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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The role of metalloproteinase ADAM17 in regulating ICOS ligand-mediated humoral immune responses.

doi: 10.4049/jimmunol.1302893

Figure Lengend Snippet: FIGURE 5. ICOSL is downregulated by ADAM17 on the surface of B cells. (A and B) Flow cytometry analysis of cell surface levels of the indicated costimulatory mole- cules in splenocytes of unimmunized ADAM17+/+ and ADAM17ex/ex mice that were incubated in vitro with one of the fol- lowing: 250 ng/ml PMA, 10 mg/ml anti-IgM F(ab9)2, 1:1 with L or L-ICOS cells for 2 h. (C) Flow cytometry analysis of the cells isolated from the draining LNs and spleens of mice immunized with OVA plus anti- CD40 mAbs and poly(I:C). The percentage of CD40+ or ICOSL+ cells among the gated B cells (B220+) is shown in histograms. Data are from 1 experiment and are repre- sentative of at least 5 experiments (A and B), or from 4–10 experiments, with a horizontal line showing the mean in each group (C). **p , 0.01 by t test.

Article Snippet: One hundred nanograms of recombinant murine ICOSL-IgG fusion protein was incubated with the indicated ratio of recombinant mouse (rm)ADAM17 (R&D Systems) or rhADAM17 in 20 mMHEPES or 50 mM Tris buffer for up to 5 h. The samples were resolved by SDS/6–16% PAGE under reducing conditions and visualized by ECL (BioRad) after incubation with goat anti-mouse ICOSL Abs (R&D Systems), followed by rabbit anti-goat IgG Abs conjugated to HRP (Millipore) or with goat anti-rabbit IgG conjugated to HRP (Sigma-Aldrich).

Techniques: Flow Cytometry, Incubation, In Vitro, Isolation

Journal: Nanomaterials

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: ICOS-Fc grafting on amino-MBGs surface.

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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: N 2 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: SSA BET and total pore volume of SG-Sr, SD-Sr, SG-Sr-ICOS-Fc, and SD-Sr-ICOS-Fc.

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: Pore Size

Journal: Nanomaterials

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: Difference FT-IR spectra of SG-Sr-ICOS-Fc ( a ) SD-Sr-ICOS-Fc ( b ); the spectra of SG-Sr and SD-Sr have been subtracted, respectively.

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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: Strontium release profiles 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: ELISA-like assay on ( a ) SG-Sr-ICOS-Fc, SD-Sr-ICOS-Fc, SG-Sr, SD-Sr, and ( b ) unb ICOS-Fc after grafting reaction. The binding and the amount of ICOS-Fc were detected by ELISA-like assay using the human ICOSL-His as the capture protein and anti-human-Hrp as detection antibody. Data are reported as Optical Density (OD) values and as concentrations expressed in μg/mL (mean and standard error were obtained from three independent samples evaluated in duplicate).

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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: Stability test of ICOS-Fc binding to MBG surface. The amount of ICOS-Fc released was evaluated for both SG-Sr-ICOS-Fc and SD-Sr-ICOS-Fc at time steps of 3, 7, 14 and 21 days.

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: Binding Assay

Journal: Nanomaterials

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: Cells were plated onto the apical side of Matrigel-coated filters in the presence and absence of either SD-Sr-ICOS-Fc, SG-Sr-ICOS-Fc, SD-Sr, and SG-Sr. ICOS-Fc was used as positive control. FBS 20% was loaded in the basolateral chamber as a chemotactic stimulus. Data are expressed as mean± SEM ( n = 5) of the percentage of migration versus control migration (FBS 20%). * p < 0.05; ** p < 0.01 SG-Sr-ICOS-Fc vs. SG-Sr and SD-Sr-ICOS-Fc vs. SD-Sr.

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: Positive Control, Migration, Control

Journal: Nanomaterials

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: 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: Nanomaterials

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

doi: 10.3390/nano11020321

Figure Lengend Snippet: Effect of ICOS-grafted MBGs on MDOC differentiation using the T0–21 treatments. Monocytes were induced to differentiate to MDOCs in the presence and absence of SG-Sr-ICOS-Fc and SD-Sr-ICOS-Fc from day 0 (T0–21 treatment). ( a ) Phase-contrast microscopy of cells at T21 observed at X20 original magnification ( b ) Microphotographs of TRAP staining at T21 were observed at original magnification X20. ( c ) Bar graphs show the percentage of the multinuclear TRAP+ cells at T21. Data are expressed as the mean± SEM of the percentage of inhibition versus the control (set at 100%) by counting 10 fields per sample (** p < 0.01 versus the control; Dunnett’s test). Scale bar 125 μm.

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: Microscopy, Staining, Inhibition, Control

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet: Validation of co-signaling receptor modulation in independent mouse models, related to Figure 2 (A) Experimental setup. 10 6 naive Cor93 T (Cor93 T N ) cells were adoptively transferred into HBV-Tg mice (lineage MUP-core). 24 h later, indicated groups of mice were injected intraperitoneally with PBS or 100 μg of monoclonal antibodies (mAbs) blocking PD-1, LAG-3, or CTLA-4. Livers were collected and analyzed at day 5. (B) Total numbers of intrahepatic leukocytes (IHLs) isolated from the indicated mice. (C) Total numbers of Cor93 T cells in the livers of the indicated mice. (D) Representative density plots of IFN-γ expression among Cor93 T cells in the liver of the indicated mice. Numbers represent the percentage of cells within the indicated gates. (E) Total number of IFN-γ-producing Cor93 T cells in the livers of the indicated mice upon ex vivo cognate peptide stimulation. n = 3–4; one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction. Each group was compared with PBS-injected controls. (F) Amount of serum alanine transaminases (sALTs) in the serum of the indicated groups of mice at the indicated time points. (G) Experimental setup. HBV replication-competent transgenic mice (lineage 1.3.32) were injected intraperitoneally with PBS or with 100 μg of agonist mAbs activating OX40 or 4-1BB. Livers were collected and analyzed at day 4. (H) Total numbers of IHL isolated from the indicated mice. (I) Representative micrographs of liver sections from the indicated groups of mice. The upper panels show hematoxylin-eosin (H&E) staining, the middle panels show immunohistochemical staining for cleaved caspase 3 (ΔCas3, brown), and the lower panels show immunohistochemical staining for HBcAg (brown). Scale bar represents 100 μm. (J) HBV DNA quantification by southern blot analysis of liver lysates from the indicated mice. Bands corresponding to the expected size of the integrated transgene (Tg), relaxed circular (RC), double-stranded (DS) linear, and single-stranded (SS) HBV DNAs are indicated. (K) Amount of sALT in the serum of the indicated groups of mice at the indicated time points. (L) Experimental setup. 10 6 Cor93 T N cells were adoptively transferred into HBV-Tg mice (lineage MUP-core). 24 h later, selected groups of mice were injected intraperitoneally with PBS or 100 μg of mAbs activating ICOS, OX40, or 4-1BB. Livers were collected and analyzed at day 5. (M) Total numbers of IHL isolated from the indicated mice. (N) Numbers of Cor93 T cells isolated from the liver of the indicated mice. (O) Representative density plots of IFN-γ expression among Cor93 T cells in the liver of the indicated mice. (P) Total number of IFN-γ-producing Cor93 T cells in the livers of the indicated mice upon ex vivo cognate peptide stimulation. n = 3–4; one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with control. (Q) Amount of sALT in the serum of the indicated group of mice at the indicated time points. n = 3–4; two-way ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with control group (simple effect within row). (R) Experimental setup. 10 6 naive Env28 CD8 + TCR transgenic cells (Env28 T N ) were adoptively transferred into HBV replication-competent transgenic mice (lineage 1.3.32; background C57BL/6 × BALB/c H-2 bxd hybrids). 24 h later, selected groups of mice were injected intraperitoneally with PBS or 100 μg of mAbs activating OX40 or 4-1BB. Livers were collected and analyzed at day 5. (S) Numbers of IHL isolated from the indicated mice. (T) Numbers of Env28 T cells isolated from the liver of the indicated mice. (U) Representative density plots of IFN-γ expression among Env28 T cells in the liver of the indicated mice. (V) Percentages of IFN-γ-producing Env28 T cells in the livers of the indicated mice upon ex vivo cognate peptide stimulation. n = 3–4; one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction. Each group was compared with PBS-injected controls. (W) Amount of sALT in the serum of the indicated groups of mice at the indicated time points. n = 3–4; two-way ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with PBS-injected controls (simple effect within row). (X) Representative micrographs of liver sections from the indicated groups of mice. The upper panels show staining for HBcAg, and the lower panels show staining for cleaved caspase 3 (ΔCas3). Scale bar represents 100 μm. (Y and Z) Representative histograms (Y) and percentages (Z) of in vitro differentiated Cor93 T effector (Cor93 T E ) or Env28 T effector (Env28 T E ) cells producing IFN-γ upon cognate in vitro peptide stimulation at the indicated concentrations. Results are representative of two independent experiments giving similar results.

Article Snippet: InVivoMAb anti-mouse/human/rat/monkey ICOS (CD278) , Bio X Cell , Cat# BE0353; RRID: AB_2894772.

Techniques: Biomarker Discovery, Injection, Bioprocessing, Blocking Assay, Isolation, Expressing, Ex Vivo, Transgenic Assay, Staining, Immunohistochemical staining, Southern Blot, Control, In Vitro

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet:

Article Snippet: InVivoMAb anti-mouse/human/rat/monkey ICOS (CD278) , Bio X Cell , Cat# BE0353; RRID: AB_2894772.

Techniques: Purification, Virus, Recombinant, Staining, Saline, Fluorsave, Sequencing, DNA Labeling, Cell Isolation, Sample Prep, Reverse Transcription, Software, Microscopy

Journal: Nature

Article Title: T FH -derived dopamine accelerates productive synapses in germinal centres

doi: 10.1038/nature23013

Figure Lengend Snippet: a , b , Representative immunohistochemistry for CgB (brown) of human lymph node ( a ) and spleen ( b ). (n=10). c , Quantification of CD3 + CgB + cells in human tonsils, lymph nodes (n=10) and spleens (n=5). d , Percentage of CgB + T cells in human reactive and neoplastic conditions. c,d , ns, not significant, *p ≤ 0.05 and **p ≤ 0.01; nonparametric Mann-Whitney test (U test). e , Representative double immunohistochemistry for CgB (left) and CD3 (middle) after colour deconvolution. Pseudo-colour image (right) showing signal colocalisation. Original magnification 40X. Scale bar 100 μm (n=3). f , Representative immunofluorescence images for CD3 (green) and ICOS (red) in human GCs.

Article Snippet: Sections were then stained using CD3 (LN10 or polyclonal, Dako), PD-1 (NAT105, CNIO, Madrid), CXCR5 (51505, R&D), ICOS (AF169, R&D), BCL6 (LN22, Novocastra), CgB (H300, Santa Cruz), DRD1 (L205G1, BioLegend) for 1h at RT in the dark, followed by anti-mouse Alexa Fluor 488 (A-21202, Invitrogen) and anti-rabbit Alexa Fluor 594 (A-21207, Invitrogen) for 30 min at RT in the dark.

Techniques: Immunohistochemistry, MANN-WHITNEY, Immunofluorescence

Journal: Nature

Article Title: T FH -derived dopamine accelerates productive synapses in germinal centres

doi: 10.1038/nature23013

Figure Lengend Snippet: a , Activated human T cells that express ICOS and CD40L were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3) as a basal condition with a ring of ICAM-1 surrounding a central cluster enriched in T cell receptor enriched extracellular vesicles by 15 minutes . This condition resulted in low presentation of CD40L in punctate structures detected by anti-CD40L mAb that accumulated in the same central synapse with the TCR enriched extracellular vesicles. Addition of ICOSL the SLB resulted in strong central accumulation of fluorescent ICOSL with the TCR enriched extracellular vesicles, but no increase in CD40L presentation. Addition of CD40 the SLB resulted in a significant increase in CD40L accumulation, which we refer to as reception because its receptor dependent. When ICOSL and CD40 were added the reception of CD40L was further significantly enhanced over the level observed with CD40 alone. Thus, ICOSL ligation in the centre of the immunological synapse increases CD40L reception. All levels are shown in gray scale except CD40L panels, for which the pseudocolor scale is indicated. Scale bar 5 µm. b , Human T FH cells were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3). Addition of ICOSL resulted in increased accumulation of CgB at the synapse centre. Addition of CD40 did not further increased CgB accumulation.

Article Snippet: Sections were then stained using CD3 (LN10 or polyclonal, Dako), PD-1 (NAT105, CNIO, Madrid), CXCR5 (51505, R&D), ICOS (AF169, R&D), BCL6 (LN22, Novocastra), CgB (H300, Santa Cruz), DRD1 (L205G1, BioLegend) for 1h at RT in the dark, followed by anti-mouse Alexa Fluor 488 (A-21202, Invitrogen) and anti-rabbit Alexa Fluor 594 (A-21207, Invitrogen) for 30 min at RT in the dark.

Techniques: Incubation, Ligation

Journal: Nature

Article Title: T FH -derived dopamine accelerates productive synapses in germinal centres

doi: 10.1038/nature23013

Figure Lengend Snippet: a , Representative ICAM-1 area quantification. b , ICAM-1 area expressed as relative units (r.u.). c-e , Interactions (white) among untreated (green), or FSK-stimulated (blue) T FH cells and allogeneic GC B cells (red) cultured in the same well. For each interaction two frames are shown, numbers indicate time after starting imaging (see corresponding Supplementary Video 1 ). Plots represent quantification of T:B interaction duration ( d ) and contact area ( e ). b,d,e , ns, not significant, ***p ≤ 0.001; Mann-Whitney test. f , Impact of the speed of ICOSL upregulation (Δt ICOSL ; fast, black and grey lines; and slow, colored lines) in GC B cells onto GC characteristics (mean affinity ( left ) and produced output ( right )) estimated with computer simulations. Simulations were repeated with short (black, red, orange lines) and long (grey, magenta, cyan lines) periods of search for T FH cells. Slow ICOSL upregulation had the tendency to shrink GCs (see Ω in Table M2 , red and magenta lines), therefore, the GC strength was restored by parameter adaptation (see , orange and cyan lines). Lines show mean of 100 simulations, grey shades show the standard deviations (details in supplementary methods). g , Graphic model of the proposed positive feedback between human T FH and GC B cells. Upon cognate interactions between T FH and GC B cells ( 1 ), dopamine (DA) is released from CgB + granules ( 2 ). DA activates dopamine receptor 1 (DRD1) on GC B cells ( 3 ) and induces increase ICOSL surface expression ( 4 ), which in turn binds to ICOS on T FH cells, inducing CD40L membrane relocation ( 5 ) and CgB + granule formation ( 6 ).

Article Snippet: Sections were then stained using CD3 (LN10 or polyclonal, Dako), PD-1 (NAT105, CNIO, Madrid), CXCR5 (51505, R&D), ICOS (AF169, R&D), BCL6 (LN22, Novocastra), CgB (H300, Santa Cruz), DRD1 (L205G1, BioLegend) for 1h at RT in the dark, followed by anti-mouse Alexa Fluor 488 (A-21202, Invitrogen) and anti-rabbit Alexa Fluor 594 (A-21207, Invitrogen) for 30 min at RT in the dark.

Techniques: Cell Culture, Imaging, MANN-WHITNEY, Produced, Expressing, Membrane

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Secretion of the ICOS-SV from ICOS-SV–overexpressing cells. A, RT-PCR to detect the ICOS-SV in human T cells. Total RNA was isolated from purified T cells in PBMCs of a healthy donor. Expression of the ICOS variant was examined by RT-PCR with specific primers. RT-PCR of ICOS-FL is shown as a control. B, DNA sequencing of ICOS-SV cDNA from human T cells. The cDNA of the PCR product was cloned into a TOPO TA vector. The ICOS-SV was confirmed by DNA sequencing using an M13 forward primer. Splicing point is shown by the red arrow. C, DNA sequencing of ICOS-FL cDNA is shown as a control. No splicing occurs in the region corresponding to the position in ( B ), as shown in blue. D, Expression of ICOS-SV and ICOS-FL in 293T cells determined by immunoblot. 293T cells were transduced with recombinant GFP retroviral vector encoding either ICOS-SV or ICOS-FL as a positive control. The parental cells and the GFP-empty vector–transduced cells were used as negative controls. Sample loading was normalized to actin. E, Cell culture supernatants were collected from the cells described in ( A ). Secreted ICOS-SV was examined by ELISA. Two-tailed unpaired t test was used to compare ICOS secretion between the two groups. ***, P < 0.001. Standard deviation of the mean (SD) is shown. F, To validate the secreted ICOS variant protein in the cell supernatant, immunoprecipitation, SDS-PAGE, and immunoblotting assays were performed. Sample loading was normalized to cell numbers.

Article Snippet: In brief, for sICOS in patient samples, 4 μg/mL of anti-ICOS capture antibody (R&D Systems) was coated on Costar ELISA plates (Corning) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Purification, Expressing, Variant Assay, Control, DNA Sequencing, Clone Assay, Plasmid Preparation, Western Blot, Transduction, Recombinant, Retroviral, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Standard Deviation, Immunoprecipitation, SDS Page

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Suppressive effects of sICOS-SV on the costimulation of T cells. A, CHO-K1 cells were transduced with either ICOSL-expressing lentiviral vector or GFP-empty vector (CHO-ICOSL − ) as a negative control. Expression of ICOSL on the cell surface of CHO-K1 was examined by flow cytometry using anti-ICOSL (open histogram with solid line) or lentivirus GFP-empty vector (open histogram with dotted line). Isotype antibody was used as a negative control (filled histogram). B, ICOSL-expressing CHO-K1 cells were stained with ICOS-SV Ig (red line), control IgG Ig (blue line), or PD-1 Ig (orange line) and analyzed by flow cytometry. Both soluble control Ig and PD-1 Ig were used as negative controls. GFP-empty vector-expression CHO-K1 cells stained with ICOS-SV Ig (green line) were used as another negative control for the binding assay. Isotype antibody control was used as a negative control (gray filled histogram). C, CD154 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. D, Two-tailed unpaired t test was used to compare expression of CD154 between two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. E, CD69 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. F, Two-tailed unpaired t test was used to compare expression of CD69 between the two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. G, CHO cells transduced with GFP-empty vector (CHO-ICOSL − ) were used as negative controls. Sample loading was normalized to total pan-Akt. H, T-cell proliferation was determined by a [ 3 H]-TdR thymidine incorporation assay. CHO cells transduced with empty vector (CHO-ICOSL − ) were used as negative controls. Two-tailed unpaired t test was used to compare 3 H uptake between the two groups, respectively. *, P < 0.1. Standard deviation of the mean (SD) is shown. I, Schematic diagram of ICOS/ICOSL costimulatory T-cell proliferation, as well as the blocking function of the sICOS-SV. Depicted are the cytoplasmic tail sequences of ICOS-FL and sICOS-SV isoforms. The YMFM Src Homology 2 (SH2) binding motif in the cytoplasmic tail of the ICOS-FL is highlighted in pink. Upon ICOS engagement by the ICOSL on CHO cells, the unique YMFM motif recruits a p85a and a p50a subunits of PI3K, resulting in the elevated phosphorylation of Akt, thereby inducing PI3K activity. In contrast, the ICOS-SV, a truncated isoform lacking the YMFM motif in its cytoplasmic tail, cannot elicit phosphorylation of Akt. Consequently, it fails to promote T-cell proliferation. The secreted ICOS-SV (red) competes with membrane-bound ICOS for binding to ICOSL, thereby blocking the interaction between ICOSL and membrane ICOS. As a result, the sICOS-SV suppresses phosphorylation of Akt and T-cell proliferation, leading to the inhibition of T-cell immunity. The diagram was created with BioRender.com. All data shown are representative of at least 2 independent experiments.

Article Snippet: In brief, for sICOS in patient samples, 4 μg/mL of anti-ICOS capture antibody (R&D Systems) was coated on Costar ELISA plates (Corning) overnight at 4°C.

Techniques: Transduction, Expressing, Plasmid Preparation, Negative Control, Flow Cytometry, Staining, Control, Binding Assay, Incubation, Two Tailed Test, Standard Deviation, Thymidine Incorporation Assay, Blocking Assay, Phospho-proteomics, Activity Assay, Membrane, Inhibition

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: sICOS from melanoma patients inhibits T-cell activation and proliferation induced by GM-CSF–driven DCs in MLRs. A, CD4 + T cells (responders) were stimulated by allogeneic GM-CSF–driven MoDCs (stimulators: negative control DCs, DCs generated by GM-CSF/IL4 + anti-IgG, or DCs generated by GM-CSF/IL4 + anti-CD116) in MLRs. T cells were assessed for activation via flow cytometry using anti-ICOS (blue), anti-GITR (green), or anti-CD25 (gray). Isotype control antibodies were used as negative controls. B, Statistical analysis of the percentage of CD4 + ICOS + , CD4 + GITR + , and CD4 + CD25 + T-cell populations from different groups as described above. C, Soluble ICOS levels were determined by ELISA using supernatants from the MLRs described above. D – E, Serum from sICOS-high and sICOS-negative patients were added to the MLRs, and T-cell proliferation ( D ) and CD69 expression ( E ) were evaluated. Additionally, sICOS was depleted from sICOS-high serum to assess its effects on T cells. F, Statistical analysis of the percentage of CD4 + CD69 + T-cell populations from different groups as described above. All data shown are representative of at least 2 independent experiments. Two-tailed unpaired t test was used between the two groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant ( P > 0.05). Standard deviation of the mean (SD) is shown.

Article Snippet: In brief, for sICOS in patient samples, 4 μg/mL of anti-ICOS capture antibody (R&D Systems) was coated on Costar ELISA plates (Corning) overnight at 4°C.

Techniques: Activation Assay, Negative Control, Generated, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Standard Deviation