Heart regeneration varies among vertebrates, with zebrafish serving as a reference species for efficient cardiac restoration. How this capacity diversified among teleosts is an emerging question, given the recent identification of non-regenerative cardiac repair in medaka and cavefish. Here, we investigate heart restorative capacity following cryoinjury in two livebearers, platyfish and swordtails (Xiphophorus species), belonging to the Poe-ciliidae family. We demonstrate that their hearts lack the vascularized compact myocardium, which is a ventricular layer implicated in the restorative response in zebrafish. After cryoinjury, both poeciliids failed to rapidly deposit fibrotic tissue that normally reinforces the damaged site. This deficiency led to striking wound protrusion reminiscent of pseudoaneurysm after myocardial infarction in humans. Although the remaining myocardium initially increased cell proliferation, subsequently deposited collagenous scar tissue permanently sealed the interrupted ventricle, preventing complete regeneration. Transcriptomic analysis revealed several divergently regulated pathways between cryoinjured hearts of zebrafish and platyfish, particularly in immune response regulation. This was validated by delayed leukocyte infiltration and prolonged inflammation in platyfish, compared to the rapid, resolved inflammatory response in zebrafish. Our findings demonstrate that Xiphophorus species have evolved hearts with compromised regenerative capacity, characterized by pseudoaneurysm-like protrusion and permanent scarring. These results reveal that evolutionary traits of phylogenetic lineages can fundamentally modulate regenerative competence among teleosts, with important implications for understanding the mechanistic basis of cardiac repair.

CD8+ T cell migration is central to host defense, enabling clonal selection, tissue infiltration, and elimination of infected cells. While chemokines guide these cells by activating Rac for F-actin polymerization, recent studies reveal a complementary mechanism through Rho activation. This pathway relies on tissue confinement, which deforms the nucleus, activates the actomyosin network, and allows CD8+ T cells to use physical cues for surveillance without external signals. In this review article, we explore how chemokine- and mechanosensing-dependent pathways guide CD8+ T cell surveillance and introduce a ‘front-’ versus ‘rear-driven’ motility model to illustrate their integration for effective tissue monitoring. Ultimately, the interplay of biochemical and physical cues ensures tissue-specific protection by T cells during homeostasis and inflammation.

Heart donation after circulatory death (DCD) is a promising strategy to increase graft supply. However, in contrast to conventional heart transplantation, in which organs are retrieved from heart-beating donors, DCD hearts are subjected to damaging conditions before and during functional, warm in-situ ischemia in the donor, leading to ischemia-reperfusion injury (IRI). Although sex differences have been identified in other contexts of cardiac IRI, such as myocardial infarction, they remain underexplored in DCD. Therefore, we aimed to investigate whether sex differences induce changes in the expression of genes in response to cardiac DCD conditions, including IRI, which may contribute to sexual dimorphism in graft quality. 102 animals were included in this study. Male, female, and ovariectomized (OVX) Wistar rats underwent simulated DCD with no or 22 min of functional, warm in-situ ischemia, followed by oxygenated reperfusion with left-ventricular loading. Functional recovery was assessed and left-ventricular tissue was used for RNA-sequencing. Recovery of left ventricular function was decreased by functional, warm in-situ ischemia, but significantly better in females than in males, with OVX resembling the males. Reperfusion induced inflammatory, stress-response and metabolic-related pathways in all groups. Expression of 110 genes correlated with cardiac recovery, many of which were more abundant in females compared to males, consistent with a role in improved post-ischemic ventricular function. Among these genes, Igfbp3, Fam78b, and Galnt10 were differentially expressed in females compared to males and OVX, suggesting an influence of female sex hormones. Compared to male hearts, cardiac recovery is significantly higher in female hearts after exposure to DCD conditions and is accompanied by an increased expression of genes related to quality control programs that positively correlate with ventricular function. Significantly higher expression of genes related to energy metabolism, including fatty acid metabolism, and inflammatory pathways was revealed in males compared to females and is associated with decreased recovery. This study suggests potential new therapeutic targets for optimizing cardiac DCD graft quality, and highlights the importance of underlying sex and sex-hormone differences, e.g. in inflammatory pathways and metabolic adaptations, that should be taken into consideration for the implementation of sex-specific precision therapies. Female hearts recover significantly better compared to male hearts after simulated donation after circulatory death ischemia and reperfusion. Ovariectomized (OVX) hearts recover similarly to males, suggesting a cardioprotective role for female sex hormones. Reperfusion is associated with a sex-independent upregulation of pro-inflammatory, stress response and metabolic pathway gene expression. Among hearts evaluated after DCD-induced functional, warm in-situ ischemia and reperfusion, all genes that correlated positively with cardiac recovery were more highly expressed in females, whereas genes that correlated negatively with cardiac recovery were more highly expressed in males and OVX. A subset of genes was identified as potential novel cardioprotective targets. These genes both correlate with functional recovery and demonstrate differential expression in females compared to both males and OVX, reflecting the pattern of ventricular recovery. Heart transplantation is currently the only treatment for patients with advanced heart failure to improve quality of life and survival. Nonetheless, the number of newly listed patients needing a transplant continues to increase and outpaces the supply of suitable donors. One promising approach to increase donor availability is using hearts from donors after circulatory death (DCD). In conventional heart donation, the donor is declared brain-dead, but machines keep the heart beating and oxygenated until removal. In DCD, the heart stops beating in the donor and it briefly receives less oxygen and nutrients, which can cause donor organ injury. Although outcomes with DCD hearts are excellent and transplant rates increase, further optimization of the protocol could allow more hearts to be used. In other situations where hearts temporarily lack oxygen, such as myocardial infarction, studies suggest that adult female hearts recover better than male hearts. In the DCD setting, little is known, but it has recently been shown in preclinical studies that female hearts are more tolerant to these conditions. In this study, we simulated the DCD protocol in a rat model. Afterwards, we identified 673 genes that were differentially expressed between male and female hearts. Many of these genes correlated with cardiac recovery; those more abundant in females were linked to better recovery, whereas those more abundant in males were linked to worse recovery. Taken together, our findings may help to identify treatments for the optimization of cardiac graft quality and improve heart transplantation options for both women and men.

Myeloproliferative neoplasms (MPNs) arise from the clonal acquisition of a driver mutation in a hematopoietic stem cell (HSC), often decades before the emergence of clinical disease. A defining characteristic of an HSC is its potential to give rise to both myeloid and lymphoid hematopoietic lineages. Yet the most common MPN driver mutation, JAK2 V617F, has only rarely been observed in lymphocytes. We conducted this study to determine why the JAK2 V617F clone does not contribute to mature lymphocytes. We directly measured myeloid and lymphoid lineage contribution of normal and MPN HSCs in 133 patients with JAK2 V617F MPN. Peripheral blood subpopulations were fractionated via fluorescence-activated cell sorting (FACS) and JAK2 V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR. JAK2 V617F was enriched in neutrophils and maintained in erythroid progenitors (EPs) relative to HSCs. In contrast, JAK2 V617F alleles were largely absent in T and B cells relative to HSCs. Lymphopoiesis declines with age, potentially explaining the absence of JAK2 V617F in lymphocytes from older patients with MPN even if JAK2 V617F has no effect on lymphopoiesis. This “JAK2 V617F neutral” hypothesis predicts that patients who acquired the mutation at a younger age, and those who harbor the mutation for a longer duration, have a higher likelihood of accumulating JAK2mutated lymphocytes. However, we found that JAK2 V617F MAF in T and B cells across our cohort does not correlate with either patient age at diagnosis or duration of clinical disease. Thus, predictions of the “JAK2 V617F neutral” hypothesis are not supported by patient data. The alternative explanation is that JAK2 V617F impairs lymphocyte differentiation from mutated progenitors. We tested this “JAK2 V617F adverse” hypothesis by tracking JAK2 V617F during various stages of lymphopoiesis. To assess the relative bias of JAK2 V617F HSCs during the early stages of lymphoid and myeloid commitment, we measured JAK2 V617F MAF in common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs) and HSCs isolated by FACS from the peripheral blood of 79 patients with JAK2 V617F MPN. We found that while myeloid commitment from HSC to CMP was associated with an increase in mean JAK2 V617F MAF, there was no statistically significant change in mean MAF between CLPs and the parent HSCs across the cohort. Therefore, JAK2 V617F HSCs are not completely excluded from the earliest stages of lymphoid differentiation. To further define the extent to which JAK2 V617F affects lymphopoiesis, we used FACS to isolate CLPs and CMPs from 17 patients with JAK2 V617F polycythemia vera (PV) and differentiated each subpopulation in vitro into T cell progenitors (pro- and pre-T cells) and erythroblasts (EBs), respectively. We found that all CLP samples could be differentiated into T cell progenitors. However, we observed a marked depletion of JAK2 V617F alleles in progressively mature T cell progenitors (Figure 1). In contrast, JAK2 V617F alleles were further enriched in progressively mature erythroblasts. The in vitro differentiation potential of MPN progenitors thus supports the “JAK2V617F adverse” hypothesis. To determine the effect of JAK2 V617F on lymphopoiesis in vivo, we performed competitive transplantations of lethally irradiated CD45.1 mice with congenic CD45.2 donor whole bone marrow (WBM) cells harboring JAK2 V617F or wild type JAK2 (JAK2 WT). Measuring CD45.2 chimerism by FACS in lymphocytes and neutrophils from engrafted mice revealed that only CD45.2 cells harboring JAK2 V617F were preferentially depleted in lymphocytes (Figure 2). Therefore, JAK2 V617F HSCs displayed a competitive disadvantage relative to their JAK2 WT counterparts during lymphopoiesis in vivo. Myeloid proliferation in MPN driven by JAK2 V617F is well recognized but does not fully explain certain MPN complications such as increased risk of infections and second malignancies. Our findings demonstrate impaired lymphoid differentiation from JAK2 V617F stem and progenitor cells that may also contribute to clinical MPN phenotypes. Further study is ongoing to define actionable mechanisms through which JAK2 V617F interferes with T cell differentiation.

The naked mole-rat (NMR;Heterocephalus glaber) is a eusocial subterranean rodent with a highly unusual set of physiological traits that has attracted great interest amongst the scientific community. However, the genetic basis of most of these traits has not been elucidated. To facilitate our understanding of the molecular mechanisms underlying NMR physiology and behaviour, we generated a long-read chromosomal-level genome assembly of the NMR. This genome was subsequently annotated and incorporated into multiple whole genome alignments in the Ensembl database. Our long-read assembly identified thousands of repeats and genes that were previously unassembled in the NMR and improved the results of routinely used short-read sequencing-based experiments such as RNA-seq, snRNA-seq, and ATAC-seq. We identified several spermatozoa related gene losses that may underlie the unique degenerative sperm phenotype in NMRs (IRGC,FSCB,AKAP3,MROH2B,CATSPER1,DCDC2C,ATP1A4,TEKT5, andZAN), and an additional gene loss related to the established NK-cell absence in NMRs (PILRB). We resolved several tandem duplications in genes related to pathways underlying unique NMR adaptations including hypoxia tolerance, oxidative stress, and nervous system protection (TINF2,TCP1,KYAT1). Lastly, we describe our ongoing efforts to generate a reference telomere-to-telomere assembly in the NMR which includes the resolution of complex gene families. This new reference genome should accelerate the discovery of the genetic underpinnings of NMR physiology and adaptation.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

Cephalopods are set apart from other mollusks by their advanced behavioral abilities and the complexity of their nervous systems. Because of the great evolutionary distance that separates vertebrates from cephalopods, it is evident that higher cognitive features have evolved separately in these clades despite the similarities that they share. Alongside their complex behavioral abilities, cephalopods have evolved specialized cells and tissues, such as the chromatophores for camouflage or suckers to grasp prey. Despite significant progress in genome and transcriptome sequencing, the molecular identities of cell types in cephalopods remain largely unknown. We here combine single-cell transcriptomics with in situ gene expression analysis to uncover cell type diversity in the European squid Loligo vulgaris. We describe cell types that are conserved with other phyla such as neurons, muscles, or connective tissues but also cephalopod-specific cells, such as chromatophores or sucker cells. Moreover, we investigate major components of the squid nervous system including progenitor and developing cells, differentiated cells of the brain and optic lobes, as well as sensory systems of the head. Our study provides a molecular assessment for conserved and novel cell types in cephalopods and a framework for mapping the nervous system of L. vulgaris.

The extent of interspecific gene flow and its consequences for the initiation, maintenance, and breakdown of species barriers in natural systems remain poorly understood. Interspecific gene flow by hybridization may weaken adaptive divergence, but can be overcome by selection against hybrids, which may ultimately promote reinforcement. An informative step towards understanding the role of gene flow during speciation is to describe patterns of past gene flow among extant species. We investigate signals of admixture between allopatric and sympatric populations of the two closely related European dung fly species Sepsis cynipsea and S. neocynipsea (Diptera: Sepsidae). Based on microsatellite genotypes, we first inferred a baseline demographic history using Approximate Bayesian Computation. We then used genomic data from pooled DNA of natural and laboratory populations to test for past interspecific gene flow based on allelic configurations discordant with the inferred population tree (ABBA–BABA test with D -statistic). Comparing the detected signals of gene flow with the contemporary geographic relationship among interspecific pairs of populations (sympatric vs. allopatric), we made two contrasting observations. At one site in the French Cevennes, we detected an excess of past interspecific gene flow, while at two sites in Switzerland we observed lower signals of past microsatellite genotypes gene flow among populations in sympatry compared to allopatric populations. These results suggest that the species boundaries between these two species depend on the past and/or present eco-geographic context in Europe, which indicates that there is no uniform link between contemporary geographic proximity and past interspecific gene flow in natural populations.

Similar to human monocytes, bovine monocytes can be split into CD14 high CD16 - classical, CD14 high CD16 high intermediate and CD14 -/dim CD16 high nonclassical monocytes (cM, intM, and ncM, respectively). Here, we present an in-depth analysis of their steady-state bulk- and single-cell transcriptomes, highlighting both pronounced functional specializations and transcriptomic relatedness. Bulk gene transcription indicates pro-inflammatory and antibacterial roles of cM, while ncM and intM appear to be specialized in regulatory/anti-inflammatory functions and tissue repair, as well as antiviral responses and T-cell immunomodulation. Notably, intM stood out by high expression of several genes associated with antigen presentation. Anti-inflammatory and antiviral functions of ncM are further supported by dominant oxidative phosphorylation and selective strong responses to TLR7/8 ligands, respectively. Moreover, single-cell RNA-seq revealed previously unappreciated heterogeneity within cM and proposes intM as a transient differentiation intermediate between cM and ncM.

In the venom of spiders, linear peptides (LPs), also called cytolytical or antimicrobial peptides, represent a largely neglected group of mostly membrane active substances that contribute in some spider species considerably to the killing power of spider venom. By next-generation sequencing venom gland transcriptome analysis, we investigated 48 spider species from 23 spider families and detected LPs in 20 species, belonging to five spider families (Ctenidae, Lycosidae, Oxyopidae, Pisauridae, and Zodariidae). The structural diversity is extraordinary high in some species: the lynx spider Oxyopes heterophthalmus contains 62 and the lycosid Pardosa palustris 60 different LPs. In total, we identified 524 linear peptide structures and some of them are in lycosids identical on amino acid level. LPs are mainly encoded in complex precursor structures in which, after the signal peptide and propeptide, 13 or more LPs (Hogna radiata) are connected by linkers. Besides Cupiennius species, also in Oxyopidae, posttranslational modifications of some precursor structures result in the formation of two-chain peptides. It is obvious that complex precursor structures represent a very suitable and fast method to produce a high number and a high diversity of bioactive LPs as economically as possible. At least in Lycosidae, Oxyopidae, and in the genus Cupiennius, LPs reach very high Transcripts Per Kilobase Million values, indicating functional importance within the envenomation process.

Identifying local adaptation in bottlenecked species is essential for conservation management. Selection detection methods have an important role in species management plans, assessments of adaptive capacity, and looking for responses to climate change. Yet, the allele frequency changes exploited in selection detection methods are similar to those caused by the strong neutral genetic drift expected during a bottleneck. Consequently, it is often unclear what accuracy selection detection methods have across bottlenecked populations. In this study, simulations were used to explore if signals of selection could be confidently distinguished from genetic drift across 23 bottlenecked and reintroduced populations of Alpine ibex (Capra ibex). The meticulously recorded demographic history of the Alpine ibex was used to generate comprehensive simulated SNP data. The simulated SNPs were then used to benchmark the confidence we could place in outliers identified in empirical Alpine ibex RADseq derived SNP data. Within the simulated data set, the false positive rates were high for all selection detection methods (F-ST outlier scans and Genetic-Environment Association analyses) but fell substantially when two or more methods were combined. True positive rates were consistently low and became negligible with increased stringency. Despite finding many outlier loci in the empirical Alpine ibex SNPs, none could be distinguished from genetic drift-driven false positives. Unfortunately, the low true positive rate also prevents the exclusion of recent local adaptation within the Alpine ibex. The baselines and stringent approach outlined here should be applied to other bottlenecked species to ensure the risk of false positive, or negative, signals of selection are accounted for in conservation management plans.

Darwinian evolution preferentially follows mutational pathways whose individual steps increase fitness. Alternative pathways with mutational steps that do not increase fitness are less accessible. Here, we show that mistranslation, the erroneous incorporation of amino acids into nascent proteins, can increase the accessibility of such alternative pathways and, ultimately, of high fitness genotypes. We subject populations of the beta-lactamase TEM-1 to directed evolution in Escherichia coli under both low- and high-mistranslation rates, selecting for high activity on the antibiotic cefotaxime. Under low mistranslation rates, different evolving TEM-1 populations ascend the same high cefotaxime-resistance peak, which requires three canonical DNA mutations. In contrast, under high mistranslation rates they ascend three different high cefotaxime-resistance genotypes, which leads to higher genotypic diversity among populations. We experimentally reconstruct the adaptive DNA mutations and the potential evolutionary paths to these high cefotaxime-resistance genotypes. This reconstruction shows that some of the DNA mutations do not change fitness under low mistranslation, but cause a significant increase in fitness under high-mistranslation, which helps increase the accessibility of different high cefotaxime-resistance genotypes. In addition, these mutations form a network of pairwise epistatic interactions that leads to mutually exclusive evolutionary trajectories towards different high cefotaxime-resistance genotypes. Our observations demonstrate that protein mistranslation and the phenotypic mutations it causes can alter the evolutionary exploration of fitness landscapes and reduce the predictability of evolution.

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients’ CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN.

Summary Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine‐scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors. We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing. We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally. These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

Dendritic cells (DC) and monocytes are vital for the initiation of innate and adaptive immune responses. Recently, we identifiedbona fideDC subsets in blood of cattle, revealing subset- and species-specific transcription of toll-like receptors (TLR). In the present study, we analyzed phenotypic and transcriptional responses of bovine DC subsets and monocytes to in vitro stimulation with four to six different TLR ligands. Bovine DC subsets, especially plasmacytoid DC (pDC), showed a clear increase of CCR7, CD25, CD40, CD80, CD86, and MHC-II expression both on mRNA and protein level. Flow cytometric detection of p38 MAPK phosphorylation 15 min after stimulation confirmed activation of DC subsets and monocytes in accordance with TLR gene expression. Whole-transcriptome sequencing of sorted and TLR-stimulated subsets revealed potential ligand- and subset-specific regulation of genes associated with inflammation, T-cell co-stimulation, migration, metabolic reprogramming, and antiviral activity. Gardiquimod was found to evoke strong responses both in DC subsets and monocytes, while Poly(I:C) and CpG preferentially triggered responses in cDC1 and pDC, respectively. This in-depth analysis of ligand responsiveness is essential for the rational design of vaccine adjuvants in cattle, and provides a solid basis for comparative studies on DC and monocyte biology across species.