Size control of induced pluripotent stem cells colonies in two-dimensional culture for differentiation into functional monocyte-like cells.

BACKGROUND AIMS: Monocytes, derived from hematopoietic stem cells (HSCs), play a pivotal role in the immune response to cancer. Although they are an attractive source of cell therapy for cancer, a method for ex vivo expansion has not yet been established. Monocytes differentiated from pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs), can be an alternative source of HSC-derived monocytes because of their self-renewal and pluripotency. To develop a standardized method for the generation of iPSC-derived monocytes for future clinical applications, we aim to control the size of the iPSC colony. METHODS: To this end, we developed a plate with multiple dots containing a chemical substrate for the iPSC scaffold. iPSCs placed in the plate expanded only on the dots and created colonies of the same size. The cells were then differentiated into monocytes by adding cytokines to the colonies. RESULTS: The dot plate substantially reduced variability in monocyte-like cell generation when compared with cultivating cells on a plate with the substrate covering the entire surface area. Furthermore, more monocyte-like cells were obtained by adjusting the dot size and the distance between the dots. The iPSC-derived monocyte-like cells phagocytosed cancer cells and secreted proinflammatory cytokines. The cells also expressed Fc receptors and exerted immunoglobulin G-mediated killing of cancer cells with the corresponding antibodies. CONCLUSIONS: The dot plate enabled the control of iPSC colony size in two-dimensional culture, which resulted in a reduction in the generation-variation of functional monocyte-like cells. This standardized method for generating iPSC-derived monocyte-like cells using the dot plate could also facilitate the development of an automated closed system on a large scale for clinical applications.

Phase I study of expanded natural killer cells in combination with cetuximab for recurrent/metastatic nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) cells express high levels of epidermal growth factor receptor (EGFR). Cetuximab is an anti-EGFR monoclonal antibody that promotes natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) via engagement of CD16. We studied safety and efficacy of combining cetuximab with autologous expanded NK cells in patients with recurrent and/or metastatic NPC who had failed at least two prior lines of chemotherapy. METHODS: Seven subjects (six patients) received cetuximab every 3 weeks (six doses maximum) in the pre-trial phase. Autologous NK cells, expanded by co-culture with irradiated K562-mb15-41BBL cells, were then infused on the day after administration of cetuximab. Primary and secondary objectives were to determine safety of this combination therapy and to assess tumor responses, respectively. RESULTS: Median NK cell expansion from peripheral blood mononucleated cells after 10 days of culture with K562-mb15-41BBL was 274-fold (range, 36-534, n = 10), and the median expression of CD16 was 98.4% (range, 67.8-99.7%). Skin rash, the commonest side effect of cetuximab in the pre-trial phase, was not exacerbated by NK cell infusion. No intolerable side effects were observed. Stable disease was observed in four subjects and progressive disease in three subjects. Three patients who received NK cells twice had time to disease progression of 12, 13, and 19 months. CONCLUSION: NK cells with high ADCC potential can be expanded from patients with heavily pre-treated NPC. The safety profile and encouraging clinical responses observed after combining these cells with cetuximab warrant further studies of this approach. (clinicalTrials.gov NCT02507154, 23/07/2015). PRECIS: Engaging NK cell-mediated ADCC using cetuximab plus autologous NK cells in EGFR-positive NPC was well tolerated among heavily pre-treated recurrent NPC. Promising results were observed with 3 out of 7 subjects demonstrating durable stable disease.

Suitability of NIID-MDCK cells as a substrate for cell-based influenza vaccine development from the perspective of adventitious virus susceptibility.

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses.

Determination of the potency of a cell-based seasonal quadrivalent influenza vaccine using a purified primary liquid standard.

In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine.

Upregulation of thioredoxin-1 in activated human NK cells confers increased tolerance to oxidative stress.

Adoptive transfer of immune cells, such as T lymphocytes and NK cells, has potential to control cancer growth. However, this can be counteracted by immune escape mechanisms within the tumor microenvironment, including those mediated by reactive oxygen species (ROS). Here, we determined the levels of anti-oxidant molecules in NK cells and their capacity to overcome ROS-induced immune suppression. We investigated the effect of H2O2 on resting NK cells, IL-2-activated NK cells and NK cells expanded by coculture with the K562 leukemia cell line genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL). Expression of anti-oxidant and anti-apoptotic genes was evaluated by expression array, and protein levels of anti-oxidant molecules by Western blot. Activated NK cells, IL-2-activated NK cells and NK cells expanded by K562-mb15-41BBL were significantly more resistant to H2O2-induced cell death than resting NK. Thioredoxin-1 (TXN1) and peroxiredoxin-1 (PRDX1) were also up-regulated in activated NK cells. Moreover, H2O2-induced cell death after IL-2 activation was significantly induced in the presence of an anti-TXN1-neutralising antibody. Collectively, these data document that activated NK cells can resist to H2O2-induced cell death by up-regulation of TXN1.

Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15.

Natural killer (NK) cell survival and, hence, cytotoxicity requires cytokine support. We determined whether expression of interleukin-15 (IL-15) in a nonsecretory, membrane-bound form could sustain NK cell growth. We linked the human IL15 gene to that encoding CD8α transmembrane domain (mbIL15). After retroviral transduction, human NK cells expressed mbIL15 on the cell surface; IL-15 secretion was negligible. Survival of mbIL15-NK cells without interleukin-2 (IL-2) after 7-day culture was vastly superior to that of mock-transduced NK cells (P < .001, n = 15) and of NK cells expressing nonmembrane-bound IL-15 (P = .025, n = 9); viable mbIL15-NK cells were detectable for up to 2 months. In immunodeficient mice, mbIL15-NK cells expanded without IL-2 and were detectable in all tissues examined (except brain) in much higher numbers than mock-transduced NK cells (P < .001). Expansion further increased with IL-2. The primary mechanism of mbIL15 stimulation was autocrine; it activated IL-15 signaling and antiapoptotic signaling. NK cells expressing mbIL15 had higher cytotoxicity against leukemia, lymphoma, and solid tumor cells in vitro and against leukemia and sarcoma cells in xenograft models. Thus, mbIL15 confers independent growth to NK cells and enhances their antitumor capacity. Infusion of mbIL15-NK cells would allow NK cell therapy without the potential adverse effects of cytokine administration.

Expanded and armed natural killer cells for cancer treatment.

The capacity of natural killer (NK) cells to recognize and kill transformed cells suggests that their infusion could be used to treat cancer. It is difficult to obtain large numbers of NK cells ex vivo by exposure to cytokines alone but the addition of stimulatory cells to the cultures can induce NK cell proliferation and long-term expansion. Some of these methods have been validated for clinical-grade application and support clinical trials testing feasibility and safety of NK cell administration. Early data indicate that ex vivo expansion of NK cells from healthy donors or from patients with cancer is robust, allowing multiple infusions from a single apheresis. NK cells can transiently expand in vivo after infusion. Allogeneic NK cells are not direct effectors of graft-versus-host disease but this may occur if donor NK cells are infused after allogeneic hematopoietic stem cell transplant, which may activate T cell alloreactivity. NK cells can be directed with antibodies, or engineered using either transient modification by electroporation of mRNA or prolonged gene expression by viral transduction. Thus, expanded NK cells can be armed with activating receptors that enhance their natural anti-tumor capacity or with chimeric antigen receptors that can redirect them towards specific tumor targets. They can also be induced to express cytokines that promote their autonomous growth, further supporting their in vivo expansion. With the implementation of these approaches, expanded and armed NK cells should ultimately become a powerful component of immunotherapy of cancer.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Fratricide-resistant CD7-CAR T cells in T-ALL.

T cell acute lymphoblastic leukemia (T-ALL) is difficult to treat when it relapses after therapy or is chemoresistant; the prognosis of patients with relapsed or refractory T-ALL is generally poor. We report a case series of 17 such patients who received autologous chimeric antigen receptor (CAR) T cells expressing an anti-CD7 CAR and an anti-CD7 protein expression blocker (PEBL), which prevented CAR T cell fratricide. Despite high leukemic burden and low CAR T cell dosing, 16 of the 17 patients attained minimal residual disease-negative complete remission within 1 month. The remaining patient had CD7- T-ALL cells before infusion, which persisted after infusion. Toxicities were mild: cytokine release syndrome grade 1 in ten patients and grade 2 in three patients; immune effector cell-associated neurotoxicity syndrome grade 1 in two patients. Eleven patients remained relapse-free (median follow-up, 15 months), including all nine patients who received an allotransplant. The first patient is in remission 55 months after infusion without further chemotherapy or transplantation; circulating CAR T cells were detectable for 2 years. T cells regenerating after lymphodepletion lacked CD7 expression, were polyclonal and responded to SARS-CoV-2 vaccination; CD7+ immune cells reemerged concomitantly with CAR T cell disappearance. In conclusion, autologous anti-CD7 PEBL-CAR T cells have powerful antileukemic activity and are potentially an effective option for the treatment of T-ALL.

Evaluation of a qualified MDCK cell line for virus isolation to develop cell-based influenza vaccine viruses with appropriate antigenicity.

We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.

Current circumstances and prospects on performance evaluation for infection control technologies of airborne viruses in indoorenvironments.

Respiratory infectious diseases have potential of aerosol transmission such as COVID-19. The development of new technologies for infection control against airborne viruses are further required. It is necessary for effective development to evaluate properly the effect and role of these technologies in indoor environment. Here, the author provided essential knowledge for infection control of viral aerosols, i.e., basic concept of infection control, features of COVID-19 and Influenza including the entry receptor in body of each virus, behavior of the viral aerosols released from patient bodies, and Wells-Riley model as a traditional quantitative assessment of the infection risk by aerosol transmission. Previous evaluation studies on airborne viruses were categorized into three types of experiments, namely, in vitro, in vivo, and in humans and real indoor environments. Some prospects were described, including standard evaluation methods for air cleaners, the research group to formulate guidelines for evaluating the hygienic effects of chemical substances on microbes in real indoor space, and personal opinions on evaluation concept linked to three types of experiments. This minireview may help to correctly evaluate the hygienic effects of control technologies against airborne viruses in indoor environment and to contribute development of technologies with required performance according to infection risk.

Identification of the properties of H5 influenza vaccine viruses with high hemagglutinin yields.

Manufactured influenza vaccines have to contain a defined amount of hemagglutinin (HA) antigen. Therefore, vaccine viruses with a high HA antigen yield (HAY) are preferable for manufacturing vaccines, particularly vaccines in response to a pandemic, when vaccines need to be rapidly produced. However, the viral properties associated with a high HAY have not yet been fully clarified. To identify the HAY-associated traits, we first propagated 26 H5 candidate vaccine viruses (CVVs) in eggs, which were previously developed based on genetic reassortment methods using master viruses, to determine their total protein yield (TPY), ratio of HA to total viral protein (%-HA content) and HAY. The results revealed that the HAY was correlated with the TPY but not with the %-HA content. We further found that altering the sequences of the 3' noncoding region of HA vRNA or replacing the master virus improved the HAYs and TPYs of the low-HAY CVVs to approximately double the values of the original CVVs but did not change the %-HA content, which a previous study suggested was associated with the HAY. Analyses based on real-time PCR assays and scanning electron microscopy revealed that the virus samples with an improved HAY contained more copies of the virus genome and viral particles than the original samples. The results suggest that an improvement in virus growth (i.e., an increase in the amount of viral particles) leads to an increase in the TPY and thus in the HAY, regardless of the %-HA content. The approximately twofold increase in the HAY shown in this study may not appear to represent a large improvement, but the impact will be significant given the millions of chicken eggs used to produce vaccines. These findings will be informative for developing high-HAY vaccine viruses.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies.

BACKGROUND AIMS: Retroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose. METHODS: We tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia. RESULTS: Median anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24-48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10(8), n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia. CONCLUSIONS: The method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.

Gene Transduction of Natural Killer Cells for Clinical Application.

Autologous T cells expressing chimeric antigen receptor (CAR) have produced a spectacular response in hematological malignancies. This success of cellular therapy has inspired the exploration of the therapeutic potential of other immune cell types. In this regard, natural killer (NK) cells hold great potential as they can identify tumor cells by mechanisms that are different from those used by T cells and have a high cytotoxic capacity. Their capacity to recognize tumors and killing potency can be further enhanced by genetic modification. Our laboratory has developed a clinically adaptable method to manufacture genetically modified NK cells using retroviral vectors in compliance with current good manufacturing practice regulations. Here, we describe relevant technical procedures, including isolation of peripheral blood mononucleated cells from a leukapheresis product, T-cell depletion, stimulation and transduction of NK cells, and preparation of transduced NK cells for infusion.

Asymmetric total synthesis of four bioactive lignans using donor-acceptor cyclopropanes and bioassay of (-)- and (+)-niranthin against hepatitis B and influenza viruses.

The asymmetric total synthesis of four lignans, dimethylmatairesinol, matairesinol, (-)-niranthin, and (+)-niranthin has been achieved using reductive ring-opening of cyclopropanes. Moreover, we performed bioassays of the synthesized (+)- and (-)-niranthins using hepatitis B and influenza viruses, which revealed the relationship between the enantiomeric structure and the anti-viral activity of niranthin.

Thirteen-month-old boy with malignant lymphoma having symptoms mimicking acute otitis media and mastoiditis with facial palsy.

Non-Hodgkin diffuse large B-cell lymphomas of the mastoid that extend from the nasopharynx are extremely rare in children. Moreover, in lymphoproliferative diseases, the presence of otoneurological signs prior to systemic disease involvement is rare. Here, we present a rare case of non-Hodgkin B-cell lymphoma invading the middle ear and mastoid in a 1-year-old boy that mimicked acute mastoiditis with complete facial nerve palsy. As this case illustrates, physicians should consider a diagnosis of malignant lymphoma if a patient presents with otitis media and mastoiditis accompanied by facial palsy.

Engineering of Natural Killer Cells for Clinical Application.

The clinical success of chimeric antigen receptor-directed T cells in leukemia and lymphoma has boosted the interest in cellular therapy of cancer. It has been known for nearly half a century that a subset of lymphocytes called natural killer (NK) cells can recognize and kill cancer cells, but their clinical potential as therapeutics has not yet been fully explored. Progress in methods to expand and genetically modify human NK cells has resulted in technologies that allow the production of large numbers of highly potent cells with specific anticancer activity. Here, we describe clinically applicable protocols for NK cell engineering, including expansion of NK cells and genetic modification using electroporation of messenger RNA.

NK cells for cancer immunotherapy.

Natural killer (NK) cells can swiftly kill multiple adjacent cells if these show surface markers associated with oncogenic transformation. This property, which is unique among immune cells, and their capacity to enhance antibody and T cell responses support a role for NK cells as anticancer agents. Although tumours may develop several mechanisms to resist attacks from endogenous NK cells, ex vivo activation, expansion and genetic modification of NK cells can greatly increase their antitumour activity and equip them to overcome resistance. Some of these methods have been translated into clinical-grade platforms and support clinical trials of NK cell infusions in patients with haematological malignancies or solid tumours, which have yielded encouraging results so far. The next generation of NK cell products will be engineered to enhance activating signals and proliferation, suppress inhibitory signals and promote their homing to tumours. These modifications promise to significantly increase their clinical activity. Finally, there is emerging evidence of increased NK cell-mediated tumour cell killing in the context of molecularly targeted therapies. These observations, in addition to the capacity of NK cells to magnify immune responses, suggest that NK cells are poised to become key components of multipronged therapeutic strategies for cancer.

A Multifaceted Evaluation on the Penetration Resistance of Protective Clothing Fabrics against Viral Liquid Drops without Pressure.

Healthcare workers should wear appropriate personal protective clothing (PPC) on assuming the risk of exposure to various pathogens. Therefore, it is important to understand PPC performance against pathogen penetration. Currently, standard methods to evaluate and classify the penetration resistance of PPC fabrics with pressure using synthetic blood or phi-X174 phage have been established by the International Organization for Standardization (ISO). However, the penetration of viral liquid drops (VLDrop) on the PPC without pressure is also a major exposure route and more realistic, necessitating further studies. Here, we evaluated the penetration resistance against VLDrop without pressure using phi-X174 phage on woven and nonwoven fabrics of commercially available PPC classified by the ISO, and analyzed in detail the penetration behaviors of VLDrop by quantifying the phage amounts in leak-through and migration into test fabrics. Our results showed that some nonwoven test fabrics had nearly the same penetration resistance against VLDrop, even if the ISO resistance class differed. Furthermore, the results revealed that the amount of leakage through the fabrics was correlated with the migration amount into the fabric, which was related to fluid-repellency of fabrics, suggesting the effectiveness for penetration resistance. Our study may facilitate more appropriate selection for PPC against pathogen penetration.