Outbreaks of respiratory virus infections and arbovirus infections both pose a substantial threat to global public health. Clinically, both types of infection range from mild to severe and coinfections may occur more commonly than supposed. Our previous experimental coinfection study in mice demonstrated that prior infection with the arbovirus Semliki Forest virus (SFV) negatively impacted immune responses to influenza A virus (IAV). Here, we investigate whether simultaneous coinfection impacts the outcome of immune responses or disease. Simultaneous SFV and IAV infection did not lead to exacerbated or attenuated disease compared with the single virus infection control groups. SFV brain virus titers and brain pathology, including inflammation and immune responses, were comparable in the coinfection and single infection groups. By contrast, there was enhanced IAV replication, but no exacerbated lung pathology in coinfected mice. The magnitude of IAV-specific CD8+ T-cell responses in the lungs was lower compared with IAV-only infection. Considered along with our previous study, this study provides evidence that the timing of viral coinfection is pivotal in determining effects on immune responses, pathological changes and disease outcome.
© 2025 The Author(s). Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.

ABSTRACT Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from blood of two horses, based on expression of CD172a, Flt3, CADM1 and CD14. All main DC subsets were detected based on key gene expression, including cDC type 1 (cDC1; XCR1 ), cDC2 ( FCER1A , CD1E ) and plasmacytoid DC (pDC; TCF4 ). In addition, we detected a small cluster of CD34-expressing DC progenitors. Our data confirms the previously reported phenotype of equine pDC (MHC-II low CADM1 int CD172a int ), cDC1 (MHC-II high CADM1 high CD172a low-int ) and cDC2 (MHC-II high CADM1 int CD172a high ), while also highlighting considerable CD14 expression for cDC2. Among Flt3 + cells clustering with cDC2, we identified a cluster resembling monocytes and showing a highly pro-inflammatory signature, likely representing DC type 3 (DC3). Notably, one cDC2-associated cluster had a mixed pDC/cDC2 signature ( TCF4 , SPIB , FCER1A ), indicating the presence of transitional DC (tDC), a new DC subset initially described in human and mouse, and more recently in pig. To assess cross-species conservation of DC subsets, we compared equine and porcine DC scRNA-seq datasets using SATURN, a deep learning method that combines gene expression with added biological knowledge encoded in protein language models. This enabled mapping of the most similar DC subsets between horse and pig, confirming the conservation of key transcriptomic features and supporting the identification of equine tDC. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for elucidating the role of DC subsets in immunological diseases such as type I hypersensitivity in horses.

The innate immune system plays a critical role in the rapid recognition and elimination of pathogens through pattern recognition receptors (PRRs). Among these PRRs are the C-type lectins (CTLs) langerin, mannan-binding lectin (MBL), and surfactant protein D (SP-D), which recognize carbohydrate patterns on pathogens. Each represents proteins from different compartments of the body and employs separate effector mechanisms. We have investigated their interaction with the Gram-positive opportunistic pathogen Staphylococcus aureus, a bacterium whose cell wall contains two key glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Using a langerin-expressing cell line and recombinant langerin, MBL, and SP-D, we demonstrated that langerin, MBL, and SP-D all recognize nonencapsulated S. aureus. However, the bacterium may produce CP that effectively shields S. aureus from recognition by all three CTLs. Experiments utilizing mutant S. aureus strains confirmed that WTA is a ligand for MBL, but that langerin likely interacts with an additional unknown ligand. A competition assay revealed that MBL and SP-D inhibit langerin's interaction with S. aureus, highlighting the intricate redundancy and cooperation within the innate immune system. This study highlights the dynamic interplay of langerin, MBL, and SP-D in recognizing specific surface structures on S. aureus and provides insight into how this pathogen evades innate immune recognition.
Copyright © 2025 Hymøller, Christiansen, Schlosser, Skov Sørensen, Lee and Thiel.

Autoantibodies generated in germinal centers (GCs) contribute to the pathogenesis of autoimmune diseases. GCs are controlled by specialized FoxP3+ T-follicular regulatory cells (Tfr), but their role in established autoimmunity is unclear. We generated autoimmune bone marrow chimeras in which Tfr could be specifically ablated by diphtheria toxin. Furthermore, we tracked the clonal persistence and evolution of Tfr populations using Confetti reporters. Ablation of Tfr caused increased early plasma cell output, but longer-term ablation did not increase plasma cell levels beyond those of Tfr-sufficient controls, suggesting that Tfr fail to contain chronic autoreactive GC responses. In agreement, Tfr were robustly induced in early autoreactive GCs but then waned. Moreover, we observed polyclonal Tfr expansion when ablating part of the Tfr subset. Hence, under homeostatic conditions, a polyclonal population of Tfr operates to control autoreactivity by limiting the output of plasma cells from GCs, but in chronic autoimmunity, this mechanism fails.
© 2024 The Author(s).

Antitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified.
In this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice.
Human NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors.
NR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.
© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.