Cell Surface Antigen Expression Research Articles

NK-92 cells activated by IL-2 inhibit the progression of endometriosis in vitro

Background Interleukin (IL)-2 is a key cytokine capable of modulating the immune response by activating natural killer (NK) cells. This study was recruited to explore the therapeutic potential of IL-2-activated NK-92 cells in endometriosis in vitro. Methods Ectopic endometrial stromal cells (EESCs) were isolated and co-cultured with IL-2-activated NK-92 cells at varying effector-to-target (E:T) ratios (1:0 [Control], 1:1, 1:3, and 1:9). The viability, cytotoxicity, and cell surface antigen expression of IL-2-activated NK-92 cells were assessed. The viability, apoptosis, invasion, and migration ability of EESCs co-cultured with NK-92 cells at different ratios were evaluated. The apoptosis-related proteins, invasion and migration-related proteins as well as MEK/ERK pathway were examined via western blot. Each experiment was repeated three times. Results IL-2 activation enhanced NK-92 cytotoxicity in a concentration-dependent manner. Co-culturing EESCs with IL-2-activated NK-92 cells at E:T ratios of 1:1, 1:3, and 1:9 reduced EESC viability by 20%, 45%, and 70%, respectively, compared to the control group. Apoptosis rates in EESCs increased in correlation with the NK-92 cell proportion, with the highest rate observed at a 1:9 ratio. Moreover, EESC invasion and migration were significantly inhibited by IL-2-activated NK-92 cells, with a 60% reduction in invasion and a 50% decrease in migration at the 1:9 ratio. Besides, the MEK/ERK signalling pathway was down-regulated in EESCs by IL-2-activated NK-92 cells. Conclusion IL-2-activated NK-92 cells exhibit potent cytotoxic effects against EESCs. They promote EESC apoptosis and inhibit viability, invasion, and migration through modulating the MEK/ERK signalling pathway.

Assessment of immunohistochemical expression of trophoblast cell surface antigen 2 (Trop 2) in oral squamous cell carcinoma and its clinicopathological correlation

Trophoblast cell surface antigen 2 (Trop 2) is a transmembrane glycoprotein that is involved in a variety of oncogenic cell signaling pathways. Its heterogeneous expression in normal and cancerous tissues makes it a diagnostic and prognostic marker for various malignancies, including Oral Squamous Cell Carcinoma (OSCC). To assess the immunohistochemical expression of Trop 2 in OSCC and evaluate its clinicopathological correlation. Histopathologically diagnosed 40 cases of OSCC and their respective clinical records were retrieved and subsequent clinical staging and histopathological grading was done. Formalin fixed parrafin embedded sections were subjected to immunohistochemistry with anti Trop 2 antibody using standard DAB technique. The immunohistochemical expression of Trop 2 was evaluated using immunostaining intensity and mean integrated score followed by its comparison with age, gender, anatomical site, clinical stages and histopathological grades of OSCCTrop 2 expression was positive in 97.5% of cases. There was a statistically significant difference (p value<0.001) between immunostaining intensity and integrated score of Trop 2 among various histopathological grades of OSCC. As grade of OSCC increased, integrated score decreased. Trop 2 is one of the biomarkers that may serve as a useful tool to detect the changes undergoing at the molecular level which have prognostic or therapeutic significance.

Effects of Oral Cavity Stem Cell Sources and Serum-Free Cell Culture on Hydrogel Encapsulation of Mesenchymal Stem Cells for Bone Regeneration: An In Vitro Investigation.

To develop a stem cell delivery model and improve the safety of stem cell transplantation for bone regeneration, this study aimed to determine the effects of stem cell sources, serum-free cell culture, and hydrogel cell encapsulation on the growth and osteogenic differentiation of mesenchymal stem cells (MSCs) from the oral cavity. The study groups were categorized according to stem cell sources into buccal fat pad adipose (hBFP-ADSCs) (Groups 1, 4, and 7), periodontal ligament (hPDLSCs) (Groups 2, 5, and 8), and dental pulp-derived stem cells (hDPSCs) (Groups 3, 6, and 9). MSCs from each source were isolated and expanded in three types of sera: fetal bovine serum (FBS) (Groups 1-3), human serum (HS) (Groups 4-6), and synthetic serum (SS) (StemPro™ MSC SFM) (Groups 7-9) for monolayer (m) and hydrogel cell encapsulation cultures (e). Following this, the morphology, expression of MSC cell surface antigens, growth, and osteogenic differentiation potential of the MSCs, and the expression of adhesion molecules were analyzed and compared. SS decreased variations in the morphology and expression levels of cell surface antigens of MSCs from three cell sources (Groups 7m-9m). The levels of osteoblastic differentiation of the hPDLSCs and hBFP-ADSCs were increased in SS (Groups 8m and 7m) and the cell encapsulation model (Groups 1e, 4e, 7e-9e), but the promoting effects of SS were decreased in a cell encapsulation model (Groups 7e-9e). The expression levels of the alpha v beta 3 (ITG-αVβ3) and beta 1 (ITG-β1) integrins in the encapsulated cells in FBS (Group 1e) were higher than those in the SS (Group 7e). Human PDLSCs and BFP-ADSCs were the optimum stem cell source for stem cell encapsulation by using nanohydroxyapatite-calcium carbonate microcapsule-chitosan/collagen hydrogel in serum-free conditions.

Generation and characterization of mesenchymal stem cells from the affected femoral heads of dogs with Legg Calvé Perthes disease

Background: Canine Legg Calvé Perthes disease (LCPD) occurs during the growth period, and the cause of ischemic necrosis of the femoral head during growth remains unclear. If LCPD-affected femoral head-derived mesenchymal stem cells (LCPD-MSCs) can be generated, they can be used as a new tool for the pathophysiological analysis of canine LCPD. Aim: To generate affected femoral head-derived MSCs from dogs with LCPD and investigate the mRNA expression levels of angiogenesis-related factors and osteogenic differentiation potency of LCPD-MSCs. Methods: This study was performed using affected femoral heads from dogs diagnosed with LCPD and underwent femoral head and neck ostectomy. The necrotic tissue was harvested from the LCPD-affected femoral head and cultured statically (LCPD group, n=6). Canine bone marrow-derived MSCs (BM-MSCs) were used as controls (control group, n=6). First, the morphology of the cultured cells was observed, and the expression of CD29, CD34, CD44, CD45, CD90, and major histocompatibility complex class II was analyzed using flow cytometry. Additionally, the trilineage differentiation potency of the LCPD-affected head-derived adherent cells was examined. Furthermore, the expression levels of HIF1A, VEGFA, VEGFB, and PDGFB mRNAs and the bone differentiation potency of LCPD-affected head-derived adherent cells were investigated. Results: LCPD-affected femoral head-derived adherent cells showed a fibroblast-like morphology, and the expression of cell surface antigens was similar to that of BM-MSCs. In addition, LCPD-affected femoral head-derived adherent cells showed the same trilineage differentiation potency as BM-MSCs and were consistent with MSC characteristics. Furthermore, the mRNA expression levels of angiogenesis-related factors could be objectively measured in LCPD-MSCs and those MSCs had bone differentiation potency. Conclusion: In the present study, canine LCPD-MSCs were successfully generated, suggesting their usefulness as a tool for pathological analysis of LCPD in dogs.

Comparative expression of trophoblast cell-surface antigen 2 (TROP2) in the different molecular subtypes of invasive breast carcinoma: An immunohistochemical study of 94 therapy-naive primary breast tumors

BackgroundSacituzumab govitecan, targeting trophoblast cell-surface antigen 2 (TROP2), is approved for the treatment of triple-negative and hormone receptor-positive/HER2-negative breast cancers. However, detailed studies comparing TROP2 protein expression in the different molecular subtypes of breast cancer are limited, and definitive evidence supporting the use of TROP2 as a biomarker for predicting response to this agent in patients with breast cancer is currently lacking. ObjectiveTo compare the expression of TROP2 in the different molecular subtypes of breast cancer. MethodsImmunohistochemical staining for TROP2 was performed on 94 therapy-naive primary invasive breast carcinomas, including 25 luminal A-like, 25 luminal B-like, 19 HER2-like, and 25 triple-negative tumors. ResultsIntermediate to high levels of TROP2 expression were observed in the majority of carcinomas of each molecular subtype, with a wide range of expression in each subtype. Occasional tumors with low or absent TROP2 expression were encountered, including two metaplastic carcinomas which were completely negative for TROP2. ConclusionsOur observations support the continued investigation of the efficacy of sacituzumab govitecan in all molecular subtypes of breast carcinoma. Furthermore, the observed wide range of expression of TROP2 suggests that TROP2 may have potential utility as a biomarker for predicting responsiveness to sacituzumab govitecan. If this proves to be the case, then immunohistochemical staining for TROP2 would be critical for identifying those patients whose tumors are completely negative for TROP2, since these patients may be least likely or unlikely to respond to this agent, and alternative therapies may be more appropriate in such instances.

Multimodal Atlas of Paired Diagnosis and Relapse AML Samples Enables Novel Therapeutic Targeting of Surface Antigens

Note: A.H. and M.U. share co-first authorship. AML is an aggressive clonal malignancy characterized by combinations of chromosomal abnormalities, gene mutations, and cell surface antigen (Ag) expression profiles. This heterogeneity contributes to refractory or relapsed disease that presents a major challenge when treating AML. Previous single cell sequencing studies of primary AML samples have provided remarkable insight into the clonal architecture of AML cell populations and how their mutational, transcriptomic, and surface Ag profiles vary between patients. However, information addressing clonal shifts during progression from diagnosis to relapse in large cohorts is lacking. Here, we adapted single cell RNA sequencing with surface Ag feature barcoding to analyze more than 450,000 cells from 28 paired AML patient bone marrow mononuclear cell samples collected at diagnosis and relapse (56 samples). To our knowledge, it is the largest and most comprehensive single cell AML atlas to date. This atlas contains rich clinical metadata including cytogenetics, mutation status of canonical AML genes, treatment history, and survival information. We leveraged these data to identify potential correlations with clonal heterogeneity during progression to relapse and to propose novel strategies for targeting the surface of AML cells. The feature barcoding panel consists of a comprehensive list of 81 surface Ags reported in clinicaltrials.gov or mined from literature, including large proteomics mass spectrometry datasets of AML patient bone marrow samples (deBoer et. al. Cancer Cell 2018; Jayavelu et al, Cancer Cell 2022). Feature barcoding produces antibody derived tag (ADT) counts which are interpreted as relative values but alone does not indicate absolute number of Ags per cell. We addressed this limitation by measuring absolute Ag density of AML Ags, CD33, CLL1, CD123, and EMR2 in patient samples using the flow cytometric QuantiBRITE assay. Focusing on these four Ags, we modeled the relationship between ADT expression and antibodies per cell (ABC) Ag density from QuantiBRITE. The model was applied to impute absolute Ag density for the remaining 77 Ags in all samples. UMAP visualization after sample transcriptome integration revealed distinct clustering of CD45-dim myeloblasts, T cells, B cells, and erythroid cells. Longitudinal analysis identified 28 surface Ags that were differentially expressed (absolute log 2FC > 1, p < 0.01) between relapse and diagnosis myeloblasts across multiple patients (Fig. 1A). An additional 19 Ags were differentially expressed in no more than one patient while the other 19 had no significant change in any patient, highlighting the inter-and intra- heterogeneity of AML at diagnosis and relapse in these patients. Targeting multiple antigens simultaneously may help address the issue of antigen heterogeneity of tumor cells and help avoid potential antigen escape. Because multi-targeting therapies are a potential solution to this, weleveraged the single-cell resolution of our atlas to systematically identify Ags that in combination provide maximum coverage and are significantly co-expressed in myeloblasts. From this analysis, we found 48 and 52 co-expressed Ag pairs in diagnosis and relapse blasts, respectively (r > 0.4, p < 0.01; Fig. 1B). Of note, CD44 and EMR2 were co-expressed at both diagnosis (r = 0.49 p < 0.01) and relapse (r = 0.3, p < 0.01) with >90% of their blasts expressing CD44, EMR2, or both at targetable antigen levels of >1000 Ags/cell. Furthermore, 10 samples had >40% of their blasts co-expressing both Ags simultaneously at >1000 Ags/cell at diagnosis or relapse. 6 of these patients had a >10% increase in co-expression at relapse, 3 had >10% decrease at relapse, and one patient had no co-expression change. Our comprehensive profiling of AML has provided high resolution insight on cell surface at diagnosis and during disease progression and produced novel hypotheses about targetable combinations of surface Ags based on their expression changes between timepoints, co-expression, and surface densities. Furthermore, our reference atlas can be further dissected to characterize the transcriptome and mutational profiles of myeloblasts at diagnosis and relapse thereby prodding an important resource to create surface antigen protein - transcript correlations within AML blasts and to trace molecular signatures during disease progression.

ImmunoPET imaging of Trop2 expression in solid tumors with nanobody tracers.

The high expression of the transmembrane glycoprotein trophoblast cell-surface antigen 2 (Trop2) was strongly associated with the progression of solid tumors, including pancreatic and gastric cancers. Our study aimed to construct Trop2-specific immuno-positron emission tomography (immunoPET) probes and assess the diagnostic abilities in preclinical pancreatic and gastric cancer models. The expression of Trop2 in pancreatic cancer was determined by single-cell sequencing and immunohistochemistry on tissue microarray (TMA). Flow cytometry was used to screen the expression of Trop2 in pancreatic cancer cell lines. Two nanobodies (i.e., RTD98 and RTD01) targeting Trop2 were developed and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) to construct immunoPET imaging probes. The agents were researched in cell-derived pancreatic and patient-derived gastric cancer models expressing varying Trop2. Single-cell sequencing results showed high expression of Trop2 in pancreatic ductal cells as well as acinar cells and immunohistochemical staining of TMA from pancreatic cancers showed significantly higher expression of Trop2 in cancerous than in paracancerous tissues. ImmunoPET utilizing [68Ga]Ga-NOTA-RTD98 could clearly delineate subcutaneous tumors, both in cell-derived pancreatic cancer models and patient-derived gastric cancer models, superior to imaging using [18F]-FDG or a non-specific probe [68Ga]Ga-NOTA-RTD161. Another probe with improved pharmacokinetics targeting Trop2, [68Ga]Ga-NOTA-RTD01, was further prepared and showed advantageous diagnostic capabilities in preclinical pancreatic cancer models. In the work, we reported two nanobody tracers targeting human Trop2 which may facilitate better use of Trop2-targeted therapeutics by noninvasively displaying expression dynamics of the target.

Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

Pan-Cancer Proteomics Analysis to Identify Tumor-Enriched and Highly Expressed Cell Surface Antigens as Potential Targets for Cancer Therapeutics

The National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) provides unique opportunities for cancer target discovery using protein expression. Proteomics data from CPTAC tumor types have been primarily generated using a multiplex tandem mass tag (TMT) approach, which is designed to provide protein quantification relative to reference samples. However, relative protein expression data are suboptimal for prioritization of targets within a tissue type, which requires additional reprocessing of the original proteomics data to derive absolute quantitation estimation. We evaluated the feasibility of using differential protein analysis coupled with intensity-based absolute quantification (iBAQ) to identify tumor-enriched and highly expressed cell surface antigens, employing tandem mass tag (TMT) proteomics data from CPTAC. Absolute quantification derived from TMT proteomics data was highly correlated with that of label-free proteomics data from the CPTAC colon adenocarcinoma cohort, which contains proteomics data measured by both approaches. We validated the TMT-iBAQ approach by comparing the iBAQ value to the receptor density value of HER2 and TROP2 measured by flow cytometry in about 30 selected breast and lung cancer cell lines from the Cancer Cell Line Encyclopedia. Collections of these tumor-enriched and highly expressed cell surface antigens could serve as a valuable resource for the development of cancer therapeutics, including antibody–drug conjugates and immunotherapeutic agents.

Polarization of THP-1-Derived Macrophage by Magnesium and MAGT1 Inhibition in Wound Healing.

Background Macrophages play a major role in wound healing and prevent infection from the outside. Polarization conversion of macrophages regulates aspects of inflammation, and two macrophages, M1 (classically activated) and M2 (alternatively activated), exist at both ends of broad-spectrum macrophage polarization. Thus, we aimed to investigate whether macrophage polarization can be artificially regulated. To this end, MgSO4 and small-interfering RNA (siRNA) targeting magnesium transport 1 (MAGT1) were used to investigate the effects of intracellular magnesium (Mg2+) concentrations on the differentiation of macrophages in vitro. Methods THP-1 derived macrophages maintained in a culture medium containing 5 mM MgSO4 and siRNA to inhibit the expression of MAGT1. As comparative groups, THP-1 derived macrophages polarized into M1 and M2 macrophages by treatment with M1, M2 inducer cytokine. The polarization status of each group of cells was confirmed by cell surface antigen expression and cytokine secretion. Results We found that MgSO4 treatment increased CD163 and CD206, similar to the effect noted in the M2 group. The expression of CD80 and HLA-DR was increased in the group treated with MAGT1 siRNA, similar to the effect noted in the M1 group. Functional assays demonstrated that the group treated with MgSO4 secreted higher levels of IL-10, whereas the MAGT1 siRNA-treated group secreted higher levels of IL-6 cytokines. Additionally, the conditional medium of the Mg2+ treated group showed enhanced migration of keratinocytes and fibroblasts. Conclusion Mg2+ can help to end the delay in wound healing caused by persistent inflammation in the early stages.

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1040.

Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

Bone marrow-derived dedifferentiated fat cells exhibit similar phenotype as bone marrow mesenchymal stem cells with high osteogenic differentiation and bone regeneration ability

BackgroundMesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model.MethodsBM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice.ResultsBM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model.ConclusionsWe showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

M1 macrophages may be effective adjuvants for promoting Th‑17 differentiation in HBeAg positive hepatitis patients with ALT ≤2ULN.

Hepatitis B virus (HBV) infection can activate macrophages to accelerate liver disease progression, including inflammation and fibrosis. However, the exact mechanism remains undetermined. The present study assessed the effects of macrophage polarization and the related cytokines on Th‑17 differentiation in HBeAg positive individuals with a HBV infection, and also evaluated the potential association of Th‑17 cell frequency with the severity of liver injury. A cross‑sectional study design was used to collect the clinical parameters, blood samples and liver tissue samples of patients with alanine transaminase £2x upper limit of normal and confirmed hepatitis B who underwent liver puncture in Qishan Hospital between January 2019‑December 2021. Macrophage and Th‑17 cell related factors were assayed using ELISA. The expression and quantification of cell surface antigen and intracellular markers in cells were assessed using flow cytometry. Pathological staining, including hematoxylin and eosin, reticular fiber staining and immunohistochemical staining were used to assess inflammation and fibrosis in the liver tissue. In the peripheral blood of patients with HBV infection, the number of CD14+ macrophages was significantly increased compared with the healthy control, especially in the hepatitis B e antigen (HBeAg) positive group. CD14+ macrophages were predominantly of the M1 type based on the assessment of the phenotype using flow cytometry and cytokine secretion. Furthermore, the percentage of M1 phenotype and related cytokines were positively correlated with Th‑17 differentiation. IL‑17A secreted by Th‑17 was positively correlated with the degree of liver inflammation and fibrosis, as well as with the severity of liver disease, which indicated that the differentiation of Th‑17 may be involved in the progression of liver disease. HBeAg may promote Th‑17 differentiation and IL‑17A production by M1 macrophages to accelerate the pathogenesis of liver inflammation and fibrosis in CHB patients.

A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets.

Natural killer (NK) cells are immune effector cells with outstanding features for adoptive immunotherapy. Immune effector cells with chimeric antigen receptors (CARs) are promising targeted therapeutic agents for various diseases. Because tumor cells exhibit heterogeneous antigen expression and lose cell surface antigen expression during malignant progression, many CARs fixed against only one antigen have limited efficacy and are associated with tumor relapse. To expand the utility of CAR-NK cells, we designed a split and universal cotinine-CAR (Cot-CAR) system, comprising a Cot-conjugator and NK92 cells (α-Cot-NK92 cells) engineered with a CAR containing an anti-Cot-specific single-chain variable fragment and intracellular signaling domain. The efficacy of the Cot-CAR system was assessed in vitro using a cytolysis assay against various tumor cells, and its single- or multiple- utility potential was demonstrated using an in vivo lung metastasis model by injecting A549-Red-Fluc cells. The α-Cot-NK92 cells could switch targets, logically respond to multiple antigens, and tune cytolytic activation through the alteration of conjugators without re-engineering. Therefore the universal Cot-CAR system is useful for enhancing specificity and diversity of antigens, combating relapse, and controlling cytolytic activity. In conclusion, this universal Cot-CAR system reveals that multiple availability and controllability can be generated with a single, integrated system.

Elucidation of adipogenic differentiation regulatory mechanism in human maxillary/mandibular bone marrow-derived stem cells

ObjectiveThis study aims to investigate the underlying molecular mechanisms that regulate the adipogenic differentiation of maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs). DesignMBMSCs and iliac bone marrow-derived MSCs (IBMSCs) were compared for osteogenic, chondrogenic, and adipogenic differentiation. Cell surface antigen expression was examined using flow cytometry, and stem cell marker expression was assessed using real-time polymerase chain reaction (PCR). Various adipogenic regulatory factors’ expression was evaluated using real-time PCR and western blotting. ResultsNo significant differences in cell surface antigen profiles or stem cell marker expression in MBMSCs and IBMSCs were observed. MBMSCs and IBMSCs displayed similar osteogenic and chondrogenic potentials, whereas MBMSCs showed significantly lower adipogenic potentials than those shown by IBMSCs. Expression of CCAAT/enhancer binding protein β (C/EBPβ), C/EBPδ, early B-cell factor 1 (Ebf-1), and Krüppel-like factor 5 (KLF5), which are early adipogenic differentiation factors, was suppressed in MBMSCs compared to that in IBMSCs. Peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which play important roles in the terminal differentiation of adipocytes, was lower in MBMSCs than that in IBMSCs. Furthermore, the level of zinc finger protein 423 (Zfp423), which is involved in the commitment of undifferentiated MSCs to the adipocyte lineage, was significantly lower in MBMSCs than that in IBMSCs. ConclusionsMBMSCs are negatively regulated in the commitment of undifferentiated MSCs to the adipocyte lineage (preadipocytes) as well as in the terminal differentiation of preadipocytes into mature adipocytes. These results may elucidate the site-specific characteristics of MBMSCs.

Salmonella Subpopulations Identified from Human Specimens Express Heterogenous Phenotypes That Are Relevant to Clinical Diagnosis.

Clonal bacterial cells can give rise to functionally heterogeneous subpopulations. This diversification is considered an adaptation strategy that has been demonstrated for several bacterial species, including Salmonella enterica serovar Typhimurium. In previous studies on mouse models infected orally with pure Salmonella cultures, derived bacterial cells collected from animal tissues were found to express heterogenous phenotypes. Here, we show mixed Salmonella populations, apparently derived from the same progenitor, present in human specimens collected at a single disease time point, and in a long-term-infected patient, these Salmonella were no longer expressing surface-exposed antigen epitopes by isolates collected at earlier days of the disease. The subpopulations express different phenotypes related to cell surface antigen expression, motility, biofilm formation, biochemical metabolism, and antibiotic resistance, which can all contribute to pathogenicity. Some of the phenotypes correlate with single nucleotide polymorphisms or other sequence changes in bacterial genomes. These genetic variations can alter synthesis of cell membrane-associated molecules such as lipopolysaccharides and lipoproteins, leading to changes in bacterial surface structure and function. This study demonstrates the limitation of Salmonella diagnostic methods that are based on a single-cell population which may not represent the heterogenous bacterial community in infected humans. IMPORTANCE In animal model systems, heterogenous Salmonella phenotypes were found previously to regulate bacterial infections. We describe in this communication that different Salmonella phenotypes also exist in infected humans at a single disease time point and that their phenotypic and molecular traits are associated with different aspects of pathogenicity. Notably, variation in genes encoding antibiotic resistance and two-component systems were observed from the subpopulations of a patient suffering from persistent salmonellosis. Therefore, clinical and public health interventions of the disease that are based on diagnosis of a single-cell population may miss other subpopulations that can cause residual human infections.

Expression of programmed cell death-1 on neuroblastoma cells in TH-MYCN transgenic mice.

Immunotherapy may improve the poor prognosis of high-risk neuroblastoma. Programmed cell death-1 (PD-1) is expressed in several cancers. The tyrosine hydroxylase MYCN (TH-MYCN) transgenic mouse model is widely used in neuroblastoma research, but detailed information on its immunological background is lacking. Therefore, we studied the immunological tumor microenvironment and tumor cell surface antigen expression in homozygote and hemizygote mice and effects of antibody therapy against PD-1. CD4, CD8, CD11b, and CD11c expression in immune cells from retroperitoneal lymph nodes and spleen was analyzed by flow cytometry. Tumor cell surface antigen expression was confirmed, and data from homozygote and hemizygote mice were compared. Effects of anti-PD-1 antibody were evaluated. CD4-, CD8-, CD11b-, and CD11c-positive cells were not significantly different in homozygote and hemizygote mice, and CD11b- and CD11c-positive cells were identified in the tumor microenvironment in both. Tumor cells expressed PD-1, and anti-PD-1 antibody had anti-tumor effects and significantly reduced the percentage of living tumor cells in cultures after 2h. The immunological background is similar in homozygote and hemizygote TH-MYCN transgenic mice, and both have PD-1-positive tumor cells. Anti-PD-1 antibody suppresses tumor growth. This mouse model may be a useful for studying immunotherapy of neuroblastoma.

LAMTOR1 degrades MHC-II via the endocytic in hepatocellular carcinoma.

Tumor cell surface antigen recognition is a major hallmark of cancer therapy, and loss of major histocompatibility complex class I (MHC-I) is the most common mechanism that impairs tumor cell surface antigen processing and expression. In addition to this, MHC-II regulates antigen presentation in CD4+ T cell immune responses involved in tumor killing by CD8+ T cells, whereas the regulation of endocytosis regulating MHC-II antigen presentation has not been reported. Therefore, the regulation of the endocytosis pathway on the expression of MHC-II surface level and antitumor T cell response remains to be explored. In this experiment, we found that LAMTOR1 regulates the endocytic pathway through the GTPase domain of DNM2 and triggers the formation of autophagosomes. We performed flow cytometry and western blotting analyses, which revealed that the expression of MHC-II molecules on the surface of cells is influenced by LAMTOR1 through the endocytic pathway. We showed that the expression of MHC-II molecules, which recognize CD4+ T cells on the surface of cells, was regulated by LAMTOR1 through an endocytic pathway. By coculture experiments, we showed that CD8+/CD4+ T cells exhibit substantially higher levels of tumor cell apoptosis than those observed when hepatocellular carcinoma (HCC) cells were cocultured with CD8+ T cells alone. This study revealed that LAMTOR1 decreases the expression levels of MHC-II on cell surfaces in order to reduce antigen expression, leading to a decrease in antitumor T cell responses.

The expression level of secretory mature B cell surface antigen in primary diagnosed multiple myeloma and its clinical significance

Objective: To explorer Secretory mature B cell surface antigen (sBCMA) expression level, changes during treatment and clinical significance in newly diagnosed MM patients. Methods: Clinical data of 158 MM patients admitted to the Department of Hematology, the First Affiliated Hospital of Soochow University from August 2018 to September 2020 were analyzed retrospectively. The concentration of sBCMA in the patients was determined by BCMA ELISA and compared with the normal range. The results were compared with clinical efficacy, age, type, R-ISS stage, renal impairment, and humoral immune function. Results: The median age of the patients was 57 (31-73 years old), 86 (54.5%) males and 72 (45.5%) females, mainly IgG type, 81 patients(51.2%). SBCMA value M(Q1,Q3) was 76.50 (55.50, 94.40) μg/L, 100% higher than the upper limit of normal value. According to the efficacy evaluation, the patients were divided into complete remission(CR) group, very good partial remission(VGPR) group, partial remission(PR) group and ineffiecacy group, the results showed the level of sBCMA in CR group[80.10 (58.05, 96.90) vs 15.70 (9.85, 28.65) μg/L] and VGPR group[74.60 (52.20, 93.00) vs 17.20 (13.30, 38.80) μg/L]was significantly higher than that before treatment(all P<0.001), and there was no significant difference in PR group and ineffective group before and after treatment (all P>0.05).The amount of serum intact protein M protein was positively correlated with the level of sBCMA expression in newly diagnosed patients (r=0.22, P=0.040), and there was no correlation between the proportion of bone marrow plasma cells and sBCMA expression (r=0.07, P=0.449).The correlation between sBCMA levels at initial diagnosis and MM type[IgG type, IgA type vs light chain type:(78.6±3.5), (72.4±5.4) vs (83.8±6.9)μg/L], age[≥65 vs<65 years: (73.6±5.5)vs (79.3±3.1)μg/L], R-ISS stage[stage Ⅰ, Ⅱ vs Ⅲ:(80.2±3.1) vs (69.4±6.1)μg/L], renal impairment [Creatinine clearance rate (Ccr) ≤30 vs>30 ml/min:(81.6±4.8) vs (76.5±3.4)μg/L], and high-risk karyotype[high-risk vs standard-risk:(73.6±5.7) vs (80.2±3.2)μg/L] were not associated (all P>0.05). Expression levels of sBCMA were negatively correlated with IgM levels in MM patients (r=-0.39, P=0.002) and after treatment (r=-0.25, P=0.015). Conclusions: The expression of sBCMA in MM patients is a reliable indicator of the clinical efficacy of MM and is related to the occurrence of MM immune deficiency and recovery after treatment. sBCMA can be used as a new independent marker for monitoring and predicting the efficacy of MM patients.