dy cd8a rabbit igg d8a8y fluidigm 3162035d  (fluidigm)

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