Journal:

Article Title: A Chimeric Human T-Cell Lymphotropic Virus Type 1 with the Envelope Glycoprotein of Moloney Murine Leukemia Virus Is Infectious for Murine Cells

doi: 10.1128/JVI.76.15.7883-7889.2002

Figure Lengend Snippet: Cell-free viral infections of murine cells. The HTLV-1 gag gene (205 bp, positions 1616 to 1821 in Seiki ATK1 sequence [29]) was detected by PCR amplification of DNA extracted from cells infected with cell-free chimeric viruses or cell-free HTLV-1. Amplified products were detected by Southern blot hybridization with specific oligonucleotide internal probes end labeled with 32P using 3′ terminal transferase. Radioactive signals were detected and quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software. To detect plasmid contamination, PCR was also performed with primer 5′-GGCTCGTATGTTGTGTGGAA-3′ in pUC19 and primer 5′-TTAGCCATATGCGTGCCATG-3′ in the HTLV-1 LTR. (A) PCR detection of the HTLV-1 gag gene and of control plasmid in DNA extracted from different cell lines infected by chimeric (Chim, Chim.ΔR) and HTLV-1 particles. The positive control (+) corresponds to 1 copy of the HTLV-1 genome from MT2 cells. The negative control (−) is 1 μg of DNA from naïve 293T cells. (B) Standard curve for PCR detection of HTLV-1 gag gene. Dilutions of pCS-HTLV-I plasmid (top) and MT2 cell DNA (bottom) are shown. (C) Evidence for infection of mouse lymphocytes by ΔR chimeric cell particles. PCR detection of the HTLV-1 gag gene and of control plasmid in DNA. Detection by reverse transcription-PCR of the doubly spliced viral mRNA for the Tax protein (221-bp spliced fragment, primers in positions 5098 to 7438 in Seiki ATK1 sequence). The GAPDH gene was also amplified by reverse transcription-PCR for a control. The positive controls are as follows: for the Gag panel, 1 copy of the HTLV-1 genome from MT2 cells; for the pUC panel, 1 μg of pCS-HTLV-1 plasmid; and for the Tax mRNA and GAPDH panels, MT2 RNA. (D) PCR detection of HTLV-1 gag gene in mouse lymphocytes infected with ΔR chimeric particles. Lanes 1, 2, and 3 show the spread of the infection from primary infected, male CD4+ lymphocytes placed in the lower chamber of a transwell to other cells placed in the upper chamber. Lane 1, DNA from primary infected cells; lane 2, DNA from secondary infected EL4 cells; lane 3, DNA from secondary infected female 129sv CD4+ lymphocytes. Lanes 4, 5, and 6 illustrate the spread of the infection from primary infected EL4 cells placed in the lower chamber of a transwell to other cells placed in the upper chamber. Lane 4, DNA from primary infected EL4 cells; lane 5, DNA from secondary infected male 129sv CD4+ lymphocytes; lane 6, DNA from secondary infected EL4 cells. The control primers to detect any contamination in secondary infections were 5′-GACTAGACATGTCTTAACATCTGTCC-3′ and 5′-CCTATTGCATGGACAGCAGCTTATG-3′ in the Zfy gene (murine Y chromosome [Y Chr.]). The positive control for the Gag panel is 1 copy of the HTLV-1 genome from MT2 cells, while that for the Y Chr. panel is 1 μg of DNA from male mouse splenocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The target cells used were NIH 3T3, EL4 (a murine CD4 T-cell lymphoma obtained from the American Type Culture Collection), 293T, 293T4, HOS (a human osteosarcoma-derived fibroblast cell line permissive for HTLV-1 [ 19 , 35 ]), and B5 (a cell line derived from the DBS-FRhL rhesus monkey lung fibroblast line that is permissive for HTLV-1 [ 6 ]).

Techniques: Sequencing, Amplification, Infection, Southern Blot, Hybridization, Labeling, Software, Plasmid Preparation, Control, Positive Control, Negative Control, Reverse Transcription

Journal: Cancer Immunology Research

Article Title: P. gingivalis Infection Upregulates PD-L1 Expression on Dendritic Cells, Suppresses CD8 + T-cell Responses, and Aggravates Oral Cancer

doi: 10.1158/2326-6066.CIR-22-0541

Figure Lengend Snippet: P. gingivalis infection dampens the cytotoxicity of CD8 + T cells. A, Left: density plot and dot plots showing the gating strategy for the cytotoxicity assay using Zombie Aqua staining to assess cell viability. Right: distinct CFSE-labeled target EL4 cell population. B, Representative cytometry dot plots showing the cytotoxicity determined by the percentage of lysed EL4 cells after coincubation with activated OT-I CD8 + T cells for 6 hours. C, Quantification of the percentage of specific lysis, as described in Materials and Methods, in a cytotoxic assay with different effector/target cell ratios upon the challenge with P. gingivalis ( n = 5 mice). Error bars represent the mean ± SE. Statistical significance was determined by two-way ANOVA with the Tukey multiple comparisons test. *, P < 0.05; **, P < 0.01. D, The percentage of lysed EL4 cells showing the effect of STAT3 inhibitor (WP-1066) on the cytotoxic activity of OT-I CD8 + T cells after 6 hours of coincubation. Each symbol represents an experimental replicate, and error bars represent the mean ± SE. Statistical significance was determined by a two-tailed unpaired t test. ***, P < 0.001. E, The percentage (left) and representative cytometry dot plots (right; n = 4 mice) of specific lysis of EL4 cells showing the different cytotoxic activity of CD8 + T cells after 3 days of coculturing with OVA-pulsed DCs with/without pretreatment of P. gingivalis or ΔKgp. Each symbol represents an experimental replicate, and data are shown as mean ± SE. Statistical significance was determined by one-way ANOVA with the Tukey multiple comparisons test. ***, P < 0.001. Results are representative of 3 independent experiments.

Article Snippet: The 1 μmol/L-CFSE-labelled EL4 cells were prepulsed with OVA peptide (5 μmol/L, AnaSpec Inc., cat. #AS-60193) for 12 hours, mixed with 0.1 μmol/L-CFSE-labelled unpulsed cells (used as an internal control), and then cocultured with primed CD8 + T cells at 5:1, 10:1, or 20:1 effector-to-target cell ratios in three independent replicates.

Techniques: Infection, Cytotoxicity Assay, Staining, Labeling, Cytometry, Lysis, Activity Assay, Two Tailed Test

Journal: Nature nanotechnology

Article Title: DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation

doi: 10.1038/s41565-020-00813-z

Figure Lengend Snippet: Polymeric micro-/nanoparticles with surface DNA scaffolds for protein presentation allow versatile modulation of immune cell therapies. ( a ) Schematic of biodegradable polymeric particles presenting therapeutic proteins via surface DNA scaffolds with high density and ratiometric control, and their potential use as immune cell engaging particles (ICEp) for tumor microenvironment modulation and localized activation of natural or engineered immune cells. N: normal tissue, C: cancer cell, T: T cell, P: ICEp. ( b ) Schematic showing the construction of high and controlled densities of DNA scaffolds on polymeric particles through a self-display of amphiphilic polymer-DNA molecules during the emulsion process, compared to the traditional surface conjugation method after particle fabrication. ( c ) Fluorescence-based analysis of hybridized, dye-labeled DNA duplexes on PLGA particles made from DNA-polymer conjugates of different coupling efficiencies versus dye-labeled DNA that was surface-conjugated to exposed functional groups on particles. Data are mean ± s.d. (n = 6 independent samples from 3 independent experiments), and P values were determined by one-way analysis of variance (ANOVA) and Tukey’s tests. ( d ) Photos of PLGA microparticles in Eppendorf tubes after adding Quasar670 (Q670)-labeled complementary DNA to a matched or unmatched sequence for 2 minutes at room temperature. ( e ) Schematic displaying the control of DNA scaffolds with distinct sequences at intended ratios, which is confirmed by a corresponding dye-labeled complementary DNA (compDNA) hybridization assay. ( f ) Representative confocal microscopy images of PLGA microparticles with DNA scaffolds of different sequence compositions after hybridization with corresponding dye-labeled compDNA. The merged images of particles agreed with the theoretically integrated color at different input ratios. Scale bar, 5 μm. ( g ) Fluorescence-based analysis of DNA duplexes of different sequences on particles shown in ( f ). Data are mean ± s.d. (n = 5 independent samples from 2 independent experiments), and P values were determined by one-way analysis of variance (ANOVA) and Turkey’s tests. ( h ) Correlation between hybridized duplexes of specific sequences on the surface and their corresponding DNA-polymer conjugate inputs shown in ( g ). Data are mean ± s.d. (n = 5 independent samples from 2 independent experiments), and the linear regression was created from individual replicates (R 2 = 0.8501) and the mean of each condition (R 2 = 0.9516).

Article Snippet: Murine pro-B cell line BA/F3 (DSMZ #ACC 300), T cell lymphoma line EL4 (DSMZ #ACC 831), and bone marrow line 32D (DSMZ #ACC 411) were used to test synthetic circuit activation in mouse cells.

Techniques: Activation Assay, Conjugation Assay, Fluorescence, Labeling, Functional Assay, Sequencing, Hybridization, Confocal Microscopy

Journal: Nature nanotechnology

Article Title: DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation

doi: 10.1038/s41565-020-00813-z

Figure Lengend Snippet: Local activation of AND-gate CAR T cell for tumor killing by intratumoral injection of ICEp presenting a priming antigen. ( a ) Schematic showing the use of ICEp to present synthetic priming antigens (GFP) via intratumoral (i.t.) injection to locally prime synNotch-CAR AND-gate T cells to kill tumor cells. Primary human T cells were engineered with the anti-GFP synNotch and corresponding response elements regulating anti-HER2 4–1BB CAR expression. The synNotch-CAR T cells only express the CAR after sensing ICEp-presented GFP via synNotch binding, which should reduce ON-target OFF-tumor toxicity to healthy organs also expressing the CAR antigen. ( b ) Representative confocal fluorescence microscopy image of the co-incubation of CD8+ synNotch CAR-T cells, ICEp-GFP, and HER2-overexpressing A375 cells after 24 hours (n = 2 independent experiments). Scale bar, 10 μm. ( c ) Target A375 cell killing test 48 hours after co-culture with ICEp-GFP and synNotch CAR-T cells. Data are mean ± s.d. (n = 6 independent experiment with 4 independent T cell donors), and P values were determined by one-way ANOVA and Tukey’s tests. ( d ) IL-2 secretion by primary human CD4+ T cells after a 48-hour co-culture with HER2+ A375 target cells and ICEp with different GFP densities (e.g. 1/3, 1/2 and full). The total amount of antigen presented to T cells by ICEp particles with “Full” was equal to “1/3 × 3”. Data are mean ± s.d. (n = 4 biologically independent samples from 2 independent experiments), and P values were determined by one-way ANOVA and Tukey’s tests. ( e ) Killing efficacy of HER2+ A375 cells after a 48 hour co-culture with AND-gate T cells that were primed by ICEp with varying densities of GFP from DNA scaffolds compared to a traditional conjugation chemistry. Images are representative confocal fluorescence microscopic images of ICEp. Data are mean ± s.d. (n = 16 biological replicates from 4 independent experiments), and P values were determined by one-way ANOVA and Tukey’s tests. ( f ) Schematic of ICEp-GFP versus GFP-expressing K562 cells activating murine immune cell lines that were engineered with anti-GFP synNotch and corresponding response elements regulating BFP expression. ( g ) The population of BFP+ murine immune cells that were activated by ICEp-GFP compared to GFP-expressing K562 cells and K562 cells without GFP antigen. Data are mean ± s.d. (n = 3 biological replicates), and P values were determined by one-way ANOVA and Tukey’s tests. ( h ) Schematic of the NSG mice two tumor model for selected clearance by ICEp-primed synNotch CAR-T cell activation, while ICEp-GFP and engineered human primary CD4+/CD8+ T cells were administered through i.t. and intravenous (i.v.) injection, respectively. ( i ) Comparison between the volumes of the ICEp-injected tumor and the contralateral tumor of mice treated with engineered synNotch CAR-T cells (left graph) or untransduced T cells (right graph). Data are mean ± s.e.m. (n = 8 mice, and 2 mice met the euthanasia criteria at day 13–15), and P values were determined by two-tailed paired t test. ( j ) Representative images of fixed staining of tumor sections from one mouse that was sacrificed on an earlier date (Day 15) that met euthanasia criteria (n = 10 images). Scale bar, 50 μm. ( k ) Quantification of CD3+ T cell numbers in the stained tumor sections represented in ( j ). Data are mean ± s.d. (from n = 10 images), and P values were determined by two-tailed paired t test.

Article Snippet: Murine pro-B cell line BA/F3 (DSMZ #ACC 300), T cell lymphoma line EL4 (DSMZ #ACC 831), and bone marrow line 32D (DSMZ #ACC 411) were used to test synthetic circuit activation in mouse cells.

Techniques: Activation Assay, Injection, Expressing, Binding Assay, Fluorescence, Microscopy, Incubation, Co-Culture Assay, Conjugation Assay, Two Tailed Test, Staining

Journal: Nature nanotechnology

Article Title: DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation

doi: 10.1038/s41565-020-00813-z

Figure Lengend Snippet: ICEp, capable of versatile and precisely controlled modulatory signals, regulate T cell characteristics during ex vivo expansion. ( a ) Schematic of ICEp (PLGA microparticles with anti-CD3 and anti-CD28 antibodies controlled at specific ratios by DNA scaffolds, e.g. [1:5] to [5:1]) triggering human primary T cell activation and expansion. ( b ) Representative confocal microscopy images of human primary CD8+ T cell co-incubated with ICEp-[1:1] overnight, showing ICEp-induced cell clumps (n = 3 biologically independent samples). Scale bar, 10 μm. ( c ) Cell yield of human primary CD8+ T cells 8 and 14 days after activation by ICEp-[3:1], compared to commercially available Dynabeads. Data are mean ± s.d., and P values were determined by two-tailed paired t test (n = 3 independent donors of 2 independent experiments). ( d ) Exhaustion marker analysis of CD8+ T cells after stimulation with ICEp-[3:1] or Dynabeads for 14 days. The P value was determined by nested one-way ANOVA analysis of cell population with 0 to 3 inhibitory receptors between ICEp-[3:1] and Dynabeads activation. ( e,f ) Cell yield of human primary CD4+ ( e ) and CD8+ ( f ) T cells 14 days after activation of 1.4 × 10 5 cells by ICEp with varying ratios of anti-CD3 to anti-CD28 ranging from [1:5] to [5:1]. Data are mean ± s.e.m. (n = 3 independent donors of 2 independent experiments), and P values were determined by one-way ANOVA test for linear trend. ( g ) The population of CD8+ T cells with 0 to 3 inhibitory receptors after being stimulated by ICEp-[1:5] or ICEp-[3:1] for 14 days. Data are mean ± s.d., and P values were determined by multiple t test for comparison of 0–3 subgroups and nested one-way ANOVA analysis for comparison between treatments of ICEp-[1:5] and ICEp-[3:1] (n = 3 independent donors of 2 independent experiments). ( h ) Representative two-dimensional dot plot of CCR7 and CD45RA expression on CD8+ T cells activated by ICEp-[3:1] for 7 days, and the gating strategy to analyze T cell differentiation phenotype. ( i ) CCR7 and CD45RA expression profile of CD8+ T cells stimulated by ICEp-[1:5] or ICEp-[3:1] for 7 days. Data are mean ± s.d. (n = 3 biological replicates from the same donor), and P values were determined by multiple t test with correction using the Holm-Sidak method. ( j ) Schematic of IL-2 presented on particles through its antibody (clone5355) that exposes the epitope for β and γ units of its receptor on T cells, thus promoting T cell proliferation. ( k,l ) Cell expansion profile of primary CD4+ ( k ) and CD8+ ( l ) T cells stimulated by ICEp-[3:1] for 14 days with ICEp-bound IL-2 or free IL-2 supplemented at an equivalent dose. Data are mean ± s.d. (n = 4 independent donors of 3 independent experiments), and P values were determined by two-tailed paired t test. ( m ) The population of CD4+ and CD8+ T cells with 0 to 3 inhibitory receptors stimulated by ICEp-[3:1] for 14 days with ICEp-bound IL-2 or free IL-2 supplemented at an equivalent dose. Data are mean ± s.d. (n = 4 independent donors of 3 independent experiments), and P values were determined by multiple t test with correction using the Holm-Sidak method.

Article Snippet: Murine pro-B cell line BA/F3 (DSMZ #ACC 300), T cell lymphoma line EL4 (DSMZ #ACC 831), and bone marrow line 32D (DSMZ #ACC 411) were used to test synthetic circuit activation in mouse cells.

Techniques: Ex Vivo, Activation Assay, Confocal Microscopy, Incubation, Two Tailed Test, Marker, Expressing, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Hydroxychloroquine enhances efferocytosis and modulates inflammation via MerTK/Gas6 signaling in a pristane-induced lupus mouse model

doi: 10.3389/fimmu.2025.1524315

Figure Lengend Snippet: HCQ enhances efferocytosis in vitro . (A, B) The viability of RAW264.7 cells treated with varying concentrations of HCQ (A) and chloroquine (CQ) (B) was assessed using MTT assays over 24 hours (n=3). (C, D) Representative flow cytometry images (upper) and data plots (lower) show efferocytotic capacity of RAW264.7 cells (CFSE-labeled, green) treated with subtoxic doses of HCQ (C) or CQ (D) (2.5-10 μM) in the presence of apoptotic EL4 cells (Deep Red labeled, red) for 1 hour (n=5). (E) Representative flow cytometry images (left) and data plot (right) indicate the percentage of pHrodo red-positive cells within RAW264.7 cells treated with HCQ after 4 hours of co-incubation (n=3). (F) Representative immunofluorescent images (left) and data plot (right) display the percentage of apoptotic EL4 cells phagocytosed by HCQ-treated RAW264.7 cells after 30 minutes of co-incubation (n=5). Scale bar: 170 μm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: RAW264.7 and EL4 cells were procured from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: In Vitro, Flow Cytometry, Labeling, Incubation

Journal: Frontiers in Immunology

Article Title: Hydroxychloroquine enhances efferocytosis and modulates inflammation via MerTK/Gas6 signaling in a pristane-induced lupus mouse model

doi: 10.3389/fimmu.2025.1524315

Figure Lengend Snippet: HCQ increases MerTK expression in vitro . (A, B) Gene expression of TAM receptors Mertk (A) and Axl (B) in RAW264.7 cells treated with indicated concentration of HCQ (5, 7.5, 10 μM) for 24 hours with or without co-incubation with apoptotic EL4 cells (n=3). (C, D) Gene expression of TAM ligands Gas6 (C) and Pros (D) in RAW264.7 cells treated with indicated concentration of HCQ (5, 7.5, 10 μM) for 24 hours with or without co-incubation with apoptotic EL4 cells (n=3). (E) Representative flow cytometry images (right) and data plot (left) show surface expression of MerTK on RAW264.7 cells treated with indicated concentration of HCQ (5, 7.5, 10 μM) for 24 hours with or without co-incubation with apoptotic EL4 cells (n=3). (F) Representative WB images (left) and data plot (right) show the protein expression of MerTK, p-MerTK, Gas6, p62, and LC3B in RAW264.7 cells treated with HCQ (5 and 10 μM) for 24 hours (n=3). MFI refers to the median fluorescence intensity. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: RAW264.7 and EL4 cells were procured from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Expressing, In Vitro, Gene Expression, Concentration Assay, Incubation, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Hydroxychloroquine enhances efferocytosis and modulates inflammation via MerTK/Gas6 signaling in a pristane-induced lupus mouse model

doi: 10.3389/fimmu.2025.1524315

Figure Lengend Snippet: HCQ promotes efferocytosis and anti-inflammatory signals via MerTK. (A) Representative western blot images (left) and quantitative analysis (right) show the levels of phosphorylated MerTK and total MerTK protein in RAW264.7 cells treated with HCQ (5 μM), UNC2025 (1 μM), or HCQ+UNC2025 after co-incubation with apoptotic EL4 cells for 15 minutes. Macrophages were pretreated with UNC2025 for 1 hour prior to exposure to apoptotic cells (n=3). (B) Representative flow cytometry plots (left) and quantification (right) of efferocytosis in RAW264.7 cells treated with HCQ, UNC2025, or HCQ+UNC2025 following co-incubation with apoptotic EL4 cells for 1 hour (n=3). (C) Representative flow cytometry plots (left) and quantification (right) of efferocytosis in peritoneal macrophages isolated from normal or PIL mice treated with HCQ, UNC2025, or HCQ+UNC2025, following co-incubation with apoptotic thymocytes for 30 minutes (n=3). (D-F) Relative gene expression levels of Ifnα (D) , Il6 (E) , Il10 (F) in RAW264.7 cells treated with HCQ, UNC2025, or HCQ+UNC2025 after co-incubation with apoptotic EL4 cells for 2 hours, compared to untreated controls (n=5). (G) Gene expression of Il6 in peritoneal macrophages isolated from normal or PIL mice treated as indicated, following co-incubation with apoptotic thymocytes for 1 hour (n=3). (H) Representative western blot images (left) and quantification (right) show the expression of anti-inflammatory transcription factors PPARγ and LXR in RAW264.7 cells treated with HCQ, UNC2025, or HCQ+UNC2025 after co-incubation with apoptotic EL4 cells for 2 hours (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: RAW264.7 and EL4 cells were procured from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Western Blot, Incubation, Flow Cytometry, Isolation, Gene Expression, Expressing