cd69  (Miltenyi Biotec)

Journal: Advanced Science

Article Title: KRAS Withdrawal in Cholangiocarcinoma Leads to Immune Infiltration and Tumor Regression

doi: 10.1002/advs.202511312

Figure Lengend Snippet: Kras G12D withdrawal recruits CD8 T cells and Macrophages to the tumor. A) H&E and IHC staining of TKP Kras ON and Kras OFF tumors. Kras OFF tumors were collected 8 days after doxycycline withdrawal. B) Quantification of CD8 and F4/80 in TKP Kras ON tumors (n=6) and Kras OFF tumors (n=8). Each data point represents the mean of positive cells of ten 20X microscopic fields. Lines represent the mean value. P‐values were calculated by an unpaired t‐test. *** P <0.001. C) Representative immunofluorescence images of CK19, CD8, and CD69 positive cells in TKP Kras ON and Kras OFF tumors. D) UMAP visualization of CD8 + T cell clusters identified by scRNA‐seq of 13,329 cells from one TKP Kras ON and one TKP Kras OFF tumor. E) Bar graph indicating the enrichment of CD8 + T cells, CD69 + T cells, and CD8 + CD69 + T cells in TKP Kras ON and Kras OFF tumors by scRNA‐seq.

Article Snippet: The following primary antibodies were used: CK19 (EPNCIR127B; 1:400) (Abcam); CD8 (4SM15; 1:100) (Thermo); CD69 (Cat# AF2386; 1:100) (R&D Systems).

Techniques: Immunohistochemistry, Immunofluorescence

Journal: Advanced Science

Article Title: KRAS Withdrawal in Cholangiocarcinoma Leads to Immune Infiltration and Tumor Regression

doi: 10.1002/advs.202511312

Figure Lengend Snippet: Murine IL‐15 or CCL17 delays the progression of syngeneic mouse CCA xenograft tumors. A) RIL‐175 CCA cell lines from a B6 genetic background were transduced with a lentiviral vector encoding a constitutive Ef1α promoter and mouse cytokine/chemokine cDNAs or an empty lentiviral vector as a control. Xenograft models were made by injecting 10 6 cells subcutaneously into immunocompetent B6 mice. B) Overexpression of either IL‐15 or CCL17 delayed tumor progression. Control, n=10; IL‐15‐O/E, n=9, CCL17‐O/E, n=10. Data are presented as mean ± SD. C) Tumor volume at day 13 after injection. Data are presented as mean ± SD. D–G) Flow cytometry analysis in control, IL‐15‐overexpressed, and CCL17‐overexpressed tumors at day 13 after injection. Lines represent the mean value. E) CD69, an activated T cell marker. (F) GZMB, granzyme B. (G) CD62L + CD44 + , memory T cells markers. H) CD8⁺ T cell depletion abolishes the anti‐tumor effect induced by IL‐15. Tumor growth curves of control and IL‐15 overexpressing RIL‐175 mouse CCA cell line in mice treated with vehicle, CD8‐depleting antibody, or isotype control (n=6). Data are presented as mean ± SD. I) Representative immunohistochemistry (IHC) images showing CD8⁺ T cell infiltration in spleens and tumors from vehicle control, IgG, and CD8‐depleted mice. CD8 depletion efficiency is evident by the abolition of CD8⁺ staining in the αCD8 group. P‐values were calculated by two‐way ANOVA (B, H) or one‐way ANOVA (C‐G). ns=no significance, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: The following primary antibodies were used: CK19 (EPNCIR127B; 1:400) (Abcam); CD8 (4SM15; 1:100) (Thermo); CD69 (Cat# AF2386; 1:100) (R&D Systems).

Techniques: Transduction, Plasmid Preparation, Control, Over Expression, Injection, Flow Cytometry, Marker, Immunohistochemistry, Staining