Antonio Cosma

Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection

Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4+ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4+ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4+ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4+ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.

Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five‐fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry‐based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms.

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4+ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4+ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.

Plasmacytoid dendritic cell dynamics tune interferon-alfa production in SIV-infected cynomolgus macaques.

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα+ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα+ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα− production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67+-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67+-pDC precursors, none of these being IFNα+ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors.

CD34-derived dendritic cells transfected ex vivo with HIV-Gag mRNA induce polyfunctional T-cell responses in nonhuman primates.

The pivotal role of DCs in initiating immune responses led to their use as vaccine vectors. However, the relationship between DC subsets involved in antigen presentation and the type of elicited immune responses underlined the need for the characterization of the DCs generated in vitro. The phenotypes of tissue‐derived APCs from a cynomolgus macaque model for human vaccine development were compared with ex vivo‐derived DCs. Monocyte/macrophages predominated in bone marrow (BM) and blood. Myeloid DCs (mDCs) were present in all tested tissues and were more highly represented than plasmacytoid DCs (pDCs). As in human skin, Langerhans cells (LCs) resided exclusively in the macaque epidermis, expressing CD11c, high levels of CD1a and langerin (CD207). Most DC subsets were endowed with tissue‐specific combinations of PRRs. DCs generated from CD34+ BM cells (CD34‐DCs) were heterogeneous in phenotype. CD34‐DCs shared properties (differentiation and PRR) of dermal and epidermal DCs. After injection into macaques, CD34‐DCs expressing HIV‐Gag induced Gag‐specific CD4+ and CD8+ T cells producing IFN‐γ, TNF‐α, MIP‐1β, or IL‐2. In high responding animals, the numbers of polyfunctional CD8+ T cells increased with the number of booster injections. This DC‐based vaccine strategy elicited immune responses relevant to the DC subsets generated in vitro.

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells.

Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine‐targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti‐Langerin‐HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti‐HIV immune response without requirement of an adjuvant. Although the co‐injection of the TLR‐7/8 synthetic ligand, R‐848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA‐DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine‐targeted epidermal LCs and R‐848.

The road ahead: Implementing mass cytometry in clinical studies, one cell at a time

Disease in humans involves a complicated interplay of pathology expressed on each individual’s unique genetic and phenotypic backdrop. The exponential growth of genomic, transcriptomic, and proteomic single-cell technologies provides unprecedented opportunities to capture and understand this complexity. Mass cytometry in particular—a flow cytometry platform that enables simultaneous measurement of over 50 parameters per cell (1–3)—holds significant promise to identify molecular signatures that underlie clinical outcomes (4,5), to help monitor disease progression (6), and to predict therapeutic responses (7,8). However, as the dimensionality of mass cytometry datasets increases, the development of appropriate computational approaches and standardized protocols becomes paramount (9). In this special issue published jointly with Cytometry A (10), we explore the necessary steps to transform mass cytometry from a technological tour-de-force to a valuable clinical platform. We highlight six studies that illustrate recent progress. The manuscripts by Yao et al. (11), Corneau et al. (12), and Strauss-Albee et al. (13) emphasize the utility of mass cytometry for identifying cell subsets that capture patient-specific disease attributes in the fields of pulmonology, neonatology, and virology. The studies by Abraham et al. (14) and Vendrame et al. (15) apply visualization and statistical tools in novel combinations to interpret high-dimensional data, while the manuscript by Leelatian et al. (16) describes a standardized protocol to derive isolated cancer cells from solid tissue for analysis by mass cytometry. Yao et al. (11) demonstrate the utility of multiparameter profiling of airway inflammatory cells in patients with cystic fibrosis (CF) to better understand the complex interactions between an individual’s immune state and disease severity. Using a novel assay for the mass cytometry analysis of airway inflammatory cells, the authors identified significant differences in the frequency of immune cell subsets in sputum collected from patients with CF, patients with asthma, and healthy controls. Interestingly, within each group substantial variability was observed when stimulating airway monocytes with lipopolysaccharide. These findings provide the basis for future work relating patient-specific signatures of airway inflammatory cells to disease progression and severity. Strauss-Albee et al. (13) focus on the deep immune profiling of umbilical cord blood samples to better define the natural killer (NK) cell repertoire and functional capacity of newborns. The authors provide a comprehensive overview of neonatal NK cell subsets and their functional attributes. Such characterization is important to better understand disease processes that engage the innate immune system of neonates who are uniquely vulnerable to infections and are unable to mount an efficient immune response to vaccination. In a third example emphasizing a translational application of mass cytometry, Corneau et al. (12) examine the cell cycle of CD41 T cells in the setting of HIV infection. Using a combination of cell cycle, differentiation, activation and exhaustion markers, they provide a more nuanced view of the effects of HIV infection on CD41 T cell cycling and question the traditional definitions of “resting” CD41 T cells. These results have potential clinical implications, as resting CD4 1 T cells are a reservoir for HIV infection difficult to target with commonly used treatments for HIV. Visualization of multiple cellular attributes across many cell subsets and robust statistical interpretation of high dimensional data remain critical challenges in implementing mass cytometry in clinical studies. The studies by Vendrame et al. (15) and Abraham et al. (14) illustrate these points. Vendrame et al. leverage the distinct strengths of existing computational approaches (i.e., viSNE (17) and citrus (18)] combined with custommade statistical tools (i.e., correspondence analysis and Friedman–Rafsky significance test) to extensively explore the effects of cytokines on human NK cell phenotype and function. Abraham et al. (14) introduce a radial visualization method called RADVIS, as an elegant

Mass cytometry: The time to settle down

MASS cytometry (CyTOF) technology was first described by Bandura et al. in 2009 (1) boosting the number of measurable markers per single cell and revolutionizing the flow cytometry field toward a horizon of a theoretical 100 measurements This revolution also boosted the development of other single cell multi-parameter technologies such as spectral flow cytometry (2) and chip-based cytometry (3), together with conventional flow cytometry that can now reach 50 theoretical parameters (4). The aim of this special issue is to mark the point that mass cytometry is presently a well-established technology with a large community of scientists committed to its development. It represents also a “settling moment” as described in a previous editorial (5) to next bring the technology to full maturity. This special Cytometry Part A issue includes the first OMIP describing a mass cytometry panel for the immune phenotype of human peripheral leukocytes together with a series of manuscripts introducing new reagents, protocols, quality controls and, also, a nice example of how the multidimensional nature of mass cytometry can address important biological question such as the status of a “challenged” immune system in comparison to a system kept in the clean environment of a pathogen-free laboratory. For those accustomed to traditional flow cytometry, one of the main drawbacks of mass cytometry is the absence of forward and side light scatter measurements to appreciate cell size and internal complexity. In this issue Stern et al. (this issue, page 14) use two plasma membrane staining assays based on wheat germ agglutinin and osmium tetroxide to evaluate cell size in mass cytometry experiments. Resolution is not comparable to conventional flow cytometry light scatter measurements; nevertheless, the combined use of these new membrane specific moieties, combined with phenotypic markers and the use of algorithms able to simultaneously evaluate multiple measurements hold promise for an extended use of these two reagents. To increase the assortment of available labels, Schulz et al. (this issue, page 25) introduce streptavidin coupled silver nanoparticles that can be used to include biotinylated reagents in mass cytometry panels. Of note, silver isotopes are detected in channels where at the moment no other reagents are available and hence the new reagent can be easily integrated in existing antibody panels. Wheat germ agglutinin and osmium were previously used to stain plasma membranes whereas silver nanoparticles are already in use in a wide range of immunoassays. Hence, the manuscripts by Stern et al. and Schulz et al. are nice examples of innovation created by changing the domain of usage. In the near future, mass cytometry will be used for clinical and longitudinal studies requiring an improvement of standardized protocols, methods to facilitate longitudinal analysis, and to accelerate the time needed for sample acquisition. Four manuscripts go in this direction. The OMIP-034 by Baumgart et al. (this issue, page 34) describes a basic 26 antibody panel able to identify neutrophils, eosinophils, basophils, monocytes, dendritic cells, T and B lymphocytes. Of note, the panel leaves several channels free to be completed with additional “drop-in” markers, and therefore a common backbone can be shared for multiple purposes. The panel described in OMIP-34 was designed keeping in account of the minimal but significant signal interference described by Takahashi et al. (this issue, page 39). Of interest for longitudinal studies and

SPADEVizR: an R package for visualization, analysis and integration of SPADE results

Motivation: Flow, hyperspectral and mass cytometry are experimental techniques measuring cell marker expressions at the single cell level. The recent increase of the number of markers simultaneously measurable has led to the development of new automatic gating algorithms. Especially, the SPADE algorithm has been proposed as a novel way to identify clusters of cells having similar phenotypes in high‐dimensional cytometry data. While SPADE or other cell clustering algorithms are powerful approaches, complementary analysis features are needed to better characterize the identified cell clusters. Results: We have developed SPADEVizR, an R package designed for the visualization, analysis and integration of cell clustering results. The available statistical methods allow highlighting cell clusters with relevant biological behaviors or integrating them with additional biological variables. Moreover, several visualization methods are available to better characterize the cell clusters, such as volcano plots, streamgraphs, parallel coordinates, heatmaps, or distograms. SPADEVizR can also generate linear, Cox or random forest models to predict biological outcomes, based on the cell cluster abundances. Additionally, SPADEVizR has several features allowing to quantify and to visualize the quality of the cell clustering results. These analysis features are essential to better interpret the behaviors and phenotypes of the identiÜed cell clusters. Importantly, SPADEVizR can handle clustering results from other algorithms than SPADE. Availability and Implementation: SPADEVizR is distributed under the GPL‐3 license and is available at https://github.com/tchitchek‐lab/SPADEVizR. Contact: nicolas.tchitchek@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.