Blood cells need to self-renew, proliferate, and differentiate in a balanced fashion to enable self-sustaining blood systems such as the immune system. The biochemical signaling network that regulates this balance is complex, non-intuitive, and not well understood. Adding to the complexity, blood cells exist as diverse lineages with rare, but critical, subsets within each lineage. Traditional FACS analyses with limited cell surface marker panels have created the false notion of restricted subsets, with abrupt transitions in a lineage trajectory. Such limited subset classification (and analyses of signals herein) has obstructed a full understanding of the function of biochemical networks that regulate the cellular balance between proliferation and differentiation. Our recently pioneered single-cell mass cytometry (CyTOF) method broke this impasse and has revealed that hematopoiesis in the bone marrow is a continuum with over a hundred identifiable subsets. It is known that aberrant biochemical networks can form the basis for human diseases like cancer, autoimmune diseases, or immunodeficiency Our deterministic and stochastic computational models that explored the topology of Ras signaling predicted distinct patterns of Ras activation as a function of the Ras activator proteins Rasgrp and Sos. Testing these hypotheses, we found that analog Rasgrp1- Ras-ERK or bimodal Sos-Ras-ERK signals can occur in lymphocytes. Our new mouse models now indicate that different perturbation in Rasgrp1 lead to reshaping of the Ras signals and result in cancer, autoimmune diseases, or immunodeficiency. Here we hypothesize that blood cells develop through a continuum in a balanced manner as a function of the topology and character of the Ras signaling network. In Preliminary Results, we discuss our ordinary differential equation (ODE) and Stochastic simulation compile (SSC) computational models of Ras signaling, details of our CyTOF data collection and computational SPADE and ACCENSE analysis methods, as well as our biochemical phospho-flow analyses on defined subsets of lymphocytes. We also present several lines of evidence that the Ras activator Rasgrp1 shapes the character of the Ras network to balance proliferation and differentiation. Loss of Rasgrp1 leads to immunodeficiency. We present data from our recent 2013 publications on T cell leukemia caused by oncogenic Ras mutations or overexpression of the Ras activator Rasgrp1 as well as a lupus-like autoimmune phenotype in a mouse model with a point-mutated Rasgrp1Anaef allele. In this proposal we will combine computational hypothesis generation, high-resolution analytic approaches of high-dimensional CyTOF data, and high-throughput biochemical analyses of primary blood cells from mouse models with distinct Ras signals and human leukemia samples to understand the topology of the Ras signaling network in T lymphocytes properly transitioning through the normal continuum in the bone marrow (Aim 1) and thymus (Aim 2). We will also characterize how perturbations of the network's character can lead to immunodeficiency, autoimmunity, or T cell leukemia. Using reiterative loops between the three disciplines, we focus on the T cell lineage here to ensure a productive research plan but will also generate new insights relevant for all hematopoietic blood lineages to spur future investigations.
The hematopoietic blood system is a robust and dynamic system with the critical function to create a self- sustaining immune system in which non-intuitive and non-linear biochemical signals create a balance between proliferation and differentiation. The goal of this project is to determine the identity and topology of these biochemical networks and their dynamic responses in the context of normal development of T cell lineages as well as the consequences of aberrant perturbations of these equilibrated networks, which we propose leads to human diseases like blood cancer, autoimmune diseases, or immunodeficiency. We are combining our three distinct disciplines of hypothesis-driving computation, high dimensional CyTOF cell analysis, and high- throughput quantitative biochemistry of primary blood cells, and results from these highly collaborative efforts will provide mechanistic insights into complex regulation of T cells in their developmental continuum through the bone marrow and thymus, which will be of relevance to the mission of the NIH and of interest to a broad audience of researchers studying blood diseases.
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