plko 3 thy1 1 cd90 1 vector  (Addgene inc)

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Recipient mice (Thy1.2 + ) were transferred with mixed OVA-specific Cd69 +/+ (Thy1.1 + Thy1.2 + ) and Cd69 −/− (Thy1.1 + Thy1.2 − ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.n.: intranasally; i.v.: intravenously. ( B ) Plots of Cd69 +/+ and Cd69 −/− CD45(iv) − CD4 + T cells in the lung (left) and absolute numbers ( n = 5) (right). ( C ) Confocal micrograph of the lung at day 35 (left). V: vessels. Frequency of transferred Cd69 +/+ (yellow) and Cd69 −/− (red) CD4 + T cells in iBALT ( n = 5) (right). DAPI: 4′,6-diamidino-2-phenylindole. ( D ) Recipient mice (Ly5.2 + ) were transferred with the mixed OVA-specific creER T2 Cd69 +/+ (Ly5.1 + Ly5.2 − ) and Cd69 fl/fl (Ly5.1 + Ly5.2 + ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.p.: intraperitoneally. ( E ) Plots of creER T2 Cd69 +/+ and Cd69 fl/fl cells in Ly5.1 + CD45(iv) − CD4 + T cells in the lung on day 49 and ( F ) absolute numbers (PBS, n = 8; TAM, n = 13). ( G ) Experimental protocol to investigate the functional impact of CD4 + T RM cell egress from the lungs on airway inflammation. ( H ) Absolute numbers of eosinophils (Eos.), neutrophils (Neut.), lymphocytes (Lymph.), and macrophages (Mac.) in the bronchoalveolar lavage fluid (BALF). ( I ) Representative H&E-stained lung sections. ( J ) Experimental protocol to assess the impact of CD69 ablation in CD4 + T RM cells under BCG infection. ( K ) Representative plots of CD45(iv) − CD44 hi CD69 + CD4 + T cells in the lung at day 49. ( L ) Absolute numbers of CD45(iv) − CD4 + T cells in PBS-treated ( n = 10) or TAM-treated ( n = 10) mice. Pooled data from two independent experiments. [(B), (C), (F), and (H)] Data of more than two independent experiments. Significance was determined by an unpaired U test in (B) and (C) and a one-way ANOVA in (F), (H), and (L). Data represent the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Thy1.1 + mice were purchased from the Jackson Laboratory.

Techniques: Functional Assay, Staining, Infection

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Volcano plot of the differential gene expression between CD4 + T RM cells and CD4 + T CIRC cells identified in fig. S5 (A to E). ( B ) Plots of CD69 and S1PR1 in Ly5.1 + CD45(iv) − CD4 + T cells in the lung of the OVA-treated mice at day 49 shown in fig. S6A. ( C ) Absolute numbers of CD45(iv) − S1PR1 + CD4 + T cells in the mice transferred with creER T2 Cd69 +/+ T H 2 cells ( n = 9) or creER T2 Cd69 fl/fl T H 2 cells ( n = 10). Pooled data from two independent experiments. ( D ) Absolute numbers of CD45(iv) − CD4 + T cells in the three groups ( n = 4 or 5). Data of two independent experiments. ( E ) Confocal micrograph of the lungs at day 49. B: bronchiole. ( F ) Absolute numbers of Ly5.1 + memory CD4 + T cells in iBALT. Data of two independent experiments. ( G ) H&E-stained lung section (top). Lungs were collected from the mice treated as shown in fig. S2A. The vessel wall, iBALT, and a parenchyma area located 200 μm from blood vessels were chosen to measure S1P levels by mass spectroscopy. The S1P intensity in these regions is shown (bottom). ( H ) Average peak intensities of S1P in each region. ( I ) Confocal micrograph of the lungs at day 35. Lungs were collected from the mice treated as shown in fig. S6H. ( J ) Absolute numbers of CD69 + Thy1.1 + cells (left) and CD69 − Thy1.1 + cells (right) in concentric distance zones from Lyve-1 + lymphatic vessels. Data represent the means ± SEM. Significance was determined by a one-way ANOVA in (C), (D), and (F), by paired one-way ANOVA in (H), and by an unpaired U test in (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Thy1.1 + mice were purchased from the Jackson Laboratory.

Techniques: Gene Expression, Staining, Mass Spectrometry

Journal: Nature

Article Title: Facile induction of immune tolerance by an interleukin-2–TGFβ surrogate agonist

doi: 10.1038/s41586-026-10208-0

Figure Lengend Snippet: a–d , Therapeutic effect of TGM1–IL-2 in DSS-induced colitis (PBS, n = 9 mice; TGM1–IL-2, n = 10 mice). a , Individual weight changes of mice in the indicated groups. b , Representative flow cytometry plots and quantification of IFN-γ + and IL-17a + CD4 + T-cell frequencies in the colon. c , Representative flow cytometry plots and quantification of Foxp3 + and Foxp3 - Thy1.1 + OT-II cell frequencies among total CD4 + T cells in the indicated tissues. d , Representative flow cytometry plots and quantification of Rorγt + and Gata3 + cell frequencies among Foxp3 + OT-II cells in the indicated tissues. e , UMAP plots showing the indicated transcription factor activity scores across clusters. f , Venn diagram illustrating the overlap of up- and down-regulated DEGs between the indicated groups. DEGs were filtered using an adjusted p value < 0.0001 and log 2 fold change > 1. g , Gene regulatory networks and signature genes identified by scRNA-seq analysis. The two numbers in parentheses represent the specific values from the Venn diagram shown in Extended Data Fig. 12f (right). h , Schematic illustration of antigen-specific pTreg differentiation in vivo driven by TCR stimulation, TGM1–IL-2, and key transcription factors. Created in BioRender; Sun, Q. https://BioRender.com/2rbbpii (2026). Data are presented as mean ± s.e.m. The data in a–d are representative of two independent experiments. The statistics were obtained by unpaired Welch’s t-test (two-tailed) ( b and c ).

Article Snippet: Foxp3 -GFP mice (IMSR_JAX:006772) were crossed with OT-II Thy1.1 mice.

Techniques: Flow Cytometry, Activity Assay, In Vivo, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Influence of LCN2 on the growth of lung cancer in mice. (A) Simplified flowchart outlining the establishment of an anti-PD-L1 immunotherapy tolerance model in mice with lung cancer. (B) Western blot analysis of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice. (C) Immunohistochemical detection of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice (scale bar, 100 µm). (D) Establishment of a model for anti-PD-L1 immunotherapy in LCN2-silenced mice. (E) Evaluation of LCN2 mRNA expression in tumor tissues after LCN2 gene intervention in mice. (F) Assessment of LCN2 protein expression in tumor tissues following LCN2 gene intervention in mice. (G) Tumor size in LCN2 gene intervention mice (n=10 in each group). *P<0.05, **P<0.01 and ***P<0.001. Unpaired two-tailed Student's t-test was used for (B) and (C) and one-way ANOVA with Tukey's multiple comparisons test was used for (E) and (G). LCN2, lipocalin 2; PD-L1, programmed death-ligand 1; sh-, short hairpin; NC, negative control; COL1A1, collagen α-1(I) chain; THY1, Thy-1 membrane glycoprotein; i.p., intraperitoneal.

Article Snippet: The following primary antibodies were used: Rabbit anti-LCN2 (cat. no. ab125075; 1:100; Abcam), rabbit anti-COL1A1 (cat. no ab34710; 1:15; Abcam), rabbit anti-Thy-1 membrane glycoprotein (THY1) (cat. no 13801; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-E-cadherin (cat. no 3195; 1:400; Cell Signaling Technology, Inc.), rabbit anti-Vimentin (cat. no. 5741; 1:4,000; Cell Signaling Technology, Inc.), rabbit anti-IL-6 (cat. no. ab6672; 1:400; Abcam), rabbit anti-TNF-α (cat. no. ab307164; 1:1,000; Abcam), rabbit anti-Ki-67 (cat. no. ab16667; 1:200; Abcam) and rabbit anti-cleaved caspase-3 A (cat. no. 9661; 1:400; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Two Tailed Test, Negative Control, Membrane