Sequential targeting of PARP with carboplatin inhibits primary tumour growth and distant metastasis in triple-negative breast cancer.

BACKGROUND: Patients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity. METHODS: We evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity. RESULTS: Concurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration (70.4%, P < 0.0001), invasion (56.9%, P < 0.0001), lung micrometastasis (56.4%, P < 0.0001), and less toxicity in a BRCAWT model. CONCLUSION: The sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients.

Regulatory T cell/Th17 balance in the pathogenesis of paediatric Behçet disease.

OBJECTIVES: Behçet disease (BD) is a chronic systemic inflammatory disorder of unknown aetiology. The aim of this study was to determine the orientation of T cell subpopulations in paediatric BD and more precisely to look for a regulatory T lymphocyte (Treg)/Th17 imbalance. METHODS: T cell subpopulations were analysed by flow cytometry in the peripheral blood of paediatric patients with acute BD (aBD; n = 24), remitting BD (rBD; n = 12) and in healthy controls (HCs; n = 24). Tregs (CD4+CD25hiCD127-/loFoxp3+), activated Tregs (GITR, LAP, CTLA-4 and HLA-DR expression), CD4+ and CD8+ T cells producing IFN-γ (Th1 and Tc1) or IL-17 (Th17 and Tc17) under polyclonal (OKT3/IL-2) or antigenic (Streptococcus sanguis KTH-1 peptides and heat shock protein 60) stimulation were enumerated. RESULTS: Th17 (1.9- and 5.1-fold) and Tc17 (4.0- and 2.0-fold) frequency under mitogenic stimulation was significantly increased in aBD and rBD patients as compared with HCs. Th17 frequency under antigenic stimulation was also higher in patients than in HCs. The percentage and number of Tregs and activated Tregs in patients and in HCs were similar. However, when Tregs were removed, antigen-driven differentiation into Th1 and Th17 was significantly boosted in BD but not in HC CD4+ T cells. CONCLUSION: There is a bias towards Th17 polarization in aBD and rBD in children. Although we did not observe an increase in the number of Tregs in these patients, their Tregs limit CD4+ T cell differentiation into Th1 and Th17 cells. Thus, in paediatric BD, Tregs seem to incompletely counterbalance a Th17 orientation of the Th cell response.

Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic vaccine.

The potential benefit in using IL-2 in immunotherapy for cancer and autoimmunity has been linked to the modulation of immune responses, which partly relies on a direct effect on Tregs populations. Here, we revisited the role of IL-2 in HIV infection and investigated whether its use as an adjuvant with therapeutic vaccination, impacts on HIV-specific responses. Antiretroviral therapy treated-patients were randomized to receive 4 boosts of vaccination (ALVACHIV/Lipo-6T, weeks 0/4/8/12) followed by 3 cycles of IL-2 (weeks 16/24/32) before treatment interruption (TI) at week40. IL-2 administration increased significantly HIV-specific CD4+CD25+CD134+ T-cell responses, which inversely correlated with viral load after TI (r = -0.7, p <0.007) in the vaccine/IL-2 group. IL-2 increased global CD25+CD127lowFoxP3+Tregs (p <0.05) while it decreased HIV- but not CMV- specific CD39+FoxP3+CD25+CD134+Tregs (p <0.05). HIV-specific Tregs were inversely correlated with IFN-γ producing specific-effectors (p = 0.03) and positively correlated with viral load (r = 0.7, p = 0.01), revealing their undesired presence during chronic infection. Global Tregs, but not HIV-specific Tregs, inversely correlated with a decrease in exhausted PD1+CD95+ T-cells (p = 0.001). Altogether, our results underline the negative impact of HIV-specific Tregs on HIV-specific effectors and reveal the beneficial use of IL-2 as an adjuvant as its administration increases global Tregs that impact on T-cell exhaustion and decreases HIV-specific CD39+Tregs by shifting the balance towards effectors.

Immune Cells Release MicroRNA-155 Enriched Extracellular Vesicles That Promote HIV-1 Infection.

The hallmark of HIV-1 infection is the rapid dysregulation of immune functions. Recent investigations for biomarkers of such dysregulation in people living with HIV (PLWH) reveal a strong correlation between viral rebound and immune activation with an increased abundance of extracellular vesicles (EVs) enriched with microRNA-155. We propose that the activation of peripheral blood mononuclear cells (PBMCs) leads to an increased miR-155 expression and production of miR-155-rich extracellular vesicles (miR-155-rich EVs), which can exacerbate HIV-1 infection by promoting viral replication. PBMCs were incubated with either HIV-1 (NL4.3Balenv), a TLR-7/8 agonist, or TNF. EVs were harvested from the cell culture supernatant by differential centrifugation, and RT-qPCR quantified miR-155 in cells and derived EVs. The effect of miR-155-rich EVs on replication of HIV-1 in incubated PBMCs was then measured by viral RNA and DNA quantification. HIV-1, TLR7/8 agonist, and TNF each induced the release of miR-155-rich EVs by PBMCs. These miR-155-rich EVs increased viral replication in PBMCs infected in vitro. Infection with HIV-1 and inflammation promote the production of miR-155-rich EVs, enhancing viral replication. Such autocrine loops, therefore, could influence the course of HIV-1 infection by promoting viral replication.

The relationship between residential exposure to atmospheric pollution and circulating miRNA in adults living in an urban area in northern France.

INTRODUCTION: MicroRNAs are epigenetic regulatory factors capable of silencing the expression of target genes and might mediate the effects of air pollution on health. The objective of the present population-based study was to investigate the association between microRNA expression and long-term, residential exposure to atmospheric PM10 and NO2. METHOD: We included 998 non-smoking adult participants from the cross-sectional ELISABET survey (2010-2014) in the Lille urban area of France. The mean residential annual pollution levels were estimated with an atmospheric dispersion modelling system. Ten microRNAs were selected on the basis of the literature data, together with two housekeeping microRNAs (miR-93-5p and miR-191-5p) and were quantified with RT-qPCRs. Multivariate linear regression models were used to study the association between microRNAs and air pollution. The threshold for statistical significance (after correction for the FDR) was set to p < 0.1. RESULTS: The mean annual exposure between 2011 and the year of inclusion was 26.4 ± 2.0 µg/m3 for PM10 and 24.7 ± 5.1 µg/m3 for NO2. Each 2 µg/m3 increment in PM10 exposure was associated with an 8.6% increment (95%CI [3.1; 14.3]; pFDR = 0.019) in miR-451a expression. A 5 µg/m3 increment in NO2 exposure was associated with a 5.3% increment ([0.7; 10]; pFDR = 0.056) in miR451a expression, a 3.6% decrement (95%CI [-6.1; -1.1]; pFDR = 0.052) in miR-223-3p expression, a 3.8% decrement (95%CI[-6.8; -0.7]; pFDR = 0.079) in miR-28-3p expression, a 4.3% decrement (95%CI [-7.7; -0.8]; pFDR = 0.055) in miR-146a-5p expression, and a 4.0% decrement (95% CI[-7.4; -0.4]; pFDR = 0.059) in miR-23a-5p expression. The difference between the two housekeeping microRNAs miR-93-5p and miR-191-5p was also associated with PM10 and NO2 exposure. CONCLUSION: Our results suggest that circulating miRNAs are potentially valuable biomarkers of the effects of air pollution.

EAAT expression by macrophages and microglia: still more questions than answers.

Glutamate is the main excitatory amino acid, but its presence in the extracellular milieu has deleterious consequences. It may induce excitotoxicity and also compete with cystine for the use of the cystine-glutamate exchanger, blocking glutathione neosynthesis and inducing an oxidative stress-induced cell death. Both mechanisms are critical in the brain where up to 20% of total body oxygen consumption occurs. In normal conditions, the astrocytes ensure that extracellular concentration of glutamate is kept in the micromolar range, thanks to their coexpression of high-affinity glutamate transporters (EAATs) and glutamine synthetase (GS). Their protective function is nevertheless sensitive to situations such as oxidative stress or inflammatory processes. On the other hand, macrophages and microglia do not express EAATs and GS in physiological conditions and are the principal effector cells of brain inflammation. Since the late 1990s, a number of studies have now shown that both microglia and macrophages display inducible EAAT and GS expression, but the precise significance of this still remains poorly understood. Brain macrophages and microglia are sister cells but yet display differences. Both are highly sensitive to their microenvironment and can perform a variety of functions that may oppose each other. However, in the very particular environment of the healthy brain, they are maintained in a repressed state. The aim of this review is to present the current state of knowledge on brain macrophages and microglial cells activation, in order to help clarify their role in the regulation of glutamate under pathological conditions as well as its outcome.

Expanding the Use of PARP Inhibitors as Monotherapy and in Combination in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and is known to be associated with a poor prognosis and limited therapeutic options. Poly (ADP-ribose) polymerase inhibitors (PARPi) are targeted therapeutics that have demonstrated efficacy as monotherapy in metastatic BRCA-mutant (BRCAMUT) TNBC patients. Improved efficacy of PARPi has been demonstrated in BRCAMUT breast cancer patients who have either received fewer lines of chemotherapy or in chemotherapy-naïve patients in the metastatic, adjuvant, and neoadjuvant settings. Moreover, recent trials in smaller cohorts have identified anti-tumor activity of PARPi in TNBC patients, regardless of BRCA-mutation status. While there have been concerns regarding the efficacy and toxicity of the use of PARPi in combination with chemotherapy, these challenges can be mitigated with careful attention to PARPi dosing strategies. To better identify a patient subpopulation that will best respond to PARPi, several genomic biomarkers of homologous recombination deficiency have been tested. However, gene expression signatures associated with PARPi response can integrate different pathways in addition to homologous recombination deficiency and can be implemented in the clinic more readily. Taken together, PARPi have great potential for use in TNBC patients beyond BRCAMUT status, both as a single-agent and in combination.

Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA.

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

Intradermal vaccination prevents anti-MOG autoimmune encephalomyelitis in macaques.

BACKGROUND: Autoimmune demyelinating diseases (ADD) are a major cause of neurological disability due to autoreactive cellular and humoral immune responses against brain antigens. A cure for chronic ADD could be obtained by appropriate immunomodulation. METHODS: We implemented a preclinical scheme to foster immune tolerance to myelin oligodendrocyte glycoprotein (MOG), in a cynomolgus-macaque model of experimental autoimmune encephalomyelitis (EAE), in which administration of recombinant human MOG (rhMOG) elicits brain inflammation mediated by MOG-autoreactive CD4+ lymphocytes and anti-MOG IgG. For immunotherapy, we used a recombinant antibody (Ab) directed against the dendritic cell-asialoglycoprotein receptor (DC-ASGPR) fused either to MOG or a control antigen PSA (prostate-specific antigen). FINDINGS: rhMOG and the anti-DC-ASGPR-MOG were respectively detected in CD1a+ DCs or CD163+ cells in the skin of macaques. Intradermal administration of anti-DC-ASGPR-MOG, but not control anti-DC-ASGPR-PSA, was protective against EAE. The treatment prevented the CD4+ T cell activation and proinflammatory cytokine production observed in controls. Moreover, the administration of anti-DC-ASGPR-MOG induced MOG-specific CD4+CD25+FOXP3+CD39+ regulatory lymphocytes and favoured an upsurge in systemic TGFß and IL-8 upon rhMOG re-administration in vivo. INTERPRETATION: We show that the delivery of an anti-DC-ASGPR-MOG allows antigen-specific adaptive immune modulation to prevent the breach of immune tolerance to MOG. Our findings pave the way for therapeutic vaccines for long-lasting remission to grave encephalomyelitis with identified autoantigens, such as ADD associated with anti-MOG autoantibodies. FUND: Work supported by the French ANR (ANR-11-INBS-0008 and ANR-10-EQPX-02-01), NIH (NIH 1 R01 AI 105066), the Baylor Scott and White Healthcare System funding and Roche Research Collaborative grants.

Regulatory T cells (Tregs): A major immune checkpoint to consider in combinatorial therapeutic HIV-1 vaccines.

The field of immunotherapeutics is living an exceptional time as new antibodies that take brakes off T-cells and unleash them on tumours are being approved by the US-Food and Drug Administration (FDA). For the design and development of an HIV-1 therapeutic-vaccine, one would need preferably to shift the balance T-effectors/T-regulatory cells (Teff/Tregs) towards effectors to improve vaccine-specific immune-responses. Given the success with the new immune-checkpoint-blockers (ICB), it is an appropriate time for HIV-1 field to seize this opportunity and develop new therapeutic vaccine-strategies that take into consideration ICB and other immunomodulators such as cytokines. While the vaccine is important to stimulate HIV-1-specific T-cell responses, cytokines will support the expansion of the stimulated virus-specific T-cells and ICB will reverse exhaustion and unchain cytotoxic T-cells. In this commentary, we will spotlight Tregs as another major brake for T-cell immunity and address the main stumbling-blocks that often blurs HIV-1-specific-Tregs status with regards to their role (beneficial or detrimental) and we will recall some proof-of-concept studies where therapeutic immunization skewed the HIV-1-specific response from Tregs to Teffs which impacts on the magnitude of viral replication. We will also suggest some strategies to shift the balance towards Teffs and potentiate HIV-1-specific immune-responses.

Elevated Abundance, Size, and MicroRNA Content of Plasma Extracellular Vesicles in Viremic HIV-1+ Patients: Correlations With Known Markers of Disease Progression.

BACKGROUND: Because of factors only partly understood, the generalized elevated immune activation and inflammation characterizing HIV-1-infected patients are corrected incompletely with antiretroviral therapy (ART). Extracellular vesicles (EVs) including exosomes and microvesicles released by several cell types may contribute to immune activation and dysfunction. EV size, abundance, and content appear to differ according to infection phase, disease progression, and ART. METHODS: We examined whether the size of EVs and the abundance of exosomes in plasma are associated with cell and tissue activation as well as with viral production. Acetylcholinesterase-bearing (AChE+) exosomes in plasma were quantified using an AChE assay. EV size was analyzed using dynamic light scattering. Proteins and microRNAs present in EVs were detected by Western blot and real-time polymerase chain reaction, respectively. RESULTS: Exosomes were found more abundant in the plasma of ART-naive patients. EV size was larger in ART-naive than in ART-suppressed patients, elite controllers, or healthy control subjects. Both exosome abundance and EV sizes were inversely correlated with CD4/CD8 T-cell ratio and neutrophil, platelet, and CD4 T-cell counts and positively correlated with CD8 T-cell counts. A negative correlation was found between CD4 T-cell nadir and exosome abundance, but not EV size. Levels of miR-155 and miR-223 but not miR-92 were strongly correlated negatively with EV abundance and size in ART-naive patients. CONCLUSIONS: Monitoring of circulating EVs and EV-borne microRNA is possible and may provide new insight into HIV-1 pathogenesis, disease progression, and the associated inflammatory state, as well as the efficacy of ART and the treatments intended to reduce immune activation.