Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Annual rhythm in immune functions of blood leucocytes in an ophidian, Natrix piscator.

Annual variations in animal's physiological functions are an essential strategy to deal with seasonal challenges which also vary according to the time of year. Information regarding annual adaptations in the immune-competence to cope with seasonal stressors in reptiles is scarce. The present research plan was designed to analyze the presence of circannual immune rhythms in defense responses of the leucocytes in an ophidian, Natrix piscator. Peripheral blood leucocytes were obtained, counted, and superoxide anion production, neutrophil phagocytosis, and nitrite release were tested to assess the innate immune functions. Peripheral blood lymphocytes were separated by centrifugation (utilizing density gradient) and the cell proliferation was measured. The Cosinor rhythmometry disclosed the presence of significant annual rhythms in the number of leucocytes, superoxide anion production, nitric oxide production, and proliferation of stimulated lymphocytes. The authors found that respiratory burst activity and proliferative responses of lymphocytes were crucial immune responses that showed the annual rhythm. It was summarized that the immune function of the N. piscator is a labile attribute that makes the animal competent to cope with the seasonal stressor by adjustment in the potency of response.

MNDA, a PYHIN factor involved in transcriptional regulation and apoptosis control in leukocytes.

Inflammation control is critical during the innate immune response. Such response is triggered by the detection of molecules originating from pathogens or damaged host cells by pattern-recognition receptors (PRRs). PRRs subsequently initiate intra-cellular signalling through different pathways, resulting in i) the production of inflammatory cytokines, including type I interferon (IFN), and ii) the initiation of a cascade of events that promote both immediate host responses as well as adaptive immune responses. All human PYRIN and HIN-200 domains (PYHIN) protein family members were initially proposed to be PRRs, although this view has been challenged by reports that revealed their impact on other cellular mechanisms. Of relevance here, the human PYHIN factor myeloid nuclear differentiation antigen (MNDA) has recently been shown to directly control the transcription of genes encoding factors that regulate programmed cell death and inflammation. While MNDA is mainly found in the nucleus of leukocytes of both myeloid (neutrophils and monocytes) and lymphoid (B-cell) origin, its subcellular localization has been shown to be modulated in response to genotoxic agents that induce apoptosis and by bacterial constituents, mediators of inflammation. Prior studies have noted the importance of MNDA as a marker for certain forms of lymphoma, and as a clinical prognostic factor for hematopoietic diseases characterized by defective regulation of apoptosis. Abnormal expression of MNDA has also been associated with altered levels of cytokines and other inflammatory mediators. Refining our comprehension of the regulatory mechanisms governing the expression of MNDA and other PYHIN proteins, as well as enhancing our definition of their molecular functions, could significantly influence the management and treatment strategies of numerous human diseases. Here, we review the current state of knowledge regarding PYHIN proteins and their role in innate and adaptive immune responses. Emphasis will be placed on the regulation, function, and relevance of MNDA expression in the control of gene transcription and RNA stability during cell death and inflammation.

TLRs1-10 Protein Expression in Circulating Human White Blood Cells during Bacterial and COVID-19 Infections.

INTRODUCTION: Toll-like receptors play crucial roles in the sepsis-induced systemic inflammatory response. Septic shock mortality correlates with overexpression of neutrophilic TLR2 and TLR9, while the role of TLR4 overexpression remains a debate. In addition, TLRs are involved in the pathogenesis of viral infections such as COVID-19, where the single-stranded RNA of SARS-CoV-2 is recognized by TLR7 and TLR8, and the spike protein activates TLR4. METHODS: In this study, we conducted a comprehensive analysis of TLRs 1-10 expressions in white blood cells from 71 patients with bacterial and viral infections. Patients were divided into 4 groups based on disease type and severity (sepsis, septic shock, moderate, and severe COVID-19) and compared to 7 healthy volunteers. RESULTS: We observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in septic shock neutrophils compared to the control group (p < 0.001). Severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils compared to controls (p < 0.05). Septic shock patients also showed a similar increase in TLR7 in neutrophils along with elevated intermediate monocytes (CD14+CD16+) compared to the control group (p < 0.005 and p < 0.001, respectively). However, TLR expression remained unchanged in lymphocytes. CONCLUSION: This study provides further insights into the mechanisms of TLR activation in various infectious conditions. Additional analysis is needed to assess their correlation with patient outcome and to evaluate the impact of TLR-pathway modulation during septic shock and severe COVID-19.

The role of leukocytes in myeloproliferative neoplasm thromboinflammation.

Classic myeloproliferative neoplasms lacking the Philadelphia chromosome are stem cell disorders characterized by the proliferation of myeloid cells in the bone marrow and increased counts of peripheral blood cells. The occurrence of thrombotic events is a common complication in myeloproliferative neoplasms. The heightened levels of cytokines play a substantial role in the morbidity and mortality of these patients, establishing a persistent proinflammatory condition that culminates in thrombosis. The etiology of thrombosis remains intricate and multifaceted, involving blood cells and endothelial dysfunction, the inflammatory state, and the coagulation cascade, leading to hypercoagulability. Leukocytes play a pivotal role in the thromboinflammatory process of myeloproliferative neoplasms by releasing various proinflammatory and prothrombotic factors as well as interacting with other cells, which contributes to the amplification of the clotting cascade and subsequent thrombosis. The correlation between increased leukocyte counts and thrombotic risk has been established. However, there is a need for an accurate biomarker to assess leukocyte activation. Lastly, tailored treatments to address the thrombotic risk in myeloproliferative neoplasms are needed. Therefore, this review aims to summarize the potential mechanisms of leukocyte involvement in myeloproliferative neoplasm thromboinflammation, propose potential biomarkers for leukocyte activation, and discuss promising treatment options for controlling myeloproliferative neoplasm thromboinflammation.

Albumin influences leucocyte FcRn expression in the early days of kidney transplantation.

FcRn, a receptor originally known for its involvement in IgG and albumin transcytosis and recycling, is also important in the establishment of the innate and adaptive immune response. Dysregulation of the immune response has been associated with variations in FcRn expression, as observed in cancer. Recently, a link between autophagy and FcRn expression has been demonstrated. Knowing that autophagy is strongly involved in the development of reperfusion injury in kidney transplantation and that albuminemia is transiently decreased in the first 2 weeks after transplantation, we investigated variations in FcRn expression after kidney transplantation. We monitored FcRn levels by flow cytometry in leukocytes from 25 renal transplant patients and considered parameters such as albumin concentrations, estimated glomerular filtration rate, serum creatinine, serum IgG levels, and ischaemia/reperfusion time. Two groups of patients could be distinguished according to their increased or non-increased FcRn expression levels between days 2 and 6 (d2-d6) post-transplantation. Leukocyte FcRn expression at d2-d6 was correlated with albumin concentrations at d0-d2. These results suggest that albumin concentrations at d0-d2 influence FcRn expression at d2-d6, raising new questions about the mechanisms underlying these original observations.

Determination of Cuproptosis-related Subtypes, Development of a Prognostic Model, and Characterization of Tumor Microenvironment Infiltration in Acute Myeloid Leukemia.

BACKGROUND/AIM: Acute myeloid leukemia (AML) is a severe malignancy of the bone marrow marked by an abnormal accumulation of bone marrow precursors. Cuproptosis is a recently identified type of copper-dependent regulatory cell apoptosis that relies on mitochondrial respiration. However, its participation in the development of AML remains unclear. This study analyzed the association between cuproptosis-related genes and the prognosis of AML patients. MATERIALS AND METHODS: Cases of AML were acquired from TCGA, GEO, and TARGET and the molecular subgroups characterized by genes associated with cuproptosis, besides the associated cell infiltration of the tumor microenvironment (TME) were investigated. The cuproptosis score was developed using the minor absolute shrinkage and selection operator (LASSO) tool to evaluate the cuproptosis features of a single tumor sample. RESULTS: Two distinct molecular subgroups related to cuproptosis were discovered in AML with different prognoses. The cellular infiltration assay of TME showed immunological heterogeneity between the two subtypes. The cuproptosis score predicted tumor subgroups, immunity, and prognosis. A small cuproptosis value was marked by a good prognosis, whereas the anti-PD-1/PD-L1 immunotherapy group suggested the same cuproptosis group was related to an elevated immunotherapy potency. CONCLUSION: The cuproptosis score is a biomarker important for determining the molecular subgroups, prognosis, TME cell infiltration features, and immunotherapeutic efficacy of individuals with leukemia.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

Clinical manifestations and expression of CD18 to guide the diagnosis of leukocyte adhesion deficiency type 1

Background: Leukocyte adhesion deficiency type 1 (LAD-1) is an inborn error of immunity characterized by a defect in leukocyte trafficking. Methods: Patients with clinical suspicion of LAD-1 were referred to our institution. Complete blood count and flow cytometric analysis, to identify the expression of CD18, CD11b, and the lymphocyte population phenotyping, were performed, and statistical analysis was completed. Results: We report clinical manifestations and immunological findings of six Mexican patients diagnosed with LAD-1. The diagnosis was based on typical clinical presentation, combined with laboratory demonstration of leukocytosis, and significant reduction or near absence of CD18 and its associated molecules CD11a, CD11b, and CD11c on leukocytes. We found atypical manifestations, not described in other countries, such as early-onset autoimmunity or infections caused by certain microorganisms. Conclusions: Patients with LAD-1 may present with atypical manifestations, making flow cytometry an indispensable tool to confirm the diagnosis. We present the first report of LAD-1 patients in a Latin American country (AU)

A physical wiring diagram for the human immune system.

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention.

Brain-associated innate leukocytes display diverse inflammatory states following experimental stroke.

Previous studies investigating innate leukocyte recruitment into the brain after cerebral ischemia have shown conflicting results. Using distinct cell surface and intracellular markers, the current study evaluated the contributions of innate immune cells to the poststroke brain following 1-h middle cerebral artery occlusion (tMCAO) or permanent MCAO (pMCAO), and assessed whether these cells ascribed to an inflammatory state. Moreover, we examined whether there is evidence for leukocyte infiltration into the contralateral (CL) hemisphere despite the absence of stroke infarct. We observed the recruitment of peripheral neutrophils, monocytes and macrophages into the hemisphere ipsilateral (IL) to the ischemic brain infarct at 24 and 96 h following both tMCAO and pMCAO. In addition, we found evidence of increased leukocyte recruitment to the CL hemisphere but to a lesser extent than the IL hemisphere after stroke. Robust production of intracellular cytokines in the innate immune cell types examined was most evident at 24 h after pMCAO. Specifically, brain-associated neutrophils, monocytes and macrophages demonstrated stroke-induced production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß, while only monocytes and macrophages exhibit a significant expression of arginase 1 (Arg1) after stroke. At 96 h after stroke, brain-resident microglia demonstrated production of TNF-α and IL-1ß following both tMCAO and pMCAO. At this later timepoint, neutrophils displayed TNF-α production and brain-associated macrophages exhibited elevation of IL-1ß and Arg1 after tMCAO. Further, pMCAO induced significant expression of Arg1 and IL-1ß in monocytes and macrophages at 96 h, respectively. These results revealed that brain-associated innate immune cells display various stroke-induced inflammatory states that are dependent on the experimental stroke setting.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Identification of Potential Key Biomarkers of Atrial Fibrillation and Their Correlation with Immune Infiltration in Atrial Tissue.

Objective: To identify potential key biomarkers and characterize immune infiltration in atrial tissue of patients with atrial fibrillation (AF) through bioinformatics analysis. Methods: Differentially expressed genes (DEGs) were identified by the LIMMA package in Bioconductor, and functional and pathway enrichment analyses were undertaken using GO and KEGG. The LASSO logistic regression and BORUTA algorithm were employed to screen for potential novel key markers of AF from all DEGs. Gene set variation analysis was also performed. Single-sample gene set enrichment analysis was employed to quantify the infiltration levels for each immune cell type, and the correlation between hub genes and infiltrating immune cells was analyzed. Results: A total of 52 DEGs were identified, including of 26 downregulated DEGs and 26 upregulated DEGs. DEGs were primarily enriched in the Major Histocompatibility Complex class II protein complex, glucose homeostasis, protein tetramerization, regulation of synapse organization, cytokine activity, heart morphogenesis, and blood circulation. Three downregulated genes and three upregulated genes were screened by LASSO logistic regression and the BORUTA algorithm. Finally, immune infiltration analysis indicated that the atrial tissue of AF patients contained significant infiltration of APC_co_inhibition, Mast_cell, neutrophils, pDCs, T_cell_costimulation, and Th1_cells compared with paired sinus rhythm (SR) atrial tissue, and the three downregulated genes were negatively correlated with the six kinds of immune cells mentioned above. Conclusion: The hub genes identified in this study and the differences in immune infiltration of atrial tissue observed between AF and SR tissue might help to characterize the occurrence and progression of AF.

Detection of Phosphatidylserine Exposure on Leukocytes Following Treatment with Human Galectins.

Cellular turnover represents a fundamental aspect of immunological homeostasis. While many factors appear to regulate leukocyte removal during inflammatory resolution, recent studies suggest that members of the galectin family play a unique role in orchestrating this process. Unlike cellular removal through apoptotic cell death, several members of the galectin family induce surface expression of phosphatidylserine (PS), a phagocytic marker on cells undergoing apoptosis, in the absence of cell death. However, similar to PS on cells undergoing apoptosis, galectin-induced PS exposure sensitizes cells to phagocytic removal. As galectins appear to prepare cells for phagocytic removal without actually inducing apoptotic cell death, this process has recently been coined preaparesis. Given the unique characteristics of galectin-induced PS exposure in the context of preaparesis, we will examine unique considerations when evaluating the potential impact of different galectin family members on PS exposure and cell viability.

Methods for Assessing the Effects of Galectins on Leukocyte Trafficking and Clearance.

Numerous protocols exist for investigating leukocyte recruitment and clearance both in vitro and in vivo. Here we describe an in vitro flow chamber assay typically used for studying the mechanisms underpinning leukocyte movement through the endothelium and zymosan-induced peritonitis, an acute in vivo model of inflammation that enables both leukocyte trafficking and clearance to be monitored. Insight is given as to how these models can be used to study the actions of galectins on the inflammatory process.

Neural control of immune cell trafficking.

Leukocyte trafficking between blood and tissues is an essential function of the immune system that facilitates humoral and cellular immune responses. Within tissues, leukocytes perform surveillance and effector functions via cell motility and migration toward sites of tissue damage, infection, or inflammation. Neurotransmitters that are produced by the nervous system influence leukocyte trafficking around the body and the interstitial migration of immune cells in tissues. Neural regulation of leukocyte dynamics is influenced by circadian rhythms and altered by stress and disease. This review examines current knowledge of neuro-immune interactions that regulate leukocyte migration and consequences for protective immunity against infections and cancer.

PlexinD1 Deficiency in Lung Interstitial Macrophages Exacerbates House Dust Mite-Induced Allergic Asthma.

Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)-induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow-derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.

Deep Phenotypic Analysis of Blood and Lymphoid T and NK Cells From HIV+ Controllers and ART-Suppressed Individuals.

T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56brightTCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features.

RNA-Seq of Single Fish Cells - Seeking Out the Leukocytes Mediating Immunity in Teleost Fishes.

The immune system is a complex and sophisticated biological system, spanning multiple levels of complexity, from the molecular level to that of tissue. Our current understanding of its function and complexity, of the heterogeneity of leukocytes, is a result of decades of concentrated efforts to delineate cellular markers using conventional methods of antibody screening and antigen identification. In mammalian models, this led to in-depth understanding of individual leukocyte subsets, their phenotypes, and their roles in health and disease. The field was further propelled forward by the development of single-cell (sc) RNA-seq technologies, offering an even broader and more integrated view of how cells work together to generate a particular response. Consequently, the adoption of scRNA-seq revealed the unexpected plasticity and heterogeneity of leukocyte populations and shifted several long-standing paradigms of immunology. This review article highlights the unprecedented opportunities offered by scRNA-seq technology to unveil the individual contributions of leukocyte subsets and their crosstalk in generating the overall immune responses in bony fishes. Single-cell transcriptomics allow identifying unseen relationships, and formulating novel hypotheses tailored for teleost species, without the need to rely on the limited number of fish-specific antibodies and pre-selected markers. Several recent studies on single-cell transcriptomes of fish have already identified previously unnoticed expression signatures and provided astonishing insights into the diversity of teleost leukocytes and the evolution of vertebrate immunity. Without a doubt, scRNA-seq in tandem with bioinformatics tools and state-of-the-art methods, will facilitate studying the teleost immune system by not only defining key markers, but also teaching us about lymphoid tissue organization, development/differentiation, cell-cell interactions, antigen receptor repertoires, states of health and disease, all across time and space in fishes. These advances will invite more researchers to develop the tools necessary to explore the immunology of fishes, which remain non-conventional animal models from which we have much to learn.