The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells.

Immunoglobulin E (IgE) plays a central role in the pathogenesis of Type I hypersensitivity through interaction with a high-affinity receptor (FcεRIα). For therapeutic applications, substantial attention has been focused recently on the blockade of the IgE interaction with FcεRIα. While exploring better options for preventing allergic diseases, we found that the Fab fragment of the rat anti-murine IgE antibody (Fab-6HD5) strongly inhibited passive cutaneous anaphylaxis (PCA) in vivo, as well as spleen tyrosine kinase (Syk) activity and ß-hexosaminidase release from basophilic leukemia cells in vitro. The in vivo effects of Fab-6HD5 pre-administration were maintained over a long period of time for at least 10 days. Using flow cytometry analysis, we also found that Fab-6HD5 did not recognize the IgE Cε3 domain containing specific binding sites for FcεRIα. Furthermore, deletion-mapping studies revealed that Fab-6HD5 recognized conformational epitopes on the Cε2 domain of IgE. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcRIα, our results suggest that the specific binding of Fab-6HD5 to the Cε2 domain prevents allergic reactions through destabilizing the preformed IgE-FcεRIα complex on rat mast cells. Although the present study was performed using animal models, these findings support the idea that a certain antibody directed against IgE CH domains may contribute to preventing allergic diseases through interacting with IgE-FcεRIα complex.

Breast-feeding regulates immune system development via transforming growth factor-ß in mice pups.

BACKGROUND: Breast milk contains important nutrients and immunoregulatory factors that are essential for newborn infants. Recently, epidemiological studies suggested that breast-feeding prevents a wide range of infectious diseases and lowers the incidence of infant allergic diseases. METHODS: To examine the effects of breast milk on immunological development in infancy, we established an artificial rearing system for hand-feeding mice and compared mouse pups fed with either breast milk or milk substitute. All mice were killed at 14 days of age and immune cells in the thymus, spleen, and small intestine were examined on flow cytometry. RESULTS: The number of thymocytes was higher whereas that of total immune cells of peripheral lymphoid tissues was lower in mice fed breast milk compared with milk substitute-fed mice. In peripheral lymphoid tissues, the proportion of B cells was higher and that of CD8+ T cells, macrophages, dendritic cells, and granulocytes was significantly lower in breast milk-fed mice. The same alteration in immune cells of the thymus and peripheral lymphoid tissues in milk substitute-fed mice was also observed in pups reared by mother mice treated with anti-transforming growth factor-ß (anti-TGF-ß) monoclonal antibody. CONCLUSIONS: Breast milk regulates the differentiation and expansion of innate and adaptive immune cells partly due to TGF-ß. Hence, TGF-ß in breast milk may be a new therapeutic target for innate immune system-mediated diseases of infancy.

Notch2 activation ameliorates nephrosis.

Activation of Notch1 and Notch2 has been recently implicated in human glomerular diseases. Here we show that Notch2 prevents podocyte loss and nephrosis. Administration of a Notch2 agonistic monoclonal antibody ameliorates proteinuria and glomerulosclerosis in a mouse model of nephrosis and focal segmental glomerulosclerosis. In vitro, the specific knockdown of Notch2 increases apoptosis in damaged podocytes, while Notch2 agonistic antibodies enhance activation of Akt and protect damaged podocytes from apoptosis. Treatment with triciribine, an inhibitor of Akt pathway, abolishes the protective effect of the Notch2 agonistic antibody. We find a positive linear correlation between the number of podocytes expressing activated Notch2 and the number of residual podocytes in human nephrotic specimens. Hence, specific activation of Notch2 rescues damaged podocytes and activating Notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis.

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.

INTRODUCTION: Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis. METHODS: Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb). RESULTS: Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss. CONCLUSIONS: The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.

Expression of Notch receptors and ligands on immature and mature T cells.

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.

Regulation of experimental autoimmune uveoretinitis by anti-delta-like ligand 4 monoclonal antibody.

PURPOSE: To investigate the involvement of δ-like ligand (Dll)4 in the development of experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. METHODS: B10.RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) peptide 161-180 in complete Freund's adjuvant together with intraperitoneal injection of Bordetella pertussis toxin. mRNA expressions of Notch receptors and their ligands in the eye were evaluated. To investigate the involvement of Dll in EAU, anti-Dll1, anti-Dll4, or control antibody (Ab) was intraperitoneally injected during both the induction and the effector phases or only the effector phase. Alternatively, mice were intraperitoneally injected with γ-secretase inhibitor (GSI) or the control vehicle during the induction phase. Fourteen days after immunization, the eyes and spleens were harvested. The eyes were used for histologic and/or cytokine mRNA expression analysis, whereas the spleens were used for flow cytometric analysis, and antigen-recall proliferation and cytokine assays. RESULTS: Expression of Notch1, 2, 4, and Dll4 in the eye were upregulated by EAU induction. Anti-Dll4 Ab treatment during both the induction and effector phases, but not only the effector phase, significantly reduced the severity of EAU. IFN-γ, IL-12p35, IL-17A, and TGF-ß mRNA expression in the eye were significantly attenuated by treatment with anti-Dll4 Ab. Splenocytes from anti-Dll4 Ab-treated mice showed significantly less proliferation and IL-17 production on antigen stimulation. Also, the severity of EAU was significantly reduced by γ-secretase inhibitor treatment during the induction phase. CONCLUSIONS; Dll4-mediated Notch signaling during the sensitization is critical for the development of EAU. This can be a novel prophylactic target for autoimmune uveitis.

Notch1-mediated signaling induces MHC class II expression through activation of class II transactivator promoter III in mast cells.

Mast cells constitutively express Notch1 and Notch2 on the cell surface. Notch ligand Dll1 (Delta-like 1) stimulation induces MHC class II expression in mast cells and renders them as antigen-presenting cells. However, nothing is known about the mechanism by which Notch signaling induces MHC class II expression in mast cells. MHC class II genes are regulated by the class II transactivator (CIITA). In mice, transcription of the CIITA gene is controlled by three cell type-specific promoters (pI, pIII, and pIV). Here, we show that CIITA expression induced by Dll1 stimulation in mouse bone marrow-derived mast cells (BMMCs) depends critically on the signal mediated by Notch1 and that the most dominant promoter in Notch signaling-mediated CIITA expression in BMMCs is pIII, which is a lymphoid lineage-specific promoter. ChIP assays indicated that Notch signaling increased the binding of the transcription factor PU.1 to CIITA pIII in BMMCs. The knockdown of PU.1 expression using a specific siRNA suppressed Notch signaling-mediated CIITA expression, suggesting that PU.1 contributes to the expression of MHC class II induced by Notch signaling in mast cells. Furthermore, we show that a portion of freshly isolated splenic mast cells express MHC class II and that the most dominant promoter of CIITA in mast cells is pIII. These findings indicate that activation of CIITA pIII plays an important role in MHC class II expression in mast cells.

Notch2 signaling is required for potent antitumor immunity in vivo.

CD8(+) T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8(+) T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8(+) T cells is required for antitumor CTL responses. Notch2(flox/flox) mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8(+) T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1(flox/flox) mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8(+) T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.

Development of monoclonal antibodies for analyzing immune and hematopoietic systems of common marmoset.

OBJECTIVE: Common marmosets are considered experimental animals of primates useful for medical research. We developed several monoclonal antibodies (mAbs) directed to CD molecules to gain initial insight into the immune and hematopoietic systems of this organism, and analyzed the basic cellularity and characters of marmoset lymphocytes. MATERIALS AND METHODS: Anti-marmoset CD antigen mAbs were prepared using marmoset antigen-expressing transfectants and used for flow cytometric analyses and cell fractionation. Expression of T-cell-related cytokine gene transcripts was examined in response to T-cell receptor stimulation by reverse transcription polymerase chain reaction analyses. Hematopoietic progenitor activities of marmoset bone marrow cells were examined in fractionated cells by mAbs against CD117 (c-kit) and CD34. RESULTS: CD4 and CD8 expression profiles in T-cell subsets of marmoset were essentially similar to those in mouse and human. CD4(+) and CD8(+) subsets were isolated from marmoset spleens. Detected transcripts after stimulation of T cells included Th1-, Th2-, and Th17-related cytokines in CD4(+) cells and cytotoxic proteases in CD8(+) cells, respectively. Colony-forming abilities were detected mainly in CD117 (c-kit)(+) cells, irrespective of CD34 expression. CONCLUSIONS: Marmoset immune system was basically similar to human and mouse systems.

Eotaxin-1, -2, and -3 immunoreactivity and protein concentration in the nasal polyps of eosinophilic chronic rhinosinusitis patients.

OBJECTIVES/HYPOTHESIS: Eosinophilic chronic rhinosinusitis (CRS) is characterized by the accumulation of numerous eosinophils in the sinus mucosa and nasal polyps, which are frequently difficult to control, even with surgery. The present study was designed to evaluate the expression and localization of eotaxins, which are well known to be potent and selective chemoattractants for eosinophils in CRS. STUDY DESIGN: Randomized study. METHODS: The patients were classified into eosinophilic and noneosinophilic groups. Histopathological profiles of the nasal polyp were observed with hematoxylin-eosin staining. Eotaxin-1, -2, and -3 were immunohistochemically stained in the nasal polyps. Furthermore, the protein content of eotaxin subtypes inside the nasal polyp and sinus effusion was measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: In the nasal polyps, immunoreactivities of the eotaxin subfamily, eotaxin-1, -2, and -3, were noted in most of the infiltrating eosinophils, as well as in other inflammatory cells, epithelial cells, and endothelial cells. Compared with noneosinophilic CRS groups, eosinophilic CRS groups had a significant expression of eotaxins in their eosinophils. The eotaxin concentrations of nasal polyp and sinus effusion as measured by ELISA were significantly increased in the eosinophilic CRS group compared to the noneosinophilic CRS group. CONCLUSIONS: The present findings suggest that enhanced eotaxin family production by eosinophils results in the recruitment of eosinophils into the tissue by a self-amplifying process. Laryngoscope, 2009.

Role of ephrinB2 in nonproductive angiogenesis induced by Delta-like 4 blockade.

Delta-like 4 (DLL4) is one of the Notch ligands and plays an important role in vascular development. DLL4 blockade inhibits tumor growth by promoting nonproductive angiogenesis, which is characterized by an increase in vascular density and decrease in tissue perfusion. However, a detailed mechanism remains unclear. In this study, newly developed neutralizing antibodies against mouse and human DLL4 were used to investigate the possible involvement of VEGF-DLL4-ephrinB2 cascade in nonproductive angiogenesis caused by DLL4 blockade. DLL4 blockade and soluble ephrinB2 treatment suppressed tumor growth and induced nonproductive angiogenesis. DLL4 was expressed in subcutaneous tumors, and DLL4 blockade suppressed ephrinB2 expression in the tumors. DLL4 blockade significantly promoted human umbilical vein endothelial cell (HUVEC) proliferation in vitro, and the effect was additive to that of VEGF. Both DLL4 blockade and VEGF significantly increased cord length and branch points in a tubular formation assay. Expression of ephrinB2 in HUVECs was enhanced by VEGF alone, and the enhancement was inhibited by DLL4 blockade. Moreover, when we studied the effect of ephrinB2 RNA interference on HUVEC tubular formation, knockdown of ephrinB2 mimicked the effect of DLL4. These results suggest that ephrinB2 plays a crucial role in nonproductive angiogenesis caused by DLL4 blockade.

Differential regulation of splenic CD8- dendritic cells and marginal zone B cells by Notch ligands.

The importance of Notch signaling to maintain CD8- dendritic cells (DCs) in the spleen has recently been revealed. However, the ligand responsible for this Notch signaling has not been determined yet. In this study, we demonstrated that blocking of Delta-like (Dll) 1 alone had no significant effect on the maintenance of CD8- DCs while marginal zone (MZ) B cells were significantly reduced in the spleen of mice. On the other hand, blocking of Dll1, Dll4, Jagged1 and Jagged2 significantly decreased CD8- DCs. All these Notch ligands are expressed predominantly in the red pulp of the spleen where CD8- DCs reside. These results indicate a differential regulation of CD8- DCs and MZ B cells by Notch ligands in the spleen.

Notch signaling confers antigen-presenting cell functions on mast cells.

BACKGROUND: Notch signaling is involved in cell fate determination along with the development of the immune system. However, very little is known about the role for Notch signaling in mast cells. OBJECTIVE: We investigated the role of Notch signaling in mast cell functions. METHODS: After mouse bone marrow-derived mast cells (BMMCs) or peritoneal mast cells (PMCs) were cocultured with mouse Notch ligand-expressing chinese hamster ovary cells for 5 days, we examined the mast cell surface expressions of MHC-II molecules and OX40 ligand (OX40L), Fc epsilon RI-mediated cytokine production, and the effects of the mast cells on proliferation and differentiation of naive CD4(+) T cells in vitro. RESULTS: We showed that BMMCs and PMCs constitutively expressed Notch1 and Notch2 proteins on the cell surface. We also found that Delta-like 1 (Dll1)/Notch signaling induced the expression of MHC-II and upregulated the expression level of OX40L on the surface of the mast cells. Dll1/Notch signaling augmented Fc epsilon RI-mediated IL-4, IL-6, IL-13, and TNF production by BMMCs. Dll1-stimulated MHC-II(+)OX40L(high) BMMCs promoted proliferation of naive CD4(+) T cells and their differentiation into T(H)2 cells producing IL-4, IL-5, IL-10, and IL-13. CONCLUSION: Dll1/Notch signaling confers the functions as an antigen-presenting cell on mast cells, which preferentially induce the differentiation of T(H)2.

Notch ligand Delta-like4 inhibits the development of murine experimental allergic conjunctivitis.

BACKGROUND: To investigate the involvement of Notch ligands in the development of experimental allergic conjunctivitis (EC) in mice. METHODS: To induce EC, wild-type (WT) or IFN-gamma-deficient (GKO) BALB/c mice were immunized with ragweed (RW) in alum followed by RW challenge in eye drops. Twenty-four hours after RW challenge, the conjunctivas, spleens and blood were harvested to evaluate conjunctival eosinophil infiltration, RW-specific cytokine production and serum Ig levels, respectively. Abs against Notch ligands (anti-Jagged1, anti-Jagged2, anti-Delta-like (Dll)1 and anti-Dll4) were injected intraperitoneally into EC-developing mice during the induction or effector phase. As a control, normal hamster IgG (nhIgG) was injected. RESULTS: Treatment with anti-Dll4 Ab but not the other Abs during the induction phase significantly augmented the severity of EC, as measured by the conjunctival eosinophil infiltration. Anti-Dll4 Ab treatment also significantly upregulated RW-recall IL-4 production and suppressed serum IgE and IgG1 levels. However, anti-Dll4 Ab treatment during the induction phase did not significantly affect the severity of EC in GKO mice. None of the Abs significantly affected the severity of EC, splenocyte cytokine production, or serum Ig levels when administered during the effector phase. CONCLUSIONS: These observations suggest that Dll4, a Notch ligand, plays a role in suppressing the development of EC, possibly by providing a negative signal for Th2 development during the induction phase. In addition, IFN-gamma may participate in the augmentation of EC by anti-Dll4 Ab treatment.

Highly efficient and feeder-free production of subculturable vascular endothelial cells from primate embryonic stem cells.

The vascular endothelial cell (VEC) differentiation from primate embryonic stem (ES) cells has critical problems: low differentiation efficiencies (<2%) and/or subculture incapability. We report a novel feeder-free culture method for high efficiency production of subculturable VECs from cynomolgus monkey ES cells. Spheres, which were generated from ES cells in the presence of cytokine cocktail, were cultured on gelatin-coated plates. Cobblestone-shaped cells spread out after a few days, which were followed by an emergence of a sac-like structure containing hematopoietic cells. All adherent cells including sac walls cells and surrounding cobblestone cells expressed vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Subculture of these cells resulted in a generation of homogeneous spindle-shaped population bearing cord-forming activities and a uniform acetylated low density lipoprotein-uptaking capacity with von Willbrand factor and endothelial nitric oxide synthetase expressions. They were freeze-thaw-tolerable and subculturable up to eight passages. Co-existence of pericytes or immature ES cells was ruled out. When introduced in a collagen sponge plug implanted intraperitoneally in mice, ES-derived cells recruited into neovascularity. Although percentages of surface VE-cadherin-positive population varied from 20% to 80% as assessed by flow cytometry, the surface VE-cadherin-negative population showed intracellular VE-cadherin expression and mature functions, as we call it as atypical VECs. When sorted, the surface VE-cadherin-positive population expanded as almost pure (>90%) VE-cadherin/PECAM-1-positive VECs by 160-fold after five passages. Thus, our system provides pure production of functional, subculturable and freeze-thaw-tolerable VECs, including atypical VECs, from primate ES cells.

Dendritic cell-mediated NK cell activation is controlled by Jagged2-Notch interaction.

Natural killer (NK) cells regulate various immune responses by exerting cytotoxic activity or secreting cytokines. The interaction of NK cells with dendritic cells (DC) contributes to NK cell-mediated antitumor or antimicrobial responses. However, the cellular and molecular mechanisms for controlling this interaction are largely unknown. Here, we show an involvement of Jagged2-Notch interaction in augmenting NK cell cytotoxicity mediated by DC. Enforced expression of Jagged2 on A20 cells (Jag2-A20 cells) suppressed their growth in vivo, which was abrogated by depleting NK cells. Moreover, Jag2-A20 cells exerted a suppression on the growth of nonmanipulated A20 cells in SCID mice in an NK-dependent manner. Consistently, coinoculation of A20 cells with DC overexpressing Jagged2 (Jag2-DC) suppressed the growth of A20 cells in mice. Stimulation of NK cells with Jagged2 directly enhanced their cytotoxicity, IFN-gamma production, and proliferation. Ligation of Notch2 on NK cells enhanced their cytotoxic activity, and Jag2-DC or CpG-treated DC-mediated NK cell cytotoxicity was suppressed by a gamma-secretase inhibitor. These results indicate that the Jagged2-Notch axis plays a crucial role in DC-mediated NK cell cytotoxicity. Furthermore, manipulation of this interaction may provide an approach to induce potent tumor immunity or to inhibit certain autoimmune diseases caused by NK cell activation.

Delta-like 1 is essential for the maintenance of marginal zone B cells in normal mice but not in autoimmune mice.

Notch2 and Delta-like 1 (Dll1) have been implicated in the development of marginal zone B (MZB) cells. In the present study, we characterized the expression and function of mouse Notch receptors and ligands in the spleen by using newly generated mAbs. Although Notch2 was expressed on both B and T cells in the spleen, the highest expression was observed on precursors of marginal zone B and MZB cells. Dll1 was expressed on macrophage and erythroblasts in the red pulp, but not on B cells or marginal zone macrophage. Administration of a blocking mAb against Dll1 not only blocked the development of MZB cells in juvenile mice but also gradually depleted the pre-established MZB cells in adult mice, indicating a critical role for Dll1 in the maintenance of MZB cells in the spleen of normal mice. Interestingly, Dll1 was not necessary for the maintenance of MZB cells in lupus-prone (NZB x NZW) F1 mice particularly after the onset of the disease, suggesting that the Dll1 independence may be a feature of dysregulated MZB cells producing auto-antibodies.

Involvement of TNF-like weak inducer of apoptosis in the pathogenesis of collagen-induced arthritis.

TNF-like weak inducer of apoptosis (TWEAK) is a type II membrane protein belonging to the TNF family that regulates apoptotic cell death, cellular proliferation, angiogenesis, and inflammation. However, the role of TWEAK in the pathogenesis of rheumatoid arthritis (RA) remains unclear. In this study, we have investigated the effect of neutralizing anti-TWEAK mAb on the development of collagen-induced arthritis (CIA), a well-established murine model of RA. Administration of anti-TWEAK mAb significantly ameliorated paw swelling, synovial hyperplasia, and infiltration of inflammatory cells. The levels of proinflammatory chemokines such as MCP-1 and MIP-2 in serum and knee joints were reduced by this treatment. Consistently, recombinant TWEAK enhanced the proliferation of MCP-1 and MIP-2 production by synovial cells from CIA mice in vitro. Histological examination also revealed that the treatment with anti-TWEAK mAb suppressed the development of small vessels in synovial tissues. These results indicated anti-inflammatory and antiangiogenic effects of the TWEAK blockade in CIA, which may be also beneficial for the treatment of RA.

Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production.

PD-1 is an immunoinhibitory receptor that belongs to the CD28/CTLA-4 family. B7-H1 (PD-L1) and B7-DC (PD-L2), which belong to the B7 family, have been identified as ligands for PD-1. Paradoxically, it has been reported that both B7-H1 and B7-DC co-stimulate or inhibit T cell proliferation and cytokine production. To determine the role of B7-H1 and B7-DC in T cell-APC interactions, we examined the contribution of B7-H1 and B7-DC to CD4+ T cell activation by B cells, dendritic cells, and macrophages using anti-B7-H1, anti-B7-DC, and anti-PD-1 blocking mAbs. Anti-B7-H1 mAb and its Fab markedly inhibited the proliferation of anti-CD3-stimulated naive CD4+ T cells, but enhanced IL-2 and IFN-gamma production in the presence of macrophages. The inhibition of T cell proliferation by anti-B7-H1 mAb was abolished by neutralizing anti-IFN-gamma mAb. Coculture of CD4+ T cells and macrophages from IFN-gamma-deficient or wild-type mice showed that CD4+ T cell-derived IFN-gamma was mainly responsible for the inhibition of CD4+ T cell proliferation. Anti-B7-H1 mAb induced IFN-gamma-mediated production of NO by macrophages, and inducible NO synthase inhibitors abrogated the inhibition of CD4+ T cell proliferation by anti-B7-H1 mAb. These results indicated that the inhibition of T cell proliferation by anti-B7-H1 mAb was due to enhanced IFN-gamma production, which augmented NO production by macrophages, suggesting a critical role for B7-H1 on macrophages in regulating IFN-gamma production by naive CD4+ T cells and, hence, NO production by macrophages.

Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells.

Naturally arising CD4(+)CD25(+) regulatory T (T(R)) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4(+)CD25(-) T cells also possesses regulatory activity. Programmed death-1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4(+)CD25(-)PD-1(+) T cells in the spleen of naive mice that constitutively expressed CTLA-4 and FoxP3 and was hypoproliferative in response to anti-CD3 antibody stimulation in vitro. However, the CD4(+)CD25(-)PD-1(+) T cells uniquely produced large amounts of IL-4 and IL-10 in response to anti-CD3 and anti-CD28 mAb stimulation, unlike the CD4(+)CD25(+) T(R) cells. The CD4(+)CD25(-)PD-1(+) T cells exhibited a suppressor activity against the proliferation of anti-CD3 antibody-stimulated CD4(+)CD25(-)PD-1(-) T cells in vitro, which was partially abrogated by anti-CTLA-4 mAb, but not by anti-IL-10 or anti-PD-1 mAb. Remarkably, the CD4(+)CD25(-)PD-1(+) T cells inhibited the development of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into C.B17-scid/scid mice, albeit to a lesser extent than CD4(+)CD25(+) T(R) cells, in a CTLA-4-dependent manner. These results indicate that the CD4(+)CD25(-)PD-1(+) T cells contain substantial amounts of T(R) cells that are involved in the maintenance of peripheral tolerance.