Journal: Differentiation; research in biological diversity

Article Title: Transformation of jaw muscle satellite cells to cardiomyocytes

doi: 10.1016/j.diff.2016.11.003

Figure Lengend Snippet: A-C. Induced cardiomyocytes visualized by immunostaining at day 14 of culture. A. Cardiac troponin T (cTnT). B. α-myosin heavy chain. C. Connexin 43 showing gap junctions between adjacent cells D. Expression of five genes for cardiomyocyte terminal differentiation markers (qRT-PCR). Error bars indicate ± standard error of the mean. E. Typical spontaneous action potentials for E13.5 embryonic cardiomyocytes; induced cardiomyocyte; and evoked action potential for skeletal myotube. The duration of the induced cardiomyocyte action potentials is similar to those of the embryonic cardiomyocytes and much longer than those of the myotubes. F. Effects of norepinephrine, carbachol and nifedipine on spontaneous action potentials. Colored overlayed traces indicate spontaneous action potential after addition of each drug. G. Calcium transients in induced cardiomyocytes visualized with Fluo3AM. The traces indicate the signal for the four indicated cells, which are highly synchronized. Scale bar 20μm.All measurements were repeated several times as indicated, with standard errors, in the text.

Article Snippet: Beating aggregates of induced cardiomyocytes were re-plated on glass culture slides and immunostained for cTnT (CT-3; DSHB) using a far-red tagged (Cy5) secondary antibody and visualized for co-localization with GFP.

Techniques: Immunostaining, Expressing, Quantitative RT-PCR

Journal: Differentiation; research in biological diversity

Article Title: Transformation of jaw muscle satellite cells to cardiomyocytes

doi: 10.1016/j.diff.2016.11.003

Figure Lengend Snippet: A. Evidence that induced cardiomyocytes are derived from satellite cells. Pax7 expressing cells were labeled by treating the Pax7-CreER;mT/mG mice with tamoxifen. The green label indicates that cTnT-positive cardiomyocytes formerly expressed Pax7. Scale bar 25μm. B. Evidence that induced cardiomyocytes are derived from the secondary heart field. Satellite cells were prepared from Isl1-Cre;mTmG mice and then sorted for GFP expression, and subjected to the cardiomyocyte differentiation procedure. The NKX2.5 and cTnT-positive cells must be derived from cells that had expressed Isl1 in vivo. Scale bar: 25μm.

Article Snippet: Beating aggregates of induced cardiomyocytes were re-plated on glass culture slides and immunostained for cTnT (CT-3; DSHB) using a far-red tagged (Cy5) secondary antibody and visualized for co-localization with GFP.

Techniques: Derivative Assay, Expressing, Labeling, In Vivo

Journal: The Journal of infectious diseases

Article Title: Aberrant Humoral Immune Responses in Neurosyphilis: CXCL13/CXCR5 Play a Pivotal Role for B-Cell Recruitment to the Cerebrospinal Fluid.

doi: 10.1093/infdis/jix233

Figure Lengend Snippet: Figure 5. Pathologic diagnosis of intracranial syphilitic gumma and cellular organization of follicle-like structures. HE staining of intracranial syphilitic gumma (first row). Immunostaining of spirochete in intracranial syphilitic gumma (second row). Immunostaining of CXCL13 in intracranial syphilitic gumma and follicle-like structure (third row). Immunostaining of CXCR5 in intracranial syphilitic gumma and follicle-like structure (fourth row). Immunostaining of CD20+ B cells in intra- cranial syphilitic gumma and follicle-like structure (fifth row). Immunostaining of CD3+ T cells in intracranial syphilitic gumma and follicle-like structure (sixth row). Immunostaining of CD35+ FDCs in intracranial syphilitic gumma and follicle–like structure (seventh row). Immunostaining of CD138+ plasma cells in intracranial syphilitic gumma and follicle-like structure (eighth row). Abbreviations: FDCs, follic- ular dendritic cells; HE, hematoxylin and eosin.

Article Snippet: Paraffin-embedded brain biopsy (4 μm) was stained with appropriate concentration of primary antibodies against CXCL13, CXCR5, CD20, CD3, CD35, CD138 (Maixin-Bio, Fujian, China), and spirochete (Biocare Medical, Pacheco, CA), then incubated with streptavidin-biotin complex system according to manufacturer’s instructions (Boster, Pleasanton, CA).

Techniques: Biomarker Discovery, Staining, Immunostaining, Clinical Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: Single-cell RNA-Seq reveals transcriptome heterogeneity of in vitro differentiated Th2 cells. Naïve CD4+ T cells (CD4+CD62LhiCD44lowCD25−) isolated from C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-4 and anti-IFN-γ. Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system. A total of 56 cells were collected and sequenced with an Illumina Hiseq system, and 10,000 pooled cells were sequenced using a standard protocol (Pooled cells). A, correlations of gene expression between the pooled cells and average of the 56 single cells (left panel), between two randomly selected single cells (center panel), and between the pooled cells and a single cell (right panel). 10,565 genes were detected. Gene expression was determined by TPM, and Pearson correlation coefficient (r) is depicted. B, heatmap depicting Pearson correlation coefficients between global gene expression profiles of each pair of the 56 individual cells. C, cytokine genes were abundantly expressed and variable among Th2 single cells. Average expression (μ, x axis) and standard deviation (σ, y axis) of each gene were calculated and plotted. The blue dashed lines represent the threshold of CV (highly variable, >0.28; less variable, <0.2). The black dashed line represents the threshold of abundantly expressed genes (μ > 8). Red represents cytokine genes, green denotes housekeeping genes, and gray represents other genes. D, gene ontology (GO) analysis of the most variably expressed genes (μ > 8 and CV > 0.28, left panel) and most consistently expressed genes (μ > 8 and CV < 0.2, right panel). The top five most significantly enriched GO terms (biological processes and molecular functions) are shown. The p values were Bonferroni-corrected.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: RNA Sequencing Assay, In Vitro, Isolation, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: IL4nc is constitutively expressed in Th2 cells. A, IL4nc was specifically expressed in Th2 and Th0 cells. Naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 and differentiated into various lineages under the respective conditions. Five days after differentiation, expression of IL4nc was analyzed by real-time PCR either under steady state or after being activated by anti-CD3 and anti-CD28 or PAM and ionomycin for 5 h. B, IL4nc is primarily located in the cytoplasm. D10.G4.1 cells were fractionated into the nuclear (N) fraction and cytoplasmic (C) fraction, and the presence of the IL4nc isoform was determined by real-time PCR (left panel). The protein lysates from both fractions were further analyzed by immunoblotting for the cytoplasmic marker (B-actin) and nucleus marker (histone H3) (right panel). C, expression of either IL4nc or IL4fl in resting in vitro differentiated Th2 cells was determined by real-time PCR. D, induction of IL4nc RNA precedes IL4fl following TCR stimulation. Th2 cells were differentiated as described and restimulated with anti-CD3/anti-CD28 for the indicated time. Expression of either IL4nc or IL4fl was determined by real-time PCR. Data are representative (B) or the sum (A–D) of at least three independent experiments. Error bars stand for the standard deviation of the mean. **, p < 0.01 in paired Student's t test. ns, not significantly changed.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, In Vitro, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: IL4nc RNA promotes IL-4 production in Th2 cells. A, a diagram showing the gene structure of IL4fl (top panel) or IL4nc RNAs (bottom panel). The gray boxes depict noncoding region of transcripts, and the black boxes depict coding regions. B and C, overexpression of IL4nc promotes IL-4 production in Th2 cells. Th2 cells were differentiated in vitro as described in Fig. 1 and transduced with a retrovirus containing either IL4nc RNA or empty vector (Mock). 5 days after stimulation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. Expression of IL-4 was determined by intracellular staining. Data shown are representative (B) or summary (C) of IL-4+% or IL-13+% cells from three independent experiments. D, Th2 cells were differentiated and stimulated as in B, and secreted IL-4 in the supernatant was determined by ELISA. Data are summary of three independent experiments. E–G, exon 3 of IL4nc is required for promoting IL-4 production post-transcriptionally. As above, full-length IL4nc (FL) or its mutants depleted the indicated exons (Δexon1, Δexon2, and Δexon3, respectively) and were retrovirally expressed in Th2 cells. Production of IL-4 was analyzed by intracellular staining (E and F) or ELISA (G). H, as in E. Expression of IL-4 and GATA3 mRNAs 2 h after restimulation was analyzed by real-time quantitative PCR. I, as in B. Th2 cells were transduced with a retrovirus overexpressing IL4nc RNA, and the cells were selected with puromycin for 3 days (3 μg/ml). 5 days after differentiation, expression of IL4fl was analyzed by real-time PCR after activation by anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) (left panel) or PMA (50 ng/ml) and ionomycin (500 ng/ml) (right panel) for the indicated time. Data are representative (B and E) or the sum (C, D, and F–I) of at least three independent experiments. Error bars stand for the standard deviation of the mean. *, p < 0.05; **, p < 0.01; paired Student's t test. ns, not significantly changed.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: Over Expression, In Vitro, Transduction, Plasmid Preparation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activation Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Single-cell RNA-Seq analysis identifies a noncoding interleukin 4 ( IL-4 ) RNA that post-transcriptionally up-regulates IL-4 production in T helper cells

doi: 10.1074/jbc.RA118.004111

Figure Lengend Snippet: Knockdown of IL4nc RNA represses IL-4 production. Th2 cells were differentiated in vitro as described in Fig. 1 and transduced with a retrovirus containing a scramble sequence (sh-Ctl) or two independent shRNAs against IL4nc RNA. Five days after differentiation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. A–C, production of IL-4 was determined by intracellular staining (A and B) and ELISA (C). D, as in A. The cells were restimulated with anti-CD3 and anti-CD28, and expression of IL4fl (left panel) and IL4nc (right panel) RNA was analyzed by real-time PCR at the indicated time after restimulation. E, as in A. 5 days after differentiation, the cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 3.5 h. The cells were then treated with cycloheximide (100 μg/ml), lysed, and fractionated on a 10–50% sucrose gradient. Distribution of IL4 mRNA at each fraction was analyzed by real-time PCR. Distribution of ribosomal RNA is shown in Fig. S6. F, as in E. In vitro differentiated Th2 cells were transduced with a retrovirus overexpressing IL4nc (IL4nc) or an empty vector (Mock). Distribution of IL4 mRNA at each fraction after sucrose density gradient centrifugation was determined by real-time PCR. Data are representative (A) or the sum (B–F) of at least three independent experiments. Error bars stand for the standard deviation of the mean. *, p < 0.05; **, p < 0.01; paired Student's t test.

Article Snippet: Five days after stimulation, the cells were restimulated with plate-bound anti-CD3 and anti-CD28 for 6 h, and single cells were captured with a Fluidigm C1 system.

Techniques: In Vitro, Transduction, Sequencing, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Gradient Centrifugation, Standard Deviation

Journal: Annals of Translational Medicine

Article Title: Hyperion imaging system reveals heterogeneous tumor microenvironment of oral squamous cell carcinoma patients at T1N0M0 stage

doi: 10.21037/atm-20-7194

Figure Lengend Snippet: Antibodies targeting the tumor environment of oral squamous cell carcinoma (OSCC)

Article Snippet: CD3 , Er170 , Polyclonal, C-Termina , Fluidigm , 3170019D.

Techniques:

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Optimization of γδ T cell electroporation with bicistronic CD19 CAR-GFP mRNA (A) γδ T cells express GFP after mRNA electroporation using the BioRad Gene Pulser Xcell Electroporator. γδ T cell electroporation was optimized by testing varying cell numbers and mRNA amounts in each reaction. (B) Cell yield, calculated by determining the proportion of live cells remaining 24 h after electroporation to the starting number of cells used for the electroporation reaction, was calculated for all reaction conditions and increased as the cell number increased. (C) GFP mean fluorescence intensity (MFI) was determined by flow cytometry and increased with increasing amounts of mRNA. (D) The percentage of live cells expressing GFP and the CD19 CAR was similar for all conditions and was found to be about 90% and 60%, respectively. (E) γδ T cell cytotoxicity was determined by flow cytometry to test two promising electroporation reaction conditions and found no difference when comparing different cell numbers in each reaction.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Electroporation, Fluorescence, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Electroporation of γδ T cells before freezing results in lower CAR expression and reduced cytotoxicity γδ T cells were electroporated on day 12 of expansion and were analyzed before and after freezing. (A) While GFP expression (circles) remained constant at around 90% before and after freezing, the CAR percentage (triangles) decreased from about 60% before freezing to about 30%–40% after thawing. Closed data points denote before freezing and open data points denote after thawing. (B) The cytotoxicity of CD19 CAR-expressing γδ T cells before and after freezing was also measured to determine if a freeze/thaw cycle effects the cytotoxicity of the engineered cells. Similar cytotoxicity was observed at low effector to target (E:T) ratios; however, there was a reduction at higher E:T ratios for the thawed engineered cells.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Electroporation, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: CD19 CAR- and CD22 CAR-expressing γδ T cells enhances cytotoxicity against two B-ALL cell lines Effector and target cells were cocultured at the specified E:T ratio for 4 h and the percent cytotoxicity was determined by flow cytometry. Target cells were stained with VPD450 to differentiate effector and target cell death. Mock-electroporated, CD19 CAR-, and CD22 CAR-expressing γδ T cells were tested against the B-ALL cell lines 697 (A and B) and Nalm6 (C). While the cytotoxicity of mock-electroporated γδ T cells remained constant over all E:T ratios, CD19 CAR- and CD22 CAR-expressing γδ T cells exhibited a dose-dependent increase in cytotoxicity.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Expressing, Flow Cytometry, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: γδ T cells express and secrete CD19 sBite after mRNA electroporation (A) γδ T cells were electroporated with 3 μg, 7.5 μg, and 15 μg of mRNA and the amount of CD19 sBite in the conditioned media was determined using an ELISA. (B) Unmodified and CD19 sBite-modified γδ T cells were cocultured with several CD19 + cancer cells lines to test their cytotoxic capabilities. sBite-modified γδ T cells showed increased cytotoxicity at all E:T ratios and reached about 90% at the 5:1 ratio. (C) A CD19KO 697 cell line was generated using CRISPR to test the specificity of the secreted CD19 sBite. As expected, the sBite-modified γδ T cells showed improved cytotoxicity against the naive 697 cell line; however, no increase in cytotoxicity was seen when the CD19KO 697 cell line was used as target cells. (D) To test whether the CD19 sBite can induce killing of unmodified cells, conditioned media from unmodified and sBite-modified γδ T cells was collected after 16 h of culture and mixed with either unmodified or sBite-modified γδ T cells. As expected, the sBite-modified cells showed improved cytotoxicity, regardless of the conditioned media. The unmodified cells cultured with the sBite-conditioned media showed improved cytotoxicity, compared with unmodified cells with unmodified conditioned media.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Electroporation, Enzyme-linked Immunosorbent Assay, Modification, Generated, CRISPR, Cell Culture

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: γδ T cells are not able to kill cancer cells once they extravasate from circulation (A) In this cancer model, cancer cells are injected i.v. and gradually leave the circulation and form non-vascularized nodules in various organs and other compartments, leaving few cancer cells in circulation by days 3 and 7. Based on this model, it can be predicted that the timing for γδ T cell treatment is important in treating mice bearing the 697 cancer cell line. (B) Tissue samples from blood, bone marrow, and spleen were collected 3 weeks after cancer cell injection to detect the presence of cancer cells in each compartment (left flow plots are representative). A substantial number of CD45 + CD3 − 697 cells were detected in the bone marrow, while limited numbers were found in the blood and spleen. (C) Representative hematoxylin and eosin staining images showing the presence of cancer cells with no vasculature around the cancer cells. Histopathological analysis revealed the presence of cancer cells in the brain, liver, lungs, and kidneys, with avascularized nodules found within the liver.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Injection, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Engineered γδ T cells expressing a CD19 CAR reduce tumor burden and improve survival in the 697 model (A) NSG mice were injected with 2 × 10 6 luciferase-expressing 697 cells and bioluminescence images were captured during the course of the experiment. Mice treated with CD19 CAR-expressing γδ T cells on day 1 of the experiment showed a reduction in tumor burden compared with control mice. (B) Raw total flux values were calculated and showed delayed tumor progression and significantly reduced tumor burden for mice treated with the CD19 CAR-expressing γδ T cells (triangles), compared with the control mice (circles). Statistics were performed using a 2-tailed Student’s t test to compare experimental groups at each given time point. (C) Kaplan-Meier survival curves showed significantly increased survival in mice treated with CD19 CAR-expressing γδ T cells (dashed line), compared with control mice (p = 0.02 by log rank test). Control: n = 4; CD19 CAR: n = 3; error bars indicate standard deviation; ∗∗p < 0.01.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Expressing, Injection, Luciferase, Control, Standard Deviation

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Engineering γδ T cells with CD19 CAR or sBite mRNA reduces tumor burden and improves survival in the Nalm6 model NSG mice were injected with 2 × 10 6 luciferase-expressing Nalm6 cells and were treated with unmodified, CD19 CAR-modified, or CD19 sBite-modified γδ T cells on day 1 of the experiment with a treatment regimen of twice a week for 2 weeks. (A) Before injection, unmodified or modified γδ T cells were analyzed for CAR expression and MFI using flow cytometry. CAR expression was about 60% for CD19 CAR-expressing γδ T cells and, interestingly, the CD19 sBite-modified γδ T cells bound to the CD19Fc, with an average of about 40% CD19Fc positive (left graph). Despite the CD19 sBite-modified γδ T cell binding to the CD19Fc, the MFI was minimal compared with the CD19 CAR (right graph). (B) The cytotoxicity of the unmodified and modified γδ T cells were also examined before injection at E:T ratios of 1:2 and 2:1. The CD19 CAR- and sBite-modified γδ T cells exhibited increased cytotoxicity compared with the unmodified γδ T cells. (C) Bioluminescent imaging was performed during the experiment and mice treated with unmodified γδ T cells showed a high tumor burden as early as 1 or 2 weeks after cancer cell injection. (D) Raw total flux was determined for each image and graphed over time to compare treatment with unmodified γδ T cells and CD19 CAR-expressing (top graph) or CD19 sBite-expressing γδ T cells (bottom graph). Treatment with modified γδ T cells resulted in delayed tumor progression and reduced tumor burden. Statistics were performed using a 2-tailed Student’s t test to compare experimental groups at each given time point. (E) Kaplan-Meier survival curves were generated to compare survival for each treatment group to treating with unmodified γδ T cells. Treatment with CD19 CAR- and CD19 sBite-expressing γδ T cells resulted in a significant survival benefit compared with treating with unmodified γδ T cells (p = 0.01 for CAR and sBite by log rank test). n = 5; error bars indicate standard deviation; ∗p < 0.05.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: Injection, Luciferase, Expressing, Modification, Flow Cytometry, Binding Assay, Imaging, Generated, Standard Deviation

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Longer treatment regimen does not lengthen survival benefit for Nalm6 model Despite being significant, the survival benefit for the previous in vivo experiments was not as robust as the in vitro data would suggest. (A) To test whether a more extensive treatment regimen of three doses for the first 2 weeks, two doses for the next 2 weeks, and one dose for the final 2 weeks could further improve the survival benefit. (B) NSG mice were injected with 2 × 10 6 luciferase-expressing Nalm6 cells and treated with CD19 sBite-modified γδ T cells using the more extensive treatment regimen. Bioluminescent images were taken and again showed reduced tumor burden for the sBite-treated group, compared with the control group. (C) Graph of raw total flux shows the more extensive treatment regimen delayed tumor progression and reduced tumor burden, compared with control mice. The inset shows an expansion of the first 20 days of treatment. Statistics were performed using a 2-tailed Student’s t test. (D) Kaplan-Meier survival curves were generated for the control group and more extensive treatment regimen of CD19 sBite-expressing γδ T cells. As expected, the more extensive treatment regimen resulted in a significant survival benefit compared with the control group (p = 0.01 by log rank test); however, there was no difference in survival when comparing the more extensive treatment regimen with the previous regimen of twice a week for 2 weeks (p = 0.39 by log rank test). Control: n = 3; sBite: n = 4; error bars indicate standard deviation; ∗p < 0.05.

Article Snippet: Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard.

Techniques: In Vivo, In Vitro, Injection, Luciferase, Expressing, Modification, Control, Generated, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Allograft or Recipient ST2 Deficiency Oppositely Affected Cardiac Allograft Vasculopathy via Differentially Altering Immune Cells Infiltration

doi: 10.3389/fimmu.2021.657803

Figure Lengend Snippet: The effects of recipient or graft ST2 deficiency on CD3 + T cells infiltration in cardiac allograft. (A) The allograft infiltrated CD3 + T cells identified using IHC. (B) Quantification of infiltrated CD3 + T cells (n = 4-6 per group) in the arterial wall of allografts with Image J. HTx: heart transplantation. Data are presented as mean ± SEM. Scale bars are 50 µm. P values were established by 2-way ANOVA.

Article Snippet: Subsequently, sections were incubated with antibodies against CD3 (1:200, #PB9093, Boster Ltd., Wuhan, China), CD20 (1:500, #GB11540, Servicebio, Wuhan, China), and F4/80 (1:200, #70076, Cell Signaling Technology, Inc., Danvers, MA, USA) over night at 4°C to detect the infiltration of T cell, B cell and macrophage into heart, respectively.

Techniques: Transplantation Assay