Masaaki Hashiguchi

Costimulation via Glucocorticoid-Induced TNF Receptor in Both Conventional and CD25+ Regulatory CD4+ T Cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25− CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25−CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-γ, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-κB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.

Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation

Phagocytes such as macrophages and dendritic cells (DCs) engulf apoptotic cells to maintain peripheral immune tolerance. However, the mechanism for the recognition of dying cells by phagocytes is not fully understood. Here, we demonstrate that T-cell immunoglobulin mucin-3 (Tim-3) recognizes apoptotic cells through the FG loop in the IgV domain, and is crucial for clearance of apoptotic cells by phagocytes. Whereas Tim-4 is highly expressed on peritoneal resident macrophages, Tim-3 is expressed on peritoneal exudate macrophages, monocytes, and splenic DCs, indicating distinct Tim-mediated phagocytic pathways used by different phagocytes. Furthermore, phagocytosis of apoptotic cells by CD8(+) DCs is inhibited by anti-Tim-3 mAb, resulting in a reduced cross-presentation of dying cell-associated antigens in vitro and in vivo. Administration of anti-Tim-3 as well as anti-Tim-4 mAb induces autoantibody production. These results indicate a crucial role for Tim-3 in phagocytosis of apoptotic cells and cross-presentation, which may be linked to peripheral tolerance.

CD11b+ Peyer’s Patch Dendritic Cells Secrete IL-6 and Induce IgA Secretion from Naive B Cells

Peyer’s patch (PP) dendritic cells (DCs) have been shown to exhibit a distinct capacity to induce cytokine secretion from CD4+ T cells compared with DCs in other lymphoid organs such as the spleen (SP). In this study, we investigated whether PP DCs are functionally different from DCs in the SP in their ability to induce Ab production from B cells. Compared with SP DCs, freshly isolated PP DCs induced higher levels of IgA secretion from naive B cells in DC-T cell-B cell coculture system in vitro. The IgA production induced by PP DCs was attenuated by neutralization of IL-6. In addition, the induction of IgA secretion by SP DCs, but not PP DCs, was further enhanced by the addition of exogenous IL-6. Finally, we demonstrated that only PP CD11b+ DC subset secreted higher levels of IL-6 compared with other DC subsets in the PP and all SP DC populations, and that PP CD11b+ DC induced naive B cells to produce higher levels of IgA compared with SP CD11b+ DC. These results suggest a unique role of PP CD11b+ DCs in enhancing IgA production from B cells via secretion of IL-6.

Triggering receptor expressed on myeloid cell-like transcript 2 (TLT-2) is a counter-receptor for B7-H3 and enhances T cell responses

The B7 family member B7-H3 (CD276) plays important roles in immune responses. However, the function of B7-H3 remains controversial. We found that murine B7-H3 specifically bound to Triggering receptor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2, TREML2). TLT-2 was expressed on CD8+ T cells constitutively and on activated CD4+ T cells. Stimulation with B7-H3 transfectants preferentially up-regulated the proliferation and IFN-γ production of CD8+ T cells. Transduction of TLT-2 into T cells resulted in enhanced IL-2 and IFN-γ production via interactions with B7-H3. Blockade of the B7-H3:TLT-2 pathway with a mAb against B7-H3 or TLT-2 efficiently inhibited contact hypersensitivity responses. Our results demonstrate a direct interaction between B7-H3 and TLT-2 that preferentially enhances CD8+ T cell activation.

Topical Application of Cream-emulsified CD86 siRNA Ameliorates Allergic Skin Disease by Targeting Cutaneous Dendritic Cells

Induction of the co-stimulatory molecule CD86 on dendritic cells (DCs) in the peripheral tissues is a critical event in triggering antigen-specific immune responses. In this study, we propose a new small interfering RNA (siRNA)-based therapy using cream-emulsified CD86 siRNA, targeting DCs for murine contact hypersensitivity (CH) and atopic dermatitis (AD)-like disease. Topical application of CD86 siRNA efficiently inhibited CH and markedly decreased the numbers of infiltrating CD86(+) or major histocompatibility complex (MHC) class II(+) cells in murine ear skin. The total number of cells, the percentage of hapten-carrying DCs, and their CD86 expression in the regional lymph nodes (RLNs) also significantly decreased. These results suggest that the silencing of CD86 in local DCs inhibits the recruitment and migration of DCs into the skin and RLNs, respectively, resulting in reduced antigen-specific local inflammation. The therapeutic efficacy of the CD86 siRNA was confirmed in AD-prone NC/Nga mice. Treatment produced marked amelioration in the clinical manifestations of AD and reduced the antigen-specific production of interleukin-4 (IL-4) and serum immunoglobulin E (IgE) and IgG1. Our results suggest that the targeting of cutaneous DCs by CD86 siRNA may be a promising strategy in the treatment of allergic skin disease.

Suppressive effect of dietary raffinose on T-helper 2 cell-mediated immunity.

The effects of the dietary oligosaccharide raffinose on immune responses, with special reference to its anti-allergic functions, were examined in vivo. First, feeding a diet supplemented with 50 g raffinose/kg to BALB/c mice significantly (P<0.05) increased interleukin (IL) 12 secretion from isolated Peyers patch (PP) cells in vitro compared with feeding control diet. When isolated PP cells were used as antigen-presenting cells (APC) for CD4+ T-splenocytes isolated from ovalbumin (OVA)-specific T-cell receptor transgenic (Tg) mice in the presence of OVA as antigen, significantly (P<0.05) higher levels of interferon-gamma were observed in the cultures using APC from raffinose-fed mice than those cultures using APC from control mice. Second, the diet containing 50 g raffinose/kg or control diet was fed to OVA Tg mice, and subsequently, OVA was added to each diet to prime T cells in vivo. CD4+ T-cells from the mesenteric lymph nodes of the raffinose-fed mice secreted significantly (P<0.05) higher levels of IL-2 and significantly (P<0.05) lower levels of IL-4 following in vitro antigenic stimulation compared with those of the control mice. These present results suggest that feeding raffinose may suppress differentiation of naïve T-helper (Th) cells into Th2 cells in the mesenteric lymphoid nodes. Last, feeding raffinose suppressed rises of serum immunoglobulin E levels in the Tg mice treated with long-term ingestion of OVA. In conclusion, it is suggested that dietary raffinose suppresses serum immunoglobulin E response through suppression of Th2-type immune response against oral antigen in the lymphoid organs located in or near the intestine.

B7-H1 Overexpression Regulates Epithelial–Mesenchymal Transition and Accelerates Carcinogenesis in Skin

B7-H1 (CD274) is a T-cell coinhibitory molecule that is also often induced on human carcinoma cells, where its expression has been implicated in immune escape. Under inflammatory conditions, B7-H1 is also inducible in normal epithelial cells but little is known about its involvement in conversion of normal cells to tumor cells. Here, we show that skin-specific expression of B7-H1 accelerates inflammatory carcinogenesis in a methylcholantrene (MCA)-induced model of squamous cell carcinoma (SCC). Inflammatory responses induced by MCA or phorbol ester TPA were clearly inhibited in B7-H1 transgenic mice (B7-H1tg mice). Antibody-mediated blockade of either B7-H1 or the related molecule PD-1 revealed that their ability to limit inflammation relied on ligand interactions made by B7-H1 or PD-1. Skin keratinocytes derived from B7-H1tg mice exhibited constitutive reduction of E-cadherin, and SCC induced in B7-H1tg mice also showed loss of E-cadherin along with elevated expression of the transcription factors Slug and Twist that drive epithelial-mesenchymal transition (EMT). Our results indicate that upregulation of B7-H1 in skin epithelial cells promotes EMT and accelerates carcinogenesis, revealing insights into the significance of B7-H1 overexpression on solid tumor cells and hinting at a close relationship between EMT and immune escape signaling pathways in cancer.

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25(-)CD4(+) effector (Teff) and CD25(+)CD4(+) regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4(+) T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4(+) T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.

Enhancement of T-cell-mediated anti-tumour immunity via the ectopically expressed glucocorticoid-induced tumour necrosis factor receptor-related receptor ligand (GITRL) on tumours

Glucocorticoid‐induced tumour necrosis factor receptor‐related receptor (GITR) costimulates functions of both effector and regulatory T cells. The administration of agonistic anti‐GITR monoclonal antibodies efficiently enhances various T‐cell‐mediated immune responses; however, it is unknown to what extent the ligand of GITR (GITRL) contributes to T‐cell responses. We investigated the involvement of endogenously expressed GITRL on dendritic cells and ectopically expressed GITRL on tumours in T‐cell‐mediated immunity. Expression of GITRL on dendritic cells in secondary lymphoid organs was limited, and treatment with anti‐GITRL monoclonal antibodies did not substantially affect T‐cell‐mediated immunity to alloantigens, a specific protein antigen (ovalbumin), or tumour antigens. The introduction of GITRL promoted anti‐tumour immunity in four tumour models. Tumour‐associated GITRL greatly augmented the effector function of CD8+ T cells and enhanced the contribution of CD8+ T cells. These events reduced the crucial contribution of CD25+ CD4+ regulatory T cells, which were found to inhibit immunity against tumours lacking GITRL. Peritumoral injection of GITRL tumour vaccine efficiently inhibited the growth of established tumours. Our results suggest that the ectopic expression of GITRL in tumour cells enhances anti‐tumour immunity at peripheral tumour sites. Consequently, the combined use of a GITRL tumour vaccine with methods aimed at enhancing the activation of host antigen‐presenting cells in secondary lymphoid tissues may be a promising strategy for tumour immunotherapy.

Decrease in expression of α5β1 integrin during neuronal differentiation of cortical progenitor cells

Abstract Neuronal differentiation of embryonic neural progenitor cells is regulated by both intrinsic and extrinsic signals. Since dynamic changes in cell shape typify neuronal differentiation, cell adhesion molecules could be relevant to this process. Although it has been reported that fibronectin–integrin interactions are important for the proliferation of neural progenitor cells, little is known about the contribution of integrins to neuronal differentiation. In order to address this shortfall, we examined integrin expression on cortical progenitor cells by using immunohistochemistry and FACS analysis of cells in which GFP expression was driven by regulatory (promoter) regions of the nestin gene (nestin-GFP+). We here report that high levels of nestin promoter activity correlated with high expression levels of α5β1 integrin (α5β1high cells). FACS analysis of nestin-GFP+ cortical cells revealed an additional subpopulation with reduced expression of α5β1 integrin (α5β1low cells). The size of the α5β1low subpopulation increased during cortical development. To investigate the correlation between integrin and neuronal differentiation, nestin-GFP+ cortical progenitor cells were sorted into α5β1high or α5β1low populations, and each potential to differentiate was analyzed. We show that the nestin-GFP+ α5β1high population corresponded to broadly multipotential neural progenitor cells, whereas nestin-GFP+ α5β1low cells appeared to be committed to a neuronal fate. These findings suggest that α5β1 expression on cortical progenitor cells is developmentally regulated and its downregulation is involved in the process of neuronal differentiation.