cd8 alpha d8a8y rabbit mab  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Inhibitors of oncogenic Kras specifically prime CTLA4 blockade to transcriptionally reprogram Tregs and overcome resistance to suppress pancreas cancer

doi: 10.1101/2025.02.28.640711

Figure Lengend Snippet: (A) Schematic representation of orthotopic injection and MRTX1133 treatment of KPC1 mice. ( B-C ) Dot plots of genes used to define T cell subtypes (B) and UMAP of all cell types (left) and T cell subsets in Vehicle/ MRTX1133 treated (right) KPC1 PDAC determined by scRNA seq. analysis (C) (n=3 tumors/ group). (D) Relative abundance as a percentage of T cells (left) and CD45 + cells (right) in KPC1 PDAC determined by scRNA seq. (E-F) Violin plots for B2m , Muc1 (E) and Ido1 , Ptges2 (F) in ductal/ cancer cells and fibroblasts of KPC1 mice determined by scRNA seq.. (G) Schematic representation of orthotopic injection and RMC6236, MRTX1133, αCTLA4 and αPD1 treatments of KPC2 mice. (H-I) Kaplan-Meier survival curves of Veh/Iso (n=17), MRTX1133 (n=11), MRTX1133+αCTLA4 (n=11), MRTX1133+αPD1 (n=9) treated KPC2 mice (H) , Veh/Iso (n=6), RMC6236 (n=8), RMC6236+αCTLA4 (n=10), RMC6236+αPD1 (n=8) treated KPC2 mice (I) . (J-K) Representative H&E (top panel) and CD8-CD19-DAPI immunofluorescence images (bottom panel) from MRTX1133 treated age-matched KPC2 mice (J) , with αCTLA4 or αPD1 with quantification of tissue histology (K) . KPC2 tumors treated with Veh/Iso (n=3), MRTX1133 (n=11), MRTX1133+αCTLA4 (n=7), MRTX1133+αPD1 (n=7). (L) Pancreas/ tumor weights of age-matched KPC2 mice treated with Veh/Iso (n=10), MRTX1133 (n=13), MRTX1133+αCTLA4 (n=15), MRTX1133+αPD1 (n=15). (M) Quantification of CD8 and CD19 + cells from J (n=4-6 mice/group). (N-O) Representative H&E (top panel) and CD8-CD19-DAPI immunofluorescence images (bottom panel) from RMC6236 treated age-matched KPC2 mice with αCTLA4 or αPD1 (N) , with quantification of tissue histology (O) (n=3/group). (P) Pancreas/ tumor weights of age-matched KPC2 mice treated with Veh/Iso (n=8), RMC6236 (n=7), RMC6236+αCTLA4 (n=9), RMC6236+αPD1 (n=6). (Q) Quantification of CD8 and CD19 + cells from N (n=3-4 mice/group). Data are presented as mean in D , mean + SD in K, L, M, O, P, Q and as violin plots in E, F . Significance was determined by log-rank test in H and I , two-way ANOVA in K and O , Kruskal-Wallis with Dunn’s multiple comparisons test in L and CD19 comparisons in M , one-way ANOVA with Dunnett’s multiple comparisons test in P, Q and CD8 comparisons in M . Scale bars-100μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns , not significant. See related figs. S1-S2.

Article Snippet: After blocking for 1h at RT using blocking buffer, the sections were further incubated with CD8 (D8A8Y) (Cell Signaling Technology, #85336S, 1:200) primary antibody for 3h at RT.

Techniques: Injection, Immunofluorescence

Journal: bioRxiv

Article Title: Inhibitors of oncogenic Kras specifically prime CTLA4 blockade to transcriptionally reprogram Tregs and overcome resistance to suppress pancreas cancer

doi: 10.1101/2025.02.28.640711

Figure Lengend Snippet: (A) Schematic representation of orthotopic injection and MRTX1133, αCD4 and αCD25 treatments of KPC2 mice. (B) Kaplan-Meier survival curves of Veh/Iso (n=17), MRTX1133 (n=11), MRTX1133+αCD4 (n=5), MRTX1133+αCD25 (n=10) treated KPC2 mice. Note: The Iso/Veh and MRTX1133 curves are repeated from . (C-G) Immunophenotyping analysis of KPC2 PDAC of indicated groups. CD3 + (T cells); CD3 + CD4 + (CD4 + T cells); CD3 + CD8 + (CD8 + T cells) were measured as a percentage of CD45 + cells (C) ; CD4 + Foxp3 + were measured as a percentage of CD3 + cells (D) ; CD4 + T-bet + (Th1) measured as a percentage of CD45 + cells (E) , and CD19 + B cells measured as a percentage of CD45 + cells (F) . CD8/Treg ratio and Th1/Treg ratio (G) . n=4-9 tumors/ group. (H) Schematic representation of subcutaneous injection and αCTLA4 and αPD1 treatments of MC38 mice. (I) Tumor weights of age-matched MC38 mice treated with Iso (n=8), αCTLA4 (n=11), αPD1 (n=6). (J) Growth kinetics of Iso (n=8), αCTLA4 (n=11), αPD1 (n=6) treated MC38 tumors. (K-N) Immunophenotyping analysis of MC38 tumors of indicated groups. CD3 + (T cells); CD3 + CD4 + (CD4 + T cells); CD3 + CD8 + (CD8 + T cells), CD8 + T-bet + cells, CD8 + Ki67 + cells and CD8 + CD69 + cells were measured as a percentage of CD45 + cells (K) ; CD4 + Foxp3 + were measured as a percentage of CD3 + cells (L) ; CD4 + T-bet + (Th1) and CD8 + T-bet + measured as a percentage of CD3 + cells (M) . CD8/Treg ratio and Th1/Treg ratio (N) . n=6-9 tumors/ group. Data are presented as mean + SD in C, D, E, F, G, I, K, L, M and N and as individual tumor volume measurements in J . Significance was determined by log-rank test in B , Kruskal-Wallis with Dunn’s multiple comparisons test in E and in CD4 comparisons in K , one-way ANOVA with Dunnett’s multiple comparisons test in C, D, F, G, I, K, L, M and N and CD3, CD8 comparisons in M . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns , not significant. See related figs. S6-S7.

Article Snippet: After blocking for 1h at RT using blocking buffer, the sections were further incubated with CD8 (D8A8Y) (Cell Signaling Technology, #85336S, 1:200) primary antibody for 3h at RT.

Techniques: Injection

Journal: bioRxiv

Article Title: Inhibitors of oncogenic Kras specifically prime CTLA4 blockade to transcriptionally reprogram Tregs and overcome resistance to suppress pancreas cancer

doi: 10.1101/2025.02.28.640711

Figure Lengend Snippet: (A-B) Representative H&E sections of TLS in MRTX1133, RMC6236 treated KPC2 mice with aCTLA4/ aPD1 (A) , with quantification of number of TLS (n=3-7 tumors/ group) (B) . (C-D) Representative CD8-CD19-CD31-DAPI (left) and CD21-CD23-DAPI (right) immunofluorescence images of TLS (C) , with quantification of cell numbers in the TLS regions of MRTX1133 treated KPC2 mice with aCTLA4/ aPD1 (D) (n=4-6 tumors/ group). (E-F) Representative CD8-CD19-CD31-DAPI immunofluorescence images of TLS (E) , with quantification of cell numbers in the TLS regions of RMC6236 treated KPC2 mice with aCTLA4/ aPD1 (F) (n=3-4 tumors/ group). (G-H) UMAP of all cell types (left) and B cell subsets in aCTLA4, MRTX1133, MRTX1133+aCTLA4 treated (right) KPC2 PDAC (G) , with number of cells in B cell subsets (as % of CD45 + cells) (H) determined by scRNA seq. analysis (n=3 mice/group). (I-J) UMAP of all cell types (left) and B cell subsets in Veh/Iso, RMC6236, RMC6236+aCTLA4, RMC6236+aPD1 treated (right) KPC2 PDAC (I) , with number of cells in B cell subsets (J) determined by scRNA seq. analysis (n=3 tumors/group). Data are presented as mean in H and J , mean + SD in B, D, and F . Significance was determined by Kruskal-Wallis with Dunn’s multiple comparisons test in B and D , one-way ANOVA with Dunnett’s multiple comparisons test in F . * P < 0.05, ** P < 0.01, **** P < 0.0001; ns , not significant. See related figs. S8-S10.

Article Snippet: After blocking for 1h at RT using blocking buffer, the sections were further incubated with CD8 (D8A8Y) (Cell Signaling Technology, #85336S, 1:200) primary antibody for 3h at RT.

Techniques: Immunofluorescence

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Kyn induced Siglec‐15‐mediated immune escape. (A) The chromatin immunoprecipitation (ChIP)‐PCR assay was performed using an IgG or AhR antibody after treatment with 200 µmol/L Kyn for 1 h. Two primers targeting the promoter region of SIGLEC15 and CD274 mRNA were used for RT‐qPCR analysis. (B) 293T cells were co‐transfected with SIGLEC15 / CD274 promoter‐luciferase reporter PGL3 for 24 h and treated with the indicated concentration of Kyn for another 6 h, followed by an analysis of luciferase activity. (C) 293T cells were co‐transfected with SIGLEC15 / CD274 promoter‐luciferase reporter PGL3 for 24 h and treated with 200 µmol/L Kyn for the indicated time, followed by an analysis of luciferase activity. (D) 293T cells were co‐transfected with promoter‐luciferase reporter plasmids and siAhR /siScr for 24 h and treated with 200 µmol/L Kyn for 6 h. (E) SCCVII cells were transfected with lentivirus‐ SIGLEC15 , and transfection was confirmed using Western blotting analysis. (F) Vector or SIGLEC15 ‐overexpressing SCCVII cells were subcutaneously injected into C3H/He mice ( n = 5 per group). Tumor volumes were measured once every two days. (G) Tumor weights were measured after mice were euthanized. (H‐J) H&E (H), TUNEL (I), and Ki‐67 (J) staining analyses of tumor tissues in each group. (K‐P) Tumor‐infiltrating lymphocytes (TIL) harvested from xenograft tumors, and the percentages of CD3 + CD8 + (K), Ki‐67 + (L), PD‐1 + (M), IFN‐γ + (N), granzyme B + (O), and perforin + cells (P) were analyzed using flow cytometry. (Q) The percentages of CD8 + in the indicated tumors were analyzed by multiplex immunofluorescence staining. (R) The volume and Ki‐67 expression of tumor were compared in tongue orthotopic transplant models established by vector or SIGLEC15 ‐overexpressing SCCVII cells ( n = 6 mice per group). (S) PD‐1 + CD8 + T cells were compared in tongue orthotopic transplant models ( n = 6 mice per group), based on multiplex immunofluorescence staining. Three regions of interest (ROIs) in each tumor were analyzed and measured. Data are represented as mean ± SEM (A‐D, F‐G and I‐S) based on three independent experiments (A‐D). * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: Kyn, kynurenine; ChIP, chromatin immunoprecipitation; RT‐qPCR, real‐time quantitative polymerase chain reaction; AhR, aryl hydrocarbon receptor; siScr, siScramble; H&E, hematoxylin and eosin; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; PD‐1, programmed cell death protein 1; TIL, Tumor‐infiltrating lymphocyte; IFN‐γ, interferon‐gamma; ROI, region of interest; SEM, tandard error of the mean; ns, not significant.

Article Snippet: Anti‐CD3 rabbit antibody (1:200 dilution, ab16669, Abcam, Cambridge, MA, USA) and anti‐CD8 rabbit antibody (1:200 dilution, ab209775, Abcam) for SCCVII tumors, anti‐CD8 (D8A8Y) rabbit antibody (1:200 dilution, 85336, Cell Signaling Technology) for HNSCC tissues, and anti‐PD‐1 rabbit antibody (1:200 dilution, 66220‐1‐Ig, Proteintech) were used as primary antibodies.

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Transfection, Luciferase, Concentration Assay, Activity Assay, Western Blot, Plasmid Preparation, Injection, TUNEL Assay, Staining, Flow Cytometry, Multiplex Assay, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, End Labeling

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Targeting AhR reversed Kyn‐Siglec‐15‐mediated immune escape in vivo. (A) Mice with SCCVII were treated with Kyn (100 mg/kg) or/and CH‐223191 (10 µmol/L) once every two days for three times ( n = 5 per group). Tumor volumes were measured once every two days. (B) Tumor weights were measured after mice euthanized on day 20 ( n = 5 per group). (C) Western blotting analysis of Siglec‐15 expression after Kyn stimulation in mice. (D‐F) H&E (D), TUNEL (E and F upper), and Ki‐67 (E and F lower) staining analyses of tumor tissues in each group. (G) Concentration of serum TNF‐α and IFN‐γ in mice were measured by ELISA. (H‐M) The percentage of CD3 + CD8 + (H), Ki‐67 + (I), PD‐1 + (J), IFN‐γ + (K), granzyme B + (L), and perforin + cells (M) in cytotoxic T lymphocytes (CTLs) isolated from the indicated tumors were analyzed. (N) Representative images of multiplex immunofluorescence staining of CD3 + (green) and CD8 + (red) are shown, and quantification analysis were performed in tumor tissues ( n = 10 fields of 5 mice per group). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: AhR, aryl hydrocarbon receptor; Kyn, kynurenine; H&E, hematoxylin and eosin; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; TNF‐α, tumor necrosis factor‐alpha; IFN‐γ, interferon‐gamma; ELISA, enzyme‐linked immunosorbent assay; PD‐1, programmed cell death protein 1; CTL, cytotoxic T lymphocyte; DAPI, 4',6‐diamidino‐2‐phenylindole; SEM, tandard error of the mean; ns, not significant.

Article Snippet: Anti‐CD3 rabbit antibody (1:200 dilution, ab16669, Abcam, Cambridge, MA, USA) and anti‐CD8 rabbit antibody (1:200 dilution, ab209775, Abcam) for SCCVII tumors, anti‐CD8 (D8A8Y) rabbit antibody (1:200 dilution, 85336, Cell Signaling Technology) for HNSCC tissues, and anti‐PD‐1 rabbit antibody (1:200 dilution, 66220‐1‐Ig, Proteintech) were used as primary antibodies.

Techniques: In Vivo, Western Blot, Expressing, TUNEL Assay, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Multiplex Assay, Immunofluorescence, End Labeling

Journal: Cancer Communications

Article Title: Metabolic landscape of head and neck squamous cell carcinoma informs a novel kynurenine/Siglec‐15 axis in immune escape

doi: 10.1002/cac2.12545

Figure Lengend Snippet: Siglec‐15 specific siRNA delivered by NH 2 ‐MSN nanoparticles enhances immunotherapy efficacy in vivo. (A) Synthesis routes of NH 2 ‐MSN nanoparticles loaded with siRNA. (B) Macroscopy characterization of NH 2 ‐MSNs with siS15. (C) Transmission electron microscopy images of NH 2 ‐MSNs and NP‐siS15 with indicated ratios. (D‐E) The morphology and Zeta potential of NH 2 ‐MSNs and NP‐siS15. (F) Scanning microscopy analysis of the cellular uptake of NH 2 ‐MSNs with or without the indicated siS15‐Cy5 by SCCVII cells after in vitro treatment for 24 h. (G) Western blotting analysis confirming Siglec‐15 gene‐silencing effect by siRNA released from nanoparticles in SCCVII cells. (H) Schematic diagram of the treatment strategy in SCCVII mouse model. (I) Probability of survival analysis for each group was performed. (J) Analysis of TUNEL and Ki‐67 staining of tumor tissues in each group. (K‐L) The percentage of CD3 + CD8 + (K) and PD‐1 + cells in CTLs (L) of the indicated treatment groups. (M) Analysis of multiplex immunofluorescence staining of CD3 + (green) and CD8 + (red) were shown and quantification analysis were performed in tumor tissues ( n = 10 fields of five mice per group). (N) Schematic diagram of the anti‐PD‐L1 and NP‐siS15 combination treatment strategy in C3H/He subcutaneous tumorigenesis models. (O‐P) Tumor volume and weights were measured and analyzed in the indicated groups. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Abbreviations: siS15, Siglec‐15 small interfering RNA; DAPI, 4',6‐diamidino‐2‐phenylindole; AhR, aryl hydrocarbon receptor; NP‐siS15, NH 2 ‐MSN‐si SIGLEC15 ; DAPI, 4',6‐diamidino‐2‐phenylindole; siScr, siScramble; anti‐PD‐1, anti‐programmed cell death protein 1 antibody; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling; CTL, cytotoxic T lymphocyte; anti‐PD‐L1, anti‐programmed death‐ligand 1 antibody; SEM, tandard error of the mean; ns, not significant.

Article Snippet: Anti‐CD3 rabbit antibody (1:200 dilution, ab16669, Abcam, Cambridge, MA, USA) and anti‐CD8 rabbit antibody (1:200 dilution, ab209775, Abcam) for SCCVII tumors, anti‐CD8 (D8A8Y) rabbit antibody (1:200 dilution, 85336, Cell Signaling Technology) for HNSCC tissues, and anti‐PD‐1 rabbit antibody (1:200 dilution, 66220‐1‐Ig, Proteintech) were used as primary antibodies.

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Microscopy, In Vitro, Western Blot, TUNEL Assay, Staining, Multiplex Assay, Immunofluorescence, Small Interfering RNA, End Labeling

Journal: Translational Lung Cancer Research

Article Title: Systemic immune index predicts tumor-infiltrating lymphocyte intensity and immunotherapy response in small cell lung cancer

doi: 10.21037/tlcr-23-696

Figure Lengend Snippet: The correlation between PNI score and local immune contexture in limited-stage SCLC cohort. (A) Representative images of mIF staining in low- and high-PNI tumor tissues. The dashed line represents the tumor boundary. Scale bars for all images, 100 µm. (B-D) Quantification of counts of CD4 + T-cells, CD8 + T-cells, CD68 + cells in low- and high-PNI tumors. (E-G) Proportions of PD-L1 + tumor cells, PD-L1 + CD8 + cells, and CTLA-4 + cells in low- and high-PNI tumors. PNI, prognostic nutritional index; H&E, hematoxylin and eosin; PD-L1, programmed cell death 1 ligand 1; SCLC, small-cell lung cancer; mIF, multiplex immunofluorescence.

Article Snippet: TMA sections were stained using antibodies against CD4 (clone EP204, dilution 1:100, Cell Signaling Technologies, Boston, USA), CD8 (clone D8A8Y, dilution 1:200, Cell Signaling Technologies), CD68 (clone D4B9C, dilution 1:400, Cell Signaling Technologies), programmed cell death 1 (PD-1) (clone NAT105, dilution 1:100, Abcam), PD-L1 (clone E1L3N, dilution 1:100, Cell Signaling Technologies), CTLA-4 (clone CAL49, dilution 1:200, Abcam), and cytokeratins (clone C-11, dilution 1:250, Abcam).

Techniques: Staining, Multiplex Assay, Immunofluorescence