Taku Kambayashi

IL25 elicits a multipotent progenitor cell population that promotes TH2 cytokine responses

CD4+ T helper 2 (TH2) cells secrete interleukin (IL)4, IL5 and IL13, and are required for immunity to gastrointestinal helminth infections. However, TH2 cells also promote chronic inflammation associated with asthma and allergic disorders. The non-haematopoietic-cell-derived cytokines thymic stromal lymphopoietin, IL33 and IL25 (also known as IL17E) have been implicated in inducing TH2 cell-dependent inflammation at mucosal sites, but how these cytokines influence innate immune responses remains poorly defined. Here we show that IL25, a member of the IL17 cytokine family, promotes the accumulation of a lineage-negative (Lin-) multipotent progenitor (MPP) cell population in the gut-associated lymphoid tissue that promotes TH2 cytokine responses. The IL25-elicited cell population, termed MPPtype2 cells, was defined by the expression of Sca-1 (also known as Ly6a) and intermediate expression of c-Kit (c-Kitint), and exhibited multipotent capacity, giving rise to cells of monocyte/macrophage and granulocyte lineages both in vitro and in vivo. Progeny of MPPtype2 cells were competent antigen presenting cells, and adoptive transfer of MPPtype2 cells could promote TH2 cytokine responses and confer protective immunity to helminth infection in normally susceptible Il25-/- mice. The ability of IL25 to induce the emergence of an MPPtype2 cell population identifies a link between the IL17 cytokine family and extramedullary haematopoiesis, and suggests a previously unrecognized innate immune pathway that promotes TH2 cytokine responses at mucosal sites.

TSLP promotes interleukin-3-independent basophil haematopoiesis and type 2 inflammation

CD4+ T-helper type 2 (TH2) cells, characterized by their expression of interleukin (IL)-4, IL-5, IL-9 and IL-13, are required for immunity to helminth parasites and promote the pathological inflammation associated with asthma and allergic diseases. Polymorphisms in the gene encoding the cytokine thymic stromal lymphopoietin (TSLP) are associated with the development of multiple allergic disorders in humans, indicating that TSLP is a critical regulator of TH2 cytokine-associated inflammatory diseases. In support of genetic analyses, exaggerated TSLP production is associated with asthma, atopic dermatitis and food allergies in patients, and studies in murine systems demonstrated that TSLP promotes TH2 cytokine-mediated immunity and inflammation. However, the mechanisms through which TSLP induces TH2 cytokine responses remain poorly defined. Here we demonstrate that TSLP promotes systemic basophilia, that disruption of TSLP–TSLPR interactions results in defective basophil responses, and that TSLPR-sufficient basophils can restore TH2-cell-dependent immunity in vivo. TSLP acted directly on bone-marrow-resident progenitors to promote basophil responses selectively. Critically, TSLP could elicit basophil responses in both IL-3–IL-3R-sufficient and -deficient environments, and genome-wide transcriptional profiling and functional analyses identified heterogeneity between TSLP-elicited versus IL-3-elicited basophils. Furthermore, activated human basophils expressed TSLPR, and basophils isolated from eosinophilic oesophagitis patients were distinct from classical basophils. Collectively, these studies identify previously unrecognized heterogeneity within the basophil cell lineage and indicate that expression of TSLP may influence susceptibility to multiple allergic diseases by regulating basophil haematopoiesis and eliciting a population of functionally distinct basophils that promote TH2 cytokine-mediated inflammation.

Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation

Commensal bacteria that colonize mammalian barrier surfaces are reported to influence T helper type 2 (TH2) cytokine-dependent inflammation and susceptibility to allergic disease, although the mechanisms that underlie these observations are poorly understood. In this report, we find that deliberate alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum IgE concentrations, increased steady-state circulating basophil populations and exaggerated basophil-mediated TH2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Furthermore, B cell–intrinsic expression of myeloid differentiation factor 88 (MyD88) was required to limit serum IgE concentrations and circulating basophil populations in mice. Commensal-derived signals were found to influence basophil development by limiting proliferation of bone marrow–resident precursor populations. Collectively, these results identify a previously unrecognized pathway through which commensal-derived signals influence basophil hematopoiesis and susceptibility to TH2 cytokine–dependent inflammation and allergic disease.

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo.

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRαβ+ cells have been described as being either CD4+CD8− or CD4−CD8−. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Vα14-Jα281 TCR chain in conjunction with either a Vβ2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRαβ+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1−/−, and Jα281−/− mice that lack classical NKT cells. Unlike classical NKT cells, 50–60% of these NK1.1+TCRαβ+ cells express CD8 and have a diverse TCR Vβ repertoire. Purified NK1.1−CD8α+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rβ+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.

TLR Stimulation Modifies BLyS Receptor Expression in Follicular and Marginal Zone B Cells

Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in B cell survival and differentiation. Recent evidence indicates BLyS receptor levels shift following BCR ligation, suggesting that activation cues can alter overall BLyS receptor profiles and thus ligand sensitivity. In this study, we show that TLR stimuli also alter BLyS receptor expression, but in contrast to BCR ligation, TLR9 and TLR4 signals, preferentially increase transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) expression. Although both of these TLRs act through MyD88-dependent mechanisms to increase TACI expression, they differ in terms of their downstream mediators and the B cell subset affected. Surprisingly, only TLR4 relies on c-Rel and p50 to augment TACI expression, whereas TLR9 does not. Furthermore, although all follicular and marginal zone B cells up-regulate TACI in response to TLR9 stimulation, only marginal zone B cells and a subset of follicular B cells respond to TLR4. Finally, we find that both BLyS and APRIL enhance viability among quiescent and BCR-stimulated B cells. However, although BLyS enhances viability among TLR stimulated B cells, APRIL does not, suggesting that TACI but not BLyS receptor 3 may share survival promoting pathways with TLRs.

Memory CD8+ T Cells Provide an Early Source of IFN-γ

During the non-Ag-specific early phase of infection, IFN-γ is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-γ release, IFN-γ-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-γ was also detected in a significant fraction of CD8+ T cells. CD8+ T cells represented the second major population of IFN-γ-producing cells in the spleen (∼30%) and the majority of IFN-γ+ cells in the lymph nodes (∼70%). LPS-induced IFN-γ production by CD8+ T cells was MHC class I independent and was restricted to CD44high (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8+ T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-α/β, IL-12, and IL-18. IFN-γ was also detected in memory CD8+ T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-γ.

Emergence of CD8 + T Cells Expressing NK Cell Receptors in Influenza A Virus-Infected Mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8+ T cell population at day 10 postinfection. Almost all of the CD8+NK1.1+ cells were CD8αβ+CD3+TCRαβ+ and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-γ after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1−/− mice. These results demonstrate that a significant proportion of CD8+ T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8+ T cell effector functions during viral infection.

cAMP-elevating agents suppress dendritic cell function

The administration of cAMP‐elevating agents affects a number of autoimmune and inflammatory conditions. Because dendritic cells (DCs) play a pivotal role in autoimmunity and inflammation, the isolated effects of cAMP‐elevating agents on the function of DCs was examined. In a dose‐dependent manner, 8‐Bromo cAMP, prostaglandin E2, and 3‐isobutyl‐1‐methylxanthine inhibited tumor necrosis factor α release and suppressed antigen presentation by DCs. The same effect was observed with rolipram, a specific inhibitor of phosphodiesterase type 4, but not with inhibitors of other phosphodiesterases. The decreased antigen presentation by DCs was associated with an enhanced production of interleukin (IL)‐10 and with lower major histocompatibility complex type II (MHC II) expression. Furthermore, the inhibition of antigen presentation and MHC II expression was significantly reversed by treatment of DCs with neutralizing antibody against IL‐10, suggesting the involvement of an IL‐10‐dependent mechanism. Taken together, these results might explain why certain cAMP‐elevating agents such as rolipram are effective in blocking autoimmunity and inflammation.

Inducible MHC class II expression by mast cells supports effector and regulatory T cell activation

In addition to their well-established role as regulators of allergic response, recent evidence supports a role for mast cells in influencing the outcome of physiologic and pathologic T cell responses. One mechanism by which mast cells (MCs) influence T cell function is indirectly through secretion of various cytokines. It remains unclear, however, whether MCs can directly activate T cells through Ag presentation, as the expression of MHC class II by MCs has been controversial. In this report, we demonstrate that in vitro stimulation of mouse MCs with LPS and IFN-γ induces the expression of MHC class II and costimulatory molecules. Although freshly isolated peritoneal MCs do not express MHC class II, an in vivo inflammatory stimulus increases the number of MHC class II-positive MCs in situ. Expression of MHC class II granted MCs the ability to process and present Ags directly to T cells with preferential expansion of Ag-specific regulatory T cells over naive T cells. These data support the notion that, in the appropriate setting, MCs may regulate T cell responses through the direct presentation of Ag.

Role of Mast Cells and Basophils in IgE Responses and in Allergic Airway Hyperresponsiveness

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.