cd4 depletion  (Bio X Cell)

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A) Flow cytometry analysis of melanoma antigen panel for MHC-I, MHC-II and PD-L1 in control, responder, and treatment escape groups. Tumor cells were normalized to SOX10 + cells, n=3/group. (B) Immunohistochemistry (IHC) staining and quantification of MHC-I and PD-L1 expression in control, responder, and treatment escape groups, n=3/group. H-score was used to access MHC-I expression and the percentage of positive cells for PD-L1. (C) Flow cytometry analysis of T cell panel (number of infiltrated CD4 + and CD8 + T cells, activated CD4 + /CD69 + and CD8 + /CD69 + T cells, and memory CD44 + /CD62L − /CD4 + and CD44 + /CD62L − /CD8 + T cells) of control, responder, and treatment escape groups. Cells were normalized to the total number of live cells. (D) Flow cytometry analysis of Foxp3 + CD4 + T cells (Tregs) in control, responder, and treatment escape groups. Tregs were normalized to the total number of CD4 + cells. (E) Immunohistochemistry staining and quantification of CD8, CD69 and PD-1 expression in control, responder, and treatment escape groups. n=3/group. H-score was used to access CD8 and CD69 expressions and the percentage of positive cells for PD1, according to the best fit suggested by the software. (F) Flow cytometry analysis of exhaustion markers (PD-1, TIM-3, LAG-3 and CTLA-4) in control, responder, and treatment escape groups. T cells were normalized to the total number of live cells. Scale bar = 200 μm (G) Multiplex immunofluorescence (IF) staining of control, responder, and treatment escape tumors for DAPI, CD4, CD8, CD11b, CD11c, Ly6G, Ly6C, CD34 and SOX10 expression (H) Scoring of the multiplex IF images of the whole slides. (I) Degrees of clustering of CD4 + or CD8 + T cells versus M-MDSCs (CD11b + Ly6C + Ly6G − ) and PMN-MDSCs (CD11b + Ly6C − Ly6G + ) of control, responder, and treatment escape groups, n=3/group. Cells were normalized by the number of total cells. The graphs are represented as average ± SEM. One-way ANOVA was performed followed by Tukey’s multiple comparisons test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns: non-significant; C: control (not treated cells); R: responder; TE: treatment escape.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Flow Cytometry, Control, Immunohistochemistry, Expressing, Staining, Software, Multiplex Assay, Immunofluorescence

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A-B) Tumor growth and survival curves following pre-treatment with IgG control (n=5) or anti-CD4 + anti-CD8a +/− RMC-7977 (all n=15). Vehicle or RMC-7977 treatment was initiated when tumors reached around 100mm 3 . For T cell depletion, mice were treated 3 days before the injection of the tumor with the Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P.) and then every 4 days thereafter. The T cell depleted mice were treated with Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P., every 4 days), Anti-CD4 + Anti-CD8a + RMC-7977 was treated with Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P., every 4 days) and RMC-7977 (25 mg/kg, P.O., q.d.); and RMC-7977 group was treated with only RMC-7977 (25 mg/kg, P.O., q.d.) for 7 weeks. Plots correspond to each individual mouse. Dotted lines indicate when treatment started and stopped.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Control, Injection

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A-B) The effects of IgG control, anti-PD-1, RMC-7977 or anti-PD-1+RMC-7977 on the growth of OSUMMER.13 tumors. Treatment was initiated when tumors reached around 100mm 3 . The IgG group was treated with IgG control (2µg/µL, I.P., every 5 days: n=5), the vehicle group was treated with vehicle (P.O., q.d.: n=5), the vehicle + IgG group was treated with IgG control (2µg/µL, I.P., every 5 days: n=5) and vehicle (P.O., q.d.: n=5), total of n=15M for controls, the anti-PD-1 group was treated with Anti-PD-1 (2µg/µL, I.P., every 5 days: n=7), the RMC-7977 group was treated with RMC-7977 (25 mg/kg, P.O., q.d.: n=7), and the RMC-7977 + anti-PD-1 group (n=7) was treated with a combination of RMC-7977 and Anti-PD1 as indicated for 7 weeks. Plots correspond to each individual mouse. (C) Immunohistochemistry and quantification of pERK, Ki67, Melan-A, CD8, CD69, PD-1 and MHC-I of control (vehicle), anti-PD-1, RMC-7977 and RMC-7977 + anti-PD-1 group, n=3/group. H-score was used to access pERK, Ki67 and MHC-I expressions and the percentage of positive cells for melan-A and PD1, according to the best fit suggested by the software. Scale bar = 200 μm (D) Flow cytometry analysis of the T cell panel (number of infiltrated CD4 + and CD8 + T cells, activated CD4 + /CD69 + and CD8 + /CD69 + T cells, and memory CD44 + /CD62L − /CD4 + and CD44 + /CD62L − /CD8 + T cells) of vehicle, anti-PD1, RMC-7977 and the RMC-7977 + anti-PD-1 groups. The graphs are represented as average ± SEM. One-way ANOVA was performed followed by Tukey’s multiple comparisons test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns: non-significant. Dotted lines indicate when treatment started, stopped and when rechallenge was carried out.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Control, Immunohistochemistry, Software, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 2 The anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations suppress melanoma brain metastases in the SM1 mouse melanoma model. (A) The ICI doublets were more effective than single agent ICI in the brain tumors of SM1 mouse melanoma model with 2/10 mice relapsing in each combination group. Mice received 200 µg/100 µL i.p. doses of single agents and doublets every 5 days for 80 days. (B) MRI T2 sequence of brain of mice with SM1 MBM tumors demonstrating a lack of tumor regrowth after holding the therapy for 28 days. MRIs were generated using standard T2-weighted Turbo Spring Echo. Brain tumors were manually contoured on all MRI slices using ImageJ. A custom program was used to extract voxels within the manually drawn contours and compute total volume burden, in mm3. (C) Monitoring melanoma brain metastases by MRI. Time course of MRI T2 sequences of brain of mouse melanoma brain metastases following treatment with IgG, anti-PD- 1+LAG-3 or anti-PD-1+CTLA-4. MRIs were taken twice a week and were generated using standard T2-weighted Turbo Spring Echo. Brain tumors were manually contoured on all MRI slices using ImageJ. A custom MATLAB program was used to extract voxels within the manually drawn contours and compute total volume burden, in mm3. (D–F) Kinetics of CD4+ and CD8+ T cell, Myeloid-derived suppressor cells (MDSCs) and macrophage infiltrate following treatment with each ICI doublet over a period of 28 days in SM1 brain tumors. Tumors were collected at days 7 and 14 for IgG and at days 7, 14, and 28 after treatment initiation for the doublets. The results were represented as average ±SEM with 10 mice per group for all the growth curve experiments. The results were represented as average±SEM of 3 mice per group for panels D–F. Statistical significance was assessed with one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001). ANOVA, analysis of variance; ICI, immune checkpoint inhibitor.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Sequencing, Generated, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 3 Immune landscape of flank and brain tumors in SM1 mouse model following the treatment with single agent and combination ICI therapy. A) t-SNE plots showing major cell types identified in relapsed brain tumors and responding flank SM1 tumors following treatment with single agent and combination ICI. (B) Proportion of each cell type in the responding flank tumors from the indicted treatment groups. (C) Proportion of different T-cell clusters identified in the responding flank tumors from the indicated treatment groups. (D) Heatmap showing expression of activation/exhaustion markers across the identified T-cell clusters. (E) Violin plots showing expression of T cell activation markers and immune checkpoints in each T-cell cluster. (F) IHC analysis of tumors treated with anti-PD-1+LAG-3 or anti-PD-1+CTLA-4 demonstrates increased CD4+and CD8+ T cell infiltration in responding flank tumors. The samples were stained with anti-CD4 and anti-CD8 by IHC. Tumors were harvested at day 9 for IgG and single agents and at day 19 for anti-PD-1+LAG-3 and day 25 for anti-PD-1+CTLA-4 following the initiation of treatment. ICI, immune checkpoint inhibitor.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Expressing, Activation Assay, Staining

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 4 Anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 differentially polarize CD4+T cells in the SM1 mouse melanoma model. (A) Proportion of T cell clusters and three major subsets of CD4+T cells identified in the responding flank tumors from the indicated treatment groups. (B) Expression of immune checkpoints and markers for T helper subsets and T regulatory cells in the three major subsets of CD4+T cells identified by violin plots. (C) Percentage of CD4+T cells and activated CD69+CD4+ T cells in the responding flank tumors treated with combination of anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 identified by flow cytometry. Tumors were harvested at the endpoint following the treatment initiation. (D) Flow cytometry histogram plot showing activated CD4+T cells in tumors treated with IgG, anti-PD-1+LAG-3 or anti-PD-1+CTLA-4 as evidenced by cell surface CD69 and CD4 staining. (E) Treatment with anti-PD-1+CTLA-4 increased Tregs infiltration in the responding flank tumors and relapsing brain tumors. The samples were stained with anti-FOXP3 by IHC. Tumors were harvested at day 9 for IgG and single agents and at day 19 for anti-PD-1+LAG-3 and day 25 for anti-PD-1+CTLA-4 following the initiation of treatment for sc-RNAseq and IHC. The results were represented as average±SEM of 3 mice per group for panels C and D. Statistical significance was assessed with one-way ANOVA test (***p<0.001). ANOVA, analysis of variance ; IHC, immunohistochemistry.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Expressing, Flow Cytometry, Staining, Immunohistochemistry

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 5 Antitumor responses to anti-PD-1+LAG-3 are dependent on CD4+T helper function. (A, B) Depletion of CD4+T cells demonstrates that responses to anti-PD-1+LAG-3 but not anti-PD-1+CTLA-4 are dependent on CD4+T cells in both SM1 flank and brain tumor models. The results were represented as an average±SEM of 5 mice per group. (C) Decreased percentage of tumor-infiltrating CD69+CD8+ T cells and CD8+T cells in SM1 flank tumors, respectively, following CD4+T cell depletion and anti-PD-1+LAG-3 treatment. Increased percentages of CD69+CD8+ T cells and total CD8+T cells were seen in SM1 flank tumors following CD4+T cell depletion and anti-PD-1+CTLA-4 treatment. Tumors were collected at day 10 after the treatment initiation. (D) Antitumor responses of the PD-1+LAG-3 but not the PD-1+CTLA-4 combination are dependent on CD4+T cells in B16 flank tumors. Results show the average±SEM of 5 mice per group. (E) Decreased percentage of tumor-infiltrating CD69+CD8+ T cells and CD8+T cells in B flank tumors, respectively, following CD4+T cell depletion and anti-PD-1+LAG-3 treatment. Increased percentages of CD69+CD8+ T cells and total CD8+T cells were seen in B16 flank tumors following CD4+T cell depletion and anti-PD-1+CTLA-4 treatment. Tumors were harvested for flow cytometry analysis at day 5 following the start of the treatment. (F) Responses to Anti-PD-1+LAG-3 but not Anti-PD-1+CTLA-4 are dependent on CD4+T cells in the flank tumors of D4M- UV2 mouse melanoma model. The results were represented as an average±SEM of 5 mice per group. (G) G) Percentage of CD69+CD8+ and CD8+ T cells decrease in the D4M-UV2 flank tumors significantly, following CD4+T cell depletion and Anti-PD- 1+LAG-3 treatment. Tumors were collected for flow cytometry analysis at day 16 of treatment for IgG and day 19 of treatment for the doublets. (H, I) ELISPOT assays showing decreased levels of IFNγ production following CD4+T cell depletion and treatment with the PD-1+LAG-3 combination in both the SM1 and B16 mouse melanoma models. The results were represented as average±SEM of 3 mice per group for panels D-G. Statistical significance was assessed with one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001). ANOVA, analysis of variance.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Flow Cytometry, Enzyme-linked Immunospot