Laura Carter

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.

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.

IL-21 Limits NK Cell Responses and Promotes Antigen-Specific T Cell Activation: A Mediator of the Transition from Innate to Adaptive Immunity

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.

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.

Type 1 and Type 2: a fundamental dichotomy for all T-cell subsets

Cytokine secretion is not confined to CD4+ T cells; rather, Type 1 and Type 2 populations of CD8+ and gamma delta T cells can also be generated in vitro and isolated from in vivo situations. These subsets and their physiological functions are significant.

Blockade of Programmed Death-1 Engagement Accelerates Graft-Versus-Host Disease Lethality by an IFN-γ-Dependent Mechanism

Acute graft-vs-host disease (GVHD) is influenced by pathways that can enhance or reduce lethality by providing positive or negative signals to donor T cells. To date, the only reported pathway to inhibit GVHD is the CTLA-4:B7 pathway. Because absence of the programmed death-1 (PD-1) pathway has been implicated in a predisposition to autoimmunity and hence a lack of negative signals, the effect of PD-1 pathway blockade on GVHD was explored using several distinct approaches. In each, GVHD lethality was markedly accelerated. Coblockade of CTLA-4 and PD-1 was additive in augmenting GVHD, indicating that these pathways are not fully redundant. Although neither perforin nor Fas ligand expression was required for GVHD enhancement, donor IFN-γ production was required for optimal GVHD acceleration in the absence of PD-1 ligation. These data indicate that PD-1 ligation down-regulates GVHD through modulation of IFN-γ production and suggest a novel therapeutic target for inhibiting GVHD lethality.

Single cell analyses of cytokine production.

Subsets of T lymphocytes have been defined by their secretion of polarized patterns of cytokines. Type 1 and Type 2 subsets have been characterized in a variety of model systems both in vivo and in vitro and are a useful framework for studying immune responses. Recent technical advances have made it possible to analyze cytokine production by these populations at the level of individual cells.

Regulation of T cell subsets from naive to memory.

To gain insights into the development and regulation of immune responses, we have studied the phenotype, cytokine profiles, activation requirements, and susceptibility to apoptosis of naive CD4, Th1, Th2 polarized effectors, resting memory, and memory effector cells. T cell receptor (TCR) transgenic mice were utilized as a source of enriched naive cells that could be used to generate effector and memory populations. The direct comparison of these populations, which all bear the same TCR, has revealed some interesting distinctions. When restimulated with antigen, effector populations secrete high titers of cytokines in polarized patterns. Retaining their polarized profile, memory cells secrete low levels and memory effector cells secrete very large levels of cytokine. Unlike naive CD4 T cells, effector cell proliferation is not dependent on classic co-stimulation but does require a threshold level of TCR signaling that can be enhanced by accessory interactions. Memory cells have intermediate requirements for co-stimulation/accessory interactions. However, different thresholds of activation are required for production of various cytokines, with requirements for production of interleukin (IL) 2 >> interferon-gamma > IL-4. CD4 subsets also differ dramatically in their susceptibility to apoptosis. Naive Th2 effectors and resting memory cells undergo activation-induced cell death (AICD) 4-7 days after antigen stimulation. In contrast, both primary and memory Th1 effectors undergo rapid AICD mediated by Fas/FasL within 0.5-2 days after stimulation. AICD is substantially blocked by IL-2 and transforming growth factor-beta1, resulting in impressive effector expansion. The process of memory development from effector populations remains mysterious, but these studies suggest roles for cytokines in promoting survival.

Sterol metabolism controls TH17 differentiation by generating endogenous RORγ agonists

Retinoic acid receptor-related orphan receptor γ (RORγt) controls the differentiation of naive CD4(+) T cells into the TH17 lineage, which are critical cells in the pathogenesis of autoimmune diseases. Here we report that during TH17 differentiation, cholesterol biosynthesis and uptake programs are induced, whereas their metabolism and efflux programs are suppressed. These changes result in the accumulation of the cholesterol precursor, desmosterol, which functions as a potent endogenous RORγ agonist. Generation of cholesterol precursors is essential for TH17 differentiation as blocking cholesterol synthesis with chemical inhibitors at steps before the formation of active precursors reduces differentiation. Upon activation, metabolic changes also lead to production of specific sterol-sulfate conjugates that favor activation of RORγ over the TH17-inhibiting sterol receptor LXR. Thus, TH17 differentiation is orchestrated by coordinated sterol synthesis, mobilization and metabolism to selectively activate RORγ.

Cytotoxic T-lymphocyte antigen-4 and programmed death-1 function as negative regulators of lymphocyte activation.

The B7 family of ligands and receptors plays a critical role in the modulation of immune responses. The B7/cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the more recently identified programmed death ligand/programmed death-1 (PD-L/PD-1) ligand/receptor pairs define pathways that function as rheostats of lymphocyte activation. Analysis of receptor and ligand expression patterns, as well as the phenotype of CTLA-4 or PD-1-deficient mice, strongly suggests that these pathways are nonredundant. Current data suggest that the B7/CTLA-4 pathway functions primarily to attenuate, limit, and/or terminate naïve T-cell activation in secondary lymphoid organs. The PD-L/PD-1 pathway, on the other hand, may primarily attenuate, limit, and/or terminate T-, B-, and myeloid cell activation/effector function at sites of inflammation in the periphery.