derive an improved biomarker for the detection of rejection. Our findings were subsequently confirmed in a FC-based validation cohort. Method A total of 157 urine samples from kidney transplant recipients (KTR) were collected from patients undergoing indication transplant biopsy. Of these samples, 57 were analyzed in the discovery CyTOF cohort. CD8+CD38+ T cells were identified as a potential biomarker for T cell-mediated rejection (TCMR), which was confirmed in a validation cohort (n = 100) using FC. Additionally, we analyzed a publicly available scSeq dataset of KTR biopsies and urine to investigate our biomarker and its expression in TCMR with an additional method. Results By utilizing high-dimensional mass cytometry, we identified CD8+CD38+ T cells as a potential biomarker for TCMR. Reassessment of publicly available scSeq datasets demonstrated that CD38 expression in CD8+ T cells in kidney transplant tissue corresponds to CD38 expression in urinary CD8+ T cells. We translated our finding to FC and validated urinary CD8+CD38+ T cells in a validation cohort. CD8+CD38+ T cells showed an AUC of 0.956 to diagnose TCMR and exceeded prior published biomarkers. Conclusion Urinary CD8+CD38+ T cells are a new biomarker for TCMR, validated by 3 independent methods in 3 patient cohorts.

Immune checkpoint blockade (ICB) has led to paradigm shifts in the treatment of various tumour types1-4, yet limited efficacy has been observed in patients with metastatic mismatch-repair proficient (pMMR) colorectal cancer5. Here we report clinical results and in-depth analysis of patients with early-stage pMMR colon cancer from the phase II NICHE study (ClinicalTrials.gov: NCT03026140). A total of 31 patients received neoadjuvant treatment of nivolumab plus ipilimumab followed by surgery. The response rate was 26% and included six patients with a major pathological response (≤10% residual viable tumour). One patient with an ongoing clinical complete response did not undergo surgery. Circulating tumour DNA (ctDNA) was positive in 26/31 patients at baseline, and clearance was observed in 5/6 responders prior to surgery, while 19/20 non-responders remained ctDNA+. Responses were observed despite a low tumour mutational burden in all tumours, while chromosomal genomic instability scores were significantly higher in responders compared to non-responders. Furthermore, responding tumours had significantly higher baseline expression of proliferation signatures and TCF1, and imaging mass cytometry revealed a higher percentage of Ki-67+ cancer and Ki-67+ CD8+ T cells in responders compared to non-responders. These results provide a comprehensive analysis of response to neoadjuvant ICB in early-stage pMMR colon cancers and identify potential biomarkers for patient selection.

Esophageal squamous cell carcinoma (ESCC) evolves within a highly interactive tumor microenvironment (TME) that shapes therapeutic response. We utilized mass cytometry to analyze over 10 million cells from 25 ESCC tumors, 24 adjacent nontumor tissues, and 23 peripheral blood samples, employing an extensive panel of 42 immune markers. The resulting atlas reveals a compartmentalized landscape with a reproducible paucity of CD4⁺ and CD8⁺ central memory T cells (TCM) in tumor sites. Reintroduction of patient-derived TCMs restored antitumor immunity in coculture assays, demonstrating their cytotoxic capacity in vitro and suggesting their potential relevance for future therapeutic exploration. Myeloid profiling identified PD-L1⁺ tumor-associated macrophages (TAMs) as correlates of clinical benefit; ex vivo PD-L1 blockade reprogrammed TAMs toward proinflammatory states, indicating pharmacological malleability. Notably, CD39⁺ tumor-infiltrating T cells were consistently associated with favorable prognosis and increased responsiveness to PD-1 blockade across cancer types. The functional inhibition of CD39 impaired cytotoxic T-cell activity, underscoring its dual role as a marker of immune dysfunction and a promising therapeutic target. Collectively, our findings provide a comprehensive immune landscape of ESCC, highlighting key immunological deficits and opportunities for targeted interventions. The insights gained underscore the potential of tailoring immunotherapies to the specific immune profiles of the TME, potentially revolutionizing treatment paradigms for ESCC patients. This study sets the stage for a more nuanced understanding and manipulation of the immune elements critical for optimizing cancer immunotherapy.

People with HIV (PWH) are at an increased risk for AIDS-associated non-Hodgkin lymphoma (AIDS-NHL); however, the immune signatures underlying this risk are not well understood. In this study, we utilized mass cytometry by time-of-flight (CyTOF) to analyze T-cells and monocytes in the PBMCs of treatment-naïve PWH, including those 3 to 36 months before an AIDS-NHL diagnosis (HIV-positive pre-NHL), as well as people without HIV (PWoH). Mass cytometry is an advanced single-cell analysis platform that combines flow cytometry principles with mass spectrometry. Unlike conventional flow cytometry, this technology employs antibodies conjugated to unique metal isotopes instead of fluorescent markers, enabling simultaneous measurement of over 40 distinct cellular markers per individual cell without spectral overlap limitations. Participants were enrolled at the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Unsupervised clustering and Uniform Manifold Approximation and Projection (UMAP) analysis identified CD3+ T-cell and CD14+ monocyte metaclusters, and Spearman's rank correlation assessed their relationships with B-cell subsets exhibiting aberrant phenotypes. We observed elevated levels of CD8+CD20+ T-cells, CD8+CD14+ T-cells, and M2-like CD14+CD163+ monocytes in HIV-positive pre-NHL individuals compared to HIV-negative controls. Positive correlations were found between CD19+ AICDA+ cMYC+ B-cells and M1-like CD14+cMYC+ monocytes (metacluster, MC02), and between metaclusters of CD8+PD-1+CD27+CXCR4- T-cells (MC05) and CD4+FoxP3+PD-1+CD27+CD28+CXCR4- ICOS+ T-cells (MC08). In addition, a different CD19+ B-cell metacluster (FoxP3+AICDA+cMYC+) was positively associated with a metacluster of CD8+PD-1+CD27+CD28+CXCR4+ T-cells (MC03). Moreover, the metacluster of CD8+PD-1+CD27+CXCR4- T-cells (MC05) negatively correlated with M2-like CD14+CD163+ monocytes (MC06), while CD8+CD14+ T-cells positively correlated with AICDA+ Bregs and IL-10+ B-regs in HIV-positive pre-NHL individuals. Unsupervised analysis revealed increased frequencies of CD8+CD20+ T-cells in HIV-positive individuals compared to HIV-negative controls. These immune alterations provide valuable insights into potential biomarkers for early detection, monitoring, and therapeutic strategies for AIDS-NHL.

Enthesitis, a hallmark of psoriatic arthritis (PsA), reflects the interplay between mechanical stress and immune dysregulation at tendon-bone interfaces. This study investigates the cellular and molecular responses in entheseal tissues following interleukin (IL)-17A inhibition in patients with active PsA. In this prospective, interventional phase 4 trial, we enrolled 10 patients with enthesitis of the lateral epicondyle, performed entheseal biopsies, and analysed tissues by imaging mass cytometry (IMC) and spatial transcriptomics before and after treatment. Following 24 weeks of IL-17A inhibition, 9/10 patients of the cohort clinically responded to treatment as assessed by Disease Activity in Psoriatic Arthritis (DAPSA), Spondyloarthritis research consortium of canada (SPARCC) score, and power Doppler sonography. IMC analysis showed significant reductions of enthesitis-related immune cell populations, in particular IL-17-producing CD4, CD8, CD4neg/CD8neg T cells, granulocytes, and innate lymphoid cells type 3. Spatial transcriptomics revealed that CD200+DKK3+ fibroblasts and innate lymphoid cells type 2 expanded and formed an anti-inflammatory niche upon treatment. Notably, IL-17A inhibition led to decreased osteoblast differentiation markers, suggesting a potential mechanism to inhibit pathological bone formation. These findings underline the pivotal role of IL-17A in enthesitis, showing that IL-17A inhibition profoundly modulates the tissue microenvironment of entheses in PsA.

BackgroundTo spatially characterize the single-cell tumor microenvironment (TME) of salivary gland carcinomas (SGC) and identify prognostic biomarkers.MethodsSGC, including salivary duct carcinomas (SDC), acinic cell, mucoepidermoid, and secretory carcinomas, were analyzed using a 13-marker imaging mass cytometry panel. Multichannel image data from 54 primary cases and nodal metastases were processed to generate single-cell datasets. Cell phenotypes (tumor cells, cancer-associated fibroblasts (CAFs), endothelia, immune cells) were classified using a validated CAF algorithm, followed by spatial analysis and clinicopathological correlation. Clinicopathological results were validated using a previously published independent bulk RNA-sequencing (RNA-seq) cohort of n = 67 SDC cases. Spatial transcriptomics data of three SDC cases was leveraged to understand the molecular mechanisms of spatial interactions.ResultsAmong 509,364 cells, SDC exhibited the highest fractions of Collagen- and matrix-CAFs (mCAFs). Acinic cell carcinomas (ACC) showed enriched CD4+/CD8+ T cells and antigen-presenting CAFs (apCAFs), indicating strong immune infiltration. A spatially defined cellular neighborhood (CN8) of mCAFs and endothelia was elevated in SDC, with higher CAF infiltration in androgen receptor (AR)high versus ARlow SDC. Elevated mCAF frequency and CN8 were significantly associated with reduced recurrence-free probability (RFP) and distant control rates (DCR) in SDC. Additionally, higher mCAF frequencies were an independent prognostic factor for decreased RFP and DCR in Cox regression analysis. The association between mCAFs and a decreased RFP as well as recurrence-free survival was confirmed with previously published RNA-seq data using an mCAF signature. Spatial transcriptomics confirmed enrichment of metastasis-associated gene signatures as well as platelet-derived growth factor and insulin-like growth factor pathways within mCAFhighendotheliahigh tumor niches indicating that the latter may be involved in mCAF-endothelia crosstalk.ConclusionSDC are characterized by Collagen-/mCAF-rich microenvironments and mCAF-endothelial spatial interactions that are linked to metastasis. ACC display pronounced immune infiltration, suggesting its potential for immunotherapy. mCAFs in SDC emerge as prognostic biomarkers and therapeutic targets, highlighting the importance of targeting CAF-driven metastasis in future treatments. This study provides insights into the biology of SGC and identifies novel prognostic markers.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03551-z.

Colitis-associated cancer (CAC) is the most severe complication of inflammatory bowel disease (IBD). We hypothesized that chronic inflammation activates endogenous anti-inflammatory mechanisms that promote dysplasia by undermining immunosurveillance. Our aim was to determine chronic inflammation-induced immune cell reprogramming in IBD patients at risk for developing CAC. This cohort study utilized GeoMx digital spatial profiling and imaging mass cytometry in 11 patients with either CAC or sporadic CRC (SCRC). Results from this discovery cohort were validated using IHC/IF in an independent cohort of CAC and SCRC patients (n = 10 and n = 14, respectively), as well as in an independent cohort of IBD patients with (n = 6) and without dysplasia (n = 18). Histologically uninflamed colon from patients who developed CAC displayed upregulated metabolism and stress response pathways as compared to SCRC patients, indicating ongoing epithelial stress-responses. Endogenous anti-inflammatory mechanisms included increased IL-10 expression by lamina propria IgA+ plasma cells and CD163+ macrophages. T cell recruitment and effector pathways were downregulated in CAC, which was associated with a decrease in CD8+ intraepithelial T cells (IELs) and reduced levels of Granzyme B within CD8+ IELs. Decreased CD8+ IEL density was associated with CAC susceptibility, as IBD patients that developed dysplasia showed significantly lower levels of CD8+ IELs than IBD patients that never developed dysplasia. Chronic inflammation induces endogenous mechanisms to protect from inflammation-induced damage, including increased anti-inflammatory cytokine production and decreased levels of CD8+ IELs. While this may limit inflammation, these mechanisms may also reduce immunosurveillance, favoring the development of CAC.

Flow and mass cytometry experiments are essential for profiling immune cells at single-cell resolution. Better understanding of human immunology increasingly involves analyzing studies at the scale of hundreds or thousands of samples, with data analysis a significant bottleneck. This trend increases the demand for automated analysis methods. In particular, a common preprocessing step in cytometry data analysis is distinguishing single cells from doublets (or multiplets), events in which two (or more) cells pass simultaneously through the detector. Typically, doublets are identified on two-dimensional density plots, using their high measured values for DNA intercalators (mass cytometry) or scattering channels (flow cytometry). Despite its popularity, this bivariate gating method is sometimes imprecise: for example, we show that bivariate gating of mass cytometry data can mistake single eosinophils for doublets, due to their high DNA content. Taking inspiration from methods already used in single-cell transcriptomics, but not in the cytometry community, we propose an alternative approach. Our method, called Cleanet, first simulates doublet events, then identifies true events with protein expression similar to the simulated doublets. This simple method is completely automated and detects both homotypic and heterotypic doublets. We validate it in datasets acquired with mass and flow cytometry; moreover, we verify with imaging flow cytometry data from ImageStream and Discover A8 instruments that most events predicted to be doublets truly consist of multiple cells. Cleanet can also classify doublets based on their component cell types, which potentially enables the study of cell-cell interactions, mining extra information out of doublet events that would otherwise be discarded. As a proof of concept, we demonstrate that Cleanet can detect a treatment-specific increase in interactions between two cell lines. By automating doublet detection and classification, we aim to streamline the data analysis in large cytometry studies and provide a more accurate picture of both immune cell populations and cell-cell interactions.

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are the two most common B-cell lymphomas and are characterized by a dynamic crosstalk between tumor B cells and a heterogeneous tumor-supportive microenvironment, including immune, endothelial, and stromal components. Despite their recognized impact on the pathogenesis and prognosis of B-cell lymphoma, tumor-associated macrophages (TAM) have not been extensively explored in these diseases. Herein, we investigated mononuclear phagocyte (MNP) heterogeneity at the single-cell level and the activation profile of MNP, stromal, and endothelial compartments in B-cell lymphoma lymph nodes compared to reactive secondary lymphoid organs. This was achieved using a combination of mass cytometry, single-cell RNA sequencing, in silico and spatial imaging approaches. Our findings revealed a lymphoma-specific pattern of TAM and blood endothelial cell (BEC) co-activation. Furthermore, we identified in DLBCL a spatial interaction between Annexin A1 (ANXA1)-expressing BEC and formyl-peptide receptor (FPR1/2) and S100A9-expressing monocytes/macrophages. This crosstalk is associated with an immunosuppressive tumor microenvironment and an adverse prognosis in two cohorts of DLBCL patients.

Mass cytometry (CyTOF) and Imaging Mass Cytometry (IMC) provide single-cell resolution for over 50 protein markers, enabling unprecedented exploration of tumour and immune heterogeneity. We conducted a scoping review of 61 original studies (inception–2025), spanning 17 cancer types, to map current applications, analytical strategies, and emerging biological insights. 46 studies used CyTOF alone, 12 employed IMC exclusively, and 3 combined both platforms. Median panel sizes were 33.5 markers for CyTOF and 33 for IMC. While lineage and immune checkpoint markers were universal, phospho-epitopes, metabolic enzymes, and stromal proteins appeared in more focused subsets. Most studies followed a three-step analytical workflow: (i) segmentation or gating, (ii) unsupervised clustering, and (iii) downstream spatial or functional analyses. CyTOF investigations frequently identified exhausted CD8+ T-cell subsets (e.g., PD-1+TIM-3+CD39+), suppressive myeloid populations (e.g., CD163+HLA-DR− macrophages), and metabolically reprogrammed Tregs. IMC studies uncovered spatial patterns predictive of outcome, such as tertiary lymphoid structures (TLSs) and macrophage–T cell exclusion zones. Several studies proposed predictive immune signatures or integrated CyTOF with transcriptomic or spatial datasets. We identified five recurrent immunobiological motifs, CD8+ T-cell bifurcation, CD38+ TAM barriers, TLS maturity, CTLA-4+ NK-cell signatures and metabolically defined niches, highlighting convergent axes of resistance and response. Bioinformatic pipelines converged around FlowSOM or PhenoGraph clustering, CITRUS or elastic-net feature selection, and increasingly, machine learning and agent-based spatial modelling. Collectively, CyTOF and IMC are redefining biomarker discovery, therapeutic stratification, and virtual trial design in oncology, establishing high-dimensional CyTOF as a cornerstone of next-generation precision cancer medicine.

IL-21/IL-21R signaling is crucial in various immune diseases and cellular development, however, its role in bacterial pneumonia remains unclear. Here, IL-21R knockout (IL-21R−/−) mice were more susceptible to Actinobacillus pleuropneumoniae (APP) than wild-type (WT) mice. High-dimensional mass cytometry analysis revealed that IL-21R deficiency inhibited neutrophil activation, decreased the numbers of monocytes and proinflammatory macrophages, and augmented the defective CD3low T cells in the lungs. Intracellular cytokine staining showed decreased IFN-γ/TNF-α/IL-6 production in IL-21R−/− mice, particularly in CD8⁺ T cells. Furthermore, a previously unrecognized Ly6C+Ly6G+CD4+ T cell subset emerged only in the lungs of WT mice post-APP infection, which was in an activated status with stronger secretion capacities of IL-10, IL-21, granzyme B, and perforin by flow cytometry. These cells polarized macrophages into M2- or M1- phenotype without/with infection, respectively, and enhanced proliferation, phagocytosis, and macrophage extracellular traps/ROS-mediated bactericidal activity of macrophages against-APP, Klebsiella pneumoniae, or Escherichia coli infection. Thus, our study demonstrated that IL-21 drives the differentiation of neutrophils, monocytes, and macrophages into pro-inflammatory subsets. IL-21-induced Ly6C+Ly6G+CD4+ T cells cooperate with macrophages to enhance bacterial clearance, providing a promising target for preventing bacterial pneumonia.