The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. β-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.
Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation.
The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.
TCR-modified T-cell therapy (TCR-T) is emerging as a treatment option for solid tumours containing mutant KRAS,1–4 an area of great unmet medical need. We have developed a high-precision cell-based analytical platform for scalable TCR-T product development. Herein, we describe use of the platform to perform target quantification and to generate a clinical candidate TCR recognizing the 10mer peptide derived from the KRAS G12V mutant form presented in HLA-A*11:01.
Methods
HLA-presented peptides were quantified using mass spectrometry in extracts from mono-allelic engineered antigen presenting cell lines (eAPC) ectopically expressing KRAS G12V and separately in cancer cell lines natively expressing KRAS G12V. TCRs were isolated via paired single cell sequencing following eAPC-stimulated outgrowth of HLA-matched naïve CD8 donor cells. TCRs were characterised in engineered TCR-presenting cells (eTPC) with TCR-linked reporters. Cross-reactivity and allo-reactivity was tested via contact assays using libraries of eAPCs containing epitope and/or HLA variants and eTPC reporters. TCR-T cells were constructed via gene editing of primary donor CD8 cells.
Results
A candidate TCR recognizing a 10mer peptide derived from G12V KRAS in HLA-A*11:01 was developed. This TCR recognizes the mutant epitope but not the corresponding wild-type epitope both via peptide loading (mutant EC501 nM; wild-type not recognised) and via ectopic KRAS-G12V in eAPCs as well as in cancer cell lines expressing KRAS G12V. Allo-reactivity was assessed via a panel of 82 different mono-allelic eAPCs, and cross-reactivity via a library of 200 KRAS G12V peptide variant eAPCs, showing no apparent cross-reactivity or allo-reactivity risk. Primary CD8 cells were transduced by insertional gene editing, generating a TCR-product with high levels of surface TCR and potent cytotoxic action.
Conclusions
The TCR-T product is now being advanced to clinical trial and is designed to be the first entry in a broader program covering multiple HLA restrictions and mutant KRAS epitopes.
References
Leidner R, Silva NS, Huang H, Sprott D, Zheng C, Shih Y-P, Leung A, Payne R, Sutcliffe K, Cramer J, Rosenberg S, Fox B, Urba W, Tran E. N Engl J Med 2022;386:2112–2119. Poole P, Karuppiah V, Hartt A, Haidar J, Moureau S, Dobrzycki T, Hayes C, Rowley C, Dias J, Harper H, Barnbrook K, Hock M, Coles C, Yang W, Aleksic M, Bence Lin A, Robinson R, Dukes J, Liddy N, Van der Kamp M, Plowman G, Vuidepot A, Cole D, Whale A, Chillakuri C. Nature Commun 2022;13:5333. Choi J, Goulding S, Conn B, McGann C, Dietze J, Kohler J, Lenkala D, Boudot A, Rothenberg D,Turcott P, Srouji J, Foley K, Rooney M, van Buuren M, Gaynor R, Abelin J, Addona A, Juneja V. Reports Methods 2021;1:100084. Bear A, Blanchard T, Cesare J, Ford M, Richman L, Xu C, Baroja M, McCuaig S, Costeas C, Gabunia K, Scholler J, Posey A, O'Hara M, Smole A, Powell D, Garcia B, Vonderheide R, Linette G, Carreno B. Nat Commun 2021;12:4365–4365.
Ethics Approval
Collection of healthy donor peripheral blood and leukapheresis products was undertaken with written informed consent, under ethical approvals 2019–02481/2020–03585 and 2019–02482 granted by the Swedish Ethical Review Authority
The aim of this work is to register serial in-vivo confocal microscopy images of zebrafish to enable accurate cell tracking on corresponding fluorescence images. The following problem arises during acquisition; the zebrafish tail may undergoe a series of movement and non-linear deformations, which if not corrected, adds to the motion of leukocytes being tracked. This makes it difficult to accurately assess their motion. We developed a correlation based, local affine image matching method, which is well suited to the textured DIC images of the anatomy of the zebrafish and enables accurate and efficient tracking of image regions over successive frames. Experimental results of the serial registration and tracking demonstrate its accuracy in estimating local affine motions in ze-brafish sequences.
MNK (Menkes copper-translocating P-type ATPase, or the Menkes protein; ATP7A) plays a key role in regulating copper homoeostasis in humans. MNK has been shown to have a dual role in the cell: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper from the cell. These roles can be achieved through copper-regulated trafficking of MNK. It has previously been shown to undergo trafficking from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations, and to be endocytosed from the plasma membrane to the trans-Golgi network upon the removal of elevated copper. However, the fundamental question as to whether copper influences trafficking of MNK to or from the plasma membrane remained unanswered. In this study we utilized various methods of cell-surface biotinylation to attempt to resolve this issue. These studies suggest that copper induces trafficking of MNK to the plasma membrane but does not affect its rate of internalization from the plasma membrane. We also found that only a specific pool of MNK can traffic to the plasma membrane in response to elevated copper. Significantly, copper appeared to divert MNK into a fast-recycling pool and prevented it from recycling to the Golgi compartment, thus maintaining a high level of MNK in the proximity of the plasma membrane. These findings shed new light on the cell biology of MNK and the mechanism of copper homoeostasis in general.
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