Biology subjects

Park, T.

Publications and source records attributed to Park, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Sequential assembly in vivo of the bacteriophage P22 genome translocation channel

Bacteriophage P22 ejects four internal proteins into infected cells to assemble a channel for genome translocation across the cell envelope. However, how these proteins cooperate to build the trans-envelope channel remains enigmatic. Using in situ single-particle cryogenic electron microscopy, we determined near-atomic in vivo structures of P22 infection intermediates, revealing how the tail hub protein gp10 templates the sequential assembly of three internal proteins gp7*, gp20 and gp16 into a ~50-nm trans-envelope channel that extends from the virion into the host cytoplasm. Functional analyses explain why phage DNA can be detected in supernatant fluids during infection by channel-defective mutants. Our structures also provide a mechanistic basis for the long-standing observation that a P22 virion lacking gp16 can be complemented, extra-cytoplasmically, by a co-infecting virion lacking gp20, thereby allowing the genome in the gp16-deficient particle to enter the host cytoplasm.

microbiology↗

bioRxiv2026

Sequence-to-function modeling uncovers the context-specific grammar of Drosophila chromatin insulation

Chromatin is organized into self-interacting topologically associating domains partitioned by boundary elements that insulate adjacent domains and restrict regulatory interactions. Yet, how sequence context and combinations of factors dictate boundary strength remains incompletely understood. Here we present Domino, a deep learning framework that maps genomic sequences to quantitative insulation scores defined directly from single-nucleosome resolution Drosophila melanogaster Micro-C data. Unlike traditional transcription factor motif scanning, Domino captures broad sequence context to resolve the functional contributions of individual sequence elements. We validate model predictions through experimental perturbations of insulator sequences. Model interpretation yields insulation-associated motifs genome-wide. Across 7,311 embryonic boundaries, Domino reveals a comprehensive insulation grammar defined by just 24 primary motifs that account for 59% of the boundaries, with an average of only two motifs per motif-containing boundary. Beyond known factors, we identify the zinc-finger proteins Trem, CG4854 and CG17385 as previously unreported insulation factors. We uncover distance- and orientation-dependent motif synergy, including a strict orientation preference of the prominent architectural factor M1BP. Finally, Domino traces tissue-specific shifts in the insulator landscape from the embryo to larval and adult brains, nominating new brain-specific insulation motifs. In sum, Domino provides a generalizable framework for decoding the regulatory logic of 3D genome architecture.

genomics↗

bioRxiv2026

Shared binding sites for the chromosomal architectural protein Su(Hw) mediate physical interactions between Drosophila TAD boundaries

The head-to-tail pairing of the nhomie and homie boundaries is responsible for the formation of a stem-loop TAD (topologically associated domain) that encompasses the even-skipped (eve) gene and its regulatory elements. TAD boundaries like homie and nhomie have partner preferences and one mechanism thought to be important in linking compatible boundaries is shared binding sites for the same architectural protein. Both eve boundaries have a single binding site for the polydactyl zinc finger protein Su(Hw). Here we have investigated the role of the shared Su(Hw) sites in the long-distance regulation of a dual reporter by the eve enhancers. Consistent with this mechanism, we find that when the transgene nhomie or homie boundary has a mutation in the Su(Hw) binding site, reporter activation by the eve enhancers is severely compromised. Using MicroC we show the mutating the single Su(Hw) substantially weakens the ability of the nhomie and homie boundaries in the transgene to physically pair with the two endogenous eve boundaries. Su(Hw) binding sites are not, however, sufficient as the gypsy su(Hw) insulator is unable to support long-distance regulation. In a less demanding transvection assay mutations in the nhomie and homie Su(hw) site have only a modest effect on regulatory interactions. The eve boundaries can mediate transvection with the gypsy insulator; however. they are not compatible with the dCTCF Fab-8 boundary even in the transvection assay. Finally, we show that the regulatory and physical interactions of the nhomie boundary in the transgene and the two endogenous eve boundaries are incompatible with the popular cohesin-loop extrusion model.

genetics↗

bioRxiv2026

Germ-free piglets display variable neuroinflammatory-like perturbations in prefrontal cortical microglia

Communication between gut microbiota and immune cells within the brain is essential for neurotypical development. Specifically, microglia are known to play a key role in regulating and supporting neural progenitor stem cell production during brain development, and are sensitive to changes in the maternal gut microbial composition during perinatal development. Here, we employed a germ-free (GF) porcine paradigm to examine how the absence of the microbiome affects microglial dynamics during a key epoch of brain development. We utilized automated software to evaluate microglial density and morphology across three developmentally significant regions: the ventricular/subventricular zone (VZ/SVZ), the prefrontal subcortical white matter (PFCSWM), and layers II/III of the prefrontal cortex (PFCII-III). We found no significant differences in microglial morphology or density in the VZ/SVZ or PFCSWM. In contrast, the PFCII-III of P16 piglets exhibited an increase in microglia density paired with morphologies indicative of an activated/reactive functional state. Notably, these effects were identified with no overall changes in microglial density in any of the regions assessed. Transcriptomics on RNA isolated from the PFCII-III revealed a significant upregulation of genes related to neuroinflammation, in agreement with a region-specific microglial and immune response in the absence of microbial colonization during postnatal development. Together, these findings build on the limited knowledge available on how microbiota influence brain development in large animal model organisms with high similarities to human brain anatomy and developmental trajectories. Significance StatementThe prefrontal cortex of porcine display unique, ramified microglia which are sensitive to germ-free conditions whereby they display alterations in morphology with a more transcriptionally reactive signature. These findings indicate that microglia are regionally sensitive to stimuli in the periphery, and studies in lissencephalic mammalian models may not be directly correlative to other higher-order species. The neuroanatomical heterogeneity of microglia across species is informative and understudied, but necessary, to draw conclusions on the array of perturbations spanning neurodevelopmental trajectories in health and disease.

neuroscience↗

bioRxiv2026

The origin and evolution of amphibious hearing in pinnipeds

Seals (pinnipeds) are the only mammals that can hear in both air and water. How and when they achieved the ability to negotiate such contrasting auditory media remains unknown. Here, we apply 3D shape and phylogenetic comparative analyses to a large dataset of caniform carnivorans (119 species, 217 specimens) to study the emergence of amphibious hearing in pinnipeds despite significant evolutionary constraints. We find support for the cavernous tissue as a functional and evolutionary mechanism for amphibious hearing. This tissue, which fills with blood during diving to equalise air pressure in the ear, enables a shift from in-air to underwater hearing by matching the acoustic impedance of the ear to that of the surrounding water. Early diverging freshwater pinnipeds had impaired hearing underwater. The first marine pinnipeds could hear amphibiously but were limited by a functional tradeoff between hearing abilities and the need to prevent damage from loud underwater sounds. Subsequently, otariids (eared seals) and phocids (true seals) independently acquired middle ear adaptations that expanded their underwater hearing range. This iterative evolution likely facilitated the exploration of novel auditory adaptive zones by crown pinnipeds, resulting in rare acoustic abilities like ultrasonic singing, vocal learning, and keeping rhythm.

evolutionary biology↗

bioRxiv2025

PIP2 Binding at Allosteric Site Blocks Activation in Human Rod CNG Channels

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a ubiquitous signaling lipid that regulates multiple ion channels. In human cyclic nucleotide-gated (CNG) channels, including the rod photoreceptor channel, PIP2 has been reported to exert inhibitory effects, but the underlying mechanism has remained unclear. Because this inhibition lowers the apparent cGMP sensitivity of rod CNG channels, it can play a key role in controlling the light sensitivity and dynamic range of rod photoreceptors. Here we report how PIP2 modulates the function of human CNGA1 channels, the major subunit of human rod photoreceptor CNG channels. Ensemble ion flux assays with liposome-reconstituted purified CNGA1 channels demonstrated robust inhibition by PIP2 via a reduction in apparent cGMP sensitivity, and single-channel recordings revealed PIP2 reduces the channels open probability without altering unitary conductance. To uncover the structural basis, we determined cryo-EM structures of CNGA1 in lipid nanodiscs under multiple ligand conditions. In PIP2-free conditions, closed, intermediate, and open conformations were observed, whereas in the presence of PIP2, the open state was absent. Density consistent with bound PIP2 was detected at inter-protomer grooves between the voltage-sensing and pore domains indicating that PIP2 binding stabilizes non-conductive conformations by sterically preventing C-linker elevation and outward movement of helix S6, conformational changes needed for pore dilation. Collectively, our results establish a structural mechanism for PIP2-mediated inhibition of rod CNG channels, define a mechanistic framework for phosphoinositide control of ligand-gated channels across the CNG superfamily, and provide an inhibitory allosteric binding site for future drug targeting in this channel family.

biophysics↗

bioRxiv2025

Precision at Every Scale: Efficiency in AI-Driven De Novo Antibody Design

The precise de novo design of antibodies remains a therapeutic challenge. The AI platform, GaluxDesign, was evaluated in a high-efficiency Precision-Scale Workflow by synthesizing and testing only 50 full-length IgG candidates per epitope across eight distinct epitopes from six therapeutic targets. This campaign yielded a 10.5% binder rate (estimated EC50 < 100 nM), identifying target-specific binders for seven of eight epitopes, with multiple candidates exhibiting sub-nanomolar to single-digit nanomolar dissociation constants (Kd). We further assessed the same workflow on nine shared benchmark targets selected for external comparison, where GaluxDesign identified target-specific binders for eight of nine targets, demonstrating strong target-level performance relative to previously reported de novo antibody design approaches. Together, these results establish a high-efficiency, precision-scale workflow for generating novel, high-affinity therapeutic antibodies.

bioengineering↗

bioRxiv2025

FOXA1 mutations co-opt nascent transcription factor networks in partnership with androgen receptor to enhance prostate tumorigenicity

Mutations in the pioneer transcription factor FOXA1, found in 10-40% of human prostate cancers, alter global chromatin accessibility and promote growth in prostate cells. Through analysis of a novel cohort of 874 primary and metastatic prostate tumors with somatic FOXA1 mutations, we confirm the high frequency of missense mutations (n=339) and indels (n=335) in the Wing2 region of the Forkhead domain, as well as frameshift mutations that truncate the C-terminus (n=287). To investigate the transcriptomic consequences of each mutation subgroup as well as elevated levels of wild-type FOXA1 (WT) (a fourth well documented subgroup), we performed single nucleus multiome sequencing in primary mouse organoids following inducible expression of representative alleles. Whereas each mutant induced distinct transcriptomic and DNA accessibility features, a prominent feature of all mutants was perturbed epithelial lineage specification, ranging from basal-like fates in cells expressing indel mutants to secretory (L1-like) luminal fates in cells expressing C-terminal truncation, missense mutations or excess WT. Integrated RNA-seq, ATAC-seq and ChIP-seq analysis of L1-like fate specification revealed enrichment of a composite androgen receptor-FOXA1 hybrid motif and cooperativity with the Oct family transcription factor POU2F1. Importantly, L1-like fate specification is seen in vivo tumorigenicity assays where, in combination with Trp53/Pten loss, expression of these mutants results in a histologic switch from basal-like to secretory luminal histology.

genomics↗

bioRxiv2025

Runx/Cbfβ regulates the development of tolerogenic Thetis cells

Establishing immune tolerance to gut microbiota and food antigens upon first exposures during early life is essential to prevent inflammatory bowel diseases and food allergy and depends on induction of peripherally induced Ror{gamma}t expressing regulatory T (Ror{gamma}t+ pTreg) cells1, 2, 3, 4, 5, 6. Recent studies have identified a critical role for Ror{gamma}t expressing antigen-presenting cells (APC), Thetis cells (TCs), in peripheral regulatory T (pTreg) cell differentiation and tolerance to food and commensal microbes7, 8, 9, 10, 11. TCs encompass four distinct subsets, and a subset of TCs, TC IV induces pTreg differentiation, but the transcription factors that control their differentiation are not fully known. Here, using orthogonal genetic approaches to impair Runx/Cbf{beta} activity, we show that development of specific TCs subsets is regulated by Runx/Cbf{beta} transcriptional factor complexes. While attenuated Runx3 by germline mutations resulted in a severe reduction of all Ror{gamma}t+ APCs, mice lacking one of two Cbf{beta} splicing variants, Cbf{beta}2, exhibited a loss of TC II, III and TC IV subsets with associated loss of Ror{gamma}t+ pTreg cells. Conditional inactivation of Runx1 and Runx3 genes by CD11c-Cre led to a specific loss of TC III and TC IV subsets. Strikingly, CD11c-Cre driven transgenic Runx expression, particularly Runx1, led to enhanced TC IV differentiation and thus Ror{gamma}t+ pTreg cells. Furthermore, transgenic Cbf{beta}2 by CD11c-Cre recovered only TC IV subset with restoration of Ror{gamma}t+ pTreg in Cbf{beta}2-deficient mice. Collectively, our findings establish a critical pathway for TC IV differentiation and provide new insights into therapeutic interventions to promote Ror{gamma}t+ pTreg induction in autoimmune and inflammatory diseases.

immunology↗

bioRxiv2025

Manually weighted taxonomy classifiers improve species-specific rumen microbiome analysis compared to unweighted or average weighted taxonomy classifiers

Previous research has demonstrated that applying taxonomic weights to shotgun metagenomic data can improve species identification in 16S rRNA gene-based microbiome analysis. However, such an approach does not allow for accurate analysis of samples collected from less studied habitats, such as rumen. In the present study, we developed a method to incorporate taxonomic weights based on relative abundance of species identified from shotgun sequencing and amplicon sequencing data derived from rumen. Using this weighting method, we evaluated latest versions of five prominent databases--SILVA, Greengenes2, RDP, NCBI RefSeq, and GTDB--against the BLAST 16S rRNA database, assessing classification counts, fully classified ratios (proportion of ASVs classified to a known genus and species), and error rates. Our results revealed that the use of the weighting method significantly improved both classification counts and fully classified ratios, along with a substantial (P < 0.05) reduction in error rates compared to unweighted taxonomy classifier. While GG2 and SILVA struggled with accurate classification at the species level owing to their inherent database characteristics, GTDB consistently improved all metrics using the manually weighted taxonomy classifier, achieving up to an 8% error rate reduction at the species level. NCBI RefSeq and RDP also exhibited remarkable improvement in the classification counts and fully classified ratios, along with substantial error rate reductions by up to 47% at the species level. These findings demonstrate that amplicon sequencing datasets can enhance rumen microbiome analyses through effective weighting methods. While SILVA is commonly used in metataxonomic analyses of the rumen microbiome, we recommend NCBI RefSeq for species-level classification due to its superior accuracy and minimal ambiguous classification (e.g., "uncultured" or "sp.") in future metataxonomic studies.

bioinformatics↗

bioRxiv2025

Precise, Specific, and Sensitive De Novo Antibody Design Across Multiple Cases

The precision design of antibodies, which naturally recognize diverse molecules through six variable loops, remains a critical challenge in therapeutic molecule discovery. In this study, we demonstrate that precise, sensitive, and specific antibody design can be achieved without prior antibody information across eight distinct target proteins. For each target, binders were identified from a yeast display scFv library of approximately 106 sequences, constructed by combining 102 designed light chain sequences with 104 designed heavy chain sequences. Binders with varying binding strengths were identified for all eight targets, including a case where no experimentally resolved target protein structure was available, demonstrating the highest level of precision compared to previous de novo antibody design reports. To further validate the designed antibodies, they were characterized in the IgG format for five distinct targets. The antibodies exhibited favorable developability properties, positioning them as promising hit or lead candidates. Notably, for one target in particular, the IgG-formatted antibodies exhibited affinity, activity, and developability, comparable to a commercial antibody, highlighting the sensitivity of the design method. Furthermore, binders capable of distinguishing closely related protein subtypes or mutants were identified, demonstrating that the method can achieve high molecular specificity. Cryo-EM analysis experimentally validated the designs accuracy by confirming that the key binding interactions were precisely as designed. These findings underscore the effectiveness of precision molecular design based on atomic-accuracy structure prediction. This study establishes computational antibody design as a viable approach for generating therapeutic molecules with tailored properties, with promising potential for achieving the efficacy and safety required for successful therapeutics.

bioengineering↗

bioRxiv2024

Direct expression of CPT1a enables a high throughput platform for the discovery of CPT1a modulators

Carnitine palmitoyltransferase 1 (CPT1), which catalyzes the rate-limiting step of fatty acid oxidation, has been implicated in therapeutic approaches to several human diseases characterized by aberrant lipid metabolism. Isoform-specific quantification of CPT1 activity is essential in the characterization of small molecule inhibitors of CPT1, but several existing means to quantify enzymatic activity, including the use of radioisotope labeled carnitine, are not amenable to scalable, high throughput screening. Here, we demonstrate that mitochondrial extracts from Expi293 cells transfected with a CPT1a plasmid are a reliable and robust source of catalytically active human CPT1. Moreover, with a source of catalytically active enzyme in hand, we modified a previously reported colorimetric method of coenzyme A (CoA) easily scalable to a 96-well format for the screening of CPT1a inhibitors. This assay platform was validated by two previously reported inhibitors of CPT1a: R-etomoxir and perhexiline. To further demonstrate the applicability of this method in small molecule screening, we prepared and screened a library of 87 known small molecule APIs, validating the inhibitory effect of chlorpromazine on CPT1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/628575v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f69f0corg.highwire.dtl.DTLVardef@43eb95org.highwire.dtl.DTLVardef@11fa21corg.highwire.dtl.DTLVardef@1cbe405_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Sociality shapes mitochondrial adaptations supporting hypoxia tolerance

Oxygen deprivation or hypoxia is poorly dealt with by most terrestrial species and often leads to permanent tissue damage and death. One prominent exception is the naked mole-rat (Heterocephalus glaber) which is remarkably adapted to withstand prolonged periods ([~]18 mins) of severe hypoxia, a trait likely driven by its crowded underground lifestyle. Other African mole-rat species are less social or entirely solitary like the Cape mole-rat (Georychus capensis). Here, we asked whether cellular and molecular adaptations to hypoxia map to social traits. We discovered that at the cellular level naked mole-rat fibroblasts survive >30 hours in 1% oxygen, while fibroblasts from terrestrial or non-social mole-rat species (human, mouse and Cape mole-rat) die rapidly under hypoxic conditions. We further show that naked mole-rat mitochondria have evolved morphological, functional and proteomic adaptations crucial for hypoxia resistance, remaining unaffected after long periods of severe hypoxia. We identify the mitochondrial protein Optic Atrophy 1 (OPA1) as a key player in naked mole-rat hypoxia resilience. Naked mole-rat mitochondria not only express more protective forms of OPA1, but also harbor a structurally unique isoform that likely protects cells from hypoxic damage. We show that evolutionary changes including the functionalization of a unique Opa1 exon support mitochondrial mediated cellular protection. Indeed, knockdown of OPA1 in naked mole-rat cells is sufficient to render them equally susceptible to hypoxia as human cells or cells from non-social African species. Our study demonstrates how molecular evolution drives unique adaptations that enable cells to achieve unprecedented resistance to hypoxic damage. We also show that molecular changes at the level of mitochondria are crucial in conferring hypoxia resistance. Our results thus chart a novel molecular path to understand how robust cellular hypoxia resistance can be achieved. Such knowledge may eventually inspire novel strategies to circumvent the consequences of hypoxic-damage in humans.

physiology↗

bioRxiv2024

Intracortical injection of immune checkpoint inhibitor promotes monocyte/macrophage infiltration and restores microglial function and neuronal activity in an AD mouse model

Alzheimer's disease (AD) involves complex neuroimmune interactions. However, the role of immune checkpoint pathways in regulating the glial and neuronal functions of the AD brain remains unclear. This study aims to investigate how the brain-specific modulation of the PD-1/PD-L1 axis affects glial and neuronal function in an AD mouse model, which is characterized by the significant upregulation of microglial PD-1 and astrocytic PD-L1. A single intracortical administration of anti-PD-L1 antibody reshaped the glial microenvironment, which was accompanied by the restoration of impaired microglial process convergence. Astrocyte-specific Pd-l1 knockdown using the pSico system validated the essential role of astrocytic PD-L1 in enhancing microglial responses. Notably, the blockade of the PD-1/PD-L1 signaling pathway increased the microglial P2RY12 expression, a key marker of microglial homeostasis, which likely contributed to the restoration of neuronal hyperactivity. Collectively, these findings confirmed the potential of targeting the astrocyte-microglia PD-1/PD-L1 axis to mitigate AD pathology.

neuroscience↗

bioRxiv2024

Accurate antibody loop structure prediction enables zero-shot design of target-specific antibodies

Protein loops, characterized by their versatile structures with varying sizes and shapes, can recognize a wide range of targets with high specificity and affinity. The variable loops of the antibody complementarity-determining region (CDR) are particularly crucial for immune responses and therapeutic applications due to their effective target recognition capabilities. Accurate structure prediction of these antibody loops is essential for the efficient in silico design of target-binding antibodies for therapeutic or industrial use. However, predicting antibody loop structures is challenging due to the lack of evolutionary information from related proteins. Thus, a successful ab initio structure prediction method, which operates without structural templates or related sequences, is crucial for the effective design of antibody loop-mediated interactions. This study demonstrates that highly accurate antibody loop structure prediction enables the effective zero-shot design of target-binding antibody loops. The performance of loop design has been shown to depend on the accuracy of ab initio loop structure prediction, as tested with two versions of our design model. The high affinity, diversity, novelty, and specificity of the antibody loops designed with these new methods were validated experimentally on four target proteins.

bioengineering↗

bioRxiv2024

Thetis cells induce food-specific Treg cell differentiation and oral tolerance

The intestinal immune system must establish tolerance to food antigens to prevent onset of allergic and inflammatory diseases. Peripherally generated regulatory T (pTreg) cells play an essential role in suppressing inflammatory responses to allergens; however, the antigen-presenting cell (APC) that instructs food-specific pTreg cells is not known. Here, we show that antigen presentation and TGF-{beta} activation by a subset of ROR{gamma}t+ antigen-presenting cells (APC), Thetis cells IV (TC IV), is required for food-induced pTreg cell differentiation and oral tolerance. By contrast, antigen presentation by dendritic cells (DCs) was dispensable for pTreg induction but required for TH1 effector responses, highlighting a division of labor between tolerogenic TCs and pro-inflammatory DCs. While antigen presentation by TCs was required for food-specific pTreg generation both in early life and adulthood, the increased abundance of TCs in the peri-weaning period was associated with a window of opportunity for enhanced pTreg differentiation. These findings establish a critical role for TCs in oral tolerance and suggest that these cells may represent a key therapeutic target for the treatment of food-associated allergic and inflammatory diseases.

immunology↗

bioRxiv2023

Algorithm for selecting potential SARS-CoV-2 dominant variants based on POS-NT frequency

COVID-19, currently prevalent worldwide, is caused by a novel coronavirus, SARS-CoV-2. Similar to other RNA viruses, SARS-CoV-2 continues to evolve through random mutations, creating numerous variants, such as Alpha, Beta, and Delta. It is, therefore, necessary to predict the mutations constituting the dominant variant before they are generated. This can be achieved by continuously monitoring the mutation trends and patterns. Hence, in the current study, we sought to design a dominant variant candidate (DVC) selection algorithm. To this end, we obtained COVID-19 sequence data from GISAID and extracted position-nucleotide (POS-NT) frequency ratio data by country and date through data preprocessing. We then defined the dominant dates for each variant in the USA and developed a frequency ratio prediction model for each POS-NT. Based on this model, we applied DVC criteria to develop the selection algorithm, verified for Delta and Omicron. Using Condition 3 as the DVC criterion, 69 and 102 DVC POS-NTs were identified for Delta and Omicron an average of 47 and 82 days before the dominant dates, respectively. Moreover, 13 and 44 Delta- and Omicron-defining POS-NTs were recognized 18 and 25 days before the dominant dates, respectively. We identified all DVC POS-NTs before the dominant dates, including soaring and gently increasing POS-NTs. Considering that we successfully defined all POS-NT mutations for Delta and Omicron, the DVC algorithm may represent a valuable tool for providing early predictions regarding future variants, helping improve global health. Author Summary

bioinformatics↗

bioRxiv2023

Reconciling the spectrum of RORγt+ antigen-presenting cells

Three recent contemporaneously published papers1-3 showed that antigen-presenting cells (APC) expressing the nuclear receptor ROR{gamma}t instruct peripheral regulatory T (pTreg) cell differentiation and establish tolerance to the gut microbiota. These studies identified a spectrum of ROR{gamma}t+ APCs, distinct from dendritic cells, that included type 3 innate lymphoid cells (ILC3s) as well as novel APC types. Given the discordant conclusions as to the nature of the pTreg-inducing APC and the divergent nomenclature used in these three studies, there is a clear need to reconcile these analyses and the identity of the cell types described. Our reanalysis of the single cell RNA-seq data from these studies revealed the presence of four distinct subsets of non-ILC3 ROR{gamma}t+ APCs, present in all three datasets reported, and confirmed expression of Itgb8, the critical factor for ROR{gamma}t+ APC mediated pTreg induction, in cells synonymous with the non-Aire expressing Thetis cell subset, TC IV.

immunology↗