Heterochronic development of pelvic fins in zebrafish: possible involvement of temporal regulation of pitx1 expression.

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3 weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8+ T cells against tumor.

BACKGROUND: A key to success of cancer immunotherapy is the amplification and sustenance of various effector cells. The hallmark of prominent antitumor T cells is their long-term effector function. Although interleukin (IL)-2 is an attractive cytokine, several attempts have been made towards developing IL-2 modalities with improved effectiveness and safety that enhance natural killer (NK) cells or T cells in cancer models. However, whether such IL-2 modalities can simultaneously support long-term innate and adaptive immunity, particularly stem-like memory, has not been shown. To resolve this issue, we compared the antitumor cellular mechanism with two IL-2/anti-IL-2 complexes (IL-2Cxs) administered in combination with a therapeutic cancer vaccine, which we had previously established as an in vivo dendritic cell-targeting therapy. METHODS: Two types of IL-2Cxs, CD25-biased IL-2Cx and CD122-biased IL-2Cx, together with a Wilms' tumor 1-expressing vaccine, were evaluated in a leukemic model. The immunological response and synergistic antitumor efficacy of these IL-2Cxs were then evaluated. RESULTS: When CD25-biased or CD122-biased IL-2Cxs in combination with the vaccine were assessed in an advanced-leukemia model, the CD122-biased IL-2Cx combination showed 100% survival, but the CD25-biased IL-2Cx did not. We first showed that invariant natural killer T (NKT) 1 cells are predominantly activated by CD122-biased IL-2Cx. In addition, in-depth analysis of immune responses by CD122-biased IL-2Cx in lymphoid tissues and the tumor microenvironment revealed a dramatic increase in the distinct subsets of NK and CD8+ T cells with stem-like phenotype (CD27+Sca-1hi, CXCR3hi, CD127+TCF-1+T-bet+ Eomes+). Moreover, CD122-biased IL-2Cx combination therapy maintained long-term memory CD8+ T cells capable of potent antitumor protection. After the high dimensional profiling analysis of NK and CD8+T cells, principal component analysis revealed that the stem-like-NK cell and stem-like-CD8+T cell state in the combination were integrated in the same group. CONCLUSIONS: CD122-biased IL-2Cx combined with the vaccine can induce a series of reactions in the immune cascade, including activation of not only NKT1 cells, but also NK and CD8+ T cells with a stem-like memory phenotype. Since it can also lead to a long-term, strong antitumor response, the combination of CD122-biased IL-2Cx with a vaccine may serve as a potential and competent strategy for patients with advanced cancer.

Natural Killer T and Natural Killer Cell-Based Immunotherapy Strategies Targeting Cancer.

Both natural killer T (NKT) and natural killer (NK) cells are innate cytotoxic lymphoid cells that produce inflammatory cytokines and chemokines, and their role in the innate immune response to tumors and microorganisms has been investigated. Especially, emerging evidence has revealed their status and function in the tumor microenvironment (TME) of tumor cells. Some bacteria producing NKT cell ligands have been identified to exert antitumor effects, even in the TME. By contrast, tumor-derived lipids or metabolites may reportedly suppress NKT and NK cells in situ. Since NKT and NK cells recognize stress-inducible molecules or inhibitory molecules on cancer cells, their status or function depends on the balance between inhibitory and activating receptor signals. As a recent strategy in cancer immunotherapy, the mobilization or restoration of endogenous NKT or NK cells by novel vaccines or therapies has become a focus of research. As a new biological evidence, after activation, effector memory-type NKT cells lasted in tumor-bearing models, and NK cell-based immune checkpoint inhibition potentiated the enhancement of NK cell cytotoxicity against cancer cells in preclinical and clinical trials. Furthermore, several new modalities based on the characteristics of NKT and NK cells, including artificial adjuvant vector cells, chimeric antigen receptor-expressing NK or NKT cell therapy, or their combination with immune checkpoint blockade have been developed. This review examines challenges and future directions for improving these therapies.

A single immunization with cellular vaccine confers dual protection against SARS-CoV-2 and cancer.

The efficacy of current coronavirus disease 2019 (COVID-19) vaccines has been demonstrated; however, emerging evidence suggests insufficient protection in certain immunocompromised cancer patients. We previously developed a cell-based anti-cancer vaccine platform involving artificial adjuvant vector cells (aAVCs) capable of inducing a strong adaptive response by enhancing the innate immunity. aAVCs are target antigen-transfected allogenic cells that simultaneously express the natural killer T-cell ligand-CD1d complex on their surface. In the present study, we applied this system for targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (CoV-2-S) using CoV-2-S-expressing aAVCs (aAVC-CoV-2) and evaluated the immune response in a murine model. A single dose of aAVC-CoV-2 induced a large amount of CoV-2-S-specific, multifunctional CTLs in addition to CD4+ T-cell-dependent anti-CoV-2-S-specific Abs. CoV-2-S-specific CTLs infiltrated the lung parenchyma and persisted as long-term memory T cells. Furthermore, we immunized mice with CoV-2-S- and tumor-associated antigen (TAA)-co-expressing aAVCs (aAVC-TAA/CoV-2) and evaluated whether the anti-SARS-CoV-2 and antitumor CTLs were elicited. We found that the aAVC-TAA/CoV-2-S therapy exerted apparent antitumor effects and induced CoV-2-S-specific CTLs. These findings suggest aAVC-TAA/CoV-2-S therapy as a promising vaccine candidate for preventing COVID-19, as well as enhancing the effectiveness of cancer therapies.

Cancer immunotherapy using artificial adjuvant vector cells to deliver NY-ESO-1 antigen to dendritic cells in situ.

NY-ESO-1 is a cancer/testis antigen expressed in various cancer types. However, the induction of NY-ESO-1-specific CTLs through vaccines is somewhat difficult. Thus, we developed a new type of artificial adjuvant vector cell (aAVC-NY-ESO-1) expressing a CD1d-NKT cell ligand complex and a tumor-associated antigen, NY-ESO-1. First, we determined the activation of invariant natural killer T (iNKT) and natural killer (NK) cell responses by aAVC-NY-ESO-1. We then showed that the NY-ESO-1-specific CTL response was successfully elicited through aAVC-NY-ESO-1 therapy. After injection of aAVC-NY-ESO-1, we found that dendritic cells (DCs) in situ expressed high levels of costimulatory molecules and produced interleukn-12 (IL-12), indicating that DCs undergo maturation in vivo. Furthermore, the NY-ESO-1 antigen from aAVC-NY-ESO-1 was delivered to the DCs in vivo, and it was presented on MHC class I molecules. The cross-presentation of the NY-ESO-1 antigen was absent in conventional DC-deficient mice, suggesting a host DC-mediated CTL response. Thus, this strategy helps generate sufficient CD8+ NY-ESO-1-specific CTLs along with iNKT and NK cell activation, resulting in a strong antitumor effect. Furthermore, we established a human DC-transferred NOD/Shi-scid/IL-2γcnull immunodeficient mouse model and showed that the NY-ESO-1 antigen from aAVC-NY-ESO-1 was cross-presented to antigen-specific CTLs through human DCs. Taken together, these data suggest that aAVC-NY-ESO-1 has potential for harnessing innate and adaptive immunity against NY-ESO-1-expressing malignancies.

Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2.

SARS-CoV-2-specific CD8+ T cells are scarce but detectable in unexposed healthy donors (UHDs). It remains unclear whether pre-existing human coronavirus (HCoV)-specific CD8+ T cells are converted to functionally competent T cells cross-reactive to SARS-CoV-2. Here, we identified the HLA-A24-high binding, immunodominant epitopes in SARS-CoV-2 spike region that can be recognized by seasonal coronavirus-specific CD8+ T cells from HLA-A24+ UHDs. Cross-reactive CD8+ T cells were clearly reduced in patients with hematological malignancy, who are usually immunosuppressed, compared to those in UHDs. Furthermore, we showed that CD8+ T cells in response to a selected dominant epitope display multifunctionality and cross-functionality across HCoVs in HLA-A24+ donors. Cross-reactivity of T-cell receptors isolated from them exhibited selective diversity at the single-cell level. Taken together, when stimulated well by immunodominant epitopes, selective pre-existing CD8+ T cells with high functional avidity may be cross-reactive against SARS-CoV-2.

PD-L1 Expression Affects Neoantigen Presentation.

Although PD-L1 expression on tumor is related to the prognosis of immune checkpoint blockade (ICB) therapy, a recent study also demonstrated clinical benefits even in patients without PD-L1 expression. To understand the relationship between innate resistance and antitumor cytotoxic T lymphocyte (CTL) responses especially against neoantigens, the interaction between PD-L1+ or genetically PD-L1-deleted colorectal tumors and CTLs was assessed under an ICB therapy, finding the robust CTL activation in PD-L1-deleted tumor-bearing mice. Using antigen libraries based on immunogenomics, we identified three H2-Kb-restricted, somatic-mutated immunogenic neoantigens by utilizing enhanced CTLs responses due to PD-L1 deficiency. Furthermore, we identified three T cell receptor (TCR) repertoires relevant to the neoantigens, confirming the response of TCR-gene-transduced CTLs to parental tumor cells. Notably, neoantigen-pulsed dendritic cell (DC) therapy reversed the tumor tolerance. Thus, innate resistance of tumors determines their responsiveness to neoantigens and mixed neoantigen peptides may be useful in DC therapy against innate resistance type tumor.

Evolution of the avian digital pattern.

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Cux2 refines the forelimb field by controlling expression of Raldh2 and Hox genes.

In vertebrates, two pairs of buds that give rise to the fore- and hindlimbs form at discrete positions along the rostral-caudal axis of the body. The mechanism responsible for the positioning of the limb buds is still largely unknown. Here we show a novel function for Cut homeobox transcription factor 2 (Cux2), the ortholog of Drosophila cut, in refining the forelimb field during chick development. Cux2 is expressed in the forelimb field before the emergence of the limb buds. Knocking down the expression of Cux2 using small interfering RNA (siRNA) resulted in a caudal shift of the forelimb bud, whereas misexpression of Cux2 or the constitutively active Cux2-VP16 caused a rostral shift of the forelimb bud or reduction of the forelimb field along the anterior-posterior axis. Further functional analyses revealed that expression of Hoxb genes and retinaldehyde dehydrogenase 2 (Raldh2), which are involved in limb positioning, are directly activated by Cux2 in the lateral plate mesoderm. Our data suggest that Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes in chicken embryos.

Localization of ß-Catenin and Islet in the Pelvic Fin Field in Zebrafish.

In zebrafish, pelvic fin buds appear at 3 weeks post fertilization (wpf) during the larval to juvenile transition (metamorphosis), but their fate is already determined during embryogenesis. Thus, presumptive pelvic fin cells appear to memorize their positional information for three weeks, but no factors expressed in the pelvic fin field from the embryonic to the metamorphic stages have been identified. In mice, Islet1 is proposed to promote nuclear accumulation of ß-catenin in the hindlimb field, which leads to the initiation of hindlimb bud outgrowth through activation of the Wnt/ßcatenin pathway. Here, we examined the distribution of ß-catenin and islet proteins in the pelvic fin field of zebrafish from the embryonic to the metamorphic stages. We found that transcripts of islet2a, but not islet1, are detected in the posterior lateral plate mesoderm, including the presumptive pelvic fin field, at the embryonic stage as well as in the pelvic fin bud at the metamorphic stage. Immunolocalization revealed that ß-catenin and islet proteins, which are synthesized during the embryonic stage, remain in the cytoplasm of the presumptive pelvic fin cells during the larval stage, and are then translocated into the nuclei of the pelvic fin bud at the metamorphic stage. We propose that cytoplasmic localization of these proteins in the presumptive pelvic fin cells that remained during the larval stage may underlie the mechanism by which pelvic fin cells memorize their positional information from the embryonic stage to the metamorphic stage.

Transgene Introduction into the Chick Limb Bud by Electroporation.

Electroporation enables delivering bionanomolecules, such as DNAs, RNAs, siRNAs, and morpholinos, into chick embryos in a spatially and temporally restricted fashion. Recent advances in electroporation techniques allowed us to deliver transgenes into the restricted area of the limb bud and to analyze the function of the enhancers in the limb field. Here, we describe the introduction of transgenes by electroporation in the limb field and its application on enhancer analysis.

Purification and biochemical characterization of a novel ecto-apyrase, MP67, from Mimosa pudica.

We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.

[Paraurethral leiomyoma in women].

We experienced a case of paraurethral leiomyoma in a 36-year-old woman. She had been complaining of dysuria. She had noticed the presence of a mass in anterior vagina. That mass was about 3 x 3 cm, floating moderately firm with a smooth face sited in a paraurethral region. Magnetic resonance imaging showed medium-signal intensity on T1-weighted images and a homogeneous low signal on T2-weighted images. We diagnosed it as a paraurethral leiomyoma and enucleated it by surgery. At histological examination the tumor resulted in being a leiomyoma. The diagnosis and the treatment of female paraurethral leiomyoma are discussed.