Immune profiling of multi-parameter flow cytometry using computational statistics

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DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (04) Clinical Research and specific Challenge Topic 04-AI- 102: The human immune response to infection and immunization - Profiling via modern immunological methods and systems biology. The ability to monitor complex immune responses quantitatively is essential for the development of effective vaccines and the discovery of diagnostic or prognostic biomarkers for clinical trials. Multi-parameter flow cytometry (FCM) can measure multiple immune parameters (cell phenotype, activation or maturation status, intracellular cytokine or other effector molecule concentrations) with a single peripheral blood (PB) sample, and provides a detailed snapshot of the immune response that is ideal for profiling. The purpose of this project is to develop and validate objective statistical methods to profile FCM data, and apply these methods to discover FCM-based immune correlates of efficacy in well characterized HIV and advanced melanoma cohorts. Recent advances in polychromatic FCM technology allow the simultaneous measurement of up to 20 fluorescent markers at the single cell level, and these state-of-the-art assays show tremendous promise for profiling the immune responses to infection and vaccination. However, software for analysis of FCM data has not kept pace and still relies on serial 2D gating methods that are sub-optimal for analysis of multi-dimensional data sets. As a result, FCM results can be highly variable across different institutions. Systems biological approaches that handle multi-dimensional data directly are needed to design software that can keep up with the rapid pace of FCM technological innovations. We propose to develop computational statistical models to characterize immune response profiles using multi- parameter FCM, and to implement efficient software for automated FCM analysis and discovery of predictive immune signatures. Our specific aims are to 1) develop multivariate computational statistical methods to characterize FCM data consistently across multiple samples; 2) validate automated cell subset identification on a broad set of FCM samples; and 3) identify immune signatures based on statistical models of FCM data that predict infection or vaccination outcome. The research will substantially extend the utility of FCM analysis with effective, automated statistical methods and tools for identifying heterogeneous cell subsets and immune signatures from FCM data. This will benefit anyone using multi-parameter FCM, with particular impact on vaccine development, clinical diagnostics and immune therapeutics, given their common need for objective FCM software for immune profiling. Hence, our proposal directly addresses the objectives of Challenge Topic 04-AI-102, and represents methodological advances with major potential impact on the rational design and development of safe and effective vaccines. The proposed application seeks to develop automated cell subset identification and predictive immune profiling of infection and vaccination outcomes from multi-parameter flow cytometry data. Success in this project will result in statistical methodology and software that will produce more accurate, reproducible flow cytometry analysis, as well as identify immune correlates of infection control and vaccine efficacy.