Shinsuke Taki

Multistage Regulation of Th1-Type Immune Responses by the Transcription Factor IRF-1

Eradication of a given pathogen is dependent on the selective differentiation of T helper (Th) cells into Th1 or Th2 types. We show here that T cells from mice lacking the transcription factor IRF-1 fail to mount Th1 responses and instead exclusively undergo Th2 differentiation in vitro. Compromised Th1 differentiation is found to be associated with defects in multiple cell types, namely impaired production of interleukin-12 by macrophages, hyporesponsiveness of CD4+ T cells to interleukin-12, and defective development of natural killer cells. These results indicate the involvement of IRF-1 in multiple stages of the Th1 limb of the immune response.

Requirement for IRF-1 in the microenvironment supporting development of natural killer cells

Natural killer (NK) cells are critical for both innate and adaptive immunity. The development of NK cells requires interactions between their progenitors and the bone-marrow microenvironment; however, little is known about the molecular nature of such interactions. Mice that do not express the transcription factor interferon-regulatory factor-1 (IRF-1; such mice are IRF-1−/− mice) have been shown to exhibit a severe NK-cell deficiency,. Here we demonstrate that the lack of IRF-1 affects the radiation-resistant cells that constitute the microenvironment required for NK-cell development, but not the NK-cell progenitors themselves. We also show that IRF-1−/− bone-marrow cells can generate functional NK cells whencultured with the cytokine interleukin-15 (refs 9-12) and that the interleukin-15 gene is transcriptionally regulated by IRF-1. These results reveal, for the first time, a molecular mechanism by which the bone-marrow microenvironment supports NK-cell development.

CD8+ T Cell–Mediated Skin Disease in Mice Lacking IRF-2, the Transcriptional Attenuator of Interferon-α/β Signaling

Abstract The balanced action of cytokines is known to be critical for the maintenance of homeostatic immune responses. Here, we report the development of an inflammatory skin disease involving CD8 + T cells, in mice lacking the transcription factor, interferon regulatory factor-2 (IRF-2). CD8 + T cells exhibit in vitro hyper-responsiveness to antigen stimulation, accompanied with a notable upregulation of the expression of genes induced by interferon-α/β (IFN-α/β). Furthermore, both disease development and CD8 + T cell abnormality are suppressed by the introduction of nullizygosity to the genes that positively regulate the IFN-α/β signaling pathway. IRF-2 may represent a unique negative regulator, attenuating IFN-α/β-induced gene transcription, which is necessary for balancing the beneficial and harmful effects of IFN-α/β signaling in the immune system.

Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells

Naïve T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8+ T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8+ T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

The Igα/Igβ Heterodimer on μ-Negative ProB Cells Is Competent for Transducing Signals to Induce Early B Cell Differentiation

Abstract The immunoglobulin α (Igα)/Igβ heterodimer was detected on the surface of μ-negative proB cell lines in association with calnexin. The cross-linking of Igβ on proB cells freshly isolated from bone marrow of recombination activating gene (RAG)-2–deficient mice induced a rapid and transient tyrosine-phosphorylation of Igα as well as an array of intracellular proteins including Syk, PI3-kinase, Vav, and SLP-76. It also elicited the phosphorylation and activation of a MAP kinase ERK but not JNK/SAPK or p38. When RAG-2–deficient mice were treated with anti-Igβ monoclonal antibody, developmentally arrested proB cells were induced to differentiate to the small preB cell stage as observed when the μ transgene was expressed in RAG-2–deficient mice. Thus, the cross-linking of Igβ on proB cells appears to elicit differentiation signals analogous to those delivered by the preB cell receptor in normal B cell development.

Prostaglandin D2 Reinforces Th2 Type Inflammatory Responses of Airways to Low-dose Antigen through Bronchial Expression of Macrophage-derived Chemokine

PGD2, a lipid mediator released from mast cells, is known to participate in allergic reactions. However, the mechanism by which PGD2 contributes to such reactions remains unclear. We established a novel experimental model of asthma that permitted direct assessment of the role of PGD2 in airway inflammation. Antigen-sensitized mice were exposed to aerosolized prostaglandin D2 (PGD2) 1 d before challenge with low-dose aerosolized antigen. Not only the numbers of eosinophils, lymphocytes, and macrophages but also the levels of IL-4 and IL-5 in bronchoalveolar lavage fluid were higher in PGD2-pretreated mice than in control mice. The expression of macrophage-derived chemokine (MDC), a chemoattractant for Th2 cells, was greater in PGD2-pretreated mice than in control. Injection of anti-MDC antibody into PGD2-pretreated mice markedly inhibited inflammatory cell infiltration as well as Th2 cyto-kine production after antigen challenge. These results indicate that PGD2 accelerates Th2 type inflammation by induction of MDC. Our results suggest that this mechanism may play a key role in the development of human asthma and that MDC might be a target molecule for therapeutic intervention.

Retroviral expression of the human IL-2 gene in a murine T cell line results in cell growth autonomy and tumorigenicity

In mature T lymphocytes (T cells) the regulated expression of the genes for interleukin‐2 (IL‐2) and its receptor (IL‐2R) constitutes an essential part in controlling the cell growth. Evidence has been provided which suggests the involvement of an aberrant function of the IL‐2 system in developing T cell neoplasms, particularly the adult T cell leukemia/lymphoma (ATL). As an approach to examine the extent of the IL‐2 system contribution to T cell neoplasms, we created the experimental conditions wherein both IL‐2 and IL‐2R are expressed constitutively in a murine T cell line. We made use of a retroviral vector to infect an IL‐2‐dependent CTLL‐2 line and lead to the expression of human IL‐2. Here, we show that the virus‐infected cells not only proliferate in vitro in the absence of exogenously supplied IL‐2 under certain conditions, but also develop tumors (lymphomas) in nude and syngeneic mice.

IFN regulatory factor-2 deficiency revealed a novel checkpoint critical for the generation of peripheral NK cells.

NK cell development is far less understood compared with that of T and B cells despite the critical importance of NK cells in innate immunity. Mice lacking the transcription factor IFN regulatory factor-2 (IRF-2) are known to exhibit NK cell deficiency. However, the role of IRF-2 in NK cell development has remained unclear. In this study we found that NK cell deficiency in the periphery in IRF-2-deficient mice was due to selective loss of mature NK cells, but not to maturation arrest, and NK cells in these mice exhibited very immature surface phenotypes (CD11blowDx5low) with highly compromised NK receptor expression. In contrast, IRF-2-deficient NK cells in bone marrow (BM) showed relatively mature phenotypes (CD11blowDx5high) with less compromised NK receptor repertoire. Furthermore, BM NK cells in IRF-2-deficient mice were found to proliferate almost normally, but underwent accelerated apoptosis. These observations indicated that NK cell maturation could advance up to a late, but not the final, stage in the BM, whereas these cells were incapable of contributing to the peripheral NK cell pool due to premature death in the absence of IRF-2. In contrast, NK cell numbers and Ly49 expression were much more severely reduced in BM in IL-15-deficient mice than in IRF-2−/− mice. The differential peripheral and central NK cell deficiencies in IRF-2−/− mice thus revealed a novel late checkpoint for NK cell maturation, distinct from the early IL-15-dependent expansion stage.

Functional dissection of the cytoplasmic subregions of the IL-2 receptor βc chain in primary lymphocyte populations

The interleukin 2 (IL‐2) receptor βc chain (IL‐2Rβc) is known to regulate the development and function of distinct lymphocyte populations. Thus far, the functions of the IL‐2Rβc cytoplasmic subregions have been studied extensively by using cultured cell lines; however, this approach has limitations with respect to their functions in distinct primary lymphocyte populations. In the present study, we generated mice each expressing a mutant form of an IL‐2Rβc transgene, lacking the cytoplasmic A‐ or H‐region, on an IL‐2Rβc null background. We show that lack of the H‐region, which mediates activation of the Stat5/Stat3 transcription factors, selectively affects the development of natural killer cells and T cells bearing the γδ T cell receptor. This region is also required for the IL‐2‐induced proliferation of T cells in vitro, by upregulating IL‐2Rα expression. In contrast, the A‐region, which mediates activation of the Src family protein tyrosine kinase (PTK) members, contributes to downregulation of the T cell proliferation function. The IL‐2Rβc null mutant mice develop severe autoimmune symptoms; these are all suppressed following the expression of either of the mutants, suggesting that neither the Stats nor the Src PTK members are required. Thus, our present approach offers new insights into the functions of these cytoplasmic subregions of the IL‐2Rβc chain.

Predominant Role of T Cell Receptor (TCR)-α Chain in Forming Preimmune TCR Repertoire Revealed by Clonal TCR Reconstitution System

The CDR3 regions of T cell receptor (TCR)-α and -β chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-α and -β to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-α or -β chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-β or -α chains. Surprisingly, one-third of such TCR-β containing random CDR3β from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-α. A similar dominant function of TCR-α in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus–specific TCR. Subsequently, we generated TCR-α and/or -β transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-α Tg generated CTLs with heterogeneous TCR-β, whereas TCR-β Tg-induced CTLs bearing a single TCR-α. These findings of Ag recognition with minimum involvement of CDR3β expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-α chain in determining the preimmune repertoire of Ag-specific TCR.