Plasmacytoid dendritic cells synergize with myeloid dendritic cells in the induction of antigen-specific antitumor immune responses

Plasmacytoid dendritic cells (pDC) are capable of producing high levels of type I IFNs upon viral stimulation, and play a central role in modulating innate and adaptive immunity against viral infections. Whereas many studies have assessed myeloid dendritic cells (mDC) in the induction of antitumor immune responses, the role of pDC in antitumor immunity has not been addressed. Moreover, the interaction of pDC with other dendritic cell subsets has not been evaluated. In this study, we analyzed the capacity of pDC in stimulating an Ag-specific T cell response. Immunization of mice with Ag-pulsed, activated pDC significantly augmented Ag-specific CD8(+) CTL responses, and protected mice from a subsequent tumor challenge. Immunization with a mixture of activated pDC plus mDC resulted in increased levels of Ag-specific CD8(+) T cells and an enhanced antitumor response compared with immunization with either dendritic cell subset alone. Synergy between pDC and mDC in their ability to activate T cells was dependent on MHC I expression by mDC, but not pDC, suggesting that pDC enhanced the ability of mDC to present Ag to T cells. Our results demonstrate that pDC and mDC can interact synergistically to induce an Ag-specific antitumor immune response in vivo.     

The countervailing actions of myeloid and plasmacytoid dendritic cells control autoimmune diabetes in the nonobese diabetic mouse

Islet Ag-specific CD4(+) T cells receive antigenic stimulation from MHC class II-expressing APCs. Herein, we delineate the direct in vivo necessity for distinct subsets of macrophages and dendritic cells (DC) in type 1 diabetes mellitus of the NOD mouse by using diphtheria toxin-mediated cell ablation. The ablation of macrophages had no impact on islet Ag presentation or on the induction of insulitis or diabetes in either transfer or spontaneous models. However, the ablation of CD11b(+)CD11c(+) DC led to the loss of T cell activation, insulitis, and diabetes mediated by CD4(+) T cells. When the specific myeloid DC subset was "added-back" to mice lacking total DC, insulitis and diabetes were restored. Interestingly, when NOD mice were allowed to progress to the insulitis phase, the ablation of DC led to accelerated insulitis. This accelerated insulitis was mediated by the loss of plasmacytoid DC (pDC). When pDC were returned to depleted mice, the localized regulation of insulitis was restored. The loss of pDC in the pancreas itself was accompanied by the localized loss of IDO and the acceleration of insulitis. Thus, CD11c(+)CD11b(+) DC and pDC have countervailing actions in NOD diabetes, with myeloid DC providing critical antigenic stimulation to naive CD4(+) T cells and pDC providing regulatory control of CD4(+) T cell function in the target tissue.     

TGF-beta 1 attenuates the acquisition and expression of effector function by tumor antigen-specific human memory CD8 T cells

TGF-beta1 is a potent immunoregulatory cytokine. However, its impact on the generation and effector function of Ag-specific human effector memory CD8 T cells had not been evaluated. Using Ag-specific CD8 T cells derived from melanoma patients immunized with the gp100 melanoma Ag, we demonstrate that the addition of TGF-beta1 to the initial Ag activation cultures attenuated the gain of effector function by Ag-specific memory CD8 T cells while the phenotypic changes associated with activation and differentiation into effector memory were comparable to control cultures. These activated memory CD8 T cells consistently expressed lower mRNA levels for T-bet, suggesting a mechanism for TGF-beta1-mediated suppression of gain of effector function in memory T cells. Moreover, TGF-beta1 induced a modest expression of CCR7 on Ag-activated memory CD8 T cells. TGF-beta1 also suppressed cytokine secretion by Ag-specific effector memory CD8 T cells, as well as melanoma-reactive tumor-infiltrating lymphocytes and CD8 T cell clones. These results demonstrate that TGF-beta1 suppresses not only the acquisition but also expression of effector function on human memory CD8 T cells and tumor-infiltrating lymphocytes reactive against melanoma, suggesting that TGF-beta1-mediated suppression can hinder the therapeutic benefits of vaccination, as well as immunotherapy in cancer patients.     

Human immunodeficiency virus type 1 derived from cocultures of immature dendritic cells with autologous T cells carries T-cell-specific molecules on its surface and is highly infectious

During the budding process, human immunodeficiency virus type 1 (HIV-1) acquires cell surface molecules; thus, the viral surface of HIV-1 reflects the antigenic pattern of the host cell. To determine the source of HIV-1 released from cocultures of dendritic cells (DC) with T cells, immature DC (imDC), mature DC (mDC), T cells, and their cocultures were infected with different HIV-1 isolates. The macrophage-tropic HIV-1 isolate Ba-L allowed viral replication in both imDC and mDC, whereas the T-cell-line-tropic primary isolate PI21 replicated in mDC only. By a virus capture assay, HIV-1 was shown to carry a T-cell- or DC-specific cell surface pattern after production by T cells or DC, respectively. Upon cocultivation of HIV-1-pulsed DC with T cells, HIV-1 exclusively displayed a typical T-cell pattern. Additionally, functional analysis revealed that HIV-1 released from imDC-T-cell cocultures was more infectious than HIV-1 derived from mDC-T-cell cocultures and from cultures of DC, T cells, or peripheral blood mononuclear cells alone. Therefore, we conclude that the interaction of HIV-1-pulsed imDC with T cells in vivo might generate highly infectious virus which primarily originates from T cells.     

Induction of bystander suppression by feeding antigen occurs despite normal clonal expansion of the bystander T cell population

The induction of bystander suppression, whereby the response against one Ag is suppressed when it is presented in the context of an Ag to which tolerance is already established, would be an important property of oral tolerance, because it would allow treatment of autoimmune and hypersensitivity responses where the initiating Ag is not known. Although bystander suppression has been described in oral tolerance, it is not known how its effects are mediated at the level of the bystander T cells. In addition, previous studies have not compared regimes in which Ag is fed in a tolerogenic or immunogenic manner, meaning that the possible effects of Ag competition have not been excluded. In this study we have used two populations of Ag-specific TCR transgenic CD4(+) T cells to examine the cellular basis of bystander suppression associated with oral tolerance in mice in vitro and in vivo. Our results show that bystander responses can be inhibited by feeding Ag and that these effects are more pronounced in mice fed protein in tolerogenic form than after feeding Ag with mucosal adjuvant. However, the expansion of the bystander-specific CD4(+) T cells is not influenced by the presence of oral tolerance. Thus, bystander suppression does not reflect clonal deletion or reduced clonal expansion of the bystander T cells, but may act by altering the functional differentiation of bystander T cells.     

Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC

The ex vivo priming and expansion of human CTL by APC, such as autologous monocyte-derived dendritic cells (DC), has the potential for use in immunotherapy for infectious diseases and cancer. To overcome the difficulty of obtaining sufficient number of autologous DC from patients, we have developed cell-based artificial APC (aAPC), designated Med-APC. These aAPC rapidly activate and expand the corresponding Ag-specific CD8+ T cells when pulsed with CTL epitope peptide(s) as efficiently as mature DC (mDC). We have also shown that Med-APC possess an innate cellular machinery that is sufficient to support the processing of complete Ag into immunodominant peptides, which considerably extends the usefulness of this technology. In addition, we have developed a novel expression vector system that expresses ubiquitinated Ag, resulting in an enhanced APC function of this system. Genetically encoded Ag can be easily introduced into Med-APC by transfection with this vector. Med-APC transfected with ubiquitinated Ag can efficiently expand the corresponding Ag-specific CTL without exogenous peptides. Therefore, Med-APC may have important therapeutic implications for adoptive immunotherapy and can be used for the detection of Ag-specific CTL for immunomonitoring.     

Loss of NK stimulatory capacity by plasmacytoid and monocyte-derived DC but not myeloid DC in HIV-1 infected patients

Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions.     

TGF-beta 1 regulates antigen-specific CD4+ T cell responses in the periphery

T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-beta1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11 days of age, accompanied by CD4+T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4+T cell expansion in TGF-beta1(-/-) mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR Vbeta repertoires in peripheral CD4+T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4+T cells from 11-day-old TGF-beta1(-/-) mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR Vbeta CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-beta1(-/-) CD4+ T cells than for TGF-beta1(+/-) CD4+T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-beta1(-/-) CD4+ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4+ T cells in TGF-beta1(-/-) mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-beta1 in the peripheral regulation of Ag-specific CD4+ T cell responses.     

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.     

Plasmacytoid dendritic cells take up opsonized antigen leading to CD4+ and CD8+ T cell activation in vivo

Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.     

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.     

IFN-gamma overcomes low responsiveness of myeloid dendritic cells to CpG DNA

Dendritic cells (DC) are professional APC that have an extraordinary capacity to prime naive T cells. It has been reported that human DC subsets express distinct toll-like receptor (TLR), which influences their function. In mice, we observed that plasmocytoid DC (pDC) express a higher level of TLR9 compared with myeloid DC (mDC) cultured with GM-CSF. However, we demonstrated that stimulation with IFN-gamma is capable of upregulating TLR9 expression in mDC to a level comparable with expression in pDC. Consistent with this observation, IL-12 p40 and IL-6 mRNA expression and IL-12 p70 secretion in response to CpG-oligodeoxynucleotides are enhanced in mDC pretreated with IFN-gamma compared with untreated cells. Therefore, TLR-mediated responses of DC subsets may be influenced not only by signals delivered by pathogens but also by regulatory signals from cytokines such as IFN-gamma.     

Compartmental imbalance and aberrant immune function of blood CD123+ (plasmacytoid) and CD11c+ (myeloid) dendritic cells in atopic dermatitis

Atopic dermatitis (AD) is a pruritic, chronically relapsing skin disease in which Th2 cells play a crucial role in cutaneous and extracutaneous immune reactions. In humans, CD11c+CD123- myeloid dendritic cells (mDC) and CD11c-CD123+ plasmacytoid DC (pDC) orchestrate the decision-making process in innate and acquired immunity. Since the number and function of these blood dendritic cell (DC) subsets reportedly reflect the host immune status, we studied the involvement of the DC subsets in the pathogenesis of AD. Patients with AD had an increased DC number and a low mDC:pDC ratio with pDC outnumbering mDC in the peripheral blood compared with normal subjects and psoriasis patients (a Th1 disease model group). The mDC:pDC ratio was correlated with the total serum IgE level, the ratio of IFN-gamma-producing blood cells:IL-4-producing blood cells, and the disease severity. In vitro allogeneic stimulation of naive CD4+ cells with atopic DC showed that the ability of pDC for Th1 induction was superior or comparable to that of mDC. In skin lesions, pDC infiltration was in close association with blood vessels expressing peripheral neural addressins. Therefore, compartmental imbalance and aberrant immune function of the blood DC subsets may deviate the Th1/Th2 differentiation and thus induce protracted allergic responses in AD.     

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.     

Expansion of functional endogenous antigen-specific CD4+CD25+ regulatory T cells from nonobese diabetic mice

CD4+CD25+Foxp3+ regulatory T cells (T(reg)) are critical for controlling autoimmunity. Evidence suggests that T(reg) development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the T(reg) responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific T(reg) are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific T(reg) in NOD mice. Ag-specific T(reg) from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific T(reg) expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded T(reg) were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific T(reg) were more efficient than polyclonal T(reg) in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific T(reg) in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.     

Noncovalent assembly of anti-dendritic cell antibodies and antigens for evoking immune responses in vitro and in vivo

Targeting of Ags directly to dendritic cells (DCs) through anti-DC receptor Ab fused to Ag proteins is a promising approach to vaccine development. However, not all Ags can be expressed as a rAb directly fused to a protein Ag. In this study, we show that noncovalent assembly of Ab-Ag complexes, mediated by interaction between dockerin and cohesin domains from cellulose-degrading bacteria, can greatly expand the range of Ags for this DC-targeting vaccine technology. rAbs with a dockerin domain fused to the rAb H chain C terminus are efficiently secreted by mammalian cells, and many Ags not secreted as rAb fusion proteins are readily expressed as cohesin directly fused to Ag either via secretion from mammalian cells or as soluble cytoplasmic Escherichia coli products. These form very stable and homogeneous complexes with rAb fused to dockerin. In vitro, these complexes can efficiently bind to human DC receptors followed by presentation to Ag-specific CD4(+) and CD8(+) T cells. Low doses of the HA1 subunit of influenza hemagglutinin conjugated through this means to anti-Langerin rAbs elicited Flu HA1-specific Ab and T cell responses in mice. Thus, the noncovalent assembly of rAb and Ag through dockerin and cohesin interaction provides a useful modular strategy for development and testing of prototype vaccines for elicitation of Ag-specific T and B cell responses, particularly when direct rAb fusions to Ag cannot be expressed.     

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin(-)) HLA-DR(+)CD11c(+)CD123(-) mDC and Lin(-)HLA-DR(+)CD11c(-)CD123(+) pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin(-)HLA-DR(moderate)CD11c(-)CD123(-) cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.     

Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract

APCs, including dendritic cells (DC), are central to Ag surveillance in the respiratory tract (RT). Research in this area is dominated by mouse studies on purportedly representative RT-APC populations derived from whole-lung digests, comprising mainly parenchymal tissue. Our recent rat studies identified major functional differences between DC populations from airway mucosal vs parenchymal tissue, thus seriously questioning the validity of this approach. We addressed this issue for the first time in the mouse by separately characterizing RT-APC populations from these two different RT compartments. CD11c(high) myeloid DC (mDC) and B cells were common to both locations, whereas a short-lived CD11c(neg) mDC was unique to airway mucosa and long-lived CD11c(high) macrophage and rapid-turnover multipotential precursor populations were predominantly confined to the lung parenchyma. Airway mucosal mDC were more endocytic and presented peptide to naive CD4+ T cells more efficiently than their lung counterparts. However, mDC from neither site could present whole protein without further maturation in vitro, or following trafficking to lymph nodes in vivo, indicating a novel mechanism whereby RT-DC function is regulated at the level of protein processing but not peptide loading for naive T cell activation.     

IL-5-induced hypereosinophilia suppresses the antigen-induced immune response via a TGF-beta-dependent mechanism

Although eosinophils play an essential role in allergic inflammation, their role has recently been under controversy. Epidemic studies suggest that hypereosinophilia induced by parasite infection could suppress subsequent Ag sensitization, although the mechanism has not been fully clarified. In this study, we investigated whether eosinophils could suppress the Ag-specific immune response in the airway. BALB/c mice were sensitized and airway challenged with OVA. Systemic hypereosinophilia was induced by delivery of an IL-5-producing plasmid. IL-5 gene delivery suppressed the Ag-specific proliferation and cytokine production of CD4+ T cells in the spleen. IL-5 gene delivery before OVA sensitization significantly suppressed airway eosinophilia and hyperresponsiveness provoked by subsequent OVA airway challenge, while delivery during the OVA challenge did not suppress them. This IL-5-induced immune suppression was abolished in eosinophil-ablated mice, suggesting an essential role of eosinophils. IL-5 treatment increased the production of TGF-beta1 in the spleen, and we demonstrated that the main cellular source of TGF-beta1 production was eosinophils, using eosinophil-ablated mice and depletion study. TGF-beta1, but not IL-5 itself, suppressed the Ag-specific immune response of CD4+ T cells in vitro. Furthermore, IL-5 treatment enhanced phosphorylation of Smad2 in CD4+ T cells. Finally, a TGF-beta type I receptor kinase inhibitor restored this IL-5-induced immune suppression both in vitro and in vivo. These results suggest that IL-5-induced hypereosinophilia could suppress sensitization to Ag via a TGF-beta-dependent mechanism, thus suppressed allergic airway inflammation. Therefore, hypereosinophilia could reveal an immunosuppressive effect in the early stage of Ag-induced immune response.