Transcriptome Analysis of Mycobacteria-Specific CD4+ T Cells Identified by Activation-Induced Expression of CD154

Abstract

Analysis of antigen-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. While several methods exist for identifying antigen-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC-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, antigen-specific CD4+ T cells but this approach remains underexplored, and to our knowledge has not previously been applied in mycobacteria-infected animals. Here we show that CD154 expression identifies adoptively transferred or endogenous antigen-specific CD4+ T cells induced by Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination. We confirmed that antigen-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from BCG-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 the 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 characterized by the production of interleukin-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live, antigen-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

Generation of IL-3–Secreting CD4+ T Cells by Microbial Challenge at Skin and Mucosal Barriers

“…2C), including positivity for Tbet (Fig. 2D), as previously reported with M. bovis BCG model (12). Collectively, these results suggest that in these models, IL-3–secreting T cells are closely related to conventionally defined multifunctional Th1 cells and may define a specific subset or activation state of these.…”

“…In our previous work, we have shown that IL-3–producing CD4 + T cells develop following vaccination with BCG by s.c. route or infection with M. tuberculosis by aerosol (12). We tested the possibility that i.v.…”

“…Our previous experiments with BCG-vaccinated mice have shown that IL-3–producing CD4 + T cells coexpress IFN-γ, the signature cytokine of Th1 cells, but segregate from cells that produce the canonical Th2 cytokine IL-4 or Th17 cytokine IL-17A (12). Coexpression of IL-3 with IFN-γ was also noted for CD4 + T cells from mice infected with virulent M. tuberculosis by aerosols (Supplemental Fig.…”

“…However, given the effect of IL-3 on proliferation of mast cells and basophils, its role in antiparasite immunity, and in potentiation of Th2 immunity, most studies have investigated IL-3 in the context of Th2 immune responses (8, 11). In contrast, we observed that IL-3–producing CD4 + T cells were also prominent among CD4 + T cells specific to Mycobacterium bovis bacillus Calmette-Guerin (BCG), which has generally been associated with priming of strong Th1 responses (12). This finding was surprising because IL-3 is seldom studied in the context of mycobacterial immunity and motivated us to further explore this finding.…”

Recent Trends in System-Scale Integrative Approaches for Discovering Protective Antigens Against Mycobacterial Pathogens

“…The transcriptome reflects the set of all RNA molecules or transcripts in a cell or organism. Transcriptomics aim to study all species of transcripts including mRNAs, non-coding RNAs and small RNAs produced in a cell of an organism at a specific time ( Wang et al, 2009 ; Kunnath-Velayudhan et al, 2017 ; Lowe et al, 2017 ). Transcriptomics analysis has played a central role in unraveling the gene expression during a particular physiological condition and deciphering the intricacies of regulations at the transcriptional level.…”