Clive Wood

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

Programmed death 1 (PD-1)–deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2–PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2–PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L–PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon γ treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L–PD-1 pathway in regulating T cell responses.

Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production

Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-γ-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-γ and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10-pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothelium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on adjacent normal tissue suggesting a role in attenuating antitumor immune responses.

PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2.

Programmed death‐1 (PD‐1) is an immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐containing receptor expressed upon T cell activation. PD‐1–/– animals develop autoimmune diseases, suggesting an inhibitory role for PD‐1 in immune responses. Members of the B7 family, PD‐L1 and PD‐L2, are ligands for PD‐1. This study examines the functional consequences of PD‐1:PD‐L engagementon murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti‐CD3/PD‐L1.Fc‐coated beads display dramatically decreased proliferation and IL‐2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti‐CD28 mAb can overcome PD‐1‐mediated inhibition by augmenting IL‐2 production. However, PD‐1:PD‐L interactions inhibit IL‐2 production even in the presence of costimulation and, thus, after prolonged activation, the PD‐1:PD‐L inhibitory pathway dominates. Exogenous IL‐2 is able to overcome PD‐L1‐mediated inhibition at all times, indicating that cells maintain IL‐2 responsiveness. Experiments using TCR transgenic CD4+ or CD8+ T cells stimulated with antigen‐presenting cells expressing PD‐L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8+ T cells may be more sensitive to modulation by the PD‐1:PD‐L pathway because of their intrinsic inability to produce significant levels of IL‐2.

PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3ζ signalosome and downstream signaling to PKCθ

Engagement of the immunoinhibitory receptor, programmed death‐1 (PD‐1) attenuates T‐cell receptor (TCR)‐mediated activation of IL‐2 production and T‐cell proliferation. Here, we demonstrate that PD‐1 modulation of T‐cell function involves inhibition of TCR‐mediated phosphorylation of ZAP70 and association with CD3ζ. In addition, PD‐1 signaling attenuates PKCθ activation loop phosphorylation in a cognate TCR signal. PKCθ has been shown to be required for T‐cell IL‐2 production. A phosphorylated PD‐1 peptide, corresponding to the C‐terminal immunoreceptor tyrosine‐switch motif (ITSM), acts as a docking site in vitro for both SHP‐2 and SHP‐1, while the phosphorylated peptide containing the N‐terminal PD‐1 immunoreceptor tyrosine based inhibitory motif (ITIM) associates only with SHP‐2.

Regulation of flt‐1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium

Flt‐1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt‐1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt‐1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt‐1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up‐regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt‐1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt‐1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt‐1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt‐1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt‐1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.

Characterization of murine Flt4 ligand/VEGF-C.

Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based affinity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murine flt4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptide-like sequence that is removed to generate the mature N-terminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21 000 Mr polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.

Expression of Murine Interleukin 11 and its Receptor α-Chain in Adult and Embryonic Tissues

Interleukin 11 (IL‐11) is a multifunctional cytokine that has diverse effects on blood cells and their precursors and on a number of cell types outside of the hematopoietic system. The cDNAs encoding murine IL‐11 and its receptor α‐chain (IL‐11Rα) have recently been isolated. We have used the RNase protection assay to examine the expression of murine IL‐11 and IL‐11Rα in a range of adult mouse tissues, in embryos, and during development of embryonic stem (ES) cells into cystic embryoid bodies in vitro. The testis showed a high level of IL‐11 gene expression while a much lower level of expression was detected in the lung, stomach, small intestine, and large intestine. Expression of IL‐11 was not detected between day 10.5 and day 18.5 post coitum of embryonic development or in differentiating ES cells in vitro. In contrast, the IL‐11Rα was found to be expressed in all adult tissues examined, during embryonic development, and in totipotent and differentiating ES cells.

Isolation of a receptor tyrosine kinase (DTK) from embryonic stem cells: structure, genetic mapping and analysis of expression.

Analysis of receptor tyrosine kinases expressed during mouse embryonic stem cell differentiation resulted in the cloning of a receptor designated developmental tyrosine kinase (DTK). The 850 amino acid mature receptor protein comprises an extracellular domain with two immunoglobulin-like motifs and two fibronectin type III modules, a 25 amino acid transmembrane domain and a cytoplasmic region with a catalytic kinase domain. In embryonic stem cells growing in the presence of leukemia inhibitory factor DTK is abundantly expressed and this level of expression is maintained in differentiating embryonic stem cells and cystic embryoid bodies. In mid-gestational embryos (E14.5), DTK RNA is expressed in many tissues including brain, eye, thymus, lung, heart, gut, liver, testis and limbs. In contrast, expression of DTK in adult mice becomes restricted to brain, portions of the gastrointestinal tract, bladder, testis and ovary. There is enrichment of transcripts encoding DTK in purified fetal liver hematopoietic stem cells, when compared with unfractionated fetal liver. The DTK gene maps to mouse chromosome 2, band F.

Expression of interleukin-11 and its encoding mRNA by glioblastoma cells.

Interleukin-11 (IL-11) is a pleiotropic cytokine with important effects on hematopoietic and other cells. IL-11 was originally described as a product of stromal cell lines and fibroblasts. Using RT-PCR, Northern blotting, and ELISA we demonstrated that the human U373 and U87 glioblastoma cell lines expressed IL-11 and its encoding mRNA when stimulated with IL-1 beta, phorbol ester, and calcium ionophore. The neuroblastoma cell line SH-SY5Y did not express IL-11 mRNA in response to these agents. Cerebral expression of IL-11 by glial cells is important because IL-11 has been shown to have effects on neuronal electrophysiology, has overlapping functions with the neuroactive cytokine interleukin-6, and is part of the gp130-associated neuropoietic family of cytokines.

Strategies for the application of functional genomics technology to biopharmaceutical drug discovery

We have developed a strategy to apply genomics and functional genomics‐based technologies to the discovery of novel proteins which represent potential therapeutic candidates. The core of this strategy is based on a process for the high‐throughput discovery, full‐length cDNA cloning and expression of novel human secreted proteins. The availability of full‐length cDNA sequences enables sophisticated computational sequence analysis. Expression of the encoded proteins facilitates the direct testing of each gene product in many different bioassays. We expect that a systematic approach to the analysis of this bioassay data, in combination with genetic mapping data and gene expression pattern analysis, will accelerate the discovery and functional analysis of human secreted proteins and, ultimately, the rate of development of therapeutic protein products. Drug Dev. Res. 41:173–179, 1997.