Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice.

BACKGROUND: Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, the immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. METHODS: We transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL-2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. RESULTS: The human immune cells were engrafted for more than 3 months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+ T cell responses in vitro. hiPS-CM sheets disappeared approximately 17 to 24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. CONCLUSIONS: hiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease.

BACKGROUND: The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA. METHODS AND RESULTS: We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction. CONCLUSIONS: The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.

PU.1 and IRF8 Modulate Activation of NLRP3 Inflammasome via Regulating Its Expression in Human Macrophages.

NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1ß and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1ß in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.