Journal: JCI Insight

Article Title: Protective role of tissue-resident Tregs in a murine model of beryllium-induced disease

doi: 10.1172/jci.insight.156098

Figure Lengend Snippet: Frequency (left panel) and number (right panel) of CD4 + CD25 + FoxP3 + T cells in the lungs of HLA-DP2 Tg mice sensitized with 1 (1×) or 3 (3×) doses of BeO and harvested at day 12 ( A ) or sensitized with 1 (1×), 3 (3×), or 7 (7×, sensitization/boost) BeO exposures and examined at day 21 ( B ). ( C ) Representative dot plots show CD103 and CD69 expression on CD4 + CD25 + FoxP3 + Tregs derived from the spleen (top panels) and lung (bottom panels) of mice exposed to BeO on 1 (1×), 3 (3×), or 7 (7×) occasions and examined at day 21. ( D and E ) Number of resident effector (RE, CD103 – CD69 + ) and resident memory (RM, CD103 + CD69 + ) T cells among tissue-resident Tregs in HLA-DP2 Tg mice exposed to 1, 3, and 7 doses of BeO. ( F ) Representative histograms show expression of ICOS, Nrp-1, GITR, and CTLA-4 on tissue-specific CD25 + FoxP3 + CD4 + Tregs in mice exposed to 1 (1×, red) or 3 (3×, green) doses of BeO and analyzed at day 12 or exposed to 1 (1×, blue), 3 (3×, purple), or 7 (7×, brown) doses of BeO and analyzed at day 21. ( G – I ) T cell homeostatic chemokines CXCL-10 ( G ), CCL19 ( H ), and CCL21 ( I ) were measured in the lung tissue lysates by ELISA. Data (mean ± SEM) are representative of 3 individual experiments (2–5 mice per group). Significance was determined by 1-way ANOVA ( A , B , D , and E ) and Mann-Whitney U test ( G – I ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: In ICOS or CTLA-4 blocking experiments, HLA-DP2 Tg mice were injected with 300 μg of an anti–CTLA-4 antibody (BioXcell; clone UC10-4F10-11) or an anti-ICOS antibody (BioXcell; clone 7E.17G9) on day –1.

Techniques: Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Protective role of tissue-resident Tregs in a murine model of beryllium-induced disease

doi: 10.1172/jci.insight.156098

Figure Lengend Snippet: ( A and B ) Plots show the number of CD4 + T cells (left panel) and CD44 + T effector cells (right panel) in the lungs of BeO-sensitized HLA-DP2 Tg mice on day 12 treated i.p. with either an isotype control antibody and anti–CTLA-4 ( A ) or anti-ICOS ( B ) blocking antibodies at day –1. ( C ) Percentage of FoxP3 + Tregs in the lungs of isotype and anti–CTLA-4 (left) or anti-ICOS (right) blocking antibody–treated, BeO-exposed HLA-DP2 Tg mice on day 12. ( D ) At day 12, Teff/Treg ratio was calculated by dividing the total number of tissue-specific Teffs (CD44 + ) by total Tregs (CD25 + FoxP3 + ) in anti–CTLA-4 (left) and anti-ICOS (right) antibody–treated HLA-DP2 Tg mice. ( E ) IFN-γ secretion by CD4 + T cells purified from the lungs of BeO-exposed and anti–CTLA-4 (left) or anti-ICOS (right) blocking antibody–treated HLA-DP2 Tg mice and stimulated with BeSO 4 (100 μM) in the presence of irradiated naive splenocytes. ( F ) Protein in the BALF of mice exposed with BeO (3×) and treated with anti–CTLA-4 (left) or anti-ICOS (right) blocking antibody was examined by ELISA. Data (mean ± SEM) are representative of 2 individual experiments (4–5 mice per group). Significance was determined by Mann-Whitney U test. * P < 0.05.

Article Snippet: In ICOS or CTLA-4 blocking experiments, HLA-DP2 Tg mice were injected with 300 μg of an anti–CTLA-4 antibody (BioXcell; clone UC10-4F10-11) or an anti-ICOS antibody (BioXcell; clone 7E.17G9) on day –1.

Techniques: Control, Blocking Assay, Purification, Irradiation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 1. Schematic illustration of Sr-containing mesoporous bioactive glasses (MBGs) functionalized with ICOS-Fc on osteoblast and osteoclast cells.

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 3. FE-SEM images of SG-Sr (a), SD-Sr (b), SG-Sr-ICOS-Fc (c), and SD-Sr-ICOS-Fc (d).

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 4. N2 adsorption-desorption isotherm of SG-Sr, Amino-SG_Sr and SG-Sr-ICOS-Fc (a), SD-Sr, Amino-SD_Sr and SD-Sr-ICOS-Fc (b). Pore size distribution of SG-Sr, Amino-SG_Sr and SG-Sr-ICOS-Fc (c), SD-Sr, Amino-SD_Sr, and SD-Sr- ICOS-Fc (d).

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques: Adsorption, Pore Size

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 6. FESEM images of SG-Sr-ICOS-Fc bioactivity after 1 day (a) and 7 days (b) of soaking in SBF, SD-Sr-ICOS-Fc after 1 day (c) and 7 days (d) of soaking in SBF.

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 7. XRD at different time steps of soaking in SBF of SG-Sr-ICOS-Fc (a) and SD-Sr-ICOS-Fc (b).

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 10. Cytofluorimetric analysis and MFI-R results for SG-Sr-ICOS-Fc, SD-Sr-ICOS-Fc, SG-Sr and SD-Sr. Dot plots and cytofluorimetric histograms of ICOS-Fc for each sample tested are shown. C-: unstained sample; α-ICOS: sample stained with the antibody. FSC: forward scatter; SSC: side scatter.

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques: Staining

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 11. ELISA-like assay conducted on SG-Sr-ICOS-Fc and SD-Sr-ICOS-Fc post soaking in DMEM collected at different time steps (3, 7, 14 and 21 days). The presence and the binding of ICOS-Fc were detected by ELISA-like assay using the human ICOSL-His as capture protein and anti-human-Hrpas detection antibody. The graph shows the Optical Density (OD) values (mean and standard error were obtained from three separate samples).

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 12. Cell viability studies performed on SG-Sr-ICOS-Fc (white bars) and SD-Sr-ICOS-Fc (black bars) samples with MC3T3-E1 cell line at different exposure times (a) 2 d, (b) 4 d, and (c) 7 d considering different particle concentrations. ** p < 0.01 vs. untreated cells (CTR) (Dunnett’ s test). The graphs show cell viability (%) as mean and standard error obtained from three independent experiments. Cell viability was calculated with the following formula: cell viability = absorbance of sample/absorbance of control (untreated cells) × 100.

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques: Control

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

doi: 10.3390/nano11020321

Figure Lengend Snippet: Figure 14. Clonogenic assay: (a) U2OS; (b) HOS. Cells were treated with ICOS-Fc, SD-Sr-ICOS-Fc, SD-Sr, SG-Sr-ICOS-Fc, and SG-Sr at 2 and 0.2 µg/mL concentration for 72 h. Then, the cell medium was changed, and the cells were cultured for additional 7 days in a free medium (c), (d) Colonies were then photographed. Then, the cells were treated with acetic acid to induce a completely dissolution of the crystal violet and absorbance was evaluated. Three different experiments were performed. Data are shown as mean ± SEM.

Article Snippet: After the indicated time, samples were centrifuged at 10,000 rpm for 5 min, re-suspended in PBS 1X+ 1% NGS and stained with an allophycocyanin (APC)-conjugated anti-human ICOS (R&D System) monoclonal antibody for 20 min at 4 ◦C.

Techniques: Clonogenic Assay, Concentration Assay, Cell Culture, Dissolution

Journal: Cell Reports Medicine

Article Title: Response and recurrence correlates in individuals treated with neoadjuvant anti-PD-1 therapy for resectable oral cavity squamous cell carcinoma

doi: 10.1016/j.xcrm.2021.100411

Figure Lengend Snippet:

Article Snippet: ICOS (C398.4A) , Fluidigm , Cat# 3168024B, RRID: AB_2858237.

Techniques: Recombinant, Isolation, Mass Cytometry, Software

Journal: Cell reports

Article Title: Molecular Signatures of Dengue Virus-Specific IL-10/IFN-γ Co-producing CD4 T Cells and Their Association with Dengue Disease

doi: 10.1016/j.celrep.2019.11.098

Figure Lengend Snippet:

Article Snippet: CD278/ICOS, 148Nd , Fluidigm , Cat#3148019B; Clone C398.4A; RRID: AB_2756435.

Techniques: Recombinant, Staining, Activation Assay, Software

Journal: Cell Reports

Article Title: COX2 regulates senescence secretome composition and senescence surveillance through PGE 2

doi: 10.1016/j.celrep.2021.108860

Figure Lengend Snippet:

Article Snippet: Rat anti-CD278-176Yb , Fluidigm , # 3176014B.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MiR-155 -targeted IcosL controls tumor rejection.

doi: 10.1073/pnas.2408649121

Figure Lengend Snippet: Fig. 1. MiR-155 impairs T cell antitumor response at least in part by targeting ICOSL transcripts. (A) Detection of miR-155 (dark blue) in mice with miR-155 ON and OFF using ISH. Left, miR-155 detection (dark blue) when this microRNA is turned ON in Nestin-Cre x miR-155LSLtTAoffspring; Right, Lack of miR-155 expression when this microRNA has been turned OFF in the same mice. (B) Tumor infiltrates in mice with miR-155 ON for 3 mo (Top panels) then back OFF for 1 wk (Bottom panels). Top panels: Left: H&E staining of the kidney showing infiltrates (dark blue color); Middle: B220 (red signal) and CD4 (brown signal) staining of the infiltrate from the kidney indicated by the square); Right: B220 (red signal) and CD4 (brown signal) staining of a skeletal muscle. Bottom panels, tumor infiltrates in kidneys. From left to right: H&E staining at low (first panel) and high magnification (second panel); B220 staining (red, third panel, notice lack of B220-positive cells); CD4 staining (brown, fourth panel). (C) Top panels: Left: IHC for CD4 (brown) showing T cell infiltration surrounding a lymph node. Right: Magnification of a part of the image on the Left. Bottom panels: IHC for Icos (brown) on lymphomas with miR-155 ON (Left) and then turned back OFF (Right). (D) Dual-Luciferase reporter assays performed in Raw264.7 macrophages cotransfected with a Renilla luciferase reporter vector containing the human ICOSL-3′-UTR with either the WT miR-155 binding site or its mutated version, along with either a miR-Control RNA, a human miR-155 RNA, or a human miR-155-Inhibitor RNA, as indicated. n = 6 replicates/each experimental setting; P = 5.09182E-07. (E) Raw264.7 cells transfected with either a miR-Control RNA, a mouse miR-155 RNA, or a mouse miR-155- Inhibitor RNA, as indicated, were treated with either LPS (100 ng/mL) or the vehicle 24 h after transfection. Two days posttransfection, cell lysates were analyzed by western blot for IcosL expression. GAPDH was used as a loading control. (F) Two days after transfection with either a miR-Control RNA or human-miR-155 RNA, BL cell lines BJAB and NAMALWA were analyzed for ICOSL expression by western blotting. α-tubulin and GAPDH were used as loading control. (G) Inverse correlation between miR-155 and ICOSL transcripts as measured by qRT-PCR in CLL (MEC1 and MEC2) and BL (NAMALWA, BJAB, RAJI, PH3R1, DAUDI) cell lines.

Article Snippet: The specific antibodies used (source and catalog numbers) were as follows: ICOS (ProSci; 8685), ICOSL (ProSci; 8687), T cell (CD3) (Abcam; ab16669), CD163 (Abcam; ab182422), CD31 (Abcam; ab28364); and CD20 B cells (Biogenex; AM537GP).

Techniques: Expressing, Staining, Luciferase, Plasmid Preparation, Binding Assay, Control, Transfection, Western Blot, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MiR-155 -targeted IcosL controls tumor rejection.

doi: 10.1073/pnas.2408649121

Figure Lengend Snippet: Fig. 2. Turning ON the expression of miR-155 in lymphocytes of Nestin-Cre x miR-155LSLtTA mice leads to development of B cell lymphomas lacking IcosL expression, while turning miR-155 back OFF allows the formation of B (IcosL) - T (Icos) cells synapses mostly located around blood vessels. (A) Flow cytometry analyses for B220 and IcosL expression in lymph nodes of littermates with miR-155 OFF (Left) or ON (Right) for 2 mo. Representative images. (B) First row: IcosL (green; Left) and CD20 (red; Middle) staining in a WT spleen. Colocalization (yellow; Right). Second row: Lack of IcosL (green; Left) expression by CD20-positive cells (red; Middle) in lymphomas with miR-155 ON (no yellow signal, Right). IcosL-positive cells in the left are distinct from malignant B cells. Third row: IcosL-positive cells (green; Left) in lymphomas with miR-155 ON are CD31-positive (red, Middle) with colocalization (yellow; Right). Fourth row: A few benign B cells present in the mice with miR-155 ON then OFF for 1 wk had started to re-express IcosL. CD20 (green; Left); IcosL (red; Middle); colocalization (yellow; Right). Panels are representative of staining of 3 mice/group. (C) IHC for B220 (red; Top Left), CD3 (green; Top Right) in infiltrates from mice with miR-155 ON for 10 wk then back OFF for 4 d. Scattered B220-CD3 synapses are seen as yellow fluorescence in the pictures at the Bottom Left after merging B220-CD3 signals; Bottom Right is a magnification of the square in the left picture. (D) IHC for Icos (red; Top) and IcosL (green; Middle) and coexpression (yellow; Bottom) in infiltrates from mice with miR-155 ON for 2 to 3 mo then OFF for 4 d. The immunological synapses between B cells (IcosL) and T cells (Icos) are located mostly around blood vessels (ovals). (Scale bar, 100 µm.)

Article Snippet: The specific antibodies used (source and catalog numbers) were as follows: ICOS (ProSci; 8685), ICOSL (ProSci; 8687), T cell (CD3) (Abcam; ab16669), CD163 (Abcam; ab182422), CD31 (Abcam; ab28364); and CD20 B cells (Biogenex; AM537GP).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: Cell Transplantation

Article Title: Inducible Costimulator Gene-Transduced Bone Marrow-Derived Mesenchymal Stem Cells Attenuate the Severity of Acute Graft-Versus-Host Disease in Mouse Models

doi: 10.3727/096368914x684592

Figure Lengend Snippet: Figure 2. MSCsICOS-EGFP or MSCsEGFP competitively bind to B7RP-1 molecules in vitro. (a) Expression of ICOS on MSCsICOS-EGFP or MSCsEGFP and B7RP-1 on mDCs when cultured separately. (b) Expression of ICOS on MSCsICOS-EGFP or MSCsEGFP when incubated with B7RP-1 fusion protein and the expression of B7RP-1 on mDCs when incubated with ICOS fusion protein. (c) Expression of ICOS on MSCsICOS-EGFP or MSCsEGFP and B7RP-1 on mDCs after coculture for 24 h.

Article Snippet: After mDCs (100 μl) were incubated with 5 μg mouse ICOS-IgG fusion protein (R&D systems, USA) for 45 min at 37°C, expression of B7h on the mDC surface was used as B7h positive control.

Techniques: In Vitro, Expressing, Cell Culture, Incubation

Journal: Cell Transplantation

Article Title: Inducible Costimulator Gene-Transduced Bone Marrow-Derived Mesenchymal Stem Cells Attenuate the Severity of Acute Graft-Versus-Host Disease in Mouse Models

doi: 10.3727/096368914x684592

Figure Lengend Snippet: Figure 6. Roles of MSCsICOS-EGFP on GvHD induced by CD4+ T cells. (a) CD28 and ICOS signaling positively regulates T-cell responses to alloantigens and supports GvHD development. (b) MSCs are able to modulate the immune system and suppress the acti- vation, proliferation, maturation, and effector functions of CD4+ T cells by suppressing Th1 and Th17 polarization and promoting Th2 polarization. (c) ICOS-IgG competitively binds to B7h molecules on mDCs and induces the suppression of CD4+ T cells by suppress- ing Th1 and Th17 polarization and promoting Th2 polarization. (d) MSCsICOS-EGFP are able to provide the synergistic benefits of MSCs and ICOS-IgG. They competitively bind to B7h molecules on mDCs and induce the suppression of CD4+ T cells by suppressing Th1 and Th17 polarization and promoting Th2 polarization. APC, antigen-presenting cell; TCR, T-cell receptor.

Article Snippet: After mDCs (100 μl) were incubated with 5 μg mouse ICOS-IgG fusion protein (R&D systems, USA) for 45 min at 37°C, expression of B7h on the mDC surface was used as B7h positive control.

Techniques:

Journal: Molecular Systems Biology

Article Title: Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria

doi: 10.15252/msb.202110824

Figure Lengend Snippet:

Article Snippet: 151Eu‐conjugated hamster anti‐human ICOS , Fluidigm , Clone DX29; Cat# 3151020B.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Marker, Infection, Purification, Flow Cytometry, Blocking Assay, Generated, Recombinant, Saline, Staining, Software, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Inducible Costimulator and Its Ligand Promote Proliferation and Migration of Tumor Cells in Cutaneous T-Cell Lymphoma

doi: 10.3390/ijms27031408

Figure Lengend Snippet: Expression of ICOS and ICOSL in patients with cutaneous T-cell lymphoma and healthy controls. ( a , b ) The expression of ICOS and ICOSL mRNA in the lesional skin of patients with cutaneous T-cell lymphoma (CTCL) (total n = 58; early n = 29, advanced n = 29) and the normal skin of healthy controls ( n = 12). The statistical analysis was performed using the Mann–Whitney U -test. ( c ) The correlation between serum lactate dehydrogenase (LDH) or soluble interleukin-2 receptor (sIL-2R) levels and ICOS or ICOSL mRNA expression. Each curve represents the correlation line. The correlation coefficients were determined using the Spearman’s rank correlation test. ( d ) The Kaplan–Meier analysis of the overall survival between the patients with higher and lower ICOS or ICOSL expression. The differences in overall survival were assessed using the log-rank test. The optical cut-off points were determined by a receiver operating characteristic analysis. * p < 0.05, ** p < 0.01.

Article Snippet: After rehydration, the sections were autoclaved in 10 mM of sodium citrate buffer for antigen retrieval and stained with rabbit ICOS antibody (Biorbyt, Cambridge, UK), rabbit anti-ICOS Ligand/ICOSL antibody (Abcam, Cambridge, MA, USA), and rabbit IgG, polyclonal (Abcam) as the isotype control, followed by ABC staining (Vector Lab, Burlingame, CA, USA).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Inducible Costimulator and Its Ligand Promote Proliferation and Migration of Tumor Cells in Cutaneous T-Cell Lymphoma

doi: 10.3390/ijms27031408

Figure Lengend Snippet: Immunohistochemical and flowcytometric analysis of ICOS and ICOSL in patients with cutaneous T-cell lymphoma and normal controls. ( a ) The expression of ICOS, ICOSL, and the control IgG was evaluated by immunohistochemistry in skin sections from patients with mycosis fungoides (MF) (total n = 26; early n = 11, advanced n = 15), Sézary syndrome (SS) ( n = 5), and healthy controls ( n = 9). The arrows represent the well-stained areas. Representative results are shown. The scale bars represent 50 μm. ( b ) ICOS- and ICOSL-positive cell ratios in peripheral blood T-cells between the patients with SS ( n = 5) and the healthy controls ( n = 5) were compared by flow cytometry. Black curves represent isotype. The statistical analysis was performed using the Mann–Whitney U -test. ** p < 0.01.

Article Snippet: After rehydration, the sections were autoclaved in 10 mM of sodium citrate buffer for antigen retrieval and stained with rabbit ICOS antibody (Biorbyt, Cambridge, UK), rabbit anti-ICOS Ligand/ICOSL antibody (Abcam, Cambridge, MA, USA), and rabbit IgG, polyclonal (Abcam) as the isotype control, followed by ABC staining (Vector Lab, Burlingame, CA, USA).

Techniques: Immunohistochemical staining, Expressing, Control, Immunohistochemistry, Staining, Flow Cytometry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Inducible Costimulator and Its Ligand Promote Proliferation and Migration of Tumor Cells in Cutaneous T-Cell Lymphoma

doi: 10.3390/ijms27031408

Figure Lengend Snippet: Involvement of ICOS and ICOSL in cutaneous T-cell lymphoma cell lines. ( a ) ICOS and ICOSL expression was evaluated by flow cytometry in human CTCL cell lines. ( b ) HH, Hut78, SeAx, MyLa, and MJ cells (2.5 × 10 4 cells/well) were cultured with anti-ICOS (5 µg/mL) and/or anti-ICOSL (5 µg/mL) neutralizing antibodies. The statistical analysis was performed using the Mann–Whitney U -test. ( c ) HH and Hut78 cells (2.0 × 10 5 cells/well) were cultured with anti-ICOSL (5 µg/mL) neutralizing antibody, everolimus, or the control IgG for 24 h. Apoptosis was evaluated with flow cytometry using Annexin V and 7-AAD staining. The statistical analysis was performed using the Mann–Whitney U -test. mTOR: everolimus. * p < 0.05, ** p < 0.01.

Article Snippet: After rehydration, the sections were autoclaved in 10 mM of sodium citrate buffer for antigen retrieval and stained with rabbit ICOS antibody (Biorbyt, Cambridge, UK), rabbit anti-ICOS Ligand/ICOSL antibody (Abcam, Cambridge, MA, USA), and rabbit IgG, polyclonal (Abcam) as the isotype control, followed by ABC staining (Vector Lab, Burlingame, CA, USA).

Techniques: Expressing, Flow Cytometry, Cell Culture, MANN-WHITNEY, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inducible Costimulator and Its Ligand Promote Proliferation and Migration of Tumor Cells in Cutaneous T-Cell Lymphoma

doi: 10.3390/ijms27031408

Figure Lengend Snippet: Western blotting analysis and in vivo effects. ( a ) Hut78 cells were cultured with anti-ICOSL neutralizing antibody (5 µg/mL) for 0, 6, 12, and 24 h for the phosphorylation of AKT, ERK1/2, p38 MAPK, and JNK. ( b ) Changes in the phosphorylation of the signal transduction molecules. The intensities of each signaling molecule were measured using ImageJ version 1.53e. The relative optical density of p-AKT/AKT, p-ERK/ERK, p-p38 MAPK/p38 MAPK, and p-JNK/JNK was calculated and normalized to the value of the “0 h” group. The statistical analysis was performed using the Mann–Whitney U -test. ( c ) Hut78 cells (5.0 × 10 6 cells) in 100 µL of PBS were injected subcutaneously into the abdomen of NOD/SCID interleukin-2 receptor γ-chain-deficient (NSG) mice. Anti-ICOS, anti-ICOSL neutralizing antibody, or isotype IgG were injected on days 0, 4, 7, 11. Representative images are shown. ( d ) Tumor volume was evaluated using the following equation: V = π (L1 × L2)/6, where V = volume (mm 3 ), L1 = the longest diameter (mm), and L2 = the shortest diameter (mm). The statistical analysis was performed using the Mann–Whitney U -test. * p < 0.05, ** p < 0.01.

Article Snippet: After rehydration, the sections were autoclaved in 10 mM of sodium citrate buffer for antigen retrieval and stained with rabbit ICOS antibody (Biorbyt, Cambridge, UK), rabbit anti-ICOS Ligand/ICOSL antibody (Abcam, Cambridge, MA, USA), and rabbit IgG, polyclonal (Abcam) as the isotype control, followed by ABC staining (Vector Lab, Burlingame, CA, USA).

Techniques: Western Blot, In Vivo, Cell Culture, Phospho-proteomics, Transduction, MANN-WHITNEY, Injection