bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.05.21.595010

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

Ramarapu, R.·Wulcan, J.·Chang, H.·Moore, P. F.·Vernau, W.·Keller, S. M.
View original source ↗Export metadata ↓

Abstract

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5 reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Source connections

Explore related subjects

immunologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Ramarapu, R., Wulcan, J., Chang, H., Moore, P. F., Vernau, W., Keller, S. M.. 2024-05-21. Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes. https://doi.org/10.1101/2024.05.21.595010

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Differential and Cooperative Immunomodulation by Saponin Adjuvants QS-21 and QS-7 in Human THP-1 Macrophages

QS-21, a triterpene glycoside saponin derived from Quillaja saponaria, is a potent immunostimulatory adjuvant incorporated in several FDA-approved vaccines. QS-7, a structurally related saponin fraction, has been reported to retain immunomodulatory activity with reduced hemolytic toxicity; however, its lower natural abundance has limited its systematic immunological characterization. To address this gap, we systematically compare responses to QS-21 and QS-7 in the THP-1 human macrophage model to resolve their distinct and cooperative contributions to antigen presenting cell (APC) activation. Following cytotoxicity profiling to establish safe and biologically relevant dosing, flow cytometry, proteomics, and transcriptomics were used to delineate the immunological programs engaged by each saponin alone and in combination. QS-7 alone induced minimal upregulation of cell surface activation markers and did not elicit a significant transcriptional signature, yet it drove dose-dependent secretion of a broad panel of pro-inflammatory cytokines and chemokines, revealing a dissociation between these two arms of macrophage activation. Strikingly, co-administration of QS-7 with QS-21 substantially potentiated macrophage activation across all parameters tested, amplifying surface marker upregulation, broadening cytokine and chemokine output to include IL-12p70 and IFN-{gamma}, and markedly expanding the pro-inflammatory transcriptional and pathway landscape induced by QS-21, while concurrently suppressing counter-regulatory lipid-sensing pathways (PPAR, RAR, FXR/RXR) that constrain inflammatory output. Collectively, these findings establish QS-7 not merely as an attenuated analog of QS-21, but as a functionally distinct immunomodulator that recalibrates both the magnitude and regulatory tone of QS-21-driven macrophage activation. These findings show that structurally distinct saponins elicit distinct and complementary macrophage responses, providing a foundation for structure-function studies. Together with synthetic biology and data-driven design, this knowledge can guide the development of new-to-nature saponin adjuvants.

immunology↗
bioRxiv2026

MAFB and MAF cooperatively orchestrate human anti-inflammatory macrophage identity via autocrine Interleukin-10

Macrophage lineage identity is governed by intricate transcriptional networks. Although the large MAF basic region-leucine zipper (bZIP) transcription factors MAFB and MAF are critical regulators of anti-inflammatory programs, their molecular coordination and non-redundant contributions within human macrophages remain poorly defined. Here, utilizing integrated proteomic, transcriptomic, and epigenomic profiling, we demonstrate that endogenous MAFB and MAF physically associate to form a stable heterodimeric complex on chromatin in human M-CSF-dependent monocyte-derived macrophages (M-Mac). Genome-wide CUT&Tag mapping reveals a hierarchical epigenomic architecture in which MAFB acts as the primary global anchor of M-Mac identity, whereas MAF provides localized transcriptional tuning. Mechanistically, both factors directly bind the IL10 locus, but functional IL-10 expression is primarily driven by MAFB. Neutralizing antibody assays reveal that endogenously produced IL-10 establishes an obligate autocrine feedback loop mandatory for both the initial acquisition and the maintenance of the M-Mac anti-inflammatory profile. Functionally, MAFB and MAF exert non-redundant control over core macrophage effectors: MAFB dictates apoptotic cell efferocytosis amd T cell activation, whereas MAF modulates phagocytosis, and acts as a brake suppressing acute pro-inflammatory cytokine/chemokine hyper-induction. Together, these findings establish a comprehensive molecular blueprint of how MAFB and MAF cooperatively fine-tune human monocyte-derived macrophage identity and functional plasticity.

immunology↗
bioRxiv2026

Peripheral Immune Cell Shifts Associated With Alzheimer's Disease Vary in Multi-Ancestry Cohorts

Background: Alzheimer's disease (AD) is heavily influenced by immune system dysregulation, yet the role of peripheral immune processes--and how these vary across ethnicities--remains understudied. Objective: To investigate ancestry-specific immune alterations in AD by profiling peripheral immune cell proportions using high-resolution deconvolution of whole blood RNA-sequencing data, characterizing disease associated cell types, and identifying enriched biological pathways. Methods: We analyzed whole blood RNA-seq data from non-Hispanic White (NHW), African American (AA), Caribbean Hispanic (CariHISP), and Peruvian (PERU) individuals, balanced between AD cases and cognitively normal controls. Immune cell proportions were estimated using CIBERSORTx and compared using linear models (propeller R package) adjusted for age, sex, and APOE genotypes in case-control models. Cell-type-specific differential expression was performed using CARseq, and pathway enrichment analyses performed using MSigDB gene sets. Results: Baseline immune composition differed substantially across cohorts for multiple innate and adaptive cell types. In a multi-ethnic AD versus control analysis, eight cell types reached FDR significance: AD was associated with decreased proportions naive B cells, CD56dim NK cells, B cells, intermediate monocyte I, and double-negative T cells, alongside significant expansion in neutrophils and non-classical monocytes. Interaction analyses revealed heterogeneous group-specific effects, with the broadest shifts in AA and CariHISP. Pathway enrichment indicated shared heme metabolism, interferon, and oxidative phosphorylation programs across groups, with distinct emphases: broad, strong heme metabolism enrichment and the highest oxidative phosphorylation signal (in myeloid populations) in AA; MYC targets in naive B cells and eosinophils in CariHISP; dendritic cell - and T follicular helper cell - centered interferon, inflammatory, and proliferative programs in NHW; and TGF-{beta} and apoptosis signatures, unique to PERU, alongside the broadest cell-type coverage. Conclusions: Peripheral immune dysregulation in AD is characterized by depletion of lymphoid and intermediate monocyte populations with concurrent neutrophil and non-classical monocyte expansion, and these patterns vary meaningfully by ancestry. These findings underscore the need for population-informed biomarkers and therapeutic strategies in AD.

immunology↗