Roles, function and relevance of LAG3 in HIV infection.

HIV causes several forms of immune dysfunction that need to be addressed in a functional cure for HIV. Immune exhaustion describes a dysfunctional phenotype caused by chronic cellular activation. Lymphocyte activation gene-3 (LAG3) is one of several negative coreceptors known as immune checkpoints that contribute to this exhaustion phenotype. Antibodies targeting immune checkpoints are now used clinically to restore immunity against cancer and hold promise in restoring immunity during HIV infection. Here, we summarize current knowledge surrounding LAG3 and discuss its relevance during HIV infection and the potential for LAG3-targeting antibodies in a functional HIV cure.

Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension.

BACKGROUND: Right ventricular (RV) failure is the most important factor of both morbidity and mortality in pulmonary arterial hypertension (PAH). However, the underlying mechanisms resulting in the failed RV in PAH remain unknown. There is growing evidence that angiogenesis and microRNAs are involved in PAH-associated RV failure. We hypothesized that microRNA-126 (miR-126) downregulation decreases microvessel density and promotes the transition from a compensated to a decompensated RV in PAH. METHODS AND RESULTS: We studied RV free wall tissues from humans with normal RV (n=17), those with compensated RV hypertrophy (n=8), and patients with PAH with decompensated RV failure (n=14). Compared with RV tissues from patients with compensated RV hypertrophy, patients with decompensated RV failure had decreased miR-126 expression (quantitative reverse transcription-polymerase chain reaction; P<0.01) and capillary density (CD31(+) immunofluorescence; P<0.001), whereas left ventricular tissues were not affected. miR-126 downregulation was associated with increased Sprouty-related EVH1 domain-containing protein 1 (SPRED-1), leading to decreased activation of RAF (phosphorylated RAF/RAF) and mitogen-activated protein kinase (MAPK); (phosphorylated MAPK/MAPK), thus inhibiting the vascular endothelial growth factor pathway. In vitro, Matrigel assay showed that miR-126 upregulation increased angiogenesis of primary cultured endothelial cells from patients with decompensated RV failure. Furthermore, in vivo miR-126 upregulation (mimic intravenous injection) improved cardiac vascular density and function of monocrotaline-induced PAH animals. CONCLUSIONS: RV failure in PAH is associated with a specific molecular signature within the RV, contributing to a decrease in RV vascular density and promoting the progression to RV failure. More importantly, miR-126 upregulation in the RV improves microvessel density and RV function in experimental PAH.

Role for DNA damage signaling in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with sustained inflammation known to promote DNA damage. Despite these unfavorable environmental conditions, PAH pulmonary arterial smooth muscle cells (PASMCs) exhibit, in contrast to healthy PASMCs, a pro-proliferative and anti-apoptotic phenotype, sustained in time by the activation of miR-204, nuclear factor of activated T cells, and hypoxia-inducible factor 1-α. We hypothesized that PAH-PASMCs have increased the activation of poly(ADP-ribose) polymerase-1 (PARP-1), a critical enzyme implicated in DNA repair, allowing proliferation despite the presence of DNA-damaging insults, eventually leading to PAH. METHODS AND RESULTS: Human PAH distal pulmonary arteries and cultured PAH-PASMCs exhibit increased DNA damage markers (53BP1 and γ-H2AX) and an overexpression of PARP-1 (immunoblot and activity assay), in comparison with healthy tissues/cells. Healthy PASMCs treated with a clinically relevant dose of tumor necrosis factor-α harbored a similar phenotype, suggesting that inflammation induces DNA damage and PARP-1 activation in PAH. We also showed that PARP-1 activation accounts for miR-204 downregulation (quantitative reverse transcription polymerase chain reaction) and the subsequent activation of the transcription factors nuclear factor of activated T cells and hypoxia-inducible factor 1-α in PAH-PASMCs, previously shown to be critical for PAH in several models. These effects resulted in PASMC proliferation (Ki67, proliferating cell nuclear antigen, and WST1 assays) and resistance to apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling and Annexin V assays). In vivo, the clinically available PARP inhibitor ABT-888 reversed PAH in 2 experimental rat models (Sugen/hypoxia and monocrotaline). CONCLUSIONS: These results show for the first time that the DNA damage/PARP-1 signaling pathway is important for PAH development and provide a new therapeutic target for this deadly disease with high translational potential.

Impaired angiogenesis and peripheral muscle microcirculation loss contribute to exercise intolerance in pulmonary arterial hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by significant exercise intolerance, which is multifactorial and involves skeletal muscle alterations. There is growing evidence that microRNAs (miRs) are involved in PAH pathogenesis. OBJECTIVES: We hypothesized that miR-126, an endothelial-specific, proangiogenic miR, is down-regulated in the peripheral muscles of patients with PAH, which would account for skeletal muscle microcirculation loss and exercise intolerance. MEASUREMENTS AND MAIN RESULTS: Patients with PAH displayed decreases in exercise capacity ([Formula: see text]o2max) and microcirculation loss on quadriceps muscle biopsy (in CD31(+) immunofluorescence experiments) compared to control subjects. Exercise capacity correlated with muscle capillarity (r = 0.84, P < 0.01). At the cellular level, vascular endothelial growth factor (VEGF) and VEGF receptor 2 expression were similar in both groups. Conversely, PAH was associated with a 60% decrease in miR-126 expression in a quantitative reverse transcriptase polymerase chain reaction experiment (P < 0.01), resulting in up-regulation of its targeted protein, Sprouty-related, EVH1 domain-containing protein 1 (SPRED-1), and a marked decrease in the downstream effectors of the VEGF pathway, p-Raf/Raf and p-ERK/ERK, as determined by immunoblot analysis. Using freshly isolated CD31(+) cells from human quadriceps biopsies, we found that the down-regulation of miR-126 in PAH triggered the activation of SPRED-1, impairing the angiogenic response (Matrigel assay). These abnormalities were reversed by treating the PAH cells with miR-126 mimic, whereas inhibition of miR-126 (antagomir) in healthy CD31(+) cells fully mimicked the PAH phenotype. Finally, miR-126 down-regulation in skeletal muscle of healthy rats decreased muscle capillarity in immunofluorescence assays (P < 0.05) and exercise tolerance in treadmill tests (P < 0.05), whereas miR-126 up-regulation increased them in monocrotaline PAH rats. CONCLUSIONS: We demonstrate for the first time that exercise intolerance in PAH is associated with skeletal muscle microcirculation loss and impaired angiogenesis secondary to miR-126 down-regulation.

Comparative analysis of LAG3 antibodies shows differential binding patterns by flow cytometry.

BACKGROUND: LAG3 is an immune checkpoint molecule with emerging therapeutic use. Expression of LAG3 is well studied on T cells, but the proportion of LAG3-expressing cells varies greatly by study and its comparative expression between non-T cells is lacking. METHODS/OBJECTIVES: This study uses flow cytometry to compare surface LAG3 expression between T cells, NK cells, B cells, pDCs and monocytes of healthy donors. This study also compares three monoclonal LAG3 antibodies to a commonly used polyclonal LAG3 antibody on ex vivo and PHA-blasts from healthy donors and LAG3+ and LAG3- cell lines. RESULTS: LAG3 was most highly expressed on classical and intermediate monocytes (25 % and 32 %, respectively), while LAG3 expression on B cells, NK cells and iNKT cells was negligible. Notably, the polyclonal antibody stained a higher proportion of all cell types than the monoclonal antibodies, which had similar staining patterns to one another. Further study using LAG3+ and LAG3- cell lines showed greater specificity and similar sensitivity of the monoclonal antibody T47-530 than the polyclonal antibody. CONCLUSION: Monocytes may represent an unappreciated source of LAG3 and target of LAG3 checkpoint inhibitors. Furthermore, the discrepancies between monoclonal and polyclonal LAG3 antibodies warrants consideration when designing future studies and interpreting past studies, and may explain discrepancies in the literature.

Granzyme B deficiency exacerbates lung inflammation in mice after acute lung injury.

Granzyme B (GzmB) is a serine protease with intracellular and extracellular activities capable of regulating inflammation through cytokine processing and the apoptosis of effector cells. We tested the hypothesis that GzmB expression in T regulatory cells (Tregs) is required for the control of inflammatory responses and pathology during acute lung injury. To substantiate the clinical relevance of GzmB during lung injury, we performed GzmB immunohistochemistry on lung tissue from patients with acute respiratory distress syndrome (ARDS) and healthy control subjects. We also performed in vivo experiments with wild-type (WT) C57BL/6 and GzmB(-/-) mice exposed to a single intranasal instillation of bleomycin to model lung injury. Our results demonstrate that the expression of GzmB was elevated in ARDS lung sections, relative to healthy control samples. Bleomycin-exposed GzmB(-/-) mice exhibited greater morbidity and mortality, which was associated with increased numbers of lung lymphocytes. Bleomycin induced an equal increase in CD4(+)/CD25(+)/FoxP3(+) Treg populations in WT and GzmB(-/-) mice. GzmB expression was not significant in Tregs, with the majority of the expression localized to natural killer (NK)-1.1(+) cells. The expression of GzmB in NK cells of bleomycin-exposed WT mice was associated with greater lymphocyte apoptosis, reduced total lymphocyte numbers, and reduced pathology relative to GzmB(-/-) mice. Our data demonstrate that GzmB deficiency results in the exacerbation of lymphocytic inflammation during bleomycin-induced acute lung injury, which is associated with pathology, morbidity, and mortality.

The effect of blocking immune checkpoints LAG-3 and PD-1 on human invariant Natural Killer T cell function.

Invariant Natural Killer T (iNKT) cells undergo immune exhaustion during chronic activation caused by cancer and viral infections, such as HIV. Exhaustion is marked by cell dysfunction and increased expression of immune checkpoint proteins programmed cell-death-1 (PD-1) and lymphocyte-activation-gene-3 (LAG-3). We hypothesize that blockade of PD-1 and/or LAG-3 will enhance iNKT cell function. Utilizing peripheral blood mononuclear cells from healthy donors, LAG-3 and PD-1 expression on iNKT cells was assessed using flow cytometry following in vitro stimulation with iNKT-specific stimulant α-galactosylceramide (n = 4). Efficacy of anti-LAG-3 and/or anti-PD-1 antibody blockades in enhancing iNKT cell function was assessed by determining proliferative capacity and IFN-γ production (n = 9). LAG-3 and PD-1 expression on iNKT cells peaked at Day 4 (98.8%; p ≤ 0.0001 and 98.8%; p = 0.005, respectively), followed by steep decrease by Day 10, coinciding with peak iNKT cell proliferation. In a 10-day blocking assay, both the anti-PD-1 alone and dual anti-PD-1 and anti-LAG-3 significantly increased iNKT proliferation (6 and 6.29 log2 fold-change respectively) compared to the no blockade control (ANOVA-p = 0.0005) with the dual blockade system being more effective (t-test-p = 0.013). This provides proof-of-concept for LAG-3 and PD-1 as immunotherapeutic targets to enhance human iNKT cell function, with the long-term goal of addressing immune exhaustion.

Novel in vitro invariant natural killer T cell functional assays.

BACKGROUND: Invariant Natural Killer T (iNKT) cells are innate lymphocytes bridging the innate and adaptive immune systems and are critical first responders against cancer and infectious diseases. iNKT cell phenotype and functionality are studied using in vitro stimulation assays assessing cytokine response and proliferation capabilities. The most common stimulant is the glycolipid α-Galactosyl Ceramide (α-GalCer), which stimulates iNKT cells when presented by CD1d, an MHC class I-like molecule expressed by antigen-presenting cells (APC). Another stimulant used is α-GalCer-loaded DimerX, a CD1d-Ig fusion protein which stimulates iNKT cells in an APC-independent fashion. Here, we demonstrate use of the PBS-57-loaded CD1d-tetramer as an APC-independent stimulant, where PBS-57 is an α-GalCer analogue. METHODS: Using healthy fresh (n = 4) and frozen (n = 7) peripheral blood mononuclear cells (PBMCs), 10-h cytokine response (measuring IFN-γ production) and 10-day proliferation assays were performed assessing iNKT functionality using α-GalCer, CD1d-tetramer and DimerX stimulants. RESULTS: All stimulants effectively induced IFN-γ production in both fresh and frozen PBMC. After the 10-h activation, CD1d-tetramer was significantly more effective than α-GalCer (p = 0.032) in inducing IFN-γ production in fresh PBMC and significantly more effective than both α-GalCer (p = 0.004) and DimerX (p = 0.021) in frozen PBMC. Similarly, all stimulants induced strong proliferation responses in all samples, although this was only significant in the frozen PBMC. No significant differences in proliferation were observed between stimulants. SIGNIFICANCE: This study supports PBS-57-loaded CD1d-tetramer as an effective in vitro APC-independent iNKT cell stimulant, which is comparable to or even more effective than α-GalCer and DimerX. As CD1d is downregulated during infectious disease and cancer as evasion strategies, in vitro assays which are APC-independent can assist in providing objective insight to iNKT activation by not relying on CD1d expression by APCs. Overall, the novel CD1d-tetramer stimulation equips researchers with an expanded "toolkit" to successfully assess iNKT cell function.

LAG3's Enigmatic Mechanism of Action.

LAG3 is an important immune checkpoint with relevance in cancer, infectious disease and autoimmunity. However, despite LAG3's role in immune exhaustion and the great potential of LAG3 inhibition as treatment, much remains unknown about its biology, particularly its mechanism of action. This review describes the knowns, unknowns and controversies surrounding LAG3. This includes examination of how LAG3 is regulated transcriptionally and post-translationally by endocytosis and proteolytic cleavage. We also discuss the interactions of LAG3 with its ligands and the purpose thereof. Finally, we review LAG3's mechanism of action, including the roles of LAG3 intracellular motifs and the lack of a role for CD4 competition. Overall, understanding the biology of LAG3 can provide greater insight on LAG3 function, which may broaden the appreciation for LAG3's role in disease and potentially aid in the development of targeted therapies.

Vascular remodeling process in pulmonary arterial hypertension, with focus on miR-204 and miR-126 (2013 Grover Conference series).

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized primarily by increased proliferation and resistance to apoptosis in distal pulmonary arteries. Previous literature has demonstrated that the transcription factors NFAT (nuclear factor of activated T cells) and HIF-1α (hypoxia inducible factor 1α) are extensively involved in the pathogenesis of this disease and, more recently, has implicated STAT3 (signal transducer and activator of transcription 3) in their activation. Novel research shows that miR-204, a microRNA recently found to be notably downregulated through induction of PARP-1 (poly [ADP-ribose] polymerase 1) by excessive DNA damage in PAH, inhibits activation of STAT3. Contemporary research also indicates systemic impairment of skeletal muscle microcirculation in PAH and attributes this to a debilitated vascular endothelial growth factor pathway resulting from reduced miR-126 expression in endothelial cells. In this review, we focus on recent research implicating miR-204 and miR-126 in vascular remodeling processes, data that allow a better understanding of PAH molecular pathways and constitute a new hope for future therapy.

miRNAs in PAH: biomarker, therapeutic target or both?

Pulmonary arterial hypertension (PAH) is characterized by progressive increase in pulmonary vascular resistance leading to right ventricular hypertrophy and failure. There is a need to find new biomarkers to detect PAH at its early stages and also for new, more effective treatments for this disease. miRNAs have emerged as key players in cardiovascular diseases and cancer development and progression and, more recently, in PAH pathogenesis. In this review, we focus on the potential of miRNAs as biomarkers and new therapeutic targets for PAH.