Journal: PLoS ONE

Article Title: Th1-Like ICOS + Foxp3 + T reg Cells Preferentially Express CXCR3 and Home to β-Islets during Pre-Diabetes in BDC2.5 NOD Mice

doi: 10.1371/journal.pone.0126311

Figure Lengend Snippet: NOD.TCRα -/- mice initially received BDC2.5 CD4 + T cell (7.5X10 5 ) cells, and then cell suspensions from pancreas, draining pLN and peripheral LN of diabetic mice were examined for expression of CXCL9, CXCL10 and CXCL11 in F4/80 + (A) and CXCL10 in CD11c + (B) cells was assessed by flow cytometry. (C) NOD.TCRα -/- mice were transferred with CD4 + T cells in order to induce T1D. Following adoptive transfer, glucose levels were measured daily in order to assess diabetes onset. Pancreatic cell suspensions from recipients were obtained and CXCL10 expression (MFI) was compared among CD11c + cells from diabetic or non-diabetic recipients, as well as un-transferred (without T cell transfer) NOD.TCRα -/- mice. (D) NOD.TCRα -/- mice received T reg or T eff cells either alone or at the indicated T reg/ T eff cell ratios. When mice receiving T eff cells alone became hyperglycemic (>33mmol/L), CXCL10 levels were assessed by ELISA in supernatants from pancreatic suspensions. (E), F4/80 + and CD11c + (1X10 6 /well) cells were seeded in triplicate in lower chambers, and CD4 + (1X10 6 /well) cells in the upper chambers of 24 well Transwell plate. Following a 3 hour incubation period, the percent migrated CXCR3 + cells among ICOS + T reg cells was compared between wells containing APCs and media alone ( control ). Cell suspensions of pancreas and draining LN of 4-week-old BDC2.5 mice were obtained and CXCL10 expression (MFI) was compared (F) between pancreatic T reg cells and T eff cells (G), between T rec cells at sites indicated, and between ICOS + and ICOS + T reg cells within pancreas (H). (4A-4C n = 6, 4D n = 4, 4E n = 2, 4F-4H n = 5).

Article Snippet: DuoSet ELISA kits (R&D Systems) were used to analyze IFN-γ (DY485) and CXCL10 (DY466) in whole-pancreas protein samples.

Techniques: Expressing, Flow Cytometry, Adoptive Transfer Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: PLoS ONE

Article Title: Th1-Like ICOS + Foxp3 + T reg Cells Preferentially Express CXCR3 and Home to β-Islets during Pre-Diabetes in BDC2.5 NOD Mice

doi: 10.1371/journal.pone.0126311

Figure Lengend Snippet: β-islet cells were isolated from 4-week-old BDC2.5 mice. (A) Expression of CXCR3 chemokines was compared between insulin + cells and glucagon + cells, and unstained controls. (B), NOD.TCRα -/- mice received T reg or T eff cells either alone or at the indicated T reg/ T eff cell ratios. When mice receiving T eff cells alone became hyperglycemic (>33mmol/L), CXCL10 expression (MFI) in β-islet cells was compared between groups. (C) IFN-γR ΔMFI (ΔMFI was calculated by subtracting the isotype control MFI from IFN-γR antibody MFI) and was compared between β (insulin + ), α (glucagon + ) and δ (somatostatin + ) cells isolated from 4-week-old BDC2.5 mice. (5B, 5C n = 5).

Article Snippet: DuoSet ELISA kits (R&D Systems) were used to analyze IFN-γ (DY485) and CXCL10 (DY466) in whole-pancreas protein samples.

Techniques: Isolation, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p < 0.001. One representative out of three independent experiments is shown (B) .

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Injection, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Transfection, Control, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Migration, Control, Neutralization, Incubation, Recombinant, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Control, Migration, Neutralization, Infection, Incubation

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p < 0.001. One representative out of three independent experiments is shown (G) .

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 2. Upregulation of CXCL10 and MHC class II in human neutrophils in urine during BCG infusion therapy. (a,b) Comprehensive analysis of mRNA expression in urine-derived neutrophils compared to peripheral blood neutrophils. Blood and urine were collected from three patients after one week from the 6th BCG infusion. Comprehensive analysis of mRNA in neutrophils was performed using a DNA tip microarray. (a) Cluster analysis after adjustment and standardiza- tion. The mRNA expression in neutrophils obtained from urine samples (vertical axis) or periph- eral blood (horizontal axis) was analyzed. White lines indicate the thresholds for genes that are upregulated or downregulated > 2-fold between urine- and blood-derived neutrophils. A relatively higher expression in urine-derived neutrophils is indicated using arrows, including expression for CXCR3 ligands (CXCL9 and CXCL10) and MHC class II (HLA-DRB1, HLA-DPA1, and HLA-DQA1). (b) Volcano plot depicting the differentially expressed genes between peripheral blood-derived and urine-derived neutrophils after the 6th BCG infusion. The horizontal axis denotes the fold change in mRNA expression in neutrophils from the urine and blood, while the vertical axis represents the –log10 (p-value) for a t-test of differences in neutrophils from the blood and urine. These data represent the top 6000 genes of the –log10 (p-value). The gene expressions of CXCR3 ligands (CXCL9, CXCL10, and CXCL11) and MHC class II (HLA-DQA2, HLA-DPA1, and HLA-DQA1) were also detected as characteristic features of urine-derived neutrophils (arrows). (c,d) Representative data of intracellular- stained neutrophilic cells obtained via flow cytometric analysis. The CD33+CD15+ neutrophilic cells in the blood (c) or urine (d) samples were obtained from the same patient who was treated with 4th BCG infusions and are presented as CXCL10 MFI (upper panels) and HLA-DR MFI (lower panels). Gray-closed histograms indicate each background staining, and light blue line histograms denote the staining of CXCL10 or HLA-DR. (e–g) Comparison of intracellular expression of (e,f) CXCL10 and (g) HLA-DR in neutrophilic cells from the blood (open circle) and urine (closed circle) samples. These samples were collected after one week from the 2nd to the 6th BCG infusions (after each infusion). (e) ∆CXCL10 MFI was calculated as follows: ∆CXCL10 MFI = (MFI of PE-conjugated anti-CXCL10 mAb staining) −(MFI of PE-conjugated control IgG staining). (f) The neutrophilic cells

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Derivative Assay, Microarray, Staining, Comparison, Control

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 3. Effect of BCG on CXCL10 and MHC-II expression in human or mice neutrophilic cells in vitro. Human (a,d) or mouse (b,e) peripheral blood was diluted ten-fold in 10% FCS RPMI1640, or mouse bone marrow cells (4 × 106/mL; c,f) were incubated with or without 4 µg/mL of BCG for 20 h. Following incubation, the expression levels of CXCL10 (a–c) and MHC class II (d–f) in human (CD33+CD15+) or mouse neutrophils (CD45+Ly6G+) were analyzed, as described in Figure S2. Statistical significance was calculated with the paired t-test, * p < 0.05 (n = 3). Abbreviations: BCG, Bacillus Calmette–Guérin; CXCL10, chemokine (C-X-C motif) ligand 10; HLA-DR, human major histocompatibility complex class II cell surface receptor; MFI, mean fluorescence intensity; and I-A/I-E, mouse major histocompatibility complex class II cell surface receptor.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, In Vitro, Incubation, Immunopeptidomics, Cell Surface Receptor Assay

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 4. Upregulation of CXCL10 and MHC class II in monocytes and neutrophils in peritoneal effusion cells after BCG injections. Mice were injected with B16F10 cells (5 × 104 cells/100 µL/head), and the PECs were collected after two weeks. The PECs induced after one injection of BCG (40 µg/head) after 16 h and the PECs induced after five repeated injections of BCG (40 µg/head) after 16 h from the final injection are presented as “1-shot” and “5-shots”, respectively. These PECs were intracellularly stained with each antibody, and the relative expression (MFI) of CXCL10 and I-A/I-E was analyzed in CD45+Ly6C+ cells and CD45+Ly6G+ cells, respectively. (a–i) Representative flow cytometric analysis of mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) via flow cytometry. The (a–c) panels present flow cytometric analysis of the PECs induced 2 weeks after B16F10 cell injection (presented as “Tumor”). The (d–f) panels show representative flow cytometric analysis of the PECs induced 16 h after the administration of BCG (presented as “1-shot). The (g–i) panels indicate representative flow cytometric analyses of the PECs induced via five repeated BCG injections at one-week intervals. The PECs were collected 16 h after the final BCG admin- istration (presented as “5-shots”). The left panels (a,d,g) show CD45+ leukocytes presented with the gates of Ly6C+ cells (monocytic cells) and Ly6G+ cells (neutrophilic cells). (j–l) The number and proportion of myeloid cells (Ly6C+ and Ly6G+ cells) of the PECs. The peritoneal effusion cells obtained after injection of B16F10 cells are presented as “tumor” (open circles). The cells induced 16 h after a single administration of BCG are presented in the group “1-shot” (closed circles). The cells induced via five repeated injections of BCG are presented in the group “5-shots” (closed triangles). The (j) number of the cells in peritoneal fluid were counted using a hemocytometer, and the proportions of (k) Ly6C+ cells and (l) Ly6G+ cells in CD45+ leukocytes were analyzed via flow cytometry. (m–p) The intracellular expression levels of CXCL10 and MHC-II (I-A/I-E) in mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) after BCG injection. These PECs were intracellularly stained with each anti- body, and the relative expression (MFI) of (m,n) CXCL10 and (o,p) I-A/I-E was analyzed in (m,o) CD45+Ly6C+ cells and (n,p) CD45+Ly6G+ cells, respectively. Statistical analyses were per- formed using the Kruskal–Wallis test with the Dunn’s post-hoc test. Each bar is presented as the mean of data. * p < 0.05; ** p < 0.01; and ns, not significant. Abbreviations: PECs, peritoneal exudate cells; CXCL10, C-X-C motif chemokine ligand 10; BCG, Bacillus Calmette–Guérin; and MFI, mean fluorescence intensity.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Injection, Staining, Expressing, Cytometry

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 7. CXCL10 and MHC class II expression in neutrophils induced via BCG was inhibited via partial neutrophil depletion using anti-Ly6G mAbs. BCG (40 µg/100 µL/head) was injected into the peritoneal cavity, following which the antibodies (100 µg/50 µL/head; control mAb, open circle; or anti-Ly6G mAb, closed circle) were injected into the

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Injection, Control

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A and B ) Flow cytometry and statistical analysis of CD8 + CD45 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( C and D ) Flow cytometry and statistical analysis of IFN-γ + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( E and F ) Flow cytometry and statistical analysis of Granzyme B + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( G ) The secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in the tail skin of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups were detected by ELISA ( n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Sterility

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on fibroblasts in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRA2A expression on fibroblasts in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of fibroblasts, ADRA2A, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRA2A (red) expression in BJ cells after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells were detected by ELISA with treatment with aposcopolamine (Apos) or NE ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells with ADRA2A siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on keratinocytes in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRB2 expression on keratinocytes in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of keratinocytes, ADRB2, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRB2 (red) expression in keratinocytes after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes were detected by ELISA with treatment with NE or ICI ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes with ADRB2 siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: (A) Deletion of Tet2 in allograft tumors reduced chemokine Cxcl9, Cxcl10, and Pdl1 expression. Total mRNA was extracted from WT or Tet2-KO tumor (n = 10 for each group), and mRNA levels of genes were determined by qPCR. Data represent mean ± SD. *P < 0.05, **P < 0.01 by unpaired Student’s t test. (B and C) Knocking out Tet2 blocked IFN-γ–induced chemokines and Pdl1 gene expression in B16-OVA (B) and THP-1 (C) cells. WT or Tet2-KO cells were treated with IFN-γ for 20 hours, and total RNA was extracted. The relative mRNA levels were determined by qPCR. Error bars represent ± SD for triplicate experiments. (D) Knocking out Tet2 decreased IFN-γ–induced CXCL9 and CXCL10 protein levels in B16-OVA cells. WT or Tet2-KO B16-OVA cells were treated with IFN-γ for 72 hours; then medium was collected and subjected to ELISA analysis. Error bars represent ± SD for triplicate experiments. **P < 0.01, ***P < 0.001 by unpaired Student’s t test. (E) TET2 catalytic activity was required for IFN-γ–induced CXCL10 and PD-L1 expression. TET2-WT and catalytic mutant R1896S were overexpressed in TET2-KO THP-1 cells; then cells were treated with IFN-γ for 20 hours as indicated, and total RNA was extracted. The relative mRNA levels were determined by qPCR. Error bars represent ± SD for triplicate experiments. (F) Deletion of Tet2 impaired T cell attraction by Transwell assay. WT or Tet2-KO B16-OVA cells were treated with IFN-γ for 48 hours, and CM was collected. Triplicate independent experiments were performed for each group. Error bars represent ± SD. Bonferroni-adjusted **P < 0.01, with raw P value derived from unpaired Student’s t test.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Activity Assay, Mutagenesis, Transwell Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: WT or TET2-KO THP-1 cells were treated with 100 ng/mL IFN-γ for 20 hours, and total RNA was extracted and subjected to RNA-Seq. (A) Venn diagram of affected genes (stimulated 2-fold or more) in the whole transcriptome is shown. (B) Global gene expression analysis of WT and TET2-KO THP-1 cells stimulated with IFN-γ by RNA-Seq is shown. The region (P < 0.001 and fold change > 5) is highlighted by dashed lines. Red dots represent CXCL9, CXCL10, and CXCL11; green dot represents PD-L1. (C) Heatmap depiction of differentially expressed genes (fold change ≥ 5) between control and TET2-KO THP-1 cells.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: RNA Sequencing, Gene Expression, Control

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: (A) IFN-γ promoted TET2-STAT1 binding. THP-1 cells were treated with or without IFN-γ, and the interaction of TET2 and STAT1 was determined by IP–Western blot. (B) Y701F mutation disrupted TET2-STAT1 binding. STAT1-WT and Y701 mutant plasmids were transfected into THP-1 cells and treated with IFN-γ, and their binding to TET2 was determined by IP–Western blot. (C and D) IFN-γ promoted TET2 binding to the CXCL10 (C) and PD-L1 (D) promoters. THP-1 cells were treated with or without IFN-γ, and TET2 binding to the CXCL10 (C) and PD-L1 (D) promoters was determined by TET2 ChIP-qPCR. Error bars represent ± SD for triplicate experiments. (E and F) IFN-γ increased the 5hmC level of the CXCL10 (E) and PD-L1 (F) promoters. hMeDIP assays were performed in control and TET2-KO THP-1 cells treated or untreated with IFN-γ. 5hmC levels on the CXCL10 (E) and PD-L1 (F) promoters were determined by qPCR. Error bars represent ± SD for triplicate experiments. (G and H) JAK2 inhibitor blocked binding to (G) and demethylation of (H) the CXCL10 promoter by TET2 upon IFN-γ treatment. THP-1 cells were treated with IFN-γ and JAK2 inhibitor as indicated, and the ability of TET2 to bind to (G) and demethylate (H) the CXCL10 promoter was determined by TET2 (G) or 5hmC (H) ChIP-qPCR. Error bars represent ± SD for triplicate experiments. (I and J) JAK2 inhibitor blocked binding to (I) and demethylation of (J) the PD-L1 promoter by TET2 upon IFN-γ treatment. THP-1 cells were treated with IFN-γ and JAK2 inhibitor as indicated, and the ability of TET2 to bind to (I) and demethylate (J) the PD-L1 promoter was determined by TET2 (I) or 5hmC (J) ChIP-qPCR.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: Binding Assay, Western Blot, Mutagenesis, Transfection, ChIP-qPCR, Control

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: (A) Infiltrating lymphocyte numbers including CD3+ T cells, CD8+ cytotoxic T lymphocytes (CTLs), and CD56+ NK cells were decreased along with the loss of 5hmC in adenoma of colon. Two representative pictures show multicolor, fluorescently labeled inflammatory cells in adenomas expressing high 5hmC levels and adenomas with decreased 5hmC expression, separately. Scale bars: 100 μm. (B) Quantification of CD3+ T cells, CD8+ CTLs, and CD56+ NK cells in colon adenomas classified by high and low 5hmC staining. Four cases of adenoma with high 5hmC expression and 8 cases of adenoma with low 5hmC expression were used to count cytotoxic T cells (CD3+ and CD8+) and NK cells (CD56+). For each case, 3 areas highly infiltrated with inflammatory cells were selected. **P < 0.01 by Student’s t test. Data represent mean ± SEM. (C) Intratumoral CXCL9, CXCL10, and CXCL11 levels were correlated with 5hmC levels in colon adenomas. Representative photographs show the expression of CXCL9, CXCL10, and CXCL11 in samples with high 5hmC and in samples with low 5hmC in the same fields, on serial sections in adenoma with low-grade dysplasia, adenoma with high-grade dysplasia, and adenocarcinoma specimens. Scale bars: 50 μm. (D–F) Quantification of CXCL9 (D), CXCL10 (E), and CXCL11 (F) expression classified by high and low 5hmC staining. Five samples representing cases with low 5hmC expression and 5 with high 5hmC expression were selected separately in adenoma with low-grade dysplasia, adenoma with high-grade dysplasia, and adenocarcinoma. For each case, 5 fields were randomly selected to calculate the integrated staining density by i-Solution image analysis software. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test. Data represent mean ± SEM.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: Labeling, Expressing, Staining, Software

Journal: Gut microbes

Article Title: Lacticaseibacillus paracasei sh2020 induced antitumor immunity and synergized with anti-programmed cell death 1 to reduce tumor burden in mice.

doi: 10.1080/19490976.2022.2046246

Figure Lengend Snippet: Figure 7. L. paracasei sh2020 promoted the expression and secretion of CXCL10 in vivo and in vitro. (a) The expression of CXCL9, CXCL10 and CXCL11 in tumor tissues from control and L. paracasei sh2020-treated mice was detected by qRT-PCR. (b-c) Representative images (b) and quantification (c) of IHC staining of CXCL10 in the tumor tissues from control and L. paracasei sh2020-treated tumors. (d) Tumor growth in each group. (e) The levels of CXCL10 in the conditioned medium. (f-g) Tumor growth in the tumor-bearing mice with intratumoral injection of L. paracasei sh2020 (n = 5–6). (h-i) Representative images (h) and quantification (i) of IHC staining of CXCL10 and CD8 in each group (n = 4–5). (j) The serum levels of CXCL10 were examined by ELISA. ns, no significant difference, *P < .05, **P < .01, ***P < .001.

Article Snippet: To neutralize Cxcl10 in vivo, 50 μg of anti-mouse Cxcl10 (134013, MAB466, R&D systems) was intraperitoneally injected into mice every 3 days for 2 weeks.

Techniques: Expressing, In Vivo, In Vitro, Control, Quantitative RT-PCR, Immunohistochemistry, Injection, Enzyme-linked Immunosorbent Assay

Journal: Gut microbes

Article Title: Lacticaseibacillus paracasei sh2020 induced antitumor immunity and synergized with anti-programmed cell death 1 to reduce tumor burden in mice.

doi: 10.1080/19490976.2022.2046246

Figure Lengend Snippet: Figure 8. CXCL10 controlled CD8+ T cell migration and the effect of L. paracasei sh2020 in vivo. (a-b) Representative images of IHC staining of CD8 and CXCL10 (a), and quantification (b) for the control and L. paracasei sh2020-treated tumors (n = 6–7). (c) IHC analysis of CD8 in tumors, which were divided into two groups according to CXCL10 high and low expression. (d) Experimental design: C57BL/6 mice were implanted subcutaneously with 5.0 × 105 MC38 cells and was treated with control vehicle or anti-CXCL10 antibody by intraperitoneal injection, every 3 days starting on D3, in total three times. The mice were given L. paracasei sh2020 with a dose of 1.0 × 109 CFU by gavage starting from D0 to D13. (e) Tumor growth in tumor-bearing mice in d. (f) Quantification of IHC staining of CXCL10 and CD8 in the tumors after neutralizing CXCL10 in vivo (n = 4–5). ns, no significant difference, *P < .05, **P < .01, ***P < .001.

Article Snippet: To neutralize Cxcl10 in vivo, 50 μg of anti-mouse Cxcl10 (134013, MAB466, R&D systems) was intraperitoneally injected into mice every 3 days for 2 weeks.

Techniques: Migration, In Vivo, Immunohistochemistry, Control, Expressing, Injection