Quantitative Statistical Analysis of Cytokine Secretion Dynamics for Individual T Cells

The immune system coordinates a diverse T-cell response upon antigenic stimulation to protect the host from disease.  This response involves clonal replication and differentiation, as well as the release of many cytokines with various immunomodulatory functions.  The efficacy of the response depends, in part, on the nature of the cytokines secreted by activated T-cells.  Specifically, the types and duration of cytokine secretion highly depend on the nature of the disease and the immune system of the host.  By identifying clinical markers indicative of a functional immune response, it may be possible to predict the efficacy of treatment, or identify unique subsets of lymphocytes that enable successful outcomes.  Common analytical methods assess the mean frequencies or magnitudes of cytokine expression within a heterogeneous population of cells.  There are few assays that assign multiple functional and phenotypic characteristics to individual cells without changing their viability or response.  Microengraving is a process that uses an array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells (10⁴–10⁵ cells/assay).  The Love lab developed an approach based on this technology to quantify the rates of secretion from single immune cells, and demonstrated how microengraving can provide quantitative measurements of rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells [1].  While different combinations of these cytokines can define the functional status of individual cells at any particular time, consecutive measurements taken over time provide a dynamic profile of single-cell cytokine secretion following antigenic stimulation.  This approach to measuring functional responses of T-cells, in combination with imaging cytometry, yields integrated data on many individual viable primary immune cells that describes (i) surface-expressed markers indicating their immunophenotypes, (ii) the frequencies of cells secreting cytokines, and (iii) the rates of secretion of those cytokines. Such multidimensional profiling makes it possible to analyze the contribution of individual cells within a heterogeneous subset of cells.  For instance, we are able to compare the T-cell response of specific immunophenotypes to the aggregate behavior of greater populations.  Although the mean behavior of minority populations appears consistent, single-cell analysis suggests that certain profiles may be associated with specific differentiated subsets.  Through the use of cytokine transition matrices, we investigate the directional signaling dynamics specific to T-cell subpopulations, confirming the activation of known regulatory pathways.  Further analysis of these kinetics has the potential to identify additional regulatory mechanisms involved in the differentiation of immune cells.   

The large diversity of cells that comprise the human immune system requires methods that resolve individual contributions of specific subsets to an immunological response. Through quantitative statistical analysis of integrated single-cell data, we aim to better understand and predict the nonlinear signaling dynamics that provide qualitatively different T-cell performance.  Investigating the complex signal processing of immune cells may offer metrics to design better treatment strategies and predict the efficacy of these treatments. 

1. Han Q, Bradshaw EM, Nilsson B, Hafler DA, Love JC. Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving. Lab Chip 10: 1391-1400.