depletion antibodies anti mouse cd8α  (Bio X Cell)

Journal: Cancer cell

Article Title: Cisplatin and temozolomide combinatorial treatment triggers hypermutability and immune surveillance in experimental cancer models.

doi: 10.1016/j.ccell.2025.05.014

Figure Lengend Snippet: Figure 4. Rejection of CDDP-TMZ-primed tumors is mediated by CD8 + T cells and elicits immunological memory (A) Graphical summary for CD8 + and/or CD4 + T cell depletion experiment. A total of 5 × 10 5 CDDP-TMZ-primed CT26 cells were injected subcutaneously in the right flank of each immunocompetent (BALB/c) mouse. Mice were randomized into 4 treatment groups: control (CTRL) receiving only the vehicle, CD8 + T cell depletion (aCD8) receiving murine anti-CD8a antibody, CD4 + T cell depletion (aCD4) receiving murine anti-CD4 antibody, and CD8 + and CD4 + T cell depletion receiving both murine aCD8 and aCD4 antibodies. (B and C) Tumor growth of CDDP-TMZ-primed CT26 tumors upon T cell depletion. (B) Average of mice tumor volumes (mm 3 ) ± SEM; statistical significance evaluated by Mann-Whitney test: ****p < 0.001. (C) Single tumor volumes from (B). (D) Effect of unprimed CT26 rechallenge in immunocompetent mice that previously rejected CDDP-TMZ-primed tumors. Subsequent inoculation of CT26 un- primed or primed with single agent TMZ or CDDP (red arrow) in previously challenged (vaccinated) BALB/c mice (corresponding to tumor-free mice from Figure 3B), compared with naive BALB/c mice; n = 10 in vaccinated BALB/c group, n = 5 in naive BALB/c group. See also Figure S5.

Article Snippet: 6 Mouse CD4 depleting antibody (anti-CD4, BioXcell) and mouse CD8a depleting antibody (antiCD8a, BioXcell) were injected intraperitoneally on the same day of tumour inoculation at the dose of 200 μg/mouse, followed by a dose every other 2 days at 100 μg/mouse until the end of the experiment.

Techniques: Injection, Control, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: TNFR2/CCR8 bispecific antibody enhances antitumor activity through depleting Ti-Tregs and boosting effector CD8 + T cell function

doi: 10.1080/2162402X.2025.2497171

Figure Lengend Snippet: Assessment of expression and function of TNFR2/CCR8 dual targeting. (a) Representative flow cytometry plots showing TNFR2 and CCR8 expression in gated Treg populations from the spleen and tumor in the MC38 model. (b) Statistical analysis of TNFR2 and CCR8 expression on Tconv, CD8 and Tregs in spleen, draining lymph node and tumor tissue in MC38 and KP13 tumor models using flow cytometry ( n = 5 or 6 mice per group). (c) Statistical results of TNFR2 + CCR8 + , TNFR2 + CCR8 − , TNFR2 − CCR8 + and TNFR2 − CCR8 − expression on Tregs and CD8 + T cells in tumor tissue and Tregs in spleen in MC38 ( n = 8 mice per group) and KP13 ( n = 5 mice per group) tumor models by flow cytometry. (d) Bubble plot of Tregs functional markers in TNFRSF1B + CCR8 + tregs, TNFRSF1B + CCR8 − Tregs, TNFRSF1B − CCR8 + Tregs, and TNFRSF1B − CCR8 − Tregs in human CRC database. (e) Quantitative RT-PCR analysis of Tregs functional markers Tigit and Ki67 in Tnfrsf1b + Ccr8 + Tregs, Tnfrsf1b + Ccr8 − Tregs, Tnfrsf1b − Ccr8 + Tregs, and Tnfrsf1b − Ccr8 − in MC38 model ( n = 3 independent wells). (f) Statistical analysis of Tregs functional markers Tigit and Ki67 in TNFR2 + CCR8 + Tregs, TNFR2 + CCR8 − Tregs, TNFR2 − CCR8 + Tregs, and TNFR2 − CCR8 − Tregs measured by FACS in MC38 model ( n = 7 mice per group). Statistical significance was calculated using one-way ANOVA. Data are presented as means ± SEM, based on at least two independent experiments. Significance levels are indicated as follows: * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001 “ns” indicates no significant difference.

Article Snippet: Tumor-bearing mice were randomly assigned into three groups: Vehicle, FT10-Fab (10 mg/kg) and FT10-Fab (10 mg/kg) combined with anti-CD8a depletion antibody (5 mg/kg, Selleck#A2102).

Techniques: Expressing, Flow Cytometry, Functional Assay, Quantitative RT-PCR

Journal: Oncoimmunology

Article Title: TNFR2/CCR8 bispecific antibody enhances antitumor activity through depleting Ti-Tregs and boosting effector CD8 + T cell function

doi: 10.1080/2162402X.2025.2497171

Figure Lengend Snippet: Construction and characterization of bispecific antibody targeting TNFR2 and CCR8. (a) Schematic diagram of the anti-TNFR2, anti-CCR8 and asymmetric bispecific antibody FT10-Fab. (b) The binding activity of FT10-Fab or anti-TNFR2, anti-CCR8 antibodies to CHOK1 expressing mTNFR2 and mCCR8 analyzed by FACS. (c) The binding proportions of FT10-Fab antibody on CD8, Tconv and Tregs within the tumor of MC38 model ( n = 3 per group). (d) The binding proportions of anti-TNFR2, anti-CCR8 and FT10-Fab antibodies on Ti-Tregs in MC38 tumor model ( n = 3 per group). (e) The binding proportions of anti-TNFR2, anti-CCR8 and FT10-Fab antibodies on TNFR2 + CCR8 + Tregs, TNFR2 + CCR8 − Tregs, TNFR2 − CCR8 + Tregs, and TNFR2 − CCR8 − Tregs in MC38 tumor model ( n = 3 per group). (f) The ADCC activity on CHOK1 cells expressing mTNFR2 or mCCR8, incubated with FT10-Fab, and anti-TNFR2, anti-CCR8 in the presence of Jurkat-FcγRIIIa-luciferase cell line. (g) Schematic diagram of the mechanism of FT10-Fab-mediated ADCC activity. Cell viability was evaluated using CHOK1-mTNFR2-mCCR8 cells as target cells and PBMCs as effector cells. (h) The proportions for annexin V-PI levels of IgG1, anti-TNFR2, anti-CCR8 and FT10-Fab cytotoxicity against the CHO-mTNFR2-mCCR8 in PBMCs ( n = 4 per group). (i) The ADCC activity of Ti-Tregs from MC38 tumor model following incubation with 150 nM FT10-Fab in the presence of the Jurkat-FcγRIIIa-luciferase cell line ( n = 4 per group). (Figure c-e) Statistical significance was calculated using one-way ANOVA. (Figure i) Statistical significance was calculated using a two-tailed unpaired Student’s t-test. Data are presented as means ± SEM, based on at least two independent experiments. Significance levels are indicated as follows: * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001 “ns” indicates no significant difference.

Article Snippet: Tumor-bearing mice were randomly assigned into three groups: Vehicle, FT10-Fab (10 mg/kg) and FT10-Fab (10 mg/kg) combined with anti-CD8a depletion antibody (5 mg/kg, Selleck#A2102).

Techniques: Binding Assay, Activity Assay, Expressing, Incubation, Luciferase, Two Tailed Test

Journal: Oncoimmunology

Article Title: TNFR2/CCR8 bispecific antibody enhances antitumor activity through depleting Ti-Tregs and boosting effector CD8 + T cell function

doi: 10.1080/2162402X.2025.2497171

Figure Lengend Snippet: FT10-Fab enhanced effector T cell response and alleviated immune suppression mediated by Tregs in MC38 tumor model. (a) Schematic diagram of flow cytometry gating strategy and Treg proportions among intratumoural CD3 + T cells from MC38 tumor-bearing mice treated with FT10-Fab or other controls ( n = 7 mice per group). (b) The proportions of TNFR2 + CCR8 + and CD73 + cells among Ti-Tregs from MC38 tumor-bearing mice treated with FT10-Fab or other controls ( n = 7 mice per group). (c) Schematic diagram of flow cytometry gates and CD8 + T cells proportions among intratumour CD45.2 + lymphocytes from MC38 tumor-bearing mice treated with FT10-Fab or other controls ( n = 7 mice per group). (d) The ratio of intratumour CD8/Tregs from MC38 tumor-bearing mice treated with FT10-Fab or other controls ( n = 7 mice per group). (e) Representative immunohistochemistry images analysis of intratumour CD8 + T cells. (f) The quantitative analysis of the intratumour CD8 + T cells of the immunofluorescence images; ( n = 9 independent pictures). (g) The proportions of IL-2 + , TNF-α + , and IFN-γ + cells among intratumoral CD8 + T cells from MC38 tumor-bearing mice treated with FT10-Fab or other controls ( n = 7 mice per group). (Figure a, c, d, f, g) Statistical significance was calculated using one-way ANOVA. (Figure b) Statistical significance was calculated using a two-tailed unpaired Student’s t-test. Data are presented as means ± SEM, based on at least two independent experiments. Significance levels are indicated as follows: * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001 “ns” indicates no significant difference.

Article Snippet: Tumor-bearing mice were randomly assigned into three groups: Vehicle, FT10-Fab (10 mg/kg) and FT10-Fab (10 mg/kg) combined with anti-CD8a depletion antibody (5 mg/kg, Selleck#A2102).

Techniques: Flow Cytometry, Immunohistochemistry, Immunofluorescence, Two Tailed Test

Journal: Oncoimmunology

Article Title: TNFR2/CCR8 bispecific antibody enhances antitumor activity through depleting Ti-Tregs and boosting effector CD8 + T cell function

doi: 10.1080/2162402X.2025.2497171

Figure Lengend Snippet: The antitumor effectiveness of FT10-Fab relies on CD8 cells and fosters durable T cell memory. (a) MC38 model establishment and treatment schedule in Rag1 −/− mice. Rag1 −/− were mice received an inoculation of 5 × 10 5 MC38 tumor cells and were then administered with FT10-Fab (8.33 mg/kg on days 6, 9, 12, and 15). (b) Tumor growth curves and weights of MC38-bearing Rag1 −/− mice receiving the treatment of FT10-Fab or control ( n = 6 mice per group). (c) CD8 depletion model establishment and treatment schedule in MC38. C57BL/6 mice were inoculated with 5 × 10 5 MC38 tumor cells and treated with FT10-Fab (8.33 mg/kg) on days 7, 10, 13, and 16. Anti-CD8a (5 mg/kg) was administered one day before the start of treatment and then every other day for a total of four doses. (d) The tumor growth curves and proportions of CD8 + T cells of MC38-bearing mice receiving the treatment of FT10-Fab or control with CD8 depletion ( n = 7 mice per group). (e) Model establishment and treatment schedule of CT26 re-challenged model. (f) Tumor growth curves and weights of re-challenged model (from left to right, n = 6, 9 mice per group). (Figure b, f) Statistical significance was calculated using a two-tailed unpaired Student’s t-test. (Figure d) Statistical significance was calculated using two-way ANOVA (Tumour growth curves) and one-way ANOVA (Tumour weights). Data are presented as means ± SEM, based on at least two independent experiments. Significance levels are indicated as follows: * p < 0.05 ** p < 0.01 *** p < 0.001, **** p < 0.0001 “ns” indicates no significant difference.

Article Snippet: Tumor-bearing mice were randomly assigned into three groups: Vehicle, FT10-Fab (10 mg/kg) and FT10-Fab (10 mg/kg) combined with anti-CD8a depletion antibody (5 mg/kg, Selleck#A2102).

Techniques: Control, Two Tailed Test

Journal: Oncoimmunology

Article Title: TNFR2/CCR8 bispecific antibody enhances antitumor activity through depleting Ti-Tregs and boosting effector CD8 + T cell function

doi: 10.1080/2162402X.2025.2497171

Figure Lengend Snippet: FT10-Fab altered the expression profile of CD8 functional-related genes in MC38 model. (a) Heat map to represent the expression levels of cytotoxicity-related genes on CD8 + T cells; ( n = 2 per group). (b) Quantitative RT-PCR validation of genes associated with activation and cytotoxicity in CD8 + T cells, including Prf1, Tnf-α, Il2rα, and Il2rβ, identified via RNA-seq analysis; ( n = 3 per group). (c) Heat map to represent the expression levels of memory-associated genes on CD8 + T cells; ( n = 2 per group). (d) Quantitative RT-PCR validation of memory-associated genes, including Cd44, Il7r, Id2, and Sell, in CD8 + T cells as identified by RNA-seq analysis ( n = 3 per group). (e) GSEA of the NF-kappa-B signaling pathway on CD8 + T cells. NES, normalized enrichment score ( n = 2 per group). (f) Go ontology analysis of gene associated with CD8 + T cells functional pathway ( n = 2 per group). (Figure b, d) Statistical significance was calculated using one-way ANOVA. Data are presented as means ± SEM. Significance levels are indicated as follows: * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001 “ns” indicates no significant difference.

Article Snippet: Tumor-bearing mice were randomly assigned into three groups: Vehicle, FT10-Fab (10 mg/kg) and FT10-Fab (10 mg/kg) combined with anti-CD8a depletion antibody (5 mg/kg, Selleck#A2102).

Techniques: Expressing, Functional Assay, Quantitative RT-PCR, Biomarker Discovery, Activation Assay, RNA Sequencing

Journal: Oncoimmunology

Article Title: TNFR2/CCR8 bispecific antibody enhances antitumor activity through depleting Ti-Tregs and boosting effector CD8 + T cell function

doi: 10.1080/2162402X.2025.2497171

Figure Lengend Snippet: FT10-Fab curative antitumor efficacy in combination with anti-PD1 antibody. (a) Model establishment and treatment schedule of MC38 model. C57BL/6 mice were inoculated subcutaneously (s.C.) with 5 × 10 5 MC38 tumor cells. When tumors reached approximately 50–100 mm 3 , the mice were divided into five groups with similar mean tumor volumes and treated with vehicle (10 mg/kg), anti-PD1 (10 mg/kg), FT10-F8.33 mg/kg, molar equivalent amount) or PD1&FT10 (anti-PD1 10 mg/kg, FT10-Fab 8.33 mg/kg), respectively. (b) Tumor growth curves and weight of MC38-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (c) Survival curve graph of MC38-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 7 mice per group). (d) The proportions of Tregs among tumor CD3 + T cells from MC38 tumor-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (e) The proportions of TNFR2 + CCR8 + cells among Ti-Tregs from MC38 tumor-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (f) The proportions of CD8 + T cells among tumor CD45.2 + lymphocytes from MC38 tumor-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (g) The proportions of CD8 + T cell and the ratio of CD8/Tregs from MC38 tumor-bearing mice treated with anti-PD1, FT10-F or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (h) The proportions of CD44 − CD62L + and CD44 + CD62L − cells among CD8 + T cells from MC38 tumor-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (i) The proportions of TNF-α and IFN-γ cells among CD8 + T cells from MC38 tumor-bearing mice treated with anti-PD1, FT10-Fab or a combination of anti-PD1 and FT10-Fab ( n = 8 mice per group). (Figure b) Statistical significance was determined using two-way ANOVA (Tumour growth curves). (Figure c) Survival analysis was performed using the Kaplan-Meier method. (Figure b, d-h) Statistical significance was calculated using one-way ANOVA. Data are presented as means ± SEM, based on at least two independent experiments. Significance levels are indicated as follows: * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001 “ns” indicates no significant difference.

Article Snippet: Tumor-bearing mice were randomly assigned into three groups: Vehicle, FT10-Fab (10 mg/kg) and FT10-Fab (10 mg/kg) combined with anti-CD8a depletion antibody (5 mg/kg, Selleck#A2102).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: MAP3K1 mutations confer tumor immune heterogeneity in hormone receptor–positive HER2-negative breast cancer

doi: 10.1172/JCI183656

Figure Lengend Snippet: ( A ) Flowchart of bioinformatics analyses performed in the study. ( B ) Heatmap showing the relative abundance of immune and stromal cells calculated by MCP-Counter in each sample in CBCGA cohort ( n = 351). The 2 immunological subtypes were annotated. ( C ) Comparison of tumor mutation burden (TMB) of tumors with the ICold and IHot subtypes in CBCGA cohort ( n = 314). The center line represents the median. ( D and E ) Comparison of ki67 index ( D ) and CD274 mRNA expression level ( E ) of tumors with the ICold and IHot subtypes in CBCGA cohort ( n = 350). The center line represents the median. ( F and G ) Pathological complete response (pCR) rate of patients with the ICold and IHot subtypes in the anti-PD-L1 ( F ) and anti-PD-1 ( G ) treatment arm of the I-SPY2 clinical trial. ( H ) The somatic mutations identified in tumors with the ICold and IHot subtypes in CBCGA cohort ( n = 314). * P < 0.05. ( I ) Venn diagram showing genes with significantly different mutation prevalence between the ICold and IHot subtypes in CBCGA ( n = 314), TCGA HR + /HER2 – ( n = 475), and METABRIC HR + /HER2 – ( n = 611) breast cancer cohorts. ( J and K ) Abundance of CD8 + T cells calculated by CIBERSORT ( J ) and GZMA mRNA expression ( K ) in HR + /HER2 – breast tumors with or without MAP3K1 mutation in the TCGA cohort ( n = 481). Statistical analysis: ( C , E , J , and K ) Wilcoxon signed-rank test; ( D ) χ 2 test for trend; ( F – H ) Fisher’s exact test. ICold, immune cold subtype; IHot, immune hot subtype; TMB, tumor mutation burden; HR, hormone receptor; HER2, human epidermal growth factor receptor 2.

Article Snippet: For CD8 + T cell depletion experiments, mice were treated weekly with 200 μg of CD8a depletion antibody (InvivoMAb anti-mouse CD8, BioXcell, Cat BE0061) or its isotype control (InVivoMAb rat IgG2b isotype control, BioXcell, Cat BE0090) for 3 weeks by intraperitoneal injection.

Techniques: Comparison, Mutagenesis, Expressing

Journal: The Journal of Clinical Investigation

Article Title: MAP3K1 mutations confer tumor immune heterogeneity in hormone receptor–positive HER2-negative breast cancer

doi: 10.1172/JCI183656

Figure Lengend Snippet: ( A ) MAP3K1 mutation atlas of tumors in the CBCGA cohort and schematic diagram of full-length (WT) and kinase domain-truncated (1–1,222 aa) Map3k1 (Mut) overexpression plasmids generated for the following experiments. The mutation type and whether a stop codon was generated are annotated. ( B and C ) 67NR mouse breast cancer cells with varying Map3k1 status (overexpression based on Map3k1 -KO cell lines) were orthotopically injected into BALB/c mice ( n = 5 per group). Tumor growth curves ( B ) and tumor weights with the images ( C ) are shown. ( D ) Representative flow cytometry data of CD8 + T cell infiltration gated on CD3 + T cells in tumor tissues. ( E ) Representative IHC images of tumor tissues are shown and the numbers of CD8 + T cell are quantified. Scale bar, 50 μm. ( F and G ) MFI of IFN-γ ( F ) and TNF-α ( G ) in CD8 + T cells in the tumor tissues. Data are mean ± SD ( B – G ) ( n = 5 per group). Statistical analysis: ( B ) 2-way ANOVA with Tukey’s test; ( C – G ) 1-way ANOVA with Tukey’s test. Significance in tumor growth ( B ) and tumor weight ( C ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For CD8 + T cell depletion experiments, mice were treated weekly with 200 μg of CD8a depletion antibody (InvivoMAb anti-mouse CD8, BioXcell, Cat BE0061) or its isotype control (InVivoMAb rat IgG2b isotype control, BioXcell, Cat BE0090) for 3 weeks by intraperitoneal injection.

Techniques: Mutagenesis, Over Expression, Generated, Injection, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: MAP3K1 mutations confer tumor immune heterogeneity in hormone receptor–positive HER2-negative breast cancer

doi: 10.1172/JCI183656

Figure Lengend Snippet: ( A ) Schematic diagram of the in vitro coculture assay. OVA-expressing 67NR tumor cells with varying Map3k1 status were cocultured with splenocytes isolated from OT-I mice at a ratio of 1:10. At 24 hours after coculture, tumor cells, immune cells, and the culture medium were collected for the following analyses. ( B and C ) MFI of IFN-γ ( B ) and TNF-α ( C ) in CD8 + T cells are shown. ( D and E ) Concentration of cytokines IFN-γ ( D ) and TNF-α ( E ) in the culture medium was measured by ELISA. ( F ) T cell cytotoxicity was measured by lactate dehydrogenase (LDH) assay. ( G ) T cell cytotoxicity was assessed by the percentage of dead cells in EpCAM + tumor cells, which are indicated by black boxes. Data are mean ± SD ( B – G ) ( n = 3 per group). Statistical analysis: ( B – G ) 1-way ANOVA with Tukey’s test.

Article Snippet: For CD8 + T cell depletion experiments, mice were treated weekly with 200 μg of CD8a depletion antibody (InvivoMAb anti-mouse CD8, BioXcell, Cat BE0061) or its isotype control (InVivoMAb rat IgG2b isotype control, BioXcell, Cat BE0090) for 3 weeks by intraperitoneal injection.

Techniques: In Vitro, Co-culture Assay, Expressing, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: The Journal of Clinical Investigation

Article Title: MAP3K1 mutations confer tumor immune heterogeneity in hormone receptor–positive HER2-negative breast cancer

doi: 10.1172/JCI183656

Figure Lengend Snippet: ( A and B ) 67NR cells expressing Map3k1 -WT or Map3k1 -mut were orthotopically injected into BALB/c mice. Once tumors formed, mice were randomly assigned to receive tyramine (Tyra) or DMSO in combination with anti–PD-1 or its isotype control (IgG). Tumor growth curves ( A ) and tumor weights with the images ( B ) are shown here. ( C ) Representative flow cytometry data of CD8 + T cell infiltration in tumor tissues. ( D and E ) MFI of IFN-γ ( D ) and TNF-α ( E ) in CD8 + T cells in the tumor tissues. Data are mean ± SD ( A – E ) ( n = 5 per group). Statistical analysis: ( A ) 2-way ANOVA with Tukey’s test; ( B – E ) 1-way ANOVA with Tukey’s test. Tyra, tyramine. HR, hormone receptor; HER2, human epidermal growth factor receptor 2.

Article Snippet: For CD8 + T cell depletion experiments, mice were treated weekly with 200 μg of CD8a depletion antibody (InvivoMAb anti-mouse CD8, BioXcell, Cat BE0061) or its isotype control (InVivoMAb rat IgG2b isotype control, BioXcell, Cat BE0090) for 3 weeks by intraperitoneal injection.

Techniques: Expressing, Injection, Control, Flow Cytometry

Journal: Nature Communications

Article Title: Inhibition of glycosphingolipid synthesis with eliglustat in combination with immune checkpoint inhibitors in advanced cancers: preclinical evidence and phase I clinical trial

doi: 10.1038/s41467-024-51495-3

Figure Lengend Snippet: a Experimental design. A total of 1 × 10 5 tumour cells were subcutaneously injected and observed for tumour formation in C57BL/6 mice treated with different doses of eliglustat. b C57BL/6 mice were implanted with 1 × 10 5 MC38-OVA tumour cells, and the treatment scheme is shown in ( a ). Tumour sizes were measured every 3 days. The average and individual tumour curves ( n = 10 per group) are shown. c Survival curves of MC38-OVA tumour-bearing mice in the control and 10 mg/kg eliglustat-treated groups ( n = 10 per group). d Body weights of MC38-OVA tumour-bearing mice in the control and 10 mg/kg eliglustat-treated groups ( n = 10 per group). e Injection schedule for antibody-mediated depletion of CD4 + and CD8 + T cells and NK cells and eliglustat treatment in MC38-OVA tumour-bearing mice. f C57BL/6 mice were implanted with 1 × 10 5 MC38-OVA cells, as shown in ( e ). Tumour sizes were measured every 3 days. The average and individual tumour curves ( n = 7 per group) are shown. The data are shown as the mean ± SEM. ns, not significant. P values were calculated by unpaired two-sided t test in ( d ). P values were calculated by two-way repeated measures analysis of variance (ANOVA) in ( b ) and ( f ) and the log-rank test in ( c ). CON control, ELI eliglustst. Source data and exact p values are provided as a file.

Article Snippet: Mouse anti-CD8 depleting antibody (Clone 2.43) and anti-CD4 depleting antibody (Clone GK1.5) were purchased from BioXcell.

Techniques: Injection, Control

Journal: Nature Communications

Article Title: Inhibition of glycosphingolipid synthesis with eliglustat in combination with immune checkpoint inhibitors in advanced cancers: preclinical evidence and phase I clinical trial

doi: 10.1038/s41467-024-51495-3

Figure Lengend Snippet: a CD8 + T-cell killing assay of MC38-OVA-GFP cells cocultured at different ratios with CD8 + T cells isolated from OT-I mice that had been pretreated with anti-CD3, anti-CD28 antibodies and IL-2. MC38-OVA-GFP cells were pretreated with eliglustat. The number of viable MC38-OVA-GFP cancer cells at the end of the assay was determined and reported ( n = 3). b , CD8 + T-cell killing assay of B16f10-OVA-GFP cells cocultured at different ratios with CD8 + T cells isolated from OT-I mice. B16f10-OVA-GFP cells were pretreated with eliglustat. The number of viable B16f10-OVA-GFP cancer cells at the end of the assay was determined and reported ( n = 3). c NY-ESO-1-TCR + T-cell killing assay of CD8 + T cells isolated from healthy volunteers cocultured at different ratios with OVCAR3-GFP cells pretreated with eliglustat. The number of viable OVCAR3-GFP cancer cells at the end of the assay was determined and reported ( n = 3). d , e FACS analysis of IFN-γ-, TNF-α-, and CD107a-positive CD8 + T cells after coculture with MC38-OVA cancer cells at an effector-to-target ratio of 1:1 ( n = 3). Bar colours ( d ): T cells (red), T cells treated with eliglustat (cyan), T cells + OVA-MC38 (dark green), T cells (eliglustat) + OVA-MC38 (orange), T cells + OVA-MC38(eliglustat) (purple). f MHC-I, MHC-II and H-2Kb-SIINFEKL of MC38-OVA were measured by flow cytometry ( n = 3). Bar colours ( e , f ): control (red), 400 nM eliglustat (dark green). g OT-I T cells were stained with AF700-conjugated anti-CD8 and incubated with MC38-OVA (CFSE) cells at an E:T ratio of 2:1. Flow cytometry analysis of the proportion of OT-I T cells conjugated to MC38-OVA (CFSE) cells ( n = 3). h Experimental design. MC38-OVA cells were pretreated with eliglustat for 7 days in vitro, and 0.5 × 10 5 tumour cells were injected subcutaneously. Tumour formation was observed in C57BL/6 mice. i The tumour curves of the MC38-OVA tumour-bearing mice are shown, and the treatment scheme is shown in ( h ). j The survival curves of MC38-OVA tumour-bearing mice are shown, and the treatment scheme is shown in ( h ) ( n = 10 per group). The data represent three independent experiments with similar results ( a – g ). The data are presented as the means ± SEMs. ns, not significant. P values in ( d – f ) were calculated by unpaired two-sided t tests. P values in ( a ), ( b ), ( c ), ( g ) and ( i ) were determined by two-way repeated measures ANOVA and the log-rank test in ( j ). CON control, ELI eliglustst. Source data and exact p values are provided as a file.

Article Snippet: Mouse anti-CD8 depleting antibody (Clone 2.43) and anti-CD4 depleting antibody (Clone GK1.5) were purchased from BioXcell.

Techniques: Isolation, Flow Cytometry, Control, Staining, Incubation, In Vitro, Injection

Journal: Nature Communications

Article Title: Inhibition of glycosphingolipid synthesis with eliglustat in combination with immune checkpoint inhibitors in advanced cancers: preclinical evidence and phase I clinical trial

doi: 10.1038/s41467-024-51495-3

Figure Lengend Snippet: a Western blot analysis of the expression of UGCG in MC38-OVA with and without UGCG knockout. GAPDH was used as a protein loading control. The analysis was done thrice with biologically independent samples. b Cell proliferation of sgCONT or sgUGCG-MC38-OVA tumour cells by FACS analysis ( n = 3). c Colony formation ability of MC38-OVA tumour cells with sgCONT or sgUGCG tumour cells on day 14 ( n = 3). d Titration curves of MHC-I-specific antibodies on MC38-OVA cells. Flow cytometry charts of antibody stain as indicated by the arrow. MFI, mean fluorescence intensity ( n = 3). e CD8 + T-cell killing assay of sgUGCG-MC38-OVA tumour cells cocultured in different ratios with CD8 + T cells isolated from OT-I mice. The number of viable sgUGCG-MC38-OVA tumour cells at the end point of the assay was determined and reported ( n = 3). f FACS analysis IFN-γ, TNF-α, expression of CD8 + T cells after coculture with sgUGCG-MC38-OVA tumour cells at an effector-to-target ratio of 1:1 ( n = 3). g , sgCONT- and sgUGCG-MC38-OVA cells grown in C57BL/6 mice( n = 10). About 2 × 10 5 sgCONT- and sgUGCG-MC38-OVA cells were inoculated subcutaneously into syngeneic mice and monitored for tumour formation. h Phenotype and function of TILs from sgCONT- and sgUGCG-MC38-OVA tumours were detected by flow cytometry analysis at day 21 after tumour inoculation ( n = 3). Surface levels of CD8 + T cells, IFN-γ, TNF-α and Tetramer on tumour-infiltrating CD8 + T cells of different groups were analysed. Bar colours ( f , h ): control sgRNA (red), sgRNA targeting UGCG (dark green). The data represent three independent experiments with similar results ( b – f ). Data are shown as the mean ± SEM. ns, not significant. P values in ( b , c , f and h ) were calculated by unpaired two-sided t test. P values in ( e ) were calculated by unpaired two-sided t tests. P values in ( g ) were determined by two-way repeated measures ANOVA. Source data and exact p values are provided as a file.

Article Snippet: Mouse anti-CD8 depleting antibody (Clone 2.43) and anti-CD4 depleting antibody (Clone GK1.5) were purchased from BioXcell.

Techniques: Western Blot, Expressing, Knock-Out, Control, Titration, Flow Cytometry, Staining, Fluorescence, Isolation

Journal: Nature Communications

Article Title: Inhibition of glycosphingolipid synthesis with eliglustat in combination with immune checkpoint inhibitors in advanced cancers: preclinical evidence and phase I clinical trial

doi: 10.1038/s41467-024-51495-3

Figure Lengend Snippet: a MC38-OVA tumour samples were collected from tumour-bearing mice on Day 21 with or without eliglustat and subjected to a CyTOF assay. The t-SNE plot shows all CD45 + cells, coloured according to the type of immunocyte ( n = 3). b Proportions of CD4 + and CD8 + T cells in CD45 + T-cell clusters ( n = 3). c , d Total TCRs, clonal type, unique TCRs (D50), and average CDR3 length of T cells from both the control and eliglustat-treated tumours and spleens were determined by using TCR-β CDR3 sequencing ( n = 9). Box plots display the median (centre line) and minimum and maximum values (boxes) ( b – d ). Box colours ( b – d ): control (red), eliglustat treated (blue). e , f MC38 tumour-bearing mice 21 days after eliglustat treatment. CD8 + T cells were collected from dLNs and tumours, and the IFN-γ ELISPOT assay was performed with 4T1 or MC38 cell restimulation ( n = 4). The data represent three independent experiments with similar results ( b – f ). Box plot whiskers extend to the minimum and maximum values, with the centre line indicating the median, and the box encompassing the interquartile range. P values were determined by unpaired two-sided t tests in ( b , c , d and f ). CON control, ELI eliglustst, dLN draining lymph node. Source data and exact p values are provided as a file.

Article Snippet: Mouse anti-CD8 depleting antibody (Clone 2.43) and anti-CD4 depleting antibody (Clone GK1.5) were purchased from BioXcell.

Techniques: Control, Sequencing, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: Inhibition of glycosphingolipid synthesis with eliglustat in combination with immune checkpoint inhibitors in advanced cancers: preclinical evidence and phase I clinical trial

doi: 10.1038/s41467-024-51495-3

Figure Lengend Snippet: a Experimental design. MC38-OVA-bearing mice were administered FTY720 on Day 4 and 10 mg/kg eliglustat on Day 5, starting on Day 21 through the end of the experiment ( n = 5). b Tumour curves of MC38-OVA tumour-bearing mice; the treatment scheme is shown in Fig. a. c Flow cytometric analysis of T cells from dLNs, peripheral blood, and tumours. The percentages of CD8 + T cells in the dLNs, peripheral blood, and tumours of different groups of mice ( n = 3). d Absolute numbers of CD8 + T cells and CD8 + tetramer + T cells in different groups of mice ( n = 3). e Experimental design. MC38-OVA-bearing mice were administered 10 mg/kg eliglustat on Day 0 and were administered FTY720 on Day 7, starting on Day 21 through the end of the experiment ( n = 5). f Tumour curves of MC38-OVA tumour-bearing mice( n = 7); the treatment scheme is shown in Fig. e. g Flow cytometric analysis of T cells from dLNs, peripheral blood, and tumours. The percentages of CD8 + T cells in the dLNs, peripheral blood, and tumours of different groups of mice ( n = 3). h Absolute numbers of CD8 + T cells and CD8 + tetramer + T cells in different groups of mice ( n = 3). The data represent three independent experiments with similar results. The data are presented as the means ± SEMs. ns, not significant. P values were determined by two-way repeated measures ANOVA in ( b and f ) and by unpaired two-sided t tests in ( c , d , g and h ). CON control, ELI eliglustst, dLN draining lymph node, PB peripheral blood. Source data and exact p values are provided as a file.

Article Snippet: Mouse anti-CD8 depleting antibody (Clone 2.43) and anti-CD4 depleting antibody (Clone GK1.5) were purchased from BioXcell.

Techniques: Control

Journal: Nature Communications

Article Title: Inhibition of glycosphingolipid synthesis with eliglustat in combination with immune checkpoint inhibitors in advanced cancers: preclinical evidence and phase I clinical trial

doi: 10.1038/s41467-024-51495-3

Figure Lengend Snippet: a Experimental design. C57BL/6 mice were transplanted with 1 × 10 5 MC38-OVA cells and treated with PBS (CON group), eliglustat alone (ELI, 10 mg/kg), an anti-PD-1 antibody alone (a-PD-1, 200 mg/kg per mouse) or eliglustat plus anti-PD-1 as indicated (ELI+ a-PD-1). Tumour sizes were examined every other day. b Average and individual tumour growth curves of each treatment group are shown ( n = 7). c The phenotype and function of tumour-infiltrating lymphocytes from MC38-OVA tumours were determined by flow cytometry analysis on Day 21 after tumour inoculation ( n = 3). CD4 + T cells, CD8 + T cells, IFN-γ, TNF-α, PD-1, TIM-3 and tetramer + cell surface levels in the tumours of different treatment groups. Absolute numbers of CD4 + T cells, CD8 + T cells and IFN-γ + , TNF-α + , and tetramer + T cells per 10 6 cells ( n = 3). Bar colours ( d ): control (red), eliglustat (cyan), ant-PD-1 (dark green), eliglustat + anti-PD-1 (orange). The data represent three independent experiments with similar results. The data are presented as the means ± SEMs. ns, not significant. P values were determined by two-way repeated measures ANOVA in ( b ) and unpaired two-sided t tests in ( c ). CON control, ELI eliglustst. Source data and exact p values are provided as a file.

Article Snippet: Mouse anti-CD8 depleting antibody (Clone 2.43) and anti-CD4 depleting antibody (Clone GK1.5) were purchased from BioXcell.

Techniques: Flow Cytometry, Control

Journal: Journal for immunotherapy of cancer

Article Title: Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma.

doi: 10.1136/jitc-2021-004150

Figure Lengend Snippet: Figure 3 HSD11B1 expression confers resistance to PD-1 blockade. (A) Overview of mouse melanoma cell lines and Hsd11b1 expression (3’mRNA-seq). (B) Kinetic of 11-DHCS to CS conversion in indicated cell lines assayed by CS-specific ELISA (n=3). Dashed line indicates input (100%) of 11-DHCS. Error bars, SD. (C) 11-DHCS to CS conversion (% of input 11- DHCS) in indicated cell lines at 40 min and 3 hours assayed by CS-specific ELISA (n=3). (D) GSEA plot for indicated gene set. Comparison of CM and LN transcriptomes (3’mRNA-seq). (E) In vitro cell growth of CM vs LN cells exposed to IFN-γ. Upper panel: Quantification of n=3. Lower panel: Representative images of stained tissue culture wells. (F) Tumor growth kinetics (left) and final tumor weight at day 12 (right) of CM and LN melanomas treated with αPD-1 or IgG control. (G) Heatmap showing proliferation-associated gene expression (3’mRNA-seq) in CM and LN melanomas from (F). (H, I) Correlation of Hsd11b1 expression with T cell (cytotoxic) marker genes (H) and myeloid cell marker genes (I) in CM melanomas treated with αPD-1 or IgG control. (J) Individual tumor growth curves and (K) tumor weight (at day 8) of CM melanomas ectopically expressing Hsd11b1 (pRP.Hsd11b1) vs CM controls (pRP) treated with αPD-1 or IgG control. (L, M) Intratumoral CD8+ T cells (L) and CD4+ T cells (M) assessed by immunofluorescence from multiple representative regions. Statistics: *p<0.05, **p<0.01, ***p<0.001. Two-sided unpaired t-tests (B, F, K–M), with logarithms (C, E). Correction for multiple comparison with Benjamini and Hochberg method (E). 11-DHCS, 11-dehydrocorticosterone; CM, cutaneous melanoma; CS, corticosterone; FDR, false discovery rate; GSEA, gene set enrichment analysis; IFN-γ, interferon-γ; LN, lymph node; (N)ES,(normalized) enrichment score; r, Pearson’s correlation coefficient.

Article Snippet: Immune depletion of CD8+ T cells Anti- CD8a depleting antibody (40 mg/kg, clone 2.43, BioXcell, #BE0061) was intraperitoneally injected every 4 days starting from day −1 prior CM cells transplantation until mice were euthanized.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Comparison, In Vitro, Staining, Control, Gene Expression, Marker, Immunofluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma.

doi: 10.1136/jitc-2021-004150

Figure Lengend Snippet: Figure 6 HSD11B1 inhibition augments IFN-γ production of CD8+ T cells under PD-1 blockade. (A) Effect of HSD11B1 inhibition on IFN-γ production by intratumoral (CM) CD8+ T cells. Gating strategy for flow cytometry. (B) Representative FACS blots showing frequencies of IFN-γ +CD8+ cells in CM melanomas treated as indicated. (C) Quantification of experiment described in (B). Two-sided unpaired t-test with logarithms. (D) Effect of HSD11B1 inhibition on IFN-γ production by Pmel-1 T cells in vitro. Gating strategy for flow cytometry. (E) Representative FACS blots (IFN-γ positivity) of gp100 activated Pmel-1 T cells treated as indicated. (F) Quantification of experiments (n=3) described in (D, E). 11-DHCS, DEXA (100 nM), CBX and 10j (10 µM). (G) Experimental outline of CD8+ T cell depletion in mice bearing CM melanomas and treatment conditions. (H, I) Frequencies of CD8+ T cells in (H) tumor and (I) tumor-draining LN assessed by flow cytometry. (J) Individual CM melanoma tumor growth curves and (K) tumor volume at day 8 after inoculation treated as indicated with or without antibody-mediated depletion of CD8+ cells. Statistics: *p<0.05, **p<0.01, ***p<0.001. Two-sided unpaired t-tests (for ratios with logarithms). 11- DHCS, 11-dehydrocorticosterone; CM, cutaneous melanoma; DEXA, dexamethasone; IFN-γ, interferon-γ; tdLN, tumor-draining lymph node.

Article Snippet: Immune depletion of CD8+ T cells Anti- CD8a depleting antibody (40 mg/kg, clone 2.43, BioXcell, #BE0061) was intraperitoneally injected every 4 days starting from day −1 prior CM cells transplantation until mice were euthanized.

Techniques: Inhibition, Flow Cytometry, In Vitro