Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Identification of mitotic H2B S6ph. (A) The sites of the H2B N-terminal tail known to be modified by phosphorylation (ph), acetylation (ac), methylation (me), or ubiquination (ub) are shown in black; the novel phosphorylation of S6 is marked in red. (B) 293T cells were treated with 50 nM calyculin A for 30 min, and lysates were incubated with λ phosphatase as shown. Equal amounts of protein contained in cell lysates were analyzed by Western blotting for the occurrence and phosphorylation of H2B with specific antibodies. The positions of molecular weight markers are indicated. (C) HeLa cells were treated with the indicated reagents or the DMSO vehicle control for 16 h and lysed with SDS sample buffer. Lysates were analyzed by SDS-PAGE and Western blotting using the indicated antibodies; the cleaved form of the caspase substrate PARP is indicated by an asterisk. (D) HeLa cells were synchronized by a double thymidine block and released into S phase. Cells were lysed at the indicated time points with SDS sample buffer. Lysates were analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (E) Diploid RPE-1 cells were fixed, and immunostaining was performed with antibodies against H2B S6ph and α-tubulin. DNA was visualized with Hoechst 33342. Bars: 5 µm (main); 2 µm (magnification). 3D-SIM was used to reveal the occurrence and localization of the stained proteins during the indicated mitotic phases; the boxed areas are displayed in 4× magnification. (F) HeLa cells were synchronized by treatment with nocodazole for 16 h and released in the presence of 10 µM MG132 or DMSO. Cells were lysed at the indicated time points with SDS sample buffer and analyzed by SDS-PAGE and Western blotting using the indicated antibodies.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Modification, Phospho-proteomics, Methylation, Incubation, Western Blot, Molecular Weight, Control, SDS Page, Blocking Assay, Immunostaining, Staining

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Analysis of chromosomal localization of H2B S6ph by 3D-SIM. (A) RPE-1 cells were stained for H2B S6ph and CENP-A localization with specific antibodies, DNA was revealed by Hoechst dye. Different mitotic phases are shown. Bars: 2 µm (main); 1 µm (magnification). (B) The experiment was done as in A, with the difference that cells were stained for H2B S6ph and Aurora B. The boxed areas in A and B are displayed in 10× magnification. Bars: 2 µm (main); 1 µm (magnification). (C) RPE-1 cells were arrested at G 2 /M transition with the CDK1 inhibitor RO 3306 for 16 h and released into mitosis for 1 h in the presence of nocodazole. Chromosome spreads were produced for immunostaining against H2B S6ph, Aurora B, H3 K9 me2/3, or CENP-A as shown. Pictures show chromosome spreads of representative cells. Bars, 1 µm.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Staining, Produced, Immunostaining

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Analysis of inner centromeric H2B S6ph localization. (A) Synchronized RPE-1 cells were used to produce chromosome spreads, which were stained with antibodies specific for H2B S6ph (red) and CENP-A (green). Bar, 1 µm. The images were quantitatively analyzed with ImageJ for the distribution of H2B S6ph and CENP-A along the centromere cross section, as shown by the dotted line (top). The maximum of fluorescence intensity was set as 1, and shaded areas indicate standard deviations ( n = 8 cells/368 centromere pairs). (B) The experiment was done as in , with the difference that chromosomes were stained for H2B S6ph and the cohesin component RAD21. Representative examples are shown; the boxed area is displayed in the bottom panel in 4× magnification. Bars: 5 µm (main); 1 µm (magnification). (C) Schematic representation of the chromosomal localization of H2B S6ph on mitotic chromatids.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Comparative analysis of mitotic histone phosphorylations. (A) RPE-1 cells were stained for H2B S6ph and H3 S10ph in various phases of mitosis with specific antibodies; DNA was revealed by Hoechst dye. Different mitotic phases are shown. Bars, 2 µm. (B) RPE-1 cells were stained for H2B S6ph, H3 T3ph, and the kinetochore marker protein CENP-C during various mitotic phases. Bars, 2 µm. (C) Chromosome spreads were produced from nocodazole-arrested cells, which were stained for phosphorylation of H2B S6 and H3 S10. Bars, 1 µm. (D) RPE-1 cells were treated for 2 h with 2 µM nocodazole, and chromosome spreads were produced to stain for H2B S6ph and H3 T3ph. Bars, 1 µm.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Staining, Marker, Produced, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Analysis of H2B S6 phosphorylation networks. (A) HeLa cells were transfected with a Dharmacon siRNA library against kinase and kinase-related genes. After 42 h, cells were treated with 200 nM nocodazole for 6 h and prepared for immunofluorescence staining against phosphorylated H2B S6. Phosphorylation intensities were quantified using a wide field Nikon microscope (Eclipse Ti-E) and high-content analysis software. Volcano plot shows standard deviations of average scores from mean H2B S6 phosphorylation intensity (x axis) and their statistical significance (P values; y axis). Statistical analysis was performed using GraphPad Prism. Aurora B, BubR1, and CDK1 are shown in red. (B) GFP-BubR1 and cyclin B1-Venus were expressed in 293T cells in the absence or presence of Myc-INCENP and HA-Aurora B WT or kinase dead (KD). Cells were lysed with SDS sample buffer and analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (C) HeLa cells were arrested at prometaphase with nocodazole for 16 h and released in the presence of MG132 together with various concentrations of the indicated kinase inhibitors. Cells were lysed with SDS sample buffer and further analyzed by immunoblotting as shown. (D) Different plasmids encoding the indicated proteins, including constitutively active (AF) or kinase dead (KD) kinases, were expressed in 293T cells as shown. Aurora B was immunoprecipitated with HA antibodies and CDK1 with anti-Flag M2 affinity gel for a subsequent in vitro kinase assay with recombinant histone octamers as substrate. Samples were analyzed by SDS-PAGE, CBB staining, and immunoblotting using the indicated antibodies; unspecific IgG bands are indicated by asterisks. (E) Cyclin B1-Venus and Flag-tagged CDK1 forms were coexpressed in 293T cells. Cells were lysed with NP-40 buffer, and CDK1 was immunoprecipitated with anti-Flag M2 affinity gel for in vitro kinase assay using H2B and H3.1 as substrate proteins. Samples were analyzed by SDS-PAGE, CBB staining, and Western blotting using the indicated antibodies. (F) Lysates from nocodazole-arrested HeLa cells were used for IP of endogenous cyclin B1, followed by in vitro kinase assays to test phosphorylation of recombinant H2B. Specific antibodies were used to reveal H2B S6ph and precipitation of cyclin B1 and the associated CDK1. The asterisk indicates the position of the light chain from the precipitating antibodies.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Phospho-proteomics, Transfection, Immunofluorescence, Staining, Microscopy, High Content Screening, Software, SDS Page, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Identification of CDK1 as a direct H2B S6 kinase. (A) Flag-tagged CDK1 forms were expressed with cyclin B1-Venus in 293T cells. CDK1 was immunoprecipitated with anti-Flag M2 affinity gel and cyclin B1 with GFP-Trap beads for a subsequent in vitro kinase assay with recombinant H2B. Samples were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (B) 293T cells were transfected to express cyclin B1-Venus or GFP as shown. Cells were lysed with NP-40 buffer and GFP, and cyclin B1-Venus were immunoprecipitated with the GFP-Trap for a subsequent in vitro kinase assay using recombinant H2B as a substrate protein. The reactions were incubated in the presence or absence of 1 µg recombinant p21 CIP1 protein (Sigma) as shown, H2B S6ph was analyzed by immunoblotting. (C) 1 µg of recombinant and purified CDK1-cyclin B1 (Thermo Fisher) or 2 µg GST control (Sigma) were incubated with recombinant H2B (2 µg) in the presence of ATP. The reaction was analyzed for histone phosphorylation by Western blotting (top) and protein integrity by SDS-PAGE and CBB staining. (D) U2OS F4 2B8 cells with an array of lacO sites integrated close to the centromere of chromosome 2 were transfected with GFP-lacI alone or a nondegradable form of cyclin B1 (cyclin ND) fused to GFP-lacI and Flag-tagged forms of CDK1 AF and CDK1 KD. After 24 h, cells expressing moderate amounts of the GFP-lacI fusion proteins were fixed for immunofluorescence and analyzed for phosphorylation of H2B S6 and the H3 S10 control as well as for Flag-CDK1 localization. The left part shows representative results from the immunofluorescence studies. The right part displays the ratio of cells showing colocalization of GFP-lacI-cyclin B1 and H2B S6ph, Flag-CDK1, or H3 S10ph stained with the indicated antibodies. n > 100 cells from two independent experiments were analyzed for each condition; error bars show standard deviations between the two biological replicates. Bars, 5 µm.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, SDS Page, Western Blot, Transfection, Incubation, Purification, Control, Phospho-proteomics, Staining, Expressing, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Identification of H2B S6ph phosphatases. (A) RPE-1 cells were arrested for 16 h with RO-3306 and then released for 1 h in the presence of nocodazole and okadaic acid (1 µM), LB-100 (10 µM), or DMSO (control) for 1 h. Chromosome spreads were analyzed by immunofluorescence for H2B S6ph and CENP-A localization. Bars: 1 µm (magnification); 5 µm (main). (B) Cells were transfected with siRNAs targeting catalytic subunits of PP1α, β, and γ or control siRNAs (Origene). Knockdown efficiency was confirmed by qPRC (see Fig. S3 B). After 24 h, cells were arrested at G 1 /S by a thymidine block and released, followed by the addition of nocodazole for 6.5 h. The mitotic cells were further treated for 30 min with AZD1152 or the DMSO control and stained as shown. Bars, 2 µm. (C) The experiment was performed as in B, and fluorescence intensities (given in artitrary units; A.U.) were quantified from 10 representative cells per condition using ImageJ and GraphPad Prism. (D) The recombinant H2B protein was phosphorylated by an in vitro kinase assay as described for . CDK1-cyclin B1 was removed from the extracts by immuno-depletion and the supernatant containing 2 µg of phosphorylated H2B protein was taken and incubated with 0.2 µg of recombinant PP1α (Novus Biologicals) or GST control (Sigma) for 30 min at 37°C. The reaction was analyzed for histone phosphorylation by Western blotting (top) and protein integrity by SDS-PAGE and CBB staining as shown.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Control, Immunofluorescence, Transfection, Knockdown, Blocking Assay, Staining, Fluorescence, Recombinant, In Vitro, Kinase Assay, Immunodepletion, Incubation, Phospho-proteomics, Western Blot, SDS Page

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Identification of mitotic H2B S6ph functions. (A) Prophase LLC-PK cells were injected with anti-H2B S6ph antibodies or rabbit IgG at the onset of mitosis and further traced by live cell imaging. Pictures show representative phenotypes after injection of rabbit IgG (top) or anti-H2B S6ph antibodies (bottom). Time points after injection of antibodies are indicated (min:s), areas of microinjections are marked with asterisks (*) and the position of the cleavage furrows by arrows. Bars, 10 µm. (B) Prophase LLC-PK cells were microinjected with PBS ( n = 5), rabbit IgG ( n = 8), or anti-H2B S6ph antibodies ( n = 6). Movement rates (changes in µm/min) during anaphase are displayed. Time lapse images after microinjection of antibody or PBS were collected at 1-min intervals. Chromosome movement rates just before and during anaphase were quantified by measuring the change in distance between leading edges of the chromosome masses as they moved poleward. Each line represents the chromosome movement rates obtained from individual cells. Each cell’s data were aligned so that minute “1” represents the first minute where anaphase movements were observed. (C) Chromosome movement rates during anaphase (t = 1 min through t = 11 min) were compared between samples from individual anti-H2B S6ph injections. Samples were labeled and arranged according to their phenotypes (ranging from slight to severe chromosome segregation defects). Lines show mean movement rates and standard deviations between the time points. (D) Average chromosome movement rates for every time point during anaphase (t = 1 min through t = 11 min) were calculated and compared between the three conditions using GraphPad Prism. Lines show mean movement rates and standard deviations between the time points. The asterisk (*) indicates a significant P value < 0.04; ns = not significant (paired t test).

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Injection, Live Cell Imaging, Microinjection, Labeling

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Identification of proteins showing S6ph-dependent H2B binding. (A) HeLa cells were arrested in mitosis with nocodazole for 16 h and lysed with NP-40 buffer. The indicated biotin-coupled peptides with unmodified or modified amino acids encompassing the first 20 amino acids from H2B were used for pulldown assays using StrepTactin beads (Qiagen). One fraction of the eluates was analyzed by SDS-PAGE and silver staining. Arrows indicate phosphorylation-dependent interactors. (B) Samples from peptide pulldown experiments were analyzed by SDS-PAGE and Western blotting using SET and 14-3-3 antibodies; arrows show the SET and 14-3-3 isoforms. (C) GFP-tagged H2B WT or S6 phosphorylation site mutants were expressed in 293T cells. Cells were lysed with NP-40 buffer, sonicated, and treated with Benzonase (Millipore). H2B-GFP was immunoprecipitated using the GFP-Trap and SET binding was analyzed by immunoblotting. (D) (His)6-tagged SET (His-SET) and the indicated H2B-GST fusion proteins were purified from E. coli BL21 and then mixed as shown. Following incubation for 2 h of 1 µg GST fusion proteins or 5 µg GST and 0.5 µg his-SET, a GST pulldown was performed and the proteins were detected by CBB staining and immunoblotting as shown. (E) 293T cells were transfected to express GFP, GFP-SET, or GFP-SETΔC which lacks the 59 C-terminal amino acids. The experiment was further performed as in C, with the exception that endogenous H2B was detected by antibodies. (F) SET expression was eliminated in HCT116 cells by CRISPR-Cas9–mediated gene deletion, and a tet-inducible GFP-SET expression plasmid was stably introduced by puromycin selection. Following induction of GFP-SET expression by 1 µg/ml doxycycline for 24 h, cells were arrested with RO-3306 for 16 h and released into mitosis. Cells were stained against H2B S6ph, and the localization of H2B S6ph and GFP-SET in different mitotic phases is displayed. Bars, 2 µm.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Binding Assay, Modification, SDS Page, Silver Staining, Phospho-proteomics, Western Blot, Sonication, Immunoprecipitation, Purification, Incubation, Staining, Transfection, Expressing, CRISPR, Plasmid Preparation, Stable Transfection, Selection

Journal: The Journal of Cell Biology

Article Title: CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation

doi: 10.1083/jcb.201806057

Figure Lengend Snippet: Spatial distribution of H2B S6ph and SET during mitosis. (A) The HCT116 cells described in were induced to trigger GFP-SET expression by the addition of 1 µg/ml doxycycline for 24 h. The top and middle panels show representative examples for fixed cells that were analyzed by confocal microscopy for the distribution of GFP-SET and CENP-A in prometaphase and metaphase cells. Line scan analysis was performed for 24 centromere pairs from three different prometaphase and metaphase cells, respectively. Quantification was done using ImageJ and GraphPad Prism; the maximum of fluorescence intensity was set as 1, and the dotted lines indicate standard deviations. The bottom panel shows the comparison of H2B S6ph and GFP-SET distribution in metaphase cells; line scan analysis was done by quantitative evaluation of 48 centromere pairs from 12 different cells. Bars, 2 µm. (B) Schematic summary depicting the possible molecular mechanisms leading to spatial and temporal control of H2B S6ph. Mitotic chromosomes are shown in blue, and the position of phosphorylated H2B is indicated in red, CENP-A chromatin in yellow, and kinetochores in dark green. Black arrows represent kinase activity of CDK1-cyclin B1 and Aurora B as part of the CPC.

Article Snippet: Anti-H2B S6ph antibodies were generated by immunization of two rabbits with peptide SP120160 (Table S3) by PolyPeptide Laboratories.

Techniques: Expressing, Confocal Microscopy, Fluorescence, Comparison, Control, Activity Assay

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

Article Title: Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells

doi: 10.4049/jimmunol.1700638

Figure Lengend Snippet: (A) Kaplan-Meier survival curves for Hpb-infected or uninfected iNKT-deficient (Jα18−/−) male BMT recipients that received T cell-depleted bone marrow (TCD-BM) cells (TCD BM) only, or TCD-BM plus total splenic T (TCD-BM + T) cells, from 5–6 week old male WT C57BL/6 donor mice. Cumulative data from two independent experiments. Uninfected TCD-BM only: N = 5; Hpb-infected TCD-BM only: N = 5; uninfected TCD-BM + T: N = 6; Hpb-infected TCD-BM + T: N = 7; p<0.001 between uninfected TCD-BM + T and Hpb-infected TCD-BM + T. (B) GVHD disease score and (C) weight change of the same group of mice. Weight loss for each group of mice is displayed as percent weight change at different time points compared to initial weight. (D) Representative dot plots from MLN cells isolated 6 days after BMT, from uninfected (Uninf) or Hpb-infected Jα18−/− BMT recipients of WT C57BL/6 (WT B6) or TGFβRII DN (DN B6) splenic T cell donors. For BMT, splenic donor T cells were obtained from uninfected mice and all groups also received donor TCD-BM (T cell-depleted BM) cells from uninfected C57BL/6 mice. MLN cells were stained for CD3, CD4, H2b, H2d and Foxp3. Cells were gated on donor (H2b+) CD3+ T cells. Numbers represent the percentage of events in each quadrant and the percentage of Foxp3+ CD4 Tregs in the right upper quadrant. Representative example from 3 parallel independent experiments.

Article Snippet: Antibodies for surface staining were: anti-CD3 FITC, anti-CD3 PE-Cy7 (Clone: 145–2C11), anti-CD4 PE-Cy7 (Clone: GK1.5; eBioscience), anti-H2b PE, anti-H2d PE, and anti-H2b APC (Clones: SF1–1.1, SF1–1.1.1, AF6.88.5; BD Biosciences).

Techniques: Infection, Isolation, Staining

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

Article Title: Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells

doi: 10.4049/jimmunol.1700638

Figure Lengend Snippet: Representative dot plots from spleen (upper rows) and MLN (lower rows) cells isolated from uninfected and Hpb-infected IL4−/− or WT (BALB/c) mice 6 days after BMT. Spleen and MLN cells were stained for CD3, CD4, H2b, H2d and Foxp3. Cells were gated on donor or host CD3+ T cells. Numbers represent the percentage of events in each quadrant and the percentage of Foxp3+ CD4 Tregs in the right upper quadrant.

Article Snippet: Antibodies for surface staining were: anti-CD3 FITC, anti-CD3 PE-Cy7 (Clone: 145–2C11), anti-CD4 PE-Cy7 (Clone: GK1.5; eBioscience), anti-H2b PE, anti-H2d PE, and anti-H2b APC (Clones: SF1–1.1, SF1–1.1.1, AF6.88.5; BD Biosciences).

Techniques: Isolation, Infection, Staining

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

Article Title: Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells

doi: 10.4049/jimmunol.1700638

Figure Lengend Snippet: (A) Representative dot plots from spleen and MLN cells isolated from uninfected and Hpb-infected IL4−/− (left) or WT (BALB/c) (right) BMT recipients of WT (C57BL/6) donors, 6 days after BMT. Spleen and MLN cells were stained for CD3, CD4, H2b, Foxp3 and GATA3. Cells were gated on WT C57BL/6 (H2b+) donor CD3+ CD4+ T cells. Parallel splenocyte and MLN cell isolates were stained for CD3, CD4, H2b, Foxp3 and isotype antibody (instead of GATA3) (upper panels). Numbers represent the percentage of events in each quadrant and GATA3− and GATA3+ CD4 Tregs in left upper and the right upper quadrants, respectively. Representative example from 3 independent experiments. (B) Purified CD4 T cells from helminth-infected mice with T cell specific deficiency for GATA3 (GATA3 fl/fl x CD4 Cre+) and from helminth-infected control GATA3 sufficient mice (GATA3 fl/fl x CD4 Cre−) were stimulated plate-bound anti-CD3 and soluble anti-CD28 for 48 hours. Culture supernatants were analyzed by ELISA. Data show mean (bar) from multiple independent experiments (scatter plots) where each dot (N) represents mean value of a single independent experiment calculated from multiple (≥3) repeats (p values between GATA3 deficient and GATA3 sufficient groups as indicated in each panel; differences between groups determined by unpaired Welch’s t-test). (C) Representative dot plots of splenocytes from uninfected (Uninf) and Hpb-infected TGFβ RII DN (DN B6) or C57BL/6 WT (WT B6) mice. Cells were stained for CD3, CD4, Foxp3 and GATA3. Cells were gated on CD3+ CD4+ T cells. Representative example from 3 independent experiments.

Article Snippet: Antibodies for surface staining were: anti-CD3 FITC, anti-CD3 PE-Cy7 (Clone: 145–2C11), anti-CD4 PE-Cy7 (Clone: GK1.5; eBioscience), anti-H2b PE, anti-H2d PE, and anti-H2b APC (Clones: SF1–1.1, SF1–1.1.1, AF6.88.5; BD Biosciences).

Techniques: Isolation, Infection, Staining, Purification, Enzyme-linked Immunosorbent Assay