Standardization of Functional Immune Cell-Based Assays: An Integral Aspect to Vaccine and Biologic Development and Validation.

The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized.

However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis.

Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.