Genes and immunity. system akin to CTL cells. c-Met inhibitor 2 Overall, the gene manifestation signature of CCR6+CXCR3+CCR4? cells reveals characteristics important for controlling latent TB infections. Intro Latent tuberculosis illness (LTBI) is characterized by an often life-long containment of mycobacteria to granuloma in the lung that is mediated at least in part by IFN generating CD4+ T cells (1). We recently performed a MAP3K3 genome-wide display for epitopes of TB-specific CD4+ T cells (2). Phenotypic characterization of T cells responding to TB-specific epitopes showed that they were amazingly homogenous with more than 80% showing a CCR6+CXCR3+CCR4? phenotype (2). This T cell subset was individually explained by others to be enriched for TB-specific cells (3, 4). These c-Met inhibitor 2 cells have previously been termed Th1 co-expressing CCR6, Th17.1, Th1Th17, Th17/Th1 and Th1/17 cells (2, 5-9), mainly because they were shown to express both T-bet and RORC (3, 6, 10), the lineage-specific transcription factors of Th1 and Th17 cells, respectively. Yet how these cells differ from standard Th1 and Th17 cells has not been comprehensively characterized. Two observations support that TB-specific T cells with this phenotype (CCR6+CXCR3+CCR4?) contribute to the containment of TB in LTBI: First, CCR6+CXCR3+CCR4? cells are a favored target of HIV computer virus illness, and were shown to be diminished in chronically HIV infected individuals (6). The high rate of TB reactivation in HIV individuals could thus be a consequence of the reduction in this T cell subset. Second, we as well as others have shown that TB-specific T cells in LTBI donors are multifunctional and are major suppliers of TNF in addition to IFN (2, 11). This same phenotype has been explained for T cells in rheumatoid arthritis (5, 7), which is definitely treated with TNF blockers, which in turn has been associated with reactivation of TB (12, 13). Here, we set out to better characterize the CCR6+CXCR3+CCR4? T cell subset. We find that the rate of recurrence of CCR6+CXCR3+CCR4? cells is definitely amazingly expanded in LTBI donors compared to healthy control (HC) donors, and that these cells produce IFN, TNF, IL-2 but no IL-17 upon activation with TB derived epitopes. The transcriptional system in TB-specific T cells significantly overlaps with the general CCR6+CXCR3+CCR4? subsets of both LTBI and HC. In addition, we find a unique system of genes, indicated at significantly higher or lower levels in CCR6+CXCR3+CCR4? cells compared to both Th1 and Th17 cells, suggesting that these cells have functional characteristic unique from either Th1 or Th17 cells. These characteristics are consistent with a multi-functional hyper-activated response system that is persistently maintained and could be required to control latent TB illness. MATERIALS AND METHODS Study Subjects Leukapheresis samples from 12 adults with LTBI and 12 control donors were from the University c-Met inhibitor 2 or college of California, San Diego Antiviral Research Center clinic (age range 20-65 years). Subjects had a history of a positive tuberculin pores and skin test (TST). LTBI was confirmed by a positive QuantiFERON-TB Platinum In-Tube (Cellestis), as well as a physical examination and/or chest X-ray that was not consistent with active tuberculosis. None of them of the study subjects endorsed vaccination with BCG, or experienced laboratory evidence of HIV or Hepatitis B. The control donors experienced a negative TST, as well as a bad QuantiFERON-TB. Research carried out for this study was performed in accordance with approvals from your Institutional Review Table in the La Jolla Institute for Allergy and Immunology (FWA#00000032). All participants offered written educated consent prior to participation in the study. PBMC Isolation PBMCs were obtained by denseness gradient centrifugation (Ficoll-Hypaque, Amersham Biosciences) from 100 ml of leukapheresis sample, according to manufacturers instructions. Cell were suspended in fetal bovine serum (Gemini Bio-products) comprising 10% dimethyl sulfoxide, and cryo-preserved in liquid nitrogen. Isolation of cells and FACS analysis HLA class II tetramers conjugated using PE labeled streptavidin were provided by the Tetramer Core Laboratory at Benaroya Study Institute. CD4 T cells were purified using the Miltenyi T cell isolation kit II relating to manufacturers instructions. Purified cells were incubated in PBS comprising 0.5% BSA and 2 mM EDTA pH 8.0 (MACS buffer) having a dilution of class II tetramer (10l tetramer per 50106 CD4 T cells) for 2 h at space temperature. Cells were then stained for cell surface antigens using anti-CD4-APC EFluor780 (RPA-T4), anti-CD45RA-EFluor450 (HI100) (both from Affymetrix eBioscience), anti-CD3-Alexa Fluor.
Genes and immunity