Age-related accumulation of ploidy changes is associated with decreased expression of genes controlling chromosome segregation and cohesin functions. To determine the consequences of whole chromosome instability (W-CIN) we down-regulated the spindle assembly checkpoint component BUB1 and the mitotic cohesin SMC1A, and used four-color-interphase-FISH coupled with BrdU incorporation and analyses of senescence features to reveal the fate of W-CIN cells. We observed significant correlations between levels of not-diploid cells and senescence-associated features (SAFs). W-CIN induced DNA double strand breaks and elevated oxidative stress, but caused low apoptosis. SAFs of W-CIN cells were remarkably similar to those induced by replicative senescence but occurred in only 13 days versus 4 months. Cultures enriched with not-diploid cells acquired a senescence-associated secretory phenotype (SASP) characterized by IL1B, CXCL8, CCL2, TNF, CCL27 and other pro-inflammatory factors including a novel SASP component CLEC11A. These findings suggest that W-CIN triggers premature senescence, presumably to prevent the propagation of cells with an abnormal DNA content. Cells deviating from diploidy have the ability to communicate with their microenvironment by secretion of an array of signaling factors. Our results suggest that aneuploid cells that accumulate during aging in some mammalian tissues potentially contribute to age-related pathologies and inflammation through SASP secretion.

The significant number of people with latent and active tuberculosis infection requires further efforts to develop new vaccines or improve the Bacillus Calmette-Guérin (BCG), which is the only approved vaccine against this disease. In this study, we developed a recombinant fusion protein (PEPf) containing high-density immunodominant epitope sequences from Rv0125, Rv2467, and Rv2672 Mycobacterium tuberculosis (Mtb) proteases that proved immunogenic and used it to develop a recombinant BCG vaccine expressing the fusion protein. After challenging using Mtb, a specific immune response was recalled, resulting in a reduced lung bacterial load with similar protective capabilities to BCG. Thus BCG PEPf failed to increase the protection conferred by BCG. The PEPf was combined with Advax4 adjuvant and tested as a subunit vaccine using a prime-boost strategy. PEPf + Advax4 significantly improved protection after Mtb challenge, with a reduction in bacterial load in the lungs. Our results confirm that Mtb proteases can be used to develop vaccines against tuberculosis and that the use of the recombinant PEPf subunit protein following a prime-boost regimen is a promising strategy to improve BCG immunity.

High density cultures of the GMP compatible NK-92 natural killer cell line release heterogeneous extracellular particles (EP), notably extracellular vesicles (EV) and non-vesicular extracellular particles (NVEP). The NVEP include a small proportion of supramolecular attack particles (SMAPs), the direct cytotoxic potential of which suggests therapeutic promise, yet scalable enrichment and functional evaluation alongside other extracellular particles (EPs) have been lacking. Here, we develop a high-resolution serial size-exclusion liquid chromatography (SELC) workflow that resolves EPs into 4 fractions F1-F4, in which EV are enriched in F2 and SMAPs are enriched in F3. Multi-modal characterization using Nanoparticle Tracking Analysis (NTA), Nano Flow Cytometry (Nano FCM), Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRFM) and Proteomics) demonstrated that F2 NK-92 EVs are ∼130–150 nm particles enriched for complement proteins, whereas SMAPs (F3) are 100–110 nm particles enriched for cytotoxic proteins (PRF1, GZMB) and SMAP shell components (e.g., THBS1, THBS4). Functionally, Ca ²⁺ -stabilized SMAPs (F3) trigger caspase-3–dependent apoptosis in tumor cell lines with robust dose responses, while F2 lacks direct cytotoxicity in vitro. Ex vivo, F3 SMAPs kill patient-derived breast cancer and chronic lymphocytic leukaemia cells (CLL) in a concentration-dependent manner. In NSG mice, intra-tumoral treatment with SMAPs (F3) restrains the growth of aggressive B16F10 melanoma and PANC-1 pancreatic cancer, confirming in vivo functionality. These results establish a scalable method to isolate and compare NK-derived particle classes and provide a foundation for targeted SMAP engineering as a promising approach for treatment of solid tumours. Significance Statement: Cell therapies face barriers in solid tumours, including lack of entry and suppression. NK cells release extracellular particles (EPs), including supramolecular attack particles (SMAPs), that can traffic into tumour tissue. We define a practical, scalable chromatography process that resolves NK-derived SMAPs from other EPs and demonstrate that SMAPs induce caspase mediated apoptosis in vitro and engage in cytotoxic lymphocyte independent anti-tumour activity in vivo . These insights open a route to engineering and testing SMAP-based therapeutics to overcome limitations of whole-cell approaches in solid cancers.

Tumor necrosis factor (TNF) orchestrates immune responses but can also drive inflammation-associated tissue damage. However, the mechanisms governing tissue tolerance to TNF remain poorly understood. Here, we reveal that TNF receptor 1 (TNFR1) abundance is regulated by two upstream open reading frames (uORFs) in the 5′ untranslated region of TNFRSF1A and demonstrate that this is a key determinant of TNF tolerance. uORF2 dominantly limits TNFR1 translation, and its disruption increases TNFR1 levels, leading to excessive TNF-induced gene activation and cell death in cell culture. By contrast, uORF1 dynamically regulates TNFR1 levels in response to inflammatory and stress signals. In mice, uORF2 protects against TNF-driven systemic inflammatory response syndrome and liver pathology. We additionally report that the translation of other immune receptor messenger RNAs, including TLR4 , IFNAR1 , and IFNGR2 , is also controlled by uORFs. Thus, regulation of TNFR1 levels and possibly of other immune receptors emerges as a mechanism safeguarding against excessive immune responses and tissue damage.

Adoptive T-cell therapy (ACT) has demonstrated marked clinical benefit in hematological malignancies but has limited efficacy against solid tumors. Cancer vaccines can improve ACT, but the mechanisms underlying this combination are poorly understood. Here, we combined ACT with chimpanzee adenovirus ChAdOx1 (ChAd) and modified vaccinia Ankara (MVA) encoding tumor antigens. Whereas PD1 blockade failed to improve ACT, the combination with ChAd/MVA vaccination drove durable regression of established tumors, correlating with expansion of tumor-reactive T cells in lymphoid organs and increased tumor infiltration by T cells. Mechanistically, ChAd priming induced CXCR3 ligands (CXCL10 and CXCL9) in the vaccine-draining lymph node, thereby recruiting transferred T cells to this node independently of cognate antigen. CXCR3 expression on the transferred T cells, but not on host cells, was required for the anti-tumor effect of the combination therapy and favored T-cell accumulation in the vaccine-draining lymph node and the tumor. These findings identify CXCR3 on the transferred T cells as a determinant of ACT plus vaccination synergy and provide a rationale for combining viral-vector vaccines with TCR-T therapies, and for selecting or engineering T cells to retain CXCR3 expression. One Sentence Summary: Viral vector vaccines synergize with TCR-T cell therapy to control solid tumors in a CXCR3-dependent manner.

Glioblastoma is a lethal brain tumor that is unresponsive to current cancer immunotherapeutic approaches, including immune checkpoint blockade (ICB). This suggests that initial priming of T cells, rather than their expansion and licensing as effectors, is a restricting feature in this tumor setting. To overcome the limited initiation of CD8 ⁺ T cell responses, we employed a strong heterologous prime-boost vaccination with the simian adenovirus ChAdOx1 and poxvirus modified vaccinia Ankara (MVA). Vaccination conferred therapeutic efficacy against orthotopic, immune checkpoint-blockade (ICB)-refractory SB28 murine glioblastoma. Vaccination was effective against both the murine tumor antigen, P1A, and a newly identified glioblastoma-associated antigen, Gpr149. Additional treatment with ICB provided no additional benefit. Systemic ChAdOx1/MVA vaccination induced robust infiltration of antigen-specific T cells in tumor-challenged brains, the majority of which exhibited a CD103 ⁺ CD69 ⁺ CD8 ⁺ tissue-resident memory (TRM)-like phenotype. These cells were polyfunctional, durable in brains with sustained tumor control, and mediated tissue-specific immunological memory. Moreover, intracranial adoptive transfer of glioblastoma-derived antigen-specific TRM-like cells was sufficient to protect naïve recipients from subsequent orthotopic tumor challenge. Together, these findings establish that viral vector vaccination can generate tumor-specific TRM-like cells that mediate effective anti-glioblastoma immunity, providing a rationale for clinical evaluation of ChAdOx1/MVA-based strategies in glioblastoma.