Biology subjects

Smith, N. L.

Publications and source records attributed to Smith, N. L..

9 recordsLinked to original sources

bioRxiv2025

Humanizing a CD28 signaling domain affects CD8 activation, exhaustion and stem-like precursors

CD28 ligation provides critical signals that modulate activated T cell fate. In a human to mouse reverse-engineering approach, a single amino acid substitution adjacent to the C-terminal proline-rich domain created CD28A210P mice with enhanced signaling. CD28A210P mice experienced pro-inflammatory responses to CD28 superagonist antibody, analogous to severe cytokine storm induced in a human clinical trial, with a striking increase of activated CD8 T cells. In acute and chronic viral infections, early activation and expansion of CD28A210P CD8 effector T cells increased, with accelerated exhaustion in chronic infection. Mechanistically, CD28A210P enhanced JunB, IL-2, and inhibitory receptors driven by MEK1/2. Generation of CD28A210P stem-like progenitor (Tpex) cells was enhanced in acute and chronic infections, and further expanded by PD-L1 blockade in chronically-infected mice. Thus, humanized PYAP mice reveal key roles for CD28 signaling strength in CD8 activation, accelerating exhaustion during antigen persistence, while promoting and sustaining Tpex during acute and chronic viral infection. One sentence SummaryA single amino acid substitution adjacent to PYAP to humanize CD28 signaling enhances superagonist response, early CD8 activation and Tpex generation during viral infection while accelerating exhaustion and sustaining Tpex during chronic infection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/642460v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@13fc9f9org.highwire.dtl.DTLVardef@e50762org.highwire.dtl.DTLVardef@47c8eeorg.highwire.dtl.DTLVardef@d892d7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Humanized CD28 PYAPP enhances numbers of CD8 T cell effectors and stem-like precursors during acute viral infection, and accelerates exhaustion while sustaining increased self-renewing Tpex cells that are favored during PD-L1 blockade. C_FIG

immunology↗

bioRxiv2025

Single-cell multiomics reveals the distinct properties of neonatal and adult recent thymic emigrants

Following thymic egress, CD8+ T cells must undergo a post-thymic maturation process to transition from a recent thymic emigrant (RTE) to a mature naive T cell. Since the neonatal T cell pool is comprised of significantly more RTEs, the prevailing notion is that neonatal CD8+ T cells behave differently than their adult counterparts simply because they have undergone less post-thymic maturation. To test this theory, we leveraged a fate mapping mouse model and paired single cell transcriptome and TCR sequencing to compare neonatal and adult CD8+ RTEs that have undergone the same amount of post-thymic maturation. Interestingly, we found that neonatal and adult CD8+ RTEs exhibit distinct phenotypes, gene expression profiles, TCR usage, and functions. These data suggest that neonatal CD8+ T cells are not simply immature adult CD8+ T cells and that age-related changes in CD8+ T cell functions in early life cannot be attributed solely to differences in the amount of post-thymic maturation.

immunology↗

bioRxiv2025

Recent thymic emigrants are preferentially recruited into the memory pool during persistent infection

Cytomegalovirus (CMV) leads to a unique phenomenon known as memory inflation, where antigen-specific memory CD8+ T cells continue to accumulate in the peripheral tissues during the latent stage of infection. However, it is still not clear how the inflating pool of memory CD8+ T cells is generated and maintained. In this study, we used murine cytomegalovirus (MCMV) as a model of persistent infection and fate-mapping mice to determine the dynamics of CD8+ T cell recruitment into the memory pool. We found that neonatal exposure to CMV leads to an expansion of newly made CD8+ T cells (recent thymic emigrants, RTEs), which are maintained in the long-lived memory compartment. In contrast, CD8+ T cells made during the latent phase of infection (mature CD8+ T cells) contribute little to the memory pool. We also observed notable phenotypic differences between RTEs and mature cells. Whereas RTEs present at the time of infection gave rise to more effector memory cells, the cells produced later in infection were biased towards becoming central memory cells. Importantly, the preferential recruitment of RTEs into the effector memory pool also occurs during adult exposure to CMV. Collectively, these data demonstrate that persistent infection expands the RTE population, and timing of infection dictates whether neonatal or adult RTEs are locked in to the memory pool. Author SummaryFollowing infection with CMV, CD8+ T cells accumulate in the blood and peripheral organs over time, a feature termed memory inflation. However, it is not clear whether memory inflation is due to the continuous recruitment of cells made during the latent stage of infection or expansion of CD8+ T cells that were present at the time of infection. To address this question, we used a fate-mapping mouse model and examined the recruitment of CD8+ T cells that were produced during different stages of infection. Surprisingly, we discovered that CD8+ T cells exported from the thymus just prior to infection are preferentially recruited and maintained in the memory pool. In contrast, CD8+ T cells made during the latent stage contribute minimally to the inflating pool and exhibit a less differentiated phenotype. These results provide a new conceptual framework for understanding how the memory pool is generated and maintained after persistent viral infection.

immunology↗

bioRxiv2024

Deciphering gene regulatory programs underlying functionally divergent naive T cell subsets

Naive CD8+ T cells are a heterogeneous population, with different subsets possessing distinct functions and kinetics upon activation. However, the gene regulatory circuits differentiating these naive subsets are not well studied. In this work, we analyzed a large collection of public and newly generated RNA-seq and ATAC-seq profiles of different subsets of naive CD8+ T cells, revealing significant differences in the gene regulatory landscapes between subsets. We leveraged these data by employing a network inference algorithm, Inferelator, to identify the transcriptional regulatory circuits active in each subset. The predicted transcriptional network of the naive CD8+ T cell pool was validated by multiple orthogonal approaches, including CUT&Tag and Micro-C. Interestingly, our network analysis revealed a novel role for Eomes in promoting effector cell differentiation in specific cell subsets. Moreover, we uncovered multiple novel regulators across a variety of subsets and discovered several modules of genes that were co-regulated by shared sets of transcription factors in distinct subsets. Collectively, our data defines the gene regulatory programs differentiating naive CD8+ T cells and facilitates the identification of novel transcription factors that may alter the propensity of naive CD8+ T cells to become effector or memory cells after infection.

systems biology↗

bioRxiv2021

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 g/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

immunology↗

bioRxiv2020

A Conserved TCRβ signature dominates a highly polyclonal T-cell expansion during the acute phase of a murine malaria infection

CD4+ {beta} T-cells are key mediators of the immune response to a first Plasmodium infection, undergoing extensive activation and splenic expansion during the acute phase of an infection. However, the clonality and clonal composition of this expansion has not previously been described. Using a comparative infection model, we sequenced the splenic CD4+ T-cell receptor repertoires generated over the time-course of a Plasmodium chabaudi infection. We show through repeat replicate experiments, single-cell RNA-seq, and analyses of independent RNA-seq data, that following a first infection - within a highly polyclonal expansion - T-effector repertoires are consistently dominated by TRBV3 gene usage. Clustering by sequence similarity, we find the same dominant clonal signature is expanded across replicates in the acute phase of an infection, revealing a conserved pathogen-specific T-cell response that is consistently a hallmark of a first infection, but not expanded upon re-challenge. Determining the host or parasite factors driving this conserved response may uncover novel immune targets for malaria therapeutic purposes.

immunology↗

bioRxiv2019

Inherited Causes of Clonal Hematopoiesis of Indeterminate Potential in TOPMed Whole Genomes

Age is the dominant risk factor for most chronic human diseases; yet the mechanisms by which aging confers this risk are largely unknown.1 Recently, the age-related acquisition of somatic mutations in regenerating hematopoietic stem cell populations was associated with both hematologic cancer incidence2-4 and coronary heart disease prevalence.5 Somatic mutations with leukemogenic potential may confer selective cellular advantages leading to clonal expansion, a phenomenon termed Clonal Hematopoiesis of Indeterminate Potential (CHIP).6 Simultaneous germline and somatic whole genome sequence analysis now provides the opportunity to identify root causes of CHIP. Here, we analyze high-coverage whole genome sequences from 97,691 participants of diverse ancestries in the NHLBI TOPMed program and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid, and inflammatory traits specific to different CHIP genes. Association of a genome-wide set of germline genetic variants identified three genetic loci associated with CHIP status, including one locus at TET2 that was African ancestry specific. In silico-informed in vitro evaluation of the TET2 germline locus identified a causal variant that disrupts a TET2 distal enhancer. Aggregates of rare germline loss-of-function variants in CHEK2, a DNA damage repair gene, predisposed to CHIP acquisition. Overall, we observe that germline genetic variation altering hematopoietic stem cell function and the fidelity of DNA-damage repair increase the likelihood of somatic mutations leading to CHIP.

genomics↗

bioRxiv2019

Recovery of trait heritability from whole genome sequence data

Heritability, the proportion of phenotypic variance explained by genetic factors, can be estimated from pedigree data 1, but such estimates are uninformative with respect to the underlying genetic architecture. Analyses of data from genome-wide association studies (GWAS) on unrelated individuals have shown that for human traits and disease, approximately one-third to two-thirds of heritability is captured by common SNPs 2-5. It is not known whether the remaining heritability is due to the imperfect tagging of causal variants by common SNPs, in particular if the causal variants are rare, or other reasons such as overestimation of heritability from pedigree data. Here we show that pedigree heritability for height and body mass index (BMI) appears to be largely recovered from whole-genome sequence (WGS) data on 25,465 unrelated individuals of European ancestry. We assigned 33.7 million genetic variants to groups based upon their minor allele frequencies (MAF) and linkage disequilibrium (LD) with variants nearby, and estimated and partitioned genetic variance accordingly. The estimated heritability was 0.68 (SE 0.10) for height and 0.30 (SE 0.10) for BMI, with a range of ~0.60 - 0.71 for height and ~0.25 - 0.35 for BMI, depending on quality control and analysis strategies. Low-MAF variants in low LD with neighbouring variants were enriched for heritability, to a greater extent for protein-altering variants, consistent with negative selection thereon. Cumulatively variants with 0.0001 < MAF < 0.1 explained 0.47 (SE 0.07) and 0.30 (SE 0.10) of heritability for height and BMI, respectively. Our results imply that rare variants, in particular those in regions of low LD, is a major source of the still missing heritability of complex traits and disease.

genetics↗

bioRxiv2018

Adaptive periodicity in the infectivity of malaria gametocytes to mosquitoes

That periodicity in the biting activity of mosquito vectors explains why malaria parasites have evolved rhythms in cycles of asexual replication in the hosts blood was proposed almost 50 years ago. Yet, tests of this hypothesis have proved inconclusive. Using the rodent malaria Plasmodium chabaudi, we examine rhythms in the density and infectivity of transmission forms (gametocytes) in the hosts blood, parasite development inside mosquitoes, and onwards transmission.Moreover, we control for the confounding effects of rhythms in mosquito susceptibility. We reveal that at night, gametocytes are twice as infective to mosquitoes, despite being less numerous in the blood. This enhanced infectiousness at night interacts with mosquito rhythms to increase sporozoite burdens by almost four-fold when mosquitoes feed during their day. Thus, daytime blood-feeding (e.g. driven by the use of bed nets) may render gametocytes less infective, but this is compensated for by the greater susceptibility of mosquitoes.

microbiology↗