Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Thymus size is reduced, whereas mesenteric lymph nodes and spleen are enlarged, in BCL11B F/F CD4cre mice. Gross photographs of mutant and control thymi, lymph nodes, and spleens, indicating reduction in the thymus size and increase in the mesenteric lymph nodes and spleen of BCL11B F/F CD4cre mice. Bars, 1 cm. (bottom right) Ratios of spleen weight (g) to total body weight (g) of BCL11B F/F (WT) and BCL11B F/F CD4cre (KO) mice. P = 1.28771E-05; n = 6; 10 wk old.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Mutagenesis, Control

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Removal of BCL11B in DP thymocytes results in a severe decrease in the number of CD4 and CD8 SP thymocytes. (A) Thymocytes from BCL11B F/F CD4cre (KO) and BCL11B F/F mice (WT) were stained for CD4 and CD8 and analyzed by FACS. (B) Thymocytes were stained for CD4, CD8, and TCRβ. Histograms show TCRβ on gated CD4 or CD8 SP thymocytes. Representative data from multiple experiments are shown. (C) Thymic cellularity of BCL11B F/F CD4cre and control mice. Thymocytes were stained for CD4 and CD8 coreceptors and analyzed by FACS. Total cellularity was determined by counting the live cells. Absolute numbers of cells were calculated based on the percentage of each population and represented as values ± the SD. Cell counts should be multiplied by 10 6 . A two-tailed Student's t test was applied to determine the statistical significance. Mice in these experiments were between 5 and 6 wk of age.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Staining, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Lymph node cellularity of BCL11B F/F CD4cre mice

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Spleen cellularity of BCL11B F/F CD4cre mice

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Peripheral T lymphocytes of BCL11B F/F CD4cre mice are reduced in number and present an activated/memory-like phenotype. Lymphocytes from lymph nodes (A) and spleen (B) of BCL11B F/F CD4cre (KO) and BCL11B F/F mice (WT) were stained for CD4 and CD8 and analyzed by FACS. (C) Lymphocytes were stained for CD4, CD8, and TCRβ. Histograms show TCRβ on gated CD4 + or CD8 + T lymphocytes. (D) Surface expression of CD44 and CD62L on gated CD4 + and CD8 + T lymphocytes. Representative data from multiple experiments are shown.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Staining, Expressing

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Positive selection of BCL11B F/F CD4cre DP thymocytes is diminished without alteration in TCRα locus rearrangements. (A) Thymocytes were stained for CD4, CD8, TCRβ, and CD69 and analyzed by FACS. CD69 and TCRβ expression on gated DP thymocytes. Numbers indicate percentage of cells in each quadrant. (B) Thymocytes were stained for HSA and TCRβ and analyzed by FACS. Numbers indicate the percentage of cells in each quadrant. (C) Thymocytes were stained for CD4, CD8, TCRβ, CD69, and CD5. Histograms show TCRβ, CD69, and CD5 levels on gated DP thymocytes. (D) Genomic DNA was extracted from DP thymocytes and amplified by PCR using primers specific for the indicated Vα and Jα gene segments. Amplification of the αC region was used for normalization. Dividing lines indicate grouping of images from different parts of the same gel or different gels. This is one representative experiment out of four.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Selection, Staining, Expressing, Amplification

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Altered positive selection of CD8 and CD4 lineages in BCL11B F/F CD4cre mice expressing transgenic TCRs. (A) Positive selection analysis in OT2 mice. Thymocytes were stained for CD4, CD8, TCRVα2, TCRVβ5, and HSA (c), CD69 (d), or CD5 (e). (a) Surface expression of CD4 and CD8 on thymocytes analyzed by FACS. Boxes indicate DP, CD4 + CD8 low , and CD4 SP populations, and numbers represent the percentage of cells in the boxes. (b) Surface expression of CD4 and CD8 on gated Vα2/Vβ5TCR high thymocytes. Boxes indicate DP, CD4 + CD8 low , and CD4 SP populations. The dotted line rectangle contains all CD4 + Vα2/Vβ5TCR high thymocytes. (c) Surface expression of HSA and Vβ5TCR on gated CD4 + Vα2/Vβ5TCR high thymocytes. Vβ5TCR high /HSA low population is indicated by a rectangle. (d) Surface expression of CD69 and Vβ5TCR on DP thymocytes. (e) Histogram showing surface expression of CD5 on Vα2/Vβ5TCR high DP gated populations. (f) Surface expression of CD4 and CD8 on lymphocytes. Representative data from multiple experiments are shown. (B) Thymic cellularity of OT-2 TCR/BCL11B F/F CD4cre mice. Thymocytes were stained for CD4, CD8, TCRVα2, and TCRVβ5 and analyzed by flow cytometry. Total cellularity was determined by counting the live cells. Absolute numbers of cells were calculated based on the percentage of each population and represented as values ± the SD. Cell counts should be multiplied by 10 6 . Two-tailed Student's t test was applied to determine the statistical significance. (C) Positive selection analysis in H-Y TCR females. Thymocytes were stained for CD4, CD8, T3.70 TCR, and HSA (c), CD69 (d) or CD5 (e). (a) Surface expression of CD4 and CD8 on thymocytes. Boxes indicate DP, CD8 + CD4 low , and CD8 SP populations, and the numbers represent the percentage of cells in the boxes. (b) Surface expression of CD4 and CD8 on gated T3.70 TCR high thymocytes. Boxes indicate DP, CD8 + CD4 low , and CD8 SP populations. The dotted line rectangle contains all CD8 + T3.70 TCR high thymocytes. (c) Surface expression of HSA and T3.70 TCR on all CD8 + gated thymocytes. The T3.70 TCR high /HSA low population is indicated by a rectangle. (d) Surface expression of CD69 and T3.70 TCR on DP gated populations. (e) Histogram showing surface expression of CD5 on T3.70 TCR high DP gated populations. (f) Surface expression of CD4 and CD8 on lymphocytes. These are representative experiments out of four. (D) Thymic cellularity of H-Y TCR/BCL11B F/F CD4cre female mice. Thymocytes were stained for HY-TCR, CD4, and CD8 coreceptors and analyzed by flow cytometry. Thymic cellularity was calculated as described in B. Cell counts should be multiplied by 10 6 . Two-tailed Student's t test was applied to determine the statistical significance.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Selection, Expressing, Transgenic Assay, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: ERK phosphorylation, Ca flux, and proximal TCR signaling events are attenuated in BCL11B F/F CD4cre DP thymocytes. Immunoblot analysis of ERK phosphorylation kinetics in response to stimulation by TCR cross-linking in DP-enriched thymocytes (A) or sorted presignaled CD69 − DP thymocytes (B). In both A and B, ERK phosphorylation was evaluated with anti–phospho-p42/p44. The asterisk in A indicates a nonspecific band. (C) Ca flux analysis in CD69 − DP thymocytes. Thymocytes were loaded with Fluo4, surface stained for CD4, CD8, and CD69, and activated as described in the Supplemental materials and methods. Fluorescence was recorded for 5 min after stimulation and ionomycin was added to record the maximal calcium response. Calcium flux was analyzed in presignaled CD69 − DP thymocytes using the kinetics platform of the FlowJo and represented as Fluo-4 fluorescence over time. Notably, when anti-CD4 RM4-5 antibody was used for staining and anti-CD4 GK 1.5 antibody was used for stimulation (see Materials and methods and Supplemental materials and methods), the cross-blocking reduced the potency of the anti-CD4 stimulation. (D) Immunoblot analysis of nuclear translocation of NFAT4 in response to TCR activation. (E) Immunoblot analysis of ZAP70 and SLP76 phosphorylation. Preparation of enriched DP thymocytes and their activation is described in the Supplemental materials and methods. Representative data from multiple experiments are shown. Supplemental materials and methods are available at http://www.jem.org/cgi/content/full/jem.20070863/DC1 .

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Phospho-proteomics, Western Blot, Staining, Fluorescence, Blocking Assay, Translocation Assay, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: BCL11B F/F CD4cre DP thymocytes present enhanced apoptosis independent of TCR signaling, which is only partially rescued by transgenic BCL2. (A) Thymic cellularity of BCL11B F/F CD4cre/TCRα −/− and BCL11B F/F /TCRα −/− mice. Thymic cellularity was calculated as described in . Absolute cell numbers should be multiplied by 10 6 . Two-tailed Student's t test was applied to determine the statistical significance. Mice in these experiments were between 5 and 8 wk of age. (B) In vitro survival of BCL11B F/F CD4cre DP thymocytes. Fresh or cultured thymocytes were stained for CD4 and CD8, and with AnnexinV and 7-AAD and analyzed by FACS. Survival of DP thymocytes in vitro was estimated as the frequency of live (Annexin V − /7-AAD − ) cells after 16 h in culture reported to the input population, and represented as the mean ± the SD. n = 4 pairs of mice. P = 0.034. (C) In vitro survival of BCL11B F/F CD4cre/TCRα −/− DP thymocytes. Fresh or cultured thymocytes from BCL11B F/F CD4cre/TCRα −/− and BCL11B F/F /TCRα −/− mice were stained, analyzed, and represented as in B. n = 4 pairs of mice. P = 0.032. (D) Immunoblot analysis of the indicated proapoptotic and prosurvival factors. (E) In vitro survival of BCL11B F/F CD4cre/BCL2 DP thymocytes. Fresh or cultured thymocytes from BCL11B F/F CD4cre/BCL2 and BCL11B F/F /BCL2 mice were stained, analyzed, and represented as in B. n = 6 pairs of mice. P = 0.054. (F) Immunoblot analysis showing cleaved caspase 3 in BCL2 transgenic and nontransgenic BCL11B F/F CD4cre and BCL11B F/F DP thymocytes. Cleaved caspase 3 levels were quantified by densitometric analysis and normalized to actin. (G) Thymic cellularity of BCL-2/BCL11B F/F CD4cre mice. Thymocytes were stained for CD4 and CD8 and analyzed by FACS. Thymic cellularity was calculated as described in . Cell counts should be multiplied by 10 6 . Two-tailed Student's t test was applied to determine the statistical significance.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Transgenic Assay, Two Tailed Test, In Vitro, Cell Culture, Staining, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: BCL11B is required for positive selection and survival of double-positive thymocytes

doi: 10.1084/jem.20070863

Figure Lengend Snippet: Expression of genes implicated in positive selection, TCR signaling, and apoptosis is altered in BCL11B-deficient DP thymocytes. (A) Ratios of relative mRNA levels of genes involved in positive selection and survival/apoptosis in BCL11B-deficient DP thymocytes versus wild type. mRNA levels were quantified by qRT-PCR, as described in the Materials and methods , in BCL11B F/F CD4cre (KO) and BCL11B F/F (WT), and in BCL11B F/F CD4cre/TCRα −/− (KO) and BCL11B F/F /TCRα −/− (WT) mice. Representative data is presented from multiple experiments. PD-1 (B) and CD5 (C) surface levels in thymocyte populations. Total thymocytes from BCL11B F/F CD4cre (KO) and BCL11B F/F (WT) mice were stained for CD4, CD8, CD69, and PD-1 (B) or CD5 (C) and analyzed by flow cytometry. Histograms showing PD-1 (B) and CD5 (C) levels on gated CD69 − DP, CD69 + DP, CD4, and CD8 SP thymocytes. Data are representative of four independent experiments.

Article Snippet: BCL11B F/F mice were crossed with C57BL/6 CD4Cre transgenic mice (line 4196; Taconic) ( ) to generate conditional BCL11B F/F CD4cre mice.

Techniques: Expressing, Selection, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Development of a PCSK9-targeted nanoparticle vaccine to effectively decrease the hypercholesterolemia

doi: 10.1016/j.xcrm.2024.101614

Figure Lengend Snippet: Induction of potent immune responses by the PCSK9-NP vaccine in BALB/c mice and beagles (A) Schematic representation of the BALB/c mice vaccination protocol. Mice received two subcutaneous immunizations at W0 and W4. Serum samples were collected biweekly. (B) Time-course curve of PCSK9 catalytic domain-specific IgG titers measured in mouse sera. (C) Calculation of endpoint titers of anti-PCSK9 catalytic domain IgG1, IgG2a, and IgG2b at W6 using ELISA. (D) Detection of IgG antibodies against full-length PCSK9 at W6. (E) Flow cytometry (left) and statistical analyses (right) showing the percentages and absolute numbers of CD4 + CXCR5 + PD1 + Tfh cells (top) and CD19 + Fas + GL7 + GCB cells (bottom) in the draining lymph nodes at W2. (F) Schematic representation of the beagle vaccination protocol. (G) Time-course curve displaying PCSK9 catalytic domain-specific IgG titers in sera from beagles. (H) Flowchart illustrating the cell-based experiments conducted to evaluate the in vitro interfering activity of serum antibodies. (I and J) Flow cytometry (left) and statistical analyses (right) showing the MFI of the cell-surface receptor LDLR in Huh7 cells exposed to PCSK9 in conjunction with sera from immunized BALB/c mice (I) and from beagles at W6 (J). Experiments were conducted independently in triplicates; all data are presented as mean ± SEM; n = 4–5 per group. Data in (C–E) and (I) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. two-way ANOVA with Tukey’s (B) and Sidak’s (G) multiple comparisons test were used for statistical analysis. An unpaired Student’s t test (J) was used. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, no significance. See also Figures S1, S2, S4, and S5 .

Article Snippet: Bcl6 fl/fl or CD4 cre Bcl6 fl/fl C57BL/6 mice , GemPharmatech Co, Ltd , N/A.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, In Vitro, Activity Assay, Cell Surface Receptor Assay

Journal: Cell Reports Medicine

Article Title: Development of a PCSK9-targeted nanoparticle vaccine to effectively decrease the hypercholesterolemia

doi: 10.1016/j.xcrm.2024.101614

Figure Lengend Snippet:

Article Snippet: Bcl6 fl/fl or CD4 cre Bcl6 fl/fl C57BL/6 mice , GemPharmatech Co, Ltd , N/A.

Techniques: Recombinant, Virus, Enzyme-linked Immunosorbent Assay, Staining, Cell Stimulation, Adjuvant, Western Blot, Reverse Transcription, Plasmid Preparation, Protein Extraction, RNA Extraction, Software

Journal: European journal of immunology

Article Title: microRNAs targeting TGFβ signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function

doi: 10.1002/eji.202350548

Figure Lengend Snippet: A: B6-CD4KO mice were reconstituted with magnetically sorted CD4 T cells that were transduced with miRNA-expression construct delivering lentivirus (miR-128+212+708 and miR-NS) at 3, 5 and 7 week of age. Transduction was verified by flow cytometry through the GFP-expression of the LV construct and the reconstitution of CD4 T cells was confirmed by flow cytometry of blood ( Suppl. fig 3 ). EAE was induced using the Hooke’s kit MOG 35–55 /CFA emulsion followed by a single PTX injection on the same day (n=4 each group). B: Mean clinical scores ± SEM of neurological signs of the disease (Mann-Whitney test **: p<0.01). C: Area under the curve calculations of the clinical scores (unpaired t-test *: p<0.05). D: Disease onset as a “survival curve” as a percentage of mice without signs of EAE. E-H: At the end of the monitoring period, pooled draining lymph node cells (inguinal, axillary and cervical) were analyzed by flow cytometry for Tregs (CD25+/FoxP3+ of CD4 T cells), effector (CD62L-/CD44+ of CD4 T cells) and naïve CD4 T cells (CD62L+/CD44- of CD4 T cells). I-J : dLN cells were also stimulated by PMA + Ionomycin and CD4 T cells were analyzed for intracellular cytokines IFNγ and IL-17 after the addition of GolgiPlug. Representative plots are shown in E, G and I . Statistical analysis in F , H and J : Mann-Whitney test *: p<0.05, ns: p>0.05. Statistical analysis in J : Mean ± SEM, Mann-Whitney test *: p<0.05. A Created with BioRender.com .

Article Snippet: B10.PL, C57BL/6, C57BL/6-Foxp3 EGFP (B6.Cg-Foxp3 tm2Tch /J –#006772) [ 73 ], [ 74 ] and CD4KO (B6.129S2-Cd4 tm1Mak /J - #002663) [ 75 ] mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in a specific pathogen-free animal facility at The Ohio State University Wexner Medical Center.

Techniques: Over Expression, Transduction, Expressing, Construct, Flow Cytometry, Emulsion, Injection, MANN-WHITNEY