Loise M. Francisco

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.

The PD‐1 pathway in tolerance and autoimmunity

Summary:  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‐β 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.

Tumor-specific killer cells in paraneoplastic cerebellar degeneration.

Models for immune-mediated tumor regression in mice have defined an essential role for cytotoxic T lymphocytes (CTLs); however, naturally occurring tumor immunity in humans is poorly understood. Patients with paraneoplastic cerebellar degeneration (PCD) provide an opportunity to explore the mechanisms underlying tumor immunity to breast and ovarian cancer. Although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Here we present evidence for a specific cellular immune response in PCD patients. We have detected expanded populations of MHC class I-restricted cdr2-specific CTLs in the blood of 3/3 HLA-A2.1+ PCD patients, providing the first description, to our knowledge, of tumor-specific CTLs using primary human cells in a simple recall assay. Cross-presentation of apoptotic cells by dendritic cells also led to a potent CTL response. These results indicate a model whereby immature dendritic cells that engulf apoptotic tumor cells can mature and migrate to draining lymph organs where they could induce a CTL response to tissue-restricted antigens. In PCD, peripheral activation of cdr2-specific CTLs is likely to contribute to the subsequent development of the autoimmune neuronal degeneration.

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

Naturally occurring CD4+ regulatory T cells (TR) 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 TR in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in TR function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits TR function in vivo via direct effects on CTLA-4-expressing TR, and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits TR function, it does not reduce TR numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for TR activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional TR, 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 TR. 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 TR-mediated self-tolerance.

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

CD4+CXCR5+Foxp3+ follicular regulatory T cells (TFR cells) inhibit humoral immunity mediated by CD4+CXCR5+Foxp3− follicular helper T cells (TFH cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of TFR cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of TFR cells in the lymph nodes and that those TFR cells had enhanced suppressive ability. We also found substantial populations of TFR cells in mouse blood and demonstrated that TFR cells in the blood homed to lymph nodes and potently inhibited TFH cells in vivo. TFR 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.

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

Interaction between the inhibitory molecule PD-L2 on dendritic cells and repulsive guidance molecule b (RGMb) on lung macrophages is required to establish respiratory tolerance.

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.

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.

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

Tumors depend on a specialized pathway of regulatory T cell activation to create their immunosuppressive microenvironment, which can be blocked by inhibiting PTEN phosphatase. 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.

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.