Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: 4-1BBL + B cells in GBM patients’ peripheral blood and Ag-presenting function. (A) Box plot showing the expression of 4-1BBL in CD20 + CD19 + B cells from newly diagnosed GBM patients’ PBMCs ( n = 90). Box plots express the following values: median (line inside the box), 75th and 25th percentiles (box extremities), and maximum and minimum values (dashes). (B) Histograms representing the intracellular expression of TNFα and IFNγ and surface expression of CD69 and CD86 by 4-1BBL − (black line) and 4-1BBL + (blue line) B cells. (C) Linear regression analysis of 4-1BBL expression by B cells and CD69 expression by CD8 + T cells in newly diagnosed GBM patients’ PBMCs ( n = 68). (D and E) CD8 + T cell costimulation assay using CD8 + T cells activated with anti-CD3 and IL-2 mixed with autologous 4-1BBL − and 4-1BBL + B cells from peripheral blood of three newly diagnosed GBM patients (NU00856, NU01006, and NU00429). CD8 + T cell activation was measured as cellular expansion (D) and expression of intracellular IFNγ and GzmB (E). The experiment was performed in triplicate. (F) B Vax from GL261-OVA mice were pulsed with SIINFEKL (B Vax(SIINFEKL) ) and evaluated for SIINFEKL presentation by MHC class I (H-2K b + SIINFEKL Ab) and the coexpression of MHC class I (H-2K b ) and costimulatory molecules CD86 and 4-1BBL. A representative experiment of three independent experiments is shown. (G and H) B Vax were tested for their ability to uptake Alexa Fluor 488–conjugated OVA (B Vax[OVA] ; G) and present SIINFEKL peptide via MHC class I (H-2K b ; H). Surface transport of the H-2K b + SIINFEKL complex was inhibited using brefeldin A (BFA). A representative experiment of three independent experiments is shown. Scale bars represent 15 µm (fluorescence images) and 50 µm (fluorescence and bright light image). (I) B Naive , B Naive + IFNγ, B Vax , and DCs were pulsed with OVA and tested for their ability to promote OT-I CD8 + T cell activation assessed by cell proliferation (expansion index, x axis) and intracellular expression of GzmB (y axis). The experiment was performed in triplicate. A representative experiment of two independent experiments is shown. (J) OT-I CD8 + T cells cultured with B Naive ; B Vax pulsed with OVA and isotype control, B Vax(OVA) + IC; or MHC class I blocking Ab, B Vax(OVA) + anti-H2K b , and tested for their cellular expansion. A representative experiment of two independent experiments is shown. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Expressing, Activation Assay, Fluorescence, Cell Culture, Control, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: 4-1BBL–expressing B cells. (A) 4-1BBL expression levels by CD19 + B cells in the brain, blood, dCLNs, and superficial CLNs of CT2A glioma-bearing mice over time: no tumor (D0), 7, 14, and 21 d after tumor implantation ( n = 4 mice/time point). (B) Murine B cell ability to promote CD8 + T cell activation, measured by cell expansion and expression of intracellular GzmB, was assessed by activated B cells and compared with B cells from 4-1BBL–deficient (4-1BBL KO) mice. B cells were incubated with BAFF, anti-IgM, anti-CD40, and/or recombinant mouse (rm)IFNγ. Representative experiment of a total of three independent experiments performed in triplicate. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Expressing, Tumor Implantation, Activation Assay, Incubation, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: B Vax generation. (A) Human B cells from PBMCs were treated with 10 U/ml IFNγ for 24 h. CD86 expression was assessed by flow cytometry. Representative histogram of three independent experiments. (B) Murine B cells from WT C57BL/6 or IFNγR–deficient (IFNγR KO) mice were incubated with 5 μg/ml CD40 activating Ab ± 10 U/ml IFNγ. Expression of CD86 was assessed by flow cytometry. Representative histogram of four independent experiments. (C) Stepwise schema of B Vax generation in vitro. B Vax were produced from 4-1BBL + B cells isolated from tumor-bearing SLOs such as the spleen and deep and superficial CLNs. Cells were then activated with 5 μg/ml CD40 activating Ab and supplemented with 100 nM of B cell survival factor BAFF, which after 24 h allows the up-regulation of CD86, H-2K b , and IFNγ receptor 1 (IFNγRI) compared with only BAFF-treated B cells. The addition of IFNγ for an additional 24 h allowed further up-regulation of CD86 compared with only anti-CD40–treated B cells. Histograms represent mean ± SD of n = 3 mice/treatment. gMFI, geometric mean fluorescence intensity. (D) B Vax overexpress both IA b (MHC class II) and H-2K b compared with only BAFF-treated B cells (B Naive ). Flow cytometry dot plot representative of n = 4 mice/group. (E) B cell subsets (B Naive ± IFNγ and B Vax ) and DCs were tested for their ability to present SIINFEKL peptide to OT-I CD8 + T cells measured by cell proliferation and expression of intracellular GzmB. Representative experiment of three independent experiments performed in triplicate. (F) Same experiment as in E was performed using in addition CD8 + T cells from WT C57BL/6 mice as negative control. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Expressing, Flow Cytometry, Incubation, In Vitro, Produced, Isolation, Fluorescence, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: B Vax APC function in vivo. (A) Rag1-deficient mice were challenged intracranially with GL261-OVA. 9 d after, mice received i.v. B Naive or B Vax pulsed with OVA protein. eFluor450-labeled CD8 + T cells from WT C57BL/6 mice were concomitantly injected with B cells ( n = 4 mice/group). 7 d after the cell adoptive transfer, eFluor450 + CD8 + T cells were evaluated by flow cytometry in the tumor-bearing brains and the dCLNs. A representative experiment of two independent experiments is shown. (B) B KO mice were challenged intracranially with GL261-OVA. 9 d after, mice received i.v. B Naive , 4-1BBL − B cells activated with CD40 agonism and IFNγ (B Act ) or B Vax pulsed with OVA protein. A group of B Vax(OVA) mice was pretreated with PTX before injection ( n = 4 mice/group). 7 d after the cell adoptive transfer, SIINFEKL-specific CD8 + T cells were analyzed in the tumor-bearing brains by flow cytometry using SIINFEKL-tetramer. A representative experiment of two independent experiments is shown. (C) B KO mice were challenged intracranially with CT2A cells. 9 d after, mice received i.v. B Naive and B Vax pulsed with CT2A tumor lysates pretreated with or without PTX ( n = 5 mice/group). 7 d after the cell adoptive transfer, CD8 + T cells were evaluated for the intracellular expression of GzmB and IFNγ in the tumor-bearing brain, blood, and dCLNs. A representative experiment of three independent experiments is shown. (D) Rag1-deficient (KO) mice were challenged intracranially with CT2A cells. 9 d after, mice i.v. and concomitantly received both CellTracker red CMPTX B Vax (red) cells and CellTracker green CMFDA-labeled CD8 + T cells (green). B Vax and CD8 + T cell splenic localization was analyzed by fluorescent microscopy. Scale bars represent 100 µm (left image, 20× magnification) and 50 µm (right image, 63× magnification). Images are representative of the spleen and CLN of three mice. For all experiments shown in this figure, differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: In Vivo, Labeling, Injection, Adoptive Transfer Assay, Flow Cytometry, Expressing, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: 4-1BBL is a key marker for B Vax therapeutic effect. (A) In B cell–deficient mice, B Naive and B Vax were detected in draining dCLNs and the circulation, and B Vax were found in the tumor-bearing brains. Histogram represents mean ± SD of n = 5 mice/group. (B) CD45.1 + B Naive and B Vax were intracranially injected using cannula-guided injections 10 d after tumor implantation. 4 d after, CD45.1 + cells were magnetically isolated from tumors and tested for their ability to activate CD8 + T cells, measured by cell proliferation (expansion index) by flow cytometry. Histogram represents mean ± SD of n = 3 mice/group. (C) B Naive , B Act , and B Vax were tested for their therapeutic effect in CT2A-bearing B cell–deficient (B KO) mice ( n = 9 or 10 mice/group). (D) CT2A-bearing B cell–deficient mice treated with B Vax (pretreated with 4-1BBL–blocking Ab before injection) ± 4-1BBL–blocking Ab (500 μg/mouse × three i.p. injections after B Vax adoptive transfer) were monitored for survival ( n = 10 mice/group). Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Survival curves were generated via the Kaplan-Meier method and compared by log-rank test, and multiple comparisons were adjusted using Bonferroni method. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Marker, Injection, Tumor Implantation, Isolation, Flow Cytometry, Blocking Assay, Adoptive Transfer Assay, Generated

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: Radiotherapy favors B cell adaption in vivo. (A) CT2A-bearing mice were irradiated (RT), and CD45.1 + B Vax were adoptively transferred i.v. CD45.1 + cells were analyzed by flow cytometry in the spleens, dCLNs, and tumor-bearing brains ( n = 4 mice/group). (B) Irradiated CT2A-bearing mice were bled 36 h and 5 d after RTx ( n = 3 mice/group). Serum BAFF levels were analyzed and compared with nonirradiated (Tumor) and non–tumor-bearing mice serum (No tumor). (C) The same experiment as in A was performed using CD45.1 + B Naive , B Act (4-1BBL − B cells activated with anti-CD40 and IFNγ), or B Vax . CD45.1 + cells were analyzed by flow cytometry in the spleen and dCLN. B cell proliferative status was assessed by the expression of Ki67. A representative animal for each group ( n = 3 or 4 mice/group) is depicted. The experiment was repeated twice independently. (D) Alternatively, a group of mice received B cells pretreated with BAFF-R–blocking Ab. The treatment was also administered to the mice i.v. for 3 consecutive d after B cell transfer. (E) Irradiated CT2A-bearing mice received i.v. vehicle (Mock, black line), CD8 + T cells (gray line), pulsed B Vax (black dashed line), or combined pulsed B Vax + CD8 + T cells (pink dotted line). The experiment was performed using n = 10 mice/group. A representative experiment of three independent experiments is shown. (F) Irradiated CT2A-bearing mice received i.v. vehicle (Mock, black line), CD8 + T cells (gray line), CD8 + T cells i.v. + pulsed DC Vax administered either i.d. (DC Vax(i.d.) ; dashed black line) or i.v. (DC Vax(i.v.) ; blue line), or pulsed B Vax + CD8 + T cells (pink dotted line). The experiment was performed using n = 10 mice/group. A representative experiment of two independent experiments is shown. (G) Irradiated CT2A-bearing mice received i.v. eFluor450-labeled B Vax or DCs. DCs were administered either i.v. (DC (i.v.) ) or i.d. (DC (i.d.) ). 7 d after cell transfer, eFluor450 + cells were analyzed in the dCLN and spleen ( n = 3 or 4 mice/group). A representative experiment of two independent experiments is shown. Differences between two groups were analyzed by Student’s t test. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. Histograms are shown as mean ± SD. Survival curves were generated via the Kaplan-Meier method and compared by log-rank test, and multiple comparisons were adjusted using the Bonferroni method. Statistical significance is depicted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not statistically significant.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: In Vivo, Irradiation, Flow Cytometry, Expressing, Blocking Assay, Labeling, Generated

Journal: The Journal of Experimental Medicine

Article Title: Activation of 4-1BBL + B cells with CD40 agonism and IFNγ elicits potent immunity against glioblastoma

doi: 10.1084/jem.20200913

Figure Lengend Snippet: B Vax treatment confers tumor eradication. (A) PD-L1 membrane expression was evaluated in B Vax and B Naive cells. Representative dot plot of four independent experiments. Histograms are shown as mean ± SD. (B) CD8 + T cell infiltration in CT2A tumor in control group (14 d after tumor inoculation, n = 2). Scale bar represents 250 μm. Number of CD8 + T cells per section was assessed in the tumor area (tumor) versus nontumor (brain) area. (C) Immune cell infiltration in LTSs treated with either B Vax and CD8 + T cells (LTS-B Vax +CD8, n = 5) or only CD8 + T cells (LTS-CD8, n = 4) were compared with control CT2A-bearing (Control, n = 3) and Mock brains (No tumor, n = 2). Representative dot plot showing lymphocyte and myeloid cell compartment distribution (CD45 and CD11b expression) and CD8 + and CD4 + T cell distribution. Within the CD4 + T cell compartment, the expression of CD44 and Foxp3 was evaluated. Within the non–T cell compartment (CD8 − CD4 − lymphocytes), the CD19 + B cell compartment–expressing IFNγ and 4-1BBL was evaluated. Values of the populations of interest are shown as mean ± SD. Differences between two groups were analyzed by Student’s t test. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey's multiple comparisons test. GBM patient–derived B Vax promoted anti-tumor CD8 + T cells. (D) Schema of generation of GBM patient–derived B Vax . CD8 + T cell activation and expansion and CD8 + T cell–mediated tumor cell killing assay was performed in autologous settings. (E) Freshly resected tumors from GBM patients were cultured ex vivo as tumor spheroids. Representative picture of adherent cells after 5 d of culture. Scale bars represent 100 μm. SSC, side scatter.

Article Snippet: In a parallel experiment, B Vax were pretreated with 10 μg/ml of 4-1BBL blocking Ab (clone TKS-1; BioXCell) before injection, and mice received two i.p. injections of 500 μg/kg.

Techniques: Membrane, Expressing, Control, Derivative Assay, Activation Assay, Cell Culture, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

doi: 10.1084/jem.20132687

Figure Lengend Snippet: Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and streptavidin-APC. Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.

Article Snippet: Naive T cells were preactivated with 2 μg/ml of plate-bound anti-CD3 (2C11) and 0.5 μg/ml of soluble anti-CD28 (37N5) for 48 h. To determine secondary responsiveness, these activated/effector T cells were resorted for identical expression of 4-1BB and recultured with 0.5 μg/ml of plate-bound anti-CD3 in the presence of 10 μg/ml of control rat IgG or 10 μg/ml anti–4-1BB (3H3) without any APCs for another 24 h. In some experiments, effector CD8 T cells were recultured with varying concentrations of recombinant mouse 4-1BBL (his-tagged and cross-linked with anti-polyhistidine antibody; R&D Systems) or irradiated 4-1BBL + hybridoma cells in the presence of 10 μg/ml of control rat IgG or a blocking anti–4-1BBL (19H3; 10 μg/ml) for another 48 h. Culture supernatants were collected and cytokines measured by ELISA with antibodies to IL-2 (JES6-1A12 and biotin-JES6-5H4) and IFN-γ (R46A2 and biotin-XMG1.2; all from BD).

Techniques: Activity Assay, Expressing, In Vitro, Control, Binding Assay, Staining, Irradiation, Ex Vivo, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

doi: 10.1084/jem.20132687

Figure Lengend Snippet: Gal-9 colocalizes with 4-1BB. (a) Confocal analysis of 4-1BB (red) and Gal-9 (green) in preactivated WT CD4 + T cells cultured with agonist anti–4-1BB or rat Ig in the presence or absence of anti-CD3 for 15 min. Percent colocalization of 4-1BB and Gal-9 from confocal images is shown on the right. Cells analyzed (40 cells per group) showing zero, one, two, or more than three colocalization spots per cell in each group are shown as a percentage of total cells. Data are representative of two different experiments. Bar, 1 µm. (b) Western blot of translocated nuclear RelA in preactivated CD4 + T cells from WT and Gal-9 −/− mice cultured with agonist anti–4-1BB or without stimulation (NS) for 30 min. Lamin B was used as a nuclear loading control. Relative density of RelA to Lamin B quantified by ImageJ software (National Institutes of Health) is shown on the right. Data are representative of at least three different experiments. (c, left) 4-1BB immunoprecipitated (IP) with 4-1BBL.Fc from preactivated WT CD4 + T cells that were cultured with agonist anti–4-1BB or without stimulation (NS) for 30 min. (right) 4-1BB was immunoprecipitated with 4-1BBL.Fc or Fc control from preactivated WT or 4-1BB −/− CD4 + T cells. Western blots (WB) were performed to detect 4-1BB and Gal-9. Data are representative of two different experiments. (d) 4-1BB was immunoprecipitated with 4-1BBL.Fc from 4-1BB −/− , full-length (FL), or ΔC 4-1BB–transduced T hybridoma cells. Western blots were performed to detect 4-1BB and Gal-9 (molecular masses are the same as for c). Data are representative of two different experiments.

Article Snippet: Naive T cells were preactivated with 2 μg/ml of plate-bound anti-CD3 (2C11) and 0.5 μg/ml of soluble anti-CD28 (37N5) for 48 h. To determine secondary responsiveness, these activated/effector T cells were resorted for identical expression of 4-1BB and recultured with 0.5 μg/ml of plate-bound anti-CD3 in the presence of 10 μg/ml of control rat IgG or 10 μg/ml anti–4-1BB (3H3) without any APCs for another 24 h. In some experiments, effector CD8 T cells were recultured with varying concentrations of recombinant mouse 4-1BBL (his-tagged and cross-linked with anti-polyhistidine antibody; R&D Systems) or irradiated 4-1BBL + hybridoma cells in the presence of 10 μg/ml of control rat IgG or a blocking anti–4-1BBL (19H3; 10 μg/ml) for another 48 h. Culture supernatants were collected and cytokines measured by ELISA with antibodies to IL-2 (JES6-1A12 and biotin-JES6-5H4) and IFN-γ (R46A2 and biotin-XMG1.2; all from BD).

Techniques: Cell Culture, Western Blot, Control, Software, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

doi: 10.1084/jem.20132687

Figure Lengend Snippet: Defective 4-1BB activity in Gal-9–deficient NK cells. (a) Expression of 4-1BB (top) and NK1.1 (bottom) on IL-2–activated splenic NK cells from WT and Gal-9 −/− mice. Shaded histogram, isotype control for 4-1BB. (b and c) NK cells were preactivated as in panel a and then sorted for identical expression of 4-1BB. Cells were cultured for 2 d in the presence of varying concentrations of agonist anti–4-1BB or anti-NK1.1, or control IgG (b) or antihistidine cross-linked r4-1BBL (c), and secretion of IFN-γ was assayed. Data are means ± SEM from triplicate cultures and representative of three different experiments. (d) Flow analysis of surface expression of 4-1BB and Gal-9 on ex vivo NK cells from spleen. Shaded histograms, isotype control. (e) Confocal detection of 4-1BB (red) and Gal-9 (green) on IL-2–activated WT NK cells cultured with 5 µg/ml 4-1BBL.Fc or 5 µg/ml of control human Fc for 15 min. Data are representative of two different experiments. Bar, 1 µm. (f) 4-1BB was immunoprecipitated (IP) with 4-1BBL.Fc from IL-2–activated WT and Gal-9 −/− NK cells, followed by detection of 4-1BB and Gal-9 by Western blotting (WB). Data are representative of at least three different experiments.

Article Snippet: Naive T cells were preactivated with 2 μg/ml of plate-bound anti-CD3 (2C11) and 0.5 μg/ml of soluble anti-CD28 (37N5) for 48 h. To determine secondary responsiveness, these activated/effector T cells were resorted for identical expression of 4-1BB and recultured with 0.5 μg/ml of plate-bound anti-CD3 in the presence of 10 μg/ml of control rat IgG or 10 μg/ml anti–4-1BB (3H3) without any APCs for another 24 h. In some experiments, effector CD8 T cells were recultured with varying concentrations of recombinant mouse 4-1BBL (his-tagged and cross-linked with anti-polyhistidine antibody; R&D Systems) or irradiated 4-1BBL + hybridoma cells in the presence of 10 μg/ml of control rat IgG or a blocking anti–4-1BBL (19H3; 10 μg/ml) for another 48 h. Culture supernatants were collected and cytokines measured by ELISA with antibodies to IL-2 (JES6-1A12 and biotin-JES6-5H4) and IFN-γ (R46A2 and biotin-XMG1.2; all from BD).

Techniques: Activity Assay, Expressing, Control, Cell Culture, Ex Vivo, Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

doi: 10.1084/jem.20132687

Figure Lengend Snippet: Gal-9 binds to 4-1BB. (a) Binding of m4-1BB.Fc or mBTLA.Fc or control hIgG to plate-bound recombinant mGal-1, mGal-9, or m4-1BBL, as measured by optical density. (b) Flow cytometry detection of binding of recombinant m4-1BBL, mGal-9, or mGal-3 to saturating amounts of m4-1BB.Fc coated on protein G beads. Shaded histograms, binding to control hIgG (Fc fragment)–coated beads. (c) SDS-PAGE of coimmunoprecipitation of h/m4-1BBL, h/mGal-9, or h/mGal-4 with h4-1BB.Fc (left) or m4-1BB.Fc (right). The observed molecular masses (kilodaltons) were h/m4-1BB.Fc, 55–58; h4-1BBL, 20; m4-1BBL, 41–44; m/hGal-9, 36; and m/hGal-4, 36. Data in a–c are representative of at least three different experiments each. (d and e) Binding response of increasing concentrations (0.004–0.312 µM) of hGal-9 (d) or mGal-9 (e) to immobilized h4-1BB.Fc (d) or m4-1BB.Fc (e), as measured by SPR. Values represent the mean of three independent measurements. The response shown is reference-subtracted (unrelated Fc protein). (f) Flow analysis of competition between m4-1BBL/anti–4-1BB antibodies and mGal-9 for binding to m4-1BB.Fc. m4-1BB.Fc–coated protein G beads were incubated either with m4-1BBL (top left) or mGal-9 (bottom left) alone. For competition between m4-1BBL and mGal-9, m4-1BB.Fc–coated beads were first incubated with m4-1BBL (second left) or mGal-9 (middle). The beads were washed and incubated further with mGal-9 (second left) or m4-1BBL (middle). Binding of m4-1BBL (top) or mGal-9 (bottom) was detected using different fluorescent-labeled antibodies and analyzed by flow. Shaded histogram, binding of respective detection antibody isotype. For competition between anti–4-1BB antibodies and mGal-9, m4-1BB.Fc–coated beads were first incubated with agonistic 4-1BB antibodies, clone 3H3 (second right) or 1D8 (right). The beads were washed and further incubated with mGal-9. Open black histograms, mGal-9 binding without anti-4-1BB antibodies. Gray-shaded histograms, mGal-9 binding after anti-4-1BB antibodies. Black-shaded, beads with no mGal-9. Data are representative of two different experiments.

Article Snippet: Naive T cells were preactivated with 2 μg/ml of plate-bound anti-CD3 (2C11) and 0.5 μg/ml of soluble anti-CD28 (37N5) for 48 h. To determine secondary responsiveness, these activated/effector T cells were resorted for identical expression of 4-1BB and recultured with 0.5 μg/ml of plate-bound anti-CD3 in the presence of 10 μg/ml of control rat IgG or 10 μg/ml anti–4-1BB (3H3) without any APCs for another 24 h. In some experiments, effector CD8 T cells were recultured with varying concentrations of recombinant mouse 4-1BBL (his-tagged and cross-linked with anti-polyhistidine antibody; R&D Systems) or irradiated 4-1BBL + hybridoma cells in the presence of 10 μg/ml of control rat IgG or a blocking anti–4-1BBL (19H3; 10 μg/ml) for another 48 h. Culture supernatants were collected and cytokines measured by ELISA with antibodies to IL-2 (JES6-1A12 and biotin-JES6-5H4) and IFN-γ (R46A2 and biotin-XMG1.2; all from BD).

Techniques: Binding Assay, Control, Recombinant, Flow Cytometry, SDS Page, Incubation, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

doi: 10.1084/jem.20132687

Figure Lengend Snippet: Gal-9 binds to CRD4 of 4-1BB. (a) Coimmunoprecipitation of recombinant h4-1BBL or h/mGal-9 with full-length (CRD1–4), CRD1–3, or CRD4 h4-1BB.Fc constructs. The observed molecular masses (kilodaltons) were FL h4-1BB.Fc, 55; CRD1–3 h4-1BB.Fc, 44; CRD4 h4-1BB.Fc, 47–49; h4-1BBL, 20; and m/hGal-9, 36. Data are representative of at least three different experiments. (b and c) Binding response of a single concentration (2.5 µM) of hGal-9 or mGal-9 with the indicated immobilized CRD mutants of h4-1BB.Fc (b) or m4-1BB.Fc (c), respectively, as measured by SPR. The response shown is reference-subtracted (human Fc). Data are representative of three different experiments. (d and e) Binding response of increasing concentrations (0.004–0.312 µM) of hGal-9 (d) or mGal-9 (e) to immobilized CRD4 mutants of h4-1BB.Fc (d) or m4-1BB.Fc (e), as measured by SPR. Values represent the mean of two independent measurements.

Article Snippet: Naive T cells were preactivated with 2 μg/ml of plate-bound anti-CD3 (2C11) and 0.5 μg/ml of soluble anti-CD28 (37N5) for 48 h. To determine secondary responsiveness, these activated/effector T cells were resorted for identical expression of 4-1BB and recultured with 0.5 μg/ml of plate-bound anti-CD3 in the presence of 10 μg/ml of control rat IgG or 10 μg/ml anti–4-1BB (3H3) without any APCs for another 24 h. In some experiments, effector CD8 T cells were recultured with varying concentrations of recombinant mouse 4-1BBL (his-tagged and cross-linked with anti-polyhistidine antibody; R&D Systems) or irradiated 4-1BBL + hybridoma cells in the presence of 10 μg/ml of control rat IgG or a blocking anti–4-1BBL (19H3; 10 μg/ml) for another 48 h. Culture supernatants were collected and cytokines measured by ELISA with antibodies to IL-2 (JES6-1A12 and biotin-JES6-5H4) and IFN-γ (R46A2 and biotin-XMG1.2; all from BD).

Techniques: Recombinant, Construct, Binding Assay, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

doi: 10.1084/jem.20132687

Figure Lengend Snippet: Gal-9 binding to 4-1BB is carbohydrate dependent. (a) Coimmunoprecipitation of full length (F), C-terminal (C), or N-terminal (N) mGal-9 mutants with full-length (CRD1-4) or CRD4 m4-1BB.Fc. The observed molecular masses (kilodaltons) were FL m4-1BB.Fc, 55; CRD4 m4-1BB.Fc, 51; FL mGal-9, 36; C-terminal mGal-9, 21; and N-terminal mGal-9, 23. Data are representative of two different experiments. (b) Binding response of a single concentration (2.5 µM) of human (right) or mouse (left) Gal-9 to immobilized CRD4 of human (top) or mouse (bottom) 4-1BB.Fc, as measured by SPR. Black and gray lines indicate untreated or deglycosylated (PNGase F/Endo H treated) CRD4 4-1BB.Fc, respectively. The response shown is reference-subtracted (human Fc). Data are representative of two different experiments. (c) Binding response of 2.5 µM mouse Gal-9 (top) or 5 µM mouse 4-1BBL (bottom) to immobilized mouse 4-1BB.Fc produced in HEK 293T cells (left) or HEK 293S cells (right), as measured by SPR. The response shown is reference-subtracted (human Fc - gray line). Data are representative of three different experiments.

Article Snippet: Naive T cells were preactivated with 2 μg/ml of plate-bound anti-CD3 (2C11) and 0.5 μg/ml of soluble anti-CD28 (37N5) for 48 h. To determine secondary responsiveness, these activated/effector T cells were resorted for identical expression of 4-1BB and recultured with 0.5 μg/ml of plate-bound anti-CD3 in the presence of 10 μg/ml of control rat IgG or 10 μg/ml anti–4-1BB (3H3) without any APCs for another 24 h. In some experiments, effector CD8 T cells were recultured with varying concentrations of recombinant mouse 4-1BBL (his-tagged and cross-linked with anti-polyhistidine antibody; R&D Systems) or irradiated 4-1BBL + hybridoma cells in the presence of 10 μg/ml of control rat IgG or a blocking anti–4-1BBL (19H3; 10 μg/ml) for another 48 h. Culture supernatants were collected and cytokines measured by ELISA with antibodies to IL-2 (JES6-1A12 and biotin-JES6-5H4) and IFN-γ (R46A2 and biotin-XMG1.2; all from BD).

Techniques: Binding Assay, Concentration Assay, Produced

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: List or thermoregulatory or beige markers employed in this study and their function

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: Marker

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: List of primer/probe sets

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques:

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Markers of beige adipocytes in vitro . The transcripts for FGF21 a , P2RX5 b , PAT2 c , CITED1 d , CAR4 e , TBX1 f , CD137 g , and TMEM26 ( h ) were evaluated in white and beige adipocytes in culture treated with isoproterenol (+ Iso) or without (− Iso) for 6 hrs. before harvesting the cells. Transcripts were normalized to white – Iso. Values are means ± SEM ( n = 3). (*) different from white – Iso, (#) different from white + Iso, (+) different from beige + Iso, (@) different from beige – Iso, p ≤ 0.05, two way ANOVA followed by multiple comparisons using the Holm-Sidak method

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: In Vitro

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Summary of beigeing criteria met by the thermoregulatory genes and putative beige markers when studied under in vitro and in vivo conditions

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: In Vitro, In Vivo, Cell Culture

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Markers of beige adipocytes in inguinal adipose tissue. The transcripts for FGF21, P2RX5, PAT2, CITED1, CAR4, TBX1, CD137, and TMEM26 were evaluated in inguinal adipose tissue from mice maintained at room temperature (22 °C) or exposed to cold (5 °C) for one week in 3 separate experiments were the ages of the mice at the end of the experiment were 2 months and 10 days (Exp-1), 2 months and 23 days (Exp-2), and 4 months and 10 days (Exp-3). The levels of the transcripts at 5 °C were normalized to the transcript of the same gene at 22 °C. Values are means ± SEM ( n = 4–5). (*) different from 22 °C, p ≤ 0.05, two-tailed t-test

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: Two Tailed Test

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Markers of beige adipocytes in the adipocyte and SVF derived from inguinal adipose tissue. The transcripts for FGF21 a , P2RX5 b , PAT2 c , CITED1 d , CAR4 e , TBX1 f , CD137 g , and TMEM26 ( h ) were evaluated in the adipocyte and SVF of 4 month old mice kept at room temperature (22 °C) or exposed to the cold (5 °C) for one week. Transcripts were normalized to 22 °C, SVF. Values are means ± SEM ( n = 3–5). (*) different from SVF 22 °C, (#) different from SVF 5 °C, (+) different from adipocytes 5 °C, (@) different from adipocytes 22 °C, p ≤ 0.05, two way ANOVA followed by multiple comparisons using the Holm-Sidak method

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: Derivative Assay

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Beige markers in preadipocytes and SVF cells in vitro . The transcripts for CAR4, CITED1, PAT2, FGF21, P2RX5, CD137, TBX1, and TMEM26 were evaluated in SVF cells and beige adipocytes in culture. Transcripts were normalized to SVF cells. Values are means ± SEM ( n = 3). (*) different from SVF, p ≤ 0.05, two-tailed t-test

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: In Vitro, Two Tailed Test