Biology subjects

Nutt, S. L.

Publications and source records attributed to Nutt, S. L..

10 recordsLinked to original sources

bioRxiv2026

3D genome remodelling underlies PU.1-dependent granulocyte maturation

PU.1 is an ETS-domain transcription factor that has critical roles in many aspects of hematopoiesis and immune cell fate and function. In addition, aberrant PU.1 expression has been implicated in the development of acute myeloid leukemia (AML). Loss of PU.1 during adult murine hematopoiesis results in the expansion of immature granulocytes suggesting that PU.1 plays an important role in granulocyte maturation. To understand the molecular underpinnings of this process, we combined gene expression, transcription factor binding and 3D genome analyses with conditional deletion of PU.1 in vivo. We find that in contrast to normal granulocytes, PU.1-deficient cells possessed a transcriptome of immature granulocytes, in line with their cellular phenotype. Furthermore PU.1-deficient granulocytes display altered 3D genome architecture with a significant loss of interactivity in regions bound by PU.1 in control cells. Overall, this study implicates PU.1 as a key regulator of granulocyte maturation and lineage commitment through control of transcriptional programs and 3D chromatin architecture.

immunology↗

bioRxiv2024

Transient inhibition of type I interferon enhances CD8+ T cell stemness and vaccine protection

Developing vaccines that promote CD8+ T cell memory is a challenge for infectious disease and cancer immunotherapy. TCF-1+ stem cell-like memory T (TSCM) cells are important determinants of long-lived memory. Yet, the developmental requirements for TSCM formation are unclear. Here, we identify the temporal window for type I interferon (IFN-I) receptor (IFNAR) blockade to drive TSCM cell generation. TSCM cells were transcriptionally distinct and emerged from a transitional precursor of exhausted (TPEX) cellular state concomitant with viral clearance. TSCM differentiation correlated with T cell retention within the lymph node paracortex, due to increased CXCR3 chemokine abundance which disrupted gradient formation. These affects were due a counterintuitive increase in IFN{psi}, which controlled cell location. Combining IFNAR inhibition with mRNA-LNP vaccination promoted specific TSCM differentiation and enhanced protection against chronic infection. These finding propose a new approach to vaccine design whereby modulation of inflammation promotes memory formation and function. HIGHLIGHTSO_LIEarly, transient inhibition of the type I interferon (IFN) receptor (IFNAR) during acute viral infection promotes stem cell-like memory T (TSCM) cell differentiation without establishing chronic infection. C_LIO_LITSCM and precursor of exhausted (TPEX) cellular states are distinguished transcriptionally and by cell surface markers. C_LIO_LIDevelopmentally, TSCM cell differentiation occurs via a transition from a TPEX state coinciding with viral clearance. C_LIO_LITransient IFNAR blockade increases IFN{psi} production to modulate the ligands of CXCR3 and couple TSCM differentiation to cell retention within the T cell paracortex of the lymph node. C_LIO_LISpecific promotion of TSCM cell differentiation with nucleoside-modified mRNA-LNP vaccination elicits enhanced protection against chronic viral challenge. C_LI

immunology↗

bioRxiv2024

Divergent cytokine and transcriptional signatures control functional T follicular helper cell heterogeneity

Adaptive immune responses protect against multiple classes of pathogens, including viral, bacterial, fungal, and helminth infections. In all these settings, CD4+ T follicular helper (Tfh) cells tailor high-affinity class-switched B cells responses. How Tfh lineage sovereignty is established while allowing for this context-specific functional heterogeneity is unclear. Here, we identify Tfh transcriptional networks in response to diverse infections. While Bcl-6 is the transcriptional linchpin of the core Tfh signature, this is overlayed with pathogen-specific transcriptional modules that shape Tfh function. Cytokine-transcriptional Tfh programing in mouse and human lymphoid tissue demonstrated that type I interferon and TGF{beta} signaling direct individual Tfh subpopulations to instruct B cell output. Here, we provide a transcriptional map and cell surface resource to interrogate Tfh diversity in humans and mice. This resource can be leveraged to further understand the origins of immune flexibility, perform immune monitoring in infection and antibody-mediated diseases and to develop context-specific vaccines. ONE-SENTENCE SUMMARYDalit, Tan and colleagues provide a resource that functionally and transcriptionally profiles T follicular helper cells (Tfh) during diverse pathogen responses to reveal a blueprint for transcriptional flexibility and new tools to interrogate Tfh heterogeneity in mice and humans.

immunology↗

bioRxiv2024

BATF controls IFN I production via DC-SCRIPT in plasmacytoid dendritic cells

The basic leucine zipper ATF-like transcription factor (BATF) plays a pivotal role in coordinating various aspects of lymphoid cell biology, yet essential functions in dendritic cells (DCs) have not been reported. Here we demonstrate that BATF deficiency leads to increased interferon (IFN) I production in Toll-like receptor 9 (TLR9)-activated plasmacytoid dendritic cells (pDCs), while BATF overexpression has an inhibitory effect. BATF-deficient mice exhibit elevated IFN I serum levels early in lymphocytic choriomeningitis virus (LCMV) infection. Through ATAC-Seq analysis, BATF emerges as a pioneer transcription factor, regulating approximately one third of the known transcription factors in pDCs. Integrated transcriptomics and ChIP-Seq approaches identified the transcriptional regulator DC-SCRIPT as a direct target of BATF that suppresses IFN I promoter activity by interacting with the interferon regulatory factor 7 (IRF7). Genome-wide association study (GWAS) analyses further implicate BATF in pDC-mediated human diseases. Our findings establish a novel negative feedback axis in IFN I regulation in pDCs during anti-viral immune responses orchestrated by BATF and DC-SCRIPT, with broader implications for pDC and IFN I-mediated autoimmunity.

immunology↗

bioRxiv2023

The T2T-CHM13 reference genome has more accurate sequences for immunoglobulin genes than GRCh38

Antibody production by B-cells is essential for protective immunity. The clonal selection theory posits that each mature B cell has a unique immunoglobulin receptor generated through random gene recombination and, when stimulated to differentiate into an antibody-secreting cell, has the capacity to produce only a single antibody specificity. It follows from this "one-cell-one-antibody" dogma, that single-cell RNA-seq profiling of antibody-secreting cells should find that each cell expresses only a single form of each of the immunoglobulin heavy and light chains. However, when using GRCh38 as the genome reference, we found that many antibody-secreting cells appeared to express multiple immunoglobulin isotypes. When the newly published T2T-CHM13 genome was used instead as the genome reference, every antibody-secreting cell was found to express a unique isotype, and read mapping quality was also improved. We show that the superior performance of T2T-CHM13 was due to its European origin matching the genetic background of the query samples. On the other hand, T2T-CHM13 failed to appropriately fit the "one-cell-one-antibody" dogma when applied to data derived from East Asia. Our results show that read assignment to human immunoglobulin isotype genes is very sensitive to the ancestral origin of the genome reference.

genomics↗

bioRxiv2022

CIS calibrates GM-CSF signalling strength to regulate macrophage polarization via a STAT5-IRF8 axis

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the ability to differentiate macrophages (MOs) with opposing functions, namely proinflammatory M1-like and immunosuppressive M2-like. Despite the importance and opposing functional outcomes of these processes, the intrinsic mechanism that regulates the functional polarization of MOs under GM-CSF signaling remains elusive. Here we show that GM-CSF induced MOs polarisation resulted in the expression of the Cytokine-inducible SH2-containing protein (CIS), and that CIS deficiency diverted differentiation of monocytes into immunosuppressive M2-like MOs expression. CIS deficiency resulted in the hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting the downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss and gain of function approaches highlighted IRF8 as a critical instructor of the M1-like polarisation program. In vivo, CIS deficiency led to skewing to M2-like macrophages, which induced strong Th2 immune responses characterised by the development of severe experimental asthma. Collectively, we reveal a CIS-censored mechanism interpreting the opposing actions of GM-CSF in MO differentiation and uncovering its role in controlling allergic inflammation.

immunology↗

bioRxiv2022

NAb-seq: an accurate, rapid and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells

Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals including mice, rats, rabbits and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been "the gold standard" for antibody gene sequencing but relies on the availability of species-specific degenerate primer sets for amplification of light and heavy antibody genes, in addition to lengthy and expensive cDNA preparation. Here we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop NAb-seq: a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light and heavy chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics and therapeutics.

bioinformatics↗

bioRxiv2021

Dysregulation of PAX5 causes uncommitted B cell development and tumorigenesis in mice

PAX5 is the master transcription factor controlling B cell identity. In humans, mutations in PAX5 account for 30% of B cell acute lymphoblastic leukemia (B-ALL) cases. Investigating the causal effects of PAX5 mutations has however been difficult due to the premature lethality of Pax5-/- mice. Here we describe a novel mouse strain with a premature STOP mutation in Pax5 (Y351*) that produces a truncated protein and reduction in protein function, yet still allows for some B cell development to occur. A population of uncommitted and multipotent CD19+B220- B cells develops in the bone marrow of homozygous mice leading to the development of B-ALL. We show that the tumors frequently acquire secondary mutations in Jak3, and Ptpn11 highlighting key pathways interacting with PAX5 during malignant transformation. Analysis of the PAX5Y351* mice provide insight not only into the functional consequence of reduced PAX5 activity on B cell development and identity, but also provides an avenue in which to study PAX5-driven B-ALL in mice. One Sentence SummaryReduction in PAX5 function in mice induces the development of uncommitted B cells that have multipotent and malignant potential.

immunology↗

bioRxiv2020

The clonal and molecular aetiology of emergency dendritic cell development

Extrinsic regulation of single haematopoietic stem and progenitor cell (HSPC) fate is crucial for immune cell development. Here, we examine the aetiology of Flt3 ligand (Flt3L)-mediated emergency development of type 1 conventional dendritic cells (cDC1s), which results in enhanced immunity against infections and cancer. Using cellular barcoding, we demonstrate a predominant role of enhanced clonal expansion and moderate contribution via recruitment of additional cDC1-generating HSPCs. The selective cDC1 expansion occurs primarily via multi-/oligo-potent clones, without compromising output to other lineages. To understand the molecular hallmarks early during a Flt3L response, we develop Divi-Seq to simultaneously profile cell division history, surface phenotype and transcriptional state of single HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative early progenitor-like state, which leads to selective emergence of CD11c+cKit+ transitional precursors with high cellular output to cDC1s. These findings inform the mechanistic action of Flt3L in natural immunity and immunotherapy at a clonal level.

immunology↗

bioRxiv2019

An Erg driven transcriptional program controls B-lymphopoiesis

Summary/AbstractB-cell development is initiated by the stepwise differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating the mature B-cells that mediate protective immunity. This highly regulated process also generates clonal immunological diversity via recombination of immunoglobulin genes. While several transcription factors that control B-cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for the earliest steps in B-cell differentiation. Erg initiates a transcriptional network involving the B-cell lineage defining genes, Ebf1 and Pax5, that directly promotes the expression of key genes involved in V(D)J recombination and formation of the B-cell receptor. Complementation of the Erg-deficiency with a productively rearranged immunoglobulin gene rescued B-cell development, demonstrating that Erg is an essential and exquisitely stage specific regulator of the gene regulatory network controlling B-lymphopoiesis.

immunology↗