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The growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations. PHLEX was developed using imaging mass cytometry (IMC) in the TRACERx study, validated using published Co-detection by indexing (CODEX), IMC and orthogonal data and benchmarked against state-of-the-art approaches. We evaluated its use on different tissue types, tissue fixation conditions, image sizes and antibody panels. As PHLEX is an automated and containerised Nextflow pipeline, manual assessment, programming skills or pathology expertise are not essential. PHLEX offers an end-to-end solution in a growing field of highly multiplexed data and provides clinically relevant insights. Imaging dozens of proteins in situ and at large scales increases the complexity of data analysis. Here, the authors develop an end-to-end solution for multiplexed image analysis that facilitates data interpretation for clinically relevant insights. [ABSTRACT FROM AUTHOR]

Introduction: The autoimmune response in type 1 diabetes (T1D), in which the beta cells expressing aberrant or modified proteins are killed, resembles an effective antitumor response. Defective ribosomal protein products in tumors are targets of the anti-tumor immune response that is unleashed by immune checkpoint inhibitor (ICI) treatment in cancer patients. We recently described a defective ribosomal product of the insulin gene (INS-DRiP) that is expressed in stressed beta cells and targeted by diabetogenic T cells. T1D patient-derived INSDRiP specific T cells can kill beta cells and are present in the insulitic lesion. T cells reactive to INS-DRiP epitopes are part of the normal T cell repertoire and are believed to be kept in check by immune regulation without causing autoimmunity. Method: T cell autoreactivity was tested using a combinatorial HLA multimer technology measuring a range of epitopes of islet autoantigens and neoantigen INS-DRiP. INS-DRiP expression in human pancreas and insulinoma sections was tested by immunohistochemistry. Results: Here we report the induction of islet autoimmunity to INS-DRiP and diabetes after ICI treatment and successful tumor remission. Following ICI treatment, T cells of the cancer patient were primed against INS-DRiP among other diabetogenic antigens, while there was no sign of autoimmunity to this neoantigen before ICI treatment. Next, we demonstrated the expression of INSDRiP as neoantigen in both pancreatic islets and insulinoma by staining with a monoclonal antibody to INS-DRiP. Discussion: These results bridge cancer and T1D as two sides of the same coin and point to neoantigen expression in normal islets and insulinoma that may serve as target of both islet autoimmunity and tumor-related autoimmunity. [ABSTRACT FROM AUTHOR]

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The spatial architecture of the lymphoid tissue in follicular lymphoma (FL) presents unique challenges to studying its immune microenvironment. We investigated the spatial interplay of T cells, macrophages, myeloid cells and natural killer T cells using multispectral immunofluorescence images of diagnostic biopsies of 32 patients. A deep learning‐based image analysis pipeline was tailored to the needs of follicular lymphoma spatial histology research, enabling the identification of different immune cells within and outside neoplastic follicles. We analyzed the density and spatial co‐localization of immune cells in the inter‐follicular and intra‐follicular regions of follicular lymphoma. Low inter‐follicular density of CD8+FOXP3+ cells and co‐localization of CD8+FOXP3+ with CD4+CD8+ cells were significantly associated with relapse (p = 0.0057 and p = 0.0019, respectively) and shorter time to progression after first‐line treatment (Logrank p = 0.0097 and log‐rank p = 0.0093, respectively). A low inter‐follicular density of CD8+FOXP3+ cells is associated with increased risk of relapse independent of follicular lymphoma international prognostic index (FLIPI) (p = 0.038, Hazard ratio (HR) = 0.42 [0.19, 0.95], but not independent of co‐localization of CD8+FOXP3+ with CD4+CD8+ cells (p = 0.43). Co‐localization of CD8+FOXP3+ with CD4+CD8+ cells is predictors of time to relapse independent of the FLIPI score and density of CD8+FOXP3+ cells (p = 0.027, HR = 0.0019 [7.19 × 10−6, 0.49], This suggests a potential role of inter‐follicular CD8+FOXP3+ and CD4+CD8+ cells in the disease progression of FL, warranting further validation on larger patient cohorts. [ABSTRACT FROM AUTHOR]

Background: Urushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response. Results: Electron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562). Conclusion: Our data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols. [ABSTRACT FROM AUTHOR]

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (T regs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive T regs during primary tumor growth. Tumor-educated T regs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as T reg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that T regs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis. In line, an increased T reg /NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent. [Display omitted] • Mammary tumorigenesis drives systemic expansion of highly immunosuppressive T regs • Tumor-educated T regs promote lymph node (LN) metastasis but not lung metastasis • T regs promote LN metastasis by local suppression of NK cells in the LN niche • LNs of breast cancer patients have elevated T reg and reduced NK cell accumulation Kos et al. demonstrate that breast cancer development is accompanied by systemic expansion of immunosuppressive regulatory T cells. The phenotype and function of tumor-educated T regs are uniquely shaped by the local tissue environment. Consequently, tumor-educated T regs impair local NK cell activity to enhance metastasis to lymph nodes, but not lungs. [ABSTRACT FROM AUTHOR]

Quiescence is a state of cell cycle arrest, allowing cancer cells to evade anti-proliferative cancer therapies. Quiescent cancer stem cells are thought to be responsible for treatment resistance in glioblastoma, an aggressive brain cancer with poor patient outcomes. However, the regulation of quiescence in glioblastoma cells involves a myriad of intrinsic and extrinsic mechanisms that are not fully understood. In this review, we synthesise the literature on quiescence regulatory mechanisms in the context of glioblastoma and propose an ecological perspective to stemness-like phenotypes anchored to the contemporary concepts of niche theory. From this perspective, the cell cycle regulation is multiscale and multidimensional, where the niche dimensions extend to extrinsic variables in the tumour microenvironment that shape cell fate. Within this conceptual framework and powered by ecological niche modelling, the discovery of microenvironmental variables related to hypoxia and mechanosignalling that modulate proliferative plasticity and intratumor immune activity may open new avenues for therapeutic targeting of emerging biological vulnerabilities in glioblastoma. [ABSTRACT FROM AUTHOR]