biostudies-literatureKunnath-Velayudhan SNIAID NIH HHSNCI NIH HHSNIGMS NIH HHS2596-2606https://www.ebi.ac.uk/biostudies/studies/S-EPMC5605459biostudies-literatureUnknown199(7)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 <i>Mycobacterium bovis</i> 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 high-quality 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.Journal of immunology (Baltimore, Md. : 1950)Transcriptome Analysis of Mycobacteria-Specific CD4⁺ T Cells Identified by Activation-Induced Expression of CD154.PMC5605459T32 GM007491P30 CA013330R21 AI092448R01 AI098925P01 AI063537Ng TWJacobs WRChan JJohnson AJGoldberg MFPorcelli SAKunnath-Velayudhan SJohndrow CTSaini NKXu JfalseTranscriptome 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 <i>Mycobacterium bovis</i> 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 high-quality 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.2017-01-01T00:00:00Z2017 Oct2024-11-12T02:00:52.188Z2019-03-26T23:57:52ZS-EPMC56054592882158410.4049/jimmunol.1700654