CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses.

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell-mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell-mediated pathologic responses.

The interleukin-33-p38 kinase axis confers memory T helper 2 cell pathogenicity in the airway.

Memory CD4(+) T helper (Th) cells provide long-term protection against pathogens and are essential for the development of vaccines; however, some antigen-specific memory Th cells also drive immune-related pathology, including asthma. The mechanisms regulating the pathogenicity of memory Th cells remain poorly understood. We found that interleukin-33 (IL-33)-ST2 signals selectively licensed memory Th2 cells to induce allergic airway inflammation via production of IL-5 and that the p38 MAP kinase pathway was a central downstream target of IL-33-ST2 in memory Th2 cells. In addition, we found that IL-33 induced upregulation of IL-5 by memory CD4(+) T cells isolated from nasal polyps of patients with eosinophilic chronic rhinosinusitis. Thus, IL-33-ST2-p38 signaling appears to directly instruct pathogenic memory Th2 cells to produce IL-5 and induce eosinophilic inflammation.

Basophil-derived interleukin-4 controls the function of natural helper cells, a member of ILC2s, in lung inflammation.

Allergic asthma is an inflammatory disease characterized by lung eosinophilia controlled by type 2 cytokines. Cysteine proteases are potent triggers of allergic inflammation by causing barrier disruption in lung epithelial cells inducing the elevation of interleukin-5 (IL-5) and IL-13 from natural helper (NH) cells, a member of ILC2s, which leads to lung eosinophilia. In this study, we found that basophils play a crucial role in NH cell-mediated eosinophilic inflammation induced by protease allergens. Conditional deletion of basophils caused a resolution of the papain-induced eosinophilia and mucus production. Resolution of eosinophilia was also observed in mice lacking IL-4 specifically in basophils, indicating that basophil-derived IL-4 enhanced expression of the chemokine CCL11, as well as IL-5, IL-9, and IL-13 in NH cells, thus attracting eosinophils. These results demonstrate that IL-4 from basophils has an important role in the NH-derived cytokine and chemokine expression, subsequently leading to protease allergen-induced airway inflammation.

GATA-3 function in innate and adaptive immunity.

The zinc-finger transcription factor GATA-3 has received much attention as a master regulator of T helper 2 (Th2) cell differentiation, during which it controls interleukin-4 (IL-4), IL-5, and IL-13 expression. More recently, GATA-3 was shown to contribute to type 2 immunity through regulation of group 2 innate lymphoid cell (ILC2) development and function. Furthermore, during thymopoiesis, GATA-3 represses B cell potential in early T cell precursors, activates TCR signaling in pre-T cells, and promotes the CD4(+) T cell lineage after positive selection. GATA-3 also functions outside the thymus in hematopoietic stem cells, regulatory T cells, CD8(+) T cells, thymic natural killer cells, and ILC precursors. Here we discuss the varied functions of GATA-3 in innate and adaptive immune cells, with emphasis on its activity in T cells and ILCs, and examine the mechanistic basis for the dose-dependent, developmental-stage- and cell-lineage-specific activity of this transcription factor.

An inhibitory immunoreceptor, Allergin-1, suppresses FITC-induced type 2 contact hypersensitivity.

Although allergic contact dermatitis (ACD) is the most common T cell-mediated inflammatory responses against an allergen in the skin, the pathogenesis of ACD remains incompletely understood. In the sensitization phase in ACD, hapten-bearing dermal dendritic cells (DCs) play a pivotal role in the transport of an antigen to the lymph nodes (LNs), where they present the antigen to naïve T cells. Here we report that Allergin-1, an inhibitory immunoreceptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region, is highly expressed on dermal DCs. Mice deficient in Allergin-1 exhibited exacerbated fluorescein isothiocyanate (FITC)-induced type 2 contact hypersensitivity (CHS) such as ear swelling and skin eosinophilia. Allergin-1-deficient mice also showed larger numbers of CD4+ T cells and FITC-bearing DCs and greater expressions of type 2 cytokines, including IL-5, IL-10 and IL-13, in the draining LNs than did wild type mice. In sharp contrast, Allergin-1-deficient mice showed comparable level of type 1 CHS induced by 2,4-dinitrofluorobenzene (DNFB). These results suggest that Allergin-1 on dermal DC inhibits type 2, but not type 1, immune responses in the sensitization phase of CHS.

Airway IL-1ß associates with IL-5 production following dust mite allergen inhalation in humans.

BACKGROUND: Preclinical studies implicate interleukin (IL)-1ß as a key mediator of asthma and have shown the efficacy of IL-1 antagonism for treatment of allergic airway inflammation; human studies in this area are lacking. OBJECTIVES: Our aim was to study the relationship of airway IL-1ß to features of acute allergen-induced asthma exacerbation in humans. METHODS: Dust mite-allergic adults with mild asthma underwent inhalation challenge with Dermatophagoides farinae. Fractional exhaled nitric oxide (FeNO), induced sputum and peripheral blood samples were obtained pre- and 24 h post-challenge. Spirometry was performed before and throughout the challenge at 10-min intervals, and allergen responsiveness was defined by a 20% fall in Forced Expiratory Volume in 1 s (FEV1). Sputum samples were analyzed for inflammatory cells, cytokines and chemokines. Multiple linear regression was employed to test the association between sputum IL-1ß concentration and biomarkers of T helper type 2 (T2)-dominant inflammation. RESULTS: Fourteen volunteers underwent inhaled allergen challenge. Allergen responsive volunteers showed a greater positive change in IL-1ß in sputum following allergen challenge compared to non-responders. Higher pre-challenge sputum IL-1ß was associated with greater increase in sputum IL-5 (p = 0.004), sputum eosinophils (p = 0.001) and blood IL-5 (p = 0.003) following allergen challenge. Allergen-induced sputum IL-1ß production was significantly associated with sputum and blood IL-5 (p < 0.001 and p = 0.007, respectively), sputum IL-4 (p = 0.001), IL-13 (p = 0.026), eosinophils (p = 0.008) and FeNO (p = 0.03). CONCLUSIONS: The positive association between production of IL-1ß and biomarkers of T2 inflammation, particularly IL-5, in humans is consistent with work in animal models that demonstrates a link between IL-1ß and the pathophysiology of allergic asthma. The role of IL-1ß in human asthma warrants further study.

Transcription factor and cytokine regulation of eosinophil lineage commitment.

PURPOSE OF REVIEW: Lineage commitment is governed by instructive and stochastic signals, which drive both active induction of the lineage program and repression of alternative fates. Eosinophil lineage commitment is driven by the ordered interaction of transcription factors, supported by cytokine signals. This review summarizes key findings in the study of eosinophil lineage commitment and examines new data investigating the factors that regulate this process. RECENT FINDINGS: Recent and past studies highlight how intrinsic and extrinsic signals modulate transcription factor network and lineage decisions. Early action of the transcription factors C/EBPα and GATA binding protein-1 along with C/EBPε supports lineage commitment and eosinophil differentiation. This process is regulated and enforced by the pseudokinase Trib1, a regulator of C/EBPα levels. The cytokines interleukin (IL)-5 and IL-33 also support early eosinophil development. However, current studies suggest that these cytokines are not specifically required for lineage commitment. SUMMARY: Together, recent evidence suggests a model where early transcription factor activity drives expression of key eosinophil genes and cytokine receptors to prime lineage commitment. Understanding the factors and signals that control eosinophil lineage commitment may guide therapeutic development for eosinophil-mediated diseases and provide examples for fate choices in other lineages.

Eomesodermin controls interleukin-5 production in memory T helper 2 cells through inhibition of activity of the transcription factor GATA3.

The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.

Type 2 Cysteinyl Leukotriene Receptors Drive IL-33-Dependent Type 2 Immunopathology and Aspirin Sensitivity.

Cysteinyl leukotrienes (cysLTs) facilitate mucosal type 2 immunopathology by incompletely understood mechanisms. Aspirin-exacerbated respiratory disease, a severe asthma subtype, is characterized by exaggerated eosinophilic respiratory inflammation and reactions to aspirin, each involving the marked overproduction of cysLTs. Here we demonstrate that the type 2 cysLT receptor (CysLT2R), which is not targeted by available drugs, is required in two different models to amplify eosinophilic airway inflammation via induced expression of IL-33 by lung epithelial cells. Endogenously generated cysLTs induced eosinophilia and expanded group 2 innate lymphoid cells (ILC2s) in aspirin-exacerbated respiratory disease-like Ptges-/- mice. These responses were mitigated by deletions of either Cysltr2 or leukotriene C4 synthase (Ltc4s). Administrations of either LTC4 (the parent cysLT) or the selective CysLT2R agonist N-methyl LTC4 to allergen sensitized wild-type mice markedly boosted ILC2 expansion and IL-5/IL-13 generation in a CysLT2R-dependent manner. Expansion of ILC2s and IL-5/IL-13 generation reflected CysLT2R-dependent production of IL-33 by alveolar type 2 cells, which engaged in a bilateral feed-forward loop with ILC2s. Deletion of Cysltr1 blunted LTC4-induced ILC2 expansion and eosinophilia but did not alter IL-33 induction. Pharmacological blockade of CysLT2R prior to inhalation challenge of Ptges-/- mice with aspirin blocked IL-33-dependent mast cell activation, mediator release, and changes in lung function. Thus, CysLT2R signaling, IL-33-dependent ILC2 expansion, and IL-33-driven mast cell activation are necessary for induction of type 2 immunopathology and aspirin sensitivity. CysLT2R-targeted drugs may interrupt these processes.

Treatment of established TH2 cells with 4µ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion.

BACKGROUND: T cell activation induces ER stress and upregulates Inositol Requiring Enzyme 1 alpha (IRE1α), an activator of the unfolded protein response (UPR) pathway. Inhibition of IRE1α RNase activity in activated CD4+ splenocytes from naïve mice, via treatment of the cells with the commercially available drug 4µ8c upon activation, results in the reduction of the secretion of proteins IL-5, IL-4, and IL-13. Prior to this work, it was unknown if 4µ8c could inhibit TH2 cytokines in established TH2 cells, cells that are crucial in promoting disease in severe asthma. RESULTS: Treatment of a mouse T helper (TH)2 cell line and differentiated human TH2 cells with 4µ8c resulted in inhibition of IL-5, but not IL-4, as measured by ELISA. The reduced cytokine expression was not due to differences in mRNA stability or mRNA levels; it appears to be due to a defect in secretion, as the cells produce cytokines IL-5 as measured by flow cytometry and western blot. CONCLUSION: These data suggest that the inhibition of IL-5 was due to post-translational processes. IL-5 promotes chronic, inflammatory asthma, and 4µ8c blocks its expression in T cells in vitro. Future studies will determine if 4µ8c treatment can ameliorate the effects of the cytokine IL-5 in a disease model.

Dynamic changes in intrathymic ILC populations during murine neonatal development.

Members of the innate lymphoid cell (ILC) family have been implicated in the development of thymic microenvironments and the recovery of this architecture after damage. However, a detailed characterization of this family in the thymus is lacking. To better understand the thymic ILC compartment, we have utilized multiple in vivo models including the fate mapping of inhibitor of DNA binding-2 (Id2) expression and the use of Id2 reporter mice. Our data demonstrate that ILCs are more prominent immediately after birth, but were rapidly diluted as the T-cell development program increased. As observed in the embryonic thymus, CCR6+ NKp46- lymphoid tissue inducer (LTi) cells were the main ILC3 population present, but numbers of these cells swiftly declined in the neonate and ILC3 were barely detectable in adult thymus. This loss of ILC3 means ILC2 are the dominant ILC population in the thymus. Thymic ILC2 were able to produce IL-5 and IL-13, were located within the medulla, and did not result from ILC3 plasticity. Furthermore, in WT mice, thymic ILC2 express little RANKL (receptor activator of nuclear factor kappa-B ligand) arguing that functionally, these cells provide different signals to LTi cells in the thymus. Collectively, these data reveal a dynamic switch in the ILC populations of the thymus during neonatal development.

Hox5 Paralogous Genes Modulate Th2 Cell Function during Chronic Allergic Inflammation via Regulation of Gata3.

Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. Th2 cells contribute to the development of disease through release of the cytokines IL-4, IL-5, and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. The molecular mechanisms behind the disease pathology remain largely unknown. In this study we investigated a potential regulatory role for the Hox5 gene family, Hoxa5, Hoxb5, and Hoxc5, genes known to be important in lung development within mesenchymal cell populations. We found that Hox5-mutant mice show exacerbated pathology compared with wild-type controls in a chronic allergen model, with an increased Th2 response and exacerbated lung tissue pathology. Bone marrow chimera experiments indicated that the observed enhanced pathology was mediated by immune cell function independent of mesenchymal cell Hox5 family function. Examination of T cells grown in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression, and elevated production of IL-4, IL-5, and IL-13 in Hox5-deficient T cells compared with wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus and decreased Gata3 and IL-4 expression, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of Th2 immune cell function that demonstrates a redeployment of mesenchyme-associated developmental genes.

T Cell-Restricted Notch Signaling Contributes to Pulmonary Th1 and Th2 Immunity during Cryptococcus neoformans Infection.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen but the cell signaling pathways that drive T cell responses regulating antifungal immunity are incompletely understood. Notch is a key signaling pathway regulating T cell development, and differentiation and functional responses of mature T cells in the periphery. The targeting of Notch signaling within T cells has been proposed as a potential treatment for alloimmune and autoimmune disorders, but it is unknown whether disturbances to T cell immunity may render these patients vulnerable to fungal infections. To elucidate the role of Notch signaling during fungal infections, we infected mice expressing the pan-Notch inhibitor dominant negative mastermind-like within mature T cells with C. neoformans Inhibition of T cell-restricted Notch signaling increased fungal burdens in the lungs and CNS, diminished pulmonary leukocyte recruitment, and simultaneously impaired Th1 and Th2 responses. Pulmonary leukocyte cultures from T cell Notch-deprived mice produced less IFN-γ, IL-5, and IL-13 than wild-type cells. This correlated with lower frequencies of IFN-γ-, IL-5-, and IL-13-producing CD4+ T cells, reduced expression of Th1 and Th2 associated transcription factors, Tbet and GATA3, and reduced production of IFN-γ by CD8+ T cells. In contrast, Th17 responses were largely unaffected by Notch signaling. The changes in T cell responses corresponded with impaired macrophage activation and reduced leukocyte accumulation, leading to diminished fungal control. These results identify Notch signaling as a previously unappreciated regulator of Th1 and Th2 immunity and an important element of antifungal defenses against cryptococcal infection and CNS dissemination.

Monocyte Stabilin-1 Suppresses the Activation of Th1 Lymphocytes.

In this study, we analyzed the putative functions of stabilin-1 in blood monocytes. Microarray analysis revealed downregulation of several proinflammatory genes in the stabilin-1(high) monocytes when compared with stabilin-1(low) monocytes. When cocultured with stabilin-1(high) monocytes, IFN-γ synthesis by T cells was diminished in Ag-recall assays. Knockdown of stabilin-1 in monocytes increased the synthesis of several proinflammatory molecules, including TNF-α, and supported high IFN-γ and low IL-4 and IL-5 production by T cells in Ag-specific stimulation assays. Anti-stabilin-1 Ab treatment also led to increased IFN-γ synthesis in the recall assays. In clinical settings, the expression of stabilin-1 was diminished on blood monocytes and tissue macrophages under proinflammatory conditions. These data define stabilin-1 as a new immunosuppressive molecule and suggest that stabilin-1(high) monocytes may dampen proinflammatory reactions in vivo.

LZTFL1 Upregulated by All-Trans Retinoic Acid during CD4+ T Cell Activation Enhances IL-5 Production.

Retinoic acids, which are metabolites of vitamin A, have been shown to be involved in multiple T cell effector responses through their binding to the retinoic acid receptor, a ligand-activated transcription factor. Because the molecular mechanism of regulation by retinoic acid is still not fully uncovered, we investigated the gene expression profile of all-trans retinoic acid (ATRA)-treated human CD4(+) T cells. Leucine zipper transcription factor-like 1 (LZTFL1) was upregulated by ATRA in a dose- and time-dependent manner. The expression of LZTFL1 depended on both ATRA and TCR signaling. LZTFL1 accumulated in the plasma membrane compartment of human CD4(+) T cells, and, during immunological synapse formation, it transiently redistributed to the T cell and APC contact zone, indicating its role in T cell activation. Live-cell imaging demonstrates that at the initial stage of immunological synapse formation, LZTFL1 is concentrated at the APC contact site, and, during later stages, it relocates to the distal pole. Knockdown of LZTFL1 reduced the basal- and ATRA-induced levels of IL-5 in CD4(+) T cells, and overexpression of LZTFL1 enhanced the TCR-mediated NFAT signaling, suggesting that LZTFL1 is an important regulator of ATRA-induced T cell response. Together, these data indicate that LZTFL1 modulates T cell activation and IL-5 levels.

Characteristics of lower airway inflammatory changes in the minimal persistent inflammation of allergic rhinitis in mice.

OBJECTIVE: This study aims to establish an experimental mouse model of minimal persistent inflammation (MPI), observe the features of inflammation and hyper-responsiveness of the upper/lower airways, and explore the relationship between inflammation and hyper-responsiveness in the upper/lower airways. METHODS: Sixty-four female BALB/c mice were randomly divided into four groups: allergic rhinitis (AR) group as positive control, MPI group, negative control group and blank control group. Mice were given high and low-concentrated ovalbumin solution after basic and intensive sensitization to establish AR model and MPI model. Nasal mucosa and lung tissues were stained to observe eosinophil infiltration, goblet cell hyperplasia, and expression of intercellular adhesion molecule 1 (ICAM-1). Airway hyper-responsiveness was assessed. Levels of specific immunoglobulin E (sIgE), interleukin (IL)-4 and IL-5 in peripheral blood, nasal lavage fluid (NLF), and bronchoalveolar lavage fluid (BALF) were detected by Enzyme-linked immunosorbent assay. RESULTS: The eosinophil infiltration and expression of ICAM-1 on nasal mucosa and in lung tissues in the AR and MPI groups were significantly elevated compared to control groups. Goblet cells count increased only in the nasal mucosa and not in lung tissues. Eosinophil and neutrophil count of NLF and BALF in the AR and MPI groups increased significantly compared to control groups. Level of IL-4 did not increase significantly, but sIgE and IL-5 did. CONCLUSIONS: Mice in the MPI status exhibits lower airway inflammation and hyper-responsiveness with increase in eosinophil count, goblet cells, ICAM-1, IL-4, and IL-5. These results provide further evidence for the importance of MPI of AR in lower airway diseases.

Dysregulation of interleukin 5 expression in familial eosinophilia.

BACKGROUND: Familial eosinophilia (FE) is a rare autosomal dominant inherited disorder characterized by the presence of lifelong peripheral eosinophilia (>1500/µL). Mapped to chromosome 5q31-q33, the genetic cause of FE is unknown, and prior studies have failed to demonstrate a primary abnormality in the eosinophil lineage. OBJECTIVE: The aim of this study was to identify the cells driving the eosinophilia in FE. METHODS: Microarray analysis and real-time PCR were used to examine transcriptional differences in peripheral blood mononuclear cells (PBMC), and in purified cell subsets from affected and unaffected family members belonging to a single large kindred. Cytokine levels in serum and PBMC culture supernatants were assessed by suspension array multiplexed immunoassays. RESULTS: Whereas IL-5 mRNA expression was significantly increased in freshly isolated PBMC from affected family members, this was not accompanied by increased mRNA expression of other Th2 cytokines (IL-4 or IL-13). Serum levels of IL-5 and IL-5 receptor α, but not IgE, were similarly increased in affected family members. Of note, IL-5 mRNA expression was significantly increased in purified CD3+ CD4+, CD14+, CD19+, and ILC2 cells from affected family members, as were IL-5 protein levels in supernatants from both stimulated PBMC and ILC2 cultures. CONCLUSIONS: These data are consistent with the hypothesis that the eosinophilia in FE is secondary to dysregulation of IL-5 production in PBMC (and their component subsets).

Pathogenic memory type Th2 cells in allergic inflammation.

Immunological memory is a hallmark of adaptive immunity. Memory CD4 T helper (Th) cells are central to acquired immunity, and vaccines for infectious diseases are developed based on this concept. However, memory Th cells also play a critical role in the pathogenesis of various chronic inflammatory diseases, including asthma. We refer to these populations as 'pathogenic memory Th cells.' Here, we review recent developments highlighting the functions and characteristics of several pathogenic memory type Th2 cell subsets in allergic inflammation. Also discussed are the similarities and differences between pathogenic memory Th2 cells and recently identified type 2 innate lymphoid cells (ILC2), focusing on cytokine production and phenotypic profiles.

Spred1, a Suppressor of the Ras-ERK Pathway, Negatively Regulates Expansion and Function of Group 2 Innate Lymphoid Cells.

Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1(-/-) ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.

Presentation of high antigen-dose by splenic B220(lo) B cells fosters a feedback loop between T helper type 2 memory and antibody isotype switching.

Effective humoral immunity ensues when antigen presentation by B cells culminates in productive cooperation with T lymphocytes. This collaboration, however, remains ill-defined because naive antigen-specific B cells are rare and difficult to track in vivo. Herein, we used a defined transfer model to examine how B lymphocytes, as antigen-presenting cells, shape the development of T-cell memory suitable for generation of relevant antibody responses. Specifically, we examined how B cells presenting different doses of antigen during the initial priming phase shape the development of CD4 T-cell memory and its influence on humoral immunity. The findings indicate that B cells presenting low dose of antigen favour the development of T helper type 1 (Th1) type memory, while those presenting a high antigen dose yielded better Th2 memory cells. The memory Th2 cells supported the production of antibodies by effector B cells and promoted isotype switching to IgG1. Moreover, among the B-cell subsets tested for induction of Th2 memory, the splenic but not peritoneal B220(lo) cells were most effective in sustaining Th2 memory development as well as immunoglobulin isotype switching, and this function involved a tight control by programmed death 1-programmed death ligand 2 interactions.