Analysis of Ag-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4+ T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4+ T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from bacillus Calmette-Guérin-vaccinated mice and show that highquality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4+ CD154+ cells was distinct from that of CD154- cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grants 1R21AI092448 (to S.A.P.) and 2P01AI063537 (to W.R.J., S.A.P., and J.C.). Core resources for flow cytometry and microarray analysis were supported by the Einstein Cancer Center (Grant CA13330). Support for C.T.J. was provided by National Institutes of Health Training Grant GM07491.