Search bioRxiv⌕ 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

Exploratory positional differences in observed CDRH2 lineage-state transitions following HIV-1 germline-targeting vaccination

Background: Germline-targeting HIV-1 vaccination recruits VRC01-class B-cell precursors, but the positional patterns of observed longitudinal B-cell receptor lineage-state changes remain incompletely described. We compared CDRH2 profiles between homologous and heterologous boosting regimens using published IAVI G002 data. Methods: Participant-specific Week-8 lineages were prospectively matched to Week-24 observations. Germline-to-nongermline changes in sampled lineage-modal residues were evaluated at seven IGHV1-2*02 CDRH2 positions using participant-balanced, mean-centered profiles and exact participant-label permutation inference. Sampling-depth and sorting-probe sensitivity analyses assessed observability. A separate participant-held-out benchmark evaluated antibody-language-model representations for predicting observed Week-24 lineage detection. Results: The CDRH2 analysis included 332 lineages, 15 participants, 2,179 eligible lineage-position observations, and 241 observed state changes. The regional profile statistic was 0.027066 (exact p = 0.001709; six-region adjusted q = 0.010256), with descriptive differences at V-relative positions 51, 54, and 55. Most eligible longitudinal observations had one assessable cell per visit. In seven Group-1 participants, Week-24 dual-probe sampling recovered 181 baseline lineages with eOD-GT8 and 143 with Cg28v2, including 60 detected by both probes. In a separate cohort of 2,739 lineages from 17 participants, IgBERT and AbLang-2 representations did not improve participant-balanced average precision over a four-feature biological baseline. Conclusions: Observed CDRH2 lineage-modal-state profiles differed between regimens in this exploratory secondary analysis. Sparse sampling, potentially probe-related ascertainment, and absent linked functional validation preclude inference about mutation timing, functional improvement, or causal booster effects.

immunology↗
bioRxiv2026

Multivalent mRNA tuberculosis vaccines protect against mycobacterial infection in zebrafish, mice and guinea pigs

Despite a century of BCG use, tuberculosis remains a leading cause of death from infectious diseases and more effective vaccines are urgently needed. Here we combined antigen discovery in zebrafish with mRNA vaccine technology to generate multivalent vaccine candidates, TUmRNA4, TUmRNA6 and TUmRNA9, encoding four, six, or nine Mycobacterium tuberculosis antigens, respectively. The vaccines were well tolerated, and induced robust antigen-specific cellular immune responses, including IFN{gamma} production and expansion of memory T-cell populations. In mice, TUmRNA4 and TUmRNA6 reduced pulmonary M. tuberculosis burden and lung pathology to levels comparable to BCG vaccination and TUmRNA6 enhanced BCG-mediated protection when used as a booster vaccine. In guinea pigs, TUmRNA9 reduced bacterial burden in the lungs, spleen, and lymph nodes and decreased pulmonary lesions. These findings demonstrate that protective antigens identified in zebrafish can be translated into effective multivalent mRNA vaccines and establish a framework for tuberculosis vaccine development across multiple preclinical models.

immunology↗
bioRxiv2026

AlphaScreen profiling of extracellular vesicle-associated platelet, immune, and vascular markers reveals disease-specific signatures in ME/CFS, fibromyalgia, and long COVID

Introduction: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), fibromyalgia (FM), and long COVID (LC-19) are chronic diseases with overlapping clinical features and poorly understood pathophysiology. Increasing evidence suggests the involvement of immune dysregulation, endothelial dysfunction, and altered vascular physiology. Extracellular vesicles (EVs) are key mediators of intercellular communication and are envisioned as sensors of homeostasis and disease. The aim of this study was to determine whether EV marker profiling could provide a basis for the differential diagnosis of ME/CFS, FM and LC-19 and/or patient stratification. Methods: EV-enriched plasma from ME/CFS, FM, cases co-diagnosed with ME/CFS and FM, LC-19 patients, and matched healthy controls (n = 10 per study group) were analyzed. EV concentration and size distribution were assessed by nanoparticle tracking analysis, followed by targeted profiling of EV-associated surface markers via AlphaScreen, including tetraspanins CD9 and CD63, platelet- (CD41/CD61), myeloid- (CD13), immune- (CD74 and CX3CR1), and renin-angiotensin system- associated ACE1 (also known as CD143), and ACE2 surface markers. Results: The studied disease groups presented distinct EV signatures. LC-19 samples exhibited the most pronounced and coordinated alterations, characterized by increased levels of CD9/CD74, CD9/CX3CR1, and CD9/ACE1-positive EVs, along with reduced overall particle concentrations. Notably, platelet-derived CD9/CD41/CD61-positive EVs were significantly elevated in both LC-19 and ME/CFS, as compared to healthy controls, indicating enhanced platelet activation. Immune-associated markers revealed differential pathway activation in LC-19, with CD74-positive EVs reflecting antigen presentation and inflammatory signaling, and CX3CR1-positive EVs indicating altered leukocyte trafficking and vascular interaction. Increased ACE1-positive EVs in LC-19 further suggested a shift toward pro-inflammatory renin-angiotensin signaling. Conclusion: Collectively, these findings support a disease-specific dysregulation of platelet, immune, and vascular pathways. The integrated EV signatures distinguish LC-19 from the related chronic conditions ME/CFS and FM, highlighting the potential of EV-based profiling for differential diagnosis. Our results provide a systems-level perspective on the differential pathophysiology of these disorders and identify EV-associated markers as candidate biomarkers with clinical value.

immunology↗