Supplementary MaterialsData_Sheet_1. central/effector memory phenotypes and differentiated into polyfunctional effector cell subtypes which expressed TBX21/T-bet, antimicrobial cytokines IFN-, TNF-, GM-CSF, and cytotoxic granule molecules. Furthermore, the IL-12-expanded V2V2 T cells inhibited the growth of intracellular mycobacteria in IFN– or TNF–dependent fashion. Our findings support the concept that IL-12 drives early development BYK 204165 of fast-acting V2V2 T effector cells in antimicrobial immune responses. IFN- production and induction/maintenance of antigen-specific CD4+ Th1 cells for development of protective immunity against intracellular pathogens including resistance to (Mtb) infection (8, 9). However, little is known about whether IL-12 can promote immune response or function of other T-cell populations that do not express CD4 during Mtb or other microbial infections. T cells appear to be a non-conventional T-cell population that contributes to both innate and adaptive immune responses against microbial infections (10). V2V2 T-cell subpopulation unique in humans and nonhuman primates (NHP) constitute 65C90% of total circulating human T cells and remain the sole T-cell subset capable of recognizing phosphoantigens such as the isopentenyl pyrophosphate (IPP) metabolite (11) and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) produced by Mtb and other microbes (12). Studies in humans and NHP (13C17) have shown that IPP- or HMBPP-activated V2V2 T cells can readily produce Th1 cytokines IFN-/TNF- and cytotoxic granule molecules perforin (PRF), granzyme A/B (GZMA/B), and BYK 204165 granulysin (GNLY), and consistently exhibit antimicrobial and anti-cancer activities. On the other hand, activated V2V2 T cells can be expanded by IL-2, IL-7, IL-15, IL-21, IL-33, and Th17-related cytokines (13, 18C21). Furthermore, recent seminal studies in NHP models suggest that the phosphoantigen HMBPP-specific V2V2 T-cell subset can respond as fast-acting T cells, undergo rapid expansion and pulmonary trafficking and residence, and attenuate high-dose Mtb infection (10, 15, 16). However, whether IL-12 signaling pathway mediates fast-acting and Th1 or anti-microbial features of V2V2 T cells remains poorly defined (22, 23). In the current study, we performed mechanistic experiments to test the hypothesis that IL-12, a key innate cytokine produced by Mtb infection of macrophages/DC, plays a role in the early development of fast-acting V2V2 T effector cells. Our study provides previously-unreported data implicating signaling pathways, cytokine networks and functional mechanisms whereby IL-12 expands and differentiates HMBPP-activated V2V2 T effector cells producing multiple anti-TB cytokines and inhibiting mycobacterial growth. Materials and Methods Expansion of V2V2 T Cells by HMBPP Plus Cytokines in PBMC Culture The protocols for human blood samples for experimental procedures were evaluated and approved by the institutional review boards for human subjects’ research and institutional biosafety committees at Shanghai Pulmonary Hospital. BYK 204165 All subjects are adults and signed written Rabbit Polyclonal to ZNF280C informed consents. Human PBMC were isolated from collected fresh blood of healthy donors by density gradient centrifugation using Ficoll-Paque PLUS (GE) as described (16, 24). For expansion assay, 0.5 million PBMCs were cultured in the absence or presence of 10 ng/mL of HMBPP (provided by Dr. H. Jomaa, Germany), with/without 5 ng/mL IL-2 (R&D) or 25 ng/mL IL-12 (Miltenyi Biotech) at 200 ul in 96-U-well plate. Fresh culture media (RPMI1640 + 10% FBS, purchased from Life Technologies) with indicated cytokines was added into cultures every 2C3 day. CD4- or CD8- depleted PBMC were prepared from freshly PBMC by sorting CD4 or CD8 T cells out using MACS method (Miltenyi). In proliferation assays, CD4-depleted, CD8-depleted or undeleted PBMCs were labeled with 2 M CFSE (Life Technology), washed out, then cultured with media, HMBPP, IL-12, or HMBPP + IL-12 for 7 days. Cells were harvested at day 7, and the proliferation of V2V2 T cells was analyzed by flow cytometry. In special assays, PBMCs were co-cultured with HMBPP + IL-12 or HMBPP +.