Biology subjects

Craft, J.

Publications and source records attributed to Craft, J..

8 recordsLinked to original sources

bioRxiv2026

Human Lymph Node Cellular Senescence Atlas Reveals Age-Dependent Alteration in Germinal Center B Cell Function and Niches

Immunosenescence, the age-associated decline in immune function, is a key feature of human aging. In human lymphoid organs, however, the specific immune cell populations that acquire senescence-associated phenotypes during aging and how they influence the surrounding tissue microenvironment remain poorly understood. A spatially resolved map of these senescence-associated immune states in human lymphoid tissues could help clarify their relationship with aging and their potential contributions to the progressive decline of immune function. Here, we integrated single-cell and spatial multi-omics to systematically characterize age-related senescence in human lymph nodes (LNs). Single-cell transcriptomics of lymphoid tissues from donors aged 18 to 100 years old identified 34 immune and stromal cell types and revealed age-associated upregulation of senescence signatures in specific populations. Spatial proteomic profiling of 99 LN sections from 51 donors (18-86 years) using high-plex immunofluorescence ([~]20 million cells) mapped senescence markers (p16, p21, HMGB1, -H2AX) at single-cell resolution, revealing diverse senescent-like cell types ("senotypes") and a stepwise shift from extrafollicular to germinal center (GC) localization with age. Notably, we observed focal clonal-like senescence in GC B cells in older donor LNs. Spatial transcriptomics, epigenomics, and metabolic imaging of selected samples further elucidate the multi-omics signatures and underlying mechanisms of functional impairment, metabolic remodeling, and distinct regulatory programs in senescent-like GC B cells. This study presents a comprehensive spatial atlas of senescence-associated immune states in human lymph nodes, revealing cell-type-specific and spatial heterogeneity that may contribute to immunosenescence and the decline of immune function during aging.

immunology↗

bioRxiv2025

A T-cell Intrinsic Role for APOL1 Risk Alleles in Allograft Rejection

African Americans have an increased risk of kidney disease due to exonic variants in Apolipoprotein-L1 (G1 and G2). These prevalent variants have also been linked with kidney rejection, but outside of association with African ancestry, underpinning causal mechanisms are unknown. We investigated T-cell function using transgenic mice with physiologic expression of wild type (G0-), G1-, or G2-APOL1. Mice with variant APOL1 showed greater CD8+T-cell activation with expansion of a central memory (TCM) subset. Stimulated G1-CD8+T-cells showed enhanced proliferation and cytokine production, which reversed with APOL1 inhibition. In MHC-mismatched cardiac transplants, G1-mice demonstrated greater CD8+T-cell infiltration and reduced survival. Bulk transcriptome of G1-CD8+T-cells, and single-cell transcriptome of graft infiltrating TCMs, showed enrichment of canonical T-cell receptor (TCR) pathways including Ca2+-signaling. G1-CD8+T-cells demonstrated baseline ER-Ca2+ depletion followed by sustained increases in cytosolic-Ca2+ upon TCR stimulation. G1-CD8+T-cells were more sensitive to Ca2+ chelation, or store-operated Ca2+ entry inhibition, and relatively resistant to calcineurin antagonism vs. G0-CD8+T-cells. Analogously, in a kidney transplant cohort, APOL1-variant recipients developed rejection when they had elevated peripheral TCMs before transplantation and despite significantly higher tacrolimus levels vs G0/G0-AAs with rejection. In summary, we unravel an excitatory T-cell intrinsic mechanism for APOL1 exonic variants, causally linking them with kidney rejection.

immunology↗

bioRxiv2024

IL-4 Licenses B Cell Activation Through Cholesterol Synthesis

Lymphocyte activation involves a transition from quiescence and associated catabolic metabolism to a metabolic state with noted similarities to cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and cell division1-3. Following antigen receptor ligation, lymphocytes require spatiotemporally distinct "second signals". These include costimulatory receptor or cytokine signaling, which engage discrete programs that often involve remodeling of organelles and increased nutrient uptake or synthesis to meet changing biochemical demands4-6. One such signaling molecule, IL-4, is a highly pleiotropic cytokine that was first identified as a B cell co-mitogen over 30 years ago7. However, how IL-4 signaling mechanistically supports B cell proliferation is incompletely understood. Here, using single cell RNA sequencing we find that the cholesterol biosynthetic program is transcriptionally upregulated following IL-4 signaling during the early B cell response to influenza virus infection, and is required for B cell activation in vivo. By limiting lipid availability in vitro, we determine cholesterol to be essential for B cells to expand their endoplasmic reticulum, progress through cell cycle, and proliferate. In sum, we demonstrate that the well-known ability of IL-4 to act as a B cell growth factor is through a previously unknown rewiring of specific lipid anabolic programs, relieving sensitivity of cells to environmental nutrient availability.

immunology↗

bioRxiv2022

Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and - 9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective lysosomal trafficking but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection. Author SummaryIntracellular bacterial pathogens cause many of the worlds most deadly infectious diseases. A common requirement for nearly all intracellular pathogens is the ability to damage the endomembrane compartments in which they reside, which allows pathogens access to the nutrient-rich cytosol of the host. However, membrane damage also creates a "pattern of pathogenesis" that triggers antimicrobial immune responses. Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) act as a surveillance system for membrane damage and Gal-3 and Gal-8 inhibit bacterial growth by activating autophagy, a cellular pathway that can capture cytosolic bacteria and degrade them in lysosomes. Membrane damage-sensing galectins were hypothesized to promote bacterial killing during acute infection yet their role in the immune response of an infected animal remains unclear. Here, we show that mice deficient for Gal-3, -8, and -9 had no defects in resistance to acute infection with the pathogens Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Mycobacterium tuberculosis (Mtb), and were only modestly susceptible to chronic Mtb infection. Our data suggest that Gal-3, -8 and -9 are not critical for innate immune responses during acute infection and may play a more prominent role in the adaptive immune response. These results broaden our understanding of the role of membrane damage-sensing pathways in host defense against bacterial infection.

immunology↗

bioRxiv2021

Increased microbial diversity and decreased prevalence of common pathogens in the gut microbiomes of wild turkeys compared to domestic turkeys

Turkeys (Meleagris gallopavo) provide a globally important source of protein and constitute the second most important source of poultry meat in the world. Bacterial diseases are common in commercial poultry production causing significant production losses for farmers. Due to the increasingly recognized problems associated with large-scale/indiscriminant antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study we compared the cecal microbiota of wild and domestic turkeys, hypothesizing that environmental pressures faced by wild birds may select for a disease-resistant microbial community. Sequence analysis of 16S rRNA genes amplified from cecal samples indicate that free-roaming wild turkeys carry a rich and variable microbiota compared to domestic turkeys raised on large-scale poultry farms. Wild turkeys also had very low levels of Staphylococcus, Salmonella and E. coli when compared to domestic turkeys. E. coli strains isolated from wild or domestic turkey cecal samples also belong to distinct phylogenetic backgrounds and differ in their propensity to carry virulence genes. E. coli strains isolated from factory-raised turkeys were far more likely to carry genes for capsule (kpsII, kpsIII) or siderophore (iroN, fyuA) synthesis than those isolated from wild turkeys. These results suggest that the microbiota of wild turkeys may provide colonization resistance against common poultry pathogens. ImportanceDue to the increasingly recognized problems associated with antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study we compare the microbiota of wild and domestic turkeys. Results suggest that free ranging wild turkeys carry a distinct microbiome when compared to farm raised turkeys. The microbiome of wild birds contains very low levels of poultry pathogens compared to farm raised birds. The microbiomes of wild turkeys may be used to guide development of new ways to control disease in large scale poultry production.

microbiology↗

bioRxiv2021

High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells

T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation. One-Sentence SummaryComplementary pathways of antibody production mediate neutralizing responses to SARS-CoV-2.

immunology↗

bioRxiv2020

Neoantigen-driven B cell and CD4+ T follicular helper cell collaboration promotes robust anti-tumor CD8+ T cell responses

CD4+ T follicular helper (TFH) cells provide help to B cells, which is critical for germinal center (GC) formation, but the importance of TFH-B cell interactions in cancer is unclear. We found TFH cells correlated with GC B cells and with prolonged survival of lung adenocarcinoma (LUAD) patients. To investigate further, we developed an LUAD model, in which tumor cells expressed B-cell- and T-cell-recognized neoantigens. Interactions between tumor-specific TFH and GC B cells were necessary for tumor control, as were effector CD8+ T cells. The latter were reduced in the absence of T cell-B cell interactions or the IL-21 receptor. IL-21 was produced primarily by TFH cells, development of which required B cells. Moreover, development of tumor-specific TFH cell-responses was also reliant upon tumors that expressed B-cell-recognized neoantigens. Thus, tumor-neoantigens themselves can control the fate decisions of tumor-specific CD4+ T cells by facilitating interactions with tumor-specific B cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/424168v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f31b57org.highwire.dtl.DTLVardef@881959org.highwire.dtl.DTLVardef@1385ccdorg.highwire.dtl.DTLVardef@154ba3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2018

scFTD-seq: freeze-thaw lysis based, portable approach toward high-quality distributed single-cell 3 mRNA profiling

Cellular barcoding of 3 mRNAs enabled massively parallel profiling of single-cell gene expression and has been implemented in droplet and microwell based platforms. The latter further adds the value for compatibility with low input samples, optical imaging, scalability, and portability. However, cell lysis in microwells remains suboptimal despite the recently developed sophisticated solutions. Here, we present scFTD-seq, a microchip platform for performing single-cell freeze-thaw lysis directly toward 3 mRNA sequencing. It offers format flexibility with a simplified, widely adoptable workflow that reduces the number of preparation steps and hands-on time, with the quality of data and the cost per sample matching that of the state-of-the-art scRNA-seq platforms. Freeze-thaw, known as an unfavorable lysis method resulting in possible RNA fragmentation, turns out to be fully compatible with single-cell 3 mRNA sequencing, which detects only ~50 bases at the 3 end. We applied it to the profiling of mixed populations including whole tumors for distinguishing all major cell types and to the profiling of circulating follicular helper T cells implicated in systemic lupus erythematosus pathogenesis. Our results delineate the heterogeneity in the transcriptional programs and effector functions of these rare pathogenic T cells. As scFTD-seq decouples on-chip cell isolation and the following library preparation steps, we envision it to potentially allow the sampling (capture of cells/beads in microwells) at the distributed sites including small clinics or point-of-care settings and downstream processing at a centralized facility, which should enable wide-spread adoption beyond academic laboratories - for any users even with no experience in scRNA-seq library generation.

bioengineering↗