Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis.

INTRODUCTION: A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients. METHODS: Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry. RESULTS: Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01). CONCLUSIONS: In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.

RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

A Science-Based Methodology Framework for the Assessment of Combination Safety Risks in Clinical Trials.

Multiple components factor into the assessment of combination safety risks when two or more novel individual products are used in combination in clinical trials. These include, but are not limited to, biology, biochemistry, pharmacology, class effects, and preclinical and clinical findings (such as adverse drug reactions, drug target and mechanism of action, target expression, signaling, and drug-drug interactions). This paper presents a science-based methodology framework for the assessment of combination safety risks when two or more investigational products are used in clinical trials. The aim of this methodology framework is to improve prediction of the risks, to enable the appropriate safety risk mitigation and management to be put in place for the combination, and the development of the project combination safety strategy.

Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children worldwide. The understanding of neonatal RSV pathogenesis depends on using an animal model that reproduces neonatal RSV disease. Previous studies from us and others demonstrated that the neonatal lamb model resembles human neonatal RSV infection. Here, we provide an extensive and detailed characterization of the histopathology, viral load, cellular infiltration, and cytokine production in lungs and tracheobronchial lymph nodes of lambs inoculated with human RSV strain A2 over the course of infection. In the lung, RSV titers were low at day 3 postinfection, increased significantly by day 6, and decreased to baseline levels at day 14. Infection in the lung was associated with an accumulation of macrophages, CD4(+) and CD8(+) T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response, characterized by increased IFNγ, IL-8, MCP-1, and PD-L1, and decreased IFNß, IL-10, and TGF-ß. Laser capture microdissection studies determined that lung macrophage-enriched populations were the source of MCP-1 but not IL-8. Immunoreactivity to caspase 3 occurred within bronchioles and alveoli of day 6-infected lambs. In lung-draining lymph nodes, RSV induced lymphoid hyperplasia, suggesting an ability of RSV to enhance lymphocytic proliferation and differentiation pathways. This study suggests that, in lambs with moderate clinical disease, RSV enhances the activation of caspase cell death and Th1-skewed inflammatory pathways, and complements previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.

Respiratory syncytial virus infection is associated with an altered innate immunity and a heightened pro-inflammatory response in the lungs of preterm lambs.

INTRODUCTION: Factors explaining the greater susceptibility of preterm infants to severe lower respiratory infections with respiratory syncytial virus (RSV) remain poorly understood. Fetal/newborn lambs are increasingly appreciated as a model to study key elements of RSV infection in newborn infants due to similarities in lung alveolar development, immune response, and susceptibility to RSV. Previously, our laboratory demonstrated that preterm lambs had elevated viral antigen and developed more severe lesions compared to full-term lambs at seven days post-infection. Here, we compared the pathogenesis and immunological response to RSV infection in lungs of preterm and full-term lambs. METHODS: Lambs were delivered preterm by Caesarian section or full-term by natural birth, then inoculated with bovine RSV (bRSV) via the intratracheal route. Seven days post-infection, lungs were collected for evaluation of cytokine production, histopathology and cellular infiltration. RESULTS: Compared to full-term lambs, lungs of preterm lambs had a heightened pro-inflammatory response after infection, with significantly increased MCP-1, MIP-1α, IFN-γ, TNF-α and PD-L1 mRNA. RSV infection in the preterm lung was characterized by increased epithelial thickening and periodic acid-Schiff staining, indicative of glycogen retention. Nitric oxide levels were decreased in lungs of infected preterm lambs compared to full-term lambs, indicating alternative macrophage activation. Although infection induced significant neutrophil recruitment into the lungs of preterm lambs, neutrophils produced less myeloperoxidase than those of full-term lambs, suggesting decreased functional activation. CONCLUSIONS: Taken together, our data suggest that increased RSV load and inadequate immune response may contribute to the enhanced disease severity observed in the lungs of preterm lambs.

PD-L1+ neutrophils contribute to injury-induced infection susceptibility.

The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

T cell deficiencies as a common risk factor for drug associated progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a disease of the central nervous system caused by neuropathogenic prototypes of ubiquitous community-acquired JC virus (JCV). The disease became of particular concern following its association with certain therapies that modulate immune system function without heavy immunosuppression. Due to lack of prophylactic/treatment options and poor outcomes, which often include severe disability or death, PML is a considerable concern for development of new drugs that interfere with immune system functions. In this review of clinical and research findings, we discuss the evidence that deficiencies in CD4+ T helper cells, cytotoxic CD8+ T cells, and interferon gamma are of crucial importance for development of PML under a variety of circumstances, including those associated with use of various drugs, regardless of differences in their mechanisms of action. These deficiencies apparently enable transformation of the harmless JCV archetype into neuropathogenic prototypes, but the site(s), and the mechanisms, of this transformation are yet to be elucidated. Here we discuss the evidence for brain as one of the sites of this transformation, and propose a model of PML pathogenesis that emphasizes the central role of T cell deficiencies in the two life cycles of the JCV, one non-pathogenic and one neuropathogenic. Finally, we conclude that the development of clinical grade T cell functional tests and more consistent use of already available laboratory tests for T cell subset analysis would greatly aid the effort to more accurately predict and assess the magnitude of PML risk for concerned therapeutic interventions.

Frontline Science: Defects in immune function in patients with sepsis are associated with PD-1 or PD-L1 expression and can be restored by antibodies targeting PD-1 or PD-L1.

Sepsis is a heterogeneous syndrome comprising a highly diverse and dynamic mixture of hyperinflammatory and compensatory anti-inflammatory immune responses. This immune phenotypic diversity highlights the importance of proper patient selection for treatment with the immunomodulatory drugs that are entering clinical trials. To better understand the serial changes in immunity of critically ill patients and to evaluate the potential efficacy of blocking key inhibitory pathways in sepsis, we undertook a broad phenotypic and functional analysis of innate and acquired immunity in the same aliquot of blood from septic, critically ill nonseptic, and healthy donors. We also tested the ability of blocking the checkpoint inhibitors programmed death receptor-1 (PD-1) and its ligand (PD-L1) to restore the function of innate and acquired immune cells. Neutrophil and monocyte function (phagocytosis, CD163, cytokine expression) were progressively diminished as sepsis persisted. An increasing frequency in PD-L1+-suppressor phenotype neutrophils [low-density neutrophils (LDNs)] was also noted. PD-L1+ LDNs and defective neutrophil function correlated with disease severity, consistent with the potential importance of suppressive neutrophil populations in sepsis. Reduced neutrophil and monocyte function correlated both with their own PD-L1 expression and with PD-1 expression on CD8+ T cells and NK cells. Conversely, reduced CD8+ T cell and NK cell functions (IFN-γ production, granzyme B, and CD107a expression) correlated with elevated PD-L1+ LDNs. Importantly, addition of antibodies against PD-1 or PD-L1 restored function in neutrophil, monocyte, T cells, and NK cells, underlining the impact of the PD-1:PD-L1 axis in sepsis-immune suppression and the ability to treat multiple deficits with a single immunomodulatory agent.

Progressive multifocal leukoencephalopathy: current treatment options and future perspectives.

Progressive multifocal leukoencephalopathy (PML) is a rare but debilitating and frequently fatal viral disease of the central nervous system, primarily affecting individuals with chronically and severely suppressed immune systems. The disease was relatively obscure until the outbreak of HIV/AIDS, when it presented as one of the more frequent opportunistic infections in this immune deficiency syndrome. It attracted additional attention from the medical and scientific community following the discovery of significant PML risk associated with natalizumab, a monoclonal antibody used for treatment of relapsing-remitting multiple sclerosis. This was followed by association of PML with other immunosuppressive or immunomodulating drugs. PML is currently untreatable disease with poor outcomes, so it is a significant concern when developing new immunotherapies. Current prophylaxis and treatment of PML are focused on immune reconstitution, restoration of immune responses to JC virus infection, and eventual suppression of immune reconstitution inflammatory syndrome. This approach was successful in reducing the incidence of PML and improved survival of PML patients with HIV infection. However, the outcome for the majority of PML patients, regardless of their medical history, is still relatively poor. There is a high unmet need for both prophylaxis and treatment of PML. The aim of this review is to discuss potential drug candidates for prophylaxis and treatment of PML with a critical review of previously conducted and completed PML treatment studies as well as to provide perspectives for future therapies.

Murine CXCR1 is a functional receptor for GCP-2/CXCL6 and interleukin-8/CXCL8.

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

Interleukin 17 modulates the immune response to vaccinia virus infection.

Interleukin 17 (IL-17) is a newly identified cytokine that has a homolog in herpesvirus saimiri. We inserted murine IL-17 into vaccinia virus to study the role of IL-17 in viral infection. Vaccinia virus expressing IL-17 (vv-IL17) and its parental control virus (vv-pRB) grew to similar titers in vitro; however, vv-IL17 was more virulent in mice with a threefold lower LD(50) than for vv-pRB, and mean time to death of 2.8 days versus 4.5 days. Mice infected with vv-IL17 had higher titers of virus in the ovaries (P < 0.02) and showed a decrease in NK cell cytotoxicity (P < 0.02) on day 3 after infection. No significant difference was found in CTL activity. In addition, a significant decrease in IgG2a (P < 0.01) and increases in IgG1, IgG3, and IgA (P < 0.03) vaccinia virus-specific antibody titers were observed in mice infected with vv-IL17 versus vv-pRB, suggesting a Th-2-like response to infection. These data indicate that IL-17 modulates the immune response during virus infection.

Effect of granulocyte colony-stimulating factor combination therapy on efficacy of posaconazole (SCH56592) in an inhalation model of murine pulmonary aspergillosis.

Using an inhalation model of pulmonary aspergillosis, we observed modest differences in the survival rates of mice treated with granulocyte colony-stimulating factor (G-CSF) and posaconazole (POS) and those treated with POS alone. This finding is in contrast to a previous report that suggested that G-CSF had a significant antagonistic effect on the antifungal activity of POS.

Chronic active Epstein-Barr virus infection associated with mutations in perforin that impair its maturation.

Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease in which previously healthy persons develop severe, life-threatening illness. Mutations in the perforin gene have been found in familial hemophagocytic lymphohistiocytosis, which shares some features with CAEBV. We studied a patient who died at age 18, 10 years after the onset of CAEBV. The patient had high titers of antibodies to EBV, EBV RNA in lymph nodes, T-cell lymphoproliferative disease, and hemophagocytic lymphohistiocytosis. DNA sequencing showed novel mutations in both alleles of the perforin gene that resulted in amino acid changes in the protein. The quantity of the native form of perforin from the patient's stimulated peripheral blood mononuclear cells (PBMCs) was extremely low and immunoblotting showed accumulation of an uncleaved precursor form of perforin. Stimulated PBMCs from the patient were defective for Fas-independent cytotoxicity. These data imply that mutations in this patient resulted in reduced perforin-mediated cytotoxicity by his lymphocytes. This is the first case in which perforin mutations have been shown to result in accumulation of the uncleaved, immature form of perforin. Mutations in the perforin gene are associated with some cases of CAEBV with hemophagocytic lymphohistiocytosis.