These findings suggest the chance that circulating TN cells differentiate into T1 cells in the cells. Unlike MAIT cells, we showed that later exposure of microbial stimulation is sufficient for peripheral expansion and maintenance of T cells. cells. Upon PM exposure, IL-1-secreting neutrophils and IL-17-producing T17 cells attract each other around the airways. Accordingly, PM-induced neutrophilia was significantly relieved in T- or IL-17-deficient and germ-free mice. Collectively, these findings show that EPHB4 the commensal microbiome promotes PM-induced neutrophilia in the lung T17 cells. (B6.129P2-(B6) mice were received from Dr. Charles D. Surh (POSTECH, Korea). CD45.1/2 B6 mice were received from Dr. Sin-Hyeog Im (POSTECH, Korea). All mice were used at the age of 6-12 weeks unless indicated, and age- and sex-matched animals were used as controls. Germ-free (GF) mice were bred and used as previously described (24). All mouse experiments were performed using protocols approved by the Institutional Animal Care and Use Committees (IACUC) of the POSTECH. PM-Induced Pulmonary Inflammation RGFP966 and Treatments Mice were intranasally administered with 250 g of particulate matter (PM) in saline or saline alone as controls. PM was obtained from Sigma (PM10-like ERMCZ100-1VL and ERMCZ120-1VL) and used as a 1:1 mixture. All intranasal administration (20 l/nostril) was performed under anesthesia (i.p.) with ketamine (Yuhan)/xylazine (Rompun, BAYER) solution, as described (21). Mouse Models of Chronic HDM/PM-Induced Allergic Asthma We used a previously described house dust mite (HDM)-induced mouse asthma model with minor modification (25). HDM (the intranasal route with 250 g of PM. Parabiosis Five-week-old B6 CD45.1/2 and CD45.2/2 female mice were joined together by parabiosis for 2 RGFP966 or 7 weeks, as previously described (11). Weight-matched mice were anesthetized and shaved. An incision was made along the side of each mouse and the skin was connected using surgical clips. Flow Cytometry and Antibodies Single-cell suspensions were isolated and stained with fluorescein-conjugated antibodies. For cytokine detection experiments, lymphocytes were stimulated with RGFP966 Cell Stimulation Cocktail and protein transport inhibitors (eBioscience) for 4 hours. Cells were washed twice in FACS buffer and stained with surface markers for 30?min at 4C. For intracellular staining, single-cell suspensions were surface-stained, RGFP966 fixed, and permeabilized with the eBioscience Foxp3 staining buffer set. Following antibodies were used; anti-CD4-BUV395 (BD, GK1.5), anti-CD8-BV650 (BD, 53-6.7), anti-SiglecF-PE (BD, E50-2440), anti-CD11b-PerCP-Cy5.5 (BD, M1/70), anti-CD11c-PE-Cyanine7 (eBioscience, N418), anti-TCR-PE-CF594 (BD, H57-597), anti-Ly6G-APCCy7 (BD, IA8), anti-CD45.2-BV605 (BD, 104), anti-B220-BV711 (BD, RA3-6B2), anti-CD8-BV510 (BD, 53-6.7), anti-CD44-redFluor 710 (TONBO, IM7), RGFP966 anti-TCR-APCCy7 (BD, H57-597), anti-CD11c-BV650 (BD, HL3), anti-Thy1.2-BV786 (BD, 53-2.1), anti-GL3-BV421 (BD, GL3), anti-CD45.2-BV650 (Biolegend, 104), anti-CD11c-BV711 (BD, HL3), anti-GATA3-PE (ebioscience, TWAJ), anti-Tbet-PE-Cyanine7 (eBioscience, 4B10), anti-ROR-PE-CF594 (BD, Q31-378), anti-PLZF- Alexa Fluor 647 (BD, R17-809), Zombie-Aqua (Biolegend), anti-RORt- PerCP-Cy5.5 (BD, Q31-378), anti-IL-17A-BV650 (BD, TC11-18H10), anti-IFN-BV786 (BD, XMG1.2), anti-CD11b-BV711 (BD, M1/70), anti- IFN-PE (Invitrogen, XMG1.2), anti-proIL-1- PE-Cyanine7 (Invitrogen, NJTEN3), anti-CD11c-APC (Invitrogen, N418), anti- CD121a (IL-1R, Type I/p80)-PE (Biolegend, JAMA-147), anti-IFN-BV421 (BD, XMG1.2), anti-GL3- PE-CF594 (BD, GL3), anti-CD24-BV605 (Biolegend, M1/69), anti-?V2 TCR-BV786 (BD, UC3-10A6), anti-CD23p19-Alexa Fluor 488 (Invitrogen, fc23cpg), anti-Ki-67- PerCP-eFluor 710 (Invitrogen, SolA15), anti-GL3-PE (BD, GL3), anti-IL-17F- Alexa Fluor 488 (Biolegend, 9D3.1C8), anti-V1.1 TCR-BV421 (BD, 2.11), anti- V1.1+ V1.2 TCR-PE (Biolegend, 4B2.9), anti-V3 TCR-BV510 (BD, 536), anti-CD45.1- Pacific Blue (Biolegend, A20), anti-CD3-APCCy7 (BD, 145-2C11), anti-V6.3/2 TCR (BD, 8F4H7B7). 17D1 hybridoma (anti-V6 antibody) and biotinylated anti-V7 antibody was used as previously described (21). Cells were analyzed on an LSR (Foretessa BD Biosciences) and data were processed using FlowJo software (Tree Star). Tetramers and Cell Enrichment Biotinylated PBS57 loaded CD1d monomers and 5-OP-RU loaded MR1 monomers were obtained from the tetramer facility of the US National Institutes of Health (NIH). Biotinylated monomers were tetramerized using streptavidin-phycoerythrin (PE) (Prozyme), streptavidin-allophycocyanin (APC) (Prozyme), and streptavidin-PE-Cy7 (BD). For simultaneous enrichment of NKT, MAIT, and T cells, single cell suspensions of lung were stained with PBS57-CD1d PE-Cy7, 5-OP-RU-MR1 PE, and anti-TCR (GL3) PE-TR and enriched with anti-PE microbeads (Miltenyi) according to the manufacturers instructions. Cell Preparation Mice were sacrificed at the indicated time points and BAL fluids were collected in 1 mL PBS. To remove circulating cells, 15?ml of PBS was injected into the heart after incision of the abdominal aorta. Harvested lung tissues were minced by McIlwain Tissue Chopper and digested in 5 mL of RPMI-1640 containing collagenase D (400 Mandl Units; ROCHE) and DNase.
