Biology subjects

Lindestam Arlehamn, C. S.

Publications and source records attributed to Lindestam Arlehamn, C. S..

18 recordsLinked to original sources

bioRxiv2025

Enrichment of a CD4-CD8- NK-like cytotoxic Vδ1/3 T cell subset in tuberculosis disease

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a leading cause of global morbidity and mortality. Although gamma-delta ({gamma}{delta}) T cells have increasingly been suggested to contribute to the TB immune response, quantitative and qualitative differences in this immune cell compartment between healthy and TB diseased individuals are not well-characterized. In this study, we used single-cell RNA sequencing to provide a high-resolution characterization of CD4-CD8- {gamma}{delta} T cells in peripheral blood across healthy Mtb-non-sensitized, healthy Mtb-sensitized, and TB disease pre-/post-treatment cohorts. We found upregulation of an activated and cytotoxic gene signature in {gamma}{delta} T cells of TB disease compared to both healthy cohorts. Strikingly, these differences persisted through one year following diagnosis of TB disease (corresponding to six months after completion of anti-TB therapy). We found that these transcriptomic differences were largely mediated by an NK-like cytotoxic V{delta}1 and V{delta}3 subset that was enriched in TB disease, with a unique V{delta}3 TCR gene usage. Our findings suggest long-lasting changes in the CD4-CD8- {gamma}{delta} T cell compartment and highlight V{delta}3 cells, a previously underappreciated {gamma}{delta} T cell subset, as potentially important in TB.

immunology↗

bioRxiv2025

Differential memory enrichment of cytotoxic CD4 T cells in Parkinson's disease patients reactive to α-synuclein

Parkinsons disease (PD) is a complex neurodegenerative disease with a largely unknown etiology. Although the loss of dopaminergic neurons in the substantia nigra pars compacta is the pathological hallmark of PD, neuroinflammation also plays a fundamental role in PD pathology. We have previously reported that PD patients have increased frequencies of T cell reactive to peptides from -synuclein (-syn). However, not all PD participants respond to -syn. Furthermore, we have previously found that CD4 T cells from PD participants responding to -syn (PD_R) are transcriptionally distinct from PD participants not responding to -syn (PD_NR). To gain further insight into the pathology of PD_R participants, we investigated surface protein expression of 11 proteins whose genes had previously been found to be differentially expressed when comparing PD_R and healthy control participants not responding to -syn (HC_NR). We found that Cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2) was expressed on a significantly higher proportion of CD4 effector memory T cells (TEM) in PD_R compared to HC_NR. Single-cell RNA sequencing analysis of cells expressing or not expressing CELSR2 revealed that PD_R participants have elevated frequencies of activated TEM subsets and an almost complete loss of cytotoxic TEM cells. Flow cytometry analyses confirmed that Granulysin+ CD4 cytotoxic TEM cells are reduced in PD_R. Taken together, these results provide further insight into the perturbation of T cell subsets in PD_R, and highlights the need for further investigation into the role of Granulysin+ CD4 cytotoxic TEM in PD pathology.

neuroscience↗

bioRxiv2025

T cell responses towards PINK1 and α-synuclein are elevated in prodromal Parkinson's disease

A role of the immune system in Parkinsons disease (PD) progression has long been suspected due to the increased frequency of activated glial cells and infiltrating T cells into the substantia nigra. It was previously reported that PD donors have increased T cell responses towards PINK1 and -synuclein (-syn), two Lewy body-associated proteins. Further, T cell reactivity towards -syn was highest closer to disease onset, highlighting that autoreactive T cells might play a role in PD pathogenesis. However, whether T cell autoreactivity is present during prodromal PD is unknown. Here, we investigated T cell responses towards PINK1 and -syn in donors at high risk of developing PD (i.e. prodromal PD: genetic risk, hyposmia, and or REM sleep behavior disorder), in comparison to PD and healthy control donors. T cell reactivity to these two autoantigens was detected in prodromal PD at levels comparable to those detected in individuals with clinically diagnosed PD. Aligned with the increased incidence of PD in males, we found that males with PD, but not females, had elevated T cell reactivity compared to healthy controls. However, among prodromal PD donors, males and females had elevated T cell responses. These differing trends in reactivity highlights the need for further studies of the impact of biological sex on neuroinflammation and PD progression.

neuroscience↗

bioRxiv2024

Predicting Immunogenic CD4+ T Cell Epitopes in Bacteria Using Antigen and Peptide Features

BackgroundT cell epitope prediction methods have been broadly utilized to facilitate epitope discovery in infectious agents and help design reagents, diagnostics, and vaccines. Current prediction methods are mainly focused on peptide presentation by MHC molecules, which is a necessary but not sufficient requirement for an epitope. For complex pathogens such as bacteria, it would be desirable to make such predictions more specific to limit the number of candidates that have to be experimentally tested. ObjectiveTo develop a machine learning-based prediction model that integrates both peptide-level and antigen-level features to improve the specificity of CD4+ T cell epitope predictions for bacteria. MethodsWe used a dataset of 20,216 peptides from Mycobacterium tuberculosis (Mtb), tested for T cell recognition in Mtb-infected participants, that led to the discovery of n = 144 peptide epitopes. For each peptide, we calculated six peptide-level features (e.g. MHC class II binding predictions and conservation scores) and six antigen-level features (e.g. including RNA expression levels and subcellular localization scores). Three machine learning algorithms--Random Forest, Gradient Boosting, and XGBoost--were trained using stratified, 5-fold cross-validation and combined into an ensemble model. Experimental validation was performed on Streptococcus pneumoniae peptides, using ex vivo IFN{gamma} assays to confirm the predictive performance. ResultsThe ensemble model achieved an ROC-AUC of 0.91 in predicting immunogenic peptides in the Mycobacterium tuberculosis (Mtb) dataset. Gene expression and conservation were identified as the most impactful features, followed by MHC class II binding predictions. When validated on an independent Bordetella pertussis dataset, the model demonstrated accurate predictive capability, especially for peptides with broad recognition in the participant cohort (ROC-AUC up to 0.82). Prospectively applying the model to Streptococcus pneumoniae, we synthesized peptides predicted by our ensemble model to be immunogenic or non-immunogenic. Ex vivo testing with PBMCs from healthy participants showed that peptides predicted to be immunogenic elicited significantly higher IFN{gamma} responses than non-immunogenic peptides, validating the model. ConclusionsOur machine learning approach, integrating both peptide and antigen features, effectively predicts immunogenic CD4+ T cell epitopes across different bacterial pathogens. This method enhances epitope selection efficiency, aiding vaccine development and immunological research by reducing the need for extensive experimental screening.

immunology↗

bioRxiv2024

Rare Variable M. tuberculosis Antigens induce predominant Th17 responses in human infection

CD4 T cells are essential for immunity to M. tuberculosis (Mtb), and emerging evidence indicates that IL-17-producing Th17 cells contribute to immunity to Mtb. While identifying protective T cell effector functions is important for TB vaccine design, T cell antigen specificity is also likely to be important. To identify antigens that induce protective immunity, we reasoned that as in other pathogens, effective immune recognition drives sequence diversity in individual Mtb antigens. We previously identified Mtb genes under evolutionary diversifying selection pressure whose products we term Rare Variable Mtb Antigens (RVMA). Here, in two distinct human cohorts with recent exposure to TB, we found that RVMA preferentially induce CD4 T cells that express RoR{gamma}t and produce IL-17, in contrast to classical Mtb antigens that induce T cells that produce IFN{gamma}. Our results suggest that RVMA can be valuable antigens in vaccines for those already infected with Mtb to amplify existing antigen-specific Th17 responses to prevent TB disease.

immunology↗

bioRxiv2024

PINK1 is a target of T cell responses in Parkinson's disease

Parkinsons disease (PD) is associated with autoimmune T cells that recognize the protein alpha-synuclein in a subset of individuals. Multiple neuroantigens are targets of autoinflammatory T cells in classical central nervous system autoimmune diseases such as multiple sclerosis (MS). Here, we explored whether additional autoantigenic targets of T cells in PD. We generated 15-mer peptide pools spanning several PD-related proteins implicated in PD pathology, including GBA, SOD1, PINK1, parkin, OGDH, and LRRK2. Cytokine production (IFN{gamma}, IL-5, IL-10) against these proteins was measured using a fluorospot assay and PBMCs from patients with PD and age-matched healthy controls. This approach identified unique epitopes and their HLA restriction from the mitochondrial-associated protein PINK1, a regulator of mitochondrial stability, as an autoantigen targeted by T cells. The T cell reactivity was predominantly found in male patients with PD, which may contribute to the heterogeneity of PD. Identifying and characterizing PINK1 and other autoinflammatory targets may lead to antigen-specific diagnostics, progression markers, and/or novel therapeutic strategies for PD.

immunology↗

bioRxiv2023

Identification of differentially recognized T cell epitopes in the spectrum of Mtb infection

Tuberculosis caused by Mycobacterium tuberculosis is one of the leading causes of death from a single infectious agent. Identifying dominant epitopes and comparing their reactivity in different tuberculosis (TB) infection states can help design diagnostics and vaccines. We performed a proteome-wide screen of 20,610 Mtb derived peptides in 21 Active TB (ATB) patients 3-4 months post-diagnosis of pulmonary TB (mid-treatment) using an IFN{gamma} and IL-17 Fluorospot assay. Responses were mediated exclusively by IFN{gamma} and identified a total of 137 unique epitopes, with each patient recognizing, on average, 8 individual epitopes and 22 epitopes (16%) recognized by 2 or more participants. Responses were predominantly directed against antigens part of the cell wall and cell processes category. Testing 517 peptides spanning TB vaccine candidates and ESAT- 6 and CFP10 antigens also revealed differential recognition between ATB participants mid-treatment and healthy IGRA+ participants of several vaccine antigens. An ATB-specific peptide pool consisting of epitopes exclusively recognized by participants mid-treatment, allowed distinguishing participants with active pulmonary TB from healthy interferon-gamma release assay (IGRA)+/- participants from diverse geographical locations. Analysis of longitudinal samples indicated decreased reactivity during treatment for pulmonary TB. Together, these results show that a proteome-wide screen of T cell reactivity identifies epitopes and antigens that are differentially recognized depending on the Mtb infection stage. These have potential use in developing diagnostics and vaccine candidates and measuring correlates of protection.

immunology↗

bioRxiv2023

M. tuberculosis antigen-responsive IL17+ CD4 T cells are disproportionately spared in ART-suppressed HIV

BackgroundInterleukin 17 producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with Human Immunodeficiency Virus (HIV) disproportionately affects distinct Th17 cell subsets that respond to Mtb is incompletely defined. MethodsWe performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by LC/MS on plasma and tested the hypothesis that tryptophan catabolism influences Th17 cell frequencies in this context. ResultsWe identified two subsets of Th17 cells: subset 1 defined as CD4+V7.2-CD161+CD26+ and subset 2 defined as CD4+V7.2-CCR6+CXCR3- cells of which subset 1 was significantly reduced in LTBI with HIV-ART, yet Mtb-responsive IL17-producing CD4 T cells were preserved; we found that IL17-producing CD4 T cells dominate the response to Mtb antigen but not CMV antigen or staphylococcal enterotoxin B (SEB); and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17 cell frequencies. ConclusionsWe found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

immunology↗

bioRxiv2022

Profile of Mycobacterium tuberculosis-specific CD4 T cells at the site of disease and blood in pericardial tuberculosis

Our understanding of the immune response at the site of disease in extra-pulmonary tuberculosis (EPTB) is still limited. In this study, using flow cytometry, we defined the pericardial fluid (PCF) cellular composition and compared the phenotypic and functional profile of Mycobacterium tuberculosis (Mtb)-specific T cells between PCF and whole blood in 16 patients with pericardial TB (PCTB). We found that lymphocytes were the predominant cell type in PCF in PCTB, with a preferential influx of CD4 T cells. The frequencies of TNF- producing myeloid cells and Mtb-specific T cells were significantly higher in PCF compared to blood. Mtb-specific CD4 T cells in PCF exhibited a distinct phenotype compared to those in blood, with greater GrB expression and lower CD27 and KLRG1 expression. We observed no difference in the production IFN{gamma}, TNF or IL-2 by Mtb-specific CD4 T cells between the two compartments, but MIP-1{beta} production was lower in the PCF T cells. Bacterial loads in the PCF did not relate to the phenotype or function of Mtb-specific CD4 T cells. Upon anti-tubercular treatment completion, HLA-DR, Ki-67 and GrB expression was significantly decreased, and relative IL-2 production was increased in peripheral Mtb-specific CD4 T cells. Overall, using a novel and rapid experimental approach to measure T cell response ex vivo at site of disease, these results provide novel insight into molecular mechanisms and immunopathology at site of TB infection of the pericardium.

immunology↗

bioRxiv2022

Peripheral blood T-cell deficiency and hyperinflammatory monocyte responses associate with MAC lung disease

RationaleAlthough nontuberculous mycobacterial (NTM) disease is a growing problem, available treatments are suboptimal and diagnostic tools are inadequate. Immunological mechanisms of susceptibility to NTM disease are poorly understood. ObjectiveTo understand NTM pathogenesis, we evaluated innate and antigen-specific adaptive immune responses to Mycobacterium avium complex (MAC) in individuals with MAC lung disease (MACDZ). MethodsWe synthesized 15mer MAC-, NTM-, or MAC/Mtb-specific peptides and stimulated peripheral blood mononuclear cells (PBMC) with pools of these peptides. We measured T-cell responses by cytokine production, expression of surface markers, and analysis of global gene expression in 27 MACDZ individuals and 32 healthy controls. We also analyzed global gene expression in Mav-infected and uninfected peripheral blood monocytes from 17 MACDZ and 17 healthy controls. Measurements and Main ResultsWe were unable to detect T-cell responses against the peptide libraries or Mav lysate that has increased reactivity in MACDZ subjects compared to controls. T-cell responses to non-mycobacteria derived antigens were preserved. MACDZ individuals had a lower frequency of Th1 and Th1* T-cell populations. By contrast, global gene expression analysis demonstrated upregulation of proinflammatory pathways in uninfected and Mav-infected monocytes derived from MACDZ subjects compared to controls. ConclusionsPeripheral blood T-cell responses to Mycobacterial antigens and the frequency of Th1 and Th1* cell populations are diminished in individuals with MAC disease. In contrast, MACDZ subjects had hyperinflammatory monocyte responses. Together, these data suggest a novel immunologic defect which underlies MAC pathogenesis and includes concurrent innate and adaptive dysregulation.

immunology↗

bioRxiv2021

CD4 T cells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection

The HIV-mediated decline in circulating CD4 T cells correlates with increased the risk of active tuberculosis (TB)1-4. However, HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals also have an increased incidence of TB prior to loss of CD4 T cells in blood3,5, raising the possibility that HIV co-infection leads to disruption of CD4 T cell responses at the site of lung infection before they are observed systemically. Here we used a rhesus macaque model of SIV/Mtb co-infection to study the early effects of acute SIV infection on CD4 T cells in pulmonary Mtb granulomas. Two weeks after SIV co-infection CD4 T cells were dramatically depleted from granulomas, before significant bacterial outgrowth, disease reactivation as measured by PET-CT imaging, or CD4 T cell loss in blood, airways, and lymph nodes. Mtb-specific CD4 T cells, CCR5-expressing, in granulomas were preferentially depleted by SIV infection. Moreover, CD4 T cells were preferentially depleted from the granuloma core and lymphocyte cuff relative to B cell-rich regions, and live imaging of granuloma explants showed that SIV co-infection reduced T cell motility. Thus, Mtb-specific CD4 T cells in pulmonary granulomas may be decimated before many patients even experience the first symptoms of acute HIV infection.

immunology↗

bioRxiv2021

CD4+CCR6+ T cells dominate the BCG-induced transcriptional signature

The century-old Mycobacterium bovis Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine against tuberculosis (TB). Despite this, there is still a lot to learn about the immune response induced by BCG, both in terms of phenotype and specificity. Here, we investigated immune responses in adult individuals pre and 8 months post BCG vaccination. We specifically determined changes in gene expression, cell subset composition, DNA methylome, and the TCR repertoire induced in PBMCs and CD4 memory T cells associated with antigen stimulation by either BCG or a Mycobacterium tuberculosis (Mtb)-derived peptide pool. Following BCG vaccination, we observed increased frequencies of CCR6+ CD4 T cells, which includes both Th1* and Th17 subsets, and mucosal associated invariant T cells (MAITs). A large number of immune response genes and pathways were upregulated post BCG vaccination with similar patterns observed in both PBMCs and memory CD4 T cells, thus suggesting a substantial role for CD4 T cells in the cellular response to BCG. These upregulated genes and associated pathways were also reflected in the DNA methylome. We described both qualitative and quantitative changes in the BCG-specific TCR repertoire post vaccination, and importantly found evidence for similar TCR repertoires across different subjects. The immune signatures defined herein can be used to track and further characterize immune responses induced by BCG, and can serve as reference for benchmarking novel vaccination strategies.

immunology↗

bioRxiv2021

Transcriptional analysis of peripheral memory T cells reveals Parkinson's disease-specific gene signatures

Parkinsons disease (PD) is a multi-stage neurodegenerative disorder with largely unknown etiology. Recent findings have identified PD-associated autoimmune features including roles for T cells. To further characterize the role of T cells in PD, we performed RNA sequencing on PBMC and peripheral CD4 and CD8 memory T cell subsets derived from PD patients and age-matched healthy controls. When the groups were stratified by their T cell responsiveness to alpha-synuclein (-syn) as a proxy for ongoing inflammatory autoimmune response, the study revealed a broad differential gene expression profile in memory T cell subsets and a specific PD associated gene signature. We identified a significant enrichment of transcriptomic signatures previously associated with PD, including for oxidative stress, phosphorylation, autophagy of mitochondria, cholesterol metabolism and inflammation, and the chemokine signaling proteins CX3CR1, CCR5 and CCR1. In addition, we identified genes in these peripheral cells that have previously been shown to be involved in PD pathogenesis and expressed in neurons, such as LRRK2, LAMP3, and aquaporin. Together, these findings suggest that features of circulating T cells with -syn-specific responses in PD patients provide insights into the interactive processes that occur during PD pathogenesis and suggest potential intervention targets.

immunology↗

bioRxiv2021

A novel Mycobacterium tuberculosis-specific subunit vaccine provides synergistic immunity upon co-administration with Bacillus Calmette-Guerin

Tuberculosis (TB) remains a global health crisis. Following encouraging clinical results of subunit vaccination and revaccination with Bacillus Calmette-Guerin (BCG), it has been suggested to combine BCG and subunit vaccines for increased efficacy. Current subunit vaccines are almost exclusively designed as BCG boosters. The goal of this study was to design a subunit vaccine that does not share antigens with BCG and explore the advantages of a BCG+subunit vaccine co-administration strategy, where the two vaccines do not cross-react. Eight protective antigens were selected to create a Mycobacterium tuberculosis (Mtb)-specific subunit vaccine, named H107. Whereas subunit vaccines with BCG-shared antigens displayed cross-reactivity to BCG in vivo in both mice and humans, H107 showed no cross-reactivity and did not inhibit BCG colonization in mice. Encouragingly, co-administering H107 with BCG (BCG+H107) led to increased adaptive immune responses against both H107 and BCG leading to improved BCG-mediated immunity. In contrast to subunit vaccines with BCG-shared antigens, boosting BCG with H107 led to substantial expansion of clonal diversity in the T cell repertoire, and BCG+H107 co-administration conferred significantly increased Th17 responses and less differentiated CD4 T cells. CD4 T cells induced by BCG+H107 maintained functional superiority after Mtb infection, and BCG+H107 provided significantly increased long-term protection compared to both BCG and H107 alone, as well as, BCG co-administered with a subunit vaccine composed of antigens shared with BCG. Overall, we identify several advantages of combining an Mtb-specific subunit vaccine with BCG and introduce H107 as a BCG-complementing vaccine with distinctive value for co-administration with BCG.

immunology↗

bioRxiv2021

Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit in M. tuberculosis infected macaques. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.

immunology↗

bioRxiv2020

The challenge of distinguishing cell-cell complexes from singlet cells in non-imaging flow cytometry and single-cell sorting

Our recent work has highlighted that care needs to be taken when interpreting single cell data originating from flow cytometry acquisition or cell sorting: We found that doublets of T cells bound to other immune cells are often present in the live singlet gate of human peripheral blood samples acquired by flow cytometry. This hidden contamination generates atypical gene signatures of mixed cell lineage in what is assumed to be single cells, which can lead to data misinterpretation, such as the description of novel immune cell types. Here, based on the example of T cell-monocyte complexes, we identify experimental and data analysis strategies to help distinguishing between singlets and cell-cell complexes in non-imaging flow cytometry and single-cell sorting. We found robust molecular signatures in both T cell-monocyte and T cell-B cell complexes that can distinguish them from singlets at both protein and mRNA levels. Imaging flow cytometry with appropriate gating strategy (matching the one used in cell sorting) and direct microscopy imaging after cell sorting were the two methods of choice to detect the presence of cell-cell complexes in suspicious dual-expressing cells. We finally applied this knowledge to highlight the likely presence of T cell-B cell complexes in a recently published dataset describing a novel cell population with mixed T cell and B cell lineage properties.

immunology↗

bioRxiv2018

No cell is an island: circulating T cell:monocyte complexes are markers of immune perturbations

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artefacts and thus ignored in data acquisition and analysis, are the result of true biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell:monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be revisited.

immunology↗

bioRxiv2017

DAFi: A Directed Recursive Filtering and Clustering Approach to Data-Driven Identification of Cell Populations from Polychromatic Flow Cytometry Data

Computational methods for identification of cell populations from high-dimensional flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. We found that combining recursive filtering and clustering with constraints converted from the user manual gating strategy can effectively identify overlapping and rare cell populations from smeared data that would have been difficult to resolve by either a single run of data clustering or manual segregation. We named this new method DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell-based biomarkers, but also makes the results interpretable to experimental scientists as in supervised classification through mapping and merging the high-dimensional data clusters into the user-defined 2D gating hierarchy. By recursive data filtering before clustering, DAFi can uncover small local clusters which are otherwise difficult to identify due to the statistical interference of the irrelevant major clusters. Quantitative assessment of cell type specific characteristics demonstrates that the population proportions calculated by DAFi, while being highly consistent with those by expert centralized manual gating, have smaller technical variance than those from individual manual gating analysis. Visual examination of the dot plots showed that the boundaries of the DAFi-identified cell populations followed the natural shapes of the data distributions. To further exemplify the utility of DAFi, we show that DAFi can incorporate the FLOCK clustering method to identify novel cell-based biomarkers. Implementation of DAFi supports options including clustering, bisecting, slope-based gating, and reversed filtering to meet various auto-gating needs from different scientific use cases.

bioinformatics↗