Journal: Journal for immunotherapy of cancer

Article Title: Depletion of myeloid-derived suppressor cells sensitizes murine multiple myeloma to PD-1 checkpoint inhibitors.

doi: 10.1136/jitc-2024-008979

Figure Lengend Snippet: Figure 2 The composition of immunosuppressive cells in TME differs between Vk*MYC-bearing and 5TGM1-bearing mice. CyTOF analysis showing the representative t-SNE displays of protein expression in the BM of (A) tumor-free or Vk*MYC- bearing MM mice or (B) Vk*MYC-bearing or 5TGM1-bearing MM mice. (C) PD-L1 expression on CD138+ plasma cells in the BM of tumor-free (TF), Vk*MYC-bearing or 5TGM1-bearing mice (n=4–5). (D) Percentages of CD3+ T cells, LAG3+ PD-1+ CD8+ T cells in the BM of mice intravenously inoculated with 2×106 5TGM1-luc cells and treated with intraperitoneal injections of control IgG or αPD-1 on day 14 and day 17. Analysis was done on day 18 after the tumor injection (n=5). (E) Percentages of CD3+ T cells and LAG3+ PD-1+ CD8+ T cells in the BM of mice intravenously inoculated with 0.5×106 Vk*MYC cells and treated with intraperitoneal injections of control IgG or αPD-1 on day 14 and day 17. Analysis was done on day 18 after the tumor injection (n=6). (F) CyTOF analysis showing the representative t-SNE display of cell composition in the BM of Vk*MYC-bearing or 5TGM1-bearing mice (n=3). Data are presented as mean±SD. *P<0.05; **p<0.01; ***p<0.001. BM, bone marrow; CyTOF, cytometry by time of flight; MM, multiple myeloma; NK, natural killer; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand 1; TME, tumor microenvironment; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: C57BL/6J, LysmCre×Csf1rLsL- DTR C57BL/6 mice (MMDTR mice), Foxp3 DTR mice (C57BL/6- Tg (Foxp3- HBEGF/ EGFP)23.2Spar/Mmjax), CD4 KO mice (B6.129S2Cd4tm1Mak/J), and CD8 KO mice (B6.129S2Cd8atm1Mak/J) were purchased from the Jackson laboratory.

Techniques: Expressing, Clinical Proteomics, Control, Injection, Cytometry, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Depletion of myeloid-derived suppressor cells sensitizes murine multiple myeloma to PD-1 checkpoint inhibitors.

doi: 10.1136/jitc-2024-008979

Figure Lengend Snippet: Figure 7 CD8+ T cells are required for the restored PD-1 inhibitor response induced by MDSCs depletion in Vk*MYC MM. (A) WT or CD8−/− C57BL/6J mice were inoculated intravenously with 0.5×106 Vk*MYC cells and randomly received intraperitoneal injections of irrelevant peptides or peptibodies on day 10, followed by the treatment with IgG or αPD-1. (B) Serum IgG levels were measured by ELISA on day 9 and day 18 after tumor cell inoculation (n=5). (C) Kaplan-Meyer survival plots of treated mice (n=5–7). (D–E) PMN-MDSCs (CD45+ CD11b+ Ly6G+ Ly6C−) and M-MDSCs (CD45+ CD11b+ Ly6G− Ly6C+) were sorted out from Vk*MYC tumor-bearing (MM) or tumor-free mice and cocultured with tumor-free mouse-derived CD8+ T cells stimulated by CD3/CD28 antibodies. After a 72-hour coculture, the proliferation (CFSE) inhibition and cytokine (IFNγ and TNFα) secretion of CD8+ T cells were determined. (F) C57BL/6J mice were inoculated intravenously with 0.5×106 Vk*MYC cells or PBS, and the percentage of CD84+ PMN-MDSCs and CD84+ M-MDSCs in the BM of tumor-free or Vk*MYC MM-bearing mice. (G) CD84+ MDSCs and CD84– MDSCs were sorted out, cocultured with splenocytes at a ratio of (MDSCs: splenocytes) 1:4 and treated with αPD-1. After a 72-hour coculture, the percentages of intracellular IFNγ and TNFα positive CD8+ T cells were determined. Data are presented as mean±SD. *P<0.05; **p<0.01; ns, not significant. BM, bone marrow; CFSE, carboxyfluorescein diacetate succinimidyl ester; IFNγ interferon γ; MDSCs, myelood-derived suppressor cells; MM, multiple myeloma; PD-1, programmed cell death protein 1; PMN, polymorphonuclear; TNFα, tumor necrosis factor α.

Article Snippet: C57BL/6J, LysmCre×Csf1rLsL- DTR C57BL/6 mice (MMDTR mice), Foxp3 DTR mice (C57BL/6- Tg (Foxp3- HBEGF/ EGFP)23.2Spar/Mmjax), CD4 KO mice (B6.129S2Cd4tm1Mak/J), and CD8 KO mice (B6.129S2Cd8atm1Mak/J) were purchased from the Jackson laboratory.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Inhibition

Journal: Journal for immunotherapy of cancer

Article Title: Depletion of myeloid-derived suppressor cells sensitizes murine multiple myeloma to PD-1 checkpoint inhibitors.

doi: 10.1136/jitc-2024-008979

Figure Lengend Snippet: Figure 8 Expressions of MDSCs and cytotoxic CD8+ T cells signature genes are negatively correlated in human patients with MM. (A) Analyses of gene-profiling data of CD138– BM aspirate cells from GSE104171. Heatmap shows a negative correlation between the cytotoxic CD8+ T cell signature genes and MDSCs signature genes in patients with MM. (B) Analyses of gene- profiling data from GSE136324 show the expressions of CD38, the ratio of cytotoxic CD8+ T cell to PMN-MDSCs signature genes, and the ratio of cytotoxic CD8+ T cell to M-MDSCs signature genes in pretreated and treated post-maintenance patients with MM. Data are presented as mean±SD. *P<0.05; ***p<0.001. BM, bone marrow; MDSCs, myeloid-derived suppressor cells; MM, multiple myeloma; PMN, polymorphonuclear.

Article Snippet: C57BL/6J, LysmCre×Csf1rLsL- DTR C57BL/6 mice (MMDTR mice), Foxp3 DTR mice (C57BL/6- Tg (Foxp3- HBEGF/ EGFP)23.2Spar/Mmjax), CD4 KO mice (B6.129S2Cd4tm1Mak/J), and CD8 KO mice (B6.129S2Cd8atm1Mak/J) were purchased from the Jackson laboratory.

Techniques: Derivative Assay

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: (a) Siglec-15 mRNA relative levels in human tissues and immune cell subsets from BioGPS database. A dash line was added to indicate the flow cytometry detection threshold (verified by Siglec-15 negative staining on CD8 T cells). Data are mean ± s.e.m. (tissues, n = 2; monocyte, n = 6; macrophage, n = 10; other immune subsets, n = 4 samples). (b) Siglec-15 mRNA expression in mouse tissues was tested by RT-PCR, quantified by ImageJ software (NIH) and normalized to the levels of reference gene GAPDH. The 293T-cells overexpressing Siglec-15 were used as a positive control. E7, day 7 embryos.

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Flow Cytometry, Negative Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Software, Positive Control

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: ( a , b ) Quantitative PCR (Q-PCR) estimation on Siglec-15 mRNA levels in human macrophages derived from CD14 + peripheral blood monocytes with 100ng/ml M-CSF ( a ), or LPS-treated mouse BMDMs or BMDCs ( b ) at indicated time points. Data are mean ± s.e.m. after normalization to reference gene GAPDH ( a ), or RPL13a ( b ), and representative of two independent experiments. ( c ) Flow cytometry analysis of Siglec-15 expression by anti-Siglec-15 mAb (clone m03) staining on mouse myeloid cell subsets from blood, spleen, bone marrow (BM) or peritoneal cavity of S15KO and WT mice. MΦ , macrophage. Data are representative of three independent experiments. ( d ) Human macrophages were generated from CD14 + peripheral blood monocytes from two healthy donors (D#1 and D#2) with M-CSF for 7 days. Alternatively, monocytes were cultured with M-CSF for 4 days followed by M-CSF + IFN-γ for 3 more days. Siglec-15 expression was analyzed by flow cytometry with anti-Siglec-15 (clone 1H3) or an isotype control mAb staining.Data are representative of two independent experiments. ( e - g ) The % of divided human peripheral CD8 + ( e ) or CD4 + T-cells ( f ) as indicated by CFSE dilution, as well as IFN-γ in the culture medium ( g ) after stimulation with 0.1 μg/ml of anti-CD3 in the presence of 5 μg/ml human Siglec-15 fusion protein (hS15-hIg) or control (hIg) for 3 days. ( e and f , n = 6 cell cultures; g , n = 3 cell cultures from the same donor) ( h , i ) OT-I T-cells pre-activated with OVA 257-264 (1×10 5 /well) were co-cultured with irradiated 293T-K b OVA cells stably expressing Siglec-15 (293T-K b OVA-S15 + ) or mock (293T-K b OVA-Control) (2×10 4 /well) in a 96-well plate. OT-I T-cell proliferation was determined by 3 H-thymidine incorporation at 72 hrs ( h ). The cytokine levels in the culture medium were analyzed at 48 hrs ( i ). (n = 4 cell cultures from the same mouse) ( j ) The % of divided OT-I T-cells as indicated by CFSE dilution after co-cultured for 3 days with S15KO or WT peritoneal macrophages pulsed with OVA 257-264 at the indicated concentrations. (n = 3 cell cultures) In e - j , data are presented as mean ± s.e.m. and representative of two or three independent experiments. P values by two-tailed unpaired t -test. See also - .

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Flow Cytometry, Expressing, Staining, Generated, Cell Culture, Control, Irradiation, Stable Transfection, Two Tailed Test

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: (a) The effect of plate-coated hSiglec-15-hIg or control hIg (5 μg/ml) on human T-cell proliferation in the presence of plate-coated anti-human CD3 mAb at the indicated concentrations. Proliferation of T-cells was indicated by 3H-thymidine incorporation at 72 hrs. (b) The effect of plate-coated mSiglec-15-mIg or control mIg (5 μg/ml) on mouse splenic T-cell proliferation in the presence of plate-coated anti-mouse CD3 mAb (1 μg/ml). Proliferation of T-cells was indicated by 3H-thymidine incorporation at 72 hrs. (c, d) The effect of soluble mSiglec-15-mIg or control mIg (5 μg/ml) on mouse splenic CD8+ T-cells in the presence of coated anti-mouse CD3 mAb (1 μg/ml). The cell proliferation as indicated by CSFE dilution (c) and IFN-γ in the culture medium (d) at 72 hrs are shown. (e) The 293T-K b OVA-S15+ or S15 negative control cells were placed in a 384-well plate at 1×104/well for 24 hrs, followed by the addition of OT-I (1×104/well) pre-activated with OVA 257-264 . Real-time survival of target cells was monitored by the xCELLigence cellular impedance assay (left panel) and normalized by the value right before adding OT-I cells (normalized cell index). Data at 72 hrs are shown as a bar in the right panel. All data above are mean ± s.e.m. (n = 3 or 4 cell cultures) and representative of two independent experiments. P values by two-tailed unpaired t-test.

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Control, Negative Control, Two Tailed Test

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: (a) Tissue histological analysis was performed on 18-month-old Siglec-15 KO mice and WT littermate and is shown as the pathological score (see Materials and Methods). The indicated tissues were fixed in formalin, embedded with paraffin, and stained with hematoxylin and eosin. The inflammatory status of tissues was evaluated based on a semi-quantitative method that describes the level of immune infiltration. Data are presented as mean. (b-d) The body (b), spleen (c) and liver (d) weight of Siglec-15 KO and WT mice. Data are presented as mean ± s.d. (e, f) The levels of anti-dsDNA IgG antibodies (e) and anti-nuclear antibodies (ANA) (f) in sera of 18-month-old Siglec-15 KO and WT mice were quantified by specific sandwich ELISA. Data are presented as mean ± s.e.m. In a-f, data are analyzed by two-tailed unpaired t-test (WT n = 27 mice; KO n = 34 mice; n.s., not significant). (g) Gating strategy for OT-I T-cell EdU incorporation and apoptosis analysis by flow cytometry. (h) Siglec-15 KO or WT BMDCs pulsed with OVA 257-264 peptide were injected i.p. into WT mice at 5×105/mouse followed by i.p. injection of OT-I T-cells at 2×106/mouse 6 hrs later. The OT-I in the blood at the indicated time-points were analyzed by flow cytometry. The results are shown as the percentage of OT-I among total CD8+ T-cells. Data are mean ± s.e.m. (n= 5 mice per group). Data are analyzed by two-way ANOVA.

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Staining, Sandwich ELISA, Two Tailed Test, Flow Cytometry, Injection

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: Splenic cells from OT-I/Rag-1 KO mice were injected i.v. into WT, Siglec-15 KO or LysM-Cre KO mice on day −1. On day 0, mice were immunized i.p. with 100 μg OVA 257-264 peptide plus 100μg poly(I:C). OT-I T-cells in blood on day 4 ( a ) and in spleen on day 5 ( b ) were analyzed by flow cytometry with H-2K b OVA 257-264 tetramer (OT-I tetramer) and CD8 mAb staining. Representative flow cytometry analysis and quantification of OT-I T-cells among total CD8 + T-cells are shown. Data are mean ± s.e.m. (n = 3 mice per group) and representative of three independent experiments. P values by two-tailed unpaired t -test. In some experiments, WT and KO were fed with EdU at 0.8mg/ml in drinking water from day 0 of immunization. On day 5, the % of EdU + OT-I T-cells ( c ) and Annexin V + OT-I T-cells in the spleen ( d ) were analyzed by flow cytometry. Data are mean ± s.e.m. (n = 5 mice per group) and representative of two independent experiments. P values by two-tailed unpaired t -test (n.s., not significant; P = 0.5566). ( e , f ) The kinetics of OT-I T-cells in the blood ( e ) and IL-10 levels in the plasma ( f ) of WT, Siglec-15 KO and LysM-Cre KO mice after OVA 257-264 /poly(I:C) immunization are shown. Data are mean ± s.e.m. (WT n = 4 mice; KO or LysM-Cre KO n=3 mice) and representative of three independent experiments. P values by two-way ANOVA (n.s.; P = 0.1475). ( g ) WT and KO mice were treated with 200 μg anti-IL-10 mAb or isotype control mAb daily after OT-I T-cell transfer. The % of OT-I T-cells among total CD8 T-cells in blood at day 5 is shown. Data are mean ± s.e.m. (control, n = 3 mice per group; α-IL-10, n = 5 mice per group). P values by two-tailed unpaired t -test (n.s., not significant, P = 0.1581). See also .

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Injection, Flow Cytometry, Staining, Two Tailed Test, Clinical Proteomics, Control

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: ( a, b ) B16-GMCSF tumor cells at 1.5×10 6 were injected s.c. into Siglec-15 WT and KO mice. Tumor incidence and growth in individual mouse ( a ) and percentage of survival ( b ) are shown. (WT, n = 21 mice; KO, n =17 mice; results were pooled from two independent experiments). P values by two-sided Log-rank test. ( c-e ) Mass cytometry analysis of tumor-infiltrating leukocytes at day 14 after B16-GMCSF tumor inoculation. t-SNE plot of tumor infiltrating leukocytes overlaid with color-coded clusters ( c ). Heatmap displaying normalized marker expression of each immune cluster ( d ). Frequency of clusters of indicated immune cell subsets ( e ). Data are mean ± s.e.m. (n = 3 mice per group). P values by two-tailed unpaired t -test. (f-h) The ex vivo function analysis of B16-GMCSF tumor-infiltrating T-cells or myeloid cells. T-cells ( f ) or myeloid cells ( g , h ) were isolated from B16-GMCSF tumors from WT and KO mice at day 14. The % of IFN-γ and TNF-α producing T-cells were analyzed by intracellular staining after 4 hrs re-stimulation with PMA and ionomycin ( f ). CD11b + myeloid cells were co-cultured with CSFE-labeled naïve splenic CD8 + T-cells stimulated with anti-CD3. T-cell proliferation ( g ) and cytokine production ( h ) was analyzed at 48 hrs. Data are mean ± s.e.m. (n = 4 mice per group) and representative of two independent experiments. P values by two-tailed unpaired t -test. See also - .

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Injection, Mass Cytometry, Marker, Expressing, Two Tailed Test, Ex Vivo, Isolation, Staining, Cell Culture, Labeling

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: (a-c) Mass cytometry analysis of tumor-infiltrating leukocytes isolated at day 14 after B16-GMCSF tumor cell inoculation as described in (n = 3 mice per group). t-SNE plot of tumor infiltrating leukocytes overlaid with the expression of indicated markers (a). Density t-SNE plots of an equal number of CD45+ tumor-infiltrating leukocytes in Siglec-15 KO and WT mice (b). The normalized expression value (mean mass intensity) of checkpoint receptors on tumor-infiltrating CD8+ T-cells (c). (d, e) On day 14 after B16-GMCSF tumor cell inoculation, spleens and lymph nodes (LN) from WT and KO mice were dissected (d). The percentage of CD4+ and CD8+ T-cells in the draining and non-draining lymph nodes (LN) was analyzed by flow cytometry (e). Data are mean ± s.e.m. (n = 4 mice per group). P values by two-tailed unpaired t-test. (f) B16-GMCSF tumor cells were injected s.c. into Siglec-15 WT and KO at 1.5×10 6 /mouse. Mice were treated with 200μg anti-CD8 antibody every 3 to 4 days since 3 days before tumor inoculation. Tumor growth was measured regularly and is shown as the mean tumor diameter ± s.e.m. (n=5 mice per group). P values by two-way ANOVA (n.s., not significant; P = 0.9372).

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Mass Cytometry, Isolation, Expressing, Flow Cytometry, Two Tailed Test, Injection

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: (a, b) GL261-luc cells were injected i.c. into Siglec-15 WT, KO or LysM-Cre KO mice at 4×10 5 /mouse. Mice were subsequently treated with a 4Gy whole brain radiation on day 4. Tumor volume in mice was measured by the IVIS imaging system every 4 to 5 days. Tumor growth in individual Siglec-15 WT or KO mice (left) and imaging at day 13 and 18 after tumor inoculation (right) are shown in (a) (n=10 mice per group). Data are representative of two independent experiments. The GL261-luc tumor growth in Siglec-15 WT, KO and LysM-Cre KO mice is mean bioluminescence in radiance ± s.e.m. over time (b) (WT, n = 10 mice; KO, n = 10 mice; LysM-Cre KO, n=8 mice). P values by two-tailed Mann-Whitney test. (c-e) Flow cytometry analysis of tumor-infiltrating immune cells at day 14 after GL261 tumor inoculation (n=4 per group). CD8+ T-cells, CD4+ T-cells, CD11b+ CD45high macrophages (MØ), CD11b+ CD45low microglia, and CD11c+ dendritic cells (DC) in brain (c) or spleen (d) were quantified by flow cytometry. Brain mononuclear cells were further re-stimulated with irradiated GL261-luc cells for 5 days. Total number of IFN-γ-producing CD8+ T-cells and CD4+ T-cells was determined by live cell counting and intracellular staining (e). Data are mean ± s.e.m. (n = 4 mice per group) and representative of two independent experiments. P values by two-tailed unpaired t-test (n.s., not significant; c, P = 0.1937; d, P = 0.0916 and 0.0624; e, P = 0.4820).

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Injection, Imaging, Two Tailed Test, MANN-WHITNEY, Flow Cytometry, Irradiation, Cell Counting, Staining

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: (a) Binding of PE-labeled α-S15 (5G12) to 293T cells overexpressing human or mouse Siglec-15. 293T parental cells served as controls. Data are representative of three independent experiments. (b, c) Human PBMCs were stimulated by coated OKT3 (0.1 μg/mL) in 96-well plates for 3 days in the presence of 5 μg/ml hS15-hIg or control hIg with or without α-S15 at 12 μg/ml. The proliferation of CD4+ T-cell (b) and CD8+ T-cell (c) was indicated by CFSE dilution. Data are mean ± s.e.m. (n = 6 cell cultures) and representative of three independent experiments. P values by two-tailed unpaired t-test. (d) B16-GMCSF tumor cells were s.c. injected into WT C57BL/6 mice at 1.5×10 6 /mouse and subsequently treated with 200 μg α-S15 or isotype control mAb at day 5, 9, 13 and 17 (n= 7 mice per group). P values by two-tailed unpaired t-test. (e) MC38 tumor cells (3×105) mixed with or without WT or KO BMDMs (2×105) were s.c. injected into C57BL/6 mice (n= 5 mice per group). P values by two-way ANOVA (n.s., not significant; P = 0.4920). (f) MC38 tumor cells (3×105) mixed with Siglec-15 KO BMDMs (2×105) were s.c. injected into C57BL/6 mice and subsequently treated with 200 μg α-S15 or isotype control mAb at day 5, 9, 13 and 17 (n= 7 mice per group). P values by two-way ANOVA (n.s.; P = 0.9727). (g) CT26 tumor cells (1.5×105) mixed with Balb/c BMDMs (1.5×105) were s.c. injected into Balb/c mice and subsequently treated with 200 μg α-S15 or isotype control mAb as described in the methods (n= 10 mice per group). Data are representative of two independent experiments. P values by two-way ANOVA. (h, i) On day 15 after CT26 tumor inoculation as described in (g), tumor infiltrating CD8+ T-cells (h) and CT26 tumor-specific CD8+ T-cells (i) were stained with anti-CD8 mAb and AH1 dextramer+ and analyzed by flow cytometry (control, n = 5 mice; α-S15, n = 3 mice). P values by two-tailed unpaired t-test. (j) CT26 tumor cells (1.5×105) mixed with Balb/c BMDMs (1.5×105) were s.c. injected into Balb/c mice. Mice were treated with 200 μg α-S15 or isotype control mAb and/or 100 μg anti-PD-1 mAb as described in the methods (n= 10 mice per group). P values by two-way ANOVA. In d-j, data are presented as mean ± s.e.m. (k) Expression of Siglec-15 on transduced MC38 cells (MC38-S15+) or parental cells (MC38-WT) as determined by staining with m03 mAb or control antibody and flow cytometry analysis. Data are representative of three independent experiments. (l) OT-I T-cells from OT-I/Rag-1 KO mice were injected i.v. into C57BL/6 mice that are subsequently immunized with OVA 257-264 peptide and adjuvant as described in . Spleen cells were isolated on day 5 and stained by mouse Siglec-15 recombinant fusion protein or by control Ig for flow cytometry analysis. Data are shown in a histogram as specific binding to OT-I T-cells gated by anti-CD8 mAb and OT-I tetramer positive staining. Data are representative of two independent experiments.

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Binding Assay, Labeling, Control, Two Tailed Test, Injection, Staining, Flow Cytometry, Expressing, Adjuvant, Isolation, Recombinant

Journal: Nature medicine

Article Title: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

doi: 10.1038/s41591-019-0374-x

Figure Lengend Snippet: ( a ) The % of divided OT-I T-cells (1×10 5 /well) was analyzed by CFSE dilution after co-culture with irradiated 293T-K b OVA-S15 + or control cells (2×10 4 /well) in a 96-well plate for 5 days with 10 μg/ml Siglec-15 antibody (α-S15) or isotype control mAb (Control). ( b ) The % of divided OT-I T-cells (2×10 5 /well) was analyzed by CFSE dilution after co-culture for 3 days with S15KO or WT BMDMs (2×10 4 /well) pulsed with 0.1ng/ml OVA 257-264 in the presence of 10 μg/ml α-S15 or isotype control antibody. In a and b , data are mean ± s.e.m. (n = 3 cell cultures). P values by two-tailed unpaired t -test. ( c ) MC38 tumor cells (3×10 5 ) mixed with WT BMDMs (2×10 5 ) were s.c. injected into WT C57BL/6 mice. Mice were treated with 200μg α-S15 or control antibody from day 5, every 4 days for 4 doses in total. Data are mean ± s.e.m. (n = 5 mice per group). P values by two-way ANOVA. ( d , e ) Naïve splenic CD8 + T-cells isolated from PD-1 KO mice were labeled with CFSE and co-cultured with WT BMDMs in the presence of anti-CD3 and α-S15 or control antibody. T-cell proliferation ( d ) and cytokine production ( e ) was analyzed at 72 hrs. Data are mean ± s.e.m. (n = 3 or 4 cell cultures). P values by two-tailed unpaired t -test. ( f , g ) IL-2 ( f ) and TNF-α ( g ) production from human PBMCs after stimulation with anti-CD3 and SEB in the presence of α-S15, α-PD-1 (Nivolumab) or their control antibodies for 72hrs. Data are mean ± s.e.m. (n = 5 cell cultures). P values by two-tailed unpaired t -test (n.s., not significant; P = 0.1759). ( h ) MC38-S15 + cells were s.c. injected into WT C57BL/6 mice (4×10 5 /mouse). Mice were treated with 200μg α-S15 or control antibody from day 6, every 4 days for 4 doses in total. Data are mean ± s.e.m. (n = 5 mice per group). P values by two-way ANOVA. ( i ) MC38-S15 + cells were i.v. injected into WT C57BL/6 mice (1×10 5 /mouse). Mice were treated with 400 μg α-S15 or control antibody from day 2, every 4 days for 6 doses in total. Lungs were harvested on day 28 and tumor nodules were counted. Data are mean ± s.e.m. (n = 8 mice per group). P values by two-tailed unpaired t -test. All data are representative of two or three independent experiments. See also .

Article Snippet: OT-I were purified from spleen of OT-I/Rag-1 KO mice using a CD8 + T-cell isolation kit (StemCell, Vancouver, Canada) by negative selection.

Techniques: Co-Culture Assay, Irradiation, Control, Two Tailed Test, Injection, Isolation, Labeling, Cell Culture

Journal: Cancer Research

Article Title: GFER Represents a Target for Dual Disruption of Redox Homeostasis and Reactivation of the Immune Response in Pancreatic Adenocarcinoma

doi: 10.1158/0008-5472.CAN-24-4211

Figure Lengend Snippet: Immunogenicity increases upon GFER depletion in PDAC cells. A–E, Gene set enrichment analysis of RNA-seq data reveals the upregulation of the IFNα response ( A ), IFNγ response ( B ), TNFα signaling via NFκB ( C ), inflammatory response ( D ), and IL2–STAT5 signaling ( E ) pathways in GFER-depleted PATC 124 cells infected with sgGFER. A total of 100,000 PATC cells were cultured in six-well plates for 7 days and then measured. F, RNA-seq analysis demonstrates significant changes in gene expression in orthotopic tumor samples derived from 10,000 KPC (p53 mut) cells infected with nontargeting shRNA ( n = 2) as well as shGFER (shGFER#4, n = 2; shGFER#5, n = 3). Tumors were harvested when all tumors were of comparable size. Immune system–related genes are highlighted in red boxes. G, Protein levels of chemokines ( CXCL9, CXCL10 , and CXCL11 ) related to CD8 + T-cell infiltration in orthotopic shCTRL and shGFER KPC (p53 mut) tumors. Statistical analysis was performed using a two-tailed Student unpaired t test. Data represent the mean ± SD; n = 4 mice. H and I, Immunophenotyping of orthotopic shCTRL and shGFER KPC tumors. A total of 10,000 KPC cells were injected into the pancreas, and tumors were harvested for multicolor flow cytometry analysis. The percentage of CD8 + T cells, CD4 + T cells, NK cells (NK1.1+ cells), B cells (B220+ cells), macrophages (F4/80+ cells), and dendritic cells (DC; CD11c + cells) were identified in CD45 + cells ( H ). The percentage of PD-1–positive and granzyme B–positive cells were identified in CD8 + and CD8 + PD-1 + cells, respectively ( I ). Fibroblasts were identified as CD45 − EpCAM − αSMA + cells ( J ). Statistical analysis was performed using a two-tailed Student unpaired t test. Data represent the mean ± SD; n = 5 mice. K, Kaplan–Meier survival analysis of C57BL/6 mice orthotopically implanted with 10,000 KPC (p53 mut) cells infected with shCTRL or shGFER. Mice received either anti-CD8 antibody (10 mg/kg) or rat IgG2a starting 1 day prior to surgery and then intraperitoneally every 7 days for four cycles. Animals were euthanized when tumor volume reached ∼2000 mm 3 . Survival curves were analyzed by one-way ANOVA ( n = 10 per group). L, Growth curves of 10,000 KPC (p53 mut) cells subcutaneously implanted into C57BL/6 mice. Prior to implantation, KPC cells were infected with shCTRL or shGFER. Tumor-bearing mice were treated with IgG control or anti–PD-1 antibodies. Tumor volume was measured every 4 days for 36 days. Statistical analysis was performed using one-way ANOVA. Data represent the mean ± SD; n = 4–7 per group. M, Pancreatic cancer patient with anti–PD-1/anti-CD40 treatment prior to chemotherapy were selected from the PRINCE trial and divided in to GFER-high and -low groups. The overall survival analysis was done with log-rank test ( N = 17).

Article Snippet: We used 8-week-old male or female nude mice (Strain #002019, The Jackson Laboratory) and immunocompetent C57BL/6J mice (Strain #000664, The Jackson Laboratory) and CD8 knockout (KO) mice (Strain #002665, The Jackson Laboratory) to establish subcutaneous and orthotopic models, with n = 3–10 mice per treatment group.

Techniques: Immunopeptidomics, RNA Sequencing, Infection, Cell Culture, Gene Expression, Derivative Assay, shRNA, Two Tailed Test, Injection, Flow Cytometry, Control

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: Increases in PRMT5 expression were found to be linked to adverse prognosis in cervical cancer. ( A ) PRMT5 expression was conducted in both healthy cervical tissues and tumor tissues obtained from the TCGA database (num (T) = 306; num (N) = 13). ( B ) Correlation between PRMT5 expression and immune cells from TCGA database (ssgsea test). ( C ) The analyses were performed to investigate the association among high/low PRMT5 expression levels and T cells/CD8 + T cells/Macrophages/NK/DC in cervical cancer patients derived from TCGA database. ( D ) The relationship between PRMT5 expression and T cells, CD8 + T cells, Macrophages, NK, DC using the TCGA database (ssgsea test). OS ( E ), DSS ( F ) and PFI ( G ) analyses were conducted on cervical cancer patients with high/low levels of PRMT5 using data from the TCGA database. The data represent the mean ± SEM. Blue represents low PRMT5 expression, red represents high PRMT5 expression. ns = no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Expressing, Derivative Assay

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: Disruption of PRMT5 suppressed less tumor growth in CD8 KO mice. 6-week-old female C57BL/6 mice and CD8 KO mice received subcutaneous injections of either control cells or U14 cells with PRMT5 knockdown ( n = 5 per group). ( A – C ) The mice were euthanized, and the tumors that were resected were subsequently analyzed on day 16 after inoculation. ( A ) The tumor growth curve observed on the mice is depicted in a line graph. On day 16, images ( B ) and weight measurements ( C ) of the excised tumor were recorded. ( D ) A survival curve was generated for tumor-bearing mice that received subcutaneous injections of control and U14 cells with PRMT5 knockdown. The data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Disruption, Control, Knockdown, Generated

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: Disruption of PRMT5 enhanced CXCL10 secretion by tumor cells. ( A ) Venn analysis comparing control cells and U14 cells with PRMT5 knockdown using RNA-seq. ( B ) Comparison of chemokine expression profiles between control cells and U14 cells with PRMT5 knockdown analyzed through RNA-seq. ( C ) Real-time PCR experiments were conducted to examine CCL5, CCL11, CCL4, CXCL9, and CXCL10 expression in control cells and U14 cells with PRMT5 knockdown. ( D ) CXCL10 level was measured by ELISA. ( E ) Correlation between CXCL10 expression and immune cell populations was assessed using the Kruskal–Wallis test from TCGA database. ( F ) Analysis of high PRMT5 expression enrichment compared to low PRMT5 expression in T cells and CD8 + T cells of cervical cancer patients from TCGA database. Blue represents low CXCL10 expression, red represents high CXCL10 expression. Correlation between CXCL10 expression and T cells ( G )/CD8 + T cells ( H ) was evaluated using the Kruskal–Wallis test from TCGA database. The data represent the mean ± SEM. ns = no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Disruption, Control, Knockdown, RNA Sequencing, Comparison, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: PRMT5 regulated CD8 + T cells recruitment through CXCL10/CXCR3 axis. Control cells and PRMT5 knockdown U14 cells were subcutaneously injected into 6-week-old female C57BL/6 mice and CXCR3 KO mice. Mice were euthanized at day 18 after inoculation ( n = 4 per group). The tumor single cell suspension was prepared and analyzed by flow cytometry. ( A ) A line graph shows the tumor growth curve of mice. Images ( B ) and weight ( C ) of the resected tumor at day 18 after inoculation. ( D ) The percentage of CD4 + and CD8 + T cells in CD45 + cells. The data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Control, Knockdown, Injection, Suspension, Flow Cytometry

Journal: Biomolecules

Article Title: Enhancing CD8 + T Cells Infiltration Through the Protein Arginine Methyltransferase 5 (PRMT5)/CXCL10 Axis Restricts Cervical Cancer Progression

doi: 10.3390/biom15121717

Figure Lengend Snippet: EPZ015666 effectively suppressed cervical cancer growth by enhancing intracellular cytokine expression in T cells. ( A – I ) 6-week-old female C57BL/6 mice received daily intraperitoneal injections of EPZ015666 (150 mg kg −1 ) 3 days after inoculation of U14 cells ( n = 5 per group). ( A ) The growth curve of tumors in the mice is illustrated by a line graph. On day 12 post-inoculation, images ( B ) and weight measurements ( C ) of the excised tumors were collected. ( D ) Analysis was conducted to determine PRMT5 expression and SDMA levels within the tumor. ( E ) The proportion of CD4 + and CD8 + T cells among CD45 + cells was determined. ( F ) IFN-γ, TNF-α and granzyme B expression levels were assessed in CD8 + T cells. ( G ) PD-1, TIM-3 and LAG-3 expression levels were revealed through surface examination on CD8 + T cells. ( H ) IFN-γ, TNF-α and Foxp3 expression levels were examined in CD4 + T cells. ( I ) PD-1, TIM-3 and LAG-3 expression levels was assessed on CD4 + T cells. The data represent the mean ± SEM. * p < 0.05 and *** p < 0.001.

Article Snippet: B6.129P2-Cd8atm1Mak/J (CD8 KO) mice and B6.129P2-Cxcr3 tm1Dgen /J (CXCR3 KO) mice were acquired from Jackson Laboratory.

Techniques: Expressing

Journal:

Article Title: A Mage3/Heat Shock Protein70 DNA Vaccine Induces both Innate and Adaptive Immune Responses for the Antitumor Activity

doi: 10.1016/j.vaccine.2009.09.119

Figure Lengend Snippet: Mice were immunized i.m. with 100 μg of different DNA constructs twice at a two-week interval. Data were mean +/− s.d. of triplicate wells. A, Splenocytes were harvested and incubated with TC-1/Mage3 cell lysate for 20 hr. The frequency of T cells producing IFN-γ was determined by using Elispot. B, Specificity of splenocytes from pc-sMage3Hsp-immunized mice responding to TC-1/Mage3 vs. parental TC-1 cells, and Mage3 protein vs. irrelevant CD20 protein was determined by using Elispot. C, Response of CD4+ T cells to Mage3-pulsed DCs was determined by using Elispot. D, CTL response of splenocytes to TC-1/Mage3 was determined by using a 51Cr-release assay.

Article Snippet: Six- to eight-week-old female C57BL/6 mice, CD4 + knockout (KO) mice, and CD8 + KO mice were purchased from Harlan Sprague Dawley, Taconic, and Jackson Laboratory, respectively.

Techniques: Construct, Incubation, Enzyme-linked Immunospot, Release Assay

Journal:

Article Title: A Mage3/Heat Shock Protein70 DNA Vaccine Induces both Innate and Adaptive Immune Responses for the Antitumor Activity

doi: 10.1016/j.vaccine.2009.09.119

Figure Lengend Snippet: A, Expression and secretion of Mage3 from pc-Mage3 or pc-sMage3-transfected 293 T cells, tested by western blotting (the upper panel) or ELISA (the lower panel). B. Antibody reponse from pc-Mage3 or pc-sMage3 immunized mice evaluated by ELISA. C. CD4+ and CD8+ T cell responses from pc-Mage3 or pc-sMage3 immunized mice evaluated by intracellular IFN-γ staining. The data are the representative of the two independent experiments.

Article Snippet: Six- to eight-week-old female C57BL/6 mice, CD4 + knockout (KO) mice, and CD8 + KO mice were purchased from Harlan Sprague Dawley, Taconic, and Jackson Laboratory, respectively.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal:

Article Title: A Mage3/Heat Shock Protein70 DNA Vaccine Induces both Innate and Adaptive Immune Responses for the Antitumor Activity

doi: 10.1016/j.vaccine.2009.09.119

Figure Lengend Snippet: A C57BL/6 wild-type, CD4+ KO, or CD8+ KO mice were inoculated with newly generated TC-1/Mage3 cells. Three days later, the mice were immunized by i.m. injection with 100 μg of pc-sMage3Hsp DNA or PBS twice at a one-week interval.. The percentage of tumor-free mice was calculated on the indicated days. B. C57BL/6 wild-type, CD4+ KO, or CD8+ KO mice were immunized by i.m. injection with 100 μg of pc-sMage3Hsp DNA twice at a two-weeks interval. Two weeks after the second injection, the mice were inoculated s.c. with in vitro maintained TC-1/Mage3 tumor cells. The percentage of tumor-free mice was calculated on the indicated days. C. C57BL/6 wild-type and CD8+ KO mice were immunized by i.m. injection with 100 μg of pc-Mage3Hsp DNA twice at a two-week interval. Two weeks after the second injection, the mice were inoculated s.c. with TC-1/Mage3 tumor cells. The CD8+ KO mice and a half of the C57BL/6 wild-type mice received PK136 treatment prior to and after TC-1/Mage3 challenge. The remaining half of wild-type mice received control IgG administration. The percentage of tumor-free mice was calculated on the indicated days. D. C57BL/6 mice were immunized by i.m. injection with 100 g of pc-Mage3Hsp DNA twice at a two-week interval. Two weeks after the second injection, the mice were inoculated s.c. with TC-1 tumor cells. Half of mice received PK136 treatment, and half received control IgG administration. The percentage of tumor-free mice was calculated on the indicated days. E. C57BL/6 mice were immunized by i.m. injection with 100 μg of pc-sHsp, pc-Mage3Hsp DNA or PBS twice at a two-week interval. Two weeks after the second injection, the mice were inoculated s.c. with the newly generated TC-1/Mage3 tumor cells. Half of pc-sHsp-immunized mice and all pc-sMage3Hsp-immunized mice received PK136 treatment. The percentage of tumor-free mice was calculated on the indicated days.

Article Snippet: Six- to eight-week-old female C57BL/6 mice, CD4 + knockout (KO) mice, and CD8 + KO mice were purchased from Harlan Sprague Dawley, Taconic, and Jackson Laboratory, respectively.

Techniques: Generated, Injection, In Vitro, Control

Journal: Journal of Clinical Investigation

Article Title: Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance

doi: 10.1172/jci64742

Figure Lengend Snippet: Figure 2 Immunity to the cancer-driving oncogene following virus-induced activation. (A) The amount of TAg-specific IgG antibodies was determined in serum obtained from B6 (n = 4) and LoxP-TAg mice (Tg) 3 (n = 14) and 20 weeks (n = 7) after i.v. injection of Ad.Cre (1 × 109 PFUs). Bars indicate mean values. As controls, LoxP-TAg mice were i.v. injected with 1 × 109 PFUs of Ad.Luc (n = 4). LoxP-TAg mice analyzed 20 weeks after Ad.Cre injection had macroscopically visible tumors (see Figure 1, B and D). (B) LoxP-TAg mice develop TAg-specific antibodies of IgG1, IgG2a, and IgG2b isotypes upon Ad.Cre-mediated TAg activation. Amounts of TAg-specific IgG1, IgG2a, IgG2b, and IgG3 were determined in serum obtained from individual mice 3 and 20 weeks after Ad.Cre application. IgG3 was not detectable in any serum sample (not shown). Each number represents an individual mouse. LTB, large tumor bearing. (C) CD4+ T cell–deficient (Cd4–/– × LoxP-TAg; n = 6) and CD8+ T cell–deficient mice (Cd8–/– × LoxP- TAg; n = 4) received 1 × 109 PFUs of Ad.Cre and were monitored for HCC development. Ad.Cre-treated T cell–competent LoxP-TAg mice (WT × LoxP-TAg; n = 5) served as controls. (D) Ad.Cre-treated Rag2–/–cg–/– × LoxP-TAg mice (n = 7) were monitored for HCC development. LoxP-TAg mice (n = 6) served as control. (E) TAg-tolerant Vil-Cre × LoxP-TAg mice (n = 6) were injected with 1 × 109 PFUs of Ad.Cre and monitored for HCC development. LoxP-TAg mice (n = 9) served as controls. (C–E) Time after adenovirus infection is given.

Article Snippet: Rag2–/– mice (B6.129S6-Rag2tm1Fwa) and Rag2–/–cg–/– mice (C57BL/6J × C57BL/10SgSnAi)-[KO]γc-[KO]Rag2) were purchased from Taconics Farms; Cd4–/– mice (B6.129S2-Cd4tm1Mak/J) and Cd8–/– mice (B6.129S2-Cd8atm1Mak/J) were purchased from The Jackson Laboratory; B6 mice were obtained from Charles River; and CD45.1 congenic mice were bred in our animal facilities.

Techniques: Virus, Activation Assay, Injection, Control, Infection