Journal: Cell Reports Medicine

Article Title: Pan-cancer mapping of single CD8 + T cell profiles reveals a TCF1:CXCR6 axis regulating CD28 co-stimulation and anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101640

Figure Lengend Snippet:

Article Snippet: Anti-Mouse/Human TOX APC Clone REA473 , Miltenyi Biotec , Cat:# 130-118-335 RRID: AB_2751485.

Techniques: Virus, Recombinant, Purification, SYBR Green Assay, Staining, Reporter Assay, In Vitro, Activation Assay, Generated, Luciferase, Plasmid Preparation, Sequencing, Software, CRISPR

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell Stem Cell

Article Title: Selective advantage of mutant stem cells in human clonal hematopoiesis is associated with attenuated response to inflammation and aging

doi: 10.1016/j.stem.2024.05.010

Figure Lengend Snippet:

Article Snippet: APC anti-human CD271 (clone ME20.4-1.H4) , Miltenyi , Cat# 130-113-418; RRID: AB_2733363.

Techniques: Recombinant, Reverse Transcription, DNA Library Preparation, DNA Sequencing, RNA Sequencing, Purification, Sequencing, Software, Sterility

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: Knockdown of SChLAP1 inhibits the growth of triple negative breast cancer cells. (A) MDA-MB-231 and SKBR3 cells were transfected with control-siRNA or SChLAP1-siRNA. The knockdown of SChLAP1 was confirmed via reverse transcription-quantitative PCR analysis. A Cell Counting Kit-8 assay was performed to determine the viability of (B) MDA-MB-231 and (C) SKBR3 cells after transfection with siRNA-SChLAP1. (D) Colony formation ability was suppressed in MDA-MB-231 and SKBR3 cells upon knockdown of SChLAP1 (magnification, ×40). (E) Knockdown of SChLAP1 significantly promoted the apoptosis of both MDA-MB-231 and SKBR3 cells compared with cells expressing a control vector. ***P<0.001 vs. siRNA-Control. OD, optical density; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Knockdown, Transfection, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Expressing, Plasmid Preparation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: SChLAP1 acts as a sponge of miR-524-5p. (A) Predicted binding site of miR-524-5p on the sequence of SChLAP1. Luciferase activity of (B) MDA-MB-231 and (C) SKBR3 cells co-transfected with miR-524-5p mimics and WT-SChLAP1 was significantly decreased, while the activity of cells expressing miR-524-5p and Mut-SChLAP1 was not obviously affected. (D) Knockdown of SChLAP1 increased the expression of miR-524-5p in MDA-MB-231 and SKBR3 cells. (E) Expression of miR-524-5p in TNBC tissues was significantly downregulated compared with that of adjacent non-cancerous tissues. (F) Expression level of miR-524-5p was negatively correlated with SChLAP1 expression in TNBC tissues. ***P<0.001 vs. corresponding control. NC, negative control; miR, microRNA; WT, wild-type; Mut, mutant; TNBC, triple negative breast cancer; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Transfection, Expressing, Knockdown, Control, Negative Control, Mutagenesis, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: HMGA2 is a target of miR-524-5p in triple negative breast cancer. (A) MDA-MB-231 and SKBR3 cells were transfected with miR-524-5p mimic and mimic-NC, the expression of miR-524-5p was validated via reverse transcription-quantitative PCR. Cell Counting Kit-8 assay showed the decreased viability of (B) MDA-MB-231 and (C) SKBR3 cells induced by miR-524-5p overexpression. (D) Predicted binding sites of miR-524-5p in the 3′-UTR of HMGA2. Relative luciferase activities in (E) MB-231 and (F) SKBR3 cells co-transfected with miR-524-5p mimics or mimic-NC and luciferase reporter plasmid containing WT or Mut 3′-UTR of HMGA2. Overexpression of miR-524-5p decreased the (G) mRNA and (H) protein expression levels of HMGA2 in TNBC cells. The protein expression level of HMGA2 was semi-quantified by normalizing the level of GAPDH using ImageJ software. **P<0.01. ***P<0.001 vs. mimic-NC. UTR, untranslated region; NC, negative control; miR, microRNA; WT, wild-type; Mut, mutant; HMGA2, High Mobility Group AT-Hook 2; OD, optical density.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Over Expression, Binding Assay, Luciferase, Plasmid Preparation, Software, Negative Control, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR-524-5p/HMGA2 axis

doi: 10.3892/mmr.2021.12085

Figure Lengend Snippet: Reintroduction of HMGA2 reverses SChLAP1 knockdown-induced viability defects in TNBC cells. Knockdown of SChLAP1 decreased both the (A) mRNA and (B) protein expression levels of HMGA2 in MDA-MB-231 and SKBR3 cells. The protein expression level of HMGA2 was semi-quantified by normalizing the level of GAPDH using the ImageJ software. (C) Expression of Flag-tagged HMGA2 was detected via western blotting with anti-Flag antibody. Overexpression of HMGA2 significantly attenuated the effect of SChLAP1 knockdown on the viability of (D) MDA-MB-231 and (E) SKBR3 cells. (F) Co-transfection of HMGA2 promoted the colony formation of TNBC cells compared with cells expressing siRNA-SChLAP1 (magnification, ×40). ***P<0.001 vs. control group. HMGA2, High Mobility Group AT-Hook 2; TNBC, triple negative breast cancer; siRNA, small interfering RNA; SChLAP1, second chromosome locus associated with prostate-1; OD, optical density.

Article Snippet: Human TNBC cell lines, including MDA-MB-231, SKBR3, BT-549 and HCC-1937, and the normal breast epithelial cell line MCF10A were purchased from the American Type Culture Collection.

Techniques: Knockdown, Expressing, Software, Western Blot, Over Expression, Cotransfection, Control, Small Interfering RNA

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 1 Expression of CD30 in the T cells and B cells and generation of CD30-deficient mice. (A) The indicated hematopoietic cells were sorted from wild-type mice. The level of CD30 mRNA expression was measured by qRT-PCR, using β-Actin as internal control. (B) The frequency of CD30+ T cells in CD4 T cells upon TCR stimulation (left). The overlays show CD30 surface expression of CD4 T cells upon TCR stimulation (right). (C) The frequency of CD30+ T cells in CD8 T cells upon TCR stimulation (left). The overlays show CD30 surface expression of CD8 T upon TCR stimulation (right). (D) CD30 expression level was detected by qRT-PCR. (E) Western blotting analysis of CD30 expression in splenic T cells. Purified splenic T cells were stimulated for 48 h on tissue culture plate coated with anti-CD3 (10 μg/mL) plus anti-CD28 (5 μg/mL) and IL-2 (100 ng/mL). The cell lysates were subjected to direct Western blot analysis with the indicated antibodies. Data shown are representative of or obtained from 3 (A–E) independent experiments. Mean ± SD is shown.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Purification

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 3 Serum immunoglobulin levels and serum antibody response to TD antigens in CD30-KO and wild-type mice. (A) Serum immunoglobulin isotype levels in wild-type and CD30-KO mice were measured by ELISA. (B) Serum NP-specific immunoglobulin isotype response to the TD antigen NP-KLH immunization in wild-type and CD30-KO mice. Sera were collected at the indicated time points after immunization and antigen-specific antibodies were measured by ELISA. (C) CD30-KO and wild-type mice immunized with NP-KLH. Splenocytes from CD30-KO and wild-type mice were stained with anti-B220, anti-CD95, and anti-GL7 at day 9 after immunization and analyzed by FACS. Percentages indicate cells in the gated B220+ cells. (D) Dot plots show percentages and numbers of GC B cells in the spleen of CD30-KO and wild-type mice. (E) Splenic mature B cells were stimulated with LPS for 48 h, and cell lysates were subjected to direct Western blot analysis with the indicated antibodies. (F) The intensities of protein bands were quantitated using Image-J software. Data shown are representative of or obtained from 6 (A), 4 (B, E, F), 5 (C, D), or 4 (E, F) CD30-KO and wild-type littermates. Mean ± SD is shown. *p < .05, **p < .01.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Software

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 4 CD30 deficiency has no effect on T-cell development. (A) Dot plots show numbers of total thymocytes in CD30-KO and wild- type mice. (B) Thymocytes from CD30-KO and wild-type mice were stained with anti-CD4 and anti-CD8 and analyzed by FACS. Percentages indicate cells in the gated live cells. (C) Dot plots show percentages and numbers of DN, DP, CD4, and CD8 T cells in the thymocytes of CD30-KO and wild-type mice. (D) Thymocytes from CD30-KO and wild-type mice were stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD44 and analyzed by FACS. Percentages indicate cells in the gated DN T cells. (E) Dot plots show percentages and numbers of DN1, DN2, DN3, and DN4 T cells in the DN T cells of CD30-KO and wild-type mice. (F) Splenocytes from CD30-KO and wild-type mice were stained with anti-CD4 and anti-CD8 and analyzed by FACS. Percentages indicate cells in the gated live cells. (G) Dot plots show percentages and numbers of CD4 and CD8 T cells in the spleen of CD30-KO and wild-type mice. Data shown are representative of or obtained from 10 (A–G) CD30-KO and wild-type littermates. Mean ± SD is shown.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Staining

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 5 CD30 deficiency reduces TCR-mediated proliferation. (A) Splenic T cells were labeled with CFSE, according to the manufacturer's recommendation, and stimulated as indicated for 72 h before analysis for cell proliferation (left). Dot plots show the percentages of proliferate rate (right). (B) Splenic T cells were stimulated as indicated for 72 h, stained with PI, and analyzed for cell cycle profile by FACS (left). Dot plots show percentages of cells in S/G2/M phase after stimulation (right). Data shown are representative of or obtained from 3 (A, B) independent experiments. Mean ± SD is shown. *p < .05, ** p < .01.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Labeling, Staining

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 6 CD30 deficiency reduces TCR-mediated T-cell activation. (A) Total thymocytes were stimulated with anti-CD3 (10 μg/ mL) for the indicated time and cell lysates were subjected to direct Western blot analysis with the indicated antibodies. (B) The intensities of protein bands were quantitated using Image-J software. (C, D) Splenic T cells from CD30-KO and wild-type littermates were stimulated with anti-CD3 for 72 h and analyzed by FACS and ELISA. (C) Splenic T cells were stained with anti-CD4, anti-CD8, anti-CD25, and anti- CD69 upon TCR stimulation. Dot plots show percentages of cells in CD4 and CD8 T cells after stimulation. (D) ELISA analysis of IL-6 and IFN-γ released in the splenic T cell culture supernatant. Data shown are representative of or obtained from 6 (A, B) or 3 (C, D) independent experiments. Mean ± SD is shown. *p < .05.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 7 Analysis of CD30-KO gene expression signature in TCR-mediated T cells. (A, B) Splenic T cells from CD30-KO and wild- type littermates were sorted and stimulated with anti-CD3/anti-CD28/IL-2 for 48 h and subjected to high-throughput RNA-sequencing. (A) Volcano plot of differentially expressed genes between CD30-KO and wild-type T cells. (B) Significantly downregulation pathways from the KEGG pathway database in CD30-KO and wild-type T cells. (C) The relative mRNA levels of LC3 and Atg7 were quantified by qRT-PCR in CD30-KO and wild-type T cells with anti-CD3/anti-CD28/IL-2 stimulation. The relative mRNA levels of the indicated genes in CD30-KO and wild-type T cells were normalized to β-Actin. (D) Splenic T cells were stimulation with anti-CD3/anti-CD28/IL-2 and cell lysates were subjected to direct Western blot analysis with the indicated antibodies (left). The intensities of protein bands were quantitated using Image-J software (right). Data shown are representative of or obtained from 2 (A, B) or 3 (C, D) independent experiments. Mean ± SD is shown. *p < .05, **p < .01.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Gene Expression, High Throughput Screening Assay, RNA Sequencing, Quantitative RT-PCR, Western Blot, Software

Journal: Immunity

Article Title: T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis

doi: 10.1016/j.immuni.2022.12.010

Figure Lengend Snippet:

Article Snippet: InVivoMAb rat IgG2a (RG7/1.30) , BioXcell , Cat# BE0251; RRID: AB_2687732.

Techniques: Purification, Control, Virus, Recombinant, Red Blood Cell Lysis, Transfection, Flow Cytometry, DNA Extraction, Gel Extraction, Plasmid Preparation, cDNA Synthesis, Cell Differentiation, Staining, Cell Isolation, Conjugation Assay, Sequencing, CRISPR, Retroviral, Software

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Antigenic stimulation of human splenic mononuclear cells mimics the T CD4 + response of GC reaction (A) Splenic mononuclear cells (splenocytes) were stimulated with CytoStim and cultured for 3 days in the presence of cytokines (IL-7, IL-12, activin) (A). CXCR5 and PD-1 expressions among CD4+ T cells were assessed. Representative flow plots showing CXCR5 and PD-1 expression on CD4 + T cells from ex vivo splenocytes and splenocytes cultured for 3, 5, and 10 days (left) and the percentage of Tfh among CD4+ T cells (right). (B) Representative flow plots showing IL-21 production by Tfh (left) and the percentage of IL-21-positive cells among Tfh (right). (C) Representative histogram showing ICOS expression among Tfh (left) and relative expression of ICOS among Tfh (right). (D) Gating strategy allowing the identification of PD-1 neg Tfh, non-GC Tfh, and GC Tfh among total CXCR5 + PD-1 + cells ex vivo or after 3 days of stimulation of splenocytes or PBMCs in polarizing cytokines. (E) Percentage of total CXCR5 + PD-1 + cells including PD-1 neg Tfh, non-GC Tfh, and GC Tfh among CD4 + T cells ex vivo or after 3 days of culture using splenocytes or PBMCs (n = 7–14). (F and G) Mean fluorescence intensity of PD-1 (F) and CXCR5 (G) expression on ex vivo GC Tfh and GC Tfh D3 splenic cells. (H) Representative flow plots showing IL-21 and IFNγ production by non-GC Tfh D3 and GC Tfh D3 cells 3 days after splenocyte stimulation. (I) Percentage of IL-21- and/or IFNγ-positive cells among non-GC Tfh D3 cells and GC Tfh D3 . (J) Gating strategy for analysis of Bcl6 expression in CD4 + T cells and histograms showing Bcl6 mean fluorescence intensity for GC Tfh D3 , non-GC Tfh D3 , and CXCR5 − PD-1 - CD4 + T cell subsets (n = 14). Each symbol represents an individual donor. A Wilcoxon matched pairs test was performed; ∗, p < 0.05; ∗∗, p < 0.005.

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques: Cell Culture, Expressing, Ex Vivo, Fluorescence

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: CyTOF antibody panel

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques:

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Ex vivo and induced Tfh display distinct phenotypic landscapes and differentiation trajectories (A) Deep immunophenotyping was performed after 3 days of culture (n = 2 independent donors). CD4 + CXCR5 + T cell selection was based on the expression of CD45, CD8, CD11c, CD56, and CD19. The Uniform Manifold Approximation and Projection (UMAP) algorithm was used to represent the whole set of CD4 + CXCR5 + T cells in a multiparametric manner at D0 and D3 after antigenic stimulation of splenocytes from two donors. (B) Projection of 8 clusters determined by k-means on the UMAP representation of D0 and D3 CD4 + CXCR5 + T cells. (C) Heatmap representing the mean expression of 29 markers by 8 cell clusters and their relative abundance, defined among D0 and D3 CD4 + CXCR5 + T cells. (D and E) Trajectory and pseudotime analysis on total D0 (D) and D3 (E) CD4 + T cells. Tree plots (left) show cluster trajectories, cell number, and metacluster assignment, and density plots (right) show the density of pseudotime across metaclusters.

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques: Ex Vivo, Selection, Expressing

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Naive and memory CD4 + can orient toward a Tfh cell profile (A) Four CD4 + T cell subsets are defined at day 0: (1) naive CD4 + CD45 RA + T cells, (2) memory CD4 + CD45 RA - PD-1 - T cells (MemPD-1 neg ), (3) memory CD4 + CD45 RA - PD-1 + T cells (MemPD-1 pos ), and (4) Tfh. (B) At day 0, D4 + T cell subsets were sorted according to the gating strategy presented in (A). Isolated CD4 + T cell subsets were stained with cell trace violet (CTV) and mixed back into the negative splenocyte fraction. Stimulation and culture were next performed as previously described ( Figure 1 A). (C) Representative flow plots of CTV tracking for each stimulated CD4 + T cell subset 3 days after antigenic stimulation (top) combined with flow cytometry analysis of CXCR5 and PD-1 expression among CTV + cells at day 3 (bottom). (D) Percentage of CXCR5 + PD-1 + cells (Tfh D3 ) among traced CD4 + T cell subsets after 3 days of antigenic stimulation. (E) Percentage of Tfh D3 cells according to the divisions of stimulated CD4 + T cell subsets. (F) Representative histograms of Bcl6 expression for Tfh D3 (colored line) compared with CXCR5 − PD-1 + (black line) and CXCR5 − PD-1 - (gray line) deriving from respective CD4 + T cell subsets naive (blue), MemPD-1 neg (turquoise blue), MemPD-1 pos (green), and Tfh (orange). (G) RNA sequencing was performed on CD4 + T cell subsets (Day 0) and the corresponding derived Tfh D3 counterparts (n = 2). Multidimensional scaling was used to better visualize transcriptomic proximity of different CD4 + T cells. (H) Venn diagrams were used to highlight Tfh-associated genes among differentially expressed genes that were shared during transition from D0 CD4 + T cell subsets to their Tfh D3 counterparts (D and E). Each symbol represents an individual donor. A Wilcoxon matched pairs test was performed; ∗, p < 0.05; ∗∗, p < 0.005.

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques: Isolation, Staining, Flow Cytometry, Expressing, RNA Sequencing, Derivative Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Distinct CD4 + T cell subsets contribute to the generation of Tfh with heterogeneous functional profiles (A) Mean fluorescence intensity of the CXCR5 marker expressed by CXCR5 + PD-1 + cells derived from (1) naive, (2) MemPD-1 neg , (3) MemPD-1 neg and Tfh. (B) Frequency of IL-21- and/or IFNγ-positive cells among CXCR5 + PD-1 + cells at day 3. (C – E) Representative flow plots showing CXCR3, ICOS, and CD40L expression by CXCR5 + PD-1 + cells (left panel) and frequency of CXCR3-, ICOS-, and CD40L-positive cells among CXCR5 + PD-1 + cells at day 3 (right panel). (F) Ex vivo cells or their respective Tfh D3 counterparts obtained after 3 days of splenocyte culture were co-cultured with autologous B cells for 7 days. (G) Box plots represent the frequency of CD27 + CD38 + cells among CD19 + cells, the concentration of total immunoglobulins and the absolute number of live B cells after co-culture. (H) Quantification of IgG1, IgG4, and IgA in the co-culture supernatants. Each symbol (A–H) represents an individual donor. (A–H) A Wilcoxon matched pairs test was performed, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques: Functional Assay, Fluorescence, Marker, Derivative Assay, Expressing, Ex Vivo, Cell Culture, Concentration Assay, Co-Culture Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: HIV infection shapes Tfh cell differentiation and functions (A) Splenocytes were stimulated according to the previously described protocol in the presence of HIV lab strain (Yu2b). (B) Representative flow plots of p24 staining among splenocytes after 3 days of culture with HIV or not. (C) RNA sequencing was performed on Tfh D3 cells derived from distinct CD4 + T cell subsets with and without HIV. Multidimensional scaling was performed to visually cluster different CD4 + T cell populations based on their transcriptional profile (8,593 genes). (D) RNA sequencing was performed on Tfh derived from each CD4 + T cell subset in the presence of HIV-1 infection or not. Differentially expressed genes were analyzed between Tfh and their original counterpart. Venn diagram representing (un)shared downregulated and upregulated genes. Genes specifically involved in Tfh cell biology were analyzed (referred to in ). (E) Contribution of each CD4 T cell subset to total Tfh generated after 3 days of splenocyte culture (%). Data are plotted as the mean percentage contribution of each ex vivo CD4 + T cell subset: (1) naive CD4 + T cells, (2) (3) memPD-1 neg/pos , and (4) Tfh to total Tfh D3 cells after splenocyte culture. (F) Representative flow plot of p24 staining in Tfh derived from distinct CD4 + T cell subsets. (G) Frequency of CD127-, CD27-, CD38-, FAS-, and ki67-positive cells among Tfh that are infected (p24 pos ) or not (p24 neg ). Each symbol (A–E) represents an individual donor. (E and G) A paired Student’s t test was performed, ∗p < 0.05, ∗∗p < 0.01. (F and G) A Wilcoxon matched pairs test was performed; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques: Infection, Cell Differentiation, Staining, RNA Sequencing, Derivative Assay, Generated, Ex Vivo

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: APC-Vio770 anti-human CD4 , Miltenyi Biotec , Cat# 130-113-223; RRID:AB_2726034.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software

Journal: Journal of Virology

Article Title: Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a pH- and Cysteine Protease-Independent Fc R Pathway

doi: 10.1128/jvi.00671-11

Figure Lengend Snippet: FIG. 2. Anti-Spike antibodies trigger SARS-CoV infection of human Raji B cells. VeroE6 or human Raji cells were infected (MOI 1) with SARS-CoV strain HK39849 (SCoV) in the presence of either control or Anti-Spike serum for 1 h. Samples were fixed with 4% paraformaldehyde at 15 h postinfection. A mouse monoclonal antibody specific for the SCoV nucleocapsid (N) and rabbit polyclonal antibodies specific for SCoV membrane (M) protein were visualized using TRITC-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit antibodies, respectively. Cell nuclei were labeled with DAPI, and separate channel images were acquired. (A) As expected, SCoV infection of VeroE6 (I) was completely blocked by anti-Spike serum (1/500, II). (B) In contrast, SCoV viral proteins were detected in Raji cells only when they were incubated with anti-Spike (1/500, IV) but never with control serum (III). Quantification of infection (right-hand portions in panels A and B) was done by counting positive cells in 30 randomly chosen fields (630, final magnification), using Metamorph as described in Materials and Methods. The results are shown as means the SD of the number of fields.

Article Snippet: The cells were then washed, and the primary antibody binding was revealed by staining at 4°C for 30 min with FITC-conjugated goat anti-mouse antibodies (Jackson Immunoresearch).

Techniques: Infection, Control, Membrane, Labeling, Incubation

Journal: Journal of Virology

Article Title: Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a pH- and Cysteine Protease-Independent Fc R Pathway

doi: 10.1128/jvi.00671-11

Figure Lengend Snippet: FIG. 5. Blockade of FcRII abrogates antibody-mediated infection of human immune cell lines. (A) Total RNAs from the indicated cell lines were extracted for RT-PCR amplification as described under Materials and Methods. cDNAs and negative RT controls (RT) were probed for detection of FcεR1-chain, FcRIA (CD64a), FcRIIA (CD32a), FcRIIB (CD32b), FcRIIIA (CD16a), ACE2, and GAPDH. DNA plasmids containing the coding sequence of the human Fc receptors or the human ACE2 (104 and 106 copies) were used as positive controls. Amplicons were visualized by ethidium bromide staining after agarose gel electrophoresis. For reference, a 1-kb molecular-weight standard is also shown. Due to the murine origin of the macrophage-like P388D1 and J774A.1 cell lines and because the primers were designed for the detection of human FcR (Table 2), no FcR transcripts were detected in P388D1 and J774A.1 cells. (B) Hematopoietic cell lines displaying a detectable level of FcR transcript in panel A were subjected to flow cytometry. Cell lines were stained with either isotype-matched control antibody (gray-filled histograms) or FcR-specific mouse monoclonal antibody (open histograms) as indicated, followed by secondary FITC-conjugated goat anti-mouse antibodies. Labeled samples were then read by flow cytometry and post-acquisition analysis was performed using FlowJo software. Insets show the median of fluorescence intensity of the anti-FcR staining. When not visible, the open histograms were superimposed on the gray-filled ones. The data are representative of two independent experiments with similar results. (C) Effect of FcR blockage on susceptibility of the cells to ADE infection. Prior to infection, cells were treated with anti-FcR blocking antibodies. At 3 days postinfection, luciferase substrate reagent was added to wells, and the luminescence was measured. The data were normalized to control conditions, viz., cells infected in the presence of IgG isotype-matched control antibody, and expressed as a percentage of the changes in luminescence. For all conditions, values of background luminescence (never exceeding 3% of control) were subtracted prior to normalization. The results are the means the SD of nine measurements from three independent experiments. ‡, P 0.05; *, P 0.001 (unpaired Student t test).

Article Snippet: The cells were then washed, and the primary antibody binding was revealed by staining at 4°C for 30 min with FITC-conjugated goat anti-mouse antibodies (Jackson Immunoresearch).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Staining, Agarose Gel Electrophoresis, Molecular Weight, Cytometry, Control, Labeling, Software, Blocking Assay, Luciferase

Journal: Journal of Virology

Article Title: Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a pH- and Cysteine Protease-Independent Fc R Pathway

doi: 10.1128/jvi.00671-11

Figure Lengend Snippet: FIG. 6. Human FcRIIA and B1, but not huFcRIA nor huFcRIIIA, are responsible for ADE of SARS-CoV pseudoparticles (SARS-CoVpp) infection. (A) Human Raji, parental, and monoclonal FcR-transduced human ST486 B cell lines were stained with either isotype-matched control antibody (gray-tinted histogram) or FcR-specific mouse monoclonal antibody (open histogram) as indicated, followed by secondary FITC- conjugated goat anti-mouse antibodies. Labeled samples were then read by flow cytometry and postacquisition analysis was performed using FlowJo software. The inset shows the median of fluorescence intensity of the anti-FcR staining. When not visible, the open histograms were superimposed on the gray-filled ones. (B) Potency of FcR(s) to trigger antibody-mediated entry of SARS-CoVpp was assessed by using ST486 cell lines stably expressing a single FcR. SARS-CoVpp were incubated in the presence or absence of different dilutions (1/1,000, 1/2,000, and 1/4,000) of either control (solid bars) or anti-Spike (hatched bars) serum for 1 h prior to addition to the cells. At 3 days postinfection, luciferase substrate reagent was added to wells, and the luminescence was measured. The data were normalized to control conditions, viz., cells incubated with SARS-CoVpp in the absence of any serum (black bar, taken as unity) and expressed as fold changes in luminescence. Because results for SARS-CoVpp control serum were virtually identical with all cell lines, for the sake of clarity only one dilution (1/4,000) is shown. The results are the means the SD of nine measurements from three independent experiments. Statistical significance was assessed by comparing the appropriate dilutions of mock and anti-Spike serum. ‡, P 0.05; *, P 0.01 (unpaired Student t test).

Article Snippet: The cells were then washed, and the primary antibody binding was revealed by staining at 4°C for 30 min with FITC-conjugated goat anti-mouse antibodies (Jackson Immunoresearch).

Techniques: Infection, Staining, Control, Labeling, Cytometry, Software, Stable Transfection, Expressing, Incubation, Luciferase

Journal: Cell reports

Article Title: Differential Functions of Splicing Factors in Mammary Transformation and Breast Cancer Metastasis

doi: 10.1016/j.celrep.2019.10.110

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MTT Assay Cell proliferation assays were performed with an ATCC MTT Assay kit (ATTC).

Techniques: Recombinant, SYBR Green Assay, MTT Assay, Sequencing, shRNA, Software