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