Lori Fitz

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

We have identified and purified a novel cytokine, NK cell stimulatory factor (NKSF), from the cell-free supernatant fluid of the phorbol diester-induced EBV-transformed human B lymphoblastoid cell line RPMI 8866. NKSF activity is mostly associated to a 70-kD anionic glycoprotein. The purified 70-kD protein, isolated from an SDS-PAGE gel, yields upon reduction two small species of molecular masses of 40 and 35 kD, suggesting that this cytokine is a heterodimer. When added to human PBL, purified NKSF preparations induce IFN-gamma production and synergize with rIL-2 in this activity, augment the NK cell-mediated cytotoxicity of PBL preparations against both NK-sensitive and NK-resistant target cell lines, and enhance the mitogenic response of T cells to mitogenic lectins and phorbol diesters. The three activities remain associated through different purification steps resulting in a 9,200-fold purification, and purified NKSF mediates the three biological activities at concentrations in the range of 0.1-10 pM. These data strongly suggest that the same molecule mediates these three activities, although the presence of traces of contaminant peptides even in the most purified NKSF preparations does not allow us to exclude the possibility that distinct biologically active molecules have been co-purified. The absence of other known cytokines in the purified NKSF preparations, the unusual molecular conformation of NKSF, the high specific activity of the purified protein, and the spectrum of biological activities distinguish NKSF from other previously described cytokines.

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.

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 CD3zeta. In addition, PD-1 signaling attenuates PKCtheta activation loop phosphorylation in a cognate TCR signal. PKCtheta 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.

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 engagement on 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.