Plasticity and lineage commitment of individual TH1 cells are determined by stable T-bet expression quantities.

T helper 1 (TH1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated TH1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus-induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the TH2 lineage: T-bet quantities were inversely correlated with the ability to express the TH2 lineage-specifying transcription factor GATA-3 and TH2 cytokines. Reprogramed TH1 cells acquired graded mixed TH1 + TH2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated TH1 cells was essential to ensure TH1 cell stability. Thus, innate cytokine signals regulate TH1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.

The aMPPle differentiation potential of TH2 cells in human allergy.

Single-cell studies of human tissues reveal a stem-like TH2 subset as progenitors of key effectors in chronic type 2 inflammation.

Triggers for eosinophilic esophagitis (EoE): The intersection of food allergy and EoE.

Eosinophilic esophagitis and IgE-mediated food allergy are both food-triggered diseases that are increasing in prevalence. They share many clinical links, including significant comorbidity and similar food triggers, and as atopic diseases, they likely share upstream mechanisms related to barrier function and signals leading to TH2 skewing. In this review, we focus on links between eosinophilic esophagitis and IgE-mediated food allergy with an emphasis on what insights may be derived from overlapping food triggers and immune phenotypes. Through further investigation of these connections, we may be able to better understand not only IgE-mediated food allergy and eosinophilic esophagitis but also general atopic response to food proteins and evolution of allergic response to food.

Canine peripheral non-conventional TCRαß+ CD4-CD8α- double-negative T cells show T helper 2-like and regulatory properties.

The dog is an important companion animal and also serves as model species for human diseases. Given the central role of T cells in immune responses, a basic understanding of canine conventional T cell receptor (TCR)αß+ T cells, comprising CD4+ single-positive (sp) T helper (Th) and CD8α+ sp cytotoxic T cell subsets, is available. However, characterization of canine non-conventional TCRαß+ CD4+CD8α+ double-positive (dp) and TCRαß+ CD4-CD8α- double-negative (dn) T cells is limited. In this study, we performed a comprehensive analysis of canine dp and dn T cells in comparison with their conventional counterparts. TCRαß+ T cells from peripheral blood of healthy dogs were sorted according to their CD4/CD8α phenotype into four populations (i.e. CD4+ sp, CD8α+ sp, dp, and dn) and selected surface markers, transcription factors and effector molecules were analyzed ex vivo and after in vitro stimulation by RT-qPCR. Novel characteristics of canine dp T cells were identified, expanding the previously characterized Th1-like phenotype to Th17-like and Th2-like properties. Overall, mRNA expression of various Th cell-associated cytokines (i.e. IFNG, IL17A, IL4, IL13) in dp T cells upon stimulation highlights their versatile immunological potential. Furthermore, we demonstrated that the CD4-CD8α- dn phenotype is stable during in vitro stimulation. Strikingly, dn T cells were found to express highest mRNA levels of type 2 effector cytokines (IL4, IL5, and IL13) upon stimulation. Their strong ability to produce IL-4 was confirmed at the protein level. Upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn than in the conventional CD4+ sp population. Constitutive transcription of IL1RL1 (encoding IL-33Rα) further supports Th2-like properties within the dn T cell population. These data point to a role of dn T cells in type 2 immunity. In addition, the high potential of dn T cells to transcribe the gene encoding the co-inhibitory receptor CTLA-4 and to produce the inhibitory cytokine IL-10 indicates putative immunosuppressive capacity of this population. In summary, this study reveals important novel aspects of canine non-conventional T cells providing the basis for further studies on their effector and/or regulatory functions to elucidate their role in health and disease.

Type-2 immunity associated with type-1 related skin inflammatory diseases: friend or foe?

Chronic inflammatory skin diseases are multifactorial diseases that combine genetic predisposition, environmental triggers, and metabolic disturbances associated with abnormal immune responses. From an immunological perspective, the better understanding of their physiopathology has demonstrated a large complex network of immune cell subsets and related cytokines that interact with both epidermal and dermal cells. For example, in type-1-associated diseases such as alopecia areata, vitiligo, and localized scleroderma, recent evidence suggests the presence of a type-2 inflammation that is well known in atopic dermatitis. Whether this type-2 immune response has a protective or detrimental impact on the development and chronicity of these diseases remains to be fully elucidated, highlighting the need to better understand its involvement for the management of patients. This mini-review explores recent insights regarding the potential role of type-2-related immunity in alopecia areata, vitiligo, and localized scleroderma.

Plasma polyamines during pregnancy and their relationships with maternal allergies and the immune response of the neonates.

BACKGROUND: Some studies have reported that polyamine levels may influence immune system programming. The aim of this study was to evaluate the polyamine profile during gestation and its associations with maternal allergy and cytokine production in cord blood cells in response to different allergenic stimuli. METHODS: Polyamines were determined in plasma of pregnant women (24 weeks, N = 674) and in umbilical cord samples (N = 353 vein and N = 160 artery) from the Mediterranean NELA birth cohort. Immune cell populations were quantified, and the production of cytokines in response to different allergic and mitogenic stimuli was assessed in cord blood. RESULTS: Spermidine and spermine were the most prevalent polyamines in maternal, cord venous, and cord arterial plasma. Maternal allergies, especially allergic conjunctivitis, were associated with lower spermine in umbilical cord vein. Higher levels of polyamines were associated with higher lymphocyte number but lower Th2-related cells in cord venous blood. Those subjects with higher levels of circulating polyamines in cord showed lower production of inflammatory cytokines, especially IFN-α, and lower production of Th2-related cytokines, mainly IL-4 and IL-5. The effects of polyamines on Th1-related cytokines production were uncertain. CONCLUSIONS: Spermidine and spermine are the predominant polyamines in plasma of pregnant women at mid-pregnancy and also in umbilical cord. Maternal allergic diseases like allergic conjunctivitis are related to lower levels of polyamines in cord vein, which could influence the immune response of the newborn. Cord polyamine content is related to a decreased Th2 response and inflammatory cytokines production, which might be important to reduce an allergenic phenotype in the neonate.

SLAM-family receptors promote resolution of ILC2-mediated inflammation.

Type 2 innate lymphoid cells (ILC2) initiate early allergic inflammation in the lung, but the factors that promote subsequent resolution of type 2 inflammation and prevent prolonged ILC2 activation are not fully known. Here we show that SLAM-family receptors (SFR) play essential roles in this process. We demonstrate dynamic expression of several SFRs on ILC2s during papain-induced type 2 immunity in mice. SFR deficiency exacerbates ILC2-driven eosinophil infiltration in the lung, and results in a significant increase in IL-13 production by ILC2s exclusively in mediastinal lymph nodes (MLN), leading to increased dendritic cell (DC) and TH2 cell numbers. In MLNs, we observe more frequent interaction between ILC2s and bystander T cells, with T cell-expressed SFRs (especially SLAMF3 and SLAMF5) acting as self-ligands to suppress IL-13 production by ILC2s. Mechanistically, homotypic engagement of SFRs at the interface between ILC2s and T cells delivers inhibitory signaling primarily mediated by SHIP-1. This prevents activation of NF-κB, driven by IL-7 and IL-33, two major drivers of ILC2-mediated type 2 immunity. Thus, our study shows that an ILC2-DC-TH2 regulatory axis may promote the resolution of pulmonary type 2 immune responses, and highlights SLAMF3/SLAMF5 as potential therapeutic targets for ameliorating type 2 immunity.

Fermented Gracilaria lemaneiformis polysaccharides alleviate food allergy by regulating Treg cells and gut microbiota.

Food allergy has a significant impact on the health and well-being of individuals, affecting both their physical and mental states. Research on natural bioactive compounds, such as polysaccharides extracted from seaweeds, holds great promise in the treatment of food allergies. In this study, fermented Gracilaria lemaneiformis polysaccharides (F-GLSP) were prepared using probiotic fermentation. Probiotic fermentation of Gracilaria lemaneiformis reduces the particle size of polysaccharides. To compare the anti-allergic activity of F-GLSP with unfermented Gracilaria lemaneiformis polysaccharides (UF-GLSP), an OVA-induced mouse food allergy model was established. F-GLSP exhibited a significant reduction in OVA-specific IgE and mMCP levels in allergic mice. Moreover, it significantly inhibited Th2 differentiation and IL-4 production and significantly promoted Treg differentiation and IL-10 production in allergic mice. In contrast, UF-GLSP only reduced OVA-specific IgE and mMCP in the serum of allergic mice. Furthermore, F-GLSP demonstrated a more pronounced regulation of intestinal flora abundance compared to UF-GLSP, significantly influencing the populations of Firmicutes, Bacteroidetes, Lactobacillus, and Clostridiales in the intestines of mice with food allergy. These findings suggest that F-GLSP may regulate food allergies in mice through multiple pathways. In summary, this study has promoted further development of functional foods with anti-allergic properties based on red algae polysaccharides.

Combination of androgen and estrogen improves asthma by mediating Runx3 expression.

Objective: Asthma is a chronic heterogeneous airway disease, and imbalanced T-helper type 1 (Th1) and Th2 cell-mediated inflammation contribute to its pathogenesis. Although it has been suggested that androgen and estrogen were involved in development of asthma, the underlying mechanisms remained largely unclear. Studies have demonstrated that Runx3 could promote naive CD4+ T cells to differentiate into Th1 cells. Hence, our study aimed to explore the potential regulatory mechanism of androgen and estrogen on asthma via modulating Runx3. Methods: First, clinical assessments and pulmonary function tests were conducted on 35 asthma patients and 24 healthy controls. The concentrations of androgen, estrogen, and androgen estrogen ratios were assessed in peripheral blood samples of asthma patients and healthy controls. Then, a murine asthma model was established to explore the effects of estrogen and androgen (alone or in combination) on asthma. Third, an in vitro assay was used to explore the mechanism of combination of androgen and estrogen in asthma. Results: We observed decreased androgen and increased estrogen levels in asthma patients compared with healthy controls. In mice with experimental asthma, there were increased serum concentrations of estrogen and decreased serum concentrations of androgen, intervention with combination of androgen and estrogen alleviated airway inflammations, increased Runx3 expressions and elevated Th1 differentiation. In CD4+ T cells co-cultured with bronchial epithelial cells (BECs), treatment with androgen plus estrogen combination promoted Th1 differentiation, which was mitigated by Runx3 knockdown in BECs and enhanced by Runx3 overexpression. Conclusion: These findings suggest that androgen estrogen combination modulate the Th1/Th2 balance via regulating the expression of Runx3 in BECs, thereby providing experimental evidence supporting androgen and estrogen combination as a novel therapy for asthma.

Mapping the immune cell landscape of severe atopic dermatitis by single-cell RNA-seq.

BACKGROUND: Efforts to profile atopic dermatitis (AD) tissues have intensified, yet comprehensive analysis of systemic immune landscapes in severe AD remains crucial. METHODS: Employing single-cell RNA sequencing, we analyzed over 300,000 peripheral blood mononuclear cells from 12 severe AD patients (Eczema area and severity index (EASI) > 21) and six healthy controls. RESULTS: Results revealed significant immune cell shifts in AD patients, including increased Th2 cell abundance, reduced NK cell clusters with compromised cytotoxicity, and correlated Type 2 innate lymphoid cell proportions with disease severity. Moreover, unique monocyte clusters reflecting activated innate immunity emerged in very severe AD (EASI > 30). While overall dendritic cells (DCs) counts decreased, a distinct Th2-priming subset termed "Th2_DC" correlated strongly with disease severity, validated across skin tissue data, and flow cytometry with additional independent severe AD samples. Beyond the recognized role of Th2 adaptive immunity, our findings highlight significant innate immune cell alterations in severe AD, implicating their roles in disease pathogenesis and therapeutic potentials. CONCLUSION: Apart from the widely recognized role of Th2 adaptive immunity in AD pathogenesis, alterations in innate immune cells and impaired cytotoxic cells have also been observed in severe AD. The impact of these alterations on disease pathogenesis and the effectiveness of potential therapeutic targets requires further investigation.

Bronchom assuages airway hyperresponsiveness in house dust mite-induced mouse model of allergic asthma and moderates goblet cell metaplasia, sub-epithelial fibrosis along with changes in Th2 cytokines and chemokines.

Background: Asthma is a common obstructive airway disease with an inflammatory etiology. The main unmet need in the management of asthma is inadequate adherence to pharmacotherapy, leading to a poorly-controlled disease state, necessitating the development of novel therapies. Bronchom is a calcio-herbal formulation, which is purported to treat chronic asthma. The objective of the current study was to examine the in-vivo efficacy of Bronchom in mouse model of allergic asthma. Methods: Ultra high performance liquid chromatography was utilized to analyze the phytocompounds in Bronchom. Further, the in-vivo efficacy of Bronchom was evaluated in House dust mite (HDM)-induced allergic asthma in mice. Mice were challenged with aerosolized methacholine to assess airway hyperresponsiveness. Subsequently, inflammatory cell influx was evaluated in bronchoalveolar lavage fluid (BALF) followed by lung histology, wherein airway remodeling features were studied. Simultaneously, the levels of Th2 cytokines and chemokines in the BALF was also evaluated. Additionally, the mRNA expression of pro-inflammatory and Th2 cytokines was also assessed in the lung along with the oxidative stress markers. Results: Phytocompounds present in Bronchom included, gallic acid, protocatechuic acid, methyl gallate, rosmarinic acid, glycyrrhizin, eugenol, 6-gingerol and piperine. Bronchom effectively suppressed HDM-induced airway hyperresponsiveness along with the influx of leukocytes in the BALF. Additionally, Bronchom reduced the infiltration of inflammatory cells in the lung and it also ameliorated goblet cell metaplasia, sub-epithelial fibrosis and increase in α-smooth muscle actin. Bronchom decreased Th2 cytokines (IL-4 and IL-5) and chemokines (Eotaxin and IP-10) in the BALF. Likewise, it could also suppress the mRNA expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-33), and IL-13. Moreover, Bronchom restored the HDM-induced diminution of endogenous anti-oxidants (GSH and SOD) and the increase in pro-oxidants (GSSG and MDA). Furthermore, Bronchom could also decrease the nitrosative stress by lowering the observed increase in nitrite levels. Conclusion: Taken together, the results of the present study data convincingly demonstrate that Bronchom exhibits pharmacological effects in an animal model of allergic asthma. Bronchom mitigated airway hyperresponsiveness, inflammation and airway remodeling evoked by a clinically relevant allergen and accordingly it possesses therapeutic potential for the treatment of asthma.

Distinct T cell signatures are associated with Staphylococcus aureus skin infection in pediatric atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin condition with a childhood prevalence of up to 25%. Microbial dysbiosis is characteristic of AD, with Staphylococcus aureus the most frequent pathogen associated with disease flares and increasingly implicated in disease pathogenesis. Therapeutics to mitigate the effects of S. aureus have had limited efficacy and S. aureus-associated temporal disease flares are synonymous with AD. An alternative approach is an anti-S. aureus vaccine, tailored to AD. Experimental vaccines have highlighted the importance of T cells in conferring protective anti-S. aureus responses; however, correlates of T cell immunity against S. aureus in AD have not been identified. We identify a systemic and cutaneous immunological signature associated with S. aureus skin infection (ADS.aureus) in a pediatric AD cohort, using a combined Bayesian multinomial analysis. ADS.aureus was most highly associated with elevated cutaneous chemokines IP10 and TARC, which preferentially direct Th1 and Th2 cells to skin. Systemic CD4+ and CD8+ T cells, except for Th2 cells, were suppressed in ADS.aureus, particularly circulating Th1, memory IL-10+ T cells, and skin-homing memory Th17 cells. Systemic γδ T cell expansion in ADS.aureus was also observed. This study suggests that augmentation of protective T cell subsets is a potential therapeutic strategy in the management of S. aureus in AD.

The role of Interleukin-21 (IL-21) in allergic disorders: Biological insights and regulatory mechanisms.

In recent decades, allergic diseases subsequent from an IgE-mediated response to specific allergens have become a progressively public chronic disease worldwide. They have shaped an important medical and socio-economic burden. A significant proportion of allergic disorders are branded via a form 2 immune response relating Th2 cells, type 2 natural lymphoid cells, mast cells and eosinophils. Interleukin-21 (IL-21) is a participant of the type-I cytokine family manufactured through numerous subsets of stimulated CD4+ T cells and uses controlling properties on a diversity of immune cells. Increasingly, experimental sign suggests a character for IL-21 in the pathogenesis of numerous allergic disorders. The purpose of this review is to discuss the biological properties of IL-21 and to summaries current developments in its role in the regulation of allergic disorders.

SDH, a novel diarylheptane compound, alleviates dextran sulfate sodium (DSS)-induced colitis by reducing Th1/Th2/Th17 induction and regulating the gut microbiota in mice.

Ulcerative colitis, a chronic inflammatory condition affecting the rectum and colon to varying degrees, is linked to a dysregulated immune response and the microbiota. Sodium (aS,9R)-3-hydroxy-16,17-dimethoxy-15-oxidotricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexene-9-yl sulfate hydrate (SDH) emerges as a novel diarylheptane compound aimed at treating inflammatory bowel diseases. However, the mechanisms by which SDH modulates these conditions remain largely unknown. In this study, we assessed SDH's impact on the clinical progression of dextran sodium sulfate (DSS)-induced ulcerative colitis. Our results demonstrated that SDH significantly mitigated the symptoms of DSS-induced colitis, reflected in reduced disease activity index scores, alleviation of weight loss, shortening of the colorectum, and reduction in spleen swelling. Notably, SDH decreased the proportion of Th1/Th2/Th17 cells and normalized inflammatory cytokine levels in the colon. Furthermore, SDH treatment modified the gut microbial composition in mice with colitis, notably decreasing Bacteroidetes and Proteobacteria populations while substantially increasing Firmicutes, Actinobacteria, and Patescibacteria. In conclusion, our findings suggest that SDH may protect the colon from DSS-induced colitis through the regulation of Th1/Th2/Th17 cells and gut microbiota, offering novel insights into SDH's therapeutic potential.

Allergenicity and Linear Epitope Analysis of Scy p 8, an Allergen from Mud Crab.

Scy p 8 (triosephosphate isomerase) as a crab allergen in inducing distinct T-helper (Th) cell differentiation and a linear epitope associated with allergenicity remain elusive. In this study, mice sensitized with Scy p 8 exhibited significantly upregulated levels of IgE, IgG1, and IL-4 release, inducing a Th2 immune response. Moreover, the release of IFN-γ (Th1) and the levels of Treg cells were downregulated, while IL-17A (Th17) was upregulated, indicating that Scy p 8 disrupted the Th1/Th2 balance and Th17/Treg balance in mice. Furthermore, bioinformatics prediction and serum samples from crab-allergic patients and mice enabled the discovery of 8 linear epitopes of Scy p 8. Meanwhile, the analysis of peptide similarity and tertiary superposition revealed that 8 epitopes of Scy p 8 exhibited conservation across various species, potentially resulting in cross-reactivity. These findings possess the potential to enhance the comprehension of crab allergens, thereby establishing a foundation for investigating cross-reactivity.

A Th2-type immune response and low-grade systemic inflammatory reaction as potential immunotoxic effects in intensive agriculture farmers exposed to pesticides.

Pesticides are chemicals widely used in agriculture to keep crops healthy and prevent them from being destroyed by pests, thus contributing to a sustainable food and feed production. However, long-term exposure to these compounds may be harmful to human health as they can affect the function of various organs systems, including the immune system. There is growing evidence that pesticides may increase the risk of developing immune-based diseases and inflammation. This study assessed whether greenhouse farmers occupationally exposed to pesticides presented alterations in immunoregulatory proteins, used as surrogate biomarkers of immune function. The study population consisted of 175 greenhouse workers occupationally exposed to pesticides and 91 non-exposed controls. Serum levels of 27 cytokines, chemokines and growth factors were measured using a magnetic bead-based immunoassay in a subpopulation of 111 greenhouse workers and 79 non-exposed controls. Since analytical determinations were performed in two periods of the same crop season with different use of pesticides (period of high and low pesticide exposure), linear mixed models for repeated measures were used to optimize statistical inference. The increase in IL-13, IL-4 and IL-6 observed in greenhouse workers compared to controls, and in the period of high exposure to pesticides relative to that of low exposure, suggest an altered Th1/Th2 balance towards the Th2 response. This finding points to a type-2 inflammation commonly presented as allergic inflammation, which has often been reported in farm-workers and in which pesticide exposure is considered a risk factor. Furthermore, the increase in IL-1ß and VEGF, mediators of inflammation and angiogenesis, may suggest a low-grade systemic inflammation that might underlie chronic pathological conditions linked to pesticide exposure.

Association of IL-4 polymorphism with severe periodontitis in a sample of Iraqi population.

INTRODUCTION: Specific bacterial plaque and environmental factors cannot be considered the only cause of periodontitis. Still, several genetic factors affect the host response to the bacteria, like gene polymorphisms in anti-inflammatory cytokines. Several studies have reported that clones of T-helper 2 lymphocytes (TH2) are generated in response to dental plaque in periodontitis patients, while in healthy individuals, they are regulated by T-helper 1 (TH1) lymphocytes. Accordingly, such patients consistently produce more IL-4 (TH2) in response to bacterial stimulation, whereas healthy controls with intact periodontal tissues produce a significantly higher level of TH1.

Intestinal microflora promotes Th2-mediated immunity through NLRP3 in damp and heat environments.

Background: With the worsening of the greenhouse effect, the correlation between the damp-heat environment (DH) and the incidence of various diseases has gained increasing attention. Previous studies have demonstrated that DH can lead to intestinal disorders, enteritis, and an up-regulation of NOD-like receptor protein 3 (NLRP3). However, the mechanism of NLRP3 in this process remains unclear. Methods: We established a DH animal model to observe the impact of a high temperature and humidity environment on the mice. We sequenced the 16S rRNA of mouse feces, and the RNA transcriptome of intestinal tissue, as well as the levels of cytokines including interferon (IFN)-γ and interleukin (IL)-4 in serum. Results: Our results indicate that the intestinal macrophage infiltration and the expression of inflammatory genes were increased in mice challenged with DH for 14 days, while the M2 macrophages were decreased in Nlrp3 -/- mice. The alpha diversity of intestinal bacteria in Nlrp3 -/- mice was significantly higher than that in control mice, including an up-regulation of the Firmicutes/Bacteroidetes ratio. Transcriptomic analysis revealed 307 differentially expressed genes were decreased in Nlrp3 -/- mice compared with control mice, which was related to humoral immune response, complement activation, phagocytic recognition, malaria and inflammatory bowel disease. The ratio of IFN-γ/IL-4 was decreased in control mice but increased in Nlrp3 -/- mice. Conclusions: Our study found that the inflammation induced by DH promotes Th2-mediated immunity via NLRP3, which is closely related to the disruption of intestinal flora.

Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.