PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance.

Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1-deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1-deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K-AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1-deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity.

The PD-1 Pathway Regulates Development and Function of Memory CD8+ T Cells following Respiratory Viral Infection.

The PD-1 pathway regulates dysfunctional T cells in chronic infection and cancer, but the role of this pathway during acute infection remains less clear. Here, we demonstrate that PD-1 signals are needed for optimal memory. Mice deficient in the PD-1 pathway exhibit impaired CD8+ T cell memory following acute influenza infection, including reduced virus-specific CD8+ T cell numbers and compromised recall responses. PD-1 blockade during priming leads to similar differences early post-infection but without the defect in memory formation, suggesting that timing and/or duration of PD-1 blockade could be tailored to modulate host responses. Our studies reveal a role for PD-1 as an integrator of CD8+ T cell signals that promotes CD8+ T cell memory formation and suggest PD-1 continues to fine-tune CD8+ T cells after they migrate into non-lymphoid tissues. These findings have important implications for PD-1-based immunotherapy, in which PD-1 inhibition may influence memory responses in patients.

The PTEN pathway in Tregs is a critical driver of the suppressive tumor microenvironment.

The tumor microenvironment is profoundly immunosuppressive. We show that multiple tumor types create intratumoral immune suppression driven by a specialized form of regulatory T cell (Treg) activation dependent on the PTEN (phosphatase and tensin homolog) lipid phosphatase. PTEN acted to stabilize Tregs in tumors, preventing them from reprogramming into inflammatory effector cells. In mice with a Treg-specific deletion of PTEN, tumors grew slowly, were inflamed, and could not create an immunosuppressive tumor microenvironment. In normal mice, exposure to apoptotic tumor cells rapidly elicited PTEN-expressing Tregs, and PTEN-deficient mice were unable to maintain tolerance to apoptotic cells. In wild-type mice with large established tumors, pharmacologic inhibition of PTEN after chemotherapy or immunotherapy profoundly reconfigured the tumor microenvironment, changing it from a suppressive to an inflammatory milieu, and tumors underwent rapid regression. Thus, the immunosuppressive milieu in tumors must be actively maintained, and tumors become susceptible to immune attack if the PTEN pathway in Tregs is disrupted.

RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance.

We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb), which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, whereas lung dendritic cells express PD-L2. Blockade of the RGMb-PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2-deficient mice showed that PD-L2 expression on non-T cells was critical for respiratory tolerance, but expression on T cells was not required. Because PD-L2 binds to both PD-1, which inhibits antitumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer, and other immune-mediated disorders. Understanding this pathway may provide insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events.

The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood.

CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (T(FR) cells) inhibit humoral immunity mediated by CD4(+)CXCR5(+)Foxp3(-) follicular helper T cells (T(FH) cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of T(FR) cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of T(FR) cells in the lymph nodes and that those T(FR) cells had enhanced suppressive ability. We also found substantial populations of T(FR) cells in mouse blood and demonstrated that T(FR) cells in the blood homed to lymph nodes and potently inhibited T(FH) cells in vivo. T(FR) cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.

Interleukin-27 priming of T cells controls IL-17 production in trans via induction of the ligand PD-L1.

Interleukin-27 (IL-27) is a key immunosuppressive cytokine that counters T helper 17 (Th17) cell-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in T cells rapidly induced by IL-27. We found that IL-27 priming of naive T cells upregulated expression of programmed death ligand 1 (PD-L1) in a signal transducer and activator of transcription 1 (STAT1)-dependent manner. When cocultured with naive CD4(+) T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through a PD-1-PD-L1 interaction. In vivo, coadministration of naive TCR transgenic T cells (2D2 T cells) with IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Thus, these data identify a suppressive activity of IL-27, by which CD4(+) T cells can restrict differentiation of Th17 cells in trans.

Neuronal programmed cell death-1 ligand expression regulates retinal ganglion cell number in neonatal and adult mice.

OBJECTIVES: During mouse retina maturation, the final number of retinal ganglion cells (RGCs) is determined by highly regulated programmed cell death. Previous studies demonstrated that the immunoregulatory receptor programmed cell death-1 (PD-1) promotes developmental RGC death. To identify the functional signaling partner(s) for PD-1, we identified retinal expression of PD-1 ligands and examined the effect of PD-1 ligand expression on RGC number. We also explored the hypothesis that PD-1 signaling promotes the development of functional visual circuitry. METHODS: Characterization of retinal and brain programmed cell death-1 ligand 1 (PD-L1) expression were examined by immunofluorescence on tissue sections. The contribution of PD-ligands, PD-L1, and programmed cell death-1 ligand 2 (PD-L2) to RGC number was examined in PD-ligand knockout mice lacking 1 or both ligands. Retinal architecture was assessed by spectral-domain optical coherence tomography, and retinal function was analyzed by electroretinography in wild-type and PD-L1/L2 double-deficient mice. RESULTS: PD-L1 expression is found throughout the neonatal retina and persists in adult RGCs, bipolar interneurons, and Müller glia. In the absence of both PD-ligands, there is a significant numerical increase in RGCs (34% at postnatal day 2 [P2] and 18% in adult), as compared to wild type, and PD-ligands have redundant function in this process. Despite the increased RGC number, adult PD-L1/L2 double-knockout mice have normal retinal architecture and outer retina function. CONCLUSION: This study demonstrates that PD-L1 and PD-L2 together impact the final number of RGCs in adult mice and supports a novel role for active promotion of neuronal cell death through PD-1 receptor-ligand engagement.

Physiologic control of IDO competence in splenic dendritic cells.

Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.

Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF.

CD8(+) T cells in chronic viral infections such as HIV develop functional defects including loss of interleukin-2 (IL-2) secretion and decreased proliferative potential that are collectively termed 'exhaustion'. Exhausted T cells express increased amounts of multiple inhibitory receptors, such as programmed death-1 (PD-1), that contribute to impaired virus-specific T cell function. Although reversing PD-1 inhibition is therefore an attractive therapeutic strategy, the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling. It is not known whether PD-1 also acts by upregulating genes in exhausted T cells that impair their function. Here we analyzed gene expression profiles from HIV-specific CD8(+) T cells in individuals with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8(+) T cells from humans and mice. This program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, whereas BATF knockdown reduced PD-1 inhibition. Silencing BATF in T cells from individuals with chronic viremia rescued HIV-specific T cell function. Thus, inhibitory receptors can cause T cell exhaustion by upregulating genes--such as BATF--that inhibit T cell function. Such genes may provide new therapeutic opportunities to improve T cell immunity to HIV.

The PD-1 pathway in tolerance and autoimmunity.

Regulatory T cells (Tregs) and the PD-1: PD-ligand (PD-L) pathway are both critical to terminating immune responses. Elimination of either can result in the breakdown of tolerance and the development of autoimmunity. The PD-1: PD-L pathway can thwart self-reactive T cells and protect against autoimmunity in many ways. In this review, we highlight how PD-1 and its ligands defend against potentially pathogenic self-reactive effector T cells by simultaneously harnessing two mechanisms of peripheral tolerance: (i) the promotion of Treg development and function and (ii) the direct inhibition of potentially pathogenic self-reactive T cells that have escaped into the periphery. Treg cells induced by the PD-1 pathway may also assist in maintaining immune homeostasis, keeping the threshold for T-cell activation high enough to safeguard against autoimmunity. PD-L1 expression on non-hematopoietic cells as well as hematopoietic cells endows PD-L1 with the capacity to promote Treg development and enhance Treg function in lymphoid organs and tissues that are targets of autoimmune attack. At sites where transforming growth factor-beta is present (e.g. sites of immune privilege or inflammation), PD-L1 may promote the de novo generation of Tregs. When considering the consequences of uncontrolled immunity, it would be therapeutically advantageous to manipulate Treg development and sustain Treg function. Thus, this review also discusses how the PD-1 pathway regulates a number of autoimmune diseases and the therapeutic potential of PD-1: PD-L modulation.

PD-L1 regulates the development, maintenance, and function of induced regulatory T cells.

Both the programmed death (PD) 1-PD-ligand (PD-L) pathway and regulatory T (T reg) cells are instrumental to the maintenance of peripheral tolerance. We demonstrate that PD-L1 has a pivotal role in regulating induced T reg (iT reg) cell development and sustaining iT reg cell function. PD-L1(-/-) antigen-presenting cells minimally convert naive CD4 T cells to iT reg cells, showing the essential role of PD-L1 for iT reg cell induction. PD-L1-coated beads induce iT reg cells in vitro, indicating that PD-L1 itself regulates iT reg cell development. Furthermore, PD-L1 enhances and sustains Foxp3 expression and the suppressive function of iT reg cells. The obligatory role for PD-L1 in controlling iT reg cell development and function in vivo is illustrated by a marked reduction in iT reg cell conversion and rapid onset of a fatal inflammatory phenotype in PD-L1(-/-)PD-L2(-/-) Rag(-/-) recipients of naive CD4 T cells. PD-L1 iT reg cell development is mediated through the down-regulation of phospho-Akt, mTOR, S6, and ERK2 and concomitant with the up-regulation of PTEN, all key signaling molecules which are critical for iT reg cell development. Thus, PD-L1 can inhibit T cell responses by promoting both the induction and maintenance of iT reg cells. These studies define a novel mechanism for iT reg cell development and function, as well as a new strategy for controlling T reg cell plasticity.

B7-1/2, but not PD-L1/2 molecules, are required on IL-10-treated tolerogenic DC and DC-derived exosomes for in vivo function.

Costimulatory molecules, such as B7-1/2 and PD-L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7-1/2 and PD-L1/2 to the function of IL-10-treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL-10 treatment of DC significantly downregulated surface expression of MHC II, B7-1, B7-2, and decreased levels of MHC I and PD-L2. IL-10 treatment of DC resulted in a modified costimulatory profile of DC-secreted exosomes with a reduction in B7-1, PD-L1 and PD-L2. We further demonstrate that absence of B7-1 or B7-2 on donor DC results in a loss of ability of IL-10-treated DC and their exosomes to suppress the delayed-type hypersensitivity response, whereas IL-10-treated DC deficient in PD-L1/2 as well as their secreted exosomes retained the ability to suppress delayed-type hypersensitivity responses. We conclude that B7-1 and B7-2, but not PD-L1 and PD-L2, on IL-10-treated DC and DC-derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.

Role of the immune modulator programmed cell death-1 during development and apoptosis of mouse retinal ganglion cells.

PURPOSE: Mammalian programmed cell death (PD)-1 is a membrane-associated receptor regulating the balance between T-cell activation, tolerance, and immunopathology; however, its role in neurons has not yet been defined. The hypothesis that PD-1 signaling actively promotes retinal ganglion cell (RGC) death within the developing mouse retina was investigated. METHODS: Mature retinal cell types expressing PD-1 were identified by immunofluorescence staining of vertical retina sections; developmental expression was localized by immunostaining and quantified by Western blot analysis. PD-1 involvement in developmental RGC survival was assessed in vitro using retinal explants and in vivo using PD-1 knockout mice. PD-1 ligand gene expression was detected by RT-PCR. RESULTS: PD-1 is expressed in most adult RGCs and undergoes dynamic upregulation during the early postnatal window of retinal cell maturation and physiological programmed cell death (PCD). In vitro blockade of PD-1 signaling during this time selectively increases the survival of RGCs. Furthermore, PD-1-deficient mice show a selective increase in RGC number in the neonatal retina at the peak of developmental RGC death. Lastly, gene expression of the immune PD-1 ligand genes Pdcd1lg1 and Pdcd1lg2 was found throughout postnatal retina maturation. CONCLUSIONS: These findings collectively support a novel role for a PD-1-mediated signaling pathway in developmental PCD during postnatal RGC maturation.

Intestinal tolerance is converted to autoimmune enteritis upon PD-1 ligand blockade.

The B7 family member programmed death-1 ligand (PD-L1) has been shown to play an inhibitory role in the regulation of T cell responses in several organs. However, the role of PD-L1 in regulating tolerance to self-Ags of the small intestine has not been previously addressed. In this study, we investigated the role of PD-L1 in CD8(+) T cell tolerance to an intestinal epithelium-specific Ag using the iFABP-tOVA transgenic mouse model, in which OVA is expressed as a self-Ag throughout the small intestine. Using adoptive transfer of naive OVA-specific CD8(+) T cells, we show that loss of PD-1:PD-L1 signaling, by either Ab-mediated PD-L1 blockade or transfer of PD-1(-/-) T cells, leads to considerable expansion of OVA-specific CD8(+) T cells and their differentiation into effector cells capable of producing proinflammatory cytokines. A fatal CD8(+) T cell-mediated inflammatory response develops rapidly against the small bowel causing destruction of the epithelial barrier, severe blunting of intestinal villi, and recruitment and activation of myeloid cells. This response is highly specific because immune destruction selectively targets the small intestine but not other organs. Collectively, these results indicate that loss of the PD-1:PD-L1 inhibitory pathway breaks CD8(+) T cell tolerance to intestinal self-Ag, thus leading to severe enteric autoimmunity.

PD-1 and its ligands in T-cell immunity.

The past year has seen significant advances in our understanding of the critical roles of negative immunoregulatory signals delivered by the programmed death 1 (PD-1)-PD-1 ligand (PD-L) pathway in regulating T-cell activation and tolerance. Emerging evidence indicates that PD-Ls play an essential role on dendritic cells (DCs), both directly during DC-T cell interactions and indirectly through signaling into the DC. Recent studies point to a novel role for PD-L1 in maintaining tissue tolerance. Finally, PD-1 has recently been shown to be highly expressed on exhausted T cells during chronic viral infection, and blockade of PD-1 or PD-L1 can revive exhausted T cells, enabling them to proliferate and produce effector cytokines.

Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo.

Naturally occurring CD4+ regulatory T cells (T(R)) that express CD25 and the transcription factor FoxP3 play a key role in immune homeostasis, preventing immune pathological responses to self and foreign Ags. CTLA-4 is expressed by a high percentage of these cells, and is often considered as a marker for T(R) in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in T(R) function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits T(R) function in vivo via direct effects on CTLA-4-expressing T(R), and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits T(R) function, it does not reduce T(R) numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for T(R) activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional T(R), suggesting that under some circumstances other immune regulatory mechanisms, including the production of IL-10, are able to compensate for the loss of the CTLA-4-mediated pathway. This study provides direct evidence that CTLA-4 has a specific, nonredundant role in the function of normal T(R). This role has to be taken into account when targeting CTLA-4 for therapeutic purposes, as such a strategy will not only boost effector T cell responses, but might also break T(R)-mediated self-tolerance.

A set of genes selectively expressed in murine dendritic cells: utility of related cis-acting sequences for lentiviral gene transfer.

Professional antigen presenting cells such as dendritic cells (DC) and macrophages (Mphi) share similar characteristics; however, they differ in their ability to initiate an immune response. DCs are much more potent in priming and stimulating nai;ve T-cells. Thus, DCs are good targets for the expression of foreign genes to elicit and specifically modify immune responses. To identify DC markers cDNA subtraction was performed using murine MHC class II(high), B7(high) bone marrow derived DCs as tester and interferon-gamma/E. coli lipopolysaccaride (LPS) treated bone marrow derived macrophages as driver. Analysis of 114 resulting clones revealed a diverse pattern of DC selective (DC(DeltaMphi)) gene expression including known genes whose expression in DCs had not been previously demonstrated as well as multiple novel genes. For several identified DC(DeltaMphi) genes, proximal promoter elements were isolated and incorporated into self-inactivating lentiviral GFP reporter vectors. Promoter activity was measured in bone marrow derived macrophages or dendritic cells. Of the promoters analyzed those for B7-DC and CCL17 drove strong GFP expression in DCs but not in resting or activated macrophages. The CCL17 promoter offered the highest level of expression in DCs and was further activated by culture with LPS or interleukin-4 (IL-4). In contrast, the B7-DC promoter was induced by IL-4 but not by LPS. Endogenous CCL17 and B7-DC mRNAs were increased similarly in IL-4 cultured DCs but only CCL17 was induced by LPS. Additionally, IL-4 increased cell surface expression of B7-DC in both immature and mature DCs.