Conference Agenda

Systems Immunology at the Single Cell Level

Garry Nolan

Stanford University, United States of America

Single cell analysis of functional cell states via flow cytometry has been an essential tool for dissecting the functional complexity of hematopoiesis. We have focused upon the development of intracellular assays of signaling systems can be correlated to functional subsets of cells in complex populations based on active kinase states or other signaling junctions. Such correlations could be important to document and order the immune system and its disorders into understandable progressions.

Using a next-generation single-cell ‘mass cytometry’ platform we quantify surface and cytokine or drug responsive indices of kinase targets with 34 parameters analysis (e.g. 31 antibodies, viability, DNA content and relative cell size). In healthy human blood and bone marrow, we identified cell subsets spanning the hematopoietic hierarchy using a panel of up to 31 surface markers and used an automated algorithm to delineate all cell subpopulations into a progressive heirarchy. Additionally, 18 simultaneous functional markers and a battery of ex vivo stimuli and inhibitors were used to interrogate signaling. This novel algorithm for unsupervised high-dimensional clustering of single-cell data revealed a continuum of phenotypically contiguous subpopulations. This continuum was demarcated by surface marker expression, but paralleled by gradual changes in intracellular signaling architecture.

The application of these multidimensional approaches to human acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) samples, as well as differentiating ES cells will be presented. The simultaneous measurement of surface marker, cell cycle, apoptosis, and intracellular signaling parameters has revealed substantial “order” within the presumed heterogeneity in the populations of these progressions of cell states. This information is used to provide a high-content view of differential in vitro drug activity across these distinct blast subpopulations for cancer—again indicating there exists remarkable “structure” within the tumor that is suggestive of either an adulterated differentiation cascade or a pseudo-hierarchy of differentiation. For differentiating ES cells we find signatures of pluripotent marker states that predict dead ends for differentiation or indicate a progression towards a defined lineage. Collectively, these results have exposed unappreciated layers of human hematopoietic organization, and provide an opportunity to re-evaluate diseases and pharmacological therapeutics as specific perturbations to this inherent order.