Systemic inflammation, innate immunity and pathogenesis after Zika virus infection in cynomolgus macaques are modulated by strain-specificity within the Asian lineage.

Zika virus (ZIKV) is an emerging arbovirus with recent global expansion. Historically, ZIKV infections with Asian lineages have been associated with mild disease such as rash and fever. However, recent Asian sub-lineages have caused outbreaks in the South Pacific and Latin America with increased prevalence of neurological disorders in infants and adults. Asian sub-lineage differences may partially explain the range of disease severity observed. However, the effect of Asian sub-lineage differences on pathogenesis remains poorly characterized. Current study conducts a head-to-head comparison of three Asian sub-lineages that are representative of the circulating ancestral mild Asian strain (ZIKV-SG), the 2007 epidemic French Polynesian strain (ZIKV-FP), and the 2013 epidemic Brazil strain (ZIKV-Brazil) in adult Cynomolgus macaques. Animals infected intervenously or subcutaneously with either of the three clinical isolates showed sub-lineage-specific differences in viral pathogenesis, early innate immune responses and systemic inflammation. Despite the lack of neurological symptoms in infected animals, the epidemiologically neurotropic ZIKV sub-lineages (ZIKV-Brazil and/or ZIKV-FP) were associated with more sustained viral replication, higher systemic inflammation (i.e. higher levels of TNFα, MCP-1, IL15 and G-CSF) and greater percentage of CD14+ monocytes and dendritic cells in blood. Multidimensional analysis showed clustering of ZIKV-SG away from ZIKV-Brazil and ZIKV-FP, further confirming sub-lineage differences in the measured parameters. These findings highlight greater systemic inflammation and monocyte recruitment as possible risk factors of adult ZIKV disease observed during the 2007 FP and 2013 Brazil epidemics. Future studies should explore the use of anti-inflammatory therapeutics as early treatment to prevent ZIKV-associated disease in adults.

A decrease in PPD specific CD4 T cell CD38 and HLA-DR expression in pulmonary tuberculosis patients after 8 weeks of therapy correlates with successful anti-tuberculosis treatment.

Tuberculosis (TB) is a major health problem in Indonesia with a million new cases each year. The CD4 T cell adaptive immune response against Mycobacterium tuberculosis (MTB) is central to the control of this disease. We investigated whether standard therapy of TB causes changes to these cells in the early stages of treatment. To do this we took blood samples from 2 groups of TB patients in Banda Aceh, Indonesia; one from a group of patients before treatment, and the other from a group who become smear negative after 8 weeks treatment. MTB specific CD4 T cells were identified by ex vivo stimulation with PPD and flow cytometric measurement of intracellular cytokines and surface markers. We found no difference in total PPD specific CD4 T cells between the groups, but that the proportion of these cells CD38 + HLA-DR+ was significantly lower in the treatment group. This decrease was not specific to Interferon gamma (IFNg), Interleukin-2 (IL-2) or Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) producing cells. Our findings show that anti-MTB treatment affects the adaptive immune response, and that measuring the decrease of the PPD specific CD4 T cell CD38+HLA-DR+ phenotype could be a useful parameter for determination of treatment success.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Full Spectrum Flow Cytometry as a Powerful Technology for Cancer Immunotherapy Research.

The Nobel Prize-deserving concept of blocking inhibitory pathways in T cells, to unleash their anti-tumoral capacity, became one of the pillars of cancer treatment in the last decade and has resulted in durable clinical responses for multiple cancer types. Currently, two of the most important goals in cancer immunotherapy are to understand the mechanisms resulting in failure to checkpoint blockade and to identify predictive immunological biomarkers that correlate to treatment response, disease progression or adverse effects. The identification and validation of biomarkers for routine clinical use is not only critical to monitor disease or treatment progression, but also to personalize and develop new therapies. To achieve these goals, powerful research tools are needed. Flow cytometry stands as one of the most successful single-cell analytical tools used to characterize immune cell phenotypes to monitor solid tumors, hematological malignancies, minimal residual disease or metastatic progression. This technology has been fundamental in diagnosis, treatment and translational research in cancer clinical trials. Most recently, the need to evaluate simultaneously more features in each cell has pushed the field to implement more powerful adaptations beyond conventional flow cytometry, including Full Spectrum Flow Cytometry (FSFC). FSFC captures the full emission spectrum of fluorescent molecules using arrays of highly sensitive light detectors, and to date has enabled characterization of 40 parameters in a single sample. We will summarize the contributions of this technology to the advancement of research in immunotherapy studies and discuss best practices to obtain reliable, robust and reproducible FSFC results.

Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis.

Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones. Angiogenesis is dysregulated in various pathologies, including age-related macular degeneration, arthritis, and cancer. Inhibiting pathological angiogenesis therefore represents a promising therapeutic strategy for treating these disorders, highlighting the need to study angiogenesis in more detail. To this end, identifying the genes essential for blood vessel formation and elucidating their function are crucial for a complete understanding of angiogenesis. Here, focusing on potential candidate genes for angiogenesis, we performed a morpholino-based genetic screen in zebrafish and identified Cavin-2, a membrane-bound phosphatidylserine-binding protein and critical organizer of caveolae (small microdomains in the plasma membrane), as a regulator of angiogenesis. Using endothelial cells, we show that Cavin-2 is required for in vitro angiogenesis and also for endothelial cell proliferation, migration, and invasion. We noted a high level of Cavin-2 expression in the neovascular tufts in the mouse model of oxygen-induced retinopathy, suggesting a role for Cavin-2 in pathogenic angiogenesis. Interestingly, we also found that Cavin-2 regulates the production of nitric oxide (NO) in endothelial cells by controlling the stability and activity of the endothelial nitric-oxide synthase (eNOS) and that Cavin-2 knockdown cells produce much less NO than WT cells. Also, mass spectrometry, flow cytometry, and electron microscopy analyses indicated that Cavin-2 is secreted in endothelial microparticles (EMPs) and is required for EMP biogenesis. Taken together, our results indicate that in addition to its function in caveolae biogenesis, Cavin-2 plays a critical role in endothelial cell maintenance and function by regulating eNOS activity.

Human regulatory B cells combine phenotypic and genetic hallmarks with a distinct differentiation fate.

Regulatory B cells (B-reg) produce IL-10 and suppress inflammation in both mice and humans, but limited data on the phenotype and function of these cells have precluded detailed assessment of their contribution to host immunity. In this article, we report that human B-reg cannot be defined based on a phenotype composed of conventional B cell markers, and that IL-10 production can be elicited in both the CD27(+) memory population and naive B cell subset after only a brief stimulation in vitro. We therefore sought to obtain a better definition of IL-10-producing human B-regs using a multiparameter analysis of B cell phenotype, function, and gene expression profile. Exposure to CpG and anti-Ig are the most potent stimuli for IL-10 secretion in human B cells, but microarray analysis revealed that human B cells cotreated with these reagents resulted in only ∼0.7% of genes being differentially expressed between IL-10(+) and IL-10(-) cells. Instead, connectivity map analysis revealed that IL-10-secreting B cells are those undergoing specific differentiation toward a germinal center fate, and we identified a CD11c(+) B cell subset that was not capable of producing IL-10 even under optimal conditions. Our findings will assist in the identification of a broader range of human pro-B-reg populations that may represent novel targets for immunotherapy.