A cell competition system with one gene expression from a single-copy gene in one cell.

Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method.

Complete genome sequence of the type strain of Mycobacterium montefiorense, strain ATCC BAA-256.

Mycobacterium montefiorense, a nontuberculous mycobacterium, is a causative agent of mycobacteriosis in aquatic animals, its type strain M. montefiorense ATCC BAA-256 being isolated from a moray eel. In this study, we report the complete ATCC BAA-256 genome sequence with a 5,693,452-bp-containing circular chromosome, 65.2% GC content, and 5,407 coding sequences.

Degranulation and expression of cytokines were modulated by diazinon in activated mast cells.

Diazinon is an organophosphorus (OP) insecticides used in agriculture, home gardening and indoor pest control in Japan. It can activate macrophages and induce pro-inflammatory responses and has been reported to cause airway hyper-reactivity, suggesting the possibility of asthma exacerbation from exposure to OP insecticides. Despite the correlation between insecticide use and the pathogenesis of allergic diseases, there have been no reports on the effects of diazinon on mast cell function. Therefore, in this study, we investigated the effects of diazinon on mast cell function in rat basophilic leukemia (RBL)-2H3 cells. Surprisingly, we found that diazinon inhibited mast cell activation, although the degree of inhibition varied with concentration. Diazinon induced reactive oxygen species (ROS) generation and HO-1 expression at a concentration of 150 µM without affecting cell viability. Diazinon inhibited A23187-mediated degranulation and Tnf and Il4 expression in RBL-2H3 cells but did not affect calcium influx. Suppression of degranulation by diazinon was reversed when the culture supernatant was removed. As a signaling event downstream of calcium influx, diazinon inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) induced by A23187, whereas the phosphorylation of p38 had little effect. IgE cross-linking-mediated degranulation as well as the induction of Tnf and IL4 expression was significantly inhibited by diazinon, while diazinon had little effect on calcium influx. In conclusion, diazinon inhibited mast cell activation, including degranulation and cytokine expression. When evaluating the in vivo effects of diazinon, its potential to inhibit mast cell activation should be considered in the pathophysiology and development of allergic diseases in terms of basic and clinical aspects, respectively, although the effect of diazinon varies depending on the cell type.

Targeting PAK1 is effective against cutaneous squamous cell carcinoma in a syngenic mouse model.

By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.

The integration of metabolic and proteomic data uncovers an augmentation of the sphingolipid biosynthesis pathway during T-cell differentiation.

Recent studies have highlighted the significance of cellular metabolism in the initiation of clonal expansion and effector differentiation of T cells. Upon exposure to antigens, naïve CD4+ T cells undergo metabolic reprogramming to meet their metabolic requirements. However, only few studies have simultaneously evaluated the changes in protein and metabolite levels during T cell differentiation. Our research seeks to fill the gap by conducting a comprehensive analysis of changes in levels of metabolites, including sugars, amino acids, intermediates of the TCA cycle, fatty acids, and lipids. By integrating metabolomics and proteomics data, we discovered that the quantity and composition of cellular lipids underwent significant changes in different effector Th cell subsets. Especially, we found that the sphingolipid biosynthesis pathway was commonly activated in Th1, Th2, Th17, and iTreg cells and that inhibition of this pathway led to the suppression of Th17 and iTreg cells differentiation. Additionally, we discovered that Th17 and iTreg cells enhance glycosphingolipid metabolism, and inhibition of this pathway also results in the suppression of Th17 and iTreg cell generation. These findings demonstrate that the utility of our combined metabolomics and proteomics analysis in furthering the understanding of metabolic transition during Th cell differentiation.

Effects of Anti-Fibrotic Drugs on Transcriptome of Peripheral Blood Mononuclear Cells in Idiopathic Pulmonary Fibrosis.

Two anti-fibrotic drugs, pirfenidone (PFD) and nintedanib (NTD), are currently used to treat idiopathic pulmonary fibrosis (IPF). Peripheral blood mononuclear cells (PBMCs) are immunocompetent cells that could orchestrate cell-cell interactions associated with IPF pathogenesis. We employed RNA sequencing to examine the transcriptome signature in the bulk PBMCs of patients with IPF and the effects of anti-fibrotic drugs on these signatures. Differentially expressed genes (DEGs) between "patients with IPF and healthy controls" and "before and after anti-fibrotic treatment" were analyzed. Enrichment analysis suggested that fatty acid elongation interferes with TGF-ß/Smad signaling and the production of oxidative stress since treatment with NTD upregulates the fatty acid elongation enzymes ELOVL6. Treatment with PFD downregulates COL1A1, which produces wound-healing collagens because activated monocyte-derived macrophages participate in the production of collagen, type I, and alpha 1 during tissue damage. Plasminogen activator inhibitor-1 (PAI-1) regulates wound healing by inhibiting plasmin-mediated matrix metalloproteinase activation, and the inhibition of PAI-1 activity attenuates lung fibrosis. DEG analysis suggested that both the PFD and NTD upregulate SERPINE1, which regulates PAI-1 activity. This study embraces a novel approach by using RNA sequencing to examine PBMCs in IPF, potentially revealing systemic biomarkers or pathways that could be targeted for therapy.

Circulating microRNA Panel for Prediction of Recurrence and Survival in Early-Stage Lung Adenocarcinoma.

Early-stage lung adenocarcinoma (LUAD) patients remain at substantial risk for recurrence and disease-related death, highlighting the unmet need of biomarkers for the assessment and identification of those in an early stage who would likely benefit from adjuvant chemotherapy. To identify circulating miRNAs useful for predicting recurrence in early-stage LUAD, we performed miRNA microarray analysis with pools of pretreatment plasma samples from patients with stage I LUAD who developed recurrence or remained recurrence-free during the follow-up period. Subsequent validation in 85 patients with stage I LUAD resulted in the development of a circulating miRNA panel comprising miR-23a-3p, miR-320c, and miR-125b-5p and yielding an area under the curve (AUC) of 0.776 in predicting recurrence. Furthermore, the three-miRNA panel yielded an AUC of 0.804, with a sensitivity of 45.8% at 95% specificity in the independent test set of 57 stage I and II LUAD patients. The miRNA panel score was a significant and independent factor for predicting disease-free survival (p < 0.001, hazard ratio [HR] = 1.64, 95% confidence interval [CI] = 1.51-4.22) and overall survival (p = 0.001, HR = 1.51, 95% CI = 1.17-1.94). This circulating miRNA panel is a useful noninvasive tool to stratify early-stage LUAD patients and determine an appropriate treatment plan with maximal efficacy.

A case of coagulation factor V inhibitor induced by immune checkpoint inhibitor therapy.

A 73-year-old woman with lung adenocarcinoma (cT4N3M1a: Stage IVA) was treated with atezolizumab as the eighth line of therapy. Four weeks after the fourth dose of atezolizumab, the prothrombin time (PT) and activated thromboplastin time (APTT) were prolonged. Coagulation factor V (FV) activity was decreased, and FV inhibitors were observed. There was no history of PT or APTT prolongation or bleeding before the use of atezolizumab. Atezolizumab-induced coagulation FV inhibitor was diagnosed. After 2 weeks, the PT and APTT spontaneously normalized. FV activity improved and the FV inhibitors disappeared after 6 and 9 weeks, respectively.

Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.

Functional Roles of CD26/DPP4 in Bleomycin-Induced Pulmonary Hypertension Associated with Interstitial Lung Disease.

Pulmonary hypertension (PH) with interstitial lung diseases (ILDs) often causes intractable conditions. CD26/Dipeptidyl peptidase-4 (DPP4) is expressed in lung constituent cells and may be related to the pathogenesis of various respiratory diseases. We aimed to clarify the functional roles of CD26/DPP4 in PH-ILD, paying particular attention to vascular smooth muscle cells (SMCs). Dpp4 knockout (Dpp4KO) and wild type (WT) mice were administered bleomycin (BLM) intraperitoneally to establish a PH-ILD model. The BLM-induced increase in the right ventricular systolic pressure and the right ventricular hypertrophy observed in WT mice were attenuated in Dpp4KO mice. The BLM-induced vascular muscularization in small pulmonary vessels in Dpp4KO mice was milder than that in WT mice. The viability of TGFß-stimulated human pulmonary artery SMCs (hPASMCs) was lowered due to the DPP4 knockdown with small interfering RNA. According to the results of the transcriptome analysis, upregulated genes in hPASMCs with TGFß treatment were related to pulmonary vascular SMC proliferation via the Notch, PI3K-Akt, and NFκB signaling pathways. Additionally, DPP4 knockdown in hPASMCs inhibited the pathways upregulated by TGFß treatment. These results suggest that genetic deficiency of Dpp4 protects against BLM-induced PH-ILD by alleviating vascular remodeling, potentially through the exertion of an antiproliferative effect via inhibition of the TGFß-related pathways in PASMCs.

Artificial intelligence-based analysis of the spatial distribution of abnormal computed tomography patterns in SARS-CoV-2 pneumonia: association with disease severity.

BACKGROUND: The substantial heterogeneity of clinical presentations in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia still requires robust chest computed tomography analysis to identify high-risk patients. While extension of ground-glass opacity and consolidation from peripheral to central lung fields on chest computed tomography (CT) might be associated with severely ill conditions, quantification of the central-peripheral distribution of ground glass opacity and consolidation in assessments of SARS-CoV-2 pneumonia remains unestablished. This study aimed to examine whether the central-peripheral distributions of ground glass opacity and consolidation were associated with severe outcomes in patients with SARS-CoV-2 pneumonia independent of the whole-lung extents of these abnormal shadows. METHODS: This multicenter retrospective cohort included hospitalized patients with SARS-CoV-2 pneumonia between January 2020 and August 2021. An artificial intelligence-based image analysis technology was used to segment abnormal shadows, including ground glass opacity and consolidation. The area ratio of ground glass opacity and consolidation to the whole lung (GGO%, CON%) and the ratio of ground glass opacity and consolidation areas in the central lungs to those in the peripheral lungs (GGO(C/P)) and (CON(C/P)) were automatically calculated. Severe outcome was defined as in-hospital death or requirement for endotracheal intubation. RESULTS: Of 512 enrolled patients, the severe outcome was observed in 77 patients. GGO% and CON% were higher in patients with severe outcomes than in those without. Multivariable logistic models showed that GGO(C/P), but not CON(C/P), was associated with the severe outcome independent of age, sex, comorbidities, GGO%, and CON%. CONCLUSION: In addition to GGO% and CON% in the whole lung, the higher the ratio of ground glass opacity in the central regions to that in the peripheral regions was, the more severe the outcomes in patients with SARS-CoV-2 pneumonia were. The proposed method might be useful to reproducibly quantify the extension of ground glass opacity from peripheral to central lungs and to estimate prognosis.

Risk Factors for Concussion in Under 18, Under 22 and Professional Men's Rugby Union: A Video Analysis of 14,809 Tackles.

OBJECTIVE: This study aimed to identify the risk factors for tackle-related concussion observed in matches involving under (U) 18, U 22 and professional men's Rugby Union players through video analysis. STUDY DESIGN: Descriptive epidemiology study. METHODS: Twenty Rugby Union matches each for high school (U18), university/college (U22) and professional (Elite) were randomly selected from 202 matches in the 2018/2019 season. Both one-on-one and tackles involving multiple tacklers were analyzed for the 60 matches. The 28 categorical and continuous variables (e.g., tackle characteristics and duration before the tackle) were applied as risk factors to a least absolute shrinkage and selection operator (Lasso) regression analysis. To identify high-risk situations, a simulation model with coefficients obtained from the Lasso regression was used. Statistical analysis was conducted according to tackle direction. RESULTS: A total of 14,809 tackles and 41 concussions involving 1800 players were included in the analyses. The incidence rate of concussions (injuries/1000 tackles) was greater in Elite players (4.0) compared with U18 (1.9) and U22 (2.4) players. The factors most highly associated with concussions were head-in-front tackles (where the tackler's head is placed forward, impeding a ball carrier's forward movements, 11.26/1000 tackles), and were more often observed among U18 players. A simulation model predicted that the highest risk tackle situation in Elite players was a head-in-front, side-on tackle below the hip of the ball carrier (predicted incidence rate 18.07/1000 tackles). CONCLUSION: The risk factors associated with concussion need to be assessed cautiously. Avoiding head-in-front, side-on tackles to the lower extremities of a ball carrier should be considered to reduce injury risks.

1-Oleoyl-lysophosphatidylethanolamine stimulates RORγt activity in TH17 cells.

Metabolic fluxes involving fatty acid biosynthesis play essential roles in controlling the differentiation of T helper 17 (TH17) cells. However, the exact enzymes and lipid metabolites involved, as well as their link to promoting the core gene transcriptional signature required for the differentiation of TH17 cells, remain largely unknown. From a pooled CRISPR-based screen and unbiased lipidomics analyses, we identified that 1-oleoyl-lysophosphatidylethanolamine could act as a lipid modulator of retinoid-related orphan receptor gamma t (RORγt) activity in TH17 cells. In addition, we specified five enzymes, including Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2, suggestive of the requirement of glycerophospholipids with monounsaturated fatty acids being required for the transcription of Il17a. 1-Oleoyl-lysophosphatidylethanolamine was reduced in Pla2g12a-deficient TH17 cells, leading to the abolition of interleukin-17 (IL-17) production and disruption to the core transcriptional program required for the differentiation of TH17 cells. Furthermore, mice with T cell-specific deficiency of Pla2g12a failed to develop disease in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus, our data indicate that 1-oleoyl-lysophosphatidylethanolamine is a lipid metabolite that promotes RORγt-induced TH17 cell differentiation and the pathogenicity of TH17 cells.

Adjustment of creatinine clearance for carboplatin dosing in Calvert's formula and clinical efficacy for lung cancer.

BACKGROUND: The Cockcroft-Gault formula is commonly used as a substitute for glomerular filtration rate (GFR) in Calvert's formula for carboplatin dosing, where adjusting serum creatinine measured using the enzymatic method with 0.2 mg/dL has been suggested in Japan. However, the effects of these adjustments on efficacy in patients with non-small-cell lung cancer remain unknown. METHODS: We conducted a post hoc analysis of the PREDICT1 study (CJLSG1201), a multicenter prospective observational trial of carboplatin-pemetrexed. Glomerular filtration rate values in Calvert's formula were back-calculated from the administered dosages of carboplatin and the reported value of the target area under the curve. We estimated the serum creatinine adjustments and divided the patients into crude and adjusted groups. RESULTS: Patients in the crude group (N = 169) demonstrated similar efficacy to those in the adjusted group (N = 104) in progression-free survival (PFS) and overall survival (OS) (hazard ratio [HR], 1.02; 95% confidence interval [CI], 0.76-1.35; p = 0.916 vs. HR, 0.87; 95% CI, 0.65-1.17; p = 0.363), with higher grade 3-4 hematologic toxicity. Among patients aged ≥75 years, the crude group (N = 47) showed superior efficacy compared with the adjusted group (N = 17) in PFS and OS (HR, 0.37; 95% CI, 0.20-0.69; p = 0.002 vs. HR, 0.43; 95% CI, 0.23-0.82; p = 0.010). CONCLUSIONS: Serum creatinine adjustment may be associated with similar efficacy compared to the crude serum creatinine value. In older patients, the adjustment should be cautiously applied owing to the potential for reduced efficacy.

Augmented interferon regulatory factor 7 axis in whole tumor cell vaccines prevents tumor recurrence by inducing interferon gamma-secreting B cells.

Among cancer immunotherapy, which has received great attention in recent years, cancer vaccines can potentially prevent recurrent tumors by using the exquisite power and specificity of the immune system. Specifically, whole tumor cell vaccines (WTCVs) based on surgically resected tumors have been considered to elicit robust anti-tumor immune responses by exposing various tumor-associated antigens to host immunity. However, most tumors have little immunogenicity because of immunoediting by continuous interactions with host immunity; thus, preparing WTCVs based on patient-derived non-modified tumors cannot prevent tumor onset. Hence, the immunogenicity of tumor cells must be improved for effective WTCVs. In this study, we indicate the importance of the interferon regulatory factor 7 (Irf7) axis, including Irf7 and its downstream factors, within tumor cells in regulating immunogenicity. Indeed, WTCVs that augmented the Irf7 axis have exerted remarkable recurrence-preventive effects when vaccinated after tumor inactivation by radiation. Most notably, vaccination with murine colon cancer cells that enhanced the Irf7 axis prevented the development of challenged tumors in all mice and resulted in a 100% survival rate during the observation period. Furthermore, the mechanism leading to vaccine effectiveness was mediated by interferon-gamma-producing B cells. This study provides novel insights into how to enhance tumor immunogenicity and use WTCVs as recurrence prophylaxis.

Identification of a new gibberellin receptor agonist, diphegaractin, by a cell-free chemical screening system.

Gibberellin (GA) is a phytohormone that regulates various developmental processes during the plant life cycle. In this study, we identify a new GA agonist, diphegaractin, using a wheat cell-free based drug screening system with grape GA receptor. A GA-dependent interaction assay system using GA receptors and DELLA proteins from Vitis vinifera was constructed using AlphaScreen technology and cell-free produced proteins. From the chemical compound library, diphegaractin was found to enhance the interactions between GA receptors and DELLA proteins from grape in vitro. In grapes, we found that diphegaractin induces elongation of the bunch and increases the sugar concentration of grape berries. Furthermore, diphegaractin shows GA-like activity, including promotion of root elongation in lettuce and Arabidopsis, as well as reducing peel pigmentation and suppressing peel puffing in citrus fruit. To the best of our knowledge, this study is the first to successfully identify a GA receptor agonist showing GA-like activity in agricultural plants using an in vitro molecular-targeted drug screening system.

Calcineurin inhibitor inhibits tolerance induction by suppressing terminal exhaustion of donor T cells after allo-HCT.

Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD (cGVHD) in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory Tex), expressing both inhibitory receptors and effector molecules, into terminal Tex, and inhibited tolerance induction. Adoptive transfer of transitory Tex, but not terminal Tex, into secondary recipients developed cGVHD. Transitory Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory Tex and not terminal Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.

Leukocyte Depletion and Size-Based Enrichment of Circulating Tumor Cells Using a Pressure-Sensing Microfiltration Device.

Considering the challenges in isolating circulating tumor cells (CTCs) pertaining to cellular stress and purity, we report the application of a blood microfiltration device as an optimal approach for noninvasive liquid biopsy to target CTCs. We experimentally analyzed the filtration behavior of the microfilter using pressure sensing to separate tumor cells from leukocytes in whole blood. This approach achieved an average recovery of >96% of the spiked tumor cells and depletion of >99% of total leukocytes. Furthermore, we carried out genomic profiling of the CTCs using the blood microfiltration device. The method was also applied in a clinical setting; DNA amplification was performed using a small number of microfiltered CTCs and epidermal growth factor receptor mutations were successfully detected to characterize the efficacy of molecularly targeted drugs against lung cancer. Overall, the proposed method can provide a tool for evaluating efficient filtration pressure to concentrate CTCs from whole blood.

Induction of allograft tolerance by adoptive transfer of donor B cells: an immune regulatory strategy for transplantation using MHC-matched iPS cells.

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.