Marta Grzelak

The human public T-cell receptor (TCR) repertoire provides a shared immunological framework across individuals, yet whether it possesses a conserved higher-order structural organization and how this organization is affected by lifelong immune ageing remain unknown. Here, we constructed a structural atlas of the human public TCRβ repertoire from 1.33 million unique public CDR3β sequences derived from healthy Adults (80 years). The atlas comprised 64 reproducible Structural Sequence Modules (SSMs) representing sequence-coherent neighborhoods supported by quantitative model selection and independent topological validation. Projection of individual repertoires onto this common framework demonstrated complete preservation of all SSMs across both cohorts. Although 18 modules exhibited significant differences in unweighted occupancy, the global module hierarchy remained highly conserved, indicating that exceptional longevity is associated primarily with quantitative redistribution among pre-existing structural compartments rather than loss or replacement of repertoire organization. Rarefaction analysis demonstrated that unequal cohort size strongly influenced public-clonotype discovery but had minimal impact on the direction and magnitude of module-level occupancy differences, whereas frequency-weighted analysis indicated that remodeling was not primarily driven by expansion of highly abundant public clonotypes. Susceptibility to remodeling was associated mainly with module size and within-module CDR3β length variability. Module publicness was highly conserved across age groups and associated with coordinated sequence, germline and network properties, while TRBJ usage was substantially more strongly associated with SSM identity than TRBV usage. Finally, curated antigen annotations were non-randomly distributed across SSMs, demonstrating module-level functional biases, although structural proximity did not generally predict similarity in antigen-recognition profiles. Together, these findings identify higher-order structural organization as a robust property of the human public TCRβ repertoire. Exceptional longevity preserves this organization while selectively remodeling the occupancy of pre-existing Structural Sequence Modules, distinguishing age-associated quantitative redistribution from cohort-size effects and clonal expansion. The structural atlas therefore provides a reproducible framework for investigating the organization and remodeling of adaptive immune repertoires across ageing and immune-mediated conditions.

Background. Graves' disease (GD) is an autoimmune thyroid condition driven by thyroid-stimulating hormone receptor (TSHR) autoantibodies (TSI), frequently leading to hyperthyroidism and vision-threatening Graves' orbitopathy (GO) or Thyroid Eye Disease (TED). Despite its prevalence, the biological mechanisms driving disease onset, T-cell-mediated pathogenesis, and treatment resistance remain unclear. Because individual T-cell receptor (TCR) repertoires are highly private, cross-sectional snapshots often obscure these mechanistic and pathogenic signals. Longitudinal immune tracking overcomes this by using intra-individual baselines to track disease initiation, evolution, and therapeutic response. Methods and Results. In a longitudinal tracking study, we followed a patient who developed dry eyes ~7 months postpartum, initially misdiagnosed as routine conjunctivitis. She was subsequently diagnosed with TSI-positive GD. Approximately 10 months later, she developed active, steroid-refractory GO/TED. Normalization of thyroid hormones with thiamazole failed to halt orbital disease progression. Subsequent high-dose pulse corticosteroid therapy failed to control ocular inflammation and induced elevated intraocular pressure. Switching to an anti-IL-6 receptor antagonist (tocilizumab) achieved rapid and complete clinical remission of orbital inflammation. Uniquely, peripheral blood mononuclear cells (PBMCs) had been banked across a 5-year window (2021-2026), enabling continuous longitudinal immune monitoring (osLifetime), spanning pre-onset to post-remission checkpoints. Conclusion. osLifetime provided a real-time view of autoimmune T-cell activity. By monitoring the adaptive immune system across the entire clinical journey, it established a true pre-symptomatic baseline to benchmark disease progression and therapeutic transitions, capturing T-cell remodeling (manifesting as an acute drop in Immune Age alongside rising Inflammaging score), and even distinguishing corticosteroid failure from tocilizumab response. osLifetime turns complex immune dynamics into actionable quantitative signals, especially where routine biochemistry fails to reflect ongoing disease trajectory, paving the way for precision immunomonitoring in GO/TED and beyond.

Injury risk prediction in elite football relies almost exclusively on external load metrics derived from GPS tracking, overlooking the molecular state of the athlete. We monitored 26 male players from FC Barcelona’s first team across the 2025 calendar year, integrating GPS-derived training load with longitudinal blood-based immune monitoring (systemic inflammation and TCR-derived immune age). Immune age acceleration and inflammation were elevated in the 14 days preceding musculoskeletal injuries. A logistic regression model combining external load, inflammation, immune age acceleration, and career injury history reached an overall AUC of 0.678 and a mean per-player AUC of 0.754 (SD 0.146), improving on a GPS-only baseline of 0.541. Applied to 2026 data, the frozen model ranked players who later sustained non-contact musculoskeletal injuries high in the risk distribution. Together, our data suggest multimodal immune monitoring in elite football to reveal the athlete’s internal physiological state, which carries injury-relevant information that external load alone does not capture.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Approximately 90% of colorectal cancer (CRC) deaths are caused by tumors ability to migrate into the adjacent tissues and metastase into distant organs. More than 40 genes have been causally linked to the development of CRC but no mutations have been associated with metastasis yet. To identify molecular basis of CRC metastasis we performed whole-exome and genome-scale transcriptome sequencing of 7 liver metastases along with their matched primary tumours and normal tissue. Multiple, spatially separated fragments of primary tumours were analyzed in each case. Uniformly malignant tissue specimen were selected with macrodissection, for three samples followed with laser microdissection. > 100 sequencing coverage allowed for detection of genetic alterations in subpopulation of tumour cells. Mutations in KRAS, APC, POLE, and PTPRT, previously associated with CRC development, were detected in most patients. Several new associations were identified, including PLXND1, CELSR3, BAHD1 and PNPLA6. We confirm the essential role of inflammation in CRC progression but question the mechanism of matrix metalloproteinases activation described in other work. Comprehensive sequencing data made it possible to associate genome-scale mutation distribution with gene expression patterns. To our knowledge, this is the first work to report such link in CRC metastasis context.

Characterising the hierarchy of mammary epithelial cells (MECs) and how they are regulated during adult development is important for understanding how breast cancer arises. Here we report the use of single-cell RNA sequencing to determine the gene expression profile of MECs across four developmental stages; nulliparous, mid gestation, lactation and post involution. Our analysis of 23,184 cells identifies 15 clusters, few of which could be fully characterised by a single marker gene. We argue instead that the epithelial cells—especially in the luminal compartment—should rather be conceptualised as being part of a continuous spectrum of differentiation. Furthermore, our data support the existence of a common luminal progenitor cell giving rise to intermediate, restricted alveolar and hormone-sensing progenitors. This luminal progenitor compartment undergoes transcriptional changes in response to a full pregnancy, lactation and involution. In summary, our results provide a global, unbiased view of adult mammary gland development.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. Publisher9s Note: There is an Inside Blood Commentary on this article in this issue. Key Points Combined loss of Ssb1/Ssb2 induces rapid lethality due to replication stress–associated loss of hematopoietic stem and progenitor cells. Functionally, loss of Ssb1/Ssb2 activates p53 and IFN pathways, causing enforced cell cycling in quiescent HSPCs and apoptotic cell loss.

Genome-wide mechanisms that coordinate expression of subsets of functionally related genes are largely unknown. Recent studies show that receptor tyrosine kinases and components of signal transduction cascades including the extracellular signal-regulated protein kinase (ERK), once thought to act predominantly in the vicinity of plasma membrane and in the cytoplasm, can be recruited to chromatin encompassing transcribed genes. Genome-wide distribution of these transducers and their relationship to transcribing RNA polymerase II (Pol2) could provide new insights about co-regulation of functionally related gene subsets. Chromatin immunoprecipitations (ChIP) followed by deep sequencing, ChIP-Seq, revealed that genome-wide binding of epidermal growth factor receptor, EGFR and ERK pathway components at EGF-responsive genes was highly correlated with characteristic mitogen-induced Pol2-profile. Endosomes play a role in intracellular trafficking of proteins including their nuclear import. Immunofluorescence revealed that EGF-activated EGFR, MEK1/2 and ERK1/2 co-localize on endosomes. Perturbation of endosome internalization process, through the depletion of AP2M1 protein, resulted in decreased number of the EGFR containing endosomes and inhibition of Pol2, EGFR/ERK recruitment to EGR1 gene. Thus, mitogen-induced co-recruitment of EGFR/ERK components to subsets of genes, a kinase module possibly pre-assembled on endosome to synchronize their nuclear import, could coordinate genome-wide transcriptional events to ensure effective cell proliferation.