Mass cytometry for the multiplexed quantification and characterization of target expression on circulating cells in whole blood.

Characterization of target abundance on cells has broad translational applications. Among the approaches for assessing membrane target expression is quantification of the number of target-specific antibody (Ab) bound per cell (ABC). ABC determination on relevant cell subsets in complex and limited biological samples necessitates multidimensional immunophenotyping, for which the high-order multiparameter capabilities of mass cytometry provide considerable advantages. In the present study, we describe the implementation of CyTOF® for the concomitant quantification of membrane markers on diverse types of immune cells in human whole blood. Specifically, our protocol relies on establishing Bmax of Ab saturable binding on cells, then converted into ABC according to a metal's transmission efficiency and number of metal atoms per Ab. Using this method, we calculated ABC values for CD4 and CD8 within the expected range for circulating T cells and in concordance with the ABC obtained in the same samples by flow cytometry. Furthermore, we successfully conducted multiplex measurements of the ABC for CD28, CD16, CD32a, and CD64, on >15 immune cell subsets in human whole blood samples. We developed a high-dimensional data analysis workflow enabling semi-automated Bmax calculation in all examined cell subsets to facilitate ABC reporting across populations. In addition, we investigated impacts of the type of metal isotope and acquisition batch effect on the ABC evaluation with CyTOF®. In summary, our findings demonstrate mass cytometry is a valuable tool for concurrent quantitative analysis of multiple targets in specific and rare cell types, thus increasing the numbers of biomeasures obtained from a single sample.

Microphysiologic Human Tissue Constructs Reproduce Autologous Age-Specific BCG and HBV Primary Immunization in vitro.

Current vaccine development disregards human immune ontogeny, relying on animal models to select vaccine candidates targeting human infants, who are at greatest risk of infection worldwide, and receive the largest number of vaccines. To help accelerate and de-risk development of early-life effective immunization, we engineered a human age-specific microphysiologic vascular-interstitial interphase, suitable for pre-clinical modeling of distinct age-targeted immunity in vitro. Our Tissue Constructs (TCs) enable autonomous extravasation of monocytes that undergo rapid self-directed differentiation into migratory Dendritic Cells (DCs) in response to adjuvants and licensed vaccines such as Bacille Calmette-Guérin (BCG) or Hepatitis B virus Vaccine (HBV). TCs contain a confluent human endothelium grown atop a tri-dimensional human extracellular matrix substrate, employ human age-specific monocytes and autologous non heat-treated plasma, and avoid the use of xenogenic materials and exogenous cytokines. Vaccine-pulsed TCs autonomously generated DCs that induced single-antigen recall responses from autologous naïve and memory CD4+ T lymphocytes, matching study participant immune-status, including BCG responses paralleling donor PPD status, BCG-induced adenosine deaminase (ADA) activity paralleling infant cohorts in vivo, and multi-dose HBV antigen-specific responses as demonstrated by lymphoproliferation and TCR sequencing. Overall, our microphysiologic culture method reproduced age- and antigen-specific recall responses to BCG and HBV immunization, closely resembling those observed after a birth immunization of human cohorts in vivo, offering for the first time a new approach to early pre-clinical selection of effective age-targeted vaccine candidates.

Super resolution microscopy reveals that caveolin-1 is required for spatial organization of CRFB1 and subsequent antiviral signaling in zebrafish.

Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. Caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. This study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. A novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. Viral infection leads to a downregulation in Caveolin-1b (Cav-1b), disrupting clusters of CRFB1, a zebrafish type I interferon receptor (-R) subunit. Super-resolution microscopy has enabled the first single-molecule imaging of CRFB1 association with cav-1b-containing membrane domains. Strikingly, downregulation of Cav-1b, the major protein component of caveolae, caused CRFB1 clusters to disperse. Dispersal of CRFB1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. This response strongly suggests that CRFB1 organization within cav-1b-containing membrane domains is critical for IFN-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter IFN signaling and the antiviral immune response.