An Integrated Approach to Analyzing Activation Profiles in Immune Cells by Combining Cytomic and Proteomic Techniques of Cell Analysis/Sorting and Protein Fractionation.

Understanding complex responses in biological systems is important to the development of new biomarkers, diagnostic assays, drugs and biologics required for early disease detection, prognosis, monitoring, and treatment. Although several cutting edge technologies have been introduced in the last decade, they have not significantly impacted the speed and economics of new drug applications and introduction of relevant diagnostic tests. In order to overcome this predicament, a Systems Biology approach is necessary to interrogate complex biological responses. This requires that assays that have so far been carried out in isolation, for e.g. gene expression, protein synthesis, phenotype and function, etc. need to be integrated and evaluated in a complementary fashion.

In the current study, the authors have attempted such an evaluation by integrating well-known techniques of flow cytometry-based phenotypic analysis and cell sorting with protein fractionation and analysis to evaluate immune cellular activation profiles. To evaluate changes in protein profiles associated with the activation of an immune response, peripheral blood mononuclear cells (PBMCs) were stimulated with the superantigen, Staphylococcus enterotoxin B (SEB) and anti-CD28 or left un-treated for 24 hours. Lysates of PBMC populations from each culture were directly fractionated by two-dimensional gel-free liquid chromatography using isoelectric point (pI) and hydrophobicity. Alternatively, flow-based sorting techniques were 1^st utilized to isolate T cells of interest from activated and control cultures and then fractionated as described above. Cell free supernatants from activated and non activated cultures were also fractionated and analyzed.

The 1^st dimension fractions obtained by separation based on pI alone did not reveal significant differences in activated and non activated lysates. The majority of the proteins in both the sorted and non-sorted lysates eluted in the acidic fractions of the pH gradient. However, using advanced differential display software, the hydrophobicity-dependent second dimension separations of the 1^st dimension pI fractions did reveal significant quantitative and qualitative differences for several peaks in both the acidic and basic regions of the gradient in the activated and non activated fractions. Further combination of sorting of T cell populations with two-dimensional gel-free liquid chromatography permitted the characterization of an increased number of proteins that were up or down regulated by activation as compared to non sorted cell cultures. Thus the cumulative approach of sorting and 2-dimensional analysis was the most sensitive in discovering changes associated with activation. Such profiles could potentially be used as surrogate markers of efficacy in vaccine development and immunotoxicity in drug development. Further combinations of genomic, proteomic and cytomic profiling will further advance and ultimately simplify the discovery process and enable the mapping of biological pathways to accomplish a unified evaluation of relevant biomarkers in this response and other clinical models.