gray rjm  (Worthington Biochemical)

Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: Impact of RGMb–PD-L2 blockade on tolerance induction is not caused by depletion of APC. (a–c) Mice immunized with OVA in ALUM on day 0 received OVA i.n. on days 7–9 and were treated with PD-L2 mAb 2C9 or isotype control (500 µg i.p.) on day 8. Lung cells were dispersed on day 10 and the expression of PD-L2 analyzed on AMs (F4/80 + CD11c + ), DCs (F4/80 − CD11c + ), and IMs (F4/80 + CD11c lo ). (a) Gating strategy used in b–d. (b) Solid line indicates staining with PD-L2 mAb TY25; shaded histogram indicates isotype control. One mouse/group representative of three is shown. (c) The number of PD-L2 + macrophages and DCs was quantified. Data are representative of two experiments.

Article Snippet: To induce tolerance, lightly anesthetized WT BALB/cByJ or PD-L2−/− mice received 100 µg of LPS-free OVA (Worthington Biochemical) in PBS or PBS alone (control) by intranasal instillation on days 0, 1, and 2.

Techniques: Mouse Assay, Expressing, Affinity Magnetic Separation, Staining

Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: Blockade of PD-L2–RGMb interaction reduces the initial expansion of antigen-specific T cells after intranasal OVA administration. (a) Experimental protocol for Fig. 9 . Mice received DO11.10 T cells on day 0 and OVA i.n. The number of KJ1-26 + cells in mediastinal LNs was determined on the indicated days. (b) Mice treated with RGMb mAb 9D1 or control mAb on day −1 received 5 × 10 6 DO11.10 T cells. Left, TCRβ + KJ1-26 + CD4 + ; Right, FoxP3 + TCRβ + KJ1-26 + CD4 + cells. For each group, mediastinal LNs of 3–5 mice were combined; data are representative of two experiments. (c–e) Mice received 2 × 10 5 DO11.10 T cells after (c) RGMb mAb 9D1 or control mAb; (d) RGMb mAb 9D1, PD-L2 mAb 3.2, or control mAb; (e) PD-L2 mAb 3.2 or control mAb. (f) WT DO11.10 T cells were transferred into WT or PD-L2 −/− mice; (g) WT or PD-L2 −/− DO11.10 T cells were transferred into WT mice. (h–j) Mice received 2 × 10 5 DO11.10 T cells after (h) PD-L2 mAb 2C9 or control mAb; (i) PD-L1 mAb 9G2 or control mAb; (j) PD-L2 mAb 3.2, PD-1 mAb 1A12, or control mAb. The number of KJ1-26 + T cells in mediastinal lymph nodes on day 5 (h–j) was determined by flow cytometry. (b–d) Each point represents a pool of LNs from three mice. Data are representative of two to four experiments. (e–j) Each point represents the mean and SEM of KJ1-26 + T cells in LNs of two or three groups of two mice each. Data are representative of two experiments. **, P

Article Snippet: To induce tolerance, lightly anesthetized WT BALB/cByJ or PD-L2−/− mice received 100 µg of LPS-free OVA (Worthington Biochemical) in PBS or PBS alone (control) by intranasal instillation on days 0, 1, and 2.

Techniques: Mouse Assay, Flow Cytometry, Cytometry

Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: Blocking capacities of RGMb and PD-L2 mAbs and a model for RGMb and PD-L2 interactions. (a–c) The blocking capacity of RGMb mAbs and PD-L2 mAbs was determined by cell conjugation assay. After cells were stained with dyes, (a) 300-mRGMb cells were incubated with the indicated concentrations of RGMb mAbs before incubation with 300-mPD-L2 cells; (b) 300-mPD-L2 cells were incubated with the indicated concentrations of PD-L2 mAbs before incubation with 300-mRGMb cells; or (c) before incubation with 300-mPD-1 cells. (d) mRGMb-HIS was preincubated with mAbs or Ig fusion proteins and then added to BMP-4–coated plates. Binding of mRGMb-HIS was detected by anti–penta-HIS-HRP in an ELISA. Similar results were seen with BMP-2 (not depicted). (e) mPD-L2-hIgG or control-hIgG were preincubated alone or with monomeric mRGMb-HIS, and then added to BMP-4–coated plates. Binding of Ig fusion proteins was detected with anti–hIgG-HRP in an ELISA. Similar results were also seen for BMP-2 (not depicted). (f) mNeogenin- or mPD-L2–transfected 300 cells were stained with indicated Ig fusion proteins or control-Ig and analyzed by flow cytometry. (g) Binding of 300-mRGMb cells to 300-mPD-L2 or 300-mNeogenin cells was analyzed by cell conjugation assay. Cells stained with a red dye were incubated with cells stained with a green dye and binding was measured by flow cytometry. (h) Molecular model depicting PDL2–BMP–BMPR–RGMb–neogenin and PD-L2–PD-1 pathways. All data are representative of two or more independent experiments.

Article Snippet: To induce tolerance, lightly anesthetized WT BALB/cByJ or PD-L2−/− mice received 100 µg of LPS-free OVA (Worthington Biochemical) in PBS or PBS alone (control) by intranasal instillation on days 0, 1, and 2.

Techniques: Blocking Assay, Conjugation Assay, Staining, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Cytometry

Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: Expression of RGMb, PD-L2, BMPs, BMPRs, and related molecules in resting lung cell populations and airway epithelial cells. Expression of indicated mRNAs in resting lung cell populations and airway epithelial cells by qRT-PCR. (a–h) IMs (F4/80 + CD11c − ), AMs (F4/80 + CD11c + ), DCs (F4/80 − CD11c + ), and other cells (F4/80 − CD11c − ) were sorted from lung cells pooled from five to eight mice; CD4 + T cells (TCRβ + CD4 + ) and CD8 + T cells (TCRβ + CD8 + ) were sorted from lung cells pooled from two mice. (i) AECs and TECs were pooled from three and eight mice, respectively. Data are mean ± SEM; n = 3; *, P

Article Snippet: To induce tolerance, lightly anesthetized WT BALB/cByJ or PD-L2−/− mice received 100 µg of LPS-free OVA (Worthington Biochemical) in PBS or PBS alone (control) by intranasal instillation on days 0, 1, and 2.

Techniques: Expressing, Quantitative RT-PCR, Affinity Magnetic Separation, Mouse Assay

Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: RGMb binds to PD-L2, but not PD-L1 or other related molecules. (a and b) mRGMb- or mPD-L2–transfected 300 cells, untransfected 300 cells, and PD-1–transfected Jurkat T cells were stained with the indicated Ig fusion proteins (red) or control-Ig (blue) and analyzed by flow cytometry. MFI, mean fluorescence intensity. (c and d) Binding of mPD-L2-Ig or control-Ig to plates coated with mRGMb-HIS, hRGMb-HIS, mRGMa-HIS or mRGMc-HIS, was analyzed by ELISA. (e) Biacore analysis of the interaction of RGMb with PD-L2 and of PD-1 with PD-L2. (f–k) Cell–cell binding of the indicated transfected cells was analyzed by cell conjugation assay. In f, h, and j, the top panels show the FSC-SSC plots and the bottom panels show the corresponding dot plots. g, i, and k show only the dot plots. All data are representative of 2–11 independent experiments.

Article Snippet: To induce tolerance, lightly anesthetized WT BALB/cByJ or PD-L2−/− mice received 100 µg of LPS-free OVA (Worthington Biochemical) in PBS or PBS alone (control) by intranasal instillation on days 0, 1, and 2.

Techniques: Transfection, Staining, Flow Cytometry, Cytometry, Fluorescence, Binding Assay, Enzyme-linked Immunosorbent Assay, Conjugation Assay

Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: PD-L2 or RGMb blockade inhibits induction of respiratory tolerance. (a) Experimental protocol for induction of respiratory tolerance. (b–d) PD-L2–deficient or WT mice in b, and WT mice injected with the indicated mAb in c and d, were exposed to OVA i.n. (tolerized) or PBS (nontolerized) and subsequently received 50 µg OVA/ALUM i.p. T cell proliferation (top) and IL-4 production (bottom) in response to restimulation with OVA in vitro are shown. n = 2–3. Data are representative of two to five experiments. Data are mean ± SEM, *, P

Article Snippet: To induce tolerance, lightly anesthetized WT BALB/cByJ or PD-L2−/− mice received 100 µg of LPS-free OVA (Worthington Biochemical) in PBS or PBS alone (control) by intranasal instillation on days 0, 1, and 2.

Techniques: Mouse Assay, Injection, In Vitro

Journal: NPJ Breast Cancer

Article Title: Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

doi: 10.1038/s41523-020-0171-1

Figure Lengend Snippet: PD-L1 expression increases in response to 4H-CTX or paclitaxel (PTX) treatment in vitro. a EMT6-CDDP cells were treated with increasing concentrations of 4H-CTX, which resulted in increased expression of PD-L1 analyzed by flow cytometry. b EMT6-CDDP, c EMT6/P, or d SP1-AC2M2 cells were treated with 20 µM 4-HCTX or 1 µM of PTX; values represent mean fluorescence intensity of PD-L1 analyzed by flow cytometry with subtraction of mean fluorescence intensity of isotype IgG. One-way analysis of variance with Tukey’s multiple comparison test. Unpaired t -test for SP1-AC2M2 analysis. * p

Article Snippet: For analysis of in vivo PD-L1 expression, tumors were dissociated with the following enzymatic buffer (1% BSA, 12,500 units collagenase II (Worthington), 12,500 units collagenase IV (Worthington), and DNase I (cat. no. LS006333, Worthington) for 45 min and then a single-cell suspension gained.

Techniques: Expressing, In Vitro, Flow Cytometry, Fluorescence