Biology subjects

Ching, K.

Publications and source records attributed to Ching, K..

6 recordsLinked to original sources

bioRxiv2025

Therapeutic Blockade of Type 2 Cytokines and PD1 Unleashes Anti-Tumor Immunity Through Coordinated Reprogramming of Innate and Adaptive Immune Surveillance

BackgroundCheckpoint inhibitors improve survival in patients with several types of tumors. However, resistance to checkpoint inhibitors creates an opportunity for patients to benefit from novel immunotherapies. The type 2 cytokines IL-4, IL-13 and TSLP have been implicated in suppressing anti-tumor immune responses through T and myeloid cells. Our current study tested whether combined therapeutic blockade of IL-4, IL-13 and TSLP improved anti-tumor immunity alone and in combination with PD1 antagonism. MethodsWe used in vitro experiments with primary cells to identify cell types likely to participate in controlling tumors upon IL-4, IL-13, TSLP and PD1 blockade. Therapeutic blockade in the subcutaneous CT26 model tested in vivo tumor growth inhibition and associated immunological changes. Bioinformatic analysis of human tumor bulk RNA sequencing data probed for survival associations with IL-4/IL-13 and TSLP transcriptional responses. ResultsIn vitro, IL-4 suppressed T cell-mediated tumor growth inhibition and reduced monocyte-derived dendritic cell expression of proteins associated with anti-tumor immunity. In vivo, blocking IL-4, IL-13, TSLP and PD1 improved tumor growth inhibition by creating "hotter" tumors. This was associated with repolarization of CD4 and CD8 T cells and shifts in monocyte, conventional type 1 and type 2 (or monocyte-derived) dendritic cell programs. Transcriptional responses to IL-4/IL-13 and TSLP were associated with poor survival outcomes across patients with several types of cancers. ConclusionTherapeutic blockade of IL-4, IL-13 and TSLP may drive immunological tumor growth inhibition in subsets of cancer patients alone and in combination with checkpoint inhibitors. Improved tumor growth inhibition was likely driven through augmented cytotoxic T cell priming in secondary lymphoid organs and improved reactivation by repolarized monocytes and dendritic cells in tumors.

cancer biology↗

bioRxiv2025

Scaling unlocks broader generation and deeper functional understanding of proteins

Generative protein language models (PLMs) are powerful tools for designing proteins purpose-built to solve problems in medicine, agriculture, and industrial processes. Recent work has trained ever larger language models, but there has been little systematic study of the optimal training distributions and the influence of model scale on the sequences generated by PLMs. We introduce the ProGen3 family of sparse generative PLMs, and we develop compute-optimal scaling laws to scale up to a 46B-parameter model pre-trained on 1.5T amino acid tokens. Pro-Gen3s pre-training data is sampled from an optimized data distribution over the Profluent Protein Atlas v1, a carefully curated dataset of 3.4B full-length proteins. We evaluate for the first time in the wet lab the influence of model scale on the sequences generated by PLMs, and we find that larger models generate viable proteins for a much wider diversity of protein families. Finally, we find both computationally and experimentally that larger models are more responsive to alignment with laboratory data, resulting in improved protein fitness prediction and sequence generation capabilities. These results indicate that larger PLMs like ProGen3-46B trained on larger, well-curated datasets are powerful foundation models that push the frontier of protein design.1

synthetic biology↗

bioRxiv2024

Caliber of sensory axons in vivo varies spatially and temporally and is influenced by the cellular microenvironment

Cell shape is crucial to cell function, particularly in neurons. The cross-sectional diameter, also known as caliber, of axons and dendrites is an important parameter of neuron shape, best appreciated for its influence on the speed of action potential propagation. Many studies of axon caliber focus on cell-wide regulation and assume that caliber is static. Here, we have characterized local variation and dynamics of axon caliber in vivo using the peripheral axons of zebrafish touch-sensing neurons at embryonic stages, prior to sex determination. To obtain absolute measurements of caliber in vivo, we paired sparse membrane labeling with super- resolution microscopy of neurons in live fish. We observed that axon segments had varicose or "pearled" morphologies, and thus vary in caliber along their length, consistent with reports from mammalian systems. Sister axon segments originating from the most proximal branch point in the axon arbor had average calibers that were uncorrelated with each other. Axon caliber also tapered across the branch point. Varicosities and caliber, overall, were dynamic on the timescale of minutes, and dynamicity changed over the course of development. By measuring the caliber of axons adjacent to dividing epithelial cells, we found that skin cell division is one aspect of the cellular microenvironment that may drive local differences and dynamics in axon caliber. Our findings support the possibility that spatial and temporal variation in axon caliber could significantly influence neuronal physiology. Significance StatementAxon caliber directly influences how quickly neurons send messages to other cells and likely plays a role in neurons overall health. In the peripheral nervous system, where neurons cover particularly long distances, cell shape can determine whether an animal successfully executes behaviors such as escape responses. We found that axon caliber can vary between locations within the same cell and is highly dynamic. Taking these variations into account may allow neuroscientists to better estimate transmission speeds for cells in neural circuits. We observed that axon caliber is distorted when nearby skin cells change shape. Thus, cells not classically considered part of the nervous system can also contribute to caliber dynamics, broadening our view of axon caliber determinants.

neuroscience↗

bioRxiv2022

Cas9+ conditionally immortalized neutrophil progenitors as a tool for genome wide CRISPR screening for neutrophil differentiation and function

Neutrophils are short-lived cells of the innate immune system that play numerous roles in defense against infection, regulation of immune responses, tissue damage and repair, autoimmunity, and other non-communicable diseases. Understanding neutrophil function at a mechanistic level has been hampered by the difficulty of working with primary neutrophils, which die rapidly upon isolation, and the relative paucity of neutrophil cell lines. Murine neutrophil progenitors that are immortalized with estrogen-regulated expression of Hoxb8 differentiate into neutrophils upon withdrawal of estrogen and facilitate the quantitative production of neutrophils in vitro. Here we report the creation of a Cas9+ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils for the first time. By editing progenitors via transduction with sgRNAs, and then withdrawing estrogen, Cas9 edited neutrophils are produced with high efficiency. Importantly, neutrophil differentiation of edited progenitors occurs both in vitro in cell culture and when transferred into murine recipients. To demonstrate the utility of Cas9+ER-Hoxb8 progenitors for forward genetics, we performed a pooled CRISPR screen to identify factors required for survival during neutrophil differentiation. This screen identified hundreds of genes both negatively and positively selected under differentiation conditions. One of the top hits from this screen was Cebpe, a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using the progenitor cell line, we also confirmed that Cepbe is required for neutrophil differentiation in vivo, validating the utility of this cell line both for screening and for studying in vivo phenotypes. The genome-wide screen also identified all components of the WASH complex as being required for neutrophil differentiation, a finding that extends the known role of WASH in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, we demonstrate that Cas9+ER-Hoxb8 immortalized neutrophils can be used to study neutrophil function both in vitro and in vivo. This new resource will enable the analysis of the role of neutrophils in numerous disease states using genetics for the first time.

immunology↗

bioRxiv2021

Long-range migration of centrioles to the apical surface of the olfactory epithelium

Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50 to 100 m from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 m/min. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium prior to other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.

cell biology↗

bioRxiv2019

Centrioles are amplified via rosette formation in cycling progenitors of olfactory sensory neurons

Olfaction in most animals is mediated by neurons bearing cilia that are accessible to the environment. Olfactory sensory neurons (OSNs) in chordates usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how the epithelium generates cells with many centrioles--about 16 in mouse--is not yet understood. We show that centrioles are amplified via centriole rosette formation in both embryonic development and turnover of the olfactory epithelium in adults, and rosette-bearing cells often have free centrioles in addition. Cells with amplified centrioles can go on to divide, with centrioles clustered at each pole. Additionally, we found that immediate neuronal precursors amplify centrioles concomitantly with elevation of mRNA for Plk4 and Stil, key regulators of centriole duplication. Our findings highlight the importance of accounting for centriole amplification in neuron regeneration therapies derived from olfactory epithelia.

cell biology↗