Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the full length and mature amino acid sequences and numbering (subscript) and the identified N - and O -glycosylation sites (occupied sites in bold, red; unoccupied sites in bold, black) of human IL-15 and sIL-15Rα. The same amino acid sequences of IL-15 and sIL-15Rα were used for the two large-scale preparations of hetIL-15 ( i.e. the cGMP and EN lots)

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques:

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the expression, cellular presentation, function and analysis of the soluble human hetIL-15 complex. a. Engineered human IL-15 and IL-15Rα were co-expressed and secreted by HEK293 cells as a soluble heterodimeric complex (hetIL-15) after proteolytic cleavage from the cell surface. The complex binds to the IL-2Rβ/γ receptor complex located on target cells, where it initiates a cellular response. b. The hetIL-15 complex was isolated and its purity monitored using reducing (upper) and native (lower) SDS-PAGE with Coomassie blue staining (left gels) and Western blotting using anti-IL-15 and anti-IL-15Rα antibodies (right gels). The two clinically relevant preparations of hetIL-15 (i.e. the EN and cGMP lots) are shown. c. IL-15 and sIL-15Rα were separated from their heterodimeric complex by non-reductive RP-HPLC and individually subjected to LC-MS/MS-based glycan (top), glycopeptide (bottom) and glycoprotein (right) profiling in order to characterize their N- and O-glycosylation in a detailed and site-specific manner

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Expressing, Isolation, SDS Page, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Glycome profiling demonstrates extensive and reproducible N-and O-glycosylation of IL-15 and sIL-15Rα in the large-scale preparations of hetIL-15 (cGMP and EN lots). N-glycans of IL-15 (a) and sIL-15Rα (b) were structurally characterized and quantitatively profiled using PGC-LC-ESI-negative ion-CID-MS/MS. c. Several isobaric N-glycan isomers were identified as exemplified by the extracted ion chromatogram (EIC) of the abundant Man3GlcNAc5Fuc1 composition (m/z 832.9 [M – 2 H]2−, upper panel) and the corresponding CID-MS/MS (bottom panel, see Fig. 3 for key to monosaccharide symbols) demonstrating three isobaric GlcNAc-terminating N-glycan isomers i.e. N-glycan structure 5a (shown), 5b and 5c (fragment spectra for the two latter N-glycans are presented in Supplementary Fig. S1). ‘*’ represents a non-glycan signal interference. d. The O-glycome profiling of sIL-15Rα showed less micro-heterogeneity. *Structure 2a/2b could not be consistently separated and were thus combined for quantitation purposes. No O-glycosylation was detected for IL-15 (data not shown). The N- and O-glycosylation profiles of IL-15 and sIL-15Rα of the EN (red bars) and cGMP (blue bars) lots of hetIL-15 were similar as evaluated by their high correlation coefficients (R2 = 0.815–0.982). The relative glycan quantities are averages of technical duplicates (see Supplementary Table S1 and S2 for exact values). The corresponding N- and O-glycan structures and their biosynthetic relationship are depicted in Fig. 3

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Tandem Mass Spectroscopy, Quantitation Assay

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Structures and biosynthetic relationship of the observed IL-15 and sIL-15Rα N- and O-glycans. The designated numbers of the individual N-linked (left) and O-linked (right) glycans correspond to the numbering used in Fig. 2, Fig. 5 and Supplementary Tables S1–S2. Their biosynthetic interconnectivity is presented with arrows symbolizing single glycosylation enzyme reactions. The most abundant N- and O-glycans are shaded in dark grey. Monosaccharide symbols are presented according to the Essentials of Glycobiology/Consortium for Functional Glycomics nomenclature. Key: fucose (red triangle), mannose (green circle), GlcNAc (blue square), sialic acid (NeuAc) (purple diamond), galactose (yellow circle) and HexNAc (unspecified GlcNAc or GalNAc) (open square)

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Functional Assay

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Site-specific O-glycoprofiling of sIL-15Rα of clinical-grade hetIL-15 (cGMP lot) using RP (C18)-LC-ESI-positive ion-CID/ETD-MS/MS. a. The MS1 level profile (right) indicated multiple Thr81- and Thr86 - glycoforms on the tryptic O - glycopeptide R - ] 74 PA P PAPPSTVTTAGVTPQPESLSPSGK97[−E. ETD and CID fragmentation (left spectra) confirmed the O-glycosylation sites and the structure of the two conjugated core 1-type O-glycansans (HexHexNAcNeuAc, corresponding to structure 2b, Fig. 3), m/z 905.4 (4+). Additional examples of ETD-MS/MS fragment spectra of two other sIL-15Rα tryptic O-glycopeptides i. e. b. The N-terminal-1ITCPPPMSVEHADIWVK17-[S peptide conjugated with a single HexNAc (corresponding to structure i, Fig. 3) m/z 728.9 (3+) and c. the C-terminal peptide K-]152NWELTASASHQPPGVYPQG170[−conjugated with two core 1-type O-sialoglycans (HexHexNAcNeuAc, corresponding to structure 2a or 2b, latter shown) and one core 1-type O-asialoglycan (HexHexNAc, structure ii, Fig. 3) m/z 930.8 (4+). Key fragment ions for exact site localization are presented in red. See Fig. 3 for monosaccharide key

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Tandem Mass Spectroscopy

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the identified sIL-15Rα tryptic O -glycopeptides. The modified amino acid residues are underlined where known or listed as ND where unknown. The theoretical glycopeptide masses are based on carbamidomethylated cysteine residues. For peptides where the non-glycosylated variants were observed, the non-enriched LC-MS/MS data were used to establish the relative site-occupancy; otherwise glycopeptide-enriched LC-MS/MS data were used to establish the relative glycoform distribution. See for examples of assigned ETD/CID-MS/MS O -glycopeptide spectra

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Modification, Tandem Mass Spectroscopy

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Spatial map of the N- and O-glycosylation sites of hetIL-15 in its quaternary complex with IL-2Rβ and IL-2Rγ. The three putative N-glycosylation sites of IL-15 (green) are shown in blue. Only Asn79 was found to be occupied; Asn71 and Asn112 found on the interface to IL-2Rβ (cyan) and IL-2Rγ (magenta), respectively, were not utilized as N-glycosylation sites when expressed in HEK293. The available crystal structure (PDB: 4GS7) covered only the lightly O-glycosylated N-terminal region of the sIL-15Rα polypeptide chain (yellow) [34]. The occupied O-glycosylation site at Thr2 covered by this region is shown in orange. See also Fig. 1a. for schematic illustration of the quaternary complex

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques:

Journal: Nature

Article Title: Programme of Self-Reactive Innate-Like T Cell-Mediated Cancer Immunity

doi: 10.1038/s41586-022-04632-1

Figure Lengend Snippet: a-b, Flow cytometric analysis of FCER1G and CD122 expression in TCRβ+CD1d−NK1.1−CD4−CD8α− thymocytes from Il15−/− (n = 5) and Il15+/+ (n = 5) mice. c, GFP expression by CD45−EpCAM+ cells from IL-15-GFP reporter control (n = 5) and PyMT mice (n = 5). d, Correlation between frequency of FCER1G+ cells among CD45+TCRβ+CD4− cells and IL-15 expression level in tumor tissues from colon carcinoma patients. e-f, Flow cytometric analysis of FCER1G and CD122 expression in TCRβ+CD1d−NK1.1−CD4−CD8α− thymocytes and CD45+TCRβ+CD4− tumor-infiltrating cells from S100a8-CreIl15fl/flPyMT (n = 5) or control PyMT (n = 5) mice. Bottom panel shows expression of NK1.1 and granzyme B in FCER1G+CD122+ cells. g, Tumor burden in mice of indicated genotypes. Control PyMT (n = 4) and S100a8-CreIl15fl/flPyMT (n = 8). h-i, Flow cytometric analysis of adoptively transferred CD45.1+CD45.2+ αβILTCk progenitors (αβILTCkP, n = 4) or CD8 single positive T cells (CD8SP, n = 8) cells among TCRβ+CD8α+ tumor-infiltrating cells and NK1.1 as well as granzyme B expression in donor cells. j, Tumor burden in mice adoptively transferred with no cells (n = 5), Ubc-CreERRosa26LSL-Stat5b-CA/+ thymic CD8SPs (n = 8) or αβILTCkPs (n = 5). All statistical data are shown as mean ± S.D (biologically independent mice in a-c, e-g, h-j). Two-tailed un-paired t-test (b-c,f,i), linear regression (d) and two-way analysis of variance (ANOVA) (g,j) with post hoc Bonferroni t-test. *P < 0.05; **P < 0.01; ***P < 0.001 and n.s.: not significant.

Article Snippet: Fresh human tumors were fixed in Periodate-Lysine-Paraformaldehyde (PLP) for 16–24 hours, 30% sucrose for 24 hours, then frozen in OCT. Tissue was sectioned at 20 μm thickness, blocked and permeabilized in buffer containing 0.1 M Tris, 1% BSA, 1% FBS, 0.3% Triton-X100, 2% normal mouse/rat/goat serum for 30 minutes and stained with anti-IL-15 (MAB647, R&D), AF594-conjugated anti-CHD1 (DECMA-1, Biolegend) overnight at 4°C.

Techniques: Expressing, Two Tailed Test

Journal: Nature

Article Title: Programme of Self-Reactive Innate-Like T Cell-Mediated Cancer Immunity

doi: 10.1038/s41586-022-04632-1

Figure Lengend Snippet: a, Immunofluorescence images showing expression of IL-15 and CDH1 in tumor tissues from patients with colon carcinoma (left panel). Flow cytometric analysis of FCER1G and PD-1 expression in TCRβ+CD4− cells from the same tumor tissue (right panel). Data are representative of two independent experiments. b, Correlation between frequency of PD-1+ cells among CD45+TCRβ+CD4− cells and IL-15 expression level in tumor tissues from patients with colon carcinoma. Each dot denotes an independent patient sample. c, Statistical analysis showing relative mRNA expression of Il15 in sorted CD45−EpCAM+ cancer cells from S100a8-CreIl15fl/flPyMT (n = 3) and control PyMT mice (n = 3). d, Flow cytometric analysis of PD-1, NK1.1, and granzyme B expression in thymic αβILTCk progenitors (αβILTCkPs) one, three, or five days after culturing in the presence of 100 ng/ml IL-15/IL-15Ra complex. Data are representative of three independent experiments. e, A schematic diagram showing αβILTCk-based adoptive cellular transfer experiment. A constitutively active form of Stat5b (Stat5b-CA) was induced in thymic αβILTCkPs by tamoxifen administration one week after adoptive transfer into lymphocyte-deficient PyMT recipient mice with a total tumor burden of 300 – 400 mm3. f, Expression of Ly6G and TCRβ by tumor-infiltrating CD45+ cells. g, Statistical analysis of the frequency of donor-derived Ubc-CreERRosa26+/+ (n = 5) or Ubc-CerERRosa26LSL-Stat5b-CA/+ (n = 5) TCRβ+ cells among tumor-infiltrating CD45+ cells. h, Flow cytometric analysis of NK1.1 and granzyme B expression by donor-derived TCRβ+ cells in the tumor. i, Frequency of NK1.1+Granzyme B+ cells among transferred TCRβ+ cells. j, Total tumor burden in mice adoptively transferred with no cells or thymic αβILTCkPs of indicated genotypes. Data are pooled from three independent experiments. No transfer (n=4), Ubc-CreERRosa26+/+ (n=8), and Ubc-CreERRosa26Stat5b-CA/+ (n=7). k, A schematic diagram showing αβILTCk-based adoptive cellular transfer experiment. A constitutively active form of Stat5b (Stat5b-CA) was induced in thymic CD45.1+CD45.2+ αβILTCk progenitors or CD8 single positive T cells by tamoxifen administration one week after adoptive transfer into congenically distinct CD45.2+ PyMT recipient mice. All statistical data are shown as mean ± S.D (independent human samples in a-b, biologically independent mice in c,f-j and independent cell culture samples in d). Linear regression (b), two-tailed unpaired t-test (c,g,i) and two-way analysis of variance (ANOVA) (j) with post hoc Bonferroni t-test.. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and n.s.: not significant.

Article Snippet: Fresh human tumors were fixed in Periodate-Lysine-Paraformaldehyde (PLP) for 16–24 hours, 30% sucrose for 24 hours, then frozen in OCT. Tissue was sectioned at 20 μm thickness, blocked and permeabilized in buffer containing 0.1 M Tris, 1% BSA, 1% FBS, 0.3% Triton-X100, 2% normal mouse/rat/goat serum for 30 minutes and stained with anti-IL-15 (MAB647, R&D), AF594-conjugated anti-CHD1 (DECMA-1, Biolegend) overnight at 4°C.

Techniques: Adoptive Transfer Assay, Immunofluorescence, Expressing, Derivative Assay, Cell Culture, Two Tailed Test

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Example of alternative splicing analysis of Lcn-2 ( A , B ) and Cdkn1a ( C , D ) genes expressed in response to restraint stress. Analysis of mean signal intensities read from probe sets annotated to 5′, 3′ untranslated regions (UTRs) and exons of Lcn-2 (a’ and a” respectively) and Cdkn1a transcripts (c’ and c’’ respectively) revealed no alternative splicing events in Lcn-2 transcript. Analysis of Cdkn1a transcript revealed reduced level of expression of probe set 5530406 annotated to untranslated exon 2 (Cdkn1a transcript variant 2, NM_001111099) in both control and stressed animals suggesting tissue specific alternative splicing unrelated to stress. Scatter plots of summarised intensities of all probe sets annotated to Lcn-2 ( B ) and Cdkn1a ( D ) transcripts revealed two relatively separate populations of stress related data points and no outliers significantly affecting splice variant analysis. Each point represents the mean of all probe set intensities annotated to the gene of interest from one array. Data presented as mean ±SEM.

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Expressing, Variant Assay

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Psychological stress induces lipocalin-2 gene expression N = 5, ** p<0.01 ( A ) followed by protein synthesis ( B and C ); R-Lcn-2– recombinant lipocalin-2 N = 3, ** p<0.01. Data are expressed as mean ± SEM. Panel C consist representative Western blot. Triple immunohistochemistry revealed ( D ) that Lcn-2 (green) is localised mostly within and nearby of neurons (a and e) co-localised with neuronal marker (b and f) NeuN (red) and to lesser extend with astrocyte marker (purple) GFAP (c and g) in the nucleus of basolateral amygdala. The secondary antibody showed no signal resulting from nonspecific binding (h). LA, Lateral Amygdala; BLA, Basolateral Amygdala; CA, Central Amygdala. Quantitative RT-PCR reaction confirmed lack of expression of Lcn-2 gene in Lcn-2 −/− animals ( E ).

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Expressing, Recombinant, Western Blot, Immunohistochemistry, Marker, Binding Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Dendritic spine density in DiI-labeled neurons was analyzed in basolateral amygdala of wild-type and Lcn-2−/− mice before and after restraint stress. ( A ) Stress caused an increase in spine density in the neurons of BLA in wild-type mice reaching density observed in Lcn-2-deficient stress naïve mice. ( B ) Stress induced also significant decrease in proportion of mushroom spines observed in both wild-type and Lcn-2−/− strains. Those changes were accompanied by increase in other morphological groups of spines (B). Panel C represents the example of DiI stained neurons. *p<0.05; **p<0.01; ***p<0.001. Data are expressed as mean ± SEM.

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Current-clamp experiments revealed that neuronal firing rate in the basolateral amygdala is higher in Lcn-2−/− mice when compared to Lcn-2+/+ animals. Voltage responses were recorded by current steps from −100 to +600 pA in 50 pA (starting membrane potential −80 mV) from principal neurons of the basal nucleus of the amygdala. Number of action potential spikes was counted as a function of depolarizing current injection ( A ). Disruption of the lipocalin-2 gene significantly increased action potential firing rate in Lcn-2−/− animals (p<0.01 at 150 pA; p<0.05 at 200 pA). Panel B shows a significant increase in the mean input resistance in Lcn-2−/− mice compared to wild-type animals. *p<0.05; Panel C is example of neuronal firing trace. Data are expressed as mean ± SEM.

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Injection