To address the considerable challenges posed by frequent Streptococcus agalactiae infections in tilapia aquaculture, we previously developed a strain of tilapia with enhanced resistance to this pathogen. Understanding the immune mechanisms behind this resistance is essential for advancing selective breeding strategies. This study concentrates on T-cell activation and effector function to explore the reasons for the more robust immunity in S. agalactiae-resistant tilapia. Through RNA-seq and immunological assays, we demonstrated that resistant tilapia possessed a higher proportion of T cells and enhanced T-cell effector functions compared to susceptible tilapia. Upon in vitro stimulation with anti-tilapia CD38 plus CD28 monoclonal antibodies (mAbs), or in vivo infection with S. agalactiae, spleen leukocytes from resistant tilapia showed significantly enhanced degrees of Erk1/2 and NF-kappa B p65 phosphorylation, which are classical signaling pathway of T-cell activation. Once activated, spleen lymphocytes from resistant tilapia expressed higher levels of inflammatory cytokines, such as TNF-alpha, IL-2, as well as cytotoxic molecule perforin A. Moreover, CD4-1+ T cells in the resistant tilapia produced greater amounts of interferon (IFN)-gamma, suggesting their superior T-cell effector functions. These findings collectively illustrate how enhanced T-cell activation and effector responses facilitate the optimized immune defenses of resistant tilapia. By shedding light on these mechanisms, our findings provide a valuable foundation for refining breeding programs aimed at increasing disease resistance in aquaculture.

B-cell responses rely on a tightly coordinated interplay between transcriptional programs and metabolic reprogramming. Upon activation, B cells remodel their metabolic profiles, with enhanced glutamine metabolism supporting biomass synthesis and proliferation. However, whether and how glutaminolysis underpins B-cell responses in early vertebrates remains largely unexplored. Here, using the Nile tilapia (Oreochromis niloticus) as a model, we demonstrate that IgM+ B cells markedly increase glutamine utilization upon activation. Glutamine deprivation impaired B-cell activation and proliferation, whereas glutamine supplementation promoted these processes. During Edwardsiella piscicida infection, pharmacological inhibition of glutamine metabolism significantly reduced the expansion of IgM+ B cells and compromised antibody secretion. Mechanistically, glutamine metabolism in tilapia IgM+ B cells was governed by the transcription factor c-Myc. Inhibition of c-Myc disrupted glutaminolysis, leading to diminished B-cell proliferation and antibody production. Upon activation, IgM+ B cells coordinated c-Myc expression and downstream glutamine metabolism through the mTORC1 and ERK signaling pathways, thereby coordinating metabolic and immune functions. Notably, this regulatory mechanism operated in a B cell-intrinsic manner and was independent of T-cell help. Collectively, our findings reveal that teleost B cells possess evolutionarily conserved and sophisticated immunometabolic regulatory programs. This study provides new insights into how metabolic pathways are integrated with immune signaling to control B-cell function, highlighting coordinated immunometabolic regulation as a fundamental mechanism underlying vertebrate B-cell immunity.

T cells utilize diverse T cell receptors (TCRs) to recognize antigenic peptides and mediate adaptive immunity. However, the organization and function of αβ T cells in early vertebrates remain poorly understood. Here, we systematically characterized the TCRα locus and αβ T cell responses in Nile tilapia (Oreochromis niloticus) during Edwardsiella piscicida infection. Genomic analysis revealed that the TCRα locus in Nile tilapia exhibits a distinctive Vα(61)-Jα(82)-Cα(1)-Vα(65) structure with conserved synteny in the flanking regions, while the constant region retains essential residues for TCR/CD3 complex assembly. We further generated monoclonal antibody against tilapia TCRα and TCRβ and confirmed their coexpression by the majority of CD3ε+ T cells, validating these cells as bona fide αβ T cells. Functional assays showed that CD3ε/CD28 monoclonal antibody stimulation induced robust αβ T cell activation, as evidenced by enhanced phosphorylation of S6, NF-κB, and ERK1/2, together with vigorous cellular proliferation. In vivo, E. piscicida infection triggered a pronounced expansion of αβ T cells, indicating their active involvement in antibacterial immunity. Importantly, depletion of αβ T cells severely impaired pathogen clearance and significantly increased host mortality. Together, these findings elucidate the indispensable role of αβ T cells in the antibacterial immunity of teleosts and provide critical insights into the functional mechanisms of adaptive immunity in early vertebrates.

Jawless vertebrates employ variable lymphocyte receptors (VLRs) to mount adaptive immunity. Although six VLRs (VLRA-F) and T/B-like lymphocytes are identified in lampreys, it remains unclear how VLR diversity is organized at the cellular level and whether individual lymphocytes may integrate multiple VLRs. Using single-cell transcriptomics, we identify a germline-encoded, mature, non-diversified VLR-like receptor (VLR-like) in lamprey Lethenteron reissneri. VLR-like retains structural features of canonical VLRs, and is a GPI-anchored receptor expressed by T-like cells. We generate a VLR-like monoclonal antibody, enabling the identification of VLR-like+ lymphocytes across multiple tissues. Upon PHA stimulation, VLR-like+ lymphocytes exhibit robust ERK and JNK phosphorylation and proliferation, consistent with T-like cell features. In parallel, VLR-like+ cells fail to phagocytose, present antigen, or secrete soluble VLR-like, indicating the absence of B-like characteristics. Notably, VLR-like+ cells are distinct from VLRA+ lymphocytes but comprise both VLR-like+VLRC+ and VLR-like+VLRC- populations. Consistently, VLR-like interacts with VLRC but not VLRA. Together, we demonstrate that VLR-like defines a distinct T-like population with heterogeneous VLRC co-expression, and suggest a mode of receptor organization in which an invariant VLR-like coordinates a diversified VLR within an individual T-like cell, potentially contributing to adaptive immunity in lamprey.

Streptococcus agalactiae infection remains a major constraint on the productivity of Nile tilapia (Oreochromis niloticus), a species of considerable economic importance in global aquaculture. Although selective breeding programs have successfully produced tilapia strains with enhanced resistance to this pathogen, the B cell-mediated immune mechanisms underlying this resistance remain unclear. In this study, we systematically investigated differences in IgM + B cell-mediated immune responses between S. agalactiae-resistant and -susceptible Nile tilapia. Transcriptomic analysis of splenic leukocytes revealed that genes associated with immunoglobulin production and B cell differentiation were markedly upregulated in resistant tilapia following infection. Consistent with these findings, flow cytometry further demonstrated a more pronounced expansion of splenic IgM+ B cells in resistant tilapia, which was primarily driven by significantly increased proliferative activity. Importantly, this expansion coincided with elevated activation-induced apoptosis, suggesting accelerated B cell turnover that could facilitate the selection and maintenance of functionally competent cells during the immune response. Functionally, IgM+ B cells from resistant tilapia exhibited sustained enhancement of antigen processing capacity following infection. Meanwhile, macrophage-mediated phagocytosis of S. agalactiae increased progressively and became particularly evident at later stages of infection. Moreover, IgM+ B cells in resistant tilapia maintained relatively stable intracellular ROS levels, whereas susceptible tilapia exhibited persistent oxidative stress. Collectively, these findings indicate that resistance to S. agalactiae infection in Nile tilapia is closely associated with coordinated functional remodeling of IgM + B cells, characterized by efficient proliferation, functional maturation, and stringent homeostatic regulation. This refined B cell-mediated immunity likely contributes to effective pathogen clearance and provides an immunological basis for the evaluation and selective breeding of disease-resistant tilapia strains.

Functional feed additives are increasingly incorporated into aquafeeds to improve growth performance, metabolic status, and overall health of farmed fish. However, their regulatory effects on adaptive immunity, particularly T cell-mediated immune responses, remain poorly characterized. In this study, we evaluated the effects of dietary ferulic acid supplementation on T cell immunity and antioxidant capacity in Nile tilapia (Oreochromis niloticus). Fish were fed diets supplemented with 0, 200, or 400 mg/kg ferulic acid and subsequently challenged with Aeromonas hydrophila. Compared with the basal diet group, supplementation with 200 mg/kg ferulic acid significantly enhanced T cell proliferation and survival during infection, accompanied by increased proportions and absolute numbers of CD4-1+ T cells. In parallel, ferulic acid supplementation elevated expression of key T cell functional molecules, including interferon-γ (IFN-γ), perforin A, tumor necrosis factor α (TNF-α), and Fas ligand (Fas-L), together with reduced bacterial burden, alleviated hepatic inflammatory infiltration and lesions, and improved survival. In addition, ferulic acid markedly enhanced antioxidant capacity in lymphocytes, as evidenced by increased activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), elevated total antioxidant capacity (T-AOC), and reduced malondialdehyde (MDA) levels. Consistently, intracellular reactive oxygen species (ROS) accumulation in T cells and the expression of pro-inflammatory genes were significantly reduced following 200 mg/kg ferulic acid supplementation. Taken together, we demonstrate that dietary ferulic acid supplementation functionally enhances T cell-mediated immune responses and antioxidant capacity in tilapia during bacterial infection, with 200 mg/kg identified as the optimal concentration, supporting its potential application as a functional feed additive in aquaculture.

CD8 + T cells are indispensable effectors of adaptive immunity. While cytokine-mediated regulation of CD8 + T cell responses has been extensively characterized in mammals, the underlying mechanisms in fish remain largely unexplored. Here, we identified and characterized an interleukin-21 (IL-21) gene from Nile tilapia ( Oreochromis niloticus). In vitro stimulation with IL-21 significantly upregulated the transcription and expression of key CD8 + T cell effector molecules, including Granzyme B and interferon-gamma (IFN-γ), in splenic leukocytes, indicating a crucial role of IL-21 in modulating CD8 + T cell function in teleosts. Using a monoclonal antibody (mAb) against Nile tilapia IL-21, we further demonstrated that activated CD4-1 + T cells are the primary producers of this cytokine in fish. Intraperitoneal injection of recombinant IL-21 into Edwardsiella piscicida-infected Nile tilapia enhanced both proliferation and apoptosis of CD8 + T cells. Moreover, IL-21 elevated the expression of Granzyme B, IFN-γ, and IL-2, and boosted CD8 + T cell cytotoxicity, thereby strengthening their effector functions. In contrast, blockade of IL-21 signaling with the mAb severely impaired CD8 + T cell responses. Mechanistically, Nile tilapia IL-21 strongly induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), which was required for IL-21-mediated CD8 + T cell proliferation in vitro. Inhibition of STAT3 phosphorylation in vivo attenuated CD8 + T cell responses and antibacterial immunity, underscoring its functional importance. Collectively, our findings establish IL-21 as a pivotal regulator of CD8 + T cell immunity in fish and provide evolutionary insight into conserved cytokine pathways shaping adaptive immune responses.

Under intensive aquaculture conditions, fish are exposed to multiple stressors that lead to oxidative stress and increased disease susceptibility. N-acetylcysteine (NAC) is a sulfur-containing antioxidant that enhances antioxidant and anti-inflammatory capacity, but its impact on T cell immunity in fish remains unclear. Our findings demonstrate that the improved pathogen clearance and survival in dietary NAC supplementation-treated Nile tilapia (Oreochromis niloticus) are associated with enhanced T cell immunity. Supplementation with 6 g/kg NAC enhanced the activation, proliferation, survival and cytotoxic function of T cells following Edwardsiella piscicida infection. NAC also increased the expression of the anti-inflammatory cytokines IL-10 and TGF-beta 1, while suppressing the production of IFN-gamma in tilapia CD4+ and CD8+ T cells, thereby promoting immune homeostasis. Furthermore, this dosage increased the enzyme activities of T-SOD and GST, reduced ROS accumulation in CD4+ and CD8+ T cells. Mechanistically, NAC activated the KEAP1-NRF2-mediated antioxidant pathway by suppressing KEAP1 expression and promoting nuclear translocation of NRF2. In contrast, 8 g/kg NAC did not confer stronger protection. This study systematically elucidates the mechanism by which NAC enhances disease resistance in tilapia through regulating T-cell immunity and antioxidant capacity, providing a theoretical basis for the rational application of NAC in aquafeeds.

The increasing intensity of aquaculture production has exposed farmed fish to persistent pathogen challenges and oxidative stress, highlighting the urgent need for safe and effective nutritional immunostimulants. Quercetin, a natural polyphenolic compound, is known for its antioxidant and anti-inflammatory properties; however, its regulatory effects on fish T-cell immunity remain poorly defined. In this study, we investigated the immunomodulatory role of dietary quercetin in Nile tilapia (Oreochromis niloticus), with a particular focus on Tcell-mediated immunity. Our results demonstrated that quercetin markedly alleviated T-cell damage induced by oxidative stress and bacterial infection by reducing intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels and suppressing apoptosis. This protective effect was primarily associated with the activation of endogenous antioxidant enzyme systems. Moreover, quercetin enhanced the phosphorylation of multiple signaling pathway components, thereby promoting T-cell activation and proliferative capacity. At the same time, quercetin effectively inhibited the NF-kappa B and JNK inflammatory signaling pathways, downregulating pro-inflammatory cytokines while upregulating anti-inflammatory factors, thus contributing to immune homeostasis. Functionally, quercetin supplementation increased the expression of T-cell cytotoxic effector molecules, improved pathogen clearance, and significantly reduced mortality in tilapia. Notably, a dietary dose of 400 mg/ kg consistently outperformed 800 mg/kg across most evaluated parameters. Therefore, this study demonstrates that quercetin enhances piscine T-cell immunity through coordinated antioxidant, anti-inflammatory, and immune-activating mechanisms, providing both a theoretical foundation and a practical dosage reference for the application of quercetin as an eco-friendly immunostimulant in aquaculture.

Mature T cells are functionally specialized through differential expression of CD4 or CD8, with CD4+ cells designated as helper T cells (Th cells) and CD8+ cells as cytotoxic T lymphocytes (CTLs). However, due to the lack of antibody tools, the functional mechanisms of T cell subsets in teleost remain poorly understood. In this study, we established NIH/3T3 cell lines expressing grass carp (Ctenopharyngodon idella) CD4-1 and CD8α through retroviral transduction, and used these as immunogens to generate monoclonal antibodies (mAbs) targeting the T cell co-receptors. Characterization results demonstrated that our mAbs exhibited high specificity in accurately identifying grass carp CD4-1+ and CD8α+ T cells. Subsequent applications revealed that CD4-1+ and CD8α+ T cells were widely distributed in immune-related tissues including spleen, head kidney, peripheral blood, and liver. Upon PHA stimulation, these T cell subsets showed enhanced phosphorylation levels of NF-κB p65 and MAPK/ERK signaling molecules. Furthermore, the proportion of CD4-1+ and CD8α+ T cells increased significantly in grass carp following infection with Edwardsiella piscicida. Overall, the mAbs developed in this study provide reliable tools for characterizing grass carp CD4-1+ and CD8α+ T cells and investigating their functions. These findings offer both technical support and theoretical foundations for understanding teleost T cell subset functionality and the evolution of adaptive immunity.

Bacterial diseases cause major economic losses in aquaculture, underscoring the need for feed additives that enhance fish immunity. Myo-inositol (MI), an essential nutrient for fish growth, possesses antioxidant and lipid-metabolism modulating properties. However, the role of MI in regulating T cell function remains unclear. In this study, Nile tilapia (Oreochromis niloticus) individuals were fed diets containing 850 or 1700 mg/kg MI to evaluate its effects on T cell immunity. The results demonstrated that dietary MI at 850 mg/kg significantly enhanced host anti-bacterial immunity in Nile tilapia by systematically improving T cell function. Specifically, during Edwardsiella piscicida infection, dietary MI increased the absolute number of T cells in Nile tilapia, and promoted their activation, proliferation, and cytokine production while reducing apoptosis. Mechanistically, MI elevated antioxidant enzymes expression to reduce intracellular reactive oxygen species (ROS) in T cells and drove fatty acid metabolic reprogramming. These coordinated enhancements collectively accelerated pathogen clearance and significantly improved host survival upon E. piscicida infection. Therefore, these findings reveal the pivotal role of dietary MI in promoting T cell mediated anti-bacterial immunity in Nile tilapia, and establish a foundation for using nutritional immunomodulation to improve health and disease resistance in farmed fish.

The programmed death ligand 1/programmed cell death 1 (PD-L1/PD-1) serves as a critical immune checkpoint in T cell-mediated immune responses, playing a central role in maintaining peripheral immune tolerance and homeostasis. Although early vertebrates lack a PD-1 homolog and they retain PD-L1, the functional significance and regulatory mechanisms of PD-L1 in T-cell immunity in these species remain poorly understood. In this study, using Nile tilapia (Oreochromis niloticus) as a teleost model, we systematically investigated the immunosuppressive role of PD-L1 in T-cell immunity. PD-L1 was widely expressed in various lymphoid tissues, and both its mRNA and protein levels were significantly upregulated following T-cell activation induced by mAbs against CD3/CD28 and Edwardsiella piscicida infection. Functional analyses demonstrated that exogenous PD-L1 treatment markedly suppressed T-cell activation and promoted activation-induced apoptosis. Moreover, PD-L1 treatment significantly impaired T-cell proliferation, effector cytokine production, and cytotoxic activity, ultimately compromising antibacterial immune defense. Mechanistically, activated tilapia T cells exhibited enhanced de novo fatty acid synthesis, whereas PD-L1 disrupted this metabolic reprogramming by inhibiting key enzymatic activities and reducing acetyl-CoA accumulation, ultimately leading to impaired T-cell function. Notably, restoration of fatty acid synthesis effectively reversed the PD-L1-induced immunosuppressive effects on tilapia T cells. In summary, this study reveals a previously unrecognized mechanism by which PD-L1 suppresses T-cell immunity in an early vertebrate through modulation of lipid metabolism. These findings highlight the essential role of metabolism regulation in immune checkpoint signaling and provide new evolutionary insights into the maintenance of T-cell homeostasis.

T cells cooperate with the intestinal microbiota to coordinate antimicrobial defense, but whether this crosstalk arose as an independent innovation in mammals or represents an evolutionarily conserved feature of vertebrate immunity remains unknown. Using the teleost Nile tilapia as a model, we demonstrate that both systemic and localized infection with Edwardsiella piscicida induce enteritis, correlated with robust intestinal T cell responses. Selective T cell depletion triggered excessive expression of proinflammatory cytokines, impaired mucosal architecture, and diminished host resistance to infection, underscoring the essential role of T cells in gut immunity. Strikingly, T cell depletion also caused profound alterations in gut microbial composition, characterized by a sharp decline in beneficial taxa such as Cetobacterium and the expansion of opportunistic pathogens including Klebsiella and Acinetobacter, indicating that T cells are required to maintain microbiome homeostasis. Conversely, broad-spectrum antibiotic eradication of the microbiota provoked hyperproliferation of intestinal T cells and barrier disruption, revealing reciprocal regulation between T cells and commensals. From the gut content, we isolated a C. somerae strain SH518, whose dietary supplementation for 6–8 weeks enhanced the activation, proliferation, and effector function of intestinal T cells, preserved mucosal homeostasis during E. piscicida challenge, and even boosted systemic T cell immunity in the spleen. Collectively, these findings demonstrate that teleost T cells engage in bidirectional interactions with gut microbiota to orchestrate both antimicrobial defense and mucosal homeostasis. We therefore propose that T cell–microbiota cooperation represents an evolutionarily ancient strategy predates terrestrial adaptation, offering new insights into the coevolution of mucosal T cell immunity and microbiome.

Although bony fish have CD8+ T cells, the mechanisms by which these early-evolved cytotoxic cells combat intracellular pathogens remain unclear. In the present study, using Nile tilapia as a model, we investigated the detailed function, mechanism, and evolutionary pattern concerning CD8+ T cells. By depleting CD8+ T cells, they are found essential in combating Edwardsiella piscicida infection. Using siRNA interference, we propose that unlike the strategy predominantly relying on perforin/granzyme in mammals, CD8+ T-cell effector function is mediated by both FasL and perforin/granzyme in fish. Upon E. piscicida infection, FasL is induced to express in CD8+ T cells; both recombinant FasL and adoptively transferred FasL+CD8+ T cells facilitate the apoptosis of target cells. Meanwhile, tilapia FasL also triggers the apoptosis of T cells to archive homeostasis. Since advances in mammals highlight the indispensable role of FasL in maintaining CD8+ T-cell homeostasis, rather than in effector function or anti-infective immunity, we therefore propose the unique dual function of FasL in executing effector function and maintaining homeostasis in fish. Mechanistically, tilapia T cells utilize mTORC1/c-Myc axis to regulate pathogen-induced FasL expression, which binds to Fas and activates caspase-8/caspase-3 pathway, mediating apoptosis in target cells and T cells themselves. This represents a novel mechanism underpinning CD8+ T-cell function in fish. Our findings demonstrate that CD8+ T cells reshaped the FasL-dependent strategy throughout evolution, thereby enhancing the precision and specificity of adaptive immunity.

The integration of metabolic programs with T cell signaling establishes a molecular foundation for immune metabolism. As a key metabolic regulator, GSK3β's activity is dynamically modulated by phosphorylation at Ser9 and Tyr216. However, the contribution of these phosphorylation sites on metabolism-driven T cell response remains unclear. Using tilapia and mouse models, we investigated the regulation of GSK3β on T cell metabolism and its evolutionary variation. In tilapia, T cell activation induces GSK3β signaling, linking to both glycolysis and oxidative phosphorylation (OXPHOS). Tyr216 phosphorylation preferentially promotes glycolysis, facilitating T cell activation, proliferation, and antibacterial immunity; while inhibition of Ser9 phosphorylation specifically enhances OXPHOS to sustain T cell responses. Differently, Tyr216 phosphorylation supports both glycolysis and OXPHOS in mouse, ensuring CD4+ T and CD8+ T cell activation, proliferation, and cytokine production. Although Ser9 phosphorylation controls OXPHOS, its inhibition impairs rather than enhances OXPHOS and CD4+ T cell responses in mouse. We thus revealed a previously unknown mechanism underlying T cell metabolism and proposed that, through evolution, GSK3β has restructured the regulatory strategy, enabling bidirectional control of T cell metabolism and immunity in mammals and enhancing the flexibility of the adaptive immune system.

T and B lymphocytes are the primary effectors of the immune system's specific anti-infective functions, abundantly concentrated in immune-related tissues and organs. The two cell types work in different mechanisms to combat pathogens through precision guidance and jointly accomplish the immune response. Previous studies have elucidated the critical role of splenic T cells in antimicrobial infections in Nile tilapia (Oreochromis niloticus). On this basis, this study aimed to further reveal the distribution and effector functions of T and B lymphocytes in the head kidney and peripheral blood of teleosts. Here, the distribution and effector functions of T cells and IgM+ B cells were examined using a model of Edwardsiella piscicida infection in Nile tilapia. The results verified that T and B cells were distributed in the peripheral blood and spleen of Nile tilapia, with primary and secondary infection induced remarkable increases of T cells and IgM+ B cells in the head kidney and peripheral blood. Notably, T cells and IgM+ B cells proliferated rapidly in secondary infection than initial immune response, implying that immune memory as a hallmark feature of adaptive immunity was already present in early vertebrates. More importantly, the inhibition of T cells during bacterial infection impaired the expression of perforin A and granzyme B. Thus, this study indicated the distribution and effector functions of T cells and IgM+ B cells during specific immunity in Nile tilapia, and provided theoretical support for understanding the evolutionary basis of the adaptive immune system.

The adiponectin (ADPN) receptor (AdipoR) modulates T-cell responses, but its effects remain controversial since signaling can either promote or inhibit T-cell function. Interaction with the ligand ADPN inhibits T-cell responses, but given the existence of multiple AdipoR ligands, we hypothesize that ligand diversity underlies its differential effect in T-cell immunity. To test this, we use tilapia and mouse models. Tilapia encodes AdipoR1 but lacks ADPN. Instead, an alternative adipokine, CTRP9, engages AdipoR1. We find CTRP9–AdipoR1 interaction triggers Ca 2+ influx and activates the CaM–CaMKKβ–AMPK pathway, facilitating crosstalk with TCR signaling. This cascade enhances T-cell activation, proliferation, and antimicrobial immunity by promoting glycolysis. In mice, CTRP9 similarly enhances T-cell activation, proliferation, and cytokine production and improves the efficacy of anti-CD19 CAR-T cells in eliminating B-cell lymphoma in vitro. These findings reveal an evolutionarily conserved role of CTRP9 in promoting T-cell immunity, in contrast to the inhibitory effect exerted by ADPN. Mechanistically, CTRP9 and ADPN exert distinct effects on T-cell metabolism; CTRP9 enhances T-cell glycolysis, whereas ADPN suppresses it. We therefore propose ligand selectivity as a determinant of AdipoR1-dependent T-cell immune outcomes.

As a hallmark molecule of T cells, CD3ε forms a complex with T-cell receptor (TCR) to transduce antigen signals and drive T cell activation, playing a pivotal role in T cell-mediated immune response. However, the lack of specific monoclonal antibodies (mAbs) targeting CD3ε in fish has substantially impeded the study on adaptive immunity in these species. In this study, we constructed NIH/3T3 cells expressing CD3ε of the large yellow croaker (Larimichthys crocea) via retroviral transduction. Using these cells as immunogen to immune mice, we generated a mAb that specifically recognized a population of spleen leukocytes after cell fusion and screening. This identified population specifically expressed CD3ε, CD4-1 or CD8α, suggesting that it was T cell. Moreover, immunofluorescence demonstrated that the mAb could bind to the surface of some leukocytes, and it was detected as the IgG1 type. These results confirmed the specificity of this CD3ε mAb and its applicability to identify T cells in the large yellow croaker. Subsequently, we revealed the widespread distribution of CD3ε+ T cells in immune-related tissues including spleen, liver, head kidney, gill and peripheral blood by using this mAb. Upon PHA stimulation, the phosphorylation of mTORC1 and MAPK/ERK were enhanced in CD3ε+ T cells. More importantly, this CD3ε mAb could mimic antigenic signaling to induce T cell activation in vitro, since its treatment activated the mTORC1, MAPK/ERK and Ca2+ pathways which were crucial for T cell activation. Therefore, we generated a CD3ε mAb, which could not only identify but also in vitro activate T cells of the large yellow croaker, providing critical tools for investigating T-cell immune in teleost.

Interleukin-4/13 (IL-4/13) is a kind of bony fish cytokine, which is the homolog of Th2 cytokines IL-4 or IL-13 in mammals. Produced by activated T cells, these Th2 cytokines regulate B cell proliferation, survival, and antibody responses. Although IL-4/13 has been shown to participate in immune responses in bony fish, whether and how it regulates the teleost-specific IgT+ B cell subset, a lineage unique to bony fish remains unclear. In present study, we identified two conserved IL-4/13 from the Nile tilapia Oreochromis niloticus (OnIL-4/13A1 and OnIL-4/13A2), and suggested their wide expression in immunological tissues. OnIL-4/13A1 transcription was markedly upregulated in spleen and liver leukocytes upon bacterial infection, and can be dramatically induced by PHA- or CD3 mAb-induced T cell activation, suggesting activated T cells are a key cellular source of this cytokine. More importantly, we found that 2- or 4-days' in vivo administration of the recombinant OnIL-4/13A1 (rOnIL-4/13A1) caused an obvious expansion of IgT+ but not IgM+ B cells in tilapia. Further investigation demonstrated that this expansion was due to cellular proliferation, because the BrdU incorporation in IgT+ B cells was obviously increased, while the Erk1/2 and NF-κB phosphorylation that contributing to the proliferation was also enhanced in IgT+ B cells. Interestingly, as revealed by the Annexin V staining, we found that rOnIL-4/13A1 treatment elevated the apoptosis of IgT+ B cells, probably by initiating the Caspase-8/Caspase-3 pathway. Therefore, our results suggest that IL-4/13A1 involves in the anti-bacterial immune response of tilapia by promoting IgT+ B cell proliferation and maintaining IgT+ B cell hemostasis, thus shed novel light on the B cells response in bony fish.

Immunoglobulin T (IgT) is a class of Ig unique to teleost, with current research largely supporting its role in mucosal immunity. However, the precise function of IgT in many kinds of teleost remains poorly understood due to a lack of appropriate antibody tools. In this study, we prepared the recombinant protein of Oreochromis niloticus IgT heavy chain (rOn-IgT) and generated a monoclonal antibody (mAb). Through enzyme-linked immunosorbent assay (ELISA) and flow cytometry screening, the 1B3H4 hybridoma was identified as producing an IgG1-type antibody, which can recognize IgT and identify IgT + B cells in tilapia. The facts that this candidate mAb generates a lymphocyte population distinct with both IgM + B cells and CD3 + T cells, and IgT was exclusively expressed in IgT + but not IgT - lymphocytes, validates the specificity of this tilapia IgT mAb. IgT mRNA and protein is widely distributed across various secondary immune and mucosal tissues in tilapia, with IgT + B cells present in varying proportions. Upon lipopolysaccharide (LPS) stimulation, IgT expression in spleen leukocytes was significantly increased at both the mRNA and protein levels, accompanied by a marked rise in the proportion of IgT + B cells. In addition, LPS stimulation also led to an increase in the phosphorylation levels of NF-κB and ERK1/2 in IgT + B cells. Moreover, we suggested that tilapia IgT + B cells possess robust phagocytic capabilities towards Edwardsiella piscicida, suggesting a potential role in immune response. Overall, we generated an mAb for tilapia IgT, investigated the expression of IgT and distribution of IgT + B cells both in various tissues and upon LPS stimulation, and revealed the phagocytosis of IgT + B cells in tilapia. This study would provide value tool and technical support to understand the immune function of IgT + B cells in teleost.