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A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Triple-negative breast cancers (TNBCs) exhibit heightened T cell infiltration, contributing to an enhanced response to immune checkpoint blockade (ICB) compared with other subtypes. An immune-rich immune microenvironment correlates with improved prognosis in early and advanced TNBC. Combination chemotherapy and ICB is now the standard of care in early- and late-stage TNBC. Although programmed death ligand-1 (PD-L1) positivity predicts ICB response in advanced stages, its role in early-stage disease remains uncertain. Despite neoadjuvant ICB becoming common in early-stage TNBC, the necessity of adjuvant ICB after surgery remains unclear. Understanding the molecular basis of the immune response in breast cancer is vital for precise biomarkers for ICB and effective combination therapy strategies., Competing Interests: Declaration of interests S.L. receives research funding to institution from Novartis, Bristol Myers Squibb, MSD, Puma Biotechnology, Eli Lilly, Nektar Therapeutics, Astra Zeneca/Daiichi Sankyo, and Seattle Genetics. S.L. has acted as consultant (not compensated) to Seattle Genetics, Novartis, Bristol Myers Squibb, MSD, AstraZeneca/Daiichi Sankyo, Eli Lilly, Pfizer, Gilead Therapeutics, Nektar Therapeutics, PUMA Biotechnologies, and Roche-Genentech. S.L. has acted as a consultant (paid to institution) to Novartis, GlaxoSmithKline, Roche-Genentech, Astra Zeneca/Daiichi Sankyo, Pfizer, Gilead Therapeutics, Seattle Genetics, MSD, Tallac Therapeutics, Eli Lilly, and Bristol Myers Squibb. V.C.R. has received honoraria from AstraZeneca and has received travel and education funding from MSD. R.S. serves on an advisory board and/or has a consultancy role for BMS, Roche, and Owkin. R.S. has received research funding by Roche, Puma, and Merck. R.S. has received travel and congress registration support from Roche, Merck, and AstraZeneca., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

T cells are predominantly produced by the thymus and play a significant role in maintaining our adaptive immune system. Physiological involution of the thymus occurs gradually with age, compromising naive T cell output, which can have severe clinical complications. Also, T cells are utilized as therapeutic agents in cancer immunotherapies. Therefore, there is an increasing need for strategies aimed at generating naive T cells. The majority of in vitro T cell generation studies are performed in two-dimensional (2D) cultures, which ignore the physiological thymic microenvironment and are not scalable; therefore, we applied a new three-dimensional (3D) approach. Here, we use a gelatin-based 3D microgel system for T lineage induction by co-culturing OP9-DL4 cells and mouse fetal-liver-derived hematopoietic stem cells (HSCs). Flow cytometric analysis revealed that microgel co-cultures supported T lineage induction similar to 2D cultures while providing a 3D environment. We also encapsulated mouse embryonic thymic epithelial cells (TECs) within the microgels to provide a defined 3D culture platform. The microgel system supported TEC maintenance and retained their phenotype. Together, these data show that our microgel system has the capacity for TEC maintenance and induction of in vitro T lineage differentiation with potential for scalability.

One of the main consequences of thymus aging is the decrease in naïve T cell output. This condition accelerates at the onset of puberty, and presents as a major clinical complication for cancer patients who require cytoablative therapy. Specifically, the extensive use of chemotherapeutics, such as cyclophosphamide, in such treatments damage thymic structure and eliminate the existing naïve T cell repertoire. The resulting immunodeficiency can lead to increased incidence of opportunistic infections, tumor growth relapse and/or autoimmune diseases, particularly in older patients. Thus, strategies aimed at rejuvenating the aged thymus following chemotherapeutic damage are required. Previous studies have revealed that sex hormone deprivation in male mice is capable of regenerating the thymic microenvironment following chemotherapy treatment, however, further investigation is crucial to identify gender-based differences, and the molecular mechanisms involved during thymus regeneration. Through phenotypic analyzes, we identified gender-specific alterations in thymocytes and thymic epithelial cell (TEC) subsets from the onset of puberty. By middle-age, females presented with a higher number of thymocytes in comparison to males, yet a decrease in their Aire + medullary TEC/thymocyte ratio was observed. This reduction could be associated with an increased risk of autoimmune disease in middle-aged women. Given the concurrent increase in female Aire + cTEC/thymocyte ratio, we proposed that there may be an impediment in Aire + mTEC hi differentiation, and Aire + cTEC hi as its upstream precursor. The regenerative effects of LHRH receptor antagonist, degarelix, on TEC subsets was also less pronounced in middle-aged females compared to males, possibly due to slower progression of thymic involution in the former, which presented with greater TEC hi proportions. Furthermore, following cyclophosphamide treatment, degarelix enhanced thymocyte and mature TEC subset recovery, with faster recovery kinetics observed in females. These events were found to involve both reactivation and proliferation of thymic epithelial progenitor cells. Taken together, the findings from this study portray a relationship between gender disparity and thymus aging, and highlight the potential benefits of LHRH receptor antagonist treatment for thymic regeneration. Further research is required, however, to determine how gender may impact on the mechanisms underpinning these events., (Copyright © 2020 Hun, Wong, Gunawan, Alsharif, Quinn and Chidgey.)

A key feature of immune functional impairment with age is the progressive involution of thymic tissue responsible for naive T cell production. In this study, we identify two major phases of thymic epithelial cell (TEC) loss during aging: a block in mature TEC differentiation from the pool of immature precursors, occurring at the onset of puberty, followed by impaired bipotent TEC progenitor differentiation and depletion of Sca-1 lo cTEC and mTEC lineage-specific precursors. We reveal that an increase in follistatin production by aging TECs contributes to their own demise. TEC loss occurs primarily through the antagonism of activin A signaling, which we show is required for TEC maturation and acts in dissonance to BMP4, which promotes the maintenance of TEC progenitors. These results support a model in which an imbalance of activin A and BMP4 signaling underpins the degeneration of postnatal TEC maintenance during aging, and its reversal enables the transient replenishment of mature TECs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1 eGFP/+ knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1 + cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications.