Data-Driven Compensation for Flow Cytometry of Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 339)

This bibliography lists 105 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during July 1990. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data.

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry

To Be, or Not to Be: Cellular Homeostasis to Mechanical Perturbations

Mechanical homeostasis is an emerging mechanobiology concept that describes the critical biological process to maintain whole-cell/tissue physiology against forces and deformation arising both intra- and extracellularly. Dysregulation of mechanical homeostasis has important implications in pathophysiological conditions such as developmental defect, cardiovascular and pulmonary diseases, and cancer. Mechanical homeostasis has been commonly investigated at molecular, cellular, tissue levels and beyond. However, in mechanical homeostasis collective dynamics at smaller scales and its functional relationship with emergent system-level properties at larger scales remains elusive. The major contribution of this dissertation is to provide a detailed picture of the functional link between molecular and subcellular events and apparent cellular behaviors under mechanical perturbations. A novel suite of technologies, involving microfabrication, live-cell imaging, high-throughput and multidimensional image processing, and mechanical characterization, have been developed and implemented in this research for the live-cell study of both subcellular and cellular aspects of mechanical homeostasis. By utilizing these techniques, we performed cell stretch experiments and quantitative measurements of biomechanical and biochemical responses with a spatiotemporal resolution to examine cell behaviors upon mechanical perturbation. Our data have revealed that cellular mechanical homeostasis is an emergent phenomenon driven by collective and graduated, yet non-homeostatic, subcellular behaviors (“subcellular rheostasis”) that follow distinct mechanosensitive compensatory paths. We have for the first time shown that subcellular dynamics would observe patterns different from that at the single-cell level. Further investigations have revealed that impairment to the extracellular matrix (ECM) – focal adhesion (FA) – cytoskeleton (CSK) mechanical linkage can lead to an effective exit from cellular mechanical homeostasis by skewing the subcellular rheostasis pattern of FAs, which might be a sensitive gating mechanism of cellular homeostasis. Lastly, a mechano-biophysical model has been constructed in this work to quantitatively recapitulate experimental observations of subcellular rheostasis and its perturbation by different drug treatments. Cross-examination of experimental and theoretical modeling results has unveiled the regulatory roles of different mechanosensitive machineries including catch-slip bonds and myosin motor activity in governing the emergence of cellular mechanical homeostasis.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135780/1/shinuow_1.pd

TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection.

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion

Compensation between CSF1R+ macrophages and Foxp3+ Treg cells drives resistance to tumor immunotherapy.

Redundancy and compensation provide robustness to biological systems but may contribute to therapy resistance. Both tumor-associated macrophages (TAMs) and Foxp3+ regulatory T (Treg) cells promote tumor progression by limiting antitumor immunity. Here we show that genetic ablation of CSF1 in colorectal cancer cells reduces the influx of immunosuppressive CSF1R+ TAMs within tumors. This reduction in CSF1-dependent TAMs resulted in increased CD8+ T cell attack on tumors, but its effect on tumor growth was limited by a compensatory increase in Foxp3+ Treg cells. Similarly, disruption of Treg cell activity through their experimental ablation produced moderate effects on tumor growth and was associated with elevated numbers of CSF1R+ TAMs. Importantly, codepletion of CSF1R+ TAMs and Foxp3+ Treg cells resulted in an increased influx of CD8+ T cells, augmentation of their function, and a synergistic reduction in tumor growth. Further, inhibition of Treg cell activity either through systemic pharmacological blockade of PI3Kδ, or its genetic inactivation within Foxp3+ Treg cells, sensitized previously unresponsive solid tumors to CSF1R+ TAM depletion and enhanced the effect of CSF1R blockade. These findings identify CSF1R+ TAMs and PI3Kδ-driven Foxp3+ Treg cells as the dominant compensatory cellular components of the immunosuppressive tumor microenvironment, with implications for the design of combinatorial immunotherapies.D. Gyori was funded by a research grant from Karus Therapeutics. E.L. Lim was supported by a Yousef Jameel Scholarship (Cambridge Trust). R. Roychoudhuri and K. Okkenhaug received institute support from Biotechnology and Biological Sciences Research Council (BBSRC) (BBS/E/B/000 -C0407, -C0409, -C0427 and -C0428). K. Okkenhaug was also supported by Wellcome Trust grant 095198/Z/10/Z. L.R. Stephens and P.T. Hawkins were supported by and institute grant from the BBSRC (BB/J004456/1). R. Roychoudhuri is supported by the Wellcome Trust/Royal Society (Grant 105663/Z/14/Z), the UK Biotechnology and Biological Sciences Research Council (Grant BB/N007794/1), and Cancer Research UK (Grant C52623/A22597)

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers

The effects of ageing on microenvironment-contextual epithelial cell signalling

Age is the greatest risk factor for developing cancer. Two-thirds of cancer diagnoses occur in people over the age of 65. This increased vulnerability to tumourigenesis is attributed to intrinsic cellular changes, in particular the agerelated telomere shortening and the accumulation of mutations over time and. We propose that extrinsic factors comprising age-related alterations in the tissue microenvironment are also important in cancer development. We sought to elucidate how the microenvironmental affects cells, and how this is related to cancer development. This knowledge can be utilized to improve prevention, diagnosis and treatment of cancer in our ageing population. Cellular function is coordinated by microenvironmental factors such as growth factors, cytokines as well as extracellular matrix proteins. Soluble factormediated signal transduction is strongly influenced by microenvironmental context. To allow single cell level measurement of the microenvironmental contextual effect on cell signaling, we developed a novel flow cytometry method: microsphere cytometry. Single normal or neoplastic cells were adhered to uniform microspheres that display mimetic-microenvironments comprising surface combinations of extracellular matrix (ECM) in the presence of soluble agonists or antagonists. Temporal signaling responses were measured with fluorophore-conjugated antibodies that recognize responsedependent epitopes by multiparametric flow cytometry. Using this approach, we demonstrated that microenvironment-mimetic combinations of growth factors and extracellular matrix proteins generate distinct cellular signaling signatures from normal and patient biopsy-derived neoplastic cells. We asked whether the ageing process affects how human mammary epithelial cells (HMEC) respond to microenvironmental signals, and if this altered response results in increased susceptibility to oncogenic transformation. Using microsphere cytometry we analyzed age-dependent changes in human mammary myoepithelial and luminal epithelial cells exposed to different ECM and growth factors. We found that ECM–mediated MAP kinase and PI3K pathway activation levels in HMEC are attenuated with age, and that the diminished signaling magnitude in HMEC from ageing women correlated with reduced probability of activating oncogene-induced senescence. Our results suggest that attenuated cell signaling response may reduce the likelihood of activating oncogene induced senescence, for cells in ageing women. We hypothesize this is the result of age-related changes to the microenvironment that support age-emergent cellular phenotypes with increased cancer susceptibility