Biology subjects

Michaels, Y. S.

Publications and source records attributed to Michaels, Y. S..

10 recordsLinked to original sources

bioRxiv2025

Uncovering Non-Monotonic Antagonistic and Synergistic Combinations (UNMASC), a robust method with applications to T cell differentiation in vitro

Induced pluripotent stem cells (iPSCs) have been proposed as an alternative T cell source for CAR-T cell therapies, as they can be differentiated and matured into T cells in vitro using cytokines. These assays benefit from computational and mathematical models to design appropriate experimental protocols. However, models are limited by typical monotonic dose-responses, preventing them from being used for cytokine effects that are largely non-monotonic. To address this shortcoming, we developed Uncovering Non-Monotonic Antagonistic and Synergistic Combinations (UNMASC), a novel mathematical model describing non-monotonic dose-response surfaces of cytokine interactions that distinguishes synergy of efficacy and potency. We showed that our approach successfully recapitulates non-monotonic observed dose-response surfaces characterizing in vitro T cell progenitor differentiation. Our results highlighted cytokine combinations with antagonistic effects alone but synergistic in combination, particularly IL3 and IL7. Together, UNMASC accelerates the efficient cell generation assays and extends drug interaction surfaces to a broader range of dose-responses.

pharmacology and toxicology↗

bioRxiv2025

Decoding human B cell ontogeny in prenatal and adult bone marrow and in vitro models via single-cell multiomics

In humans, the bone marrow becomes the primary site for B lymphopoiesis during the second trimester of pregnancy and continues throughout life. Prenatal and adult B cell progenitors play distinct roles in the aetiology and pathology of paediatric and adult hematopoietic malignancies, though the molecular drivers of these differences remain unclear. Here, we created a comprehensive multiomics atlas of over 500k cells covering immune and stromal compartment from prenatal and adult bone marrow, enabling high-resolution analysis of the cell-intrinsic and cell-extrinsic processes that modulate prenatal and adult B lymphopoiesis. Even though B cells follow broadly similar developmental trajectories, we identify a novel postnatal lateCLP subset equivalent to prenatal preProB cells, and uncover prenatal cells carry several signatures characteristic of leukemias, including enhanced proliferation, higher RAG1/RAG2 activity, extrinsic B cell signals such as IL7, and lower retention signals in the bone marrow. We also developed and characterised, at both cellular and molecular levels, a new experimental framework for generating B cell precursors from human induced pluripotent stem cells (hiPSCs), and show that it faithfully recapitulates key stages of B cell differentiation. Together, our single-cell multiomics atlas of B lymphopoiesis in vivo and in vitro offers detailed insights into the unique molecular features of prenatal and adult B cell lymphopoiesis, and serves as a powerful resource for investigating the early events that contribute to haematological disorders.

immunology↗

bioRxiv2025

Precise engineering of chimeric antigen receptor expression levels defines T cell identity and function

Chimeric Antigen Receptor (CAR) T therapy is a potent treatment for haematological malignancies, but T cell exhaustion reduces its efficacy in many patients. Although high CAR transgene levels appear to drive T cell exhaustion, the relationship between CAR expression levels, T cell function, and transcriptional identity is yet to be mapped at high resolution. Here, we harness a high-resolution microRNA-based control system to precisely modulate CAR transgene expression levels and assess the impact on T cell activation, gene expression and function. By post-transcriptionally modulating CAR abundance, we show that differential CAR levels significantly impact T cell proliferation, cytokine production and tonic signalling. T cells with high CAR expression become strongly activated even at low target antigen densities, while those with low CAR expression are triggered only by high concentrations of their target. Single-cell RNA sequencing of primary T cells expressing a broad range of CAR transcript levels revealed global transcriptional programmes that become dysfunctional with increased CAR abundance, expanding our understanding of T cell exhaustion. Notably, we identified a narrow CAR expression range where the exhaustion transcriptional state is not triggered, demonstrating that T cell exhaustion can be controlled by fine-tuning CAR levels. This work demonstrates that CAR expression levels are key determinants of T cell transcriptional identity and function and introduces a tractable method to precisely tune CAR expression and T cell activity.

synthetic biology↗

bioRxiv2023

Time- and lineage-resolved transcriptional profiling uncovers gene expression programs and clonal relationships that underlie human T lineage specification

T cells develop from multi-potent hematopoietic progenitors in the thymus and provide adaptive protection against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors, and their subsequent specification to the T lineage, has been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single cell RNA sequencing revealed that cell-cycle exit, downregulation of the multipotent hematopoietic program, and upregulation of >90 lineage-associated transcription factors all occur within a highly co-ordinated and narrow developmental window. Computational gene-regulatory network inference elucidated the transcriptional logic of T lineage specification, uncovering an important role for YBX1. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and mathematical trajectory inference and discovered that mast and myeloid potential bifurcate from each other early in haematopoiesis, upstream of T lineage restriction. Collectively, our analyses provide a quantitative, time-resolved model of human T cell specification with relevance for regenerative medicine and developmental immunology.

immunology↗

bioRxiv2021

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8{beta}+, CD4-, CD3+, TCR{beta}+ T cells from PSCs.

bioengineering↗

bioRxiv2021

Discrete-to-analog signal conversion in human pluripotent stem cells

During development, state transitions are coordinated through changes in the identity of molecular regulators in a cell state- and dose specific manner. The ability to rationally engineer such functions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein we report the generation of synthetic gene circuits that can detect a discrete cell state, and upon state detection, produce fine-tuned effector proteins in a programmable manner. Effectively, these gene circuits convert a discrete (digital-like) cell state into an analog signal by merging AND-like logic integration of endogenous miRNAs (classifiers) with a miRNA-mediated output fine-tuning technology (miSFITs). Using an automated miRNA identification and model-guided circuit optimization approach, we were able to produce robust cell state specific and graded output production in undifferentiated hPSC. We further finely controlled the levels of endogenous BMP4 secretion, which allowed us to document the effect of endogenous factor secretion in comparison to exogenous factor addition on early tissue development using the hPSC-derived gastruloid system. Our work provides the first demonstration of a discrete-to-analog signal conversion circuit operating in living hPSC, and a platform for customized cell state-specific control of desired physiological factors, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/467377v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@150da71org.highwire.dtl.DTLVardef@135554borg.highwire.dtl.DTLVardef@72b5b3org.highwire.dtl.DTLVardef@f70f84_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

A FOXN1 mutation competitively displaces wild-type FOXN1 from higher order nuclear condensates to cause immunodeficiency

The transcription factor FOXN1 is a master regulator of thymic epithelial cell development and function. Here we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multi-molecular nuclear condensates essential for the factors transcriptional activity. FOXN1s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are significantly altered in a patient with a mutant FOXN1 which is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog, selectively impairs mouse thymic epithelial cell (TEC) differentiation revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

immunology↗

bioRxiv2019

Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells

Semaphorin-3A (Sema3A) regulates tumor angiogenesis, but its role in modulating anti-tumor immunity is unclear. We demonstrate that Sema3A secreted within the tumor microenvironment (TME) suppresses tumor-specific CD8+ T cell function via Neuropilin-1 (NRP1), a receptor that is upregulated upon activation with T cells cognate antigen. Sema3A inhibits T cell migration, assembly of the immunological synapse, and tumor killing. It achieves these functional effects through hyper-activating the acto-myosin system in T cells leading to cellular paralysis. Finally, using a clear cell renal cell carcinoma patient cohort, we demonstrate that human tumor-specific CD8+ T cells express NRP1 and are trapped in Sema3A rich regions of tumors. Our study establishes Sema3A as a potent inhibitor of anti-tumor immunity.

immunology↗

bioRxiv2018

Precise tuning of gene expression output levels in mammalian cells

Precise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

synthetic biology↗

bioRxiv2018

Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here we developed a variant screening platform to identify differential sequence determinants of human tRNA promoter and processing activities. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enabled highly specific guide RNA production from a Pol-II promoter.

bioengineering↗