Transcriptome analysis of mycobacteria-specific CD4⁺ T cells identified by activation-induced expression of CD154

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.