Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: a B220 + cells and Th17 cells in the BM niche in WT mice and BCR-ABL tTA mice ( n = 3 mice per group) were subjected to immunofluorescence staining. Representative images were shown. b The numbers of Th17 cells migrating to culture medium or to leukemia cells treated with or without 50 ng/ml rhIL-17A were determined by cell counting and FCS analysis. c Real-time PCR analysis of the relative mRNA levels of chemokine genes in SupB15 (left) and BV173 cells (right) treated with rhIL-17A (100 ng/ml) or PBS control. d The human IL-5, CXCL5, CCL5 and CXCL16 levels in the culture supernatant of SupB15 cells treated with or without rhIL-17A (100 ng/ml) were measured by ELISA. e The CXCL16 concentrations in the serum of BCR-ABL tTA mice and WT mice ( n = 5 mice per group) were quantified with a Mouse Chemokine Assay Q1 (Raybiotech). f The CXCL16 concentrations in the serum of HDs ( n = 7 samples) and primary Ph + B-ALL patients ( n = 14 samples) were measured by ELISA. g Schematic strategy for studying the differentiation of Th17 cells with or without rmCXCL16 stimulation in vitro. h Representative FACS plots and quantification of the percentage of Th17 cells in naïve CD4 + T cells with or without rmCXCL16 treatment. i Schematic strategy for studying the migration of Th17 cells cocultured with leukemia cells with or without anti-CXCL16 mAb treatment in vitro. j , k Flow cytometric analysis of the percentage of Th17 cells in the lower chambers of the inserts ( j ) and proliferation activity of leukemia cells ( k ) in a coculture system with or without anti-CXCL16 treatment. The gating strategy for isolating Th17 cells is shown in Supplementary Fig. . (b–d , h , j , k) n = 3 independent experiments. Statistical significance was calculated by ( c – f , j , k ) two-tailed Student’s t test; ( b , h ) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Immunofluorescence, Staining, Cell Counting, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vitro, Migration, Activity Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: Primary Ph + B-ALL cells were treated with or without rhIL-17A for 24 h, and relative CXCL16 mRNA level ( a ) and CXCL16 secretion ( b ) were evaluated by real-time PCR and ELISA, respectively. c Representative immunofluorescence images of B220 (red) and CXCL16 (green) staining in spleen tissue sections of WT mice and BCR-ABL tTA mice treated with or without 50 µg/kg rmIL-17A for a duration of 3 weeks (twice a week) ( n = 3 mice per group). d Flow cytometric analysis and quantification of CXCL16 expression in primary mouse B-ALL cells treated with or without rmIL-17A. e The protein levels of p65 and p-p65 in the cytoplasm and p-p65 in the nucleus of primary mouse leukemia cells treated with or without rmIL-17A were measured by Western blotting. f Immunofluorescence of p-P65 in primary B-ALL cells treated with or without rhIL-17A. g The effect of rhIL-17A treatment on NF-kB transcriptional activity. HEK 293T cells were transfected with a synthetic NF-kB luciferase reporter construct (pNF-kB-Luc) for 12 h and then treated with different concentrations of rhIL-17A for 24 h. NF-kB transcriptional activity was detected by a luciferase assay. h ChIP‒qPCR analyses of the binding of NF-kB to the CXCL16 promoter region in SupB15 cells treated with or without rhIL-17A. i – k The effect of BAY11-7082, rIL-17A or BAY11-7082 in combination with rIL-17A on the cytoplasmic and nuclear protein levels of p65, p-p65, and CXCL16 in primary Ph + B-ALL cells. i The indicated protein band intensities were quantified using ImageJ software. The relative CXCL16 mRNA level ( j ) in primary Ph + B-ALL cells and CXCL16 protein level ( k ) in the supernatant of primary Ph + B-ALL cells were measured by RT‒PCR and ELISA, respectively. (a, b , d – k ) n = 3 independent experiments. Statistical significance was calculated by ( a , b , d ) two-tailed Student’s t-test; ( e , g – k ) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing, Western Blot, Activity Assay, Transfection, Luciferase, Construct, Binding Assay, Software, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: a Flow cytometric analysis of the percentages of B220 dim CD19 + cells in BM, spleens, LNs and PB of mice with secondary transplantation treated with or without rmCXCL16 ( n = 6 mice per group). b – d Representative images of Wright-Giemsa-stained PB smears ( b , top), H&E staining of spleens ( b , bottom), spleens ( c ) and spleen weights ( d ) from the indicated mice ( n = 6 mice per group). e Representative images of Ki67 staining in the spleen tissues from the indicated mice. n = 4 fields, two different mice per group. f Flow cytometric analysis of the percentages of Th17 cells in the PB, LNs, spleens and BM from the indicated mice ( n = 6 mice per group). g Spleen tissues from the indicated mice were subjected to immunofluorescence staining for IL-17A (red), CD4 (green), and B220 (rose red). Representative images of Th17 cells were shown. n = 4 fields, two different mice per group. h Kaplan–Meier survival curves for the indicated mice ( n = 8 mice per group). i Schematic strategy for investigating the effects of anti-CXCL16 mAb alone or combined with imatinib on Ph + B-ALL progression. j – l Representative images of spleens ( j ), spleen weights ( k ), Wright-Giemsa-stained PB smears ( l, top ), and H&E staining of the spleen ( l, bottom ) from leukemia mice treated with the indicated agents ( n = 3 mice per group). m Flow cytometric analysis of the percentages of B220 dim CD19 + cells in the PB, BM, LNs and spleens from leukemia mice treated with the indicated agents ( n = 3 mice per group). n The percentage of Ki-67 + cells in the spleen was detected by immunofluorescence staining in the indicated mice. Data are presented as means ± S.E.M of eight random fields of view from three different mice per group. o Flow cytometric analysis of the percentages of Th17 cells in the PBMCs, LNs, spleens and BM of leukemia mice treated with the indicated agents ( n = 3 mice per group). (a , m ) The gating strategy for B220 dim CD19 + cells was shown in Supplementary Fig. . ( f , o ) The gating strategy for Th17 cells in the CD4 + T cells was shown in Supplementary Fig. . Statistical significance was calculated by ( a, d – g ) two-tailed Student’s t-test; ( k , m – o) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Transplantation Assay, Staining, Immunofluorescence, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: The Th17 cell population and IL-17A expression are distinctively increased in Ph + B-ALL patients, and high expression of IL-17A promotes the progression of Ph + B-ALL. IL-17A promotes the proliferation and survival of Ph + B-ALL cells by activating the BCR-ABL and IL6/JAK/STAT3 signaling pathways. Moreover, IL-17A can increase the secretion of the chemokine CXCL16 from leukemia cells by activating NF-kB, which in turn mediates the differentiation and recruitment of Th17 cells to the leukemia niche microenvironment. Targeting IL-17A or CXCL16 in the leukemia niche microenvironment attenuates the progression of Ph + B-ALL.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Expressing

Journal: Cancer Research

Article Title: High-Level Expression of Chemokine CXCL16 by Tumor Cells Correlates with a Good Prognosis and Increased Tumor-Infiltrating Lymphocytes in Colorectal Cancer

doi: 10.1158/0008-5472.can-06-3424

Figure Lengend Snippet: Figure 1. RT-PCR analysis of chemokine expression in CRC and normal mucosa. N, normal mucosa; Ca, CRC tissue. In all three cases, CXCL16 expression was stronger in cancer tissues than in normal mucosa. Other chemokines did not show such an expression pattern. GAPDH served as a loading control.

Article Snippet: Serial sections were then incubated for 1 h at room temperature with goat polyclonal antiserum against human CXCL16 (R&D Systems) or normal goat serum as a control.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: Cancer Research

Article Title: High-Level Expression of Chemokine CXCL16 by Tumor Cells Correlates with a Good Prognosis and Increased Tumor-Infiltrating Lymphocytes in Colorectal Cancer

doi: 10.1158/0008-5472.can-06-3424

Figure Lengend Snippet: Figure 3. Immunohistochemical staining of CXCL16 in CRC. A, a, nonfixed frozen section. CXCL16 expression was apparently observed on the surface of cancer cells (arrow). b, formalin-fixed frozen section. CXCL16 was expressed in the cancer membrane as well as the cytoplasm (arrow). B, a, no CXCL16 expression case (absent). b, strong expression case. CXCL16 expression was observed in the cell membrane and cytoplasm of CRC cells. c, strong expression case. Note that, in the lower right part, normal colon epithelial cells were negative for CXCL16. d, the cytoplasm and cell membrane of the colon adenoma showed strong expression of CXCL16 but normal colon epithelial cells were negative (lower part). Magnification, 200 (A), 100 (B). Bar, 200 Am.

Article Snippet: Serial sections were then incubated for 1 h at room temperature with goat polyclonal antiserum against human CXCL16 (R&D Systems) or normal goat serum as a control.

Techniques: Immunohistochemical staining, Staining, Expressing, Membrane

Journal: Cancer Research

Article Title: High-Level Expression of Chemokine CXCL16 by Tumor Cells Correlates with a Good Prognosis and Increased Tumor-Infiltrating Lymphocytes in Colorectal Cancer

doi: 10.1158/0008-5472.can-06-3424

Figure Lengend Snippet: Figure 4. Immunohistochemical staining of T cells in CRC. A, a and c, weak CXCL16 expression case. b and d, strong expression case. TILs were observed at the tumor border. c and d, double immunohistochemical staining for CD8 (blue) and CD4 (brown). In this staining, the majority of TILs were identified as CD8+ or CD4+ cells. A larger number of TILs infiltrated CXCL16 strong cases. d, 200 magnification. Bar, 200 Am. B, TIL counts. CD8+ and CD4+ TILs were counted in five randomly selected areas at the tumor border for each case. The average counts of TILs were plotted and compared between weak and strong CXCL16 expression groups. The number of CD8+ cells was significantly increased in the strong group (mean F SD, 58.2 F 25.2 per field) as compared to the weak group (29.3 F 16.2 per field). Similarly, CD4+ cells were significantly increased in the strong group (71.9 F 26.1 per field) as compared to the weak group (44.2 F 17.8 per field). C, CXCR6 mRNA expression was analyzed by RT-PCR in a strong CXCL16 expression case and a weak expression case.

Article Snippet: Serial sections were then incubated for 1 h at room temperature with goat polyclonal antiserum against human CXCL16 (R&D Systems) or normal goat serum as a control.

Techniques: Immunohistochemical staining, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction