Analysis of cellular immune responses in the peripheral blood of mice using real-time RT-PCR

Murine cancer models are commonly used in the evaluation of immunotherapeutic strategies. However, one of the major limitations in the monitoring of cellular immune responses induced by various vaccination approaches is that existing immunoassays require sacrifice of the animals for collection of the spleen or lymph nodes for analysis. We report here the development of an assay to quantitate antigen-specific T cell responses in murine blood, without euthanasia, using real-time RT-PCR for measurement of interferon-gamma mRNA levels. C57BL/6 mice were immunized with an adenoviral vector encoding the melanoma antigen gp100 (Ad2/gp100) or were left untreated. Small samples of whole blood were collected by retro-orbital puncture for analysis of T cell reactivity. The mice were then euthanized and spleen cells were isolated for comparative analyses. Blood and spleen cells were restimulated with either a peptide containing the dominant gp100 MHC Class I-restricted epitope, gp100(25-33), or a negative control peptide containing an irrelevant Class I-restricted epitope from ovalbumin. IFN-gamma mRNA was detected in gp100 peptide-pulsed whole blood as well as in spleen cells recovered from Ad2/gp100-treated mice, but not in untreated mice. In addition, there was a strong correlation in the magnitude of the gp100-specific response of spleen cells from an individual animal when measured by real-time RT-PCR with the more conventional enzyme-linked immunospot (ELISPOT) method (P<0.001). Finally, the gp100-specific immune response measured in the peripheral blood of individual animals by real-time RT-PCR or ELISPOT showed a significant correlation with the response measured in the spleen (P=0.001). We conclude that real-time RT-PCR measurement of IFN-gamma mRNA induced by antigenic stimulation is an attractive method to measure an antigen-specific cellular immune response in small samples of whole blood as it does not require euthanasia, mirrors the response observed in the spleen and correlates with the response measured using the conventional ELISPOT method.