Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Expression and internalization analyses of FcγRs. (a) Histogram overlays show expression of FcγRs (FcγRI: CD64, FcγRIIB: CD32, FcγRIII: CD16, and FcγRIV: 9E9) on splenic single lin − MHCII + CD11c + CD8 + DCs, CD11c + CD8 − DCs, and pDCs (see gating strategy in Fig. S1 a) from C57BL/6 (black) and FcγRI −/− , FcγRIIB −/− , FcγRIII −/− , and FcγRIV −/− mice (gray). This experiment was repeated three times with similar results. (b) Internalization of αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova into the indicated splenic cell populations. Splenic single-cell suspensions were incubated in media containing the antibodies labeled with an oligo containing an Atto647N fluorochrome for 10, 30, 60, or 120 min at 37°C or kept on ice (0-min time point). Extracellular fluorescence was quenched by incubation with a complementary oligo containing a specific BBQ650 quencher. Cells were gated on single CD19 + B cells, CD11c + CD8 + DCs, CD11c + CD8 − DCs, CD11c low PDCA-1 + B220 + pDCs, Ly6C high inflammatory and Ly6C low resident monocytes, T cells, and NK cells as described in Fig. S1 b. The presented experiments were repeated three times with two samples each. All data points ± SD are shown in the graph. MFI, mean fluorescence intensity.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Expressing, Incubation, Labeling, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: In vivo proliferation of CD4 + and CD8 + T cells by antigen delivery to FcγRs in the steady state. (a and b) MACS-purified, CFSE-labeled, congenic 10 6 CD8 + OT-I T cells (a) or 2 × 10 6 CD4 + OT-II T cells (b) were i.v. transferred into C57BL/6 mice. 16 h later, recipients were i.p. injected with doses of 10, 3, 1, 0.3, 0.1, and 0 µg isotype-Ova, αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova targeting antibodies in PBS. 3 d after targeting antibody injection, in vivo T cell proliferation in dependency of the injected antibody amount was measured by CFSE-dilution analysis via flow cytometry of Vα2 + CD45.1 + CD8 + (a) and Vα2 + CD45.1 + CD4 + (b) splenic T cells. All experiments were repeated at least three times with similar results.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: In Vivo, Purification, Labeling, Injection, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: T cell proliferation induced by FcγR targeting is ITAM independent, and long-term proliferative responses require additional co-stimulatory signals in vivo. (a) Injection of antigen-targeting antibodies does not change activation status of DCs in vivo. 10 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova, was i.p. injected into C57BL/6 mice. As positive control, a mixture of each 25 µg poly(I:C) and αCD40 or 10 9 HKLM was used. PBS was i.p. injected as negative control. 12 h later, splenic single lin − (CD3 − CD19 − NKp46 − ) MHCII + CD11c + CD8 + DCs, CD11c + CD8 − DCs, and CD11c low PDCA1 + pDCs were analyzed by flow cytometry for the activation markers CD80, CD86, and CD69. Data were analyzed using DIVA and FlowJo Software. This experiment was repeated more than five times with similar results and was used as quality control in the production of antigen-targeting antibodies. (b–e) MACS-purified, CFSE-labeled, congenic 10 6 CD8 + OT-I T cells (b and d) or 2 × 10 6 CD4 + OT-II T cells (c and e) were i.v. transferred into C57BL/6 mice (b–e) or NOTAM mice (b and c). 16 h later, recipients were i.p. injected with 3 µg of the targeting antibodies in PBS (b and c) or together with 25 µg αCD40 antibody and 12.5 µg poly(I:C) (pIC) or HKLM (d and e). (b and c) 3 d after targeting antibody injection, in vivo T cell proliferation was measured by CFSE-dilution analysis via flow cytometry of Vα2 + CD45.1 + CD8 + (b) and Vα2 + CD45.1 + CD4 + (c) splenic T cells. The graph shows the proliferation indices of all mice analyzed (n.s., nonsignificant; *, P < 0.05). (d and e) T cell proliferation was analyzed by cell numbers of gated Vα2 + CD45.1 + CD8 + (d) or Vα2 + CD45.1 + CD4 + (e) splenic T cells 3 d and 9 d later, when PBS, αCD40 + poly(I:C), or HKLM was coinjected. Graphs show the relative cell number expansion compared with the isotype control. (a) These experiments were repeated at least three times with similar results. (b–e) The data were generated within three independent experiments, and all data points ± SD are presented (n.s., nonsignificant; *, P < 0.05; Mann–Whitney U test).

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: In Vivo, Injection, Activation Assay, Positive Control, Negative Control, Flow Cytometry, Software, Control, Purification, Labeling, Generated, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Induction of T cell responses by FcγR targeting is dependent on the presence of FcγRs. (a–h) C57BL/6, FcγRIIB −/− (a–d), or FcγRIV −/− (e–h) mice were i.v. injected with congenic10 6 CD45.1 + CD8 + OT-I (a, b, e, and f) or 2 × 10 6 CD45.1 + CD4 + OT-II (c, d, g, and h) MACS-purified, CFSE-labeled T cells. 3 µg of the antigen-targeting antibodies αDEC205-Ova, αDCIR2-Ova, and αFcγRIV-Ova or 10 µg αFcγRIIB-Ova or isotype-Ova was i.p. injected 16 h after T cell transfer. T cell proliferation was analyzed by CFSE dilution in gated Vα2 + CD45.1 + CD8 + OT-I (a, b, e, and f) or Vα2 + CD45.1 + CD4 + OT-II (c, d, g, and h) T cells 72 h later. (a, c, e, and g) Shown are exemplary overlay histograms (gray: C57BL/6, black: [a and c] FcγRIIB −/− , [e and g] FcγRIV −/− ) of at least three independent experiments with similar results. (b, d, f, and h) Scatter plots represent the proliferation indices of all mice analyzed ± SD (gray circles: C57BL/6, black squares: [b and d] FcγRIIB −/− , [f and h] FcγRIV −/− ). (n.s., nonsignificant; **, P < 0.01; ***, P < 0.001 by Mann–Whitney U test.)

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Injection, Purification, Labeling, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Antigen targeting to FcγRIIB and FcγRIV needs receptor expression on cDCs . (a–h) Mice were i.v. injected with MACS-purified, CFSE-labeled 10 6 CD45.1 + CD8 + OT-I (a, c, e, and g) or 2 × 10 6 CD45.1 + CD4 + OT-II (b, d, f, and h) T cells. 3 µg αDEC205-Ova, αDCIR2-Ova, or αFcγRIV-Ova or 10 µg αFcγRIIB-Ova or isotype-Ova control antibodies was i.p. injected 16 h after T cell transfer. T cell proliferation was evaluated 72 h later. Shown is the CFSE dilution of Vα2 + CD45.1 + CD8 + OT-I (a, c, e, and g) or Vα2 + CD45.1 + CD4 + OT-II (b, d, f, and h) gated congenic T cells. (a and b) C57BL/6 mice and CD11c-DTR mice were treated i.p. with PBS or DT 8 h before T cell transfer. (c and d) T cell transfer into FcγRIV fl/fl , LysM-Cre × FcγRIV fl/fl , CD11c-Cre × FcγRIV fl/fl mice. (e and f) C57BL/6 mice were treated three times with 200 µg αPDCA-1 antibody 64 h, 40 h, and 16 h before T cell transfer. (g and h) T cell transfer into C57BL/6 or µMT −/− mice. All experiments were repeated at least three times with similar results.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Expressing, Injection, Purification, Labeling, Control

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Differential antigen presentation to CD8 + and CD4 + T cells induced by FcγRIIB and FcγRIV targeting to CD11c + CD8 + or CD11c + CD8 − DCs. 10 µg αDEC205-Ova, αDCIR2-Ova, or αFcγRIV-Ova or 30 µg αFcγRIIB-Ova or isotype-Ova was i.p. injected into C57BL/6 mice. 12 h later, splenocytes were sorted into CD11c + CD8 + DCs, CD11c + CD8 − DCs, pDCs, B cells, and Ly6C high and Ly6C low monocytes. (a and b) Antigen-presenting cells were co-cultured in different numbers with 10 5 MACS-enriched CD8 + OT-I T cells (a) or CD4 + OT-II T cells (b). Proliferation was evaluated by addition of 3 H-thymidine 16 h (a) or 40 h (b) after start of the co-culture. Incorporation of 3 H-thymidine was measured 24 h later. This experiment was repeated at least three times, and all data points ± SD are shown in the graph.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Immunopeptidomics, Injection, Cell Culture, Co-Culture Assay

Journal: The Journal of Experimental Medicine

Article Title: DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

doi: 10.1084/jem.20160951

Figure Lengend Snippet: Induction of effector T cell responses in naive mice mediated by antigen delivery through FcγRs. (a–d) C57BL/6 mice were i.p. injected with 10 µg of the targeting antibodies αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova together with 50 µg αCD40 antibody and 25 µg poly(I:C) after injection of PBS or 200 µg αFcγRIV w/o Ova. 14 d later, splenocytes were in vitro restimulated for 12 h with freshly isolated CD11c-positive MACS-enriched DCs loaded with a peptide pool of Ova. Intracellular IFNγ (a and c) and IL-2 (b and d) production was analyzed by flow cytometry. The graphs represent the number of cytokine-positive TCRβ + NKp46 − CD19 − CD8 + CD4 − (a and b) and TCRβ + NKp46 − CD19 − CD8 − CD4 + (c and d) T cells. This experiment was performed twice with at least five mice, and all data points were used for the analysis. Shown is the median ± interquartile range (n.s., nonsignificant; **, P < 0.01; ***, P < 0.001; Mann–Whitney U test). (e and f) C57BL/6 mice were immunized with 3 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova or 10 µg αFcγRIIB or isotype-Ova in combination with 50 µg αCD40 and 25 µg poly(I:C) (e) or 0.03, 0.10, 0.3, 1, or 3 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova or 10 µg αFcγRIIB or isotype-Ova in combination with 50 µg αCD40 and 25 µg poly(I:C) (f). 8 d later, mice were challenged i.v. with a cocktail of freshly isolated CD45.1 + splenocytes labeled with different concentrations of CFSE and/or cell trace violet and loaded with 2.4, 40, 160, or 625 nM SIINFEKL peptide (unloaded cells were used as injection control and control for specificity of the lysis). This allowed for the simultaneous analysis of target cell lysis loaded with different amounts of SIINFEKL within one mouse. 16 h later, splenocytes were analyzed for the presence of the transferred CD45.1 + cells. The data points shown in this graph have been generated within three independent experiments. Each dot represents the degree of lysis observed for splenocytes loaded with a specific amount of peptide (median ± interquartile range in e and one line for each single mouse in f). (e) This experiment was performed three times with two mice per group, and all data points are shown in the graphs. (f) This experiment was performed three times with three to five mice per group, and all data points are shown in the graphs.

Article Snippet: Wild-type and stably with FcγRIIB or FcγRIV in combination with Fc receptor γ-chain–transfected CHO cells were cultured in supplemented RPMI1640 (5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l -glutamine, and 1% nonessential amino acids). αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Injection, In Vitro, Isolation, Flow Cytometry, MANN-WHITNEY, Labeling, Control, Lysis, Generated

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: The cells were blocked in phosphate-buffered saline (PBS) containing 10% FBS and incubated with antibodies against Rab5 (1:50; Abcam ab31261), Rab7(1:20; Developmental Studies Hybridoma Bank Rab7), DCV Capsid (1:200; Abcam ab92954), dsRNA J2 (1:200; Jena Biosciences RNT-SCI-10010200) and V5 tag (1:200; ThermoFisher Scientific R960-25) for overnight at 4°C.

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation