Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells.

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity.

The ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could serve as a universal measure to overcome platelet transfusion refractoriness caused by HLA-I incompatibility. Here, we developed human induced pluripotent cell-derived HLA-I-deficient platelets (HLA-KO iPLATs) in a clinically applicable imMKCL system by genetic manipulation and assessed their immunogenic properties including natural killer (NK) cells, which reject HLA-I downregulated cells. HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in our newly established Hu-NK-MSTRG mice reconstituted with human NK cells. Additionally, HLA-KO iPLATs successfully circulated in an alloimmune platelet transfusion refractoriness model of Hu-NK-MISTRG mice. Mechanistically, the lack of NK cell-activating ligands on platelets may be responsible for evading the NK cell response. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.

Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Orally administered rutin inhibits the gene expression of Th2 cytokines in the gut and lung in aged mice.

Rutin is one of the flavonoids derived from plants such as buckwheat and is well known as a powerful antioxidant. To determine whether dietary rutin could modulate mucosal immunity, we examined the gene expression of Th1/Th2 cytokines and the receptors in the gut and lung. Aged (18 months old, 18 M) C3H/HeN female mice were orally administered rutin for 10 days. The small intestine and lung were taken and analyzed by real-time PCR for gene expression. Interleukin (IL)-13 and IL-13Rα2 gene expression was significantly low (P<0.05 respectively) in the small intestine of aged rutin-fed mice. Meanwhile, there was no change in interferon γ gene expression between control and rutin-fed mice. IL-13 gene expression was also downregulated in the lung. To examine the mechanism of the inhibitory effect of rutin on Th2 cytokines in aged mice, intestinal nitric oxide synthase (NOS) expression was evaluated. Rutin inhibited inducible NOS (NOS2) gene expression, but not neuronal NOS and endothelial NOS. Gene analysis of cells collected from the small intestine by laser capture dissection revealed that NOS2 expression was significantly inhibited in crypt regions. Thus, rutin might be effective against a Th2-dominant profile through NOS2 inhibition in aged mice.

Combined administration of (L)-cystine and (L)-theanine enhances immune functions and protects against influenza virus infection in aged mice.

Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of (L)-cystine and (L)-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of (L)-cystine and (L)-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of (L)-cystine and (L)-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of (L)-cystine and (L)-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-gamma ratio and gamma-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of (L)-cystine and (L)-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.

Cocirculation of antigenic variants and the vaccine-type virus during the 2004-2005 influenza B virus epidemics in Japan.

In the 2004-2005 season, there was a large epidemic of the influenza B virus Yamagata group in Kobe, Japan. In hemagglutination inhibition tests, most of the clinical isolates from Kobe showed antigenicities similar to those of previous isolates (the vaccine-type virus). Only a few antigenic variants were isolated around the peak of the epidemic; however, Kobe residents developed antibodies against the variants during the season. The antigenic variants showed a one-point mutation of a nucleotide in the HA1 gene (C440A or G421A), which resulted in the substitution of one amino acid in the 150 loop of the HA molecule (T147N or G141R). The 150 loop is one of four epitopes of the hemagglutinin molecule of the influenza B virus. We established a system to detect one-point differences in the nucleotides of the 150 loop by means of high-resolution melting curve analysis with LCGreen. With this system, the isolates were determined to be the vaccine-type virus, antigenic variants, or a mixture of both. Some isolates were shown to be mixtures although they had been recognized as the vaccine-type virus with the hemagglutination inhibition tests. Thus, the antigenic variants appeared in the early period of the epidemic and were cocirculating with the vaccine-type virus during the epidemic.

Detection of antigenic variants of the influenza B virus by melting curve analysis with LCGreen.

Automated, high-throughput detection methods for single-nucleotide polymorphisms have been applied to the routine genotyping of genetic polymorphisms influencing drug metabolism. Melting curve analysis with LCGreen was introduced recently as one such technique which can be performed rapidly and easily. This technique was used to detect antigenic variants of the influenza B virus. The antigenic variants and vaccine-type strains of the influenza B virus are isolated from clinical specimens of one epidemic season, and they usually differ in one nucleotide in the HA1 gene, corresponding to one amino-acid substitution. By means of melting curve analysis with LCGreen, an antigenic variant clone and a vaccine-type clone were clearly distinguished. In addition, the proportions of the antigenic variants in the mixture-type isolates were estimated. The clinical isolates were detected as the vaccine-type strains, antigenic variants, or a mixture of both. It became clear that humans were infected with a mixture of the vaccine-type strains and the antigenic variants for a certain period after which the viral antigenicities vary. This technique will contribute to the analysis of antigenic shifts in influenza B virus.