Tmem100-BAC-EGFP mice to selectively mark and purify embryonic endothelial cells of large caliber arteries in mid-gestational vascular formation.

Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.

Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.

Sema6D forward signaling impairs T cell activation and proliferation in head and neck cancer.

Immune checkpoint inhibitors (ICIs) are indicated for a diverse range of cancer types, and characterizing the tumor immune microenvironment is critical for optimizing therapeutic strategies, including ICIs. T cell infiltration and activation status in the tumor microenvironment greatly affects the efficacy of ICIs. Here, we show that semaphorin 6D (Sema6D) forward signaling, which is reportedly involved in coordinating the orientation of cell development and migration as a guidance factor, impaired the infiltration and activation of tumor-specific CD8+ T cells in murine oral tumors. Sema6D expressed by nonhematopoietic cells was responsible for this phenotype. Plexin-A4, a receptor for Sema6D, inhibited T cell infiltration and partially suppressed CD8+ T cell activation and proliferation induced by Sema6D stimulation. Moreover, mouse oral tumors, which are resistant to PD-1-blocking treatment in wild-type mice, showed a response to the treatment in Sema6d-KO mice. Finally, analyses of public data sets of human head and neck squamous cell carcinoma, pan-cancer cohorts, and a retrospective cohort study showed that SEMA6D was mainly expressed by nonhematopoietic cells such as cancer cells, and SEMA6D expression was significantly negatively correlated with CD8A, PDCD1, IFNG, and GZMB expression. Thus, targeting Sema6D forward signaling is a promising option for increasing ICI efficacy.

Tumor-derived semaphorin 4A improves PD-1-blocking antibody efficacy by enhancing CD8+ T cell cytotoxicity and proliferation.

Immune checkpoint inhibitors (ICIs) have caused revolutionary changes in cancer treatment, but low response rates remain a challenge. Semaphorin 4A (Sema4A) modulates the immune system through multiple mechanisms in mice, although the role of human Sema4A in the tumor microenvironment remains unclear. This study demonstrates that histologically Sema4A-positive non-small cell lung cancer (NSCLC) responded significantly better to anti-programmed cell death 1 (PD-1) antibody than Sema4A-negative NSCLC. Intriguingly, SEMA4A expression in human NSCLC was mainly derived from tumor cells and was associated with T cell activation. Sema4A promoted cytotoxicity and proliferation of tumor-specific CD8+ T cells without terminal exhaustion by enhancing mammalian target of rapamycin complex 1 and polyamine synthesis, which led to improved efficacy of PD-1 inhibitors in murine models. Improved T cell activation by recombinant Sema4A was also confirmed using isolated tumor-infiltrating T cells from patients with cancer. Thus, Sema4A might be a promising therapeutic target and biomarker for predicting and promoting ICI efficacy.

Bronchoalveolar lavage fluid reveals factors contributing to the efficacy of PD-1 blockade in lung cancer.

Bronchoalveolar lavage is commonly performed to assess inflammation and identify responsible pathogens in lung diseases. Findings from bronchoalveolar lavage might be used to evaluate the immune profile of the lung tumor microenvironment (TME). To investigate whether bronchoalveolar lavage fluid (BALF) analysis can help identify patients with non-small cell lung cancer (NSCLC) who respond to immune checkpoint inhibitors (ICIs), BALF and blood were prospectively collected before initiating nivolumab. The secreted molecules, microbiome, and cellular profiles based on BALF and blood analysis of 12 patients were compared with regard to therapeutic effect. Compared with ICI nonresponders, responders showed significantly higher CXCL9 levels and a greater diversity of the lung microbiome profile in BALF, along with a greater frequency of the CD56+ subset in blood T cells, whereas no significant difference in PD-L1 expression was found in tumor cells. Antibiotic treatment in a preclinical lung cancer model significantly decreased CXCL9 in the lung TME, resulting in reduced sensitivity to anti-PD-1 antibody, which was reversed by CXCL9 induction in tumor cells. Thus, CXCL9 might be associated with the lung TME microbiome, and the balance of CXCL9 and lung TME microbiome could contribute to nivolumab sensitivity in patients with NSCLC. BALF analysis can help predict the efficacy of ICIs when performed along with currently approved examinations.

Microfluidics sorting enables the isolation of an intact cellular pair complex of CD8+ T cells and antigen-presenting cells in a cognate antigen recognition-dependent manner.

Adaptive immune responses begin with cognate antigen presentation-dependent specific interaction between T cells and antigen-presenting cells. However, there have been limited reports on the isolation and analysis of these cellular complexes of T cell-antigen-presenting cell (T/APC). In this study, we successfully isolated intact antigen-specific cellular complexes of CD8+ T/APC by utilizing a microfluidics cell sorter. Using ovalbumin (OVA) model antigen and OT-I-derived OVA-specific CD8+ T cells, we analyzed the formation of antigen-specific and antigen-non-specific T/APC cellular complexes and revealed that the antigen-specific T/APC cellular complex was highly stable than the non-specific one, and that the intact antigen-specific T/APC complex can be retrieved as well as enriched using a microfluidics sorter, but not a conventional cell sorter. The single T/APC cellular complex obtained can be further analyzed for the sequences of T cell receptor Vα and Vß genes as well as cognate antigen information simultaneously. These results suggested that this approach can be applied for other antigen and CD8+ T cells of mice and possibly those of humans. We believe that this microfluidics sorting method of the T/APC complex will provide useful information for future T cell immunology research.

Development of combination adjuvant for efficient T cell and antibody response induction against protein antigen.

Most current clinical vaccines work primarily by inducing the production of neutralizing antibodies against pathogens. Vaccine adjuvants that efficiently induce T cell responses to protein antigens need to be developed. In this study, we developed a new combination adjuvant consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), D35, and an aluminum salt. Among the various combinations tested, the DOTAP/D35/aluminum salt adjuvant induced strong T cell and antibody responses against the model protein antigen with a single immunization. Adjuvant component and model antigen interaction studies in vitro also revealed that the strong mutual interactions among protein antigens and other components were one of the important factors for this efficient immune induction by the novel combination adjuvant. In addition, in vivo imaging of the antigen distribution suggested that the DOTAP component in the combination adjuvant formulation elicited transient antigen accumulation at the draining lymph nodes, possibly by antigen uptake DC migration. These results indicate the potential of the new combination adjuvant as a promising vaccine adjuvant candidate to treat infectious diseases and cancers.

Tumor microenvironment for cancer stem cells.

Tumor tissues consist of heterogeneous cancer cells including cancer stem cells (CSCs) that can terminally differentiate into cancer cells. Tissue-specific stem cells in normal organs maintain their stemness in a specific microenvironment, the stem cell niche; several studies have suggested that there are specific microenvironments that maintain CSCs in an immature phenotype. Cell types in a CSC niche vary from fibroblasts, to endothelial cells, immune cells, and so on; these non-cancer cells have been suggested to change their original features in the normal tissue/organ and to acquire a phenotype that protects CSCs from anticancer therapies. Therefore, to kill CSCs, we need to understand the cellular and molecular mechanisms involved in the maintenance of the immature phenotype of CSCs and in drug resistance.

Pharyngeal arch artery defects and lethal malformations of the aortic arch and its branches in mice deficient for the Hrt1/Hey1 transcription factor.

The aortic arch and major branch arteries are formed from the three pairs of pharyngeal arch arteries (PAAs) during embryonic development. Their morphological defects are clinically observed as isolated diseases, as a part of complicated cardiovascular anomalies or as a manifestation of multi-organ syndromes such as 22q11.2 deletion syndrome. Although numerous genes have been implicated in PAA formation and remodeling, detailed mechanisms remain poorly understood. Here we report that the mice null for Hrt1/Hey1, a gene encoding a downstream transcription factor of Notch and ALK1 signaling pathways, show perinatal lethality on the C57BL/6N, C57BL/6N × C57BL/6J or C57BL/6N × 129X1/SvJ background. Hrt1/Hey1 null embryos display abnormal development of the fourth PAA (PAA4), which results in congenital vascular defects including right-sided aortic arch, interruption of the aortic arch and aberrant origin of the right subclavian artery. Impaired vessel formation occurs randomly in PAA4 of Hrt1/Hey1 null embryos, which likely causes the variability of congenital malformations. Endothelial cells in PAA4 of null embryos differentiate normally but are structurally disorganized at embryonic day 10.5 and 11.5. Vascular smooth muscle cells are nearly absent in the structurally-defective PAA4, despite the appropriate migration of cardiac neural crest cells into the fourth pharyngeal arches. Endothelial expression of Jag1 is down-regulated in the structurally-defective PAA4 of null embryos, which may be one of the mechanisms underlying the suppression of vascular smooth muscle cell differentiation. While the direct downstream phenomena of the Hrt1/Hey1 deficiency remain to be clarified, we suggest that Hrt1/Hey1-dependent transcriptional regulation has an important role in PAA formation during embryonic development.

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps.

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.