Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor-intrinsic TTLL12 drives resistance to cancer immunotherapy via modulating myeloid-derived suppressor cells

doi: 10.1136/jitc-2024-010873

Figure Lengend Snippet: TTLL12 promotes chemokine CCL9 secretion in colorectal cancer. ( A ) The workflow to identify the cytokines bridging the connection between tumor TTLL12 and MDSCs in the tumor microenvironment. ( B ) The heatmap of 33 candidate proteins with p<0.01 and secretory ability predicted by the Human Protein Atlas database. ( C ) ELISA analysis in the supernatant of CT26 shNC and CT26 shTTLL12 cells. Values are represented as mean±SEM. P values were determined by two-tailed t-tests, ***p<0.001. ( D ) qRT-PCR analysis of CT26 shNC and CT26 shTTLL12 cells. Values are represented as mean±SEM. P values were determined by two-tailed t-tests, ***p<0.001. ( E ) Tumor image from the immunocompetent Balb/c mice subcutaneously implanted with CT26-control cells, TTLL12-overexpressing CT26 cells as well as TTLL12-overexpressing CT26 cells with CCL9 silence. ( F–G ) Tumor volume ( F ) and tumor weight ( G ) in Balb/c mice. n=5 mice for each group. Values are represented as mean±SEM. P values calculated by two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ( H ) Quantification of CCL9 concentration in tumors. Values are represented as mean±SEM. P values calculated by two-way ANOVA, ****p<0.0001. ( I ) Flow cytometry analysis to test the MDSCs infiltration in tumors. Values are represented as mean±SEM. P values calculated by two-way ANOVA, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; MDSC, myeloid-derived suppressor cells; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; TTLL12, tubulin tyrosine ligase 12.

Article Snippet: The concentrations of CCL9 in mice were measured by using a Mouse CCL9 ELISA Kit from Boster (Wuhan, China), following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Quantitative RT-PCR, Control, Concentration Assay, Flow Cytometry, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor-intrinsic TTLL12 drives resistance to cancer immunotherapy via modulating myeloid-derived suppressor cells

doi: 10.1136/jitc-2024-010873

Figure Lengend Snippet: TTLL12 induces CCL9 expression, probably by binding to its promoter region. ( A ) The intracellular location of TTLL12 was detected by IHC analysis based on colorectal cancer samples in our center. Representative images of IHC staining from three tissue samples were shown. Red arrows: cell nucleus. Scale bars: 50 µm (left panel), 25 µm (right panel). ( B–C ) Western blot analysis of the cytoplasm and nucleus of CT26 cells transfected with plasmid harboring TTLL12 overexpression or knockdown with TTLL12 antibody, α-tubulin (a cytosolic marker) antibody and Lamin-B (a marker for nuclear compartments) antibody. ( D ) The promoter region of CCL9 covered by the primer. ( E–F ) ChIP-PCR assay in DLD1/TTLL12 and CT26/TTLL12 cells. Values are represented as mean±SEM. P values were determined by two-tailed t-tests, ****p<0.0001. ChIP, chromatin iImmunoprecipitation; IHC, immunohistochemistry; TTLL12, tubulin tyrosine ligase 12.

Article Snippet: The concentrations of CCL9 in mice were measured by using a Mouse CCL9 ELISA Kit from Boster (Wuhan, China), following the manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Immunohistochemistry, Western Blot, Transfection, Plasmid Preparation, Over Expression, Knockdown, Marker, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor-intrinsic TTLL12 drives resistance to cancer immunotherapy via modulating myeloid-derived suppressor cells

doi: 10.1136/jitc-2024-010873

Figure Lengend Snippet: Working model of TTLL12-mediated antitumor immune response. Tumor-intrinsic TTLL12 depends on MDSCs to promote tumor progression and knockdown of TTLL12 can enhance the antitumor efficacy of anti-programmed cell death protein 1 therapy in an immunocompetent mouse model. Mechanistically, tumor-derived TTLL12 promotes chemokine CCL9 secretion to modulate MDSCs by inducing the transcriptional expression of CCL9, probably by binding to its promoter region. MDSC, myeloid-derived suppressor cell; NK, natural killer; TTLL12, tubulin tyrosine ligase 12.

Article Snippet: The concentrations of CCL9 in mice were measured by using a Mouse CCL9 ELISA Kit from Boster (Wuhan, China), following the manufacturer’s instructions.

Techniques: Knockdown, Derivative Assay, Expressing, Binding Assay

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet: ( A, B ) Quantification of chemokine mRNA levels by qRT-PCR in OT1 ( A ) or gBT1 ( B ) CTLs exposed to H-2K b /SIINFEKL cognate beads, or cognate SIINFEKL- or SSIEFARL-pulsed EL4 respectively, or non-cognate unpulsed EL4 tumour cells, as indicated. Data is normalised to universal mouse cDNA (positive control). Data points from four independent experiments; bars indicate mean. ND: not detected. ( C ) Quantification of absolute chemokine concentration detected in non-cognate and cognate OT1 supernatants by ELISA (for CCL1 and CCL9) or CBA (for CCL3, CCL4, CCL5, CXCL10, IFN-γ, and TNF-α). Data points from four independent experiments; red lines indicate mean. ND: not detected. ( D ) Transmigration of OT1 CTLs towards basal medium containing indicated concentrations of recombinant chemokine. Data normalised to basal medium (dashed line). Data points from three independent experiments; bars indicate mean. Error bars represent standard deviation. p- values are displayed when means are significantly different compared to the 1 ng/ml condition. ( E ) Transmigration of OT1 CTLs towards chemokine-containing basal medium in the presence of corresponding neutralising antibodies or isotype (IgG) control antibody. Data points from four independent experiments; bars indicate mean. Error bars represent standard deviation. ( F ) Transmigration of CTLs towards cognate supernatant in the presence of neutralising antibodies or IgG isotype control. Data normalised to basal medium (dashed line). Data points from at least three independent experiments; bars indicate mean. Error bars represent standard deviation. p-values are displayed when means are significantly different to both the negative control and the IgG isotype control conditions. ( G ) FMI of OT1 CTLs adjacent to tumouroids containing non-cognate or cognate tumour cells with pre-embedded tumour-reactive CTLs in the presence of chemokine-neutralising antibodies as indicated. ( H ) Time course of CCL3 and CCL4 concentrations in supernatants from CTLs sorted by FACS from 4 hr conjugations with cognate tumour cells (0 hr: completion of conjugation). Solid line: cumulative concentration. Dotted line: differential concentration per hour (difference of cumulative concentrations at consecutive timepoints divided by number of hours in-between). Error bars and shaded areas: range. ( I ) FMI of OT1 CTLs adjacent to masses containing CTLs only (no tumour cells) sorted from conjugations with cognate or non-cognate tumour cells. Comparisons of transmigration indices in ( C ) to ( E ) were performed using ANOVA and Tukey’s multiple comparisons tests. In ( G ) and ( I ), box-whiskers indicate medians and the interquartile range (IQR) with outliers outside whiskers. ns: p>0.05, p-values from two-tailed Wilcoxon signed rank test compared to a theoretical median of 0. Figure 4—figure supplement 1—source data 1. Source data file for .

Article Snippet: Commercial assay or kit , Sandwich ELISA kits (CCL9) , OriGene Technologies , Cat. #: EA100725 , .

Techniques: Quantitative RT-PCR, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transmigration Assay, Recombinant, Standard Deviation, Control, Negative Control, Conjugation Assay, Two Tailed Test

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Sandwich ELISA kits (CCL9) , OriGene Technologies , Cat. #: EA100725 , .

Techniques: In Vivo, Cell Culture, Transduction, Construct, Sequencing, Produced, Transfection, Expressing, Plasmid Preparation, Clone Assay, Control, Recombinant, In Vitro, Mass Spectrometry, Cell Isolation, Selection, Reverse Transcription, Sandwich ELISA, Cytometry, Software

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Sandwich ELISA kits (CCL9) , OriGene Technologies , Cat. #: EA100725 , .

Techniques:

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Cxcl16 −/− mice were resistant to the development of necrotizing pancreatitis. Cerulein (100 ug/kg) was injected into Cxcl16 -intact ( WT ) and Cxcl16 -deficient ( Cxcl16 −/− ) mice (n = 5 in each group) every 1 h 8 times on 2 consecutive days as decribed in Fig. ( A ) Serum amylase levels and ( B ) pathology scores of WT and Cxcl16 −/− mice. ( C ) H&E sections from WT and Cxcl16 −/− mice at 24 h and 33 h. ( D ) Immunostained (Gr1, F4/80, and CD3) sections from WT and Cxcl16 −/− mice at 33 h. ( E ) Semiquantitative assessment of infiltartion of neutrophils, macrophages, and T cells. The number of neutrophils, macrophages, and T cells were counted in 5 high powered fields in each pancreas sections of WT and Cxcl16 −/− mice at 33 h. ( F ) MPO levels determined by ELISA in the pancreatic lysates of WT and Cxcl16 −/− mice at 33 h. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Levels of Ccl9 in the sera or the pancreatic lysates were measured using a mouse Ccl9 ELISA kit (R&D Systems), and levels of myeloperoxidase (MPO) in pancreatic lysates were determined using a mouse MPO ELISA kit (ThermoFisher Scientific, Hudson, NH), according to the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Induction of Ccl9 by Cxcl16 in necrotizing acute pancreatitis. ( A ) Cytokine/chemokine array using pancreatic lysates from C57BL/6 mice and Cxcl16 −/− mice treated with necrotizing pancreatitis regimen as described in Fig. . The relative expression of cytokines and chemokines is shown together with representative images. The data presented were obtained from WT and Cxcl16 −/− mice at 33 h, and WT on 0 h. ( B ) Ccl9 , Vcam-1 , and Ccl2 mRNA expression, relative to those of WT mice at 0 h, was assessed by qPCR in the pancreas of WT and Cxcl16 −/− mice at 33 h. ( C ) Ccl9 immunostaining of pancreatic frozen sections from WT mice at 0 h and 33 h, and Cxcl16 −/− mice at 33 h. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Levels of Ccl9 in the sera or the pancreatic lysates were measured using a mouse Ccl9 ELISA kit (R&D Systems), and levels of myeloperoxidase (MPO) in pancreatic lysates were determined using a mouse MPO ELISA kit (ThermoFisher Scientific, Hudson, NH), according to the manufacturer’s instructions.

Techniques: Expressing, Immunostaining

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Expression of Ccl9 by pancreatic acinar cells. ( A ) Amylase secretion by the rat acinar cell line AR42J upon stimulation with various concentrations of cerulein. ( B ) Cxcl16 and Ccl9 mRNA expression by AR42J upon stimulation with cerulein. ( C ) Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −10 M) in combination with various concentrations of recombinant Cxcl16 protein (left). Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −7 M) in the presence of neutralizing anti-Cxcl16 Ab or control Ab (right). ( D ) Cxcl16 and Ccl9 mRNA expression by pancreatic acinar cells isolated from WT and Cxcl16 −/− mice upon stimulation with cerulein (10 −7 M). *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Levels of Ccl9 in the sera or the pancreatic lysates were measured using a mouse Ccl9 ELISA kit (R&D Systems), and levels of myeloperoxidase (MPO) in pancreatic lysates were determined using a mouse MPO ELISA kit (ThermoFisher Scientific, Hudson, NH), according to the manufacturer’s instructions.

Techniques: Expressing, Recombinant, Control, Isolation

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Therapeutic effects of neutralizing Cxcl16 Ab in the necrotizing pancreatitis model. ( A ) Injection protocol of anti-Cxcl16 Ab in necrotizing pancreatitis model. ( B ) Serum amylase level, ( C ) representative H&E sections, and pathology score of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( D ) Gr1 immunostained sections and neutrophil counts of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( E ) Pancreatic Ccl9 mRNA, pancreatic Ccl9 protein, and serum Ccl9 protein levels in control Ab or anti-Cxcl16 Ab-treated mice at 39 h. n = 5 in each group. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Levels of Ccl9 in the sera or the pancreatic lysates were measured using a mouse Ccl9 ELISA kit (R&D Systems), and levels of myeloperoxidase (MPO) in pancreatic lysates were determined using a mouse MPO ELISA kit (ThermoFisher Scientific, Hudson, NH), according to the manufacturer’s instructions.

Techniques: Injection, Control