Biology subjects

Roelofs, D.

Publications and source records attributed to Roelofs, D..

5 recordsLinked to original sources

bioRxiv2025

Adoptive cell therapy using T cell receptors equipped with ICOS yields durable anti-tumor response

Treatment with adoptively transferred T cells is challenged by limited longevity of therapeutic cells within tumors. To enhance the durability of anti-tumor T cell products, we have created T cell receptors (TCRs) with built-in co-stimulatory molecules. We observed that TCRs coupled to ICOS mediated exceptionally long-term responses including delay of tumor recurrence and cures in a mouse tumor model. TCR:ICOS T cells showed enhanced and antigen-specific production of inflammatory cytokines and resistance to exhaustion. Genetic ablation of ICOS-mediated activation of the PI3K-NF{kappa}B pathway neutralized the long-term anti-tumor effects. To translate TCR:ICOS to human T cells, we identified a single amino acid change in the cytosolic tail which was necessary for functional surface expression. Notably, the optimized receptor sustained performance of human T cells upon repeated stimulation across multiple antigen specificities. Collectively, we present a novel and uniformly applicable TCR:ICOS format that supports fitter T cell products for adoptive cell therapy. HighlightsNewly designed co-stimulatory TCR, with extracellular TCR-V and C domains coupled to CD28 transmembrane domain, and ICOS and CD3{varepsilon} intracellular domains (in short TCR:ICOS) provides:[tpltrtarr] durable anti-tumor response and T cell persistence in mouse model [tpltrtarr]inflammatory T cell phenotype and resistance to T cell exhaustion [tpltrtarr]effects via PI3K and NF{kappa}B activation [tpltrtarr]translation to human T cells upon single amino acid mutation in TCR:ICOS tail [tpltrtarr]extension to multiple clinically relevant TCRs while preserving prolonged T cell fitness O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/682056v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1ff752borg.highwire.dtl.DTLVardef@6579d9org.highwire.dtl.DTLVardef@22bdc4org.highwire.dtl.DTLVardef@d8c445_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2Apro and 3Cpro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro. Viruses with an inactive 2Apro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2Apro, but not 3Cpro, is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-/{beta} (IFN-/{beta}) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2Apro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2Apro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication.

microbiology↗

bioRxiv2024

Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

The combination of ultra-long Oxford Nanopore (ONT) sequencing reads with long, accurate PacBio HiFi reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely-studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the ultra-long reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and has the potential to provide a single-instrument solution for the reconstruction of complete genomes.

bioinformatics↗

bioRxiv2024

TCR-engineered T-cells directed against Ropporin-1 constitute a safe and effective treatment for triple-negative breast cancer in near-clinical models

Triple-negative breast cancer (TNBC) shows an urgent need for new therapies. We discovered Ropporin-1 (ROPN1) as a target to treat TNBC with T-cells. ROPN1 showed high and homogenous expression in 90% of primary and metastatic TNBC but not in healthy tissues. HLA-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCRs) from naive repertoires. Following gene introduction into T-cells and stringent selection, we retrieved a highly specific TCR directed against the epitope FLYTYIAKV that did not recognize non-cognate epitopes from alternative source proteins. Notably, this TCR mediated killing of three-dimensional tumoroids in vitro and tumor cells in vivo and outperformed standard-of-care drugs. Finally, the T-cell product expressing this TCR and manufactured using a clinical protocol fulfilled standard safety and efficacy assays. Collectively, we have identified and preclinically validated ROPN1 as a target and anti-ROPN1 TCR T-cells as a treatment for the vast majority of TNBC patients.

immunology↗

bioRxiv2019

Blastodermal cuticle formation contributes to desiccation resistance in springtail eggs: eco-evolutionary implications for insect terrestrialization

Land colonization was a major event in the history of life. Among animals, insects exerted a staggering terrestrialization success, due to traits usually associated with post-embryonic life stages, while the egg stage has been largely overlooked in comparative studies. In many insects, after blastoderm differentiation, the extraembryonic serosal tissue wraps the embryo and synthesizes the serosal cuticle, an extracellular matrix that lies beneath the eggshell and protects the egg against water loss. In contrast, in non-insect hexapods such as springtails (Collembola) the early blastodermal cells synthesize a blastodermal cuticle. Here, we investigate the relationship between blastodermal cuticle formation and egg resistance to desiccation in the springtails Orchesella cincta and Folsomia candida, two species with different oviposition environments and developmental rates. The blastodermal cuticle becomes externally visible in O. cincta and F. candida at 22 and 29% of embryogenesis, respectively. To contextualize, we describe the stages of springtail embryogenesis, exemplified by F. candida. Our physiological assays then showed that blastodermal cuticle formation coincides with an increase in egg viability in a dry environment, significantly contributing to hatching success. However, protection differs between species: while O. cincta eggs survive at least 2 hours outside a humid environment, the survival period recorded for F. candida eggs is only 15 minutes, which correlates with this species’ requirement for humid microhabitats. We suggest that the formation of this cuticle protects the eggs, constituting an ancestral trait among hexapods that predated and facilitated the process of terrestrialization that occurred during insect evolution.Research HighlightsThe formation of the blastodermal cuticle produced during early embryogenesis coincides with a higher protection against water loss in springtail (Collembola) eggs.Orchesella cincta eggs are more resistant to drought than Folsomia candida ones.The formation of a protective egg cuticle would be an ancestral trait among hexapods that facilitated their process of terrestrialization.Graphical AbstractGraphical Abstract legend: Eggs when laid uptake water but are also prone to water loss. Late eggs acquire some protection against water loss, but at different levels, depending on the species.Competing Interest StatementThe authors have declared no competing interest.View Full Text

zoology↗