牛インターロイキン-2持続発現HEK-293細胞の樹立及びその免疫学的解析と応用

宮崎大学博士（獣医学）2020年度doctoral thesi

A pooled testing system to rapidly identify cattle carrying the elite controller BoLA‐DRB3*009:02 haplotype against bovine leukemia virus infection

As genetically resistant individuals, the “elite controllers” (ECs) of human immunodeficiency virus infection have been focused on as the keys to developing further functional treatments in medicine. In the livestock production field, identifying the ECs of bovine leukemia virus (BLV) infection in cattle is desired to stop BLV transmission chains on farms. Cattle carrying the bovine leukocyte antigen (BoLA)-DRB3*009:02 allele (DRB3*009:02) have a strong possibility of being BLV ECs. Most of cattle carrying this allele maintain undetectable BLV proviral loads and do not shed virus even when infected. BLV ECs can act as transmission barriers when placed between uninfected and infected cattle in a barn. To identify cattle carrying DRB3*009:02 in large populations more easily, we developed a pooled testing system. It employs a highly sensitive, specific real-time PCR assay and TaqMan MGB probes (DRB3*009:02-TaqMan assay). Using this system, we determined the percentage of DRB3*009:02-carrying cattle on Kyushu Island, Japan. Our pooled testing system detected cattle carrying the DRB3*009:02 allele from a DNA pool containing one DRB3*009:02-positive animal and 29 cattle with other alleles. Its capacity is sufficient for herd-level screening for DRB3*009:02-carrying cattle. The DRB3*009:02-TaqMan assay showed high-discriminative sensitivity and specificity toward DRB3*009:02, making it suitable for identifying DRB3*009:02-carrying cattle in post-screening tests on individuals. We determined that the percentage of DRB3*009:02-carrying cattle in Kyushu Island was 10.56%. With its ease of use and reliable detection, this new method strengthens the laboratory typing for DRB3*009:02-carrying cattle. Thus, our findings support the use of BLV ECs in the field.Citation: Notsu K, El Daous H, Mitoma S, Norimine J, Sekiguchi S. A pooled testing system to rapidly identify cattle carrying the elite controller BoLA-DRB3*009:02 haplotype against bovine leukemia virus infection. HLA. 2022 Jan;99(1):12-24. doi: 10.1111/tan.14502. Epub 2021 Dec 19. PMID: 34837483; PMCID: PMC9543338

IncI1 Plasmid Associated with blaCTX-M-2 Transmission in ESBL-Producing Escherichia coli Isolated from Healthy Thoroughbred Racehorse, Japan

In our previous study, extended spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) were isolated from healthy Thoroughbred racehorse feces samples in Japan. Some ESBL genes were predicted to be located on the conjugative plasmid. PCR-based replicon typing (PBRT) is a useful method to monitor and detect the association of replicons with specific plasmid-borne resistant genes. This study aimed to evaluate the plasmid replicon associated with ESBLEC isolated from healthy Thoroughbred racehorses at Japan Racing Association Training Centers in Japan. A total of 24 ESBLECs isolated from 23 (10.8%) individual Thoroughbred racehorse feces samples were used in this study. ESBL gene transfer was performed using a conjugation assay. Then, replicon types of ESBLEC isolates and their transconjugants were determined using PBRT. Pulsed-field gel electrophoresis (PFGE) was performed to look at the clonality of the ESBLECs isolates. ESBLECs were detected from 10.8% of healthy Thoroughbred racehorses. The blaCTX-M-2 was identified as the dominant type of ESBL gene, followed by blaCTX-M-1 and blaTEM-116. In this study, only the blaCTX-M-2 and the IncI1 plasmid were transferred to transconjugants. The PFGE results showed that ESBL genes were distributed in diversity of ESBLECs. This finding suggested that the IncI1 plasmid was associated with the dissemination of blaCTX-M-2 in Thoroughbred racehorses in Japan.Citation: Sukmawinata, E.; Uemura, R.; Sato, W.; Mitoma, S.; Kanda, T.; Sueyoshi, M. IncI1 Plasmid Associated with blaCTX-M-2 Transmission in ESBL-Producing Escherichia coli Isolated from Healthy Thoroughbred Racehorse, Japan. Antibiotics 2020, 9, 70. https://doi.org/10.3390/antibiotics902007

Identifying Pathogen and Allele Type Simultaneously in a Single Well Using Droplet Digital PCR

In the transmission control of chronic and untreatable livestock diseases such as bovine leukemia virus (BLV) infection, the removal of viral superspreaders is a fundamental approach. On the other hand, selective breeding of cattle with BLV-resistant capacity is also critical for reducing the viral damage to productivity by keeping infected cattle. To provide a way of measuring BLV proviral load (PVL) and identifying susceptible/resistant cattle simply and rapidly, we developed a fourplex droplet digital PCR method targeting the BLV pol gene, BLV-susceptible bovine major histocompatibility complex (BoLA)-DRB3*016:01 allele, resistant DRB3*009:02 allele, and housekeeping RPP30 gene (IPATS-BLV). IPATS-BLV successfully measured the percentage of BLV-infected cells and determined allele types precisely. Furthermore, it discriminated homozygous from heterozygous carriers. Using this method to determine the impact of carrying these alleles on the BLV PVL, we found DRB3*009:02-carrying cattle could suppress the PVL to a low or undetectable level, even with the presence of a susceptible heterozygous allele. Although the population of DRB3*016:01-carrying cattle showed significantly higher PVLs compared with cattle carrying other alleles, their individual PVLs were highly variable. Because of the simplicity and speed of this single-well assay, our method has the potential of being a suitable platform for the combined diagnosis of pathogen level and host biomarkers in other infectious diseases satisfying the two following characteristics of disease outcomes: (i) pathogen level acts as a critical maker of disease progression; and (ii) impactful disease-related host genetic biomarkers are already identified.Citation: Notsu K, El Daous H, Mitoma S, Wu X, Norimine J, Sekiguchi S. Identifying Pathogen and Allele Type Simultaneously in a Single Well Using Droplet Digital PCR. mSphere. 2023 Feb 21;8(1):e0049322. doi: 10.1128/msphere.00493-22. Epub 2023 Jan 10. PMID: 36625728; PMCID: PMC9942588

Characterization of bovine interleukin-2 stably expressed in HEK-293 cells

Interleukin 2 (IL-2) is a pleotropic cytokine and well-known as a T cell growth factor in immunology. It is now known to exert both immunostimulatory and immunosuppressive effects, optimizing immunological microenvironments for effector and regulatory T cell responses. The immunomodulatory role of IL-2 is critical for deciding whether or not T cell responses against specific antigens result in protection. We have established a mammalian cell line (HEK-293) stably expressing bovine IL-2 (boIL-2) (designated as HEK-293/boIL-2), using the piggyBac transposon system. The concentration of recombinant bovine IL-2 (rboIL-2) in the culture supernatant of HEK-293/boIL-2 reached 100 ng/ml on day 7 and showed similar proliferative activity to recombinant human IL-2 (rhuIL-2) for bovine peripheral mononuclear blood cells. Although rhuIL-2 has been often used to activate bovine T cells, our results indicate that characteristics of the T cell activation through rboIL-2 and huIL-2 appear slightly but significantly different. Interestingly, the rboIL-2/anti-boIL-2 monoclonal antibody (C5) (rboIL-2/C5) complex strongly induced proliferation of bovine NKp46+cells, natural killer (NK) cells, in vitro. This indicates that the rboIL-2/C5 complex could function as an IL-2 agonist specifically to increase the NK cell population, which in turn could enhance the activity of NK cells leading to protective immunity.Citation: Mitoma S, El-Khaiat HM, Uto T, Sato K, Sekiguchi S, Norimine J. Characterization of bovine interleukin-2 stably expressed in HEK-293 cells. J Vet Med Sci. 2021 Jan 21;83(1):134-141. doi: 10.1292/jvms.20-0423. Epub 2020 Nov 11. PMID: 33177289; PMCID: PMC7870392

Relationship between Allelic Heterozygosity in BoLA-DRB3 and Proviral Loads in Bovine Leukemia Virus-Infected Cattle

Enzootic bovine leukosis is a lethal neoplastic disease caused by bovine leukemia virus (BLV), belongs to family Retroviridae. The BLV proviral load (PVL) represents the quantity of BLV genome that has integrated into the host’s genome in BLV-infected cells. Bovine leukocyte antigen (BoLA) class II allelic polymorphisms are associated with PVLs in BLV-infected cattle. We sought to identify relationships between BoLA-DRB3 allelic heterozygosity and BLV PVLs among different cattle breeds. Blood samples from 598 BLV-infected cattle were quantified to determine their PVLs by real-time polymerase chain reaction. The results were confirmed by a BLV-enzyme-linked immunosorbent assay. Restriction fragment length polymorphism-polymerase chain reaction identified 22 BoLA-DRB3 alleles. Multivariate negative binomial regression modeling was used to test for associations between BLV PVLs and BoLA-DRB3 alleles. BoLA-DRB3.2*3, *7, *8, *11, *22, *24, and *28 alleles were significantly associated with low PVLs. BoLA-DRB3.2*10 was significantly associated with high PVLs. Some heterozygous allele combinations were associated with low PVLs (*3/*28, *7/*8, *8/*11, *10/*11, and *11/*16); others were associated with high PVLs (*1/*41, *10/*16, *10/*41, *16/*27, and *22/*27). Interestingly, the BoLA-DRB3.2*11 heterozygous allele was always strongly and independently associated with low PVLs. This is the first reported evidence of an association between heterozygous allelic combinations and BLV PVLs.Citation: Daous, H.E.; Mitoma, S.; Elhanafy, E.; Thi Nguyen, H.; Thi Mai, N.; Notsu, K.; Kaneko, C.; Norimine, J.; Sekiguchi, S. Relationship between Allelic Heterozygosity in BoLA-DRB3 and Proviral Loads in Bovine Leukemia Virus-Infected Cattle. Animals 2021, 11, 647. https://doi.org/10.3390/ani1103064

Selectivity of bovine interleukin-2 mutein stimulation on bovine peripheral blood mononuclear cells

Delivery of engineered interleukin-2 (IL-2) variants (muteins) is thought to be a promising cancer therapy in humans and mice. Our previous study indicated that bovine IL-2 (boIL-2) has a great potential to elicit NK cell activity for which distribution of IL-2 receptors on the target cell surface influences signal transduction. We developed nine boIL-2 muteins and examined the influence of the muteins on bovine peripheral blood mononuclear cells in vitro. On bovine peripheral mononuclear cells, NK cells strongly expressed CD122, followed by CD8+ T cells, while CD4+ T cells and γδ T cells did not show significant CD122 expression. All boIL-2 muteins showed decreasing in binding to boIL-2 receptor α, CD25, while maintaining their ability to bind to boIL-2 receptor βγ, CD122/CD132, heterodimer. The mutein F44A and E63A suppressed CD4+ T cell expansion but maintained the NK cell expansion. These results indicate that boIL-2 muteins can alter immunological outcomes and may be used for clinical intervention for a disease progression

Crucial role of dendritic cells in the generation of anti-tumor T-cell responses and immunogenic tumor microenvironment to suppress tumor development

Dendritic cells (DCs) are known as unique professional antigen (Ag)-presenting cells (APCs) to prime naïve T cells for the initiation of adaptive immunity. While DCs are believed to play a pivotal role in generating anti-tumor T-cell responses, the importance of DCs in the protection from the progression of tumors remains elusive. Here, we show how the constitutive deficiency of CD11chi DCs influences the progression of tumors with the use of binary transgenic mice with constitutive loss of CD11chi DCs. Constitutive loss of CD11chi DCs not only enhances the progression of tumors but also reduces the responses of Ag-specific T cells. Furthermore, the congenital deficiency of CD11chi DCs generates the immunosuppressive tumor microenvironment (TME) that correlates with the marked accumulation of myeloid-derived suppressor cells (MDSCs) and the prominent productions of immunosuppressive mediators. Thus, our findings suggest that CD11chi DCs are crucial for generating anti-tumor T-cell responses and immunogenic TME to suppress the development of tumors.Citation: Moe Tominaga, Tomofumi Uto, Tomohiro Fukaya, Shuya Mitoma, Dieter Riethmacher, Kunihiko Umekita, Yoshihiro Yamashita, Katsuaki Sato, Crucial role of dendritic cells in the generation of anti-tumor T-cell responses and immunogenic tumor microenvironment to suppress tumor development, Frontiers in Immunology, 15, 2024-08-14, https://doi.org/10.3389/fimmu.2024.120046

Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay

Background Porcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd. Results In this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed. Conclusions The developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.Citation: Mai, T.N., Nguyen, V., Yamazaki, W. et al. Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay. BMC Vet Res 14, 172 (2018). https://doi.org/10.1186/s12917-018-1498-

Gut dysbiosis promotes the breakdown of oral tolerance mediated through dysfunction of mucosal dendritic cells

application/pdfWhile dysbiosis in the gut is implicated in the impaired induction of oral tolerance generated in mesenteric lymph nodes （MesLNs）, how dysbiosis affects this process remains unclear. Here, we describe that antibiotic-driven gut dysbiosis causes the dysfunction of CD11c+CD103+ conventional dendritic cells （cDCs） in MesLNs, preventing the establishment of oral tolerance. Deficiency of CD11c+CD103+ cDCs abrogates the generation of regulatory T cells in MesLNs to establish oral tolerance. Antibiotic treatment triggers the intestinal dysbiosis linked to the impaired generation of colony-stimulating factor 2 （Csf2）-producing group 3 innate lymphoid cells （ILC3s） for regulating the tolerogenesis of CD11c+CD103+ cDCs and the reduced expression of tumor necrosis factor （TNF）-like ligand 1A （TL1A） on CD11c+CD103+ cDCs for generating Csf2-producing ILC3s. Thus, antibiotic-driven intestinal dysbiosis leads to the breakdown of crosstalk between CD11c+CD103+ cDCs and ILC3s for maintaining the tolerogenesis of CD11c+CD103+ cDCs in MesLNs, responsible for the failed establishment of oral tolerance