(M1130-01-04) Evaluating the Impact of Matrix on Ex Vivo Stimulation Flow Cytometry in Bioanalysis

Purpose: First in human clinical trials are increasingly exploring ex-vivo stimulation assays to assess pharmacodynamic (PD) effect of immunomodulatory therapies. Ex-vivo stimulations can mimic the immune responses seen in a particular disease state, allowing PD effect to be evaluated in healthy volunteers who may not exhibit the aberrant immunological phenotypes targeted by the therapeutic. Such findings can support phase I safety and pharmacokinetic data while also providing early insight into the efficacy of new drugs before moving into phase II trials with patients. The sensitivity of measuring a particular PD effect relies upon consistent, high magnitude stimulation of the pathway of interest in the target cells of interest. Poor stimulation or high within-assay variability could preclude meaningful interpretation of results. Maximizing stimulation magnitude and assay precision requires tailoring a method to the biology as well as adapting for operational practicality and scalability. A principal choice in ex-vivo stimulation assay design is that of the matrix, between whole blood, isolated peripheral blood mononuclear cells (PBMCs), or isolated individual cell populations (such as CD4+ T cells). Here, we assess the impact of assay matrix on two distinct ex-vivo stimulation flow cytometry assays: polyclonal T cell receptor (TCR) stimulation and induction of CD69 expression, and cytokine stimulation and induction of intracellular phosphorylated STAT proteins.

Methods: Whole blood from the same healthy volunteers was used for isolation of PBMCs (using SepMate^TM tubes and Lymphoprep^TM), CD4+ T cells (using RosetteSep^TM Human CD4+ T cell Enrichment Cocktail), or left as whole blood. Each matrix was processed fresh on the day of stimulation (not cryopreserved). PBMCs and isolated CD4+ T cells were resuspended in culture media at a volume equivalent to the pre-isolation whole blood volume. Whole blood was inverted briefly and either diluted 1:2 in PBS + 2% FBS or left undiluted. Processed samples were added to 96-well deep well plates at 0.120 mL/well and stimulated with 17,000 U/mL universal type I IFN (cytokine stimulation) for 30 minutes or anti-CD3/anti-CD28 for 24 hours (TCR stimulation). Cytokine stimulation samples were fixed, permeabilized, stained for CD3, CD45, pSTAT1, pSTAT3, and analyzed by flow cytometry. TCR stimulation samples were stained for CD3, CD4, CD8, CD19, and CD69. In some experiments, golgi blocker was added to TCR samples for the last 4 hours and samples were permeabilized and additionally stained for intracellular IFN-gamma. In additional experiments, whole blood was incubated at ambient temperature for periods ranging from one to 48 hours to assess the impact on functional ex-vivo stimulation assays.

Results: Similar cellular proportions were observed between whole blood and PBMCs (approximately 5-8% CD19+ B cells, 90-95% CD3+ T cells). Isolated CD4+ T cells were >98% CD3+ T cells ( >95% CD4+ T cells). CD69 expression following TCR stimulation was highest in whole blood, followed by diluted whole blood, isolated CD4+ T cells, and lastly in PBMCs. Within-donor between-replicate precision of CD69 expression was comparable across all matrices, but between-donor variability was appreciably greater in whole blood compared to PBMCs. For cytokine stimulation, pSTAT1 and pSTAT3 induction following treatment with universal type I IFN was similar across all matrices. However, average background pSTAT1 and pSTAT3 was noticeably higher in PBMCs and isolated CD4+ T cells compared to whole blood or diluted whole blood. With both TCR stimulation and cytokine stimulation, whole blood remained similarly responsive within 6 hours post blood draw. However, whole blood was stable for up to 48 hours if used for PBMC isolation and TCR stimulation for analysis of CD69 and intracellular IFN-gamma expression.

Conclusion: The choice of cellular matrix for functional ex-vivo stimulation bioanalysis assays depends upon the biology of the target pathway as well as operational and scalability constraints. While whole blood yielded the greatest stimulation magnitude and high within-assay precision in both assays, limited post-draw/pre-assay blood stability compared to PBMCs could preclude the use of whole blood in large scale bioanalysis. In addition, the known capacity for PBMCs to withstand long-term cryopreservation could make them the superior choice for some study designs. Isolated CD4+ T cells are highly enriched and sufficiently responsive to TCR stimulation, making this approach favorable for situations where other potentially interfering cell types are undesirable. However, both PBMCs and isolated CD4+ T cells showed high background pSTAT in unstimulated conditions, thereby reducing sensitivity of measuring PD effects of therapeutics targeted at inhibiting these pathways. Collectively, these findings demonstrate the importance of understanding the advantages and limitations of each matrix in ex-vivo stim assays in bioanalysis.


TCR stimulation and induction of CD69 expression on CD4+ T cells across each matrix.


Cytokine stimulation and induction of pSTAT3 in T cells across each matrix.


Stability of whole blood prior to PBMC isolation for TCR stimulation intracellular cytokine flow cytometry.