Lab

Cell-state transitions during development are shaped by multiple concurrent processes, yet single-cell transcriptomic analyses often rely on static molecular profiles or infer time without an experimental anchor. We developed mCanonicalTockySeq, a systems-level framework that reconstructs temporally resolved developmental state spaces by combining signalling history with single-cell RNA sequencing. Using the Nr4a3-Tocky Fluorescent Timer system in developing thymic T cells, we combined scRNA-seq with a molecular clock of strong T-cell receptor signalling to establish an experimentally anchored temporal reference. Using Timer-defined landmark populations, mCanonicalTockySeq constructs a shared state space in which temporal progression and developmental maturation are jointly represented, identifying a Tocky-defined temporal manifold while resolving progression toward CD4 and CD8 single-positive states. This framework recovered biologically coherent dynamics of immediate TCR-response genes, lineage-associated regulators, and agonist-selection-associated programmes. We then extended the framework across species by translating human thymic single-cell transcriptomes into one-to-one mouse ortholog space and projecting them into the mouse Nr4a3-Tocky reference. The projected human cells occupied an interpretable temporal-developmental geometry, and the inferred Tocky-equivalent temporal coordinate showed a significant donor-level association with chronological age. Together, these results establish mCanonicalTockySeq as a general framework for modelling how signalling history and developmental progression are jointly organised in single-cell state space, and illustrate how experimentally anchored reference systems can support comparative analyses across species.

In cancer immunotherapy, T-cell functional states change dynamically, and no single marker or gene fully defines them. Static molecular profiles may therefore be insufficient to resolve these states. Capturing signalling history together with ongoing activity could provide a temporal framework for their dissection. Here, we developed CanonicalTockySeq, which integrates a molecular clock of T-cell receptor (TCR) signalling based on the Fluorescent Timer reporter Nr4a3-Tocky (Timer-of-cell-kinetics-and-activity) with scRNA-seq to establish an experimentally anchored temporal reference. Using landmark Tocky fractions as biological ground truths, CanonicalTockySeq constructs a transcriptomic manifold in canonical space with conical geometry. In a murine melanoma model treated with combination anti-PD-L1 and anti-CTLA-4, this framework separates temporal progression (geodesic angle) from signalling strength (radial intensity), enabling time-resolved analysis of gene-expression programmes at single-cell resolution. Application to scRNA-seq data from patients with melanoma further identifies distinct temporal programmes associated with clinical response. These analyses indicate that effective combination immunotherapy is associated with decreased persistence of antigen engagement, suppression of exhaustion-associated TCR signalling programmes, and maintenance of progenitor-like features linked to durable antitumour responses. Collectively, our findings identify temporal-state control as a key component of immunotherapy outcome and establish CanonicalTockySeq as a framework for resolving T-cell response states in vivo.

Concomitant chemoradiotherapy (CRT) is a standard-of-care for unresectable locally-advanced head and neck squamous cell carcinoma (LA-SCCHN), but its immune effects, particularly compared to radiotherapy (RT) alone, remain unclear. Using syngeneic murine models, we integrated Nr4a3-Tocky reporter analysis with single-cell transcriptomics and T-cell receptor clonotyping comprehensively to profile intratumoural CD8+ T-cells following RT/CRT. We show that CRT uniquely drives robust antigen-specific clonal expansion and biases differentiation toward progenitor (or precursor) exhausted (TPEX) phenotypes, while RT favours terminal exhaustion (TEX). Single-cell analyses reveal CRT-induced clonal dominance within TPEX subsets, suggesting the potential for enhanced immune reinvigoration. In peripheral blood mononuclear cells (PBMCs) from patients treated with CRT, high levels of TEX cells were found early and at 3 months post-treatment, with delayed peripheral TPEX expansion at 3 months, indicating phased progenitor recovery. These findings demonstrate distinct immunological remodelling by CRT versus RT and underscore the critical importance of treatment timing for optimising combination immunotherapy strategies in LA-SCCHN.

Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein, the Timer-of-cell-kinetics-and-activity system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer fluorescence data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a convolutional neural network-based method that incorporates image conversion and class-specific feature visualisation for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3 fluorescent Timer reporter mice lacking the enhancer Conserved Non-coding Sequence 2, which revealed new roles of this enhancer in regulating Foxp3 transcription frequency under specific conditions. Furthermore, analysis of wild-type Foxp3 fluorescent Timer reporter mice at different ages uncovered distinct patterns of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3+ T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, single-cell dynamics analysis, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.

Advancements in cytometry technologies have led to a remarkable increase in the number of markers that can be analyzed simultaneously, presenting significant challenges in data analysis. Traditional approaches, such as dimensional reduction techniques and computational clustering, although popular, often face reproducibility challenges due to their heavy reliance on inherent data structures. This reliance prevents the direct translation of their outputs into gating strategies for downstream experiments. Here, we propose the novel Gating Tree methodology, a pathfinding approach that investigates the multidimensional data landscape to unravel group-specific features without the use of dimensional reduction. This method employs novel measures, including enrichment scores and gating entropy, to effectively identify group-specific features within high-dimensional cytometric data sets. Our analysis, applied to both simulated and real cytometric data sets, demonstrates that the Gating Tree not only identifies group-specific features comprehensively but also produces outputs that are immediately usable as gating strategies for pinpointing key cell populations. Furthermore, by integrating machine learning methods, including Random Forest, we have benchmarked Gating Tree against existing methods, demonstrating its superior performance. A range of supervised and unsupervised methods implemented in Gating Tree thus provides effective visualization and output data, which can be immediately used as successive gating strategies for downstream study.

Fluorescent Timer proteins undergo a time-dependent shift from blue to red fluorescence after translation, providing a temporal record of transcriptional activity in Timer reporter systems. While Timer proteins are well suited for studying dynamic cellular processes such as T cell activation using the Timer-of-Cell-Kinetics-and-Activity (Tocky) framework, quantitative analysis of Timer-based flow cytometry data has yet to be fully standardized. In this study, we optimize quantitative analysis methods for the key parameter within the Tocky framework, Timer Angle, and introduce TockyLocus, an open-source R package that implements a five-category scheme based on biologically grounded angular intervals (designated as Tocky Loci). This approach is validated using both simulated and experimental datasets and enables downstream statistical testing and visualization of transcriptional dynamics in flow cytometry data. Using computational modelling of Timer protein kinetics, we define transcriptional dynamics in relation to key anchoring points in Timer Angle values at 0°, 45°, and 90°. Comprehensive simulations with synthetic spike-in datasets further demonstrate the robustness of the five-locus approach, which captures the three key points and the intermediate regions between these points. Building on the TockyPrep preprocessing framework, we systematically evaluated categorization schemes ranging from three to seven loci on real-world datasets from Nr4a3-Tocky and Foxp3-Tocky mice. The five-locus model emerged as optimal, showing significant advantages in balancing biological interpretability and statistical robustness. Optimized algorithms implemented in the TockyLocus package now standardize quantitative analysis of Timer Angle data, enabling reproducible interpretation without reliance on arbitrary gating or complex assumptions. In summary, the five-locus categorization of Timer Angle data effectively links underlying biological dynamics to the percentage of cells in each Tocky Locus, providing a robust and interpretable framework for investigating transcriptional dynamics in immunology and related fields.

Fluorescent Timer proteins, which display time-dependent changes in their emission spectra, are invaluable for analyzing the temporal dynamics of cellular events at the single-cell level. We previously developed the Timer-of-cell-kinetics-and-activity (Tocky) tools, utilizing a specific Timer protein, Fast-FT, to monitor temporal changes in cellular activities. Despite their potential, the analysis of Timer fluorescence in flow cytometry is frequently compromised by variability in instrument settings and the absence of standardized preprocessing methods. The development and implementation of effective data preprocessing methods remain to be achieved.

The Foxp3⁺ regulatory T (Treg) cell repertoire carries age-dependent biases, with neonatal subsets enriched for highly self-reactive clones. However, the thymocyte features distinguishing neonatal from adult Treg selection remain unclear. Here, we show that neonatal double-positive mouse thymocytes, unlike their adult counterparts, fail to upregulate Zap70 during thymic selection, creating a calcium signaling bottleneck. This attenuated Zap70-dependent signaling limits negative selection, allowing highly self-reactive clones to evade deletion. Modulating Zap70 expression alters this balance; reducing Zap70 in adults rescues development of these clones, whereas increasing Zap70 in neonates enforces their deletion. Similarly, enhancing neonatal calcium signaling via increased LAT Y136-mediated PLCγ1 activation promotes clonal deletion. Analysis of pediatric human thymi reveals that ZAP70 expression remains low during the first year of life, aligning with the peak window for thymic Treg cell development. These findings suggest that age-dependent Zap70 expression regulates negative selection and thymic Treg cell development.

Despite hugely promising randomized phase II study data, combined CRT plus xevinapant failed in the TrilynX phase III clinical trial in locally advanced SCCHN. We show that adding xevinapant to chemoradiotherapy in vivo dysregulates antitumor lymphocyte function, acute-phase proteins, and cell death pathways, with net immunosuppressive effects on the tumor-immune microenvironment.

Retroviruses integrate their genetic material into the host genome, enabling persistent infection. Human T cell leukaemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) share similarities in genome structure and target cells, yet their infection dynamics differ drastically. While HIV-1 leads to high viral replication and immune system collapse, HTLV-1 establishes latency, promoting the survival of infected cells and, in some cases, leading to leukaemia. The mechanisms underlying this latency preference remain unclear. Here we analyse blood samples from people with HTLV-1 and identify an open chromatin region within the HTLV-1 provirus that functions as a transcriptional silencer and regulates transcriptional burst. The host transcription factor RUNX1 binds to this open chromatin region, repressing viral expression. Mutation of this silencer enhances HTLV-1 replication and immunogenicity, while its insertion into HIV-1 suppresses viral production. These findings reveal a strategy by which HTLV-1 ensures long-term persistence, offering potential insights into retroviral evolution and therapeutic targets.

Progenitor cells initiate development upon receiving key signals, dynamically altering gene and protein expression to diverge into various lineages and fates. Despite the use of several experimental approaches, including the Fluorescent Timer-based method Timer-of-cell-kinetics-and-activity (Tocky), analysing time-dependent processes at the single-cell level in vivo remains challenging. This study introduces a novel integrated experimental and computational approach, using an advanced multidimensional toolkit. This toolkit facilitates the simultaneous examination of temporal progression and T-cell profiles using high-dimensional flow cytometric data. Employing novel algorithms based on Canonical Correspondence Analysis (CCA) and network analysis, our toolkit identifies developmental trajectories and analyses dynamic changes in developing cells. The efficacy of this approach is demonstrated through analysing thymic T-cells from Nr4a3-Tocky mice, which monitor activities downstream of the T-cell receptor (TCR) signal. Further validation was achieved by deleting the proapoptotic gene Bcl2l11 (Bim) in Nr4a3-Tocky mice. This revealed dynamic changes in thymic T-cells during cellular development and negative selection following TCR signalling. Overall, this study establishes a new method for analysing the temporal dynamics of individual developing cells in response to in vivo signalling cues.

Fluorescent Timer proteins, which spontaneously change their emission spectra over time, are valuable tools for analyzing temporal changes in cellular activities at the single-cell level. Traditional analysis of Fluorescent Timer data has mostly relied on conventional flow cytometric methods, which lacks the sophistication needed for detailed quantitative analysis. Recently, we developed the Timer-of-Cell-Kinetics-and-Activity (Tocky) tools, employing transgenic reporter systems using an mCherry mutant Timer protein, Fast-FT, to analyze these changes, implementing data preprocessing methods for Timer fluorescence. Despite this advancement, the computational implementation of effective quantitative analysis methods for Fluorescent Timer data has been lacking. In this study, we introduce rigorous algorithms for a data categorization method, designated as the Tocky locus approach, which uses normalized and trigonometric-transformed Fluorescent Timer data, suitable for quantitative analysis. We found that the five-locus approach, which categorizes Timer fluorescence into five categories, optimally captures the dynamics of Timer profiles, thus enabling effective quantitative statistical analysis and biological interpretation. The current study has developed comprehensive tools for data categorization, visualization, and statistical analysis optimized to Tocky Locus data. These algorithms have been implemented in the TockyLocus package, a new R package designed to facilitate the quantitative analysis of Timer fluorescence data. This toolkit is expected to significantly enhance the utility of experimental systems using Fluorescent Timer proteins, particularly the Tocky tools, by standardizing methods for analyzing dynamic cellular events and responses.

Background: Fluorescent Timer proteins, which display time-dependent changes in their emission spectra, are invaluable for analyzing the temporal dynamics of cellular events at the single-cell level. We previously developed the Timer-of-cell-kinetics-and-activity (Tocky) tools, utilizing a specific Timer protein, Fast-FT, to monitor temporal changes in cellular activities. Despite their potential, the analysis of Timer fluorescence in flow cytometry is frequently compromised by variability in instrument settings and the absence of standardized preprocessing methods. The development and implementation of effective data preprocessing methods remain to be achieved. Results: In this study, we introduce the R package that automates the data preprocessing of Timer fluorescence data from flow cytometry experiments for quantitative analysis at single-cell level. Our aim is to standardize Timer data analysis to enhance reproducibility and accuracy across different experimental setups. The package includes a trigonometric transformation method to elucidate the dynamics of Fluorescent Timer proteins. We have identified the normalization of immature and mature Timer fluorescence data as essential for robust analysis, clarifying how this normalization affects the analysis of Timer maturation. These preprocessing methods are all encapsulated within the TockyPrep R package. Conclusions: TockyPrep is available for distribution via GitHub at this https URL, providing tools for data preprocessing and basic visualization of Timer fluorescence data. This toolkit is expected to enhance the utility of experimental systems utilizing Fluorescent Timer proteins, including the Tocky tools.

Advancements in cytometry technologies have led to a remarkable increase in the number of markers that can be analyzed simultaneously, presenting significant challenges in data analysis. Traditional approaches, such as dimensional reduction techniques and computational clustering, although popular, often face reproducibility challenges due to their heavy reliance on inherent data structures, preventing direct translation of their outputs into gating strategies to be used in downstream experiments. Here we propose the novel Gating Tree methodology, a pathfinding approach that investigates the multidimensional data landscape to unravel group-specific features without the use of dimensional reduction. This method employs novel measures, including enrichment scores and gating entropy, to effectively identify group-specific features within high-dimensional cytometric datasets. Our analysis, applied to both simulated and real cytometric datasets, demonstrates that the Gating Tree not only identifies group-specific features comprehensively but also produces outputs that are immediately usable as gating strategies for unequivocally identifying cell populations. In conclusion, the Gating Tree facilitates a comprehensive analysis of the multidimensional data landscape and provides experimentalists with practical, successive gating strategies that enhance cross-experimental comparisons and downstream analyses such as flow cytometric sorting.

Despite extensive regulatory T cell (Treg) research, fundamental questions on in vivo dynamics remain to be answered. The current study aims to dissect several interwoven concepts in Treg biology, highlighting the ‘self-reactivity’ of Treg and their counterparts, namely naturally-arising memory-phenotype T-cells, as a key mechanism to be exploited by a human retroviral infection. We propose the novel key concept, Periodic T cell receptor (TCR)-signalled T-cells, capturing self-reactivity in a quantifiable manner using the Nr4a3-Timer-of-cell-kinetics-and-activity (Tocky) technology. Periodic and brief TCR signals in self-reactive T-cells contrast with acute TCR signals during inflammation. Thus, we propose a new two-axis model for T-cell activation by the two types of TCR signals or antigen recognition, elucidating how Foxp3 expression and acute TCR signals actively regulate Periodic TCR-signalled T-cells. Next, we highlight an underappreciated branch of immunological research on Human T-cell Leukemia Virus type 1 (HTLV-1) that precedes Treg studies, illuminating the missing link between the viral infection, CD25, and Foxp3. Based on evidence by single-cell analysis, we show how the viral infection exploits the regulatory mechanisms for T-cell activation and suggests a potential role of periodic TCR signalling in infection and malignant transformation. In conclusion, the new perspectives and models in this study provide a working framework for investigating Treg within the self-reactive T-cell spectrum, expected to advance understanding of HTLV-1 infection, cancer, and immunotherapy strategies for these conditions.

Summary The transcription factor Foxp3 controls the differentiation and function of regulatory T-cells (Treg). Studies in the past decades identified numerous Foxp3-interacting protein partners. However, it is still not clear how Foxp3 produces the Treg-type transcriptomic landscape through cooperating with its partners. Here I show the current understanding of how Foxp3 transcription factor complexes regulate the differentiation, maintenance and functional maturation of Treg. Importantly, T-cell receptor (TCR) signalling plays central roles in Treg differentiation and Foxp3-mediated gene regulation. Differentiating Treg will have recognized their cognate antigens and received TCR signals before initiating Foxp3 transcription, which is triggered by TCR-induced transcription factors including NFAT, AP-1 and NF-κB. Once expressed, Foxp3 seizes TCR signal-induced transcriptional and epigenetic mechanisms through interacting with AML1/Runx1 and NFAT. Thus, Foxp3 modifies gene expression dynamics of TCR-induced genes, which constitute cardinal mechanisms for Treg-mediated immune suppression. Next, I discuss the following key topics, proposing new mechanistic models for Foxp3-mediated gene regulation: (i) how Foxp3 transcription is induced and maintained by the Foxp3-inducing enhanceosome and the Foxp3 autoregulatory transcription factor complex; (ii) molecular mechanisms for effector Treg differentiation (i.e. Treg maturation); (iii) how Foxp3 activates or represses its target genes through recruiting coactivators and corepressors; (iv) the ‘decision-making’ Foxp3-containing transcription factor complex for Th17 and Treg differentiation; and (v) the roles of post-translational modification in Foxp3 regulation. Thus, this article provides cutting-edge understanding of molecular biology of Foxp3 and Treg, integrating findings by biochemical and genomic studies.

Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T‐cell responses. Using Foxp3‐Timer of cell kinetics and activity (Tocky) mice, which report real‐time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA‐4 and effector Treg signature genes. Using RNA‐seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti‐TNFRII antibody, and high‐frequency Foxp3 expressors are targeted by anti‐OX40 antibody. Collectively, our study dissects time‐dependent mechanisms behind Foxp3‐driven T‐cell regulation and establishes the Foxp3‐Tocky system as a tool to investigate the mechanisms behind T‐cell immunotherapies.

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.

Thymus-derived regulatory T cells (Tregs) are considered to be a distinct T-cell lineage that is genetically programmed and specialised for immunosuppression. This perspective is based on the key evidence that CD25+ Tregs emigrate to neonatal spleen a few days later than other T cells and that thymectomy of 3-day-old mice depletes Tregs only, causing autoimmune diseases. Although widely believed, the evidence has never been reproduced as originally reported, and some studies indicate that Tregs exist in neonates. Thus we examine the consequences of the controversial evidence, revisit the fundamental issues of Tregs and thereby reveal the overlooked relationship of T-cell activation and Foxp3-mediated control of the T-cell system. Here we provide a new model of Tregs and Foxp3, a feedback control perspective, which views Tregs as a component of the system that controls T-cell activation, rather than as a distinct genetically programmed lineage. This perspective provides new insights into the roles of self-reactivity, T cell–antigen-presenting cell interaction and T-cell activation in Foxp3-mediated immune regulation.

CD4+ T cells that express the transcription factor FOXP3 (FOXP3+ T cells) are commonly regarded as immunosuppressive regulatory T cells (Tregs). FOXP3+ T cells are reported to be increased in tumor-bearing patients or animals and are considered to suppress antitumor immunity, but the evidence is often contradictory. In addition, accumulating evidence indicates that FOXP3 is induced by antigenic stimulation and that some non-Treg FOXP3+ T cells, especially memory-phenotype FOXP3low cells, produce proinflammatory cytokines. Accordingly, the subclassification of FOXP3+ T cells is fundamental for revealing the significance of FOXP3+ T cells in tumor immunity, but the arbitrariness and complexity of manual gating have complicated the issue. In this article, we report a computational method to automatically identify and classify FOXP3+ T cells into subsets using clustering algorithms. By analyzing flow cytometric data of melanoma patients, the proposed method showed that the FOXP3+ subpopulation that had relatively high FOXP3, CD45RO, and CD25 expressions was increased in melanoma patients, whereas manual gating did not produce significant results on the FOXP3+ subpopulations. Interestingly, the computationally identified FOXP3+ subpopulation included not only classical FOXP3high Tregs, but also memory-phenotype FOXP3low cells by manual gating. Furthermore, the proposed method successfully analyzed an independent data set, showing that the same FOXP3+ subpopulation was increased in melanoma patients, validating the method. Collectively, the proposed method successfully captured an important feature of melanoma without relying on the existing criteria of FOXP3+ T cells, revealing a hidden association between the T cell profile and melanoma, and providing new insights into FOXP3+ T cells and Tregs.

BACKGROUND: Currently, in the era of post-genomics, immunology is facing a challenging problem to translate mutant phenotypes into gene functions based on high-throughput data, while taking into account the classifications and functions of immune cells, which requires new methods. RESULTS: Here we propose a novel application of a multidimensional analysis, Canonical Correspondence Analysis (CCA), to reveal the molecular characteristics of undefined cells in terms of cellular differentiation programmes by analysing two transcriptomic datasets. Using two independent datasets, whether RNA-seq or microarray data, CCA successfully visualised the cross-level relationships between genes, cells, and differentiation programmes, and thereby identified the immunological features of mutant cells (Gata3-KO T cells and Stat3-KO T cells) in a data-oriented manner. With a new concept, differentiation variable, CCA provides an automatic classification of cell samples, which had a high sensitivity and a comparable performance to other classification methods. In addition, we elaborate how CCA results can be interpreted, and reveal the features of CCA in comparison with other visualisation techniques. CONCLUSIONS: CCA is a visualisation tool with a classification ability to reveal the cross-level relationships of genes, cells and differentiation programmes. This can be used for characterising the functional defect of cells of interest (e.g. mutant cells) in the context of cellular differentiation. The proposed approach fits with common hypothesis-oriented studies in immunology, and can be used for a wide range of molecular and genomic studies on cellular differentiation mechanisms.

The understanding of pathological processes is based on the comparison between physiological and pathological conditions, and transcriptomic analysis has been extensively applied to various diseases for this purpose. However, the way in which the transcriptomic data of pathological cells relate to the transcriptomes of normal cellular counterparts has not been fully explored, and may provide new and unbiased insights into the mechanisms of these diseases. To achieve this, it is necessary to develop a method to simultaneously analyse components across different levels, namely genes, normal cells, and diseases. Here we propose a multidimensional method that visualises the cross-level relationships between these components at three different levels based on transcriptomic data of physiological and pathological processes, by adapting Canonical Correspondence Analysis, which was developed in ecology and sociology, to microarray data (CCA on Microarray data, CCAM). Using CCAM, we have analysed transcriptomes of haematological disorders and those of normal haematopoietic cell differentiation. First, by analysing leukaemia data, CCAM successfully visualised known relationships between leukaemia subtypes and cellular differentiation, and their characteristic genes, which confirmed the relevance of CCAM. Next, by analysing transcriptomes of myelodysplastic syndromes (MDS), we have shown that CCAM was effective in both generating and testing hypotheses. CCAM showed that among MDS patients, high-risk patients had transcriptomes that were more similar to those of both haematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors (MEP) than low-risk patients, and provided a prognostic model. Collectively, CCAM reveals hidden relationships between pathological and physiological processes and gene expression, providing meaningful clinical insights into haematological diseases, and these could not be revealed by other univariate and multivariate methods. Furthermore, CCAM was effective in identifying candidate genes that are correlated with cellular phenotypes of interest. We expect that CCAM will benefit a wide range of medical fields.