In-depth analysis of human virus-specific CD8+ T cells delineates unique phenotypic signatures for T cell specificity prediction.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Antitumor effects of CD40 ligand-expressing endothelial progenitor cells derived from human induced pluripotent stem cells in a metastatic breast cancer model.

Given their intrinsic ability to home to tumor sites, endothelial progenitor cells (EPCs) are attractive as cellular vehicles for targeted cancer gene therapy. However, collecting sufficient EPCs is one of the challenging issues critical for effective clinical translation of this new approach. In this study, we sought to explore whether human induced pluripotent stem (iPS) cells could be used as a reliable and accessible cell source to generate human EPCs suitable for cancer treatment. We used an embryoid body formation method to derive CD133(+)CD34(+) EPCs from human iPS cells. The generated EPCs expressed endothelial markers such as CD31, Flk1, and vascular endothelial-cadherin without expression of the CD45 hematopoietic marker. After intravenous injection, the iPS cell-derived EPCs migrated toward orthotopic and lung metastatic tumors in the mouse 4T1 breast cancer model but did not promote tumor growth and metastasis. To investigate their therapeutic potential, the EPCs were transduced with baculovirus encoding the potent T cell costimulatory molecule CD40 ligand. The systemic injection of the CD40 ligand-expressing EPCs stimulated the secretion of both tumor necrosis factor-α and interferon-γ and increased the caspase 3/7 activity in the lungs with metastatic tumors, leading to prolonged survival of the tumor bearing mice. Therefore, our findings suggest that human iPS cell-derived EPCs have the potential to serve as tumor-targeted cellular vehicles for anticancer gene therapy.

Induced pluripotent stem cell-derived neural stem cells transduced with baculovirus encoding CD40 ligand for immunogene therapy in mouse models of breast cancer.

The interaction between CD40 ligand (CD40L) and CD40 can directly inhibit growth of CD40-positive carcinoma cells and may indirectly inhibit tumor growth through coordination of immune responses. Many efforts in CD40L cancer gene therapy have been focused on direct CD40L gene transfer into malignant target cells. This in vivo gene therapy approach relies on high-efficiency gene transfer and could be technically challenging for the treatment of certain cancers, especially multisite metastases. We report herein an alternative means of using the tumor-homing property of neural stem cells (NSCs) to deliver CD40L molecules into tumor tissues. NSCs were derived from human induced pluripotent stem cells, transduced in vitro with a baculoviral vector encoding CD40L, and intravenously injected into immunocompetent mice with orthotopic and metastatic breast cancers. Through a bystander mechanism of intercellular transfer of CD40L from the donor NSCs to tumor target cells, the treatment impeded tumor growth, leading to prolonged survival of the tumor-bearing mice. We further showed that compared with the stem cell-based gene therapy that employed a suicide gene, the CD40L immunogene therapy did not cause liver and kidney injury in the treated mice. This new approach may be particularly valuable for metastatic cancer treatments after systemic stem cell administration.

Systemic delivery of fusogenic membrane glycoprotein-expressing neural stem cells to selectively kill tumor cells.

Intravenously injected neural stem cells (NSCs) can infiltrate both primary and metastatic tumor sites; thus, they are attractive tumor-targeting vehicles for delivering anticancer agents. However, because the systemic distribution of the injected NSCs involves normal organs and might induce off-target actions leading to unintended side effects, clinical applications of this approach is impeded. Given that the vesicular stomatitis virus glycoprotein (VSV-G) can promote the formation of multinucleated syncytia to kill cells in a pH-dependent manner, we engineered a pH sensor of VSV-G and generated a novel VSV-G mutant that efficiently promotes syncytium formation at the tumor extracellular pH (pHe) but not at pH 7.4. Using transduced NSCs derived from induced pluripotent stem cells (iPSCs), the VSV-G mutant was delivered into mice with metastatic breast cancers in the lung through tail vein injection. Compared with the conventional stem cell-based gene therapy that uses the herpes simplex virus thymidine kinase (HSVtk) suicide gene, this treatment did not display toxicity to normal non-targeted organs while retaining therapeutic effects in tumor-bearing organs. Our findings demonstrate the effectiveness of a new approach for achieving tumor-selective killing effects following systemic stem cell administration. Its potential in stem cell-based gene therapy for metastatic cancer is worthy of further exploration.

Inhibitory effects of neural stem cells derived from human embryonic stem cells on differentiation and function of monocyte-derived dendritic cells.

Neural stem cells (NSCs) possess immunosuppressive characteristics, but effects of NSCs on human dendritic cells (DCs), the most important antigen presenting cells, are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14(+) monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line, as well as human bone marrow derived mesenchymal stem cells (MSCs), were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14(+) monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a, whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However, when effects on the function of derived DCs were investigated, NSCs suppressed the elevation of the DC maturation marker CD83, although not the up-regulation of costimulatory molecules CD80, CD86 and CD40, and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs, these stem cells can still affect the function of DCs, ultimately regulating specific immune responses.

Inhibition of neuronal nitric oxide synthase activity promotes migration of human-induced pluripotent stem cell-derived neural stem cells toward cancer cells.

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells, and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy.

Mitf is a transcriptional activator of medaka germ genes in culture.

Germ cells express a unique subset of genes called germ genes mostly encoding RNA-binding proteins such as Dazl, Dnd and Vasa. How germ gene expression is controlled remains illusive, because in no organism has a transcription factor been identified that regulate expression of these genes. Microphthalmia-associated transcription factor (Mitf) has been reported to show expression in male mouse germ cells of the adult testis. Here we report in the fish medaka (Oryzias latipes) that Mitf is a transcription activator of germ gene expression. Mitf is a master regulator of melanocyte development, which activates melanogenic genes through binding to the E-box containing consensus CANNTG. The E-box was found to be present in 23-26 copies in the promoters of medaka germ genes dazl, dnd and vasa. Importantly, forced Mitf expression enhanced the transcriptional activity of the three gene promoters by up to more than 10 fold and remarkably increased the level of endogenous dazl, dnd and vasa transcripts in cell culture. Transfection of Mitf expression vectors was sufficient to induce directed differentiation of medaka embryonic stem cells into melanocytes. Fluorescence in situ hybridization revealed the expression of both medaka mitf genes in adult germ cells of male and female gonads. Mitf is well-known as the melanocyte master regulator. Our results offer first evidence that Mitf may act as a transcriptional activator of germ gene expression in medaka.

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells.

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.