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Bio X Cell depletion antibodies anti mouse cd8α
Depletion Antibodies Anti Mouse Cd8α, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 97/100, based on 1147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+cd8a+depleting+antibody/Anti-CD8+Cd8A+Antibody/pm41758652-307-0-6
Average 97 stars, based on 1147 article reviews

depletion antibodies anti mouse cd8α - by Bioz Stars, 2026-10

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Article Title: Loss of NEDD8 in cancer cells causes vulnerability to immune checkpoint blockade in triple-negative breast cancer.
Article Snippet: When tumors were palpable or established EO771 tumor-bearing mice were injected intraperitoneally (i.p.) with an anti-PD-1 antibody (clone RMP1-14, BioXcell), or a Rat isotype IgG2a control (clone 2A3, BioXCell) every 3 days (50μg per mouse). .. To deplete CD8+ T cells, an anti-CD8a depleting antibody (200μg, clone 2.43, BioXCell) or an IgG2b isotype control (clone LTF-2, BioXCell) were infused i.p. 4 days after tumor inoculation every 3 days, followed by treatment with 100μg anti-PD-1 or Rat IgG2a isotype antibody on days 7, 10, 13. .. Human breast cancer cell line, MDA-MB-231 (92020424, Sigma Aldrich), and HEK293T cells (CRL-3216, American Type Culture Collection, ATCC) were purchased.

Article Title: Loss of NEDD8 in cancer cells causes vulnerability to immune checkpoint blockade in triple-negative breast cancer
Article Snippet: When tumors were palpable or established EO771 tumor-bearing mice were injected intraperitoneally (i.p.) with an anti-PD-1 antibody (clone RMP1-14, BioXcell), or a Rat isotype IgG2a control (clone 2A3, BioXCell) every 3 days (50 μg per mouse). .. To deplete CD8+ T cells, an anti-CD8a depleting antibody (200 μg, clone 2.43, BioXCell) or an IgG2b isotype control (clone LTF-2, BioXCell) were infused i.p. 4 days after tumor inoculation every 3 days, followed by treatment with 100 μg anti-PD-1 or Rat IgG2a isotype antibody on days 7, 10, 13. .. Human breast cancer cell line, MDA-MB-231 (92020424, Sigma Aldrich), and HEK293T cells (CRL-3216, American Type Culture Collection, ATCC) were purchased.

Article Title: NEDD8 loss causes cancer vulnerability to immune checkpoint blockade in triple-negative breast cancer
Article Snippet: 609 When tumors were palpable or established EO771 tumor-bearing mice were injected 610 intraperitoneally (i.p.) with an anti-PD-1 antibody (clone RMP1-14, BioXcell), or a Rat isotype 611 IgG2a control (clone 2A3, BioXCell) every 3 days (50 μg per mouse). .. To deplete CD8+ T cells, 612 an anti-CD8a depleting antibody (200 μg, clone 2.43, BioXCell) or an IgG2b isotype control 613 (clone LTF-2, BioXCell) were infused i.p. 4 days after tumor inoculation every 3 days, followed 614 by treatment with 100 μg anti-PD-1 or Rat IgG2a isotype antibody on days 7, 10, 13. .. 615 Human cell lines 616 Human breast cancer cell line, MDA-MB-231, and HEK293T cells, were purchased from 617 American Type Culture Collection (ATCC).

Image Search Results

(A) Representative histograms of IL-18R1 expression on NK cells, CD44 + CD8 T-cells, and CD44 + CD4 T-cells from day 20 dLN. (B) Mean EAE clinical score of WT (n=13), Il18tg (n=14), and Il18tg;Il18r1 ΔT (n=12) mice. (C) Flow cytometric quantification of splenic leukocytes at days 20-21. (D) Quantitation of individual cytokine production by splenic CD4 T-cells assessed by ICS following PMA/Iono stimulation. (E) Percent and absolute number of T-cells and (F) FOXP3 + CD4T reg in spinal cords by flow cytometry. (G) Quantitation of individual cytokine production by spinal cord CD4 T-cells by ICS following PMA/Iono stimulation. (A) Representative of 3 experiments. Data pooled from (C-G) 2 or (B) 3 experiments. Error bars = SEM. Statistical analysis: (B) Kruskal-Wallis of AUC with Dunns post-test of pairwise comparisons, (C, F) one-way ANOVA with Dunnett’s post-test of pairwise comparisons to Il18tg , (D, E, G) 2way ANOVA with Dunnett’s post-test of pairwise comparisons to Il18tg . Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: The innate cytokine IL-18 inhibits CNS autoimmunity through preferential activation of protective CD8 T-cells

doi: 10.64898/2026.01.26.701759

Figure Lengend Snippet: (A) Representative histograms of IL-18R1 expression on NK cells, CD44 + CD8 T-cells, and CD44 + CD4 T-cells from day 20 dLN. (B) Mean EAE clinical score of WT (n=13), Il18tg (n=14), and Il18tg;Il18r1 ΔT (n=12) mice. (C) Flow cytometric quantification of splenic leukocytes at days 20-21. (D) Quantitation of individual cytokine production by splenic CD4 T-cells assessed by ICS following PMA/Iono stimulation. (E) Percent and absolute number of T-cells and (F) FOXP3 + CD4T reg in spinal cords by flow cytometry. (G) Quantitation of individual cytokine production by spinal cord CD4 T-cells by ICS following PMA/Iono stimulation. (A) Representative of 3 experiments. Data pooled from (C-G) 2 or (B) 3 experiments. Error bars = SEM. Statistical analysis: (B) Kruskal-Wallis of AUC with Dunns post-test of pairwise comparisons, (C, F) one-way ANOVA with Dunnett’s post-test of pairwise comparisons to Il18tg , (D, E, G) 2way ANOVA with Dunnett’s post-test of pairwise comparisons to Il18tg . Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ***p<0.001.

Article Snippet: Anti-CD8a depleting antibody (200ug, YTS169.4, BioXcell) on days −3, 0, 3, and 6.

Techniques: Expressing, Quantitation Assay, Flow Cytometry

Expression of (A) effector markers and (B) putative suppressive markers on splenic CD8 T-cells from WT and Il18bp KO mice at day 12. (C) IFNg production by day 12 splenic CD8 T-cells following PMA/Ionomycin or MOG 35-55 stimulation by ICS. (D) Representative flow plots of day 18 spinal cord T-cells. (E) Quantification of spinal cord CD4 and CD8 T-cells and the corresponding CD4:CD8 ratio. (F) Expression of key effector surface markers on spinal cord CD8 T-cells. (G) Representative flow plots and quantification of dual-expressing CD38 + PD-1 + CD8 T-cells. (H) Mean EAE clinical score of WT and Il18bp KO mice receiving isotype (n=5) or CD8-depleting antibodies (n=7; 200ug, i.p., days −3, 0, 3, and 6) ( I) Representative plots of CD4 versus CD8 expression on splenic T-cells at day 25. (A,B, E, G, H) Data pooled from 2 experiments. (C,F) Data representative of 2-3 experiments. Error bars = SEM. Statistical analysis: (A-C, E-G) unpaired t-tests, p-value with Holm-Sidak correction with multiple comparisons, (H) Kruskal-Wallis of AUC with Dunn’s post-test of pairwise comparisons of Il18bp KO isotype vs anti-CD8. Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ****p<0.0001.

Journal: bioRxiv

Article Title: The innate cytokine IL-18 inhibits CNS autoimmunity through preferential activation of protective CD8 T-cells

doi: 10.64898/2026.01.26.701759

Figure Lengend Snippet: Expression of (A) effector markers and (B) putative suppressive markers on splenic CD8 T-cells from WT and Il18bp KO mice at day 12. (C) IFNg production by day 12 splenic CD8 T-cells following PMA/Ionomycin or MOG 35-55 stimulation by ICS. (D) Representative flow plots of day 18 spinal cord T-cells. (E) Quantification of spinal cord CD4 and CD8 T-cells and the corresponding CD4:CD8 ratio. (F) Expression of key effector surface markers on spinal cord CD8 T-cells. (G) Representative flow plots and quantification of dual-expressing CD38 + PD-1 + CD8 T-cells. (H) Mean EAE clinical score of WT and Il18bp KO mice receiving isotype (n=5) or CD8-depleting antibodies (n=7; 200ug, i.p., days −3, 0, 3, and 6) ( I) Representative plots of CD4 versus CD8 expression on splenic T-cells at day 25. (A,B, E, G, H) Data pooled from 2 experiments. (C,F) Data representative of 2-3 experiments. Error bars = SEM. Statistical analysis: (A-C, E-G) unpaired t-tests, p-value with Holm-Sidak correction with multiple comparisons, (H) Kruskal-Wallis of AUC with Dunn’s post-test of pairwise comparisons of Il18bp KO isotype vs anti-CD8. Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ****p<0.0001.

Article Snippet: Anti-CD8a depleting antibody (200ug, YTS169.4, BioXcell) on days −3, 0, 3, and 6.

Techniques: Expressing

(A) Mean clinical score of WT (n=12), Il18tg (n=10), and Il18tg Il18r1 ΔCD8 (n=9) mice treated with tamoxifen at days 4 and 6. (B) IL-18R1 expression on activated (CD44 + ) CD4Tconv and CD8 T-cells. (C) Expression of CD44 on CD4T conv in dLN. ( D) Quantification of T-cell subsets in day 23 spinal cords as percent of CD45, CD4:CD8 ratio, and absolute number. ( E) CD8 T-cell activation (defined as CD62L-) and expression of putative CD8Tsupp markers in dLN. (F ) CD8 T-cell expression of putative CD8Tsupp markers and ( G) quantification of FOXP3+CD4T reg in day 23 spinal cords. (H) Serum IFNg concentration of the indicated genotypes at day 23. (A) Data pooled from 2 experiments and representative of 3 total. (B-H) Data representative of 3 experiments. Error bars = SEM. Statistical analysis: (A) Kruskal-Wallis of AUC with Dunn’s post-test of pairwise comparisons, (B-H) one-way ANOVA with Dunnett’s post-test of pairwise comparisons, (D, E, G) 2way ANOVA with Dunnett’s post-test of pairwise comparisons. All comparisons made to Il18tg control. Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Journal: bioRxiv

Article Title: The innate cytokine IL-18 inhibits CNS autoimmunity through preferential activation of protective CD8 T-cells

doi: 10.64898/2026.01.26.701759

Figure Lengend Snippet: (A) Mean clinical score of WT (n=12), Il18tg (n=10), and Il18tg Il18r1 ΔCD8 (n=9) mice treated with tamoxifen at days 4 and 6. (B) IL-18R1 expression on activated (CD44 + ) CD4Tconv and CD8 T-cells. (C) Expression of CD44 on CD4T conv in dLN. ( D) Quantification of T-cell subsets in day 23 spinal cords as percent of CD45, CD4:CD8 ratio, and absolute number. ( E) CD8 T-cell activation (defined as CD62L-) and expression of putative CD8Tsupp markers in dLN. (F ) CD8 T-cell expression of putative CD8Tsupp markers and ( G) quantification of FOXP3+CD4T reg in day 23 spinal cords. (H) Serum IFNg concentration of the indicated genotypes at day 23. (A) Data pooled from 2 experiments and representative of 3 total. (B-H) Data representative of 3 experiments. Error bars = SEM. Statistical analysis: (A) Kruskal-Wallis of AUC with Dunn’s post-test of pairwise comparisons, (B-H) one-way ANOVA with Dunnett’s post-test of pairwise comparisons, (D, E, G) 2way ANOVA with Dunnett’s post-test of pairwise comparisons. All comparisons made to Il18tg control. Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Anti-CD8a depleting antibody (200ug, YTS169.4, BioXcell) on days −3, 0, 3, and 6.

Techniques: Expressing, Activation Assay, Concentration Assay, Control

(A) Mean EAE clinical score of WT mice treated with PBS (n=19) or DR-18 (2ug, Subq, q3d; n=16) from days 0 to 15. (B) Mean EAE clinical score of WT and E8i cre ;Il18r1 fl/fl mice treated with PBS or DR-18 (n=9; 2ug, treated every 3 days from 0 to 30). (C) Mean EAE clinical score of WT mice treated with PBS or DR-18 at various timepoints indicated below the graph (treatment every 3 days from 0 to 15, colored dot corresponds to DR-18 treatment while no dot indicates PBS; n=10 for all groups). (D-I) 2.5×10 5 splenic CD4 T-cells from naïve 2D2 mice were transferred to WT and Il18bp KO mice on day −1. Half of the WT mice were randomized to receive DR-18 (n=8 per timepoint) or PBS (n=8 per timepoint) on days 9, 12, & 15. (D) Quantification of transferred splenic 2D2 T-cells, (E) CD49d expression on day 12 splenic 2D2, (F) and CD4T conv and 2D2 in day 18 spinal cord. (G) Absolute number of splenic FOXP3+ CD4T reg and CD8T eff (CD44 + CD62L − ) cells over time. (H) Expression of various surface markers and (I) CD38/PD-1 co-expression on splenic CD8 T-cells at day 12 and 18 from WT mice treated with PBS and DR-18. Data pooled from (B-I) 2 or (A) 3 experiments. Error bars = SEM except for box and whisker plot. Statistical analysis: (A) Mann-Whitney of AUC, (B,C) Kruskal-Wallis of AUC with Dunn’s post-test of pairwise comparisons to DR-18 (B) or PBS control (C), (D, G) unpaired t-tests of WT vs WT+DR-18 on days 12 and 18 only with Holm-Sidak correction of p-value, (E) one-way ANOVA with Dunnett’s post-test of pairwise comparisons (H,I) unpaired t-tests with Holm-Sidak correction of p-value. Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Journal: bioRxiv

Article Title: The innate cytokine IL-18 inhibits CNS autoimmunity through preferential activation of protective CD8 T-cells

doi: 10.64898/2026.01.26.701759

Figure Lengend Snippet: (A) Mean EAE clinical score of WT mice treated with PBS (n=19) or DR-18 (2ug, Subq, q3d; n=16) from days 0 to 15. (B) Mean EAE clinical score of WT and E8i cre ;Il18r1 fl/fl mice treated with PBS or DR-18 (n=9; 2ug, treated every 3 days from 0 to 30). (C) Mean EAE clinical score of WT mice treated with PBS or DR-18 at various timepoints indicated below the graph (treatment every 3 days from 0 to 15, colored dot corresponds to DR-18 treatment while no dot indicates PBS; n=10 for all groups). (D-I) 2.5×10 5 splenic CD4 T-cells from naïve 2D2 mice were transferred to WT and Il18bp KO mice on day −1. Half of the WT mice were randomized to receive DR-18 (n=8 per timepoint) or PBS (n=8 per timepoint) on days 9, 12, & 15. (D) Quantification of transferred splenic 2D2 T-cells, (E) CD49d expression on day 12 splenic 2D2, (F) and CD4T conv and 2D2 in day 18 spinal cord. (G) Absolute number of splenic FOXP3+ CD4T reg and CD8T eff (CD44 + CD62L − ) cells over time. (H) Expression of various surface markers and (I) CD38/PD-1 co-expression on splenic CD8 T-cells at day 12 and 18 from WT mice treated with PBS and DR-18. Data pooled from (B-I) 2 or (A) 3 experiments. Error bars = SEM except for box and whisker plot. Statistical analysis: (A) Mann-Whitney of AUC, (B,C) Kruskal-Wallis of AUC with Dunn’s post-test of pairwise comparisons to DR-18 (B) or PBS control (C), (D, G) unpaired t-tests of WT vs WT+DR-18 on days 12 and 18 only with Holm-Sidak correction of p-value, (E) one-way ANOVA with Dunnett’s post-test of pairwise comparisons (H,I) unpaired t-tests with Holm-Sidak correction of p-value. Only p adj <0.05 is shown. *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Anti-CD8a depleting antibody (200ug, YTS169.4, BioXcell) on days −3, 0, 3, and 6.

Techniques: Expressing, Whisker Assay, MANN-WHITNEY, Control

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.

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

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.

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

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.

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.

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

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.

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.

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

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.

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.

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.

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

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.

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.

Techniques:

( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

21h10 improves antitumor efficacy in in vivo B16F10 murine melanoma and ex vivo human PDOTs models, with non-neutralizing anti-drug antibodies, and toxicity alleviated with TNFα blockade. ( A ) Naïve CD8 + TRP1 high/low melanoma-specific T cells were adoptively transferred to mice prior to B16F10 inoculation. Cytokine therapy began on day 5 and continued every day until the stop of treatment (dashed line). ( B ) Mice received no prior T cell adoptive transfer before cytokine therapy. ( C ) Same as ( B ), but with adoptive transfer of naïve CD8 + TRP1 high/low T cells. ( D ) Scheme of PDOTS preparation. ( E ) Viability assessment of ex vivo human melanoma PDOTS following treatment of ICB (anti-PD-1, pembrolizumab, or anti-PD-1/anti-LAG-3), 21h10, or untreated control. ( F ) Representative images of PDOTS viability assessment shown in ( E ). PI-dead cells in red and Hoechst-nuclei in blue. ( G ) Mice died due to toxicity or after 40 days of treatment were bled and their serum was analyzed by ELISA for anti-drug antibodies against 21h10. Positivity (dotted line) was determined as two standard deviations above the mean of samples from mice treated only with PBS. Fractions indicate the number of mice classified as “positive” for anti-drug antibodies out of the total mice in each treatment group. ( H ) Mice with various titer levels of anti-21h10 antibodies were inoculated with MC38 tumors. Starting on day 6, mice were treated with 21h10 or PBS daily for 17 days. ( I ) Wild-type (WT), Rag2-/- , or Rag2-/- Il2rg-/- mice were treated with PBS or 21h10 daily and isotype or anti-NK1.1 depleting antibodies every three days. Mice were sacrificed when weight loss exceeded 20% of the initial starting weight. ( J ) Serum TNFα level was measured in C57BL/6 and Rag2-/- mice with PBS or 21h10 treatments. ( K ) Antitumor activity comparison on B16F10 melanoma between 21h10 and 21h10+aTNFα treatment groups.

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: 21h10 improves antitumor efficacy in in vivo B16F10 murine melanoma and ex vivo human PDOTs models, with non-neutralizing anti-drug antibodies, and toxicity alleviated with TNFα blockade. ( A ) Naïve CD8 + TRP1 high/low melanoma-specific T cells were adoptively transferred to mice prior to B16F10 inoculation. Cytokine therapy began on day 5 and continued every day until the stop of treatment (dashed line). ( B ) Mice received no prior T cell adoptive transfer before cytokine therapy. ( C ) Same as ( B ), but with adoptive transfer of naïve CD8 + TRP1 high/low T cells. ( D ) Scheme of PDOTS preparation. ( E ) Viability assessment of ex vivo human melanoma PDOTS following treatment of ICB (anti-PD-1, pembrolizumab, or anti-PD-1/anti-LAG-3), 21h10, or untreated control. ( F ) Representative images of PDOTS viability assessment shown in ( E ). PI-dead cells in red and Hoechst-nuclei in blue. ( G ) Mice died due to toxicity or after 40 days of treatment were bled and their serum was analyzed by ELISA for anti-drug antibodies against 21h10. Positivity (dotted line) was determined as two standard deviations above the mean of samples from mice treated only with PBS. Fractions indicate the number of mice classified as “positive” for anti-drug antibodies out of the total mice in each treatment group. ( H ) Mice with various titer levels of anti-21h10 antibodies were inoculated with MC38 tumors. Starting on day 6, mice were treated with 21h10 or PBS daily for 17 days. ( I ) Wild-type (WT), Rag2-/- , or Rag2-/- Il2rg-/- mice were treated with PBS or 21h10 daily and isotype or anti-NK1.1 depleting antibodies every three days. Mice were sacrificed when weight loss exceeded 20% of the initial starting weight. ( J ) Serum TNFα level was measured in C57BL/6 and Rag2-/- mice with PBS or 21h10 treatments. ( K ) Antitumor activity comparison on B16F10 melanoma between 21h10 and 21h10+aTNFα treatment groups.

Techniques: In Vivo, Ex Vivo, Adoptive Transfer Assay, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

( A ) Mice were treated as in , A and C, but on day 15 of tumor growth, tumors were processed for single-cell RNA-sequencing. UMAP of all samples are combined. ( B ) scRNAseq cluster composition average across treatment groups. ( C ) T cells in each scRNAseq sample from ( B ). Different colors for each bar indicate individual mice from each group. ( D ) Flow cytometry quantification of CD8 + T cell infiltration. ( E ) TRP1 high and TRP1 low melanoma antigen-specific cells captured by sequencing. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS groups. ( F ) T cells were sub-clustered from all samples from ( A ). The average T cell sub-cluster composition across treatment groups is shown on the right. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( G ) IFN-𝛾-response score total UMAP from ( A ) based on scaled and summed Hallmark IFN-𝛾-response genes for each cell. ( H ) Heatmap from pseudo-bulk differential gene expression analysis across T cell sub-clusters. Significant differences compared to PBS samples. ( I ) Similar to ( H ), but for TRP1 cells only, values from individual mice are shown. P-values displayed vs. PBS samples. ( J ) Flow cytometry quantification of CD8 + IFN-𝛾/granzyme B levels in endogenous or transferred CD8 + TRP1 high/low T cells. ( K ) CD4 + T cell sub-clustering UMAP and cluster composition. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( L ) Flow cytometry of IFN-𝛾 + CD4 + T cells. ( M ) Quantification and representative flow cytometry plots of Foxp3-GFP Treg cells. One-way ANOVA with Dunnett’s multiple comparisons for flow quantification and scRNA-seq total T cell comparison. For all dot plots with error bars: dots indicate individual mice, bars are S.E.M. *p ≤ 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001, or p-values displayed.

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Mice were treated as in , A and C, but on day 15 of tumor growth, tumors were processed for single-cell RNA-sequencing. UMAP of all samples are combined. ( B ) scRNAseq cluster composition average across treatment groups. ( C ) T cells in each scRNAseq sample from ( B ). Different colors for each bar indicate individual mice from each group. ( D ) Flow cytometry quantification of CD8 + T cell infiltration. ( E ) TRP1 high and TRP1 low melanoma antigen-specific cells captured by sequencing. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS groups. ( F ) T cells were sub-clustered from all samples from ( A ). The average T cell sub-cluster composition across treatment groups is shown on the right. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( G ) IFN-𝛾-response score total UMAP from ( A ) based on scaled and summed Hallmark IFN-𝛾-response genes for each cell. ( H ) Heatmap from pseudo-bulk differential gene expression analysis across T cell sub-clusters. Significant differences compared to PBS samples. ( I ) Similar to ( H ), but for TRP1 cells only, values from individual mice are shown. P-values displayed vs. PBS samples. ( J ) Flow cytometry quantification of CD8 + IFN-𝛾/granzyme B levels in endogenous or transferred CD8 + TRP1 high/low T cells. ( K ) CD4 + T cell sub-clustering UMAP and cluster composition. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( L ) Flow cytometry of IFN-𝛾 + CD4 + T cells. ( M ) Quantification and representative flow cytometry plots of Foxp3-GFP Treg cells. One-way ANOVA with Dunnett’s multiple comparisons for flow quantification and scRNA-seq total T cell comparison. For all dot plots with error bars: dots indicate individual mice, bars are S.E.M. *p ≤ 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001, or p-values displayed.

Techniques: RNA Sequencing Assay, Flow Cytometry, Sequencing, Expressing, Comparison

( 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.

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

( 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.

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.

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

( 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.

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.

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

( 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.

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.

Techniques: Expressing, Injection, Control, Flow Cytometry

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