Individual mouse analysis of the cellular immune response to tumor antigens in peripheral blood by intracellular staining for cytokines.

Among the experimental animal models, mice remain the most widely used for the evaluation of immunotherapeutic strategies. Vaccines against parasites and viral antigens are commonly administered to the appropriate mouse strain which also allows testing of the therapeutic effect. Similarly, in mice transgenic for human tumor associated antigens (TAA), cancer vaccines must lead to breakage of immune tolerance to elicit a significant effect on the tumor. However, one of the major drawbacks in the monitoring of cellular immune responses induced by vaccination is that functional immunological assays require suppression of the animals to collect the spleen or lymph nodes for analysis. Here, we report the application of a rapid intracellular staining (ICS) method to quantify antigen-specific T cells responses in small volumes of murine blood. Genetic vaccination with plasmid DNA followed by electroporation (DNA-EP) and the use of adenoviral vectors (Ad) encoding CEA as a model target antigen were applied to different strains of mice. Optimal blood volume, number of lymphocytes, sensitivity and reproducibility of intracellular staining for IFN-gamma were determined both in non-tolerant/wild type mice as well as in tolerant CEA transgenic mice upon restimulation of PBMCs with CEA peptides. Groups of vaccinated mice were then sacrificed and PBMCs and splenocytes from individual animals were compared for intracytoplasmic detection of IFN-gamma and TNF-alpha. A significant correlation was observed between splenic and blood immune responses. Finally, the cellular immune response was followed over time in groups of vaccinated mice. The kinetics of IFN-gamma producing effectors were measured after priming and successive boosting with adenoviral vectors. We show that intracellular staining for mouse PBMCs is a rapid and simple method to measure antigen-specific immune responses. It does not require animal euthanasia and mirrors the response observed in lymphoid organs such as the spleen.