Nagahiro Minato

Development of Lupus-like Autoimmune Diseases by Disruption of the PD-1 Gene Encoding an ITIM Motif-Carrying Immunoreceptor

PD-1, a 55 kDa transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif, is induced in lymphocytes and monocytic cells following activation. Aged C57BL/6(B6)-PD-1(-/-) congenic mice spontaneously developed characteristic lupus-like proliferative arthritis and glomerulonephritis with predominant IgG3 deposition, which were markedly accelerated by introduction of a Fas mutation (lpr). Introduction of a PD-1 null mutation into the 2C-TCR (anti-H-2Ld) transgenic mice of the H-2(b/d) background resulted in the chronic and systemic graft-versus-host-like disease. Furthermore, CD8+ 2C-TCR+ PD-1(-/-) T cells exhibited markedly augmented proliferation in vitro in response to H-2d allogenic cells. Collectively, it is suggested that PD-1 is involved in the maintenance of peripheral self-tolerance by serving as a negative regulator of immune responses.

Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade

PD-1 is a receptor of the Ig superfamily that negatively regulates T cell antigen receptor signaling by interacting with the specific ligands (PD-L) and is suggested to play a role in the maintenance of self-tolerance. In the present study, we examined possible roles of the PD-1/PD-L system in tumor immunity. Transgenic expression of PD-L1, one of the PD-L, in P815 tumor cells rendered them less susceptible to the specific T cell antigen receptor-mediated lysis by cytotoxic T cells in vitro, and markedly enhanced their tumorigenesis and invasiveness in vivo in the syngeneic hosts as compared with the parental tumor cells that lacked endogenous PD-L. Both effects could be reversed by anti-PD-L1 Ab. Survey of murine tumor lines revealed that all of the myeloma cell lines examined naturally expressed PD-L1. Growth of the myeloma cells in normal syngeneic mice was inhibited significantly albeit transiently by the administration of anti-PD-L1 Ab in vivo and was suppressed completely in the syngeneic PD-1-deficient mice. These results suggest that the expression of PD-L1 can serve as a potent mechanism for potentially immunogenic tumors to escape from host immune responses and that blockade of interaction between PD-1 and PD-L may provide a promising strategy for specific tumor immunotherapy.

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.

Rap1 Is a Potent Activation Signal for Leukocyte Function-Associated Antigen 1 Distinct from Protein Kinase C and Phosphatidylinositol-3-OH Kinase

ABSTRACT To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (α, βI, βII, and δ) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.

NEDD8 recruits E2-ubiquitin to SCF E3 ligase.

NEDD8/Rub1 is a ubiquitin (Ub)‐like post‐translational modifier that is covalently linked to cullin (Cul)‐family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul‐1, ROC1 and F‐box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul‐1 enhances recruitment of Ub‐conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8‐modifying system accelerates the formation of the E2–E3 complex, which stimulates protein polyubiquitylation.

Analyses of Peripheral Blood Mononuclear Cells in Operational Tolerance After Pediatric Living Donor Liver Transplantation

Operational tolerance (graft acceptance in an immunosuppression (IS)‐free environment) after living‐donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vδ1/Vδ2 γδT‐cells ratio in operationally tolerant patients (Gr‐tol; n = 12), compared with those from age‐matched volunteers (Gr‐vol; n = 24) or patients on IS (Gr‐IS; n = 19). The frequency of NK cells was decreased in Gr‐tol, compared with those in Gr‐IS or Gr‐vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr‐vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.

4F2 (CD98) Heavy Chain Is Associated Covalently with an Amino Acid Transporter and Controls Intracellular Trafficking and Membrane Topology of 4F2 Heterodimer

4F2, also termed CD98, is an integral membrane protein consisting of a heavy chain linked to a light chain by disulfide bond. We have generated a monoclonal antibody to the mouse 4F2 light chain and cloned the cDNA. It encodes a mouse counterpart of rat L-type amino acid transporter-1, and induces system L amino acid transport in Xenopus oocytes in the presence of 4F2 heavy chain. Transfection studies in mammalian cells have indicated that the 4F2 heavy chain is expressed on the plasma membrane on its own, whereas the 4F2 light chain can be transported to the surface only in the presence of 4F2 heavy chain. 4F2 heavy chain is expressed diffusely on the surface of fibroblastic L cells, whereas it is localized selectively to the cell-cell adhesion sites in L cells expressing cadherins. These results indicate that the 4F2 heavy chain is associated covalently with an amino acid transporter and controls the cell surface expression as well as the membrane topology of the 4F2 heterodimer. Although 4F2 heavy and light chains are expressed coordinately in most tissues, the light chain is barely detected by the antibody in kidney and intestine, despite the presence of heavy chain in a complex form. The results predict the presence of multiple 4F2 light chains.

Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.

SMAD4-deficient intestinal tumors recruit CCR1+ myeloid cells that promote invasion.

Inactivation of TGF-β family signaling is implicated in colorectal tumor progression. Using cis-Apc+/Δ716 Smad4+/− mutant mice (referred to as cis-Apc/Smad4), a model of invasive colorectal cancer in which TGF-β family signaling is blocked, we show here that a new type of immature myeloid cell (iMC) is recruited from the bone marrow to the tumor invasion front. These CD34+ iMCs express the matrix metalloproteinases MMP9 and MMP2 and the CC-chemokine receptor 1 (CCR1) and migrate toward the CCR1 ligand CCL9. In adenocarcinomas, expression of CCL9 is increased in the tumor epithelium. By deleting Ccr1 in the background of the cis-Apc/Smad4 mutant, we further show that lack of CCR1 prevents accumulation of CD34+ iMCs at the invasion front and suppresses tumor invasion. These results indicate that loss of transforming growth factor-β family signaling in tumor epithelium causes accumulation of iMCs that promote tumor invasion.

Pivotal Role of CXCR3 in Melanoma Cell Metastasis to Lymph Nodes

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis to specific organs. Here we show that mouse B16F10 melanoma cells constitutively express chemokine receptor CXCR3, and that its ligands CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC induce cellular responses in vitro, such as actin polymerization, migration, invasion, and cell survival. To determine whether CXCR3 could play a role in metastasis to lymph nodes (LNs), we constructed B16F10 cells with reduced CXCR3 expression by antisense RNA and investigated their metastatic activities after s.c. inoculations to syngeneic hosts, C57BL/6 mice. The metastatic frequency of these cells to LNs was markedly reduced to ∼15% (P < 0.05) compared with the parental or empty vector-transduced cells. On the other hand, pretreatment of mice with complete Freund’s adjuvant increased the levels of CXCL9 and CXCL10 in the draining LNs, which caused 2.5–3.0-fold increase (P < 0.05) in the metastatic frequency of B16F10 cells to the nodes with much larger foci. Importantly, such a stimulation of metastasis was largely suppressed when CXCR3 expression in B16F10 cells was reduced by antisense RNA or when mice were treated with specific antibodies against CXCL9 and CXCL10. We also demonstrate that CXCR3 is expressed on several human melanoma cell lines as well as primary human melanoma tissues (5 of 9 samples tested). These results suggest that CXCR3 inhibitors may be promising therapeutic agents for treatment of LN metastasis, including that of melanoma.