Biology subjects

Murray, J. B.

Publications and source records attributed to Murray, J. B..

3 recordsLinked to original sources

bioRxiv2026

Deep immune profiling of the peripheral blood reveals disease- and sex-associated immune cell signatures in patients with systemic sclerosis

ObjectiveSystemic sclerosis (SSc) predominantly affects females but exhibits greater disease severity in males, suggesting sex differences underlying SSc pathogenesis. We sought to define sex-associated alterations in the peripheral immune landscape of patients with SSc. MethodsWe performed high-dimensional immune profiling of PBMCs from 37 healthy donors (68% female) and 37 patients with SSc (11 limited, 26 diffuse; 68% female) using 30-color spectral flow cytometry, quantifying 56 immune cell subsets per donor. We conducted sex-stratified comparisons and correlation analysis, and used principal component analysis followed by linear discriminant analysis to derive a sex-discriminant immune cellular module. ResultsDiffuse cutaneous SSc (dcSSc) was associated with a distinct immune landscape characterized by increased monocyte and decreased natural killer-like and B cell frequencies, suggesting a myeloid-skewed peripheral immunophenotype. Males exhibited greater enrichment of innate immune subsets, including monocyte and dendritic cell subsets, while females exhibited greater enrichment of adaptive immune subsets. Among T cells, dcSSc was associated with coordinated remodeling across CD4+ and CD8+ subsets, including expansion of stem cell memory T cells (Tscm), and increased regulatory T cells, Th17 skewing, and decreased effector-memory CD8+ subsets. Females exhibited greater proportions of naive- and Tscm, and males exhibited higher proportions of effector-memory subsets. Integrating these data, we identified a sex-discriminant immune module comprised of 20 cell types that distinguishes males and females with dcSSc. ConclusionsSSc is associated with sex-specific differences in the peripheral immune landscape. A sex-associated immune program, further amplified in disease, may contribute to the paradox of female-biased susceptibility and male-biased severity in SSc.

immunology↗

bioRxiv2026

Interrogating the Mechanisms of Cas9-mediated Allele Conversion

Allele conversion describes a process where a heterozygous variant is made homozygous. Recently, it has been shown that allele conversion can be triggered by DNA damage at the heterozygous site. This process has the potential to repair pathogenic heterozygous mutations; however, the efficiency is low. Here, we endeavoured to understand the mechanism underlying allele conversion, ultimately to raise allele conversion efficiency to functionally relevant levels. To test this, we developed a Compound Heterozygous Allele Conversion Reporter (CHACR) cell line. This line comprises knocked-in fluorescent protein encoding genes, with heterozygous inactivating mutations resulting in different fluorescence profiles from each allele. These mutations create protospacer adjacent motifs (PAM) for Cas9 recognition, where allele-specific gRNAs (AS-gRNAs) target the heterozygous mutations. We showed that applying these AS-gRNAs with either Cas9 nuclease or Cas9(D10A) nickase can recover mCherry fluorescence. Sorting and sequencing these fluorescent cells revealed wild-type sequences, suggesting allele conversion repaired the mutation using the homologous allele as a template. Allele conversion can also be triggered using an adenine base editor with an AS-gRNA, and this allele conversion mechanism can be manipulated by inhibiting DNA-PKcs or overexpressing RAD51. This work introduces a model for measuring allele conversion, and modifiers of this mechanism.

molecular biology↗

bioRxiv2025

30-Color Longitudinal Full-Spectrum Immunophenotyping and Sorting of Human Circulating Immune Cell Subsets Implicated in Systemic Autoimmune Rheumatic Diseases

Purpose and Appropriate Sample TypesThis 30-color panel was developed to enable the enumeration and purification of distinct circulating immune cell subsets implicated in the pathogenesis of systemic autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc; scleroderma), Sjogrens disease (SjD), idiopathic inflammatory myopathy (IIM), and others. While designed for application to peripheral blood mononuclear cells, the inclusion of CD45 coupled with the ability to extract cellular autofluorescence spectral signatures enables the application of this panel to other tissue types. Of the 30 total markers, this panel employs 18 markers to profile T cell subsets consisting of different memory subsets and T helper polarities, > 10 markers to profile B cell subsets including double-negative B cells, and a total of 8 lineage markers to identify immune lineages including monocyte and natural killer cell subsets, conventional dendritic cells, plasmacytoid dendritic cells, and basophils. This panel reproducibly identifies target populations with excellent resolution over several months of data acquisition with minimal batch effects, offering investigators a practical approach to sort immune cell subsets of interest for downstream applications while simultaneously collecting high parameter immunophenotypic information using a limited sample quantity.

immunology↗