Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A – C Female C57BL/6 mice ( n = 5) were inoculated with 5 × 10 5 MC38 cells. CD8 + T cells were sorted for single-cell RNA sequencing 19 days after tumor inoculation. A UMAP plot of 2276 CD8 + T cells exhibiting four cell subsets. B Expression of selected markers among CD8 + T cells by 2D UMAP visualization. C Heatmap plot showing average relative expression of selected markers in four CD8 + T cell subsets. D Female C57BL/6 mice ( n = 6) were inoculated with 1 × 10 6 MC38-OVA cells. The tumor tissues were collected 19 days after tumor inoculation. TIM3 expressions on Tetramer + CD8 + T cells in tumors from MC38-OVA tumor-bearing mice. E Female C57BL/6 mice ( n = 6) were inoculated with 5 × 10 5 MC38 cells. The tumor tissues were collected 19 days after tumor inoculation. Bcl-2 expressions on TIM3 - CD8 + T cells or TIM3 + CD8 + T cells in tumors from MC38 tumor-bearing mice. The proliferation and function of OT-I CD8 + T cells primed by spleen DC (sDC) or TIDC ( n = 3/group) were assessed using CFSE dilution ( F ) and IFN-γ production ( G ). H , I MC38-bearing Female C57BL/6 mice ( n = 7/group) were i.p. treated with 200 μg anti-TIM3 and/or 25 μg Fc-IFNα on day 9, 12 and 15 after tumor inoculation. Tumor volume ( H ) and mice weight ( I ) were measured as indicated. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by paired two-tailed t tests ( D , E ), unpaired two-tailed t tests ( F , G ) or One-way ANOVA with Tukey’s test ( H , I ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Single Cell, RNA Sequencing, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A Schematic diagram of TIM3-IFNα fusion protein in homodimer or heterodimer format. B – D MC38 tumor-bearing female C57BL/6 mice ( n = 11/group) were i.p. treated with BiDT (25 μg), or a mixture of Fc-IFNα and anti-TIM3 (12.5 μg + 12.5 μg), on day 13, 16 and 19 post inoculation. Mice weight ( B ) and tumor volume ( C ) were measured as indicated and the corresponding mouse survival curve was shown in ( D ). E MC38-OVA tumor-bearing female C57BL/6 mice ( n = 6/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 post inoculation. At day15 after treatment, splenocytes from different treatment groups were isolated and stimulated with either OT-I peptide or freeze-thawing MC38-OVA tumor cells. Antigen-specific T cells were detected by ELISpot assay. The splenocyte quantification data are shown in ( E ). F , G MC38-bearing female C57BL/6 mice were i.p. treated with 25 μg BiDT on day 13, 16 and 19 post inoculation. At 15 day after treatment, cured mice ( n = 5/group) were re-challenged with 2 × 10 6 MC38 tumor cells ( F ), 2 × 10 7 splenocytes from cured mice were i.v. transferred to MC38-bearing Rag1 −/− mice ( n = 5/group) 10 days after inoculation ( G ). Tumor volume was measured as indicated. H – J Female C57BL/6 mice ( n = 10/group) were subcutaneously inoculated with 3 × 10 5 B16F10 tumor cells and injected with 2 × 10 6 B16F10 tumor cells through the tail vein on day 10. The subcutaneous tumor was intratumorally treated with 5 μg BiDT on days 11, 14, and 17. The mice were euthanized on day 21 post inoculation. The lungs were collected ( H ). The subcutaneous B16F10 tumor growth curve ( I ). The number of B16F10 tumor metastatic nodes in the lung ( J ). Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by One-way ANOVA with Tukey’s test ( B , C ) or unpaired two-tailed t tests ( E – G , I , J ). Survival curve was compared using the log-rank test ( D ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Isolation, Enzyme-linked Immunospot, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A MC38-bearing female Rag1 −/− mice ( n = 6/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. Tumor volume was measured as indicated. B MC38-bearing female C57BL/6 mice ( n = 7/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. Anti-CD4 or anti-CD8 was administrated twice a week for 2 weeks. Tumor volume was measured as indicated. C MC38-bearing female C57BL/6 mice ( n = 7/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. Anti-CSF1R or anti-NK1.1 was administrated twice a week for 2 weeks. D , E Female C57BL/6 mice were lethally irradiated (10 Gy). Bone marrow was isolated from CD11c-DTR donor mice. Irradiated C57BL/6 mice as recipient were i.v. transferred with 2 × 10 6 CD11c-DTR bone marrow cells. CD11c-DTR chimeric mice were rested for 7 weeks and then were inoculated with MC38 cells. MC38-bearing CD11c-DTR chimeric mice ( n = 5/group) were i.p. treated with 25 μg BiDT on day 18, 21 and 24 after tumor inoculation. DT was administrated at 200 ng two days before treatment initiation and then every other day for 2 weeks ( D ). Tumor volume was measured as indicated ( E ). F MC38-bearing female C57BL/6 mice ( n = 7/group) were i.p. treated with 25 μg BiDT on day 13, 16 and 19 after tumor inoculation. FTY720 was administrated at 20 μg every other day for 2 weeks. G Female C57BL/6 mice were inoculated with 5 × 10 5 MC38 cells. The mice were euthanized on day 16 after tumor inoculation. 120 mm 3 MC38 tumor tissues were collected and s.c. transferred to Rag1 −/− mice ( n = 6/group). The subcutaneous tumor was intratumorally treated with 25 μg BiDT on days 0, 3, and 6 after tumor transplanting. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by One-way ANOVA with Tukey’s test ( B , C , E , F ) or unpaired two-tailed t tests ( A , G ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Irradiation, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A Female C57BL/6 mice ( n = 6/group) were inoculated with 1 × 10 6 Ifnar1 −/− MC38 cells and treated with 25 µg BIDT on day 13, 16 and 19 after tumor inoculation. Tumor volume was measured as indicated. B MC38-bearing female Ifnar1 −/− mice ( n = 6/group) were i.p. treated with 25 µg BiDT on day 10, 13 and 16 after tumor inoculation. C , D 3 × 10 6 CD3 + T cells from spleen of female C57BL/6 mice or Ifnar1 −/− mice were adoptively transferred to MC38-bearing Rag1 −/− mice ( n = 6/group) 2 days after tumor inoculation. 25 µg BiDT was i.p. administrated on day10, 13 and 16 after tumor inoculation ( C ). Tumor volume was measured as indicated ( D ). E Female Ifnar1 fl/fl mice and Zbtb46 - Cre Ifnar1 fl/fl mice ( n = 8/group) were inoculated with 5 × 10 5 MC38 tumor cells and i.p. treated with 25 µg BiDT on day 13, 16 and 19 after tumor inoculation. F Female Havcr2 fl/fl mice, Zbtb46 - Cre Havcr2 fl/fl mice or Lyz2 - Cre Havcr2 fl/fl mice ( n = 6/group) were inoculated with 5 × 10 5 MC38 tumor cells and i.p. treated with 25 µg BiDT on day 13, 16 and 19 after tumor inoculation. G Female Havcr2 fl/fl mice or Cd4-Cre Havcr2 fl/fl mice ( n = 6/group) were inoculated with 5 × 10 5 MC38 tumor cells and i.p. treated with 25 µg BiDT on day 13, 16 and 19 after tumor inoculation. H MC38-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16. Tumor tissues were collected 72 h after injection. The number of intratumoral total CD8 + T cells/TIM3 + CD8 + T cells and TIM3 - CD8 + T cells ( H ) were measured by FACS. I MC38-OVA tumor-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16. Tumor tissues were collected 72 h after injection. The number of total Tetramer + , TIM3 + Tetramer + CD8 + T cells and TIM3 - Tetramer + CD8 + T cells ( I ) were measured by FACS. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by unpaired two-tailed t tests ( A , B , H , I ) or One-way ANOVA with Tukey’s test ( D – G ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A MC38 tumor-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16. Tumor tissues were collected 72 h after injection. Expression of CD80 in tumor-infiltrating DCs was measured by FACS. B MC38-bearing female C57BL/6 mice ( n = 7/group) were treated with 25 μg BiDT on days 13, 16 and 19 after tumor inoculation. For blocking CD80/86, anti-CD80 and anti-CD86 were administrated twice a week for 2 weeks. C – G MC38 tumor-bearing female C57BL/6 mice ( n = 6/group) were treated with 25 µg BiDT on day 16 after tumor inoculation. For blocking CD80/86, anti-CD80 and anti-CD86 were administrated on day 16 after tumor inoculation. Tumor tissues were collected on day 19 after tumor inoculation. The percentage of active caspase3 + in TIM3 + CD8 + T cells ( C ), the percentage of Bcl-2 + in TIM3 + CD8 + T cells ( D ), the percentage of CD25 in total CD8 + T cells/TIM3 + CD8 + T cells and TIM3 - CD8 + T cells ( E ), the percentage of IL-2 + in TIM3 + CD8 + T cells ( F ), the percentage of IFN-γ + in TIM3 + CD8 + T cells ( G ) were measured by FACS. H MC38-bearing female C57BL/6 mice ( n = 7/group) were treated with BiDT, or BiDT + anti-CD122 on days 13, 16 and 19 after tumor inoculation. I The female C57BL/6 mice ( n = 6/group) were euthanized, and tumor tissues were collected 72 h after BiDT treatment. Expression of PD-L1 in DC was measured by FACS. J , K B16F10-bearing female C57BL/6 mice ( n = 10/group) were i.p. treated with 50 μg BiDT and/or 150 μg anti-PD-L1 on days 9, 12 and 15 after tumor inoculation. Tumor volume ( J ) was measured as indicated, and the mouse survival curve was shown in ( K ). L 30 days after treatment with BiDT and anti-PD-L1, cured B16F10-bearing female mice ( n = 5/group) were re-challenged with 2 × 10 6 B16F10 cells. Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by unpaired two-tailed t tests ( A , I , L ) or One-way ANOVA with Tukey’s test ( B – G , H , J ). Survival curve was compared using the log-rank test ( K ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Injection, Expressing, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Reactivating exhausted tumor-infiltrating T cells by a bispecific DC-T cell engager in mice

doi: 10.1038/s41467-026-70876-4

Figure Lengend Snippet: A Schematic diagram of Pro-BiDT. B , C MC38-bearing female C57BL/6 mice ( n = 6/group) were treated i.p. with equal molar amounts (600 pmol) of BiDT or Pro-BiDT on days 13, 16 and 19 post-inoculation. Tumor volumes ( B ) and Body weights ( C ) were measured as indicated. D – F MC38-bearing female mice ( n = 6/group) were treated i.p. with equal molar amounts (600 pmol) of BiDT or Pro-BiDT on day 13 after tumor inoculation. Blood samples were collected 24 h after the first injection. Serum MCP-1 levels were measured by cytometric bead assay ( D ). Complete blood count was measured ( E , F ). G B16F10-bearing female C57BL/6 mice ( n = 6/group) were i.p. treated with equal molar amounts (600 pmol) of BiDT or Pro-BiDT on days 8, 11 and 14 post-inoculation. Tumor volumes were measured as indicated ( G ). Data are shown as mean ± SEM and are representative of at least two independent experiments. P value was determined by One-way ANOVA with Tukey’s test ( B – G ). Source data are provided as a Source Data file.

Article Snippet: MC38 cell line was purchased from Cytion (#305223).

Techniques: Injection

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: C57BL/6 mice (UNTR n=7, RT n=9) were injected subcutaneously with 6x105 MC38 cells in matrigel:PBS (1:2). Fifteen days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Tumors were analyzed nine days after radiotherapy. (B) Tumor size (length x breadth) was measured by caliper. (C) Tumor weight. (D) Flow cytometry gating strategy for intratumoral CD8+ T-cells. (E) Number of CD8+ T-cells per gram of tumor. (F) Percentage of CD62L, PD-1, PD-1 and TIM-3 expressing CD8+ T-cells. (G) Percentage of GZMB-expressing CD8+ T-cells. (H) Percentage of Ki-67 expressing CD8+ T-cells. (I) Uniform manifold approximation and Projection (UMAP) representation of CD8+ T-cell clusters. (J) The heatmap illustrates the relative expression of key markers across different clusters. (K) Percentage of UMAP clusters 4, 7 and 11. Each symbol represents one mouse. The bar indicates the mean ± SD. Groups were statistically compared using the Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: C57BL/6 mice (UNTR n=8 per group, RT n=10 per group) were injected subcutaneously with 8x105 MC38-OVA cells in matrigel:PBS (1:2). Fifteen days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, CD4 or CD8 cells were depleted by intraperitoneal injection of 500 µg of anti-CD4 (GK1.5) or anti-CD8 (YTS169.4), followed by a second dose 12 days after radiotherapy. (B) Depletion validation was performed two days after antibody injection by measuring percentages of CD4+ and CD8+ cells among CD45+ cells in the blood. (C) Tumor size (length x breadth) was measured by caliper. Each symbol represents one mouse. The bar indicates the mean ± SD. Groups were statistically compared using one-way ANOVA with Tukey’s multiple comparison correction. (D) Kaplan-Meier survival curves. Groups were statistically compared using the Mantel-Cox test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Comparison

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: C57BL/6 mice (RT+FTY720 n=10, the rest n=9 per condition) were injected subcutaneously with 8x105 MC38-OVA cells in matrigel:PBS (1:2). Fifteen days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, some groups received 20 µg FTY720 per os every second day until the endpoint. Tumors were analyzed nine days after radiotherapy. (B) Tumor size (length x breadth) was measured by caliper. (C) Tumor weight (D) Number of CD8+ T-cells per gram of tumor. (E) Percentage of CD62L, PD-1, PD-1 and TIM-3 expressing CD8+ T-cells. (F) Percentage of GZMB and Ki-67 expressing CD8+ T-cells and SIINFEKL-specific CD8+ T-cells, determined by H-2Kb OVA tetramer. (G) Percentage of GZMB and Ki-67 expressing SIINFEKL-specific CD8+ T-cells. The bar indicates the mean ±SD. Groups were statistically compared using one- way ANOVA with Tukey’s multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Expressing, Comparison

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: C57BL/6 mice (n=8 per condition) were injected subcutaneously with 8x105 MC38-OVA cells in matrigel:PBS (1:2). Fifteen days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, some groups received 20 µg FTY720 per os every second day until the endpoint. Tumors were analyzed nine days after radiotherapy. (B) Tumor size (length x breadth) was measured by caliper. (C) Tumor weight (D) Number of CD8+ T- cells per gram of tumor. (E) Number of PD-1, PD-1 and TIM-3 expressing CD8+ T-cells per gram of tumor. (F) Number of GZMB and Ki-67 expressing CD8+ T-cells and SIINFEKL-specific (determined by H-2Kb OVA tetramer) CD8+ T-cells per gram of tumor, determined by H-2Kb OVA tetramer. The bar indicates the mean ± SD. Groups were statistically compared using one-way ANOVA with Tukey’s multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Expressing, Comparison

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: C57BL/6 mice (n=9 per condition) were injected subcutaneously with 8x105 MC38-OVA cells in matrigel:PBS (1:2). Fifteen (d3) or seventeen (d1) days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, all groups received 20 µg FTY720 per os every second day until the endpoint. Tumors were analyzed one or three days after radiotherapy. (B) Tumor weight. (C) Number of CD8+ T-cells per gram of tumor. (D) Experimental setup: C57BL/6 mice (UNTR n=11, the rest n=10 per condition) were injected subcutaneously with 8x105 MC38-OVA cells in matrigel:PBS (1:2). Fifteen (d9) or eighteen (d6) days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, all groups received 20 µg FTY720 per os every second day until the endpoint. Tumors were analyzed six or nine days after radiotherapy. (E) Tumor size (length x breadth) was measured by caliper. (F) Tumor weight (G) Number of CD8+ T-cells per gram of tumor. (H) Percentage of CD62L, PD-1, PD-1 and TIM-3 expressing CD8+ T-cells. (I) Percentage of GZMB and Ki-67 expressing CD8+ T-cells and SIINFEKL-specific CD8+ T-cells, determined by H-2Kb OVA tetramer. The bar indicates the mean ±SD. Groups were statistically compared using one-way ANOVA with Tukey’s multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Expressing, Comparison

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: C57BL/6 mice (UNTR+FTY720 n=7, the rest n=8 per condition) were injected subcutaneously with 8x105 MC38-OVA cells in matrigel:PBS (1:2). Fifteen days later, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, all groups received 20 µg FTY720 per os every second day until the endpoint. Six days after radiotherapy, tumor samples were hashed and CD8+ T-cells were sorted using flow cytometry. Samples were prepared using the BD Rhapsody platform and sequenced on the Illumina NovaSeq X. (B) UMAP representation of annotated CD8+ T-cell subsets, determined by ProjectTILs package. (C) Pie chart showing frequencies of CD8+ T-cell subsets per condition. (D) UMAP representation of stem-like CD8+ T-cell clusters. (E) Heatmap illustrates signature scores (determined by UCell package) across different stem-like CD8+ T-cell clusters. (F) Percentage of UMAP clusters 1 and 3. (G) Percentage of proliferating stem-like CD8+ T-cells (defined by a G1S or G2M score >0.1), determined by UCell package. (H) Mki67 expression shown as log-normalized counts. The bar indicates the mean ±SD. Groups were statistically compared using one-way ANOVA with Tukey’s multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Flow Cytometry, Expressing, Comparison

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: Tcf7DTR-GFP→C57BL/6 mice (UNTR, RT n=8, UNTR+DTX n=7, RT+DTX n=9) bone marrow transplant was performed as described (22685313). After 8 weeks, the mice were injected subcutaneously with 8x105 MC38-OVA-GFP cells in matrigel:PBS (1:2). One day before tumor injection and one day before radiotherapy, the remaining host T-cells were depleted by a double intraperitoneal injection of 500 µg of anti-Thy1.2 (30H12). Fifteen days after tumor injection, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, all groups received 20 µg FTY720 per os every second day, some groups received 250 ng diphtheria toxin (DTX) intraperitoneal injection twice per week, whereas the other groups received PBS until the endpoint. Tumors were analyzed nine days after radiotherapy. (B) Tumor size (length x breadth) was measured by caliper. (C) Tumor weight (D) Number of CD8+ T-cells per gram of tumor. (E) Number of PD-1 and PD-1 and TIM-3 expressing CD8+ T-cells per gram of tumor. (F) Number of GZMB and Ki-67 expressing CD8+ T-cells, SIINFEKL-specific (determined by H-2Kb OVA tetramer) and p15-specific (determined by H-2Kb p15E tetramer) CD8+ T-cells per gram of tumor. Each symbol represents one mouse. The bar indicates the mean ±SD. Groups were statistically compared using one-way ANOVA with Tukey’s multiple comparison correction. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Expressing, Comparison

Journal: bioRxiv

Article Title: Pre-existing intratumoral stem-like CD8 + T-cells drive radiotherapy-induced tumor control

doi: 10.1101/2024.10.16.618635

Figure Lengend Snippet: (A) Experimental setup: Tcf7DTR-GFP→Tcrb-/- mice (UNTR n=4, the rest n=11 per condition) bone marrow transplant was performed. After 8 weeks, the mice were injected subcutaneously with 8x105 MC38-OVA-GFP cells in matrigel:PBS (1:2). Fifteen days after tumor injection, some groups received radiotherapy (RT; 20 Gy) or not (UNTR). Starting 1 day before radiotherapy, all groups received 20 µg FTY720 per os every second day. Some groups received 250 ng diphtheria toxin (DTX) intraperitoneal injection twice per week, the rest received PBS until the endpoint. (B) Bone marrow transplantation success was evaluated by measuring Thy1.1+ (left) and GFP+ (right) cells among CD8+ cells in the blood one day before tumor injection. (C) FTY720 validation was performed by measuring the percentage of CD3+ cells among CD45+ cells in the blood one day after radiotherapy. (D) Depletion of TCF-1+ (GFP+) cells was validated by measuring the percentage of GFP+ cells among CD8+ cells in the spleen upon sacrifice. (E) Tumor size (length x breadth) was measured by caliper. Each symbol represents one mouse. The bar indicates the mean ± SD. Groups were statistically compared using one-way ANOVA with Tukey’s multiple comparison correction. (F) Kaplan-Meier survival curves. Groups were statistically compared using the Mantel-Cox test. (G) Pie chart showing radiotherapy responders (tumor size below 50 mm2 at day 50) and non-responders. **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: MC38 colon adenocarcinoma was obtained from Kerafast, LLC and B16F10 melanoma cells were obtained from ATCC.

Techniques: Injection, Transplantation Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE-induced activation of T cells and cytotoxic effect of T cell-mediated cancer cells. (A, B) The cell viability was performed using the CCK-8 assay. Splenocytes were isolated from hPD-L1 MC38 cell-bearing hPD-1 knockin mice. Murine CRC hPD-L1 MC38 cells (A) and hPD-1 mice splenocytes (B) were treated with SRE for 72 hours. (C) Cocultured hPD-L1 MC38 cell viability tested by crystal violet staining; (D) Lactate dehydrogenase (LDH) released by damaged cells, detected via LDH cytotoxicity assay; (E) Relative interleukin-2 (IL-2) level, determined using the mouse IL-2 ELISA set. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Article Snippet: The hPD-L1 MC38 tumor-infiltrating CD8 + T cells was purified by immunomagnetic negative selection (#19853, STEMCELL Technologies, Inc., Vancouver, Canada).

Techniques: Activation Assay, CCK-8 Assay, Isolation, Knock-In, Staining, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE elevated the activation of hPD-1 + CD8 + T cells and the CD8 + T cell-mediated killing effect on hPD-L1 MC38 cancer. (A) Cocultured hPD-L1 MC38 cell viability, tested by crystal violet staining. Cocultured hPD-L1 MC38 cells detected with fluorescence microscopy (× 200) (B) and determined by fluorescent-activated cell sorting analysis (C) . (D) LDH released from damaged cells; (E) Relative perforin 1 (PRF1) level, determined with use of the mouse PRF1 ELISA kit. Data are presented as the mean ± SD. ** p < 0.01 and *** p < 0.001 compared to the vehicle group.

Article Snippet: The hPD-L1 MC38 tumor-infiltrating CD8 + T cells was purified by immunomagnetic negative selection (#19853, STEMCELL Technologies, Inc., Vancouver, Canada).

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, FACS, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: Sanguisorbae Radix extract reduced tumor growth in the hPD-L1 MC38 cell allograft hPD-1 mouse model. (A) Body weight (grams); (B) Tumor volume after 18 days; (C) Tumor weight after 18 days; (D) Images of tumor tissues (bar indicates 5 mm); (E) hPD-L1 MC38 tumor-bearing mice 18 days after treatment; (F) Representative microscopic images (×400) of CD8 and PRF1-positive area of tumor tissues calculated using immunohistochemical analysis. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the vehicle group.

Article Snippet: The hPD-L1 MC38 tumor-infiltrating CD8 + T cells was purified by immunomagnetic negative selection (#19853, STEMCELL Technologies, Inc., Vancouver, Canada).

Techniques: Immunohistochemical staining, Standard Deviation

Journal: JCI Insight

Article Title: Multipanel mass cytometry reveals anti–PD-1 therapy–mediated B and T cell compartment remodeling in tumor-draining lymph nodes

doi: 10.1172/jci.insight.132286

Figure Lengend Snippet: (A) MC38 tumor cells were injected subcutaneously at day 0 (D0) in the right hind limb of C57BL/6 mice, and either isotype (ISO) or PD-1 antibody (PD-1) was administered intraperitoneally on D14 and D18. Right inguinal lymph nodes were harvested on D21 for analysis. Lymph nodes at the same location from normal, non–tumor-bearing mice (NL) were used as additional control comparators. (B) Lymph nodes were weighed on D21 upon harvest, and (C) absolute number of cells was counted from each lymph node. Mean ± SD (n = 5) are shown for both plots. (D) Schematic shows how lymph nodes from each respective group (Normal LN, non–tumor-bearing normal mice; tdLN Isotype, isotype-treated tumor-bearing mice; tdLN PD-1, PD-1–antibody–treated tumor-bearing mice) were barcoded with a specific CD45 antibody tagged with a unique metal to be multiplexed and stained with a T or B cell subtyping mass cytometry panel. Results for repeated-measures ANOVA followed by pairwise testing are shown as FDR-adjusted *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.005.

Article Snippet: MC38 (Kerafast) cells were maintained in DMEM-based medium containing 10% FBS, 1% l -glutamine, 100 U/mL penicillin/streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (Life Technologies) in 5% CO 2 at 37°C.

Techniques: Injection, Staining, Mass Cytometry

Journal: EMBO Molecular Medicine

Article Title: Unexpected PD‐L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies

doi: 10.15252/emmm.202012716

Figure Lengend Snippet: A Chi‐G4S‐DR5 stable 4T1 cell were analyzed for DR5 expression using clinical tigatuzumab antibody in flow cytometry assay. B Chi‐G4S‐DR5 stable 4T1 tumors were treated with either single antibodies (IgG1, CD8 cells depleting anti‐CD8a, avelumab, lexatumumab) or in combinations (avelumab + lexatumumab or avelumab + lexatumumab+ anti‐CD8a). 6, 100 μg dose of each antibodies was injected, and tumors were harvested and imaged at same time (See Fig for tumor weight quantitation). C Chi‐G4S‐DR5 stable 4T1 tumors were treated with IgG1, lexatumumab, and lexatumumab in combination of either avelumab (anti‐PD‐L1) or BMS202, a PD‐1 inhibitor. After six doses, tumors were harvested and imaged at same time. D Quantitation of average tumor weight as shown in (C). E Chi‐G4S‐DR5 stable 4T1 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab + ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS–PAGE followed by immunoblotting using indicated CD8, CD4, Foxp3, and granzyme‐b antibody. GAPDH is loading control. F FACS histogram showing binding of anti‐mouse DR5 antibody (MD5‐1) and anti‐mouse cross‐reactive clinical PD‐L1 antibody (avelumab) to 4T1 cells. G FACS histogram showing binding of anti‐mouse DR5 antibody (MD5‐1) and anti‐mouse cross‐reactive clinical PD‐L1 antibody (avelumab) to MC38 cells. H Cell killing assay of MC38 cells treated with murine DR5 agonist MD5‐1 and bispecific avelu‐MD5 antibody. Data information: Mean ± SD. Statistical significance in (D) was determined using two‐tailed Mann–Whitney test (* P < 0.05).

Article Snippet: To establish chimeric G4S DR‐5 stable MC38 tumor (Chi‐G4S‐DR5), first 5 × 10 5 Chi‐G4S‐DR5 MC38 cells subcutaneously injected along with Matrigel in the right flank of 7‐ to 8‐week‐old female C57Bl/6 mice and allowed for tumor growth.

Techniques: Expressing, Flow Cytometry, Injection, Quantitation Assay, Control, SDS Page, Western Blot, Binding Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Unexpected PD‐L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies

doi: 10.15252/emmm.202012716

Figure Lengend Snippet: Chi‐G4S‐DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, lexatumumab + ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Mouse tumors were collected at 100–200 mm 3 & embedded in O.C.T. to make blocks. Tumor sections were stained with antibodies (CD8, CD4, FoxP3) and counter‐stained with hematoxylin as described in Methods section. One representative tumor from Fig ( n = 3) was processed for IHC studies after indicated treatments. Multiple images were taken from IHC samples, and one representative image is shown for each treatment. Since tumors were almost negative against FoxP3, a marker of regulatory T cells (see Figs and ), animal spleen tissue was processed for IHC analysis as a positive control. Images were acquired at 50 × magnification for each tumor sample, and scale bar at the bottom of image is 20 μm.

Article Snippet: To establish chimeric G4S DR‐5 stable MC38 tumor (Chi‐G4S‐DR5), first 5 × 10 5 Chi‐G4S‐DR5 MC38 cells subcutaneously injected along with Matrigel in the right flank of 7‐ to 8‐week‐old female C57Bl/6 mice and allowed for tumor growth.

Techniques: Control, Staining, Marker, Positive Control

Journal: EMBO Molecular Medicine

Article Title: Unexpected PD‐L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies

doi: 10.15252/emmm.202012716

Figure Lengend Snippet: A Schematic and genetic construction of two chimeric human‐mouse DR5 (Chi‐DR5, Chi‐G4S‐DR5) with human extracellular domain and mouse transmembrane (TM) and intracellular domains (ICD). B–D FACS plots confirming expression of human DR5 (huDR5) and Chi‐G4S‐DR5 in mouse 4T1 cells. Chi‐DR5 was not expressed on cell surface (C). E Cell viability analysis of huDR5 and Chi‐G4S‐DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 ( n = 3). F Comparison of tumor growth of grafted huDR5 and Chi‐G4S‐DR5 stable 4T1 cells in BALB/c mice ( n = 3). G Chi‐G4S‐DR5 stable 4T1 tumors (after reaching ~150–200 mm 3 ) were treated with four doses (100 μg) of IgG1, lexatumumab, lexatumumab + avelumab followed by tumor recovery and surface PD‐L1 analysis using flow cytometry similar to described in Fig ( n = 3–4). See also Appendix Fig . H Chi‐G4S‐DR5 stable 4T1 tumors (after reaching ~100 mm 3 ) were treated with six doses of indicated treatment. Isolated tumors were imaged together ( n = 4–5). ROCK1i indicates GSK269962A and was administered to animals via intra‐tumor injections (2 mg/kg). I Same as H, except average tumor weight is shown ( n = 4). J, K Confirmation of selective CD8 + T cell population depletion in mouse spleen and grafted tumors after injecting animals with either anti‐PD‐L1 avelumab alone or avelumab + anti‐CD8a antibody. CD4 + T cells remained unchanged. L Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i, and anti‐CD8 + lexatumumab + ROCK1i ( n = 4–6, 50 μg lexatumumab, 2 mg/kg ROCK1i, six doses). ROCK1i indicates GSK269962A and was administered to animals via intra‐tumor injections. Various treatments were started when tumors were ~100 mm 3 size. Also see Fig . M Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab + avelumab, and anti‐CD8 + lexatumumab + avelumab ( n = 4–6). N 6‐ to 8‐week‐old C57BL/6‐bearing Chi‐G4S‐DR5‐expressing MC38 tumors were intraperitoneally (i.p.) injected with 50 μg of indicated antibody every third day ( n = 4–6). Various treatments were started when tumors were ~100 mm 3 size. Tumor volumes were quantified at indicated days by caliper measurements. O Same as H, except KMTR2 (8 doses) antibody instead of lexatumumab was used as the DR5 agonist for the experiment. P Average of tumor weights of data shown in (O). Q Kaplan–Meier plot depicting the survival of syngeneic Chi‐G4S‐DR5 4T1 tumor‐bearing animals injected i.p. with 100 μg of indicated antibodies such as IgG1, KMTR2, and avelumab. Animals were injected with GSK269962A 2 mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated. Data information: Mean ± SD. Statistical significance in (E), (G), (I), (L), (M), and (P) was determined by unpaired two‐tailed t ‐test and in (N) using two‐tailed paired Wilcoxon–Mann–Whitney test ( n = 4–6; * P < 0.05, ** P < 0.005, *** P < 0.0001, **** P < 0.00005). Source data are available online for this figure.

Article Snippet: To establish chimeric G4S DR‐5 stable MC38 tumor (Chi‐G4S‐DR5), first 5 × 10 5 Chi‐G4S‐DR5 MC38 cells subcutaneously injected along with Matrigel in the right flank of 7‐ to 8‐week‐old female C57Bl/6 mice and allowed for tumor growth.

Techniques: Expressing, Comparison, Flow Cytometry, Isolation, Injection, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Unexpected PD‐L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies

doi: 10.15252/emmm.202012716

Figure Lengend Snippet: A Chi‐G4S‐DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab + ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100 μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2 mg/kg dose (6 total). Various treatments were started when tumors were ~400 mm 3 size. Harvested tumors were grouped together and size‐matched (3 independent sets: n = 2–6 tumors in each set) followed by TIL isolation (see methods). CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry. The data shown are from a single set of experiment. For addition sets, see also Appendix Fig 0 ( n = 3). B Plots showing % of total double positive TILs (CD8 + CD45 + + CD4 + CD45 + ) in right upper quadrant after combining three independent experiments. Indicated treatments are shown at the bottom of bars ( n = 3). C Ratio of CD8 + CD45 + /CD4 + CD45 + isolated TILs from tumors in each indicated treatment ( n = 3). D Similar to (A) Chi‐G4S‐DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab + ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS–PAGE followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase‐3, and granzyme‐b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase‐3 p19 and p17 fragments. For additional Chi‐G4S‐DR5 stable 4T1 tumors western data, see Fig . E Schematic of anti‐PD‐L1 (avelumab) and anti‐muDR5 (MD5‐1) IgG1 antibodies. Highlighted blue area depicts avelumab’s antigen binding variable domain, and dark red area indicates MD5‐1’s antigen binding variable domain. F Schematic and genetic construction of avelu‐MD5‐1 bispecific antibody that contains anti‐PD‐L1 (avelumab) and anti‐muDR5 (MD5‐1) heavy/light (VH/VL) variable domains (blue and dark red), respectively. Both monospecific and bispecific antibodies contain LALA mutation to avoid interference with Fc‐effector function. CK: C‐kappa, CH1: Constant heavy chain 1, CH2: Constant heavy chain 2, CH3: Constant heavy chain 3. Dark blue dotted line under CH3 and between MD5‐1 VH and VL depicts flexible linker. G Working mechanism of avelu‐MD5‐1 bispecific antibody where surface stabilized PD‐L1 acts as an anchor to enhance avidity optimized binding and clustering of DR5 receptor‐mediated apoptotic signaling. H Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5‐1 and bispecific avelu‐MD5 antibody. I BALB/c mouse harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50 μg dose and mice were imaged after five doses. J 6‐ to 8‐week‐old C57BL/6 mouse bearing MC38 tumors were intraperitoneally (i.p.) injected with 50 μg of indicated antibody every third day ( n = 4–6). Indicated treatments were started when tumors were ~100 mm 3 size. Tumor volumes were quantified at indicated days by caliper measurements. K ~150–200 mm 3 size MC38 tumor‐bearing C57BL/6 mice were treated with indicated MD5‐1, avelumab and bispecific antibodies along with control IgG1, six total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS–PAGE followed by immunoblotting using indicated CD8, CD4, caspase‐3, and granzyme‐b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase‐3 p19 and p17 fragments. L 6‐ to 8‐week‐old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150–200 mm 3 , animals were intraperitoneally (i.p.) injected with 50 μg of indicated antibody every third day. On day 18, tumors were harvested, size‐matched and pooled by treatment group, and exposed to collagenase/DNase and were single‐cell suspensions enriched for CD8 + cells. Enriched CD8 + T cells from various treatments were restimulated with anti‐CD3 (OKT3) antibody for four additional hours. CD8‐gated cells were next analyzed for IFN‐γ intracellular expression using flow cytometry. The data shown are from a single set of experiment. See also Appendix Fig ( n = 3). M Percentage of IFN‐γ + CD8 + double positive cells from three independent experiments. For supporting flow cytometry data, see also Appendix Fig 1 ( n = 4). Data information: Mean ± SD. Statistical significance in (B) and (C) was determined by Mann–Whitney two‐tailed test and in (J) using two‐tailed paired Wilcoxon–Mann–Whitney test. Statistical significance in (M) was determined by unpaired t ‐test (* P < 0.05, ** P < 0.005). Source data are available online for this figure.

Article Snippet: To establish chimeric G4S DR‐5 stable MC38 tumor (Chi‐G4S‐DR5), first 5 × 10 5 Chi‐G4S‐DR5 MC38 cells subcutaneously injected along with Matrigel in the right flank of 7‐ to 8‐week‐old female C57Bl/6 mice and allowed for tumor growth.

Techniques: Injection, Isolation, Expressing, Flow Cytometry, Control, Quantitation Assay, SDS Page, Western Blot, Binding Assay, Mutagenesis, Luciferase, MANN-WHITNEY, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Unexpected PD‐L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies

doi: 10.15252/emmm.202012716

Figure Lengend Snippet:

Article Snippet: To establish chimeric G4S DR‐5 stable MC38 tumor (Chi‐G4S‐DR5), first 5 × 10 5 Chi‐G4S‐DR5 MC38 cells subcutaneously injected along with Matrigel in the right flank of 7‐ to 8‐week‐old female C57Bl/6 mice and allowed for tumor growth.

Techniques: Transformation Assay, Generated, Expressing, Knock-Out, Ubiquitin Proteomics, Plasmid Preparation, Endotoxin Assay, Transfection, Modification, Sterility, Protease Inhibitor, Bioassay, Software, Recombinant