Novel Transcript Markers of T Cell Activity
BardatinLutfiAifa
Abstract
Background.The T cell has an essential role in cellular immune response, particularly against intracellular pathogens such as viruses and intracellular bacteria. Following the first exposure of a naive host to an intracellular pathogen, the T cells undergo clonal expansion as well as differentiation into effector and memory cells. The antigen-specific memory T cells are activated after re-exposure to the same antigen and exert effector functions such as producing IFN-γ and other cytokines. A number of assays have been utilised to measure cellular immune response such as Enzyme-Linked Immunospot (ELISPOT) and real-time Polymerase Chain Reaction (real-time PCR) that use a few markers of T cell activity including IFN-γ, CXCL9, and CXCL10. By use of a human gene expression microarray, a pilot study by Dr. Michael Griffiths et al. has shown a significant increase in expression of a subset of genes including UBD, CXCL11, GBP1, IFI27, and IDO1 in IFN-γ stimulated PBMCs. These potential new transcript markers of T cell activity need to be validated by use of real-time PCR assay.
Methods.7 pairs of unstimulated and IFN-γ-stimulated whole blood samples were employed to validate the up-regulation of UBD, CXCL11, GBP1, IFI27, and IDO1 genes using real-time PCR assay. Interleukin-1 receptor-associated kinase 3 (IRAK3), an invariant gene identified from array data, was used as the internal control. The differences in median Cq values between the two groups were analysed using the Mann-Whitney test. The four most up-regulated genes were tested in CMV-stimulated and unstimulated PBMCs from 3 subjects with history of previous CMV exposure (CMV positive) and 1 subject without previous CMV exposure (CMV negative). The production of IFN-γ was also confirmed by use of ELISPOT assay.
Results.All genes except IRAK3 showed significantly higher transcript abundance in IFN-γ-stimulated whole blood samples than in unstimulated whole blood samples. The four most up-regulated genes included CXCL9, CXCL11, UBD, and GBP1. All of the 4 target genes demonstrated significantly higher transcript abundance in CMV-stimulated samples than in unstimulated samples. In contrast, all genes demonstrated no significant difference of transcript abundance between CMV-stimulated and unstimulated samples in CMV negative subject. When confirmed with the ELISPOT assay, the results were positive in all CMV-stimulated samples and negative in all unstimulated samples in 3 CMV positive subjects. Contrastingly, the ELISPOT results were negative in CMV-stimulated and unstimulated samples in the CMV negative subject.
Conclusion.Our study has shown that CXCL11, UBD, and GBP1 are potential novel transcipt markers of T cells activity and may be utilised as alternatives to CXCL9 and CXCL10. By use of real-time PCR assay, these markers could be measured in either whole blood or PBMCs after stimulation with IFN-γ or re-exposure to specific pathogen (i.e. CMV).