(D) Mean SEM percent proliferation of gated H-2Kb+CD4+Foxp3+ nTreg at 72 hours following co-culture with sorted WT Gr-1lowCD11c+ cells with or without specific treatments

(D) Mean SEM percent proliferation of gated H-2Kb+CD4+Foxp3+ nTreg at 72 hours following co-culture with sorted WT Gr-1lowCD11c+ cells with or without specific treatments. and iNKT cell-deficient J18?/? BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b+ cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1lowCD11c+ cells to J18?/? BALB/c recipients of TLI/ATS + BMT restored day time 6 donor Foxp3+ nTreg proliferation and safety from CD8 effector T cell-mediated GVHD. Blockade of PD-L1 or PD-L2, but not CD40, TGF-, Arginase 1, or iNOS inhibited nTreg proliferation in co-cultures of recipient-derived Gr-1lowCD11c+ cells with donor nTreg. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory DCs are expanded after non-myeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance. development of donor-type naturally occurring regulatory CD4+CD25+Foxp3+ cells (nTreg) (11). nTreg expanded then regulate the donor effector CD8+ T-cell driven lethal acute GVHD seen TPA 023 when identical transplants are performed into standard total body irradiation (TBI)-conditioned recipients. Our earlier studies founded that TLI/ATS results in post-BMT development of Foxp3+ nTreg and not merely peripheral development of induced Treg (iTreg), as CD25-depletion of the graft prior to BMT was confirmed at day time 6 to result in loss of all expanding CD4+Foxp3+ cells at day time 6 after BMT (11). Although earlier publications suggested that IL-4-driven STAT6 signaling could down-regulate gene manifestation in induced Treg (12,13), more recent TPA 023 publications support our findings by demonstrating that GATA3 may actually stabilize Foxp3 protein manifestation in nTreg (14,15). We wanted to determine specific mechanisms by which recipient iNKT-derived IL-4 signaling could induce nTreg proliferation after TLI/ATS and allogeneic BMT. Defining the specific mechanism by which iNKT cells and Th2 polarizing conditioning in the recipient generate dono-type nTreg proliferation with this model would lay the foundation for future conditioning strategies designed to augment nTreg maintenance and development after allogeneic BMT. Here we demonstrate that the effect of recipient IL-4 on donor nTreg development early after TLI/ATS and BMT is not direct, but rather occurs via a essential recipient B220negCD11b+Gr-1lowCD11c+ regulatory dendritic cell (DC) subset fitted the immune phenotype of myeloid-derived immunomodulatory cells, maintenance and development of which after TLI/ATS + BMT is definitely Rabbit Polyclonal to TOP2A STAT6- and iNKT-dependent. Donor-type nTreg proliferation happens self-employed of common regulatory pathways explained in other CD11b+Gr-1low populations, including CD40/CD154 (CD40L), TGF- STAT6 signaling, Arginase 1 (Arg1), or inducible nitric oxide synthase (iNOS), but requires contact-dependent signaling through PD-1 ligands. These recipient DCs induce potent proliferation of donor-type nTreg cells with stable manifestation of Foxp3, and blockade of the PD-1 ligand axis using monoclonal antibody treatment of recipients abrogates donor nTreg cell development after TLI/ATS and allogeneic BMT. Our studies link for the first time this regulatory TNF- and iNOS-producing DC human population with development of Foxp3+ nTreg both and and determine a novel means by which non-myeloablative Th2-polarizing recipient conditioning may preserve durable donor-recipient immune tolerance after allogeneic BMT. Materials and Methods Mice Wild-type (WT) (CD45.2+), CD45 congenic (CD45.1+), Arginase-1flox/flox (ARG1lipopolysaccharide (LPS) (“type”:”entrez-nucleotide”,”attrs”:”text”:”L26390″,”term_id”:”432297″,”term_text”:”L26390″L26390, Sigma-Aldrich) for 72 hrs. Supernatant cytokine concentrations were analyzed using the mouse Milliplex? MAP (Millipore). For assays of intracellular cytokine manifestation by FACS, the above sorted cell populations were stimulated for 12 hours with 1 ug/mL LPS with GolgiPlug? (BD Biosciences) added TPA 023 after 7 h of tradition. Cells were fixed, permeabilized (Fixation/Permeabilization kit, eBioscience) and stained with unlabeled rabbit iNOS (clone M-19, Santa Cruz TPA 023 Biotechnologies) and PE conjugated anti-rabbit IgG (Southern Biotech) and FITC conjugated TNF- (clone MP6-XT22, BD Biosystems). Light microscopy Sorted CD11b+ human population subsets were stained for morphological assessment using Protocol Hema 3 Giemsa Stain (Fisher Healthcare, Thermo Fisher Scientific, Waltham, MA) according to the manufacturers protocol. Photomicrographs were aquired having a 100 Strategy APO 1.4/NA lens and a Nikon DXM 1200 camera. TPA 023 Images were prepared using NIS Elements AR software (NIKON Tools, Melville, NY). In vivo Gr-1+ cell depletion Recipient BALB/c mice were conditioned with TLI and ATS. Antibody clone RB6-8C5 (18) (BioXCell, Western Lebanon, NH) or isotype bad control antibody (Rat IgG2b, BioXcell) was diluted in PBS to a final concentration of 200 g/ml, and recipient mice injected intraperitoneally with 500 l (100 g/dose/mouse) on days ?10, ?8, ?6, and ?4 prior to BMT with WT C57BL/6 bone marrow cells (50 106) and spleen cells (60 106) injected via lateral tail vein on day time 0. On day time 6 after BMT, recipients were euthanized and cells.