anti cd8  (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: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , STING agonist ADU-S100 (2 μg intratumoral) and/or anti-LTβR agonistic antibody (100 μg, intraperitoneal (i.p.)) were administered to mice bearing subcutaneous KPC tumors as monotherapy or in combination as indicated. Immunohistochemistry of B cells (CD19, brown) and HEVs (MECA-79, magenta) shows numerous TLS induced by combination therapy (day 14). Scale bars, 1 mm (low magnification) and 100 μm (high magnification). b , CD19 and MECA-79 staining of TLS-rich human breast adenocarcinoma for comparison. Scale bars, 1 mm (low magnification) and 100 μm (high magnification). c , KPC tumor sections of different treatment groups were stained for CD19 and MECA-79, scanned for the whole tumor area, and the number of TLS and HEV endothelial cells (HEV-ECs) were quantified per tumor area (mm 2 ) by Halo image analyses. The results of two independent experiments were combined to generate the graphs. A total of N = 17–19 tumors were analyzed. * P < 0.05, *** P < 0.001, **** P < 0.0001. d , Left: immunofluorescence confocal image ( z -stack) of TLS induced by combination therapy. Scale bar, 100 μm. Right: the TLS area indicated by the arrow in the left image is shown in higher magnification (three-dimensional confocal image). Scale bar, 10 μm. Nuclear staining of germinal center/follicular cell transcription factor Bcl6 (green) is surrounded by B cell surface marker CD19 (white) or helper T cell marker CD4 (red), indicating germinal center B cells or T FH cells, respectively. Arrows indicate T FH cells found among the dense B cell cluster, demonstrating the intimate interaction between the T FH cell surface (red) and the B cell surface (white). e , Proliferating B cells were detected in TLS by Ki-67 and CD19 double staining. Scale bar, 100 μm. f , Triple immunofluorescence staining of LTβR monotherapy-induced TLS for CD4, CD19 and CD23 (a follicular cell marker) shown in merged and individual color channels. Blue, DAPI. Scale bar, 100 μm. g , Combination therapy on KPC tumors grown in wild-type or T cell-deficient nude mice (C57BL/6 background) demonstrating that neither TLS nor HEVs formed in tumors in the absence of T cells. A representative image of 16 tumors is shown for each genotype. Scale bar, 100 μm. h , CD4 + T cells and/or CD8 + T cells are depleted in wild-type mice by i.p. injection of depleting antibodies one or two days before KPC tumor inoculation as described in Extended Data Fig. . Isotype IgG was injected for the no-depletion control group. Alternatively, KPC tumors were grown in B cell-deficient CD79a knockout mice and treated as shown in a . The development of TLS and HEVs was studied in these mice after combination therapy. A representative image of six tumors is shown for each group. Scale bar, 100 μm. i , The number of TLS and HEV-ECs were quantified per tumor area (mm 2 ). N = 6. P < 0.0001. Representatives of at least three independent experiments are shown in d – f . Data are presented as the mean ± s.e.m. and analyzed using one-way analysis of variance (ANOVA) with Tukey’s test for statistical significance. All replicates represent biological replicates. NS, not significant.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Immunohistochemistry, Staining, Comparison, Immunofluorescence, Marker, Double Staining, Injection, Control, Knock-Out

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , Treatment schedule for subcutaneous KPC tumors. CD4 + and/or CD8 + T cells were deleted individually or together by i.p. injection of anti-CD4 and/or CD8 IgG (200 μg) every 4 days, starting from one or two days before tumor inoculation as indicated. Isotype IgG2b was injected for the no-depletion control group. b , T cell depletion was confirmed by FACS analysis of peripheral blood lymphocytes. Rat anti-mouse CD4 (clone: GK1.5) and rat anti-mouse CD8α (clone: 2.43) were used to deplete CD4 + and CD8 + T cells, respectively. Rat IgG2b was used as an isotype control (clone: LTF-2). c , Flow cytometry analyses of spleen and peripheral blood leukocytes show the absence of CD19 + B cells in CD79a knockout mice.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Injection, Control, Flow Cytometry, Knock-Out

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , TLS formation in orthotopic KPC tumors in the pancreas. B cells (CD19), T cells (CD3), cytotoxic T cells (CD8) and NK cells (NK1.1) were stained along with HEVs (MECA-79) in serial sections of orthotopic pancreatic tumors 14 days after the start of combination therapy. The areas indicated by the squares in the first images to the far left are shown in high magnification in the images to the right. Representative images of 3–4 tumors are shown. Scale bars, 1 mm (left) and 100 μm (high-magnification images). b , TLS (brown) and HEVs (magenta) formed in orthotopic Py230 mouse mammary tumors upon agonist treatment. Representative images of 6–7 tumors are shown. Graphs show the TLS and HEV-EC densities in tumors of different treatment groups. Stained sections were scanned for the whole tumor area, and the number of TLS and HEVs were quantified per tumor area (mm 2 ) by Halo image analyses. N = 5–7 tumors from 2 independent experiments representing biological replicates. Data are presented as the mean ± s.e.m. and analyzed using one-way ANOVA with Tukey’s test for statistical significance. * P < 0.05. Scale bar, 100 μm. c , Left: TLS formation (brown, CD19) in orthotopic 76-9 rhabdomyosarcoma in the calf muscle. Arrowheads, HEVs (magenta, MECA-79). Right: CD3 staining of a consecutive section. Combination therapy was given four times as indicated. Representative images of ten treated tumors are shown. Scale bar, 100 μm.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Staining

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , Mice bearing different tumor types were treated with STING agonist ADU-S100 (2 μg intratumoral) and/or anti-LTβR agonistic antibody (100 μg, i.p.) as indicated (arrowheads), and the tumor growth curves were monitored until surgical resection. s.c., subcutaneous. N = 22–28 (KPC), N = 11–18 (Py230), N = 5 (76-9) tumors from 2 independent experiments combined, representing biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. b , Subcutaneous KPC tumor growth in wild-type (WT) or T cell-deficient nude mice treated or untreated with combination therapy. N = 10 or 12. c , CD4 + T cells and/or CD8 + T cells are depleted in combination therapy-treated and untreated mice by i.p. injection of depletion antibodies one or two days before KPC tumor inoculation (s.c.) as described in Extended Data Fig. . Isotype IgG was injected to the no-depletion control group. Tumor growth was monitored for 14 days. N = 10. d , Subcutaneous KPC tumor growth in WT or B cell-deficient CD79a knockout mice treated or untreated with combination therapy. N = 20. e , Treatment schedule of neoadjuvant agonist therapies for s.c. KPC tumors and tumor resection followed by tumor reinoculation. The primary tumors were either TLS-rich or TLS-free at the time of the resection, depending on the neoadjuvant treatment options. f , Survival curves after tumor reinoculation. Sham control mice had no primary tumors but received tumor inoculation for the first time 2 weeks after sham surgery. N = 18–23. g , Growth curves of reinoculated tumors. For a – g , the results of two or more independent experiments were combined and presented in the graphs. Data in a – d are presented as the mean ± s.e.m. and analyzed using two-way ANOVA with Tukey’s test for statistical significance. All replicates represent biological replicates.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Injection, Control, Knock-Out

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , Upper panels: The abundance of CD8 + T cells in primary KPC tumors on the day of tumor resection (Day 14) determined by flow cytometry. Analyses of total CD8 + T cells as well as CD69 and CD44 expression were conducted to determine early and late activation states of CD8 + T cells, respectively. The abundance of total and activated CD8 + T cells is presented as a % fraction of total CD3 + T cells. For each data point, three tumors were pooled and dissociated to isolate lymphocytes for FACS analysis. Total 6-9 tumors were examined. Lower panels: The number of total CD8 + T cells or activated CD8 + T cells per tumor was calculated from the total live cell count after tumor dissociation and the % fractions of these T cells as determined by flow cytometry (Fig. ). For each data point, three tumors were pooled, and the obtained cell number was divided by three to yield average T cell count/tumor. Total 6-9 tumors were examined. b , A similar analysis of tumor-draining CD4 + T cells. A representative result of two independent experiments is shown for each analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Flow Cytometry, Expressing, Activation Assay, Cell Counting

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , Upper panels: Live lymph node leukocytes were gated for CD3 + T cells, and the % fractions of total and activated CD8 + T cells in total CD3 + T cells were determined by flow cytometry. Lower panels: The numbers of total and activated CD8 + T cells in a draining lymph node were calculated from the total leukocyte count in each lymph node and the % fractions of the T cell subsets. For each data point, six draining lymph nodes were pooled and averaged. A total of 18 lymph nodes were examined for each group. A representative of two independent experiments is shown for each analysis. *p < 0.05; ***p < 0.01; **p < 0.001; ns, not significant. b , A similar analysis of CD4 + T cells in the draining lymph node. Upper panels: The % fractions of total and activated CD4 + T cells in total CD3 + T cells. Lower panels: The average numbers of total and activated CD4 + T cells in a draining lymph node.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Flow Cytometry

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , Left, Subclusters of intratumoral CD3 + T cells are compared between the treatment groups. T cell subsets are identified in these clusters. Right, Relative abundance of T cell subsets is shown as the fractions of total CD3 + T cells. b , CD8 + T cell population was further subclustered to identify subsets of CD8 + T cells infiltrating the tumors. c , Subsets of CD8 + T cells were identified by the expression of indicated marker genes. T mem , memory CD8 + T cell; T ex_prog , progenitor exhausted CD8 + T cell; T ex_term , terminally exhausted CD8 + T cell. d , Relative abundance of different CD8 + T cell subsets in total CD8 + T cells. Memory CD8 + T cell population (green) expanded in treatment groups, especially in LTβR monotherapy and combination therapy groups, compared with the untreated group.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Expressing, Marker

Journal: Nature Immunology

Article Title: Simultaneous STING and lymphotoxin-β receptor activation induces B cell responses in tertiary lymphoid structures to potentiate antitumor immunity

doi: 10.1038/s41590-025-02259-8

Figure Lengend Snippet: a , Treatment schedule of neoadjuvant agonist therapies for s.c. KPC tumors and blood plasma collection or serum transfer to naive recipients. b , Blood plasma was collected 2 weeks after tumor resection, heparinized and diluted at a 1:50 ratio with PBS, and added to cultured KPC cells. The binding of plasma IgG to KPC cells was visualized by fluorescently labeled sheep anti-mouse IgG (red). Scale bar, 1 mm. c , Similarly, blood plasma collected from different mouse groups was incubated with KPC cells in single-cell suspension, and the binding of plasma IgG to these cells was detected by the right shift of the cell population in FACS analysis (indicated by red gating). IgM binding was undetectable compared with the sham and intact mouse (non-immune plasma) controls in this assay. d , Percentage fraction of IgG-bound KPC cells in total KPC cells. Plasma samples from 10–14 different mice from each group were examined for KPC cell binding. A representative result of three experiments. ** P < 0.01. e , Bone marrow was collected from the left and right femurs and tibias of each agonist-treated or untreated mouse 2 weeks after tumor resection, and bone marrow leukocytes were analyzed by flow cytometry using Blimp1 and CD44 staining as markers for long-lived plasma cells. One experiment. N = 5 per group. * P < 0.05, ** P < 0.01, **** P < 0.0001. f , Blood serum was collected from different donor mouse groups (sham surgery, agonist untreated, combination therapy treated) and transferred to naive recipient mice several times after tumor inoculation. The tumor growth in these animals was monitored for 20 days. One experiment. N = 20 tumors were analyzed for each serum transferred group. N = 10 for controls receiving no serum transfer. *** P < 0.001. g , Neoadjuvant treatment and tumor resection followed by tumor reinoculation were carried out in wild-type or B cell-deficient CD79a knockout mice as described in Fig. . Tumor size was compared between different mouse groups on day 9 after tumor reinoculation. The results of two independent experiments were combined. N = 20 tumors were analyzed for each group. **** P < 0.0001. h , Neoadjuvant treatment and tumor resection followed by tumor reinoculation were carried out in wild-type mice as described in Fig. . Starting from one day before the reinoculation of KPC tumors, anti-NK1.1, anti-CD8 or anti-Ly6G was administered (i.p.) to deplete NK cells, CD8 + T cells or neutrophils, respectively. Tumor size was compared between different mouse groups on day 6 after tumor reinoculation. One experiment. N = 20 tumors were analyzed for each group. * P < 0.05, **** P < 0.0001. Data are presented as the mean ± s.e.m. and analyzed using one-way ( d , e , g and h ) or two-way ( f ) ANOVA with Tukey’s test for statistical significance. All replicates represent biological replicates.

Article Snippet: For T cell depletion experiments, mice were injected intraperitoneally with 200 μg of anti-mouse CD4 (clone GK1.5, BioXCell BE0003-1) and/or CD8 (clone 2.43, BioXCell BE0061) monoclonal antibodies or of isotype-matched control antibody (clone LTF-2, BioXCell, BE0090) on the indicated days.

Techniques: Clinical Proteomics, Cell Culture, Binding Assay, Labeling, Incubation, Suspension, Flow Cytometry, Staining, Knock-Out

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

Journal: Cell chemical biology

Article Title: Small-molecule MHC-II inducers promote immune detection and anti-cancer immunity via editing cancer metabolism.

doi: 10.1016/j.chembiol.2023.05.003

Figure Lengend Snippet: Figure 3. Pristane increases the tumor infil- trating T cells for anti-cancer immunity (A and B) Diagrams show the impact of the pristane treatment versus control on the enrichment and upregulation of pathways of T cell proliferation (A) and activation (B) in tumors via analyzing the RNA-seq data using GSEA. (C) Frequency of CD3+ T cells in tumors derived from PyMT mice with indicated treatment by flow cytometry. (D) Number of Tregs normalized to tumor mass in tumors derived from PyMT mice with indicated treatment by flow cytometry. (E and F) Examination on the effect of the 0.1 mL pristane treatment against established 4T1 tumors in Balb/c tumors upon blockade of CD4+ T cells by CD4 depleting antibody or blockade of CD8+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies FITC Hamster Anti-Mouse CD3e BD Pharmingen Cat# 553062; RRID: AB_394595 PerCP-CyTM5.5 Rat Anti-Mouse CD45 BD Pharmingen Cat# 550994; RRID: AB_394003 PE anti-mouse CD4 Antibody Biolegend Cat# 100407; RRID: AB_312692 FOXP3 Monoclonal Antibody, PE-Cyanine7 eBioscience Cat# 25-4776-41; RRID:AB_10805514 FITC Anti-Mouse MHC Class II (I-A/I-E) proteintech Cat# FITC-65122; RRID: AB_2883788 PE anti-mouse H-2Db Antibody Biolegend Cat# 111507; RRID: AB_313512 PE anti-mouse F4/80 Antibody Biolegend Cat# 123109; RRID: AB_893498 PE anti-mouse CD11c Antibody Biolegend Cat# 117307; RRID: AB_313776 FITC Anti-Mouse CD11b proteintech Cat# FITC-65055; RRID: AB_2883745 7-AAD Viability Staining Solution Biolegend Cat# 420403 PE anti-human HLA-A,B,C Antibody Biolegend Cat# 311405; RRID: AB_314874 FITC anti-human HLA-DR, DP, DQ Antibody Biolegend Cat# 361706; RRID: AB_2563192 CD4 Rabbit mAb CST Cat# 25229; RRID: AB_2798898 CD8a XP Rabbit mAb CST Cat# 98941; RRID: AB_2756376 Purified anti-mouse CD3ε Antibody Biolegend Cat# 100301; RRID: AB_312666 Purified anti-mouse CD28 Antibody Biolegend Cat# 102101; RRID: AB_312866 InVivoMAb anti-mouse PD-1 BioXcell Cat# BE0146 Isotype control antibody BioXcell Cat# BE0089 InVivoMAb anti-mouse CD4 BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 BioXcell Cat# BE0061 JAK1 Monoclonal antibody proteintech Cat# 66466-1-Ig; RRID: AB_2881834 Phospho-JAK1 (Tyr1022/1023) Antibody Affinity Cat# AF2012; RRID: AB_2834437 STAT1 Polyclonal antibody proteintech Cat# 10422-2-AP Phospho-STAT1 (Tyr701) Antibody Affinity Cat# AF3300; RRID: AB_2834719 Bacterial and virus strains BL21-CodonPlus (DE3) This paper N/A DH5a competent cells This paper N/A Chemicals, peptides, and recombinant proteins Ni-NTA Agarose QIAGEN Cat# 30210 FASN-MAT-WT Protein This paper N/A FASN-MAT-N644 mutant Protein This paper N/A FASN-MAT-T650 mutant Protein This paper N/A RPMI 1640 WISENT Cat# 350-000-CL DMEM WISENT Cat# 319-005-CL Fetal Bovine Serum WISENT Cat# 086-150 Penicillin/Streptomycin WISENT Cat# 450-201-EL Opti-MEM medium Gibco Cat# 31985070 Lipofectamine 3000 Thermo Fisher Cat# L3000015 SuperRed/GelRed Biosharp Cat# BS354B PEI YEASEN Cat# 40816ES03 DMSO Amresco Cat# 67-68-5 EcoRI-HF NEB Cat# R3101S AgeI NEB Cat# R3552S NotI-HF NEB Cat# R3189S (Continued on next page) e1 Cell Chemical Biology 30, 1076–1089.e1–e11, September 21, 2023

Techniques: Control, Activation Assay, RNA Sequencing, Derivative Assay, Cytometry