recombinant human il 2  (R&D Systems)

 
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    Name:
    Recombinant Human IL 2 Protein
    Description:
    The Recombinant Human IL 2 Protein from R D Systems is derived from E coli The Recombinant Human IL 2 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    202-il-010
    Price:
    199
    Applications:
    Bioactivity
    Purity:
    >97%, by SDS-PAGE under reducing conditions and visualized by silver stain.
    Conjugate:
    Unconjugated
    Size:
    10 ug
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Human IL-2 Protein
    Buy from Supplier


    Structured Review

    R&D Systems recombinant human il 2
    Recombinant Human IL 2 Protein
    The Recombinant Human IL 2 Protein from R D Systems is derived from E coli The Recombinant Human IL 2 Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/recombinant human il 2/product/R&D Systems
    Average 99 stars, based on 257 article reviews
    Price from $9.99 to $1999.99
    recombinant human il 2 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Langerhans-type dendritic cells electroporated with TRP-2 mRNA stimulate cellular immunity against melanoma: Results of a phase I vaccine trial"

    Article Title: Langerhans-type dendritic cells electroporated with TRP-2 mRNA stimulate cellular immunity against melanoma: Results of a phase I vaccine trial

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2017.1372081

    LC vaccines stimulate CD4 and CD8 T-cell cytokine secretion and activation epitope expression in patients with melanoma. Responses against TRP2 mRNA-electroporated LCs were measured at approximately one and three months after completion of vaccines, and compared with pre-vaccine levels. (A) CD4 and (B) CD8 T-cell pro-inflammatory cytokine (IFN-γ, IL-2, and TNF-α) secretion and activation epitope (CD107a and Mip-1-β) expression, fold-change. For panels A and B, pooled data (triplicate means ± SEM; * P
    Figure Legend Snippet: LC vaccines stimulate CD4 and CD8 T-cell cytokine secretion and activation epitope expression in patients with melanoma. Responses against TRP2 mRNA-electroporated LCs were measured at approximately one and three months after completion of vaccines, and compared with pre-vaccine levels. (A) CD4 and (B) CD8 T-cell pro-inflammatory cytokine (IFN-γ, IL-2, and TNF-α) secretion and activation epitope (CD107a and Mip-1-β) expression, fold-change. For panels A and B, pooled data (triplicate means ± SEM; * P

    Techniques Used: Activation Assay, Expressing

    2) Product Images from "T regulatory cells control T-cell proliferation partly by the release of soluble CD25 in patients with B-cell malignancies"

    Article Title: T regulatory cells control T-cell proliferation partly by the release of soluble CD25 in patients with B-cell malignancies

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2010.03308.x

    Patient T cells exhibit decreased proliferative capacity. (a) Peripheral blood mononuclear cells were stimulated with anti-CD3/interleukin-2 and cultured in triplicates in 96-well plates in Alamar Blue assays. The mean valuesof 18 patients and 23 controls are shown in the figure. (b) Healthy controls (
    Figure Legend Snippet: Patient T cells exhibit decreased proliferative capacity. (a) Peripheral blood mononuclear cells were stimulated with anti-CD3/interleukin-2 and cultured in triplicates in 96-well plates in Alamar Blue assays. The mean valuesof 18 patients and 23 controls are shown in the figure. (b) Healthy controls (

    Techniques Used: Cell Culture

    Recombinant soluble (s) CD25 inhibit T-cell proliferation. (a) Recombinant sCD25 was added to OKT-3/interleukin-2 (IL-2) stimulated peripheral blood mononumear cells in triplicates from 11 donors in an Alamar Blue assay. Cells were cultured in final concentrations of 1000 ( n = 8), 500 ( n = 8) 250 ( n = 8) or 100 pg/ml ( n = 3) sCD25. The experiment was repeated four times with similar results. (b) PBMCs from six healthy donors were unstimulated, or stimulated with anti-CD3 (OKT3), OKT3 + IL-2, OKT3 + IL-2 + sCD25 (1000 pg/ml) or OKT3 + pre-incubated IL-2/sCD25 to allow complex formation before culture. The OKT3/IL-2 T cells were significantly different from unstimulated ( P = 0·002), OKT3-stimulated ( P = 0·02), OKT3/IL-2/sCD25-stimulated ( P = 0·0152) and OKT3 + IL-2/sCD25 complex ( P = 0·02). The experiment was repeated with six additional donors with similar results. Mann–Whitney U -test was used to analyse differences among groups.
    Figure Legend Snippet: Recombinant soluble (s) CD25 inhibit T-cell proliferation. (a) Recombinant sCD25 was added to OKT-3/interleukin-2 (IL-2) stimulated peripheral blood mononumear cells in triplicates from 11 donors in an Alamar Blue assay. Cells were cultured in final concentrations of 1000 ( n = 8), 500 ( n = 8) 250 ( n = 8) or 100 pg/ml ( n = 3) sCD25. The experiment was repeated four times with similar results. (b) PBMCs from six healthy donors were unstimulated, or stimulated with anti-CD3 (OKT3), OKT3 + IL-2, OKT3 + IL-2 + sCD25 (1000 pg/ml) or OKT3 + pre-incubated IL-2/sCD25 to allow complex formation before culture. The OKT3/IL-2 T cells were significantly different from unstimulated ( P = 0·002), OKT3-stimulated ( P = 0·02), OKT3/IL-2/sCD25-stimulated ( P = 0·0152) and OKT3 + IL-2/sCD25 complex ( P = 0·02). The experiment was repeated with six additional donors with similar results. Mann–Whitney U -test was used to analyse differences among groups.

    Techniques Used: Recombinant, Alamar Blue Assay, Cell Culture, Incubation, MANN-WHITNEY

    3) Product Images from "?? T Cells in Immunity Induced by Mycobacterium bovis Bacillus Calmette-Gu?rin Vaccination "

    Article Title: ?? T Cells in Immunity Induced by Mycobacterium bovis Bacillus Calmette-Gu?rin Vaccination

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.3.1504-1511.2004

    Direct priming of γδ T cells by M. bovis BCG vaccination. γδ T-cell proliferation in CD4-depleted PBMCs was compared in M. bovis BCG-vaccinated animals and controls at 11 weeks after M. bovis BCG vaccination. PBMCs were stained with PKH-26 and then stimulated with culture filtrate protein (CFP) or heat-killed M. tuberculosis H37Rv whole cells (WC) in the presence or absence of recombinant human IL-2 (1 ng/ml). The significant increase in γδ T-cell proliferation among the CD4-depleted PBMCs of M. bovis BCG-vaccinated pigs ( n = 3) compared to that of controls ( n = 4) is indicated (*, P
    Figure Legend Snippet: Direct priming of γδ T cells by M. bovis BCG vaccination. γδ T-cell proliferation in CD4-depleted PBMCs was compared in M. bovis BCG-vaccinated animals and controls at 11 weeks after M. bovis BCG vaccination. PBMCs were stained with PKH-26 and then stimulated with culture filtrate protein (CFP) or heat-killed M. tuberculosis H37Rv whole cells (WC) in the presence or absence of recombinant human IL-2 (1 ng/ml). The significant increase in γδ T-cell proliferation among the CD4-depleted PBMCs of M. bovis BCG-vaccinated pigs ( n = 3) compared to that of controls ( n = 4) is indicated (*, P

    Techniques Used: Staining, Recombinant

    4) Product Images from "Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model"

    Article Title: Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00563-16

    ASCs that secreted immunogen-specific antibody were present in blood recovered from chinchillas immunized by TCI with a band-aid. Blood was collected 9 days after receipt of the second of two immunizing doses delivered 1 week apart, prior to NTHI challenge. Antibody production by resiquimod- and IL-2-stimulated lymphocytes was assessed by ELISPOT assay. The total numbers of ASCs that produced immunogen-specific IgM, IgG, and IgA were comparable among the cohorts. The mean ± SEM for each antibody isotype within each cohort is presented.
    Figure Legend Snippet: ASCs that secreted immunogen-specific antibody were present in blood recovered from chinchillas immunized by TCI with a band-aid. Blood was collected 9 days after receipt of the second of two immunizing doses delivered 1 week apart, prior to NTHI challenge. Antibody production by resiquimod- and IL-2-stimulated lymphocytes was assessed by ELISPOT assay. The total numbers of ASCs that produced immunogen-specific IgM, IgG, and IgA were comparable among the cohorts. The mean ± SEM for each antibody isotype within each cohort is presented.

    Techniques Used: Enzyme-linked Immunospot, Produced

    5) Product Images from "A Dominant EV71-Specific CD4+ T Cell Epitope Is Highly Conserved among Human Enteroviruses"

    Article Title: A Dominant EV71-Specific CD4+ T Cell Epitope Is Highly Conserved among Human Enteroviruses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051957

    EV71 A3 or A8-specific CD4+ T cells respond to the poliovirus 3 Sabin (PV3) homolog, A3v peptide. PBMCs from two adult subjects were cultured in the presence of EV71 A3, EV71 A8, PV3 A3v, or SP4 peptides for 4 days and then with recombinant human IL-2 for additional 3 days. The cells were washed and re-stimulated with SP4, EV71 A3 or PV A3′, then surface-stained with fluorescent antibodies against CD3 and CD4 molecules and intracellularly for the production of IFNγ. Gated CD3+ cells are shown. (A) SP4 or PV3 A3v-stimulated cells were re-stimulated with the respective peptides. (B) EV71 A3-stimulated cells were re-stimulated with SP4, EV71 A3 or PV3 A3v, respectively. (C) EV71 A8-stimulated cells were re-stimulated with SP4, EV71 A8 or PV3 A3v, respectively. The above results were representative data from three independent experiments.
    Figure Legend Snippet: EV71 A3 or A8-specific CD4+ T cells respond to the poliovirus 3 Sabin (PV3) homolog, A3v peptide. PBMCs from two adult subjects were cultured in the presence of EV71 A3, EV71 A8, PV3 A3v, or SP4 peptides for 4 days and then with recombinant human IL-2 for additional 3 days. The cells were washed and re-stimulated with SP4, EV71 A3 or PV A3′, then surface-stained with fluorescent antibodies against CD3 and CD4 molecules and intracellularly for the production of IFNγ. Gated CD3+ cells are shown. (A) SP4 or PV3 A3v-stimulated cells were re-stimulated with the respective peptides. (B) EV71 A3-stimulated cells were re-stimulated with SP4, EV71 A3 or PV3 A3v, respectively. (C) EV71 A8-stimulated cells were re-stimulated with SP4, EV71 A8 or PV3 A3v, respectively. The above results were representative data from three independent experiments.

    Techniques Used: Cell Culture, Recombinant, Staining

    Both A3- and A8-specific T cells are HLA-DR-restricted CD4+ T cells. (A) and (B) PBMCs from adult donors were stimulated with A3 or A8 peptides and then expanded with recombinant IL-2. (A) The stimulated PBMCs were stained with fluorescently labeled antibodies against CD3, CD4, CD8 and IFNγ before flow cytometric analysis. Antigen-specific IFNγ production was primarily associated with CD4+ T cells. (B) CD8+ T cells produced negligible level of IFNγ in response to A3 or A8. (C) A3 and A8 can induce strong CD4+ cell responses in HFMD patients infected by EV71. (D) and (E) ELISpot assays were performed to examine IFNγ production by PBMCs which were stimulated with A3 (D) or A8 (E) peptides alone or in presence of blocking antibodies against HLA-DR, HLA-DP, or HLA-A, B and –C (HLA-ABC). The results were shown as average spot forming units ± standard deviation (n = 3). Only anti-HLADR antibodies abrogated the ELISpot responses, demonstrating that the responses to EV71 are Class II-restricted.
    Figure Legend Snippet: Both A3- and A8-specific T cells are HLA-DR-restricted CD4+ T cells. (A) and (B) PBMCs from adult donors were stimulated with A3 or A8 peptides and then expanded with recombinant IL-2. (A) The stimulated PBMCs were stained with fluorescently labeled antibodies against CD3, CD4, CD8 and IFNγ before flow cytometric analysis. Antigen-specific IFNγ production was primarily associated with CD4+ T cells. (B) CD8+ T cells produced negligible level of IFNγ in response to A3 or A8. (C) A3 and A8 can induce strong CD4+ cell responses in HFMD patients infected by EV71. (D) and (E) ELISpot assays were performed to examine IFNγ production by PBMCs which were stimulated with A3 (D) or A8 (E) peptides alone or in presence of blocking antibodies against HLA-DR, HLA-DP, or HLA-A, B and –C (HLA-ABC). The results were shown as average spot forming units ± standard deviation (n = 3). Only anti-HLADR antibodies abrogated the ELISpot responses, demonstrating that the responses to EV71 are Class II-restricted.

    Techniques Used: Recombinant, Staining, Labeling, Flow Cytometry, Produced, Infection, Enzyme-linked Immunospot, Blocking Assay, Standard Deviation

    6) Product Images from "Type 2 innate lymphoid cells control eosinophil homeostasis"

    Article Title: Type 2 innate lymphoid cells control eosinophil homeostasis

    Journal: Nature

    doi: 10.1038/nature12526

    IL-5 and IL-13 co-expression in lung ILC2 a , Lung IL-5 and IL-13 reporter expression before and after infection. b , Flow cytometry of CD90.2+ lung cells and percent with ILC2 surface markers (CD90.2 and either KRLG1, T1/ST2 or CD25) at rest. c , ILC2 (left lung) and CCL11 concentration (right lung) after IL-2, IL-33, and IL-13 treatment. Data representative of three independent experiments with 4 (naïve R5+S13+), 5 (infected R5+S13+), or 2 (others) mice per group ( a ), pooled from three independent experiments for 6 (R5/R5 bone marrow and spleen) or 9 (all others) mice per group ( b ), or pooled from two independent experiments for 8 (R5/R5 + IL-2/IL-33), 5 (R5/R5 Deleter + IL-2/IL-33/IL-13), or 3 (others) mice per group ( c ). Represented as mean +/− SEM. huCD4, human CD4; BM, bone marrow; NS, not significant; *, p
    Figure Legend Snippet: IL-5 and IL-13 co-expression in lung ILC2 a , Lung IL-5 and IL-13 reporter expression before and after infection. b , Flow cytometry of CD90.2+ lung cells and percent with ILC2 surface markers (CD90.2 and either KRLG1, T1/ST2 or CD25) at rest. c , ILC2 (left lung) and CCL11 concentration (right lung) after IL-2, IL-33, and IL-13 treatment. Data representative of three independent experiments with 4 (naïve R5+S13+), 5 (infected R5+S13+), or 2 (others) mice per group ( a ), pooled from three independent experiments for 6 (R5/R5 bone marrow and spleen) or 9 (all others) mice per group ( b ), or pooled from two independent experiments for 8 (R5/R5 + IL-2/IL-33), 5 (R5/R5 Deleter + IL-2/IL-33/IL-13), or 3 (others) mice per group ( c ). Represented as mean +/− SEM. huCD4, human CD4; BM, bone marrow; NS, not significant; *, p

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Concentration Assay, Mouse Assay

    7) Product Images from "Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants"

    Article Title: Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13113-5

    Allogeneic endothelial cells trigger missing self-induced activation of NK cells. Primary allogeneic human endothelial cells were co-cultured with purified NK cells from 30 healthy volunteers primed with low-dose IL-2. After 4 h of culture the activation status of the NK cells was assessed at the single cell level by flow cytometry. a , b Analyses were focused on the five NK cell populations that expressed a single inhibitory KIR. a Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. b Intracellular staining for MIP-1β in NK cells after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. c , d Analyses were focused on the NK cell populations that expressed two inhibitory KIRs, one of them lacking its ligand on endothelial cells (missing self, MS). According to the nature of the second inhibitory KIR, three situations were distinguished: 1MS+1M, if endothelial cells expressed the ligand for the second inhibitory KIR; 1MS + uneduc MS, if neither endothelial cells nor NK cell donor expressed the ligand for the second inhibitory KIR, and 2MS, if endothelial cells did not express the ligands of the two inhibitory KIRs. Results are normalised over the value observed for the NK cell population that expressed the single mismatched inhibitory KIR. c Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. d Intracellular staining for MIP-1β in NK cells after 4 h co-culture. ns: p ≥ 0.05, * p
    Figure Legend Snippet: Allogeneic endothelial cells trigger missing self-induced activation of NK cells. Primary allogeneic human endothelial cells were co-cultured with purified NK cells from 30 healthy volunteers primed with low-dose IL-2. After 4 h of culture the activation status of the NK cells was assessed at the single cell level by flow cytometry. a , b Analyses were focused on the five NK cell populations that expressed a single inhibitory KIR. a Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. b Intracellular staining for MIP-1β in NK cells after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. c , d Analyses were focused on the NK cell populations that expressed two inhibitory KIRs, one of them lacking its ligand on endothelial cells (missing self, MS). According to the nature of the second inhibitory KIR, three situations were distinguished: 1MS+1M, if endothelial cells expressed the ligand for the second inhibitory KIR; 1MS + uneduc MS, if neither endothelial cells nor NK cell donor expressed the ligand for the second inhibitory KIR, and 2MS, if endothelial cells did not express the ligands of the two inhibitory KIRs. Results are normalised over the value observed for the NK cell population that expressed the single mismatched inhibitory KIR. c Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. d Intracellular staining for MIP-1β in NK cells after 4 h co-culture. ns: p ≥ 0.05, * p

    Techniques Used: Activation Assay, Cell Culture, Purification, Flow Cytometry, Cytometry, Expressing, Co-Culture Assay, Staining, Mass Spectrometry

    8) Product Images from "Itk Negatively Regulates Induction of T Cell Proliferation by CD28 Costimulation"

    Article Title: Itk Negatively Regulates Induction of T Cell Proliferation by CD28 Costimulation

    Journal: The Journal of Experimental Medicine

    doi:

    CD28 costimulation of TCR-induced proliferation and IL-2 production. ( A ) CD4 + T cells from itk −/− mice ( circles ) and itk +/− littermates ( squares ) were stimulated with plate-bound anti-CD3 at different concentrations as indicated, in the presence ( solid lines ) or absence ( broken lines ) of anti-CD28 ascites (1:500). Cell proliferation was measured 48 h after stimulation. ( B ) IL-2 production in the culture supernatant after 48 h of stimulation was measured by ELISA. No detectable IL-2 was produced from cells stimulated with anti-CD3 alone.
    Figure Legend Snippet: CD28 costimulation of TCR-induced proliferation and IL-2 production. ( A ) CD4 + T cells from itk −/− mice ( circles ) and itk +/− littermates ( squares ) were stimulated with plate-bound anti-CD3 at different concentrations as indicated, in the presence ( solid lines ) or absence ( broken lines ) of anti-CD28 ascites (1:500). Cell proliferation was measured 48 h after stimulation. ( B ) IL-2 production in the culture supernatant after 48 h of stimulation was measured by ELISA. No detectable IL-2 was produced from cells stimulated with anti-CD3 alone.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Produced

    Cell proliferation induced by TCR and CD28 is dependent on IL-2. CD4 + T cells from itk −/− mice and itk +/− littermates were stimulated with plate-bound anti-CD3 at different concentrations as indicated, in the presence or absence of anti-CD28 ascites (1:500). Anti-CD28 and anti-murine IL-2 were added with anti-murine IL-2R or with recombinant human IL-2 at the beginning of culture. Cell proliferation was measured 72 h after stimulation. a-CD28, anti-CD28 ascites; a-mIL2, anti-murine IL-2 mAb; a-mIL2R, anti-murine IL-2Rα mAb; and hIL-2, recombinant human IL-2.
    Figure Legend Snippet: Cell proliferation induced by TCR and CD28 is dependent on IL-2. CD4 + T cells from itk −/− mice and itk +/− littermates were stimulated with plate-bound anti-CD3 at different concentrations as indicated, in the presence or absence of anti-CD28 ascites (1:500). Anti-CD28 and anti-murine IL-2 were added with anti-murine IL-2R or with recombinant human IL-2 at the beginning of culture. Cell proliferation was measured 72 h after stimulation. a-CD28, anti-CD28 ascites; a-mIL2, anti-murine IL-2 mAb; a-mIL2R, anti-murine IL-2Rα mAb; and hIL-2, recombinant human IL-2.

    Techniques Used: Mouse Assay, Recombinant

    9) Product Images from "Protein tyrosine phosphatase PTP-RR regulates corticosteroid sensitivity"

    Article Title: Protein tyrosine phosphatase PTP-RR regulates corticosteroid sensitivity

    Journal: Respiratory Research

    doi: 10.1186/s12931-016-0349-0

    Effect of formoterol on immunopurified PTP-RR activity in U937 cells. a Cells were treated with formoterol (FM; 10 −10 -10 −7 M), salmeterol (SM; 10 −7 M) or salbutamol (SB; 10 −7 M) for 20 min. b Cells stimulated with IL-2/IL-4 for 48 h were treated with FM (10 −9 M) for 20 min. c Cells were preincubated with or without ICI-118551 (ICI; 10 −5 M), a selective β 2 -adrenoceptor antagonist for 30 min, followed by treatment with FM (10 −9 M) or cAMP inducer, forskolin (10 −5 M). Data are expressed as fold change against non-treatment control (NT). Values represent means of four ( a and b ) or three ( c ) experiments ± SEM: # P
    Figure Legend Snippet: Effect of formoterol on immunopurified PTP-RR activity in U937 cells. a Cells were treated with formoterol (FM; 10 −10 -10 −7 M), salmeterol (SM; 10 −7 M) or salbutamol (SB; 10 −7 M) for 20 min. b Cells stimulated with IL-2/IL-4 for 48 h were treated with FM (10 −9 M) for 20 min. c Cells were preincubated with or without ICI-118551 (ICI; 10 −5 M), a selective β 2 -adrenoceptor antagonist for 30 min, followed by treatment with FM (10 −9 M) or cAMP inducer, forskolin (10 −5 M). Data are expressed as fold change against non-treatment control (NT). Values represent means of four ( a and b ) or three ( c ) experiments ± SEM: # P

    Techniques Used: Activity Assay

    Proposal mechanism of regulation of corticosteroid sensitivity. Reduction of GR nuclear translocation by phosphorylation of GR-Ser 226 is one of key mechanisms of corticosteroid insensitivity. Severe allergic inflammation associated with production of IL-2 and IL-4 ( white arrows ) induces phosphorylation of GR-Ser 226 by JNK1 activation and PP2A inactivation (Tyr 307 phosphorylation). PTP-RR regulates corticosteroid sensitivity by dephosphorylation of JNK1/GR-Ser 226 directly and/or indirectly via PP2A
    Figure Legend Snippet: Proposal mechanism of regulation of corticosteroid sensitivity. Reduction of GR nuclear translocation by phosphorylation of GR-Ser 226 is one of key mechanisms of corticosteroid insensitivity. Severe allergic inflammation associated with production of IL-2 and IL-4 ( white arrows ) induces phosphorylation of GR-Ser 226 by JNK1 activation and PP2A inactivation (Tyr 307 phosphorylation). PTP-RR regulates corticosteroid sensitivity by dephosphorylation of JNK1/GR-Ser 226 directly and/or indirectly via PP2A

    Techniques Used: Translocation Assay, Activation Assay, De-Phosphorylation Assay

    PTP-RR function under corticosteroid insensitive condition. a GR and JNK1 expression in PTP-RR-immunoprecipitates. b , c , d , e , f , U937 cells were exposed to IL-2/IL-4 for 48 h. PTP-RR protein expression ( b ) and activity ( c ) in whole cell extracts. GR ( d ) and JNK1 ( e ) protein expressions in PTP-RR-immunoprecipitates. Effect of MG-132 on PTP-RR expression ( f ). Data in ( d ) and ( e ) are expressed as fold change against non-treatment control (NT). Values represent means of four experiments ± SEM. # P
    Figure Legend Snippet: PTP-RR function under corticosteroid insensitive condition. a GR and JNK1 expression in PTP-RR-immunoprecipitates. b , c , d , e , f , U937 cells were exposed to IL-2/IL-4 for 48 h. PTP-RR protein expression ( b ) and activity ( c ) in whole cell extracts. GR ( d ) and JNK1 ( e ) protein expressions in PTP-RR-immunoprecipitates. Effect of MG-132 on PTP-RR expression ( f ). Data in ( d ) and ( e ) are expressed as fold change against non-treatment control (NT). Values represent means of four experiments ± SEM. # P

    Techniques Used: Expressing, Activity Assay

    Regulation of PP2A by PTP-RR. a U937 cells exposed to IL-2/IL-4 for 48 h (or were not treated; NT). PP2A C was detected in PTP-RR-immunoprecipitates. b , c , d , PP2A C -Tyr 307 phosphorylation ( b ), PP2A activity ( c ) and PP2A C protein expression ( d ) were analyzed in U937 cells transfected with scramble control (SC) or PTP-RR siRNAs. e , f , In U937 cells transfected with scramble control (SC) or PP2A siRNAs, immunopurified PTP-RR activity ( e ) and PTP-RR protein expression ( f ) were analyzed. Data in ( f ) is expressed as fold change against SC. Values represent means of four ( a , d and f ) or three ( b , c and e ) experiments ± SEM. # P
    Figure Legend Snippet: Regulation of PP2A by PTP-RR. a U937 cells exposed to IL-2/IL-4 for 48 h (or were not treated; NT). PP2A C was detected in PTP-RR-immunoprecipitates. b , c , d , PP2A C -Tyr 307 phosphorylation ( b ), PP2A activity ( c ) and PP2A C protein expression ( d ) were analyzed in U937 cells transfected with scramble control (SC) or PTP-RR siRNAs. e , f , In U937 cells transfected with scramble control (SC) or PP2A siRNAs, immunopurified PTP-RR activity ( e ) and PTP-RR protein expression ( f ) were analyzed. Data in ( f ) is expressed as fold change against SC. Values represent means of four ( a , d and f ) or three ( b , c and e ) experiments ± SEM. # P

    Techniques Used: Activity Assay, Expressing, Transfection

    10) Product Images from "Enhanced suppressive function of regulatory T cells from patients with immune-mediated diseases following successful ex vivo expansion"

    Article Title: Enhanced suppressive function of regulatory T cells from patients with immune-mediated diseases following successful ex vivo expansion

    Journal: Clinical immunology (Orlando, Fla.)

    doi: 10.1016/j.clim.2010.04.014

    CD4 + CD25 + Treg cells isolated from the peripheral blood of patients with immune-mediated diseases can be expanded 100–2000 fold ex vivo with sustained purity. Human CD4 + CD25 + Treg cells enriched from the peripheral blood of patients with immune-mediated diseases were activated and expanded ex vivo with Dynal CD3/28 T cell expander beads and recombinant IL-2 as described. Cell numbers on day 7, 14 and 21 cultures were counted for each individual culture. The ex vivo Treg cell growth curves were shown in A. Each line represents a single subject. The purity of ex vivo expanded human Treg cells at week 2 was determined by intracellular Foxp3 staining, and was shown in B. Each dot represents one subject and the bar represents the average purity of expanded Treg cells (Fig. 3B, * p
    Figure Legend Snippet: CD4 + CD25 + Treg cells isolated from the peripheral blood of patients with immune-mediated diseases can be expanded 100–2000 fold ex vivo with sustained purity. Human CD4 + CD25 + Treg cells enriched from the peripheral blood of patients with immune-mediated diseases were activated and expanded ex vivo with Dynal CD3/28 T cell expander beads and recombinant IL-2 as described. Cell numbers on day 7, 14 and 21 cultures were counted for each individual culture. The ex vivo Treg cell growth curves were shown in A. Each line represents a single subject. The purity of ex vivo expanded human Treg cells at week 2 was determined by intracellular Foxp3 staining, and was shown in B. Each dot represents one subject and the bar represents the average purity of expanded Treg cells (Fig. 3B, * p

    Techniques Used: Isolation, Ex Vivo, Recombinant, Staining

    11) Product Images from "CBF-1 Promotes the Establishment and Maintenance of HIV Latency by Recruiting Polycomb Repressive Complexes, PRC1 and PRC2, at HIV LTR"

    Article Title: CBF-1 Promotes the Establishment and Maintenance of HIV Latency by Recruiting Polycomb Repressive Complexes, PRC1 and PRC2, at HIV LTR

    Journal: Viruses

    doi: 10.3390/v12091040

    Cell activation leads to fluctuation in the levels of different chromatin-associated factors that belong to PRC1 and PRC2. ChIP analyses were performed before and after activation of latently infected primary CD4+ T cells with α-CD3/-CD28 antibodies, in the presence of IL-2 for 30 min. ( a ) Structure of lentiviral vectors. mCherry was used as reporter depicted in this diagram. ChIP results in latency systems harboring proviruses with the vector pHR’-PNL-H13LTat-mCherry ( b , c ), and pHR’-PNL-wild-typeTat-mCherry ( d , e ). Error bars represent the SEM of two independent experiments and three separate qPCR measurements from each analysis. Graphs represent the average and standard deviation from three independent and replicate samples. Statistical analysis was calculated with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The p value of statistical significance was set at either; p
    Figure Legend Snippet: Cell activation leads to fluctuation in the levels of different chromatin-associated factors that belong to PRC1 and PRC2. ChIP analyses were performed before and after activation of latently infected primary CD4+ T cells with α-CD3/-CD28 antibodies, in the presence of IL-2 for 30 min. ( a ) Structure of lentiviral vectors. mCherry was used as reporter depicted in this diagram. ChIP results in latency systems harboring proviruses with the vector pHR’-PNL-H13LTat-mCherry ( b , c ), and pHR’-PNL-wild-typeTat-mCherry ( d , e ). Error bars represent the SEM of two independent experiments and three separate qPCR measurements from each analysis. Graphs represent the average and standard deviation from three independent and replicate samples. Statistical analysis was calculated with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The p value of statistical significance was set at either; p

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Standard Deviation, Software

    12) Product Images from "Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor"

    Article Title: Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056820

    Human lung cancer cells produce variable amounts of CCL2 and show sensitivity to cytocidal activity mediated by CD8 + T cells genetically engineered to express WT1-specific TCR. (A) ELISA assay revealed that 10 human lung cancer cell lines examined produced various amounts of CCL2 in the culture supernatant. Error bars represent SDs. (B) Similarly to transduction of the WT1-specific TCR gene, CD69-positive CD8 + and CD4 + T cells activated using IL-2 and OKT-3 rarely displayed cell-surface CCR2. A representative example of 5 cases is shown. (C) CD8 + T cells gene-modified to express HLA-A*2402-restricted and WT1 235–243 nonamer-specific TCR successfully displayed cytocidal activity against lung cancer cell line cells in a HLA-A*2402-restricted manner, as determined by standard 51 Cr release assay. Error bars represent SDs. MFI indicates mean fluorescence intensity, N.D indicates less than detectable. (D) Such anti-lung cancer activity (vs. LK79 and LC99A) was inhibited by anti-HLA class I framework mAb (clone w6/32), but not by anti-HLA-DR mAb (clone L243), again illustrating that the introduced WT1-specific TCR-mediated tumoricidal activity was HLA-class I-restricted. Error bars represent SDs. (E) Tetramer assay showed that effector cells used in these experiments were positive for WT1/HLA-A*2402-tetramer. HIV/HLA-A*2402 tetramer was employed as a negative control.
    Figure Legend Snippet: Human lung cancer cells produce variable amounts of CCL2 and show sensitivity to cytocidal activity mediated by CD8 + T cells genetically engineered to express WT1-specific TCR. (A) ELISA assay revealed that 10 human lung cancer cell lines examined produced various amounts of CCL2 in the culture supernatant. Error bars represent SDs. (B) Similarly to transduction of the WT1-specific TCR gene, CD69-positive CD8 + and CD4 + T cells activated using IL-2 and OKT-3 rarely displayed cell-surface CCR2. A representative example of 5 cases is shown. (C) CD8 + T cells gene-modified to express HLA-A*2402-restricted and WT1 235–243 nonamer-specific TCR successfully displayed cytocidal activity against lung cancer cell line cells in a HLA-A*2402-restricted manner, as determined by standard 51 Cr release assay. Error bars represent SDs. MFI indicates mean fluorescence intensity, N.D indicates less than detectable. (D) Such anti-lung cancer activity (vs. LK79 and LC99A) was inhibited by anti-HLA class I framework mAb (clone w6/32), but not by anti-HLA-DR mAb (clone L243), again illustrating that the introduced WT1-specific TCR-mediated tumoricidal activity was HLA-class I-restricted. Error bars represent SDs. (E) Tetramer assay showed that effector cells used in these experiments were positive for WT1/HLA-A*2402-tetramer. HIV/HLA-A*2402 tetramer was employed as a negative control.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Produced, Transduction, Modification, Release Assay, Fluorescence, Negative Control

    In vivo anti-lung cancer activity mediated by double-transfected effector cells in therapeutic xenografted mouse models. (A) The amount of CCL2 produced by luciferase -transfected LK79 cells (LK79/luc) was similar to that produced by the parent LK79 cells. (B) Nine-week-old NOG mice (n = 18) inoculated into the abdominal wall with 5×10 6 LK79/luc cells were divided into 3 cohorts. On day 4, when each tumor mass had become palpable, three mice in each cohort started to receive weekly intravenous administration of 5×10 6 double-transfected effector cells (cohort iii; black circles), WT1-si TCR single-transfected effector cells (cohort ii; gray square), or CD8 + T cells simply activated using OKT-3/IL-2 as a negative control (cohort i; clear circles), the effector cells all being generated from an identical donor. Intravenous administration was performed three times in total, and the relative mass burden was serially monitored on the basis of luciferase photon counts relative to those on day 4, before the start of therapeutic infusion. Double-transfected effector cells in cohort iii mice most effectively suppressed the growth of LK79/luc cells, notably in the immediate phase after therapeutic infusion (on day 7). In contrast, WT1-si TCR single-transfected effector cells gradually suppressed the growth of LK79/luc cells, being apparently dependent on time and the total number of effector cells infused. Effector cells that had been simply activated also displayed a marginal degree of tumor suppression, probably because of xenoreactivity. Error bars represent SDs. NGM-CD8 + T cells indicate CD8 + T cells simply activated using OKT-3/IL-2, expressing neither CCR2 nor WT1-specific TCR. (C) Serial bioluminescence images of mice in each cohort are shown. On day 28, 10 days after the last therapeutic infusion, durable growth suppression of LK79/luc cells was most evident in cohort iii mice that had received double-transfected effector cells. NGM-CD8 + T cells represent CD8 + T cells simply activated using OKT-3/IL-2, expressing neither CCR2 nor WT1-specific TCR.
    Figure Legend Snippet: In vivo anti-lung cancer activity mediated by double-transfected effector cells in therapeutic xenografted mouse models. (A) The amount of CCL2 produced by luciferase -transfected LK79 cells (LK79/luc) was similar to that produced by the parent LK79 cells. (B) Nine-week-old NOG mice (n = 18) inoculated into the abdominal wall with 5×10 6 LK79/luc cells were divided into 3 cohorts. On day 4, when each tumor mass had become palpable, three mice in each cohort started to receive weekly intravenous administration of 5×10 6 double-transfected effector cells (cohort iii; black circles), WT1-si TCR single-transfected effector cells (cohort ii; gray square), or CD8 + T cells simply activated using OKT-3/IL-2 as a negative control (cohort i; clear circles), the effector cells all being generated from an identical donor. Intravenous administration was performed three times in total, and the relative mass burden was serially monitored on the basis of luciferase photon counts relative to those on day 4, before the start of therapeutic infusion. Double-transfected effector cells in cohort iii mice most effectively suppressed the growth of LK79/luc cells, notably in the immediate phase after therapeutic infusion (on day 7). In contrast, WT1-si TCR single-transfected effector cells gradually suppressed the growth of LK79/luc cells, being apparently dependent on time and the total number of effector cells infused. Effector cells that had been simply activated also displayed a marginal degree of tumor suppression, probably because of xenoreactivity. Error bars represent SDs. NGM-CD8 + T cells indicate CD8 + T cells simply activated using OKT-3/IL-2, expressing neither CCR2 nor WT1-specific TCR. (C) Serial bioluminescence images of mice in each cohort are shown. On day 28, 10 days after the last therapeutic infusion, durable growth suppression of LK79/luc cells was most evident in cohort iii mice that had received double-transfected effector cells. NGM-CD8 + T cells represent CD8 + T cells simply activated using OKT-3/IL-2, expressing neither CCR2 nor WT1-specific TCR.

    Techniques Used: In Vivo, Activity Assay, Transfection, Produced, Luciferase, Mouse Assay, Negative Control, Generated, Expressing

    13) Product Images from "ILDR2-Fc Is a Novel Regulator of Immune Homeostasis and Inducer of Antigen-Specific Immune Tolerance"

    Article Title: ILDR2-Fc Is a Novel Regulator of Immune Homeostasis and Inducer of Antigen-Specific Immune Tolerance

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

    doi: 10.4049/jimmunol.1700326

    ILDR2-mFc downregulates Th1 and Th17 differentiation and enhances Th2 and Treg differentiation in vitro. Naive CD4 + T cells isolated from DO11.10 mice were activated with OVA 323–339 peptide (20 μg/ml) in the presence of irradiated APCs under Th1-, Th17-, or Th2-driving conditions, as described in Materials and Methods . Soluble ILDR2-mFc or control Ig (mIgG2a) (5 mg/ml) was added to these cultures. ( A–F ) Supernatants were collected after 72 h and analyzed for cytokine production via LiquiChip. Lymph node cells from PLP 139–151 /CFA primed mice were reactivated ex vivo with PLP 139–151 in the presence of ILDR2-mFc or control Ig. The level of cytokine secretion ( G ) was assessed in triplicate, and the phenotype of the resultant cells was assessed via flow cytometry ( H ). These ex vivo-reactivated blasts were transferred to naive SJL/J mice and evaluated for transfer R-EAE induction ( I ). ( J ) Freshly isolated CD4 + CD25 − T cells were activated for 4 d with plate-bound anti-CD3 (2 μg/ml) and coimmobilized with 10 μg/ml ILDR2-mFc or control Ig (mIgG2a), in the presence of soluble anti-CD28 (1 μg/ml), with IL-2 (5 ng/ml) over the indicated range of TGF-β concentrations. The number of CD25 + Foxp3 + cells was determined via FACS. Data represent mean ± SD of duplicate wells. One representative experiment of two or three independent experiments is presented for each experimental set. * p
    Figure Legend Snippet: ILDR2-mFc downregulates Th1 and Th17 differentiation and enhances Th2 and Treg differentiation in vitro. Naive CD4 + T cells isolated from DO11.10 mice were activated with OVA 323–339 peptide (20 μg/ml) in the presence of irradiated APCs under Th1-, Th17-, or Th2-driving conditions, as described in Materials and Methods . Soluble ILDR2-mFc or control Ig (mIgG2a) (5 mg/ml) was added to these cultures. ( A–F ) Supernatants were collected after 72 h and analyzed for cytokine production via LiquiChip. Lymph node cells from PLP 139–151 /CFA primed mice were reactivated ex vivo with PLP 139–151 in the presence of ILDR2-mFc or control Ig. The level of cytokine secretion ( G ) was assessed in triplicate, and the phenotype of the resultant cells was assessed via flow cytometry ( H ). These ex vivo-reactivated blasts were transferred to naive SJL/J mice and evaluated for transfer R-EAE induction ( I ). ( J ) Freshly isolated CD4 + CD25 − T cells were activated for 4 d with plate-bound anti-CD3 (2 μg/ml) and coimmobilized with 10 μg/ml ILDR2-mFc or control Ig (mIgG2a), in the presence of soluble anti-CD28 (1 μg/ml), with IL-2 (5 ng/ml) over the indicated range of TGF-β concentrations. The number of CD25 + Foxp3 + cells was determined via FACS. Data represent mean ± SD of duplicate wells. One representative experiment of two or three independent experiments is presented for each experimental set. * p

    Techniques Used: In Vitro, Isolation, Mouse Assay, Irradiation, Plasmid Purification, Ex Vivo, Flow Cytometry, Cytometry, FACS

    14) Product Images from "The T-cell pool is anergized in patients with multiple sclerosis in remission"

    Article Title: The T-cell pool is anergized in patients with multiple sclerosis in remission

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02881.x

    Proliferative capacity of circulating T cells in patients multiple sclerosis (MS). Peripheral blood mononuclear cells (PBMC) from patients with MS and healthy controls (HC) were stimulated with interleukin-2 (IL-2) and OKT-3 in an Alamar Blue™
    Figure Legend Snippet: Proliferative capacity of circulating T cells in patients multiple sclerosis (MS). Peripheral blood mononuclear cells (PBMC) from patients with MS and healthy controls (HC) were stimulated with interleukin-2 (IL-2) and OKT-3 in an Alamar Blue™

    Techniques Used: Mass Spectrometry

    15) Product Images from "Knockdown of T-bet expression in Mart-127–35-specific T-cell-receptor-engineered human CD4+ CD25− and CD8+ T cells attenuates effector function"

    Article Title: Knockdown of T-bet expression in Mart-127–35-specific T-cell-receptor-engineered human CD4+ CD25− and CD8+ T cells attenuates effector function

    Journal: Immunology

    doi: 10.1111/imm.12431

    Effect of T-bet knockdown on intracellular expression of granzyme-B and perforin-1. Primary human CD4 + CD25 − and CD8 + T cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies in the presence of interleukin-2 (IL-2). These
    Figure Legend Snippet: Effect of T-bet knockdown on intracellular expression of granzyme-B and perforin-1. Primary human CD4 + CD25 − and CD8 + T cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies in the presence of interleukin-2 (IL-2). These

    Techniques Used: Expressing

    16) Product Images from "Circulating and Tumor-Infiltrating Foxp3+ Regulatory T Cell Subset in Chinese Patients with Extranodal NK/T Cell Lymphoma"

    Article Title: Circulating and Tumor-Infiltrating Foxp3+ Regulatory T Cell Subset in Chinese Patients with Extranodal NK/T Cell Lymphoma

    Journal: International Journal of Biological Sciences

    doi:

    The biological characteristics and function of Foxp3 + Tregs subset. A. The flow cytometer profiles are representatives from the stainings for CTLA, GITR, CD45RO, CCR7, IFNγ, IL-2, IL-17, IL-10 and TGFβ in Foxp3 + Treg subset. B. The suppressive ability of CD4 + Tregs and CD8 + Tregs to naïve CD4 + T cells in vitro was measured by the T cell proliferation experiment. CSFE-labeled naïve CD4 + T cells were alone or co-cultured with CD8 + Tregs cells at ratio of 1:1 in the in complete RPMI1640 medium without IL-2 in OKT3-coated 96-well plate for 5 days and analyzed by FACS detection.
    Figure Legend Snippet: The biological characteristics and function of Foxp3 + Tregs subset. A. The flow cytometer profiles are representatives from the stainings for CTLA, GITR, CD45RO, CCR7, IFNγ, IL-2, IL-17, IL-10 and TGFβ in Foxp3 + Treg subset. B. The suppressive ability of CD4 + Tregs and CD8 + Tregs to naïve CD4 + T cells in vitro was measured by the T cell proliferation experiment. CSFE-labeled naïve CD4 + T cells were alone or co-cultured with CD8 + Tregs cells at ratio of 1:1 in the in complete RPMI1640 medium without IL-2 in OKT3-coated 96-well plate for 5 days and analyzed by FACS detection.

    Techniques Used: Flow Cytometry, Cytometry, In Vitro, Labeling, Cell Culture, FACS

    17) Product Images from "A novel immunotherapy for superficial bladder cancer by intravesical immobilization of GM-CSF"

    Article Title: A novel immunotherapy for superficial bladder cancer by intravesical immobilization of GM-CSF

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00818.x

    The SA-GM-SCF treatment could induce and enhance CTL function. Splenocytes were pooled from naïve mice, tumour-cured or tumour-bearing mice in the SA-GM-CSF-treated group, and stimulated with irradiated MB49 cells in the presence of IL-2 for 5 days to prepare effector cells. The CTL cytotoxicities were determined with LDH cytotoxicity detection kit at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. The cytotoxicities had significant differences between the tumour-cured and the tumour-bearing mice in the SA-GM-CSF-treated group ( P
    Figure Legend Snippet: The SA-GM-SCF treatment could induce and enhance CTL function. Splenocytes were pooled from naïve mice, tumour-cured or tumour-bearing mice in the SA-GM-CSF-treated group, and stimulated with irradiated MB49 cells in the presence of IL-2 for 5 days to prepare effector cells. The CTL cytotoxicities were determined with LDH cytotoxicity detection kit at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. The cytotoxicities had significant differences between the tumour-cured and the tumour-bearing mice in the SA-GM-CSF-treated group ( P

    Techniques Used: CTL Assay, Mouse Assay, Irradiation

    18) Product Images from "Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants"

    Article Title: Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13113-5

    Allogeneic endothelial cells trigger missing self-induced activation of NK cells. Primary allogeneic human endothelial cells were co-cultured with purified NK cells from 30 healthy volunteers primed with low-dose IL-2. After 4 h of culture the activation status of the NK cells was assessed at the single cell level by flow cytometry. a , b Analyses were focused on the five NK cell populations that expressed a single inhibitory KIR. a Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. b Intracellular staining for MIP-1β in NK cells after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. c , d Analyses were focused on the NK cell populations that expressed two inhibitory KIRs, one of them lacking its ligand on endothelial cells (missing self, MS). According to the nature of the second inhibitory KIR, three situations were distinguished: 1MS+1M, if endothelial cells expressed the ligand for the second inhibitory KIR; 1MS + uneduc MS, if neither endothelial cells nor NK cell donor expressed the ligand for the second inhibitory KIR, and 2MS, if endothelial cells did not express the ligands of the two inhibitory KIRs. Results are normalised over the value observed for the NK cell population that expressed the single mismatched inhibitory KIR. c Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. d Intracellular staining for MIP-1β in NK cells after 4 h co-culture. ns: p ≥ 0.05, * p
    Figure Legend Snippet: Allogeneic endothelial cells trigger missing self-induced activation of NK cells. Primary allogeneic human endothelial cells were co-cultured with purified NK cells from 30 healthy volunteers primed with low-dose IL-2. After 4 h of culture the activation status of the NK cells was assessed at the single cell level by flow cytometry. a , b Analyses were focused on the five NK cell populations that expressed a single inhibitory KIR. a Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. b Intracellular staining for MIP-1β in NK cells after 4-h co-culture. Left panel: representative flow cytometry profiles. Middle panel: individual values of NK cell populations according to their status against primary allogeneic human endothelial cells. c , d Analyses were focused on the NK cell populations that expressed two inhibitory KIRs, one of them lacking its ligand on endothelial cells (missing self, MS). According to the nature of the second inhibitory KIR, three situations were distinguished: 1MS+1M, if endothelial cells expressed the ligand for the second inhibitory KIR; 1MS + uneduc MS, if neither endothelial cells nor NK cell donor expressed the ligand for the second inhibitory KIR, and 2MS, if endothelial cells did not express the ligands of the two inhibitory KIRs. Results are normalised over the value observed for the NK cell population that expressed the single mismatched inhibitory KIR. c Expression of CD107a (LAMP-1) on NK cell surface after 4-h co-culture. d Intracellular staining for MIP-1β in NK cells after 4 h co-culture. ns: p ≥ 0.05, * p

    Techniques Used: Activation Assay, Cell Culture, Purification, Flow Cytometry, Cytometry, Expressing, Co-Culture Assay, Staining, Mass Spectrometry

    19) Product Images from "IL-2 prevents deletion of developing T-regulatory cells in the thymus"

    Article Title: IL-2 prevents deletion of developing T-regulatory cells in the thymus

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2017.38

    CD4SP CCR7+ Helios+ thymocytes that cannot respond to IL-2 or IL-15 are deleted. ( a ) Helios/Foxp3 phenotype of CCR7+ CD4SP thymocytes (top row) and CTLA-4 labelling on their Helios+ Foxp3– and Foxp3+ subsets (bottom) from wild type, Il2 –/– , Il15 –/– and Il2 –/– Il15 −/− mice on the B10.BR background examined at 28-47 days after birth. Graphs (right) show the frequency and absolute number of thymocyte subsets gated in plots (top row) or the CTLA-4 RFI (bottom row, normalised to the CCR7+ CD4SP Helios– Foxp3– subset in the same sample). ( b ) Helios/Foxp3 phenotype of CD45.2– and CD45.2+ subsets of CCR7+ CD4SP thymocytes (top row), and CTLA-4 labelling on their Helios+ Foxp3– and Foxp3+ subpopulations (bottom row), from B6. Rag1 –/– mice that were irradiated and reconstituted 8 weeks earlier with BM mixtures from CD45.1+ wild-type mice plus CD45.2+ mice that were either Il2rb +/+ or Il2rb –/– . Graphs show the frequencies of populations gated in plots (top row) or the CTLA-4 MFI (bottom row). ( c ) Chimeric mice described in ( b ) were injected with EdU 7 days before analysis and EdU+ thymocytes were examined as in ( b ). Graph shows the frequencies of populations gated in plots. Lines on graphs join measurements from an individual mouse. Data in ( a ) were compiled from four experiments, ( b and c ) from one experiment. Two-tailed Student's t -tests (unpaired in a and paired in b and c ) P -value symbols: * 0.01–0.05, ** 0.001–0.01, *** 0.0001–0.001; ****
    Figure Legend Snippet: CD4SP CCR7+ Helios+ thymocytes that cannot respond to IL-2 or IL-15 are deleted. ( a ) Helios/Foxp3 phenotype of CCR7+ CD4SP thymocytes (top row) and CTLA-4 labelling on their Helios+ Foxp3– and Foxp3+ subsets (bottom) from wild type, Il2 –/– , Il15 –/– and Il2 –/– Il15 −/− mice on the B10.BR background examined at 28-47 days after birth. Graphs (right) show the frequency and absolute number of thymocyte subsets gated in plots (top row) or the CTLA-4 RFI (bottom row, normalised to the CCR7+ CD4SP Helios– Foxp3– subset in the same sample). ( b ) Helios/Foxp3 phenotype of CD45.2– and CD45.2+ subsets of CCR7+ CD4SP thymocytes (top row), and CTLA-4 labelling on their Helios+ Foxp3– and Foxp3+ subpopulations (bottom row), from B6. Rag1 –/– mice that were irradiated and reconstituted 8 weeks earlier with BM mixtures from CD45.1+ wild-type mice plus CD45.2+ mice that were either Il2rb +/+ or Il2rb –/– . Graphs show the frequencies of populations gated in plots (top row) or the CTLA-4 MFI (bottom row). ( c ) Chimeric mice described in ( b ) were injected with EdU 7 days before analysis and EdU+ thymocytes were examined as in ( b ). Graph shows the frequencies of populations gated in plots. Lines on graphs join measurements from an individual mouse. Data in ( a ) were compiled from four experiments, ( b and c ) from one experiment. Two-tailed Student's t -tests (unpaired in a and paired in b and c ) P -value symbols: * 0.01–0.05, ** 0.001–0.01, *** 0.0001–0.001; ****

    Techniques Used: Mouse Assay, Irradiation, Injection, Two Tailed Test

    Foxp3 and CTLA-4 upregulation in CD4SP CCR7+ Helios+ cells is IL-2 dependent. ( a ) Upper plots show Helios/Foxp3 phenotype of CCR7+ CD4SP thymocytes from Il2 +/+ , Il2 +/– and Il2 –/– littermates on the B10.BR background examined at 14–15 days after birth. Lower histograms show CTLA-4 expression on Helios+ Foxp3– (red) and Foxp3+ (blue) subsets of CCR7+ CD4SP thymocytes from the same mice. Summaries (below) show the frequencies, or CTLA-4 MFI, of Helios+ Foxp3– or Foxp3+ cells as gated in the plots, with each symbol representing one mouse. ( b ) Helios/Foxp3 phenotype of CD45 1/2 and CD45 2/2 subsets of CCR7+ CD4SP thymocytes (top row), and CTLA-4 expression on their Helios+ Foxp3– and Foxp3+ subsets (bottom row), from CD45 1/1 mice that were irradiated and reconstituted 6–10 weeks earlier with BM mixtures from CD45 1/2 wild-type mice plus CD45 2/2 mice that were either Il2ra +/+ , Il2ra +/– or Il2ra –/– . Graphs show the frequencies of populations gated in plots (top row) or the CTLA-4 relative fluorescence intensity (RFI) (bottom row, normalised to the CCR7+ CD4SP Helios– Foxp3– subset in the same sample). Lines join measurements from an individual mouse. Data in ( a ) and ( b ) were compiled from two and three experiments, respectively. Two-tailed Student's t -tests (unpaired in a ; paired in b ) P -value symbols: * 0.01–0.05, ** 0.001–0.01, ****
    Figure Legend Snippet: Foxp3 and CTLA-4 upregulation in CD4SP CCR7+ Helios+ cells is IL-2 dependent. ( a ) Upper plots show Helios/Foxp3 phenotype of CCR7+ CD4SP thymocytes from Il2 +/+ , Il2 +/– and Il2 –/– littermates on the B10.BR background examined at 14–15 days after birth. Lower histograms show CTLA-4 expression on Helios+ Foxp3– (red) and Foxp3+ (blue) subsets of CCR7+ CD4SP thymocytes from the same mice. Summaries (below) show the frequencies, or CTLA-4 MFI, of Helios+ Foxp3– or Foxp3+ cells as gated in the plots, with each symbol representing one mouse. ( b ) Helios/Foxp3 phenotype of CD45 1/2 and CD45 2/2 subsets of CCR7+ CD4SP thymocytes (top row), and CTLA-4 expression on their Helios+ Foxp3– and Foxp3+ subsets (bottom row), from CD45 1/1 mice that were irradiated and reconstituted 6–10 weeks earlier with BM mixtures from CD45 1/2 wild-type mice plus CD45 2/2 mice that were either Il2ra +/+ , Il2ra +/– or Il2ra –/– . Graphs show the frequencies of populations gated in plots (top row) or the CTLA-4 relative fluorescence intensity (RFI) (bottom row, normalised to the CCR7+ CD4SP Helios– Foxp3– subset in the same sample). Lines join measurements from an individual mouse. Data in ( a ) and ( b ) were compiled from two and three experiments, respectively. Two-tailed Student's t -tests (unpaired in a ; paired in b ) P -value symbols: * 0.01–0.05, ** 0.001–0.01, ****

    Techniques Used: Expressing, Mouse Assay, Irradiation, Fluorescence, Two Tailed Test

    The CCR7+ Helios+ subset of CD4SP thymocytes is enriched in cells that can respond to IL-2. ( a ) CD24/CCR7 phenotype of Foxp3– CD4SP thymocytes from B6 mice showing gating for CD24– CCR7+, CD24+ CCR7+ and CD24– CCR7+ subsets. ( b ) CD24– CCR7– phenotype of thymic NKT cells. Left plot shows total thymocytes from a B6 mouse stained with anti-NK1.1 plus a tetramer of mouse CD1d loaded with the α -galactosylceramide analogue, PBS-57. Thymic NKT (PBS-57/CD1d+ NK1.1+) cells are analysed for CD24/CCR7 phenotype in the right plot. Results are representative of two experiments. ( c ) Foxp3-GFP– CD4SP thymocytes were FACS sorted into three subsets based on CCR7 and CD24 expression and cultured for 20 h in the presence or absence of IL-2 (denoted above plots). Top row shows side-scattered light amplitude (SSC-H) versus forward-scattered light amplitude (FSC-H) for all events in the flow cytometry datafiles and the gate used to define lymphocytes. After excluding doublets based on FSC width and SSC width (gates not shown), single lymphocytes were analysed for Helios expression in the second row. Gated Helios+ events were analysed for Foxp3/CD25 (third row) and Foxp3/CTLA-4 (fourth row) phenotypes. ( d ) Lymphocyte frequency among ungated events as shown in top row of ( c ). ( e ) Helios+ cell frequency among single lymphocytes as shown in second row of ( c ). ( f ) Foxp3+ (top) or CD25+ (bottom) cell frequency among Helios– or Helios+ cells after 20 h in culture in the presence or absence of graded concentrations of IL-2 as gated in third and fourth rows of ( c ). Columns and error bars represent mean and s.e.m. of data obtained from three mice. Similar data were obtained in a repeat experiment. Statistical analyses used were ( d ) two-way ANOVA with Sidak's post-test or ( e and f ) multiple t- tests comparing each concentration of exogenous IL-2 to the 0 ng/ml IL-2 condition for each T-cell subset, without assuming a consistent standard deviation, with Holm–Sidak post-tests. Multiplicity adjusted P- value symbols: * 0.01–0.05, ** 0.001–0.01, ****
    Figure Legend Snippet: The CCR7+ Helios+ subset of CD4SP thymocytes is enriched in cells that can respond to IL-2. ( a ) CD24/CCR7 phenotype of Foxp3– CD4SP thymocytes from B6 mice showing gating for CD24– CCR7+, CD24+ CCR7+ and CD24– CCR7+ subsets. ( b ) CD24– CCR7– phenotype of thymic NKT cells. Left plot shows total thymocytes from a B6 mouse stained with anti-NK1.1 plus a tetramer of mouse CD1d loaded with the α -galactosylceramide analogue, PBS-57. Thymic NKT (PBS-57/CD1d+ NK1.1+) cells are analysed for CD24/CCR7 phenotype in the right plot. Results are representative of two experiments. ( c ) Foxp3-GFP– CD4SP thymocytes were FACS sorted into three subsets based on CCR7 and CD24 expression and cultured for 20 h in the presence or absence of IL-2 (denoted above plots). Top row shows side-scattered light amplitude (SSC-H) versus forward-scattered light amplitude (FSC-H) for all events in the flow cytometry datafiles and the gate used to define lymphocytes. After excluding doublets based on FSC width and SSC width (gates not shown), single lymphocytes were analysed for Helios expression in the second row. Gated Helios+ events were analysed for Foxp3/CD25 (third row) and Foxp3/CTLA-4 (fourth row) phenotypes. ( d ) Lymphocyte frequency among ungated events as shown in top row of ( c ). ( e ) Helios+ cell frequency among single lymphocytes as shown in second row of ( c ). ( f ) Foxp3+ (top) or CD25+ (bottom) cell frequency among Helios– or Helios+ cells after 20 h in culture in the presence or absence of graded concentrations of IL-2 as gated in third and fourth rows of ( c ). Columns and error bars represent mean and s.e.m. of data obtained from three mice. Similar data were obtained in a repeat experiment. Statistical analyses used were ( d ) two-way ANOVA with Sidak's post-test or ( e and f ) multiple t- tests comparing each concentration of exogenous IL-2 to the 0 ng/ml IL-2 condition for each T-cell subset, without assuming a consistent standard deviation, with Holm–Sidak post-tests. Multiplicity adjusted P- value symbols: * 0.01–0.05, ** 0.001–0.01, ****

    Techniques Used: Mouse Assay, Staining, FACS, Expressing, Cell Culture, Flow Cytometry, Cytometry, Concentration Assay, Standard Deviation

    20) Product Images from "Resident Memory T Cells (TRM) Are Abundant in Human Lung: Diversity, Function, and Antigen Specificity"

    Article Title: Resident Memory T Cells (TRM) Are Abundant in Human Lung: Diversity, Function, and Antigen Specificity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016245

    Large numbers of immunocompetent and influenza-specific T cells resides in human lung. Lung T cells were extracted by lung explant method. (A–B) The cytokine secretion of effector memory T cells after overnight stimulation with artificial APCs (microbeads coated with anti-CD2, anti CD3 and antiCD28 mAbs) at 1∶1 cells: bead ratio. Brefeldin A (golgi-stop) was added 6 h prior to intracellular staining of cytokines. A representative dot plot of each cytokine is shown and 6 additional experiments produced similar results. (C) CD4+T cells were stained with TNFα, IL-2 and IFNγ after stimulation with PMA+ionomycin for 6h in presence of brefeldin A (Gate on CD4+IFNγ+ population). A representative dot plot is shown and 10 additional experiments produced similar results. (D) Lung T cells were isolated and stained for different Vbeta T cell receptors using TCR V beta repertoir kit (Beckman coulter) according to manufacturer's instructions. Diversity of V beta TCRs was analyzed by flow cytometry. Data represent Mean +/− SEM of 3 different donors. (E) CFSE labeled T cells from lung, skin and blood were cultured with heat killed influenza virus pulsed APCs in 1∶2 ratio. On day 4, T cell proliferation was measured by analyzing CFSE dilution using flow cytometry. A representative experiment is shown and 2 additional experiments produced similar results.
    Figure Legend Snippet: Large numbers of immunocompetent and influenza-specific T cells resides in human lung. Lung T cells were extracted by lung explant method. (A–B) The cytokine secretion of effector memory T cells after overnight stimulation with artificial APCs (microbeads coated with anti-CD2, anti CD3 and antiCD28 mAbs) at 1∶1 cells: bead ratio. Brefeldin A (golgi-stop) was added 6 h prior to intracellular staining of cytokines. A representative dot plot of each cytokine is shown and 6 additional experiments produced similar results. (C) CD4+T cells were stained with TNFα, IL-2 and IFNγ after stimulation with PMA+ionomycin for 6h in presence of brefeldin A (Gate on CD4+IFNγ+ population). A representative dot plot is shown and 10 additional experiments produced similar results. (D) Lung T cells were isolated and stained for different Vbeta T cell receptors using TCR V beta repertoir kit (Beckman coulter) according to manufacturer's instructions. Diversity of V beta TCRs was analyzed by flow cytometry. Data represent Mean +/− SEM of 3 different donors. (E) CFSE labeled T cells from lung, skin and blood were cultured with heat killed influenza virus pulsed APCs in 1∶2 ratio. On day 4, T cell proliferation was measured by analyzing CFSE dilution using flow cytometry. A representative experiment is shown and 2 additional experiments produced similar results.

    Techniques Used: Staining, Produced, Isolation, Flow Cytometry, Cytometry, Labeling, Cell Culture

    21) Product Images from "Heteroclitic XBP1 peptides evoke tumor-specific memory cytotoxic T lymphocytes against breast cancer, colon cancer, and pancreatic cancer cells"

    Article Title: Heteroclitic XBP1 peptides evoke tumor-specific memory cytotoxic T lymphocytes against breast cancer, colon cancer, and pancreatic cancer cells

    Journal: Oncoimmunology

    doi: 10.4161/21624011.2014.970914

    Increased cell activation and antitumor activities of heteroclictic XBP1-CTL to various solid tumor cells in an antigen-specific and HLA-A2-restricted manner. XBP1-CTL generated with a cocktail of heteroclitic XBP1 peptides were evaluated for their antitumor activities against breast, colon or pancreatic cancer cells. HLA-A2-restricted and antigen-specific antitumor responses are demonstrated as total CD107a degranulation ( A ), total IFNγ production ( B ), total IL-2 production ( C ) and poly-functional IFNγ/CD69 ( D ) or IFNγ/CD107a ( E ) against the tumor cells. Results for each single or poly-functional antitumor activity are expressed as a percentage of CD3 + CD8 + T cells.
    Figure Legend Snippet: Increased cell activation and antitumor activities of heteroclictic XBP1-CTL to various solid tumor cells in an antigen-specific and HLA-A2-restricted manner. XBP1-CTL generated with a cocktail of heteroclitic XBP1 peptides were evaluated for their antitumor activities against breast, colon or pancreatic cancer cells. HLA-A2-restricted and antigen-specific antitumor responses are demonstrated as total CD107a degranulation ( A ), total IFNγ production ( B ), total IL-2 production ( C ) and poly-functional IFNγ/CD69 ( D ) or IFNγ/CD107a ( E ) against the tumor cells. Results for each single or poly-functional antitumor activity are expressed as a percentage of CD3 + CD8 + T cells.

    Techniques Used: Activation Assay, CTL Assay, Generated, Functional Assay, Activity Assay

    High level of IFNγ production, IL-2 production and CD107a degranulation by CM XBP1-CTL subset to various solid tumor cells. XBP1-CTL memory cell subsets were analyzed for their antitumor activity in response to a variety of solid tumor cells. Representative flow cytometric analyses showed higher levels of IFNγ production ( A ), IL-2 production ( B ) and CD107a degranulation ( C ) within the CM subset of XBP1-CTL. Specific IFNγ production ( D ), IL-2 production ( E ) and CD107a degranulation ( F ) were consistently higher against the respective tumor cells within the CM as compared to the EM subset of XBP1-CTL ( n = 2). Results of the antitumor response are shown as a percentage of the parent CM or EM CD3 + CD8 + CTL population.
    Figure Legend Snippet: High level of IFNγ production, IL-2 production and CD107a degranulation by CM XBP1-CTL subset to various solid tumor cells. XBP1-CTL memory cell subsets were analyzed for their antitumor activity in response to a variety of solid tumor cells. Representative flow cytometric analyses showed higher levels of IFNγ production ( A ), IL-2 production ( B ) and CD107a degranulation ( C ) within the CM subset of XBP1-CTL. Specific IFNγ production ( D ), IL-2 production ( E ) and CD107a degranulation ( F ) were consistently higher against the respective tumor cells within the CM as compared to the EM subset of XBP1-CTL ( n = 2). Results of the antitumor response are shown as a percentage of the parent CM or EM CD3 + CD8 + CTL population.

    Techniques Used: CTL Assay, Activity Assay, Flow Cytometry

    22) Product Images from "Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma"

    Article Title: Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027627

    PP2A function in IL-2/IL-4-treated U937 cells. Effects of IL-2/IL-4 co-treatment for 48 h on IC 50 of dexamethasone on TNFα-induced IL-8 release (A), PP2A C protein expression (B), immunoprecipitated PP2A (IP-P2A) activity (C), PP2A C -Tyr 307 phosphorylation(D), GR-Ser 226 phosphorylation (E) and JNK1 phosphorylation (F). Values represent means ± SEM (n = 3–4). # P
    Figure Legend Snippet: PP2A function in IL-2/IL-4-treated U937 cells. Effects of IL-2/IL-4 co-treatment for 48 h on IC 50 of dexamethasone on TNFα-induced IL-8 release (A), PP2A C protein expression (B), immunoprecipitated PP2A (IP-P2A) activity (C), PP2A C -Tyr 307 phosphorylation(D), GR-Ser 226 phosphorylation (E) and JNK1 phosphorylation (F). Values represent means ± SEM (n = 3–4). # P

    Techniques Used: Expressing, Immunoprecipitation, Activity Assay

    23) Product Images from "Neuropoietin, a new IL-6-related cytokine signaling through the ciliary neurotrophic factor receptor"

    Article Title: Neuropoietin, a new IL-6-related cytokine signaling through the ciliary neurotrophic factor receptor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0306178101

    Physical interaction and recruitment of CNTF receptor complex by NP. ( A ) Coimmunoprecipitation of NP and CNTFRα from the surface of BAF GLC cells. BAF GLC were incubated with IL-2 (50 ng/ml)-, CNTF (50 ng/ml)-, mock-, or NP-containing culture media for 10 min at 37°C. Cells were lysed with Brij 96 buffer, and proteins were immunoprecipitated with AN-D3 anti-CNTFRα mAb and protein A beads. NP was detected by using an anti-V5 epitope mAb. ( B ) Coimmunoprecipitation of CNTF ( Upper ) or NP( Lower ) with CNTFRα-Fc, gp130-Fc, or LIFR-Fc fusion proteins. Receptor subunits were incubated at a concentration of 4 nM for 16hat4°C in the presence of 4 nM NP or CNTF before being immunoprecipitated with protein A beads. NP and CNTF were detected by using an anti-V5 epitope mAb and a polyclonal biotinylated anti-CNTF Ab, respectively. ( C ) Tyrosine phosphorylation of gp130 and LIFR in SK-N-GP cells in response to NP. After an exposure to 50 ng/ml of indicated cytokines, SK-N-GP cells were lysed with Brij 96 buffer. Then, LIFR and gp130 were coprecipitated by using the AN-E1 anti-LIFR mAb. Tyrosine phosphorylation content was determined with the 4G10 anti-phosphotyrosine mAb. ( D ) Induction of STAT3 tyrosine phosphorylation by NP in SK-N-GP neuroblastoma (gp130+, LIFR+, and CNTFRα+) and T98G glioblastoma cells (gp130+, LIFR+, and CNTFRα–). After exposure to 50 ng/ml of indicated cytokines, cells were lysed with 1% Nonidet P-40 buffer and analyzed by Western blotting by using an anti-phospho-STAT3 Ab.
    Figure Legend Snippet: Physical interaction and recruitment of CNTF receptor complex by NP. ( A ) Coimmunoprecipitation of NP and CNTFRα from the surface of BAF GLC cells. BAF GLC were incubated with IL-2 (50 ng/ml)-, CNTF (50 ng/ml)-, mock-, or NP-containing culture media for 10 min at 37°C. Cells were lysed with Brij 96 buffer, and proteins were immunoprecipitated with AN-D3 anti-CNTFRα mAb and protein A beads. NP was detected by using an anti-V5 epitope mAb. ( B ) Coimmunoprecipitation of CNTF ( Upper ) or NP( Lower ) with CNTFRα-Fc, gp130-Fc, or LIFR-Fc fusion proteins. Receptor subunits were incubated at a concentration of 4 nM for 16hat4°C in the presence of 4 nM NP or CNTF before being immunoprecipitated with protein A beads. NP and CNTF were detected by using an anti-V5 epitope mAb and a polyclonal biotinylated anti-CNTF Ab, respectively. ( C ) Tyrosine phosphorylation of gp130 and LIFR in SK-N-GP cells in response to NP. After an exposure to 50 ng/ml of indicated cytokines, SK-N-GP cells were lysed with Brij 96 buffer. Then, LIFR and gp130 were coprecipitated by using the AN-E1 anti-LIFR mAb. Tyrosine phosphorylation content was determined with the 4G10 anti-phosphotyrosine mAb. ( D ) Induction of STAT3 tyrosine phosphorylation by NP in SK-N-GP neuroblastoma (gp130+, LIFR+, and CNTFRα+) and T98G glioblastoma cells (gp130+, LIFR+, and CNTFRα–). After exposure to 50 ng/ml of indicated cytokines, cells were lysed with 1% Nonidet P-40 buffer and analyzed by Western blotting by using an anti-phospho-STAT3 Ab.

    Techniques Used: Gas Chromatography, Incubation, Immunoprecipitation, Concentration Assay, Western Blot

    24) Product Images from "Neisseria gonorrhoeae Enhances HIV-1 Infection of Primary Resting CD4+ T Cells through TLR2 Activation"

    Article Title: Neisseria gonorrhoeae Enhances HIV-1 Infection of Primary Resting CD4+ T Cells through TLR2 Activation

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

    doi: 10.4049/jimmunol.0902125

    TLR2 activation promotes HIV replication in primary resting CD4 + T cells. A and B , Primary resting CD4 + T cells were infected with X4 HIV-1 IIIB or R5 HIV-1 BaL at an MOI of 0.05 at 37°C for 2 h. After washing off unbound virus, infected cells were then treated with LPS (1 μg/ml), MDP (20 μg/ml), PGN (20 μg/ml), or GC DOV LOS (0.1 μg/ml) in the presence of IL-2 at 37°C. Infected cells without treatment were included as a control. The p24 level in the culture supernatant was determined on day 7 postinfection. C , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus at 37°C for 2 h and then treated with Pam 3 CSK 4 at 5 μg/ml with IL-2 for 4 d before measuring luciferase assay. D , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus and then exposed to Pam 3 C-Lip at various concentrations with IL-2 for 4 d before measuring luciferase assay. E , Resting CD4 + T cells were treated with PGN (20 μg/ml) for 3 d, followed by washing. PGN-treated primary CD4 + cells were infected with HIV-1 IIIB or HIV-1 BaL . The p24 level in the culture supernatant was determined on day 7 postinfection. Data are means ±SD of triplicate sample and represent seven independent experiments. The difference in HIV infection between samples with and without TLR2 agonists treatment is significant as calculated by the two-tailed, paired Student t test. * p
    Figure Legend Snippet: TLR2 activation promotes HIV replication in primary resting CD4 + T cells. A and B , Primary resting CD4 + T cells were infected with X4 HIV-1 IIIB or R5 HIV-1 BaL at an MOI of 0.05 at 37°C for 2 h. After washing off unbound virus, infected cells were then treated with LPS (1 μg/ml), MDP (20 μg/ml), PGN (20 μg/ml), or GC DOV LOS (0.1 μg/ml) in the presence of IL-2 at 37°C. Infected cells without treatment were included as a control. The p24 level in the culture supernatant was determined on day 7 postinfection. C , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus at 37°C for 2 h and then treated with Pam 3 CSK 4 at 5 μg/ml with IL-2 for 4 d before measuring luciferase assay. D , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus and then exposed to Pam 3 C-Lip at various concentrations with IL-2 for 4 d before measuring luciferase assay. E , Resting CD4 + T cells were treated with PGN (20 μg/ml) for 3 d, followed by washing. PGN-treated primary CD4 + cells were infected with HIV-1 IIIB or HIV-1 BaL . The p24 level in the culture supernatant was determined on day 7 postinfection. Data are means ±SD of triplicate sample and represent seven independent experiments. The difference in HIV infection between samples with and without TLR2 agonists treatment is significant as calculated by the two-tailed, paired Student t test. * p

    Techniques Used: Activation Assay, Infection, Luciferase, Two Tailed Test

    TLR2 activation and GC exposure promote HIV infection at the step of nuclear import. Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus and then treated with Pam 3 CSK 4 at 5 μg/ml or fixed GC (ATCC strain 43069) at an MOI of 50 in the presence of IL-2. DNA was prepared from TLR2-activated or GC-exposed HIV-infected cells at 0, 6, 12, 24, and 48 h postinfection. DNA from mock-infected primary resting CD4 + T cells was also isolated as a control. A , Quantitation of HIV-1 early strong-stop (R/U5) and late full-length (R/gag) RT products was performed by quantitative real-time PCR analysis. Data are means ± SD of triplicate sample and represent three independent experiments. Differences in the levels of early and late RT products between samples with or without exposure to GC or TLR2 agonists are not significant as calculated by the two-tailed, paired Student t test, p
    Figure Legend Snippet: TLR2 activation and GC exposure promote HIV infection at the step of nuclear import. Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus and then treated with Pam 3 CSK 4 at 5 μg/ml or fixed GC (ATCC strain 43069) at an MOI of 50 in the presence of IL-2. DNA was prepared from TLR2-activated or GC-exposed HIV-infected cells at 0, 6, 12, 24, and 48 h postinfection. DNA from mock-infected primary resting CD4 + T cells was also isolated as a control. A , Quantitation of HIV-1 early strong-stop (R/U5) and late full-length (R/gag) RT products was performed by quantitative real-time PCR analysis. Data are means ± SD of triplicate sample and represent three independent experiments. Differences in the levels of early and late RT products between samples with or without exposure to GC or TLR2 agonists are not significant as calculated by the two-tailed, paired Student t test, p

    Techniques Used: Activation Assay, Infection, Luciferase, Isolation, Quantitation Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

    Multiple GC strains enhanced HIV-1 infection of primary resting CD4 + T cells. A , Primary resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus at 37°C for 2 h. After washing off unbound virus, infected cells were exposed to various strains of fixed GC in the presence of IL-2 for 4 d before measuring luciferase activity. There is a significant difference between samples with and without GC-exposure. * p
    Figure Legend Snippet: Multiple GC strains enhanced HIV-1 infection of primary resting CD4 + T cells. A , Primary resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus at 37°C for 2 h. After washing off unbound virus, infected cells were exposed to various strains of fixed GC in the presence of IL-2 for 4 d before measuring luciferase activity. There is a significant difference between samples with and without GC-exposure. * p

    Techniques Used: Infection, Luciferase, Activity Assay

    Enhancement of HIV infection of primary resting CD4 + T cells by GC is mediated through TLR2 signaling pathway. A , Gene expression of NOD2, TLR2, and TLR4 in resting CD4 + T cells and PHA-activated CD4 + T cells was determined by RT-PCR analysis. Total RNA from CD14 + cells was included as a control for analysis of TLR4 gene expression. B , To determine the role of TLR2 in GC-mediated enhancement of HIV infection, resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus at 37°C for 2 h. After washing off unbound virus, infected cells were treated with anti-TLR2 Ab or isotype control Ab at 10 μg/ml for 1 h. Cells were exposed to fixed GC (ATCC strain 43069) at an MOI of 50 with IL-2 and cultured for 4 d before measurement of luciferase activity. C , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus and treated with anti-TLR2 Ab or isotype control Ab as described previously. Cells were then exposed to Pam 3 C-Lip at various concentrations in the presence of IL-2 for 4 d. Data are means ±SD of triplicate sample and represent two independent experiments. There is a significant difference between samples with and without GC or Pam 3 C-Lip treatment (* p
    Figure Legend Snippet: Enhancement of HIV infection of primary resting CD4 + T cells by GC is mediated through TLR2 signaling pathway. A , Gene expression of NOD2, TLR2, and TLR4 in resting CD4 + T cells and PHA-activated CD4 + T cells was determined by RT-PCR analysis. Total RNA from CD14 + cells was included as a control for analysis of TLR4 gene expression. B , To determine the role of TLR2 in GC-mediated enhancement of HIV infection, resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus at 37°C for 2 h. After washing off unbound virus, infected cells were treated with anti-TLR2 Ab or isotype control Ab at 10 μg/ml for 1 h. Cells were exposed to fixed GC (ATCC strain 43069) at an MOI of 50 with IL-2 and cultured for 4 d before measurement of luciferase activity. C , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus and treated with anti-TLR2 Ab or isotype control Ab as described previously. Cells were then exposed to Pam 3 C-Lip at various concentrations in the presence of IL-2 for 4 d. Data are means ±SD of triplicate sample and represent two independent experiments. There is a significant difference between samples with and without GC or Pam 3 C-Lip treatment (* p

    Techniques Used: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Cell Culture, Activity Assay

    The maximal HIV enhancing effect of the TLR2 activation is achieved when PGN is added at an early stage of the HIV life cycle. A , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus for 2 h. Infected cells were cultured with IL-2 and then treated with PGN at 20 μg/ml at 2, 24, 48, or 72 h postinfection before measurement of luciferase activity at day 4, 6, or 7 postinfection. There is a significant difference between samples without PGN and with PGN at 2, 24, or 48 h postinfection (* p
    Figure Legend Snippet: The maximal HIV enhancing effect of the TLR2 activation is achieved when PGN is added at an early stage of the HIV life cycle. A , Resting CD4 + T cells were infected with pseudotyped HIV-1 VSV-G luciferase reporter virus for 2 h. Infected cells were cultured with IL-2 and then treated with PGN at 20 μg/ml at 2, 24, 48, or 72 h postinfection before measurement of luciferase activity at day 4, 6, or 7 postinfection. There is a significant difference between samples without PGN and with PGN at 2, 24, or 48 h postinfection (* p

    Techniques Used: Activation Assay, Infection, Luciferase, Cell Culture, Activity Assay

    GC exposure enhances HIV-1 infection of primary resting CD4 + T cells. Primary resting CD4 + T cells were infected with HIV-1 BaL at an MOI of 0.05 at 37°C for 2 h. After washing off unbound virus, infected cells were exposed to live ( A ) or fixed ( B ) GC (ATCC strain 43069) at various MOIs in the presence of IL-2. HIV production was determined by measuring the level of HIV capsid protein p24 at day 14 after viral infection using HIV p24 ELISA. C , Primary resting CD4 + T cells (1 × 10 4 cells per well) were exposed to live GC or fixed GC at 37°C for 72 h in the presence of IL-2. Cell proliferation was examined by the MTS assay (Promega CellTiter96 aqueous one solution cell proliferation assay). Data are means ± SD of triplicate sample. Difference in HIV infection between samples without GC exposure and GC-exposed samples at an MOI of 100 is significant as calculated by the two-tailed, paired Student t test. * p
    Figure Legend Snippet: GC exposure enhances HIV-1 infection of primary resting CD4 + T cells. Primary resting CD4 + T cells were infected with HIV-1 BaL at an MOI of 0.05 at 37°C for 2 h. After washing off unbound virus, infected cells were exposed to live ( A ) or fixed ( B ) GC (ATCC strain 43069) at various MOIs in the presence of IL-2. HIV production was determined by measuring the level of HIV capsid protein p24 at day 14 after viral infection using HIV p24 ELISA. C , Primary resting CD4 + T cells (1 × 10 4 cells per well) were exposed to live GC or fixed GC at 37°C for 72 h in the presence of IL-2. Cell proliferation was examined by the MTS assay (Promega CellTiter96 aqueous one solution cell proliferation assay). Data are means ± SD of triplicate sample. Difference in HIV infection between samples without GC exposure and GC-exposed samples at an MOI of 100 is significant as calculated by the two-tailed, paired Student t test. * p

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, MTS Assay, Proliferation Assay, Two Tailed Test

    The kinetics of HIV infection in TLR2-activated resting CD4 + T cells in the presence of inhibitors for reverse transcriptase and integrase. A , Resting CD4 + T cells were infected with pseudotyped HIV- VSV-G luciferase reporter virus for 2 h. Infected cells were treated with AZT at 5 μM at 1 h before infection (−1 h), at the same time during HIV exposure (0 h) or at 2, 8, and 24 h postinfection. PGN at 20 μg/ml was added at 2 h after viral infection. Infected cells were cultured in the presence of IL-2 for 4 d before measurement of luciferase activity. B , In a single-cycle infection assay, resting CD4 + T cells were infected with pseudotyped HIV-1 reporter virus for 2 h, treated with PGN at 20 μg/ml, and cultured in complete media with IL-2. L-731,988 at 10 μM was added to cell culture at 2, 24, and 48 h postinfection. Luciferase activity was measured on day 4 postinfection. Data are means ± SD of triplicate sample and represent three independent experiments.
    Figure Legend Snippet: The kinetics of HIV infection in TLR2-activated resting CD4 + T cells in the presence of inhibitors for reverse transcriptase and integrase. A , Resting CD4 + T cells were infected with pseudotyped HIV- VSV-G luciferase reporter virus for 2 h. Infected cells were treated with AZT at 5 μM at 1 h before infection (−1 h), at the same time during HIV exposure (0 h) or at 2, 8, and 24 h postinfection. PGN at 20 μg/ml was added at 2 h after viral infection. Infected cells were cultured in the presence of IL-2 for 4 d before measurement of luciferase activity. B , In a single-cycle infection assay, resting CD4 + T cells were infected with pseudotyped HIV-1 reporter virus for 2 h, treated with PGN at 20 μg/ml, and cultured in complete media with IL-2. L-731,988 at 10 μM was added to cell culture at 2, 24, and 48 h postinfection. Luciferase activity was measured on day 4 postinfection. Data are means ± SD of triplicate sample and represent three independent experiments.

    Techniques Used: Infection, Luciferase, Cell Culture, Activity Assay

    25) Product Images from "Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction"

    Article Title: Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131333

    Induction of T-bet by IL-2 and antigen is associated with antigen-specific proliferation. PBMCs of patients with cHBV ( n = 10) were labeled with the proliferation marker PBSE and cultured for 7 d in culture medium in the presence or absence of HBV-c18-27 antigen (Ag), IL-2, or IL-12. Antigen c18-27 was added on day 0, and cytokines were administered on day 4 in the designated groups. Negative controls were cultured in medium without further supplements, while positive controls were stimulated with CD3+CD28. On day 7, cells were stained for flow cytometry. (A) Frequencies of pentamer + CD8 T cells among total CD8 T cells and their PBSE labeling intensity. (B) Expression of T-bet and the frequency of pentamer + CD8 T cells after stimulation. (C) Mean percentage of c18-27–specific CD8 T cells among total CD8 T cells. (D) Mean percentage of PBSE − /T-bet − (white) and mean percentage of PBSE − /T-bet + specific CD8 T cells (black) among total CD8 T cells. Error bars represent the SEM. Data are representative of one experiment due to limited patient material. *, P
    Figure Legend Snippet: Induction of T-bet by IL-2 and antigen is associated with antigen-specific proliferation. PBMCs of patients with cHBV ( n = 10) were labeled with the proliferation marker PBSE and cultured for 7 d in culture medium in the presence or absence of HBV-c18-27 antigen (Ag), IL-2, or IL-12. Antigen c18-27 was added on day 0, and cytokines were administered on day 4 in the designated groups. Negative controls were cultured in medium without further supplements, while positive controls were stimulated with CD3+CD28. On day 7, cells were stained for flow cytometry. (A) Frequencies of pentamer + CD8 T cells among total CD8 T cells and their PBSE labeling intensity. (B) Expression of T-bet and the frequency of pentamer + CD8 T cells after stimulation. (C) Mean percentage of c18-27–specific CD8 T cells among total CD8 T cells. (D) Mean percentage of PBSE − /T-bet − (white) and mean percentage of PBSE − /T-bet + specific CD8 T cells (black) among total CD8 T cells. Error bars represent the SEM. Data are representative of one experiment due to limited patient material. *, P

    Techniques Used: Labeling, Marker, Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    Antigen-specific interferon-γ production correlates with T-bet and can be restored by IL-2+IL-12 co-stimulation. PBMCs of patients with cHBV ( n = 11) were cultured for 3 d in culture medium (control) containing either HBV-c18-27 antigen (Ag), c18-27 antigen+IL-2 (Ag+IL-2), c18-27 antigen+IL-12 (Ag+IL-12), IL-2+IL-12, c18-27 antigen+IL-2+IL-12 (Ag+IL-2+IL-12), or CD3+CD28. On day 3 antigen-treated groups were restimulated with antigen and all groups were incubated for 6 h in the presence of Brefeldin A before intracellular flow cytometry was performed. (A) Expression of T-bet and interferon-γ by CD8 T cells. The numbers indicate the percentage of T-bet +/− and interferon-γ +/− CD8 T cells among total CD8 T cells. (B) Mean induction of T-bet by treatment with the respective cytokines. Induction was defined as the percentage of T-bet + CD8 T cells after stimulation subtracted by the percentage of T-bet + CD8 T cells in unstimulated controls. Error bars represent the SEM. (C) Mean percentage of T-bet + interferon-γ + CD8 T cells after stimulation. Error bars indicate the SEM. Data are representative of one experiment due to limited patient material. ***, P
    Figure Legend Snippet: Antigen-specific interferon-γ production correlates with T-bet and can be restored by IL-2+IL-12 co-stimulation. PBMCs of patients with cHBV ( n = 11) were cultured for 3 d in culture medium (control) containing either HBV-c18-27 antigen (Ag), c18-27 antigen+IL-2 (Ag+IL-2), c18-27 antigen+IL-12 (Ag+IL-12), IL-2+IL-12, c18-27 antigen+IL-2+IL-12 (Ag+IL-2+IL-12), or CD3+CD28. On day 3 antigen-treated groups were restimulated with antigen and all groups were incubated for 6 h in the presence of Brefeldin A before intracellular flow cytometry was performed. (A) Expression of T-bet and interferon-γ by CD8 T cells. The numbers indicate the percentage of T-bet +/− and interferon-γ +/− CD8 T cells among total CD8 T cells. (B) Mean induction of T-bet by treatment with the respective cytokines. Induction was defined as the percentage of T-bet + CD8 T cells after stimulation subtracted by the percentage of T-bet + CD8 T cells in unstimulated controls. Error bars represent the SEM. (C) Mean percentage of T-bet + interferon-γ + CD8 T cells after stimulation. Error bars indicate the SEM. Data are representative of one experiment due to limited patient material. ***, P

    Techniques Used: Cell Culture, Incubation, Flow Cytometry, Cytometry, Expressing

    IL-12 selectively induces STAT4 phosphorylation in T-bet + CD8 T cells. PBMCs of patients with cHBV ( n = 7) were cultured for 3 d with either IL-2 or medium as control. On day 3 cells were restimulated for 20 min with either IL-2 or IL-12. Controls were left unstimulated. Cells were then analyzed by flow cytometry. (A) Coexpression of T-bet and pSTAT4 in CD8 T cells of an unstimulated control (left), PBMCs cultured with IL-2 and restimulated with IL-2 (middle), and PBMCs cultured in IL-2 and restimulated with IL-12 (right). Representative plots demonstrate the percentage of T-bet +/− and pSTAT4 +/− CD8 T cells among total CD8 T cells. (B) Mean frequency of T-bet − pSTAT4 + (white) and mean frequency of T-bet + pSTAT4 + (black) CD8 T cells among total CD8 T cells after stimulation with the respective cytokines. Error bars represent the SEM. Data are representative of one experiment due to limited patient material. **, P
    Figure Legend Snippet: IL-12 selectively induces STAT4 phosphorylation in T-bet + CD8 T cells. PBMCs of patients with cHBV ( n = 7) were cultured for 3 d with either IL-2 or medium as control. On day 3 cells were restimulated for 20 min with either IL-2 or IL-12. Controls were left unstimulated. Cells were then analyzed by flow cytometry. (A) Coexpression of T-bet and pSTAT4 in CD8 T cells of an unstimulated control (left), PBMCs cultured with IL-2 and restimulated with IL-2 (middle), and PBMCs cultured in IL-2 and restimulated with IL-12 (right). Representative plots demonstrate the percentage of T-bet +/− and pSTAT4 +/− CD8 T cells among total CD8 T cells. (B) Mean frequency of T-bet − pSTAT4 + (white) and mean frequency of T-bet + pSTAT4 + (black) CD8 T cells among total CD8 T cells after stimulation with the respective cytokines. Error bars represent the SEM. Data are representative of one experiment due to limited patient material. **, P

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry

    26) Product Images from "The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin"

    Article Title: The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039072

    Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.
    Figure Legend Snippet: Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

    Both naïve and memory human CD4 T cells expressed AR during TCR activation. (A) PBMC were treated with medium alone or allogeneic EBV-transformed B cells for 10 hours and analyzed by ICS. The gating strategy to identify activated CD4+ and CD8+ T cells is shown. (B) AR, IL-2, IFNγ or IL-4 expression was measured in four subjects in CD45RA+ (open) and CD45RA- (solid) CD4+ and CD8+ T cells after allogeneic EBV-transformed B cell stimulation. Background values have been subtracted. (C) PBMC were treated with medium alone or SEB in the presence of TAPI-1 for 8 hours. Then six populations were sorted based on surface AR, CD69 and CD45RA expression (left). AR mRNA in each population was measured by RT-PCR (right). Results in (A) and (B) represent at least three experiments, (C) represents two experiments.
    Figure Legend Snippet: Both naïve and memory human CD4 T cells expressed AR during TCR activation. (A) PBMC were treated with medium alone or allogeneic EBV-transformed B cells for 10 hours and analyzed by ICS. The gating strategy to identify activated CD4+ and CD8+ T cells is shown. (B) AR, IL-2, IFNγ or IL-4 expression was measured in four subjects in CD45RA+ (open) and CD45RA- (solid) CD4+ and CD8+ T cells after allogeneic EBV-transformed B cell stimulation. Background values have been subtracted. (C) PBMC were treated with medium alone or SEB in the presence of TAPI-1 for 8 hours. Then six populations were sorted based on surface AR, CD69 and CD45RA expression (left). AR mRNA in each population was measured by RT-PCR (right). Results in (A) and (B) represent at least three experiments, (C) represents two experiments.

    Techniques Used: Activation Assay, Transformation Assay, Expressing, Cell Stimulation, Reverse Transcription Polymerase Chain Reaction

    TCR activation induced AR expression in human PBMC T cells. (A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.
    Figure Legend Snippet: TCR activation induced AR expression in human PBMC T cells. (A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.

    Techniques Used: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification

    27) Product Images from "IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor ? subunit CD25"

    Article Title: IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor ? subunit CD25

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0909384107

    Long-acting IL-2 is equal to IL-2/mAb CD122 complexes in the absence of CD25. CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to CD25 −/− mice, which then received on the same day a single injection of rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , chTNT-3/IL-2 IL-2-FP or 12 injections of PBS or rhIL-2 every 2 h for 24 h. CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells from the host spleen was determined 3 days after adoptive transfer. Numbers indicate percentage of divided cells. The data are representative of three different experiments, with each profile representing one of at least two mice.
    Figure Legend Snippet: Long-acting IL-2 is equal to IL-2/mAb CD122 complexes in the absence of CD25. CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to CD25 −/− mice, which then received on the same day a single injection of rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , chTNT-3/IL-2 IL-2-FP or 12 injections of PBS or rhIL-2 every 2 h for 24 h. CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells from the host spleen was determined 3 days after adoptive transfer. Numbers indicate percentage of divided cells. The data are representative of three different experiments, with each profile representing one of at least two mice.

    Techniques Used: Labeling, Mouse Assay, Injection, Adoptive Transfer Assay

    IL-2 requires prolonged half-life and CD25 blockade to mimic the activity of IL-2/mAb CD122 complexes. ( A ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. On days 1 and 3, host mice received injections of PBS, rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or chTNT-3/IL-2 IL-2-FP. Where indicated, mice also received daily injections of 200 μg of anti-CD25 mAb. CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells in spleen was analyzed by flow cytometry 6 days after adoptive transfer. ( B ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. Host mice then received a single injection of rhIL-2 or rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or 12 injections of PBS or rhIL-2 every 2 h for 24 h. Where indicated, mice also received daily injections of 200 μg anti-CD25 mAb. Host spleens were analyzed by flow cytometry 3 days after adoptive transfer for CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells. Numbers in A and B indicate percentage of divided cells. The data are representative of three different experiments, with each profile representing one of at least two mice.
    Figure Legend Snippet: IL-2 requires prolonged half-life and CD25 blockade to mimic the activity of IL-2/mAb CD122 complexes. ( A ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. On days 1 and 3, host mice received injections of PBS, rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or chTNT-3/IL-2 IL-2-FP. Where indicated, mice also received daily injections of 200 μg of anti-CD25 mAb. CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells in spleen was analyzed by flow cytometry 6 days after adoptive transfer. ( B ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. Host mice then received a single injection of rhIL-2 or rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or 12 injections of PBS or rhIL-2 every 2 h for 24 h. Where indicated, mice also received daily injections of 200 μg anti-CD25 mAb. Host spleens were analyzed by flow cytometry 3 days after adoptive transfer for CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells. Numbers in A and B indicate percentage of divided cells. The data are representative of three different experiments, with each profile representing one of at least two mice.

    Techniques Used: Activity Assay, Labeling, Mouse Assay, Flow Cytometry, Cytometry, Adoptive Transfer Assay, Injection

    IL-2/mAb CD122 complexes have a prolonged in vivo half-life. ( A ) Host WT mice received a single injection of PBS, 20 μg rhIL-2 or 1.5 μg rhIL-2 plus 15 μg MAB602 anti-hIL-2 mAb CD122 at the indicated time-points before adoptive transfer of CFSE-labeled Thy1.1 + MP CD8 + T cells. Host spleens were analyzed by flow cytometry 3 days after adoptive transfer. Histograms shown are gated on Thy1.1 + CD8 + donor cells. Numbers indicate percentage of divided cells. ( B and C ) WT mice received a single injection at indicated doses of rhIL-2 (▲, 1.5 μg; •, 15 μg) or rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 (▲, 0.3 μg plus 3 μg; •, 1.5 μg plus 15 μg) ( B ) or rhIL-2 plus 5344 anti-hIL-2 mAb CD25 (▲, 1.5 μg IL-2; •, 1.5 μg plus 15 μg) ( C ), and blood samples were collected at the indicated time-points after injection. Serum was assayed on CTLL-2 cells. Proliferation was determined by measuring incorporation of [ 3 H]-thymidine. The data are representative of at least three different experiments, with each profile representing one of at least two mice.
    Figure Legend Snippet: IL-2/mAb CD122 complexes have a prolonged in vivo half-life. ( A ) Host WT mice received a single injection of PBS, 20 μg rhIL-2 or 1.5 μg rhIL-2 plus 15 μg MAB602 anti-hIL-2 mAb CD122 at the indicated time-points before adoptive transfer of CFSE-labeled Thy1.1 + MP CD8 + T cells. Host spleens were analyzed by flow cytometry 3 days after adoptive transfer. Histograms shown are gated on Thy1.1 + CD8 + donor cells. Numbers indicate percentage of divided cells. ( B and C ) WT mice received a single injection at indicated doses of rhIL-2 (▲, 1.5 μg; •, 15 μg) or rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 (▲, 0.3 μg plus 3 μg; •, 1.5 μg plus 15 μg) ( B ) or rhIL-2 plus 5344 anti-hIL-2 mAb CD25 (▲, 1.5 μg IL-2; •, 1.5 μg plus 15 μg) ( C ), and blood samples were collected at the indicated time-points after injection. Serum was assayed on CTLL-2 cells. Proliferation was determined by measuring incorporation of [ 3 H]-thymidine. The data are representative of at least three different experiments, with each profile representing one of at least two mice.

    Techniques Used: In Vivo, Mouse Assay, Injection, Adoptive Transfer Assay, Labeling, Flow Cytometry, Cytometry

    In vivo activity of IL-2/mAb CD122 complexes is largely independent of Fc receptors. CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to ( A ) WT mice ( Upper ) and FcRI −/− FcRγ −/− FcRIIB −/− mice ( Lower ), or to ( B ) WT mice ( Left ) and FcRn −/− mice ( Right ). ( C ) CFSE-labeled Thy1.1 + CD4 + T cells were adoptively transferred to WT ( Left ) or FcRn −/− mice ( Right ). Host mice subsequently received three injections of rmIL-2 plus S4B6 anti-mIL-2 mAb CD122 ( A and B ) or rmIL-2 plus JES6-1 anti-mIL-2 mAb CD25 ( C ) every other day. CFSE dilution and cell recovery from lymph nodes (LN) was analyzed by flow cytometry 7 days after adoptive transfer. Histograms shown are gated on Thy1.1 + CD8 + ( A and B ) or Thy1.1 + CD25 + FoxP3 + CD4 + donor T cells ( C ). Numbers indicate percentage of divided donor cells. The data are representative of two ( A ) or three ( B and C ) different experiments with each profile representing one of at least two mice.
    Figure Legend Snippet: In vivo activity of IL-2/mAb CD122 complexes is largely independent of Fc receptors. CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to ( A ) WT mice ( Upper ) and FcRI −/− FcRγ −/− FcRIIB −/− mice ( Lower ), or to ( B ) WT mice ( Left ) and FcRn −/− mice ( Right ). ( C ) CFSE-labeled Thy1.1 + CD4 + T cells were adoptively transferred to WT ( Left ) or FcRn −/− mice ( Right ). Host mice subsequently received three injections of rmIL-2 plus S4B6 anti-mIL-2 mAb CD122 ( A and B ) or rmIL-2 plus JES6-1 anti-mIL-2 mAb CD25 ( C ) every other day. CFSE dilution and cell recovery from lymph nodes (LN) was analyzed by flow cytometry 7 days after adoptive transfer. Histograms shown are gated on Thy1.1 + CD8 + ( A and B ) or Thy1.1 + CD25 + FoxP3 + CD4 + donor T cells ( C ). Numbers indicate percentage of divided donor cells. The data are representative of two ( A ) or three ( B and C ) different experiments with each profile representing one of at least two mice.

    Techniques Used: In Vivo, Activity Assay, Labeling, Mouse Assay, Flow Cytometry, Cytometry, Adoptive Transfer Assay

    Prolonging half-life of IL-2 is not sufficient to mimic IL-2/mAb CD122 complexes. ( A and C ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. On days 1 and 3, host mice received injections of PBS, rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or chTNT-3/IL-2 hIL-2 fusion protein (IL-2-FP), as well as ( C ) IL-2-FP plus MAB602 anti-hIL-2 mAb CD122 (IL-2-FP/mAb CD122 ). CFSE dilution of cells recovered from the host spleen was analyzed by flow cytometry 6 days after adoptive transfer. Histograms shown are gated on Thy1.1 + CD8 + donor cells. Numbers indicate percentage of divided cells. ( B and D ) WT mice received a single injection of rhIL-2 (▲, IL-2), rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 or IL-2-FP (both displayed as •), as well as ( D ) IL-2-FP plus MAB602 anti-hIL-2 mAb CD122 (•, IL-2-FP/mAb CD122 ). Blood samples were collected at the indicated time-points, and serum was assayed for proliferation of CTLL-2 cells by measuring incorporation of [ 3 H]-thymidine. The data are representative of three different experiments, with each profile representing one of at least two mice.
    Figure Legend Snippet: Prolonging half-life of IL-2 is not sufficient to mimic IL-2/mAb CD122 complexes. ( A and C ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. On days 1 and 3, host mice received injections of PBS, rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or chTNT-3/IL-2 hIL-2 fusion protein (IL-2-FP), as well as ( C ) IL-2-FP plus MAB602 anti-hIL-2 mAb CD122 (IL-2-FP/mAb CD122 ). CFSE dilution of cells recovered from the host spleen was analyzed by flow cytometry 6 days after adoptive transfer. Histograms shown are gated on Thy1.1 + CD8 + donor cells. Numbers indicate percentage of divided cells. ( B and D ) WT mice received a single injection of rhIL-2 (▲, IL-2), rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 or IL-2-FP (both displayed as •), as well as ( D ) IL-2-FP plus MAB602 anti-hIL-2 mAb CD122 (•, IL-2-FP/mAb CD122 ). Blood samples were collected at the indicated time-points, and serum was assayed for proliferation of CTLL-2 cells by measuring incorporation of [ 3 H]-thymidine. The data are representative of three different experiments, with each profile representing one of at least two mice.

    Techniques Used: Labeling, Mouse Assay, Flow Cytometry, Cytometry, Adoptive Transfer Assay, Injection

    Both Fc and Fab contribute by distinct mechanisms to in vivo activity of IL-2/mAb CD122 complexes. ( A ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. Host mice then received a single injection of rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or 12 injections of PBS or rhIL-2, every 2 h for 24 h. Host spleens were analyzed by flow cytometry 3 days after adoptive transfer for CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells. Numbers indicate percentage of divided cells. ( B ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. Host mice then received a single injection of rmIL-2 plus S4B6 anti-mIL-2 mAb CD122 , rmIL-2 plus F(ab’) 2 fragments of S4B6 anti-mIL-2 mAb CD122 , or 12 injections of PBS or rmIL-2 plus S4B6 F(ab’) 2 , every 2 h for 24 h. CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells in lymph nodes was analyzed by flow cytometry 7 days after adoptive transfer. Numbers indicate percentage of divided cells. The data are representative of three different experiments, with each profile representing one of at least two mice.
    Figure Legend Snippet: Both Fc and Fab contribute by distinct mechanisms to in vivo activity of IL-2/mAb CD122 complexes. ( A ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. Host mice then received a single injection of rhIL-2, rhIL-2 plus MAB602 anti-hIL-2 mAb CD122 , or 12 injections of PBS or rhIL-2, every 2 h for 24 h. Host spleens were analyzed by flow cytometry 3 days after adoptive transfer for CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells. Numbers indicate percentage of divided cells. ( B ) CFSE-labeled Thy1.1 + MP CD8 + T cells were adoptively transferred to WT mice. Host mice then received a single injection of rmIL-2 plus S4B6 anti-mIL-2 mAb CD122 , rmIL-2 plus F(ab’) 2 fragments of S4B6 anti-mIL-2 mAb CD122 , or 12 injections of PBS or rmIL-2 plus S4B6 F(ab’) 2 , every 2 h for 24 h. CFSE dilution and cell recovery of Thy1.1 + CD8 + donor cells in lymph nodes was analyzed by flow cytometry 7 days after adoptive transfer. Numbers indicate percentage of divided cells. The data are representative of three different experiments, with each profile representing one of at least two mice.

    Techniques Used: In Vivo, Activity Assay, Labeling, Mouse Assay, Injection, Flow Cytometry, Cytometry, Adoptive Transfer Assay

    28) Product Images from "Identification of Human STAT5-dependent Gene Regulatory Elements Based on Interspecies Homology *"

    Article Title: Identification of Human STAT5-dependent Gene Regulatory Elements Based on Interspecies Homology *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M605001200

    IL-2 and IFN- α synergize to regulate NCAM2 expression a , starved NKL cells were treated with the indicated cytokines for 7 h. NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. b and c , starved NKL cells were treated with the indicated cytokine for 2 h. The expression of IFN- γ ( b ) and CIS ( c ) was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. d , ChIP assays were performed on NKL cells that had been starved and then left untreated or stimulated with the indicated cytokines for 30 min. Chromatin immunoprecipitations were performed using antibodies specific for STAT4, STAT5, or a nonspecific antibody (IgG). Data were normalized to input and expressed relative to nonspecific IgG. Each RT-PCR and ChIP experiment was repeated two times.
    Figure Legend Snippet: IL-2 and IFN- α synergize to regulate NCAM2 expression a , starved NKL cells were treated with the indicated cytokines for 7 h. NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. b and c , starved NKL cells were treated with the indicated cytokine for 2 h. The expression of IFN- γ ( b ) and CIS ( c ) was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. d , ChIP assays were performed on NKL cells that had been starved and then left untreated or stimulated with the indicated cytokines for 30 min. Chromatin immunoprecipitations were performed using antibodies specific for STAT4, STAT5, or a nonspecific antibody (IgG). Data were normalized to input and expressed relative to nonspecific IgG. Each RT-PCR and ChIP experiment was repeated two times.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    STAT5a and STAT5b activation by IL-2 leads to NCAM2 expression in NKL cells a , starved NKL cells were either treated with IL-2 for 15 min or left untreated. Immunoprecipitation of STAT5a and STAT5b using specific antibodies was performed, followed by Western blot analysis with the indicated antibodies. b , starved NKL cells were treated with IL-2 for the indicated times. NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. c , starved NKL cells were treated for 7 h with IL-2 at the indicated concentrations (units/ml). NCAM2 expression was analyzed by quantitative real time PCR. Each RT-PCR experiment was repeated two times.
    Figure Legend Snippet: STAT5a and STAT5b activation by IL-2 leads to NCAM2 expression in NKL cells a , starved NKL cells were either treated with IL-2 for 15 min or left untreated. Immunoprecipitation of STAT5a and STAT5b using specific antibodies was performed, followed by Western blot analysis with the indicated antibodies. b , starved NKL cells were treated with IL-2 for the indicated times. NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. c , starved NKL cells were treated for 7 h with IL-2 at the indicated concentrations (units/ml). NCAM2 expression was analyzed by quantitative real time PCR. Each RT-PCR experiment was repeated two times.

    Techniques Used: Activation Assay, Expressing, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Neither STAT1 nor STAT3 bind to the NCAM2 STAT binding region ChIP assays were performed on NKL cells that had been starved and then left untreated or stimulated with IL-2 for 30 min. Immunoprecipitations were performed using antibodies ( Ab ) specific for STAT1, STAT3, STAT5, or a nonspecific antibody (IgG). DNA was quantitated by real time PCR and was normalized to input and expressed relative to nonspecific IgG. Each ChIP experiment was repeated three times.
    Figure Legend Snippet: Neither STAT1 nor STAT3 bind to the NCAM2 STAT binding region ChIP assays were performed on NKL cells that had been starved and then left untreated or stimulated with IL-2 for 30 min. Immunoprecipitations were performed using antibodies ( Ab ) specific for STAT1, STAT3, STAT5, or a nonspecific antibody (IgG). DNA was quantitated by real time PCR and was normalized to input and expressed relative to nonspecific IgG. Each ChIP experiment was repeated three times.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    STAT5a, STAT5b, and components of the basal transcriptional machinery bind to the NCAM2 intronic element in vivo a , ChIP assays were performed on NKL cells that had been starved and then left untreated or stimulated with IL-2 for 30 min. Immunoprecipitations were performed using antibodies ( Ab ) to STAT5a, STAT5b, or a nonspecific antibody (IgG). The upper panel shows PCR analysis using primers specific for the NCAM2 intronic region, whereas the lower panel shows PCR analysis using primers specific for a control region. The right panels show PCR of input DNA. b , ChIP analysis was performed using antibodies to Pol II, p300, total STAT5, or a nonspecific IgG. PCR was performed on the NCAM2 intronic region using both ChIP product ( left ) and input DNA ( right ). Each ChIP experiment was repeated two times.
    Figure Legend Snippet: STAT5a, STAT5b, and components of the basal transcriptional machinery bind to the NCAM2 intronic element in vivo a , ChIP assays were performed on NKL cells that had been starved and then left untreated or stimulated with IL-2 for 30 min. Immunoprecipitations were performed using antibodies ( Ab ) to STAT5a, STAT5b, or a nonspecific antibody (IgG). The upper panel shows PCR analysis using primers specific for the NCAM2 intronic region, whereas the lower panel shows PCR analysis using primers specific for a control region. The right panels show PCR of input DNA. b , ChIP analysis was performed using antibodies to Pol II, p300, total STAT5, or a nonspecific IgG. PCR was performed on the NCAM2 intronic region using both ChIP product ( left ) and input DNA ( right ). Each ChIP experiment was repeated two times.

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    IFN- α induces expression of NCAM2 a , starved NKL cells were either treated with the indicated cytokine for 15 min or left untreated. Western blots were performed with the indicated antibodies. b , starved NKL cells were treated with IL-2 or IFN- α for 15 min or left untreated. Immunoprecipitation was performed using an antibody to STAT4. Western blot analysis was performed against phosphotyrosine ( P-Tyr ) or STAT4. c , starved NKL cells were treated with the indicated cytokines for 7 h, and NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. d , starved NKL cells were treated for the indicated times with IFN- α . NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. Each RT-PCR experiment was repeated two times.
    Figure Legend Snippet: IFN- α induces expression of NCAM2 a , starved NKL cells were either treated with the indicated cytokine for 15 min or left untreated. Western blots were performed with the indicated antibodies. b , starved NKL cells were treated with IL-2 or IFN- α for 15 min or left untreated. Immunoprecipitation was performed using an antibody to STAT4. Western blot analysis was performed against phosphotyrosine ( P-Tyr ) or STAT4. c , starved NKL cells were treated with the indicated cytokines for 7 h, and NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. d , starved NKL cells were treated for the indicated times with IFN- α . NCAM2 expression was analyzed by quantitative real time PCR. GAPDH was used as an invariant control. Each RT-PCR experiment was repeated two times.

    Techniques Used: Expressing, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "CEACAM1 regulates TIM–3–mediated tolerance and exhaustion"

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    Journal: Nature

    doi: 10.1038/nature13848

    CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P
    Figure Legend Snippet: CEACAM1 determines TIM-3 expression and function a , HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b , Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c , HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d , Cellular distribution of c , as in b . Depicted as percentage of hTIM-3. e , Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f , Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g , Analysis of CD4 + Vβ8 + T cells after SEB tolerance induction from experimental mice of indicated genotypes. h , Galectin-9 induction of apoptosis. Annexin V + propidium iodide staining of T H 1 cells polarized from Tim3 Tg or Tim3 Tg Ceacam1 −/− mice after treatment with galectin-9 (2 μg ml −1 ) for 8 h. Note decreased apoptosis in Tim3 Tg Ceacam1 −/− T cells. i , Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in . j , GST or GST–hTIM-3 staining of hCEACAM1-4L–transfected Jurkat T cells. k , Wild-type CD4 + T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m , Characterization of tolerance in SEB model. Tim3 Tg (l) and Tim3 Tg Ceacam1 −/− ( m . Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3 Tg but not Tim3 Tg Ceacam1 −/− mice. n = 3 per group. n , Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4 + T cells from indicated genotypes expressed as levels relative to Ceacam1 −/− mice. o, p , Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4 + T cells from wild-type ( o ) or Ceacam1 −/− ( p ) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1 −/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three ( f, g, k–p ) and two ( a–e, h, j ) independent experiments. * P

    Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry, Mutagenesis, Variant Assay, Mouse Assay, Staining, Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Inhibition

    30) Product Images from "CD28 costimulation independence of target organ versus circulating memory antigen-specific CD4+ T cells"

    Article Title: CD28 costimulation independence of target organ versus circulating memory antigen-specific CD4+ T cells

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200318317

    BAL CD4 + CD28 + T cells are relatively resistant to beryllium-induced cell death. Data are shown for representative experiments using fresh BAL cells ( a ) and T cell clones derived from the lung of CBD patient 12 ( b ) stimulated with either 20 U/ml IL-2 or BeSO 4 . Following 24–48 hours of culture, cells were stained for CD4, CD28, annexin V, and propidium iodide. BAL cells were gated for CD4 expression, and the number in each quadrant of the density plots represents the percentage of CD4 + T cells.
    Figure Legend Snippet: BAL CD4 + CD28 + T cells are relatively resistant to beryllium-induced cell death. Data are shown for representative experiments using fresh BAL cells ( a ) and T cell clones derived from the lung of CBD patient 12 ( b ) stimulated with either 20 U/ml IL-2 or BeSO 4 . Following 24–48 hours of culture, cells were stained for CD4, CD28, annexin V, and propidium iodide. BAL cells were gated for CD4 expression, and the number in each quadrant of the density plots represents the percentage of CD4 + T cells.

    Techniques Used: Clone Assay, Derivative Assay, Staining, Expressing

    Intracellular staining for IFN-γ and IL-2 in stimulated BAL T cells from CBD patients. Representative experiments are shown for the flow-cytometric analysis of BAL CD4 + T cells from CBD patient 1 ( a ) and patient 3 ( b ) stimulated with either medium alone, BeSO 4 , or SEB and subsequently stained for surface CD4 and CD28 and intracellular IFN-γ or IL-2. BALcells were gated on CD4 expression. The numbers in the upper-left and -right quadrants of each density plot are the percentages of CD4 + CD28 – and CD4 + CD28 + cells, respectively, that express IFN-γ or IL-2. ( c ) Percentage of BAL CD4 + CD28 – and CD4 + CD28 + cells that express intracellular IFN-γ and IL-2 after short-term stimulation with BeSO 4 . The mean percentage of Th1 cytokine produced by CD4 + CD28 – and CD4 + CD28 + cells from CBD patients is shown as a solid line: mean ± SEM for IFN-γ ( n = 12), 11.3% ± 3.3% for CD4 + CD28 – cells and 18.9% ± 3.6% for CD4 + CD28 + cells; IL-2 ( n = 10), 4.3% ± 1.1% for CD4 + CD28 – cells and 11.8% ± 2.7% for CD4 + CD28 + cells.
    Figure Legend Snippet: Intracellular staining for IFN-γ and IL-2 in stimulated BAL T cells from CBD patients. Representative experiments are shown for the flow-cytometric analysis of BAL CD4 + T cells from CBD patient 1 ( a ) and patient 3 ( b ) stimulated with either medium alone, BeSO 4 , or SEB and subsequently stained for surface CD4 and CD28 and intracellular IFN-γ or IL-2. BALcells were gated on CD4 expression. The numbers in the upper-left and -right quadrants of each density plot are the percentages of CD4 + CD28 – and CD4 + CD28 + cells, respectively, that express IFN-γ or IL-2. ( c ) Percentage of BAL CD4 + CD28 – and CD4 + CD28 + cells that express intracellular IFN-γ and IL-2 after short-term stimulation with BeSO 4 . The mean percentage of Th1 cytokine produced by CD4 + CD28 – and CD4 + CD28 + cells from CBD patients is shown as a solid line: mean ± SEM for IFN-γ ( n = 12), 11.3% ± 3.3% for CD4 + CD28 – cells and 18.9% ± 3.6% for CD4 + CD28 + cells; IL-2 ( n = 10), 4.3% ± 1.1% for CD4 + CD28 – cells and 11.8% ± 2.7% for CD4 + CD28 + cells.

    Techniques Used: Staining, Flow Cytometry, Expressing, Produced

    31) Product Images from "A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice"

    Article Title: A kinase-dead knock-in mutation in mTOR leads to early embryonic lethality and is dispensable for the immune system in heterozygous mice

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-10-28

    Proliferative capacity of mTOR+/kd T cells . (A) Proliferation of splenocytes after IL-2 stimulation. Spleen cells from mTOR+/+ and mTOR+/kd mice were stimulated with IL-2 in the presence or absence of rapamycin and proliferation was assessed by [ 3 H] thymidine incorporation, as described in Methods. (B) Proliferation of spleen cells from mTOR+/+ and mTOR+/kd littermates in response to anti-CD3 with or without soluble anti-CD28 or IL-2 was measured as in (A). Data are expressed as % control. Results shown are representative of three experiments.
    Figure Legend Snippet: Proliferative capacity of mTOR+/kd T cells . (A) Proliferation of splenocytes after IL-2 stimulation. Spleen cells from mTOR+/+ and mTOR+/kd mice were stimulated with IL-2 in the presence or absence of rapamycin and proliferation was assessed by [ 3 H] thymidine incorporation, as described in Methods. (B) Proliferation of spleen cells from mTOR+/+ and mTOR+/kd littermates in response to anti-CD3 with or without soluble anti-CD28 or IL-2 was measured as in (A). Data are expressed as % control. Results shown are representative of three experiments.

    Techniques Used: Mouse Assay

    32) Product Images from "Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus)"

    Article Title: Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus)

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-5-23

    Proliferation of deer mouse cells to recombinant cytokines. (A) After 8 days of incubation with GM-CSF, deer mouse bone marrow cells were washed and then cultured with dilutions of GM-CSF in duplicate for 48 hours, then proliferation assessed by MTS assay. The data are representative of four deer mice. (B) To assess proliferative capacity of deer mouse T cells to human IL-2, splenocytes were cultured with a suboptimal dose of PHA (2 μg/ml) and dilutions of recombinant human IL-2 in duplicate for 48 hours, and proliferation assessed by MTS assay. The data are representative of two deer mice.
    Figure Legend Snippet: Proliferation of deer mouse cells to recombinant cytokines. (A) After 8 days of incubation with GM-CSF, deer mouse bone marrow cells were washed and then cultured with dilutions of GM-CSF in duplicate for 48 hours, then proliferation assessed by MTS assay. The data are representative of four deer mice. (B) To assess proliferative capacity of deer mouse T cells to human IL-2, splenocytes were cultured with a suboptimal dose of PHA (2 μg/ml) and dilutions of recombinant human IL-2 in duplicate for 48 hours, and proliferation assessed by MTS assay. The data are representative of two deer mice.

    Techniques Used: Recombinant, Incubation, Cell Culture, MTS Assay, Mouse Assay

    33) Product Images from "A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood"

    Article Title: A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00963

    Whole blood secretory cytokine methodology, gating strategy, and quantification. Whole blood was collected from patients and 1 mL was aliquoted per stimulatory condition. Whole blood was stimulated for 24 h in the presence of PMA-ionomycin or IL-2/12. At 22 h post-stimulation, Golgiplug was added to whole blood and incubated for an additional 2 h. At 24 h post-stimulation, whole blood was incubated for 15 min with an extracellular staining mix. Lyse/fix buffer was then added and incubated for 10 min before blood was spun down. Cells were washed, resuspended in Perm III Buffer, and incubated on ice for 30 min before being spun down and resuspended in an intracellular staining mix. Cells were then incubated for an additional 30 min at 4°C prior to being resuspended and assessed by flow cytometry (A) . The lymphocyte population was gated on before excluding doublets and dead cells. CD45 + CD14 − CD56 Bright/Dim CD3 − cells were gated on to assess intracellular IFNγ based on unstimulated controls. The percentage of CD56 Bright/Dim CD3 − cells expressing INFγ after stimulation was assessed in healthy donors ( n = 11) and cancer patients ( n = 9) (B) . Shown are the median values ± IQR.
    Figure Legend Snippet: Whole blood secretory cytokine methodology, gating strategy, and quantification. Whole blood was collected from patients and 1 mL was aliquoted per stimulatory condition. Whole blood was stimulated for 24 h in the presence of PMA-ionomycin or IL-2/12. At 22 h post-stimulation, Golgiplug was added to whole blood and incubated for an additional 2 h. At 24 h post-stimulation, whole blood was incubated for 15 min with an extracellular staining mix. Lyse/fix buffer was then added and incubated for 10 min before blood was spun down. Cells were washed, resuspended in Perm III Buffer, and incubated on ice for 30 min before being spun down and resuspended in an intracellular staining mix. Cells were then incubated for an additional 30 min at 4°C prior to being resuspended and assessed by flow cytometry (A) . The lymphocyte population was gated on before excluding doublets and dead cells. CD45 + CD14 − CD56 Bright/Dim CD3 − cells were gated on to assess intracellular IFNγ based on unstimulated controls. The percentage of CD56 Bright/Dim CD3 − cells expressing INFγ after stimulation was assessed in healthy donors ( n = 11) and cancer patients ( n = 9) (B) . Shown are the median values ± IQR.

    Techniques Used: Incubation, Staining, Flow Cytometry, Expressing

    Whole blood signaling protein methodology, gating strategy, and quantification. Whole blood was collected from patients and 500 μL was aliquoted per receptor panel. After a 15-min incubation with IL-2/12 stimulation and the desired receptor panel, lyse/fix buffer was added and incubated for 10 min before blood was spun down. Cells were washed, resuspended in Perm III Buffer, and incubated on ice for 30 min before being spun down and resuspended in an intracellular staining mix. Cells were then incubated for an additional hour at room temperature prior to being resuspended and assessed by flow cytometry (A) . The lymphocyte population was gated on before excluding doublets and dead cells. CD45 + CD14 − CD56 Bright/Dim CD3 − cells were gated on to assess signaling protein phosphorylation (B) . The relative level of expression (MFI) of phospho-proteins STAT5, STAT4, p38 MAPK, and S6 was assessed in healthy donor ( n = 13) and cancer patient ( n = 9) samples (C) . Shown are the median values ± IQR.
    Figure Legend Snippet: Whole blood signaling protein methodology, gating strategy, and quantification. Whole blood was collected from patients and 500 μL was aliquoted per receptor panel. After a 15-min incubation with IL-2/12 stimulation and the desired receptor panel, lyse/fix buffer was added and incubated for 10 min before blood was spun down. Cells were washed, resuspended in Perm III Buffer, and incubated on ice for 30 min before being spun down and resuspended in an intracellular staining mix. Cells were then incubated for an additional hour at room temperature prior to being resuspended and assessed by flow cytometry (A) . The lymphocyte population was gated on before excluding doublets and dead cells. CD45 + CD14 − CD56 Bright/Dim CD3 − cells were gated on to assess signaling protein phosphorylation (B) . The relative level of expression (MFI) of phospho-proteins STAT5, STAT4, p38 MAPK, and S6 was assessed in healthy donor ( n = 13) and cancer patient ( n = 9) samples (C) . Shown are the median values ± IQR.

    Techniques Used: Incubation, Staining, Flow Cytometry, Expressing

    Plasma collection methodology for secretory cytokine quantification by ELISA. After incubation in the absence of stimulation or with either PMA-ionomycin or IL-2/12 stimulation for 24 h, 600 μL of whole blood was collected and spun down prior to being collected and stored at −80°C for subsequent use in a human IFNγ ELISA (A) . Extracellular IFNγ was quantified in response to stimuli in healthy donors ( n = 13) and cancer patients ( n = 10) (B) . Shown are the median values ± IQR.
    Figure Legend Snippet: Plasma collection methodology for secretory cytokine quantification by ELISA. After incubation in the absence of stimulation or with either PMA-ionomycin or IL-2/12 stimulation for 24 h, 600 μL of whole blood was collected and spun down prior to being collected and stored at −80°C for subsequent use in a human IFNγ ELISA (A) . Extracellular IFNγ was quantified in response to stimuli in healthy donors ( n = 13) and cancer patients ( n = 10) (B) . Shown are the median values ± IQR.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

    34) Product Images from "Maternal Decidual Macrophages Inhibit NK Cell Killing of Invasive Cytotrophoblasts During Human Pregnancy 1"

    Article Title: Maternal Decidual Macrophages Inhibit NK Cell Killing of Invasive Cytotrophoblasts During Human Pregnancy 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.112.099465

    Decidual NK cells lysed K562 targets and CTBs. A ) In 51 Cr-release assays, purified CD56 + dNK cells were potent killers of K562 targets in the presence of IL-2 (n = 3). B ) dNK cells killed CTBs isolated from the same pregnancy (semiallogeneic) or an unrelated
    Figure Legend Snippet: Decidual NK cells lysed K562 targets and CTBs. A ) In 51 Cr-release assays, purified CD56 + dNK cells were potent killers of K562 targets in the presence of IL-2 (n = 3). B ) dNK cells killed CTBs isolated from the same pregnancy (semiallogeneic) or an unrelated

    Techniques Used: Purification, Isolation

    Total decidual leukocytes and decidual CD14 + cells were potent inhibitors of cytotoxicity. A ) With CTB targets, purified IL-2-treated dNK cells had higher cytotoxic activity than unfractionated DLs, which contained other leukocytes types (* P ≤
    Figure Legend Snippet: Total decidual leukocytes and decidual CD14 + cells were potent inhibitors of cytotoxicity. A ) With CTB targets, purified IL-2-treated dNK cells had higher cytotoxic activity than unfractionated DLs, which contained other leukocytes types (* P ≤

    Techniques Used: CtB Assay, Purification, Activity Assay

    35) Product Images from "Discordant effects of interleukin-2 on viral and immune parameters in human immunodeficiency virus-1-infected monocyte-derived mature dendritic cells"

    Article Title: Discordant effects of interleukin-2 on viral and immune parameters in human immunodeficiency virus-1-infected monocyte-derived mature dendritic cells

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02143.x

    Effects of interleukin-2 (IL-2) on CD4, CCR5, and CXCR4 gene expression in monocyte-derived macrophages (MDMs) and mature dendritic cells (MDDCs). Cultures were incubated for 2- and 24 h, either unstimulated or stimulated with 100 U/ml IL-2. RNA extracts were then subjected to reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect the levels of gene expression of CD4, CCR5, CXCR4, and β-actin (as a housekeeping gene). Changes in the expression levels were calculated as percentage of baseline expression in unstimulated cells taken as 100% (indicated by the dotted horizontal line). Results represent the mean values ± standard error of the mean (s.e.m.) of six independent experiments using cells from different donors. * P
    Figure Legend Snippet: Effects of interleukin-2 (IL-2) on CD4, CCR5, and CXCR4 gene expression in monocyte-derived macrophages (MDMs) and mature dendritic cells (MDDCs). Cultures were incubated for 2- and 24 h, either unstimulated or stimulated with 100 U/ml IL-2. RNA extracts were then subjected to reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect the levels of gene expression of CD4, CCR5, CXCR4, and β-actin (as a housekeeping gene). Changes in the expression levels were calculated as percentage of baseline expression in unstimulated cells taken as 100% (indicated by the dotted horizontal line). Results represent the mean values ± standard error of the mean (s.e.m.) of six independent experiments using cells from different donors. * P

    Techniques Used: Expressing, Derivative Assay, Incubation, Reverse Transcription Polymerase Chain Reaction

    Expression of various receptors in mature dendritic cells (MDDCs) following 24 h of culture in the presence or absence of interleukin-2 (IL-2) (500 U/ml). Cells, from one representative donor, were subjected to fluorescence-activated cell sorter (FACS) analysis to detect the expression on the surface of virus receptors, CD3, IL-2 receptors, costimulatory molecules, and activation markers. Histograms illustrate the level of positive staining with a specific antibody in unstimulated (shaded) and IL-2-stimulated (unshaded) cells. Dotted unshaded histograms demonstrate staining with the isotype-matched antibody control.
    Figure Legend Snippet: Expression of various receptors in mature dendritic cells (MDDCs) following 24 h of culture in the presence or absence of interleukin-2 (IL-2) (500 U/ml). Cells, from one representative donor, were subjected to fluorescence-activated cell sorter (FACS) analysis to detect the expression on the surface of virus receptors, CD3, IL-2 receptors, costimulatory molecules, and activation markers. Histograms illustrate the level of positive staining with a specific antibody in unstimulated (shaded) and IL-2-stimulated (unshaded) cells. Dotted unshaded histograms demonstrate staining with the isotype-matched antibody control.

    Techniques Used: Expressing, Fluorescence, FACS, Activation Assay, Staining

    Effects of interleukin-2 (IL-2) on human immunodeficiency virus-1 (HIV-1) replication in monocyte-derived macrophages (MDMs) and mature dendritic cells (MDDCs). Following a 7-day differentiation period, MDMs and MDDCs obtained from three different donors were acutely infected with primary HIV-1 isolates and were then maintained for 12–14 days in the presence or absence of IL-2 (100 U/ml). Levels of virus p24 protein were evaluated in the supernatants of cultures infected with M-tropic CHR4 (a), dual-tropic CHR1 (b), and T-tropic D2 (c) strains. Different symbols reflect experiments on cells from different donors and horizontal bars represent the means.
    Figure Legend Snippet: Effects of interleukin-2 (IL-2) on human immunodeficiency virus-1 (HIV-1) replication in monocyte-derived macrophages (MDMs) and mature dendritic cells (MDDCs). Following a 7-day differentiation period, MDMs and MDDCs obtained from three different donors were acutely infected with primary HIV-1 isolates and were then maintained for 12–14 days in the presence or absence of IL-2 (100 U/ml). Levels of virus p24 protein were evaluated in the supernatants of cultures infected with M-tropic CHR4 (a), dual-tropic CHR1 (b), and T-tropic D2 (c) strains. Different symbols reflect experiments on cells from different donors and horizontal bars represent the means.

    Techniques Used: Derivative Assay, Infection

    Levels of secreted chemokines and cytokines in human immunodeficiency virus-1 (HIV-1)-infected monocyte-derived macrophage (MDM) and mature dendritic cell (MDDC) cultures that were stimulated or unstimulated with 100 U/ml of interleukin (IL)-2. Supernatants were collected after 24 and 48 h of stimulation and evaluated for the content of macrophage inflammatory protein (MIP)-1β, MIP-1α, regulated on activation, normal, T-cell expressed, and secreted (RANTES), IL-6, IL-10 and tumour necrosis factor-α (TNF-α), using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Results represent the mean values ± standard error of the mean (s.e.m.) of five independent experiments with MDMs (a) and of four experiments with MDDCs (b).
    Figure Legend Snippet: Levels of secreted chemokines and cytokines in human immunodeficiency virus-1 (HIV-1)-infected monocyte-derived macrophage (MDM) and mature dendritic cell (MDDC) cultures that were stimulated or unstimulated with 100 U/ml of interleukin (IL)-2. Supernatants were collected after 24 and 48 h of stimulation and evaluated for the content of macrophage inflammatory protein (MIP)-1β, MIP-1α, regulated on activation, normal, T-cell expressed, and secreted (RANTES), IL-6, IL-10 and tumour necrosis factor-α (TNF-α), using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Results represent the mean values ± standard error of the mean (s.e.m.) of five independent experiments with MDMs (a) and of four experiments with MDDCs (b).

    Techniques Used: Infection, Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting"

    Article Title: Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2010.03312.x

    Morphology (a, b), survival (c, d) and proliferation (e, f) comparison of negatively (a, c, e) and positively (b, d, f) selected natural killer (NK) cells from LEWIS rats after 4 days in culture with IL-2. Forward-scatter and side-scatter dot plots represent
    Figure Legend Snippet: Morphology (a, b), survival (c, d) and proliferation (e, f) comparison of negatively (a, c, e) and positively (b, d, f) selected natural killer (NK) cells from LEWIS rats after 4 days in culture with IL-2. Forward-scatter and side-scatter dot plots represent

    Techniques Used:

    37) Product Images from "Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity"

    Article Title: Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02187-8

    Phosphorylation of Ezh2 by Akt dissociates Ezh2 from the promoter regions of major TF loci . a WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2. Immunoblot analysis of Pmel-1 cells stimulated in vitro for 3 days, 5 days, and 7 days, probed with Abs against Ezh2 and phosphorylated Ezh2 S21. Unstimulated T N were used as control. b – d Immunoblot analysis of Pmel-1 CD8 + T cells stimulated in vitro for 7 days, with or without treatment of MK2206, probed with indicated Abs. e – g WT Pmel-1 cells were cultured with anti-CD3/CD28 Ab + IL-2, with or without treatment of PI103, MK2206 or rapamycin for 7 days. Real-time RT-PCR analysis of Ezh2-targeted genes ( e ). ChIP analysis of cultured Pmel-1 cells treated with PI103, or MK2206. Graphs show the deposition of Ezh2 ( f ) and H3K27me3 ( g ) at the promoter regions of Id3 , Id2 , Prdm1 , and Eomes . h – k WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab for 36 h, followed by infection with MigR1 retrovirus (GFP) or MigR1 retrovirus encoding flag-tagged Ezh2, or Ezh2 S21A . Pmel-1 cells were collected at 7 days after culture. Immunoblot analysis of GFP and Ezh2 S21A Pmel-1 cells, probed with indicated Abs ( h ). Real-time RT-PCR analysis of their expression of major TFs ( i ). ChIP analysis shows the deposition of Ezh2 ( j ) and H3K27me3 ( k ) at the promoter regions of these major TFs loci. * p
    Figure Legend Snippet: Phosphorylation of Ezh2 by Akt dissociates Ezh2 from the promoter regions of major TF loci . a WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2. Immunoblot analysis of Pmel-1 cells stimulated in vitro for 3 days, 5 days, and 7 days, probed with Abs against Ezh2 and phosphorylated Ezh2 S21. Unstimulated T N were used as control. b – d Immunoblot analysis of Pmel-1 CD8 + T cells stimulated in vitro for 7 days, with or without treatment of MK2206, probed with indicated Abs. e – g WT Pmel-1 cells were cultured with anti-CD3/CD28 Ab + IL-2, with or without treatment of PI103, MK2206 or rapamycin for 7 days. Real-time RT-PCR analysis of Ezh2-targeted genes ( e ). ChIP analysis of cultured Pmel-1 cells treated with PI103, or MK2206. Graphs show the deposition of Ezh2 ( f ) and H3K27me3 ( g ) at the promoter regions of Id3 , Id2 , Prdm1 , and Eomes . h – k WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab for 36 h, followed by infection with MigR1 retrovirus (GFP) or MigR1 retrovirus encoding flag-tagged Ezh2, or Ezh2 S21A . Pmel-1 cells were collected at 7 days after culture. Immunoblot analysis of GFP and Ezh2 S21A Pmel-1 cells, probed with indicated Abs ( h ). Real-time RT-PCR analysis of their expression of major TFs ( i ). ChIP analysis shows the deposition of Ezh2 ( j ) and H3K27me3 ( k ) at the promoter regions of these major TFs loci. * p

    Techniques Used: In Vitro, Cell Culture, Quantitative RT-PCR, Chromatin Immunoprecipitation, Infection, Expressing

    Ezh2 is dissociated from the regulatory regions of key TFs during CD8 + T-cell expansion . WT naive Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2 for 7 days. Cells were collected at 0 days, 3 days and 7 days. a , b Immunoblot analysis of Pmel-1 cells before and after TCR-activation, probed with anti-Ezh2 Ab ( a ) and anti-H3K27me3 Ab ( b ). c Real-time RT-PCR analysis of gene expression in Pmel-1 cells before and after activation at indicated time points. d Tumor size in B16 tumor-bearing B6 mice receiving no transfer (Non-T cells), transfer of WT naive Pmel-1 cells (T N ) or in vitro TCR-activated 7 days Pmel-1 cells and treatment with IL-2 and gp100-DCs for 3 days. e – g ChIP analysis of T N ( e ), 3 days and 7 days Pmel-1 cells ( f ), and T N and 7 days Pmel-1 cells ( g ). * p
    Figure Legend Snippet: Ezh2 is dissociated from the regulatory regions of key TFs during CD8 + T-cell expansion . WT naive Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2 for 7 days. Cells were collected at 0 days, 3 days and 7 days. a , b Immunoblot analysis of Pmel-1 cells before and after TCR-activation, probed with anti-Ezh2 Ab ( a ) and anti-H3K27me3 Ab ( b ). c Real-time RT-PCR analysis of gene expression in Pmel-1 cells before and after activation at indicated time points. d Tumor size in B16 tumor-bearing B6 mice receiving no transfer (Non-T cells), transfer of WT naive Pmel-1 cells (T N ) or in vitro TCR-activated 7 days Pmel-1 cells and treatment with IL-2 and gp100-DCs for 3 days. e – g ChIP analysis of T N ( e ), 3 days and 7 days Pmel-1 cells ( f ), and T N and 7 days Pmel-1 cells ( g ). * p

    Techniques Used: Activation Assay, Quantitative RT-PCR, Expressing, Mouse Assay, In Vitro, Chromatin Immunoprecipitation

    Ezh2 orchestrates the expression of genes critical for effector differentiation and memory formation . a – c WT and Ezh2 − / − Pmel-1 cells were cultured in the presence of anti-CD3/CD28 Abs and IL-2 for 3 days. Cells were collected to extract RNA for sequencing. Using one-way ANOVA analysis, we selected transcripts with p
    Figure Legend Snippet: Ezh2 orchestrates the expression of genes critical for effector differentiation and memory formation . a – c WT and Ezh2 − / − Pmel-1 cells were cultured in the presence of anti-CD3/CD28 Abs and IL-2 for 3 days. Cells were collected to extract RNA for sequencing. Using one-way ANOVA analysis, we selected transcripts with p

    Techniques Used: Expressing, Cell Culture, Sequencing

    Inhibiting Akt-mediated phosphorylation of Ezh2 enhances the generation of T CMP . Ezh2 −/− Pmel-1 cells (Thy1.1 + ) were stimulated with anti-CD3/CD28 Ab + IL-2 for 36 h, followed by infection with MigR1 retrovirus encoding GFP, Ezh2, Ezh2 S21D and Ezh2 S21A , respectively . At 7 days, after stimulation, these infected T cells were collected. a Experiment scheme. b Immunoblot analysis of Pmel-1 cells transduced with GFP, Ezh2, Ezh2 S21D and Ezh2 S21A , probed with Flag, H3K27me3 and H3. c RT-PCR analysis of gene expression in Pmel-1 cells transduced by indicated genes. d , e These 7 day-Pmel-1 cells (Thy1.1 + ) were transferred into sublethally irradiated B6 mice (Thy1.2 + ), followed by treatment with IL-2 and gp100-DCs for 3d after transfer. d Donor T cells were collected from the spleen, PB and LN at 6d after adoptive transfer, and measured by flow cytometric analysis. e Plots and graphs show the percentage of of Id3 hi , KLRG1 hi , CD62L + and IFN-γ + within donor T cells from the spleen. * p
    Figure Legend Snippet: Inhibiting Akt-mediated phosphorylation of Ezh2 enhances the generation of T CMP . Ezh2 −/− Pmel-1 cells (Thy1.1 + ) were stimulated with anti-CD3/CD28 Ab + IL-2 for 36 h, followed by infection with MigR1 retrovirus encoding GFP, Ezh2, Ezh2 S21D and Ezh2 S21A , respectively . At 7 days, after stimulation, these infected T cells were collected. a Experiment scheme. b Immunoblot analysis of Pmel-1 cells transduced with GFP, Ezh2, Ezh2 S21D and Ezh2 S21A , probed with Flag, H3K27me3 and H3. c RT-PCR analysis of gene expression in Pmel-1 cells transduced by indicated genes. d , e These 7 day-Pmel-1 cells (Thy1.1 + ) were transferred into sublethally irradiated B6 mice (Thy1.2 + ), followed by treatment with IL-2 and gp100-DCs for 3d after transfer. d Donor T cells were collected from the spleen, PB and LN at 6d after adoptive transfer, and measured by flow cytometric analysis. e Plots and graphs show the percentage of of Id3 hi , KLRG1 hi , CD62L + and IFN-γ + within donor T cells from the spleen. * p

    Techniques Used: Infection, Transduction, Reverse Transcription Polymerase Chain Reaction, Expressing, Irradiation, Mouse Assay, Adoptive Transfer Assay, Flow Cytometry

    Ezh2 helps establish memory properties in activated CD8 + T cells early during expansion . a Schematic diagram of three characteristic phases of the T-cell response and the possible role of Ezh2 in each phase. b – g WT and Ezh2 − / − naive Pmel-1 cells (Thy1.1 + ) were transferred into sublethally irradiated non-tumor-bearing B6 mice (Thy1.2 + ), followed by immediate treatment with IL-2 and gp100-DCs for 3 days. Donor T cells were recovered at 4 days and 7 days after transfer. Plots and graphs show the percentage of KLRG-1-expressing (KLRG1 hi ) T CMP and T EFF in the spleen ( b ) and PB ( c ). d Graphs show the percentage and numbers of T CMP and T EFF (left panel) in the spleen at 4 days and 7 days after transfer. The right panel shows the percentage and numbers of MPCs and SLECs measured with KLRG-1 and CD127 at 4 days and 7 days after transfer. e The percentage of Annexin V-positive cells in the subpopulation of T CMP and T EFF at 4 days and 7 days after transfer. f Real-time RT-PCR measurement of p19 Arf in the subset of T CMP and T EFF of 4 days and 7 days. g Histograms show the expression of indicated surface markers on WT and Ezh2 − / − T cells derived from the spleen at 4d after transfer. h , i WT and Ezh2 − / − naive Pmel-1 cells (Thy1.1 + ) were transferred into sublethally irradiated non-tumor-bearing B6 mice (Thy1.2 + ), followed by immediate treatment with IL-2 and gp100-DCs for 3 days. By 7 days after transfer, donor T CMP and T EFF were highly purified using FACS sorter, and transferred into sublethally irradiated secondary recipients that had been immunized with gp100-DCs 7 days earlier (described in Supplementary Fig.4). Forty-two days later, donor T cells were collected from the spleen of these secondary recipients ( h ), and further cultured ex vivo for additional 5 days ( i ). Plots and graphs show the frequency of donor T cells derived from the secondary recipients of WT and Ezh2 − / − T CMP and T EFF . * p
    Figure Legend Snippet: Ezh2 helps establish memory properties in activated CD8 + T cells early during expansion . a Schematic diagram of three characteristic phases of the T-cell response and the possible role of Ezh2 in each phase. b – g WT and Ezh2 − / − naive Pmel-1 cells (Thy1.1 + ) were transferred into sublethally irradiated non-tumor-bearing B6 mice (Thy1.2 + ), followed by immediate treatment with IL-2 and gp100-DCs for 3 days. Donor T cells were recovered at 4 days and 7 days after transfer. Plots and graphs show the percentage of KLRG-1-expressing (KLRG1 hi ) T CMP and T EFF in the spleen ( b ) and PB ( c ). d Graphs show the percentage and numbers of T CMP and T EFF (left panel) in the spleen at 4 days and 7 days after transfer. The right panel shows the percentage and numbers of MPCs and SLECs measured with KLRG-1 and CD127 at 4 days and 7 days after transfer. e The percentage of Annexin V-positive cells in the subpopulation of T CMP and T EFF at 4 days and 7 days after transfer. f Real-time RT-PCR measurement of p19 Arf in the subset of T CMP and T EFF of 4 days and 7 days. g Histograms show the expression of indicated surface markers on WT and Ezh2 − / − T cells derived from the spleen at 4d after transfer. h , i WT and Ezh2 − / − naive Pmel-1 cells (Thy1.1 + ) were transferred into sublethally irradiated non-tumor-bearing B6 mice (Thy1.2 + ), followed by immediate treatment with IL-2 and gp100-DCs for 3 days. By 7 days after transfer, donor T CMP and T EFF were highly purified using FACS sorter, and transferred into sublethally irradiated secondary recipients that had been immunized with gp100-DCs 7 days earlier (described in Supplementary Fig.4). Forty-two days later, donor T cells were collected from the spleen of these secondary recipients ( h ), and further cultured ex vivo for additional 5 days ( i ). Plots and graphs show the frequency of donor T cells derived from the secondary recipients of WT and Ezh2 − / − T CMP and T EFF . * p

    Techniques Used: Irradiation, Mouse Assay, Expressing, Quantitative RT-PCR, Derivative Assay, Purification, FACS, Cell Culture, Ex Vivo

    Ezh2 is required for CD8 + T cells to control tumor growth and promote memory precursor formation . a WT and Ezh2 − / − naive Pmel-1 cells (1 × 10 6 , Thy1.1 + ) were transferred into sublethally irradiated (5 Gy) B6 mice (Thy1.2 + ) that had pre-established B16 melanoma, followed by treatment with IL-2 (1 × 10 5 IU per injection, i.p., twice a day) and gp100-pulsed DCs (gp100-DCs, 1 × 10 6 per mouse, i.p.) for 3 days. Tumor size was monitored over time. b – f WT and Ezh2 − / − T N Pmel-1 cells (1 × 10 6 , Thy1.1 + ) were transferred into sublethally irradiated non-tumor-bearing B6 mice, followed by immunization with IL-2 and gp100-DCs for 3d. b Donor T cells were collected from the spleen 4 days, 7 days, and 35 days after adoptive transfer. Plots and graphs show the frequency and numbers of donor T cells. c Percentage of IFN-γ-producing donor T cells in the spleen. d Donor T cells were collected from the spleen of WT and Ezh2 − / − Pmel-1 cell primary recipients 42 days after transfer, and separately transferred into sublethally irradiated secondary non-tumor-bearing B6 mice (4 × 10 4 cells per mouse), followed by treatment with IL-2 and gp100-DCs at 42 days, 43 days, and 44 days. By 49 days, donor T cells were collected from the spleen of the secondary mice. Plots and graph show the percentage of donor Pmel-1 cells. e Donor T cells derived from these secondary mice were activated with anti-CD3 Ab for 5 hrs to measure their production of IFN-γ. Graph shows the number of IFN-γ + Pmel-1 cells in the spleen. f Donor T cells collected at 49d from the secondary mice were cultured ex vivo with IL-7 + IL-15 in the presence or absence of gp100 for additional 5 days. Plots and graphs show the percentage of donor T cells in cultures. * p
    Figure Legend Snippet: Ezh2 is required for CD8 + T cells to control tumor growth and promote memory precursor formation . a WT and Ezh2 − / − naive Pmel-1 cells (1 × 10 6 , Thy1.1 + ) were transferred into sublethally irradiated (5 Gy) B6 mice (Thy1.2 + ) that had pre-established B16 melanoma, followed by treatment with IL-2 (1 × 10 5 IU per injection, i.p., twice a day) and gp100-pulsed DCs (gp100-DCs, 1 × 10 6 per mouse, i.p.) for 3 days. Tumor size was monitored over time. b – f WT and Ezh2 − / − T N Pmel-1 cells (1 × 10 6 , Thy1.1 + ) were transferred into sublethally irradiated non-tumor-bearing B6 mice, followed by immunization with IL-2 and gp100-DCs for 3d. b Donor T cells were collected from the spleen 4 days, 7 days, and 35 days after adoptive transfer. Plots and graphs show the frequency and numbers of donor T cells. c Percentage of IFN-γ-producing donor T cells in the spleen. d Donor T cells were collected from the spleen of WT and Ezh2 − / − Pmel-1 cell primary recipients 42 days after transfer, and separately transferred into sublethally irradiated secondary non-tumor-bearing B6 mice (4 × 10 4 cells per mouse), followed by treatment with IL-2 and gp100-DCs at 42 days, 43 days, and 44 days. By 49 days, donor T cells were collected from the spleen of the secondary mice. Plots and graph show the percentage of donor Pmel-1 cells. e Donor T cells derived from these secondary mice were activated with anti-CD3 Ab for 5 hrs to measure their production of IFN-γ. Graph shows the number of IFN-γ + Pmel-1 cells in the spleen. f Donor T cells collected at 49d from the secondary mice were cultured ex vivo with IL-7 + IL-15 in the presence or absence of gp100 for additional 5 days. Plots and graphs show the percentage of donor T cells in cultures. * p

    Techniques Used: Irradiation, Mouse Assay, Injection, Adoptive Transfer Assay, Derivative Assay, Cell Culture, Ex Vivo

    Id3 is a downstream effector of Akt-unphosphorylated Ezh2 in CD8 + T cells . a – c WT and Ezh2 −/− T N Pmel-1 cells (Thy1.1 + ) were activated and infected with MigR1 encoding GFP, Ezh2, and Id3, respectively. By 7 days, GFP + T cells were sorted and transferred into sublethally irradiated B6 mice (Thy1.2 + , 5 × 10 5 cells per mouse). IL-2 and gp100/DCs were administered to these mice for 3 days after the transfer. Donor T cells were collected from the spleen 35 days after transfer to measure their percentage and numbers ( a ), surface phenotype ( b ) and production of IFN-γ ( c ). Dot plots and graphs in c show the percent of IFN-γ + cells within donor Thy1.1 + T cells. d 3T3 cells were transduced with an Id3 -specific pGL3 luciferase reporter ( Id3-pGL ), together with MigR1-GFP, MigR1-Ezh2 S21A or MigR1-Ezh2 S21D . Id2 -specific pGL3 luciferase reporter ( Id2-pGL ) was used as control. Graph shows the fold change of luciferase reporter activity. The data (mean±SD) are representatives of three independent experiments. e , f 3T3 cells were transduced with pL-CRISPR.EFS.GFP plasmid or pL-CRISPR.EFS.GFP plasmid encoding guide RNA targeting Ezh2. 3 days later, GFP + cells were sorted into single cell using FACS sorter to select Ezh2-knockout cell clones. Immunoloblots show the loss of Ezh2 in 3T3 cells with Ezh2 knockout ( e ). These Ezh2-null 3T3 cells were reconstituted with MigR1-GFP vector and MigR1 encoding Ezh2, Ezh2 S21A or Ezh2 H689A . Id3-pGL luciferase reporter was introduced to these cells for reporter assay. In some experiments, WT 3T3 cells were treated with DZNep to deplete Ezh2 protein. Graph shows the fold change of luciferase reporter activity ( f ). g WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2 for 7 days for ChIP analysis. Graphs show the deposition of H3K4me3, H3K27me3and Pol II at the promoter regions of Id3 and Id2. h WT and Ezh2 −/− T N Pmel-1 cells (Thy1.1 + ) were stimulated with anti-CD3/CD28 Ab + IL-2 for 7 days to perform ChIP analysis. i Graphic summary of Ezh2 effects on memory formation and function. * p
    Figure Legend Snippet: Id3 is a downstream effector of Akt-unphosphorylated Ezh2 in CD8 + T cells . a – c WT and Ezh2 −/− T N Pmel-1 cells (Thy1.1 + ) were activated and infected with MigR1 encoding GFP, Ezh2, and Id3, respectively. By 7 days, GFP + T cells were sorted and transferred into sublethally irradiated B6 mice (Thy1.2 + , 5 × 10 5 cells per mouse). IL-2 and gp100/DCs were administered to these mice for 3 days after the transfer. Donor T cells were collected from the spleen 35 days after transfer to measure their percentage and numbers ( a ), surface phenotype ( b ) and production of IFN-γ ( c ). Dot plots and graphs in c show the percent of IFN-γ + cells within donor Thy1.1 + T cells. d 3T3 cells were transduced with an Id3 -specific pGL3 luciferase reporter ( Id3-pGL ), together with MigR1-GFP, MigR1-Ezh2 S21A or MigR1-Ezh2 S21D . Id2 -specific pGL3 luciferase reporter ( Id2-pGL ) was used as control. Graph shows the fold change of luciferase reporter activity. The data (mean±SD) are representatives of three independent experiments. e , f 3T3 cells were transduced with pL-CRISPR.EFS.GFP plasmid or pL-CRISPR.EFS.GFP plasmid encoding guide RNA targeting Ezh2. 3 days later, GFP + cells were sorted into single cell using FACS sorter to select Ezh2-knockout cell clones. Immunoloblots show the loss of Ezh2 in 3T3 cells with Ezh2 knockout ( e ). These Ezh2-null 3T3 cells were reconstituted with MigR1-GFP vector and MigR1 encoding Ezh2, Ezh2 S21A or Ezh2 H689A . Id3-pGL luciferase reporter was introduced to these cells for reporter assay. In some experiments, WT 3T3 cells were treated with DZNep to deplete Ezh2 protein. Graph shows the fold change of luciferase reporter activity ( f ). g WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2 for 7 days for ChIP analysis. Graphs show the deposition of H3K4me3, H3K27me3and Pol II at the promoter regions of Id3 and Id2. h WT and Ezh2 −/− T N Pmel-1 cells (Thy1.1 + ) were stimulated with anti-CD3/CD28 Ab + IL-2 for 7 days to perform ChIP analysis. i Graphic summary of Ezh2 effects on memory formation and function. * p

    Techniques Used: Infection, Irradiation, Mouse Assay, Transduction, Luciferase, Activity Assay, CRISPR, Plasmid Preparation, FACS, Knock-Out, Clone Assay, Reporter Assay, Chromatin Immunoprecipitation

    Inhibiting Akt-mediated Ezh2 phosphorylation improves antitumor immunity . a , b Ex vivo-expanded 7 day-Pmel-1 cells (Thy1.1 + ), MK2206-treated 7 day-Pmel-1 cells (Thy1.1 + ), or 7 day-Pmel-1 cells infected with MigR1 retrovirus encoding Ezh2 S21A (Thy1.1 + , 5 × 10 5 T cells per mouse) were transferred into sublethally irradiated B6 mice that had pre-established B16 melanoma, followed by treatment with IL-2 and gp100-DCs from 0 to 3 days and repeated once more from 18 to 20 days. Tumor size was monitored over time ( a ). Some recipient mice were sacrificed at 9 days to examine the presence of donor Pmel-1 cells in the spleen using flow cytometric analysis. Graphs show the percentage of indicated total cells and cell subsets ( b ). c – h B6 mice with pre-established B16 melanoma cells received no adoptive transfer (Non-T cells), transfer of in vitro expanded 7 day-Pmel-1 cells infected with MigR1 encoding GFP, Ezh2 or Ezh2 S21A (5 × 10 5 T cells per mouse), followed by treatment with IL-2 and gp100-DCs from 0-3 days and repeated once more from 18 to 20 days. Tumor size was monitored over time ( c ). The percentage of total donor Pmel-1 cells ( d ) and KLRG1 hi Pmel-1 cells ( e ) in circulating PB from each group of mice receiving GFP-, Ezh2- and Ezh2 S21A -7 day-Pmel-1 cells over a period of 24 days after transfer. f – h In separate experiments as described in ( c – e ) above, donor T cells were recovered from the spleen and tumor at 21 days after transfer. f Plots show the percentage of transferred GFP + cells in tumor and spleen by gating on donor Thy1.1 + Pmel-1 CD8 + cells, with Pmel-1 cells prior to transfer as controls. g Graphs show the number of total donor GFP + Thy1.1 + Pmel-cells and IFN-γ + Pmel-1 cells in the spleen and tumor. h Plots show the expression of CD62L on the surface of donor GFP + Thy1.1 + T cells derived from the spleen and tumor. * p
    Figure Legend Snippet: Inhibiting Akt-mediated Ezh2 phosphorylation improves antitumor immunity . a , b Ex vivo-expanded 7 day-Pmel-1 cells (Thy1.1 + ), MK2206-treated 7 day-Pmel-1 cells (Thy1.1 + ), or 7 day-Pmel-1 cells infected with MigR1 retrovirus encoding Ezh2 S21A (Thy1.1 + , 5 × 10 5 T cells per mouse) were transferred into sublethally irradiated B6 mice that had pre-established B16 melanoma, followed by treatment with IL-2 and gp100-DCs from 0 to 3 days and repeated once more from 18 to 20 days. Tumor size was monitored over time ( a ). Some recipient mice were sacrificed at 9 days to examine the presence of donor Pmel-1 cells in the spleen using flow cytometric analysis. Graphs show the percentage of indicated total cells and cell subsets ( b ). c – h B6 mice with pre-established B16 melanoma cells received no adoptive transfer (Non-T cells), transfer of in vitro expanded 7 day-Pmel-1 cells infected with MigR1 encoding GFP, Ezh2 or Ezh2 S21A (5 × 10 5 T cells per mouse), followed by treatment with IL-2 and gp100-DCs from 0-3 days and repeated once more from 18 to 20 days. Tumor size was monitored over time ( c ). The percentage of total donor Pmel-1 cells ( d ) and KLRG1 hi Pmel-1 cells ( e ) in circulating PB from each group of mice receiving GFP-, Ezh2- and Ezh2 S21A -7 day-Pmel-1 cells over a period of 24 days after transfer. f – h In separate experiments as described in ( c – e ) above, donor T cells were recovered from the spleen and tumor at 21 days after transfer. f Plots show the percentage of transferred GFP + cells in tumor and spleen by gating on donor Thy1.1 + Pmel-1 CD8 + cells, with Pmel-1 cells prior to transfer as controls. g Graphs show the number of total donor GFP + Thy1.1 + Pmel-cells and IFN-γ + Pmel-1 cells in the spleen and tumor. h Plots show the expression of CD62L on the surface of donor GFP + Thy1.1 + T cells derived from the spleen and tumor. * p

    Techniques Used: Ex Vivo, Infection, Irradiation, Mouse Assay, Flow Cytometry, Adoptive Transfer Assay, In Vitro, Expressing, Derivative Assay

    38) Product Images from "Viral infection prevents diabetes by inducing regulatory T cells through NKT cell-plasmacytoid dendritic cell interplay"

    Article Title: Viral infection prevents diabetes by inducing regulatory T cells through NKT cell-plasmacytoid dendritic cell interplay

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20101692

    iNKT cells dampen the anti-islet CD8 + T cell response in infected Ins2 −/− NOD mice through pDCs, T reg cells, and TGF-β. (A–C) Analysis of CD8 + T cells specific for IGRP 206–214 (A–C) and NP 396–404 (A) in 5-wk-old Ins2 −/− mice 10 d after LCMV challenge (and treated or not) with α-GalCer. Pancreatic islets were harvested and cultured for 8 d in complete RPMI medium with recombinant IL-2. Cells were then stimulated for 4 h with IGRP 206–214 or NP 396–404 peptides and stained with anti–TCR-β, anti-CD8, and IFN-γ mAbs. Values correspond to the frequency of epitope-specific CD8 + T cells in the lymphoid gate. (B) Analysis of IGRP 206–214 -specific cells among CD8 + T population in pancreatic islets of Ins2 −/− mice treated with anti-CD25, 120G8, or anti–TGF-β mAbs. (C) For NRP-V7 (high affinity analogue of IGRP 206–214 tetramer staining), cells were stained with tetramers (NRP-17 and TUM control) and then surface stained with anti-CD8 mAb. Values correspond to the frequency of tetramer-specific CD8 + T cells in the lymphoid gate. Data are represented as mean values ± SD obtained in two (B and C) to six (A) independent experiments with three independent mice for each group. *, P
    Figure Legend Snippet: iNKT cells dampen the anti-islet CD8 + T cell response in infected Ins2 −/− NOD mice through pDCs, T reg cells, and TGF-β. (A–C) Analysis of CD8 + T cells specific for IGRP 206–214 (A–C) and NP 396–404 (A) in 5-wk-old Ins2 −/− mice 10 d after LCMV challenge (and treated or not) with α-GalCer. Pancreatic islets were harvested and cultured for 8 d in complete RPMI medium with recombinant IL-2. Cells were then stimulated for 4 h with IGRP 206–214 or NP 396–404 peptides and stained with anti–TCR-β, anti-CD8, and IFN-γ mAbs. Values correspond to the frequency of epitope-specific CD8 + T cells in the lymphoid gate. (B) Analysis of IGRP 206–214 -specific cells among CD8 + T population in pancreatic islets of Ins2 −/− mice treated with anti-CD25, 120G8, or anti–TGF-β mAbs. (C) For NRP-V7 (high affinity analogue of IGRP 206–214 tetramer staining), cells were stained with tetramers (NRP-17 and TUM control) and then surface stained with anti-CD8 mAb. Values correspond to the frequency of tetramer-specific CD8 + T cells in the lymphoid gate. Data are represented as mean values ± SD obtained in two (B and C) to six (A) independent experiments with three independent mice for each group. *, P

    Techniques Used: Infection, Mouse Assay, Cell Culture, Recombinant, Staining

    39) Product Images from "CD43 Expression Regulated by IL-12 Signaling Is Associated with Survival of CD8 T Cells"

    Article Title: CD43 Expression Regulated by IL-12 Signaling Is Associated with Survival of CD8 T Cells

    Journal: Immune Network

    doi: 10.4110/in.2010.10.5.153

    Attenuation of apoptosis in CD43 lo population. Spleen cell suspensions from naïve OT-I mice were cultured for 7 days with 100 nM OVAp in the presence of IL-12. Then, activated OT-I cells were MACS-purified and rested for 3 additional days without any stimulant and with IL-2. At the indicated time points, the cells were harvested and apoptotic cell death was determined by Annexin V and 7-AAD double-staining. The samples were assayed in triplicate and the error bars represent the SD values of the mean.
    Figure Legend Snippet: Attenuation of apoptosis in CD43 lo population. Spleen cell suspensions from naïve OT-I mice were cultured for 7 days with 100 nM OVAp in the presence of IL-12. Then, activated OT-I cells were MACS-purified and rested for 3 additional days without any stimulant and with IL-2. At the indicated time points, the cells were harvested and apoptotic cell death was determined by Annexin V and 7-AAD double-staining. The samples were assayed in triplicate and the error bars represent the SD values of the mean.

    Techniques Used: Mouse Assay, Cell Culture, Magnetic Cell Separation, Purification, Double Staining

    40) Product Images from "Cell generation dynamics underlying naive T-cell homeostasis in adult humans"

    Article Title: Cell generation dynamics underlying naive T-cell homeostasis in adult humans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000383

    Evidence for a link between replicative history and homeostatic proliferation potential controlled by NF-κB. (A) Memory/effector (green) and naive CD4 + T cells divided into CD31 − (red) and CD31 + (blue) cells were merged and their high-dimensional single-cell phenotypes analyzed by mass cytometry visualized using a tSNE dimensionality reduction. (B) DREVI plots depicting correlation between CD31 and p-SLP-76, p-ERK1/2, (C) p-STAT5, and (D) p-NF-κB throughout a 20-minute homeostatic stimulation with αCD3 and IL-7. Data are representative of four separate experiments on eight donors. (E) Proliferation of sort-purified CD4 + CD31 − (left) and CD4 + CD31 + (right) naive T cells in response to different stimulation conditions (blue histograms) as well as in the presence of the NF-κB inhibitor 6-amino-4–4-phenoxyphenylethylamino-quinazoline (red histograms). Data represent four experiments on eight donors. (F) Summary data from four independent donors showing percent CFSE low (proliferated) cells within the CD31 + and CD31 − fraction of naive CD4 + T cells relative to different stimulatory conditions. Cells were treated with vehicle (DMSO) or NF-κB inhibitor (“NFκBinh” 6-amino-4–4-phenoxyphenylethylamino-quinazoline) for each stimulation condition ( S3 Data ). Full Stim: αCD3/αCD28/IL-2. (G) Schematic summary of the dynamics of CD4 + naive T-cell homeostasis in adult humans. A.U., arbitrary units; CFSE, carboxyfluorescein succinimidyl ester; DREVI, conditional-density rescaled visualization; IL, interleukin; nd, no difference; NF-κB, nuclear factor κB; p-ERK1/2, phosphorylation of extracellular signal–regulated kinase 1/2; p-NF-κB, phosphorylation of NF-κB; p-SLP-76, phosphorylation of SH2 domain-containing leukocyte protein of 76 kilodaltons; p-STAT5, phosphorylation of signal transducer and activator of transcription 5; stim, stimulation; TNF, tumor necrosis factor; tSNE, t-stochastic neighborhood embedding.
    Figure Legend Snippet: Evidence for a link between replicative history and homeostatic proliferation potential controlled by NF-κB. (A) Memory/effector (green) and naive CD4 + T cells divided into CD31 − (red) and CD31 + (blue) cells were merged and their high-dimensional single-cell phenotypes analyzed by mass cytometry visualized using a tSNE dimensionality reduction. (B) DREVI plots depicting correlation between CD31 and p-SLP-76, p-ERK1/2, (C) p-STAT5, and (D) p-NF-κB throughout a 20-minute homeostatic stimulation with αCD3 and IL-7. Data are representative of four separate experiments on eight donors. (E) Proliferation of sort-purified CD4 + CD31 − (left) and CD4 + CD31 + (right) naive T cells in response to different stimulation conditions (blue histograms) as well as in the presence of the NF-κB inhibitor 6-amino-4–4-phenoxyphenylethylamino-quinazoline (red histograms). Data represent four experiments on eight donors. (F) Summary data from four independent donors showing percent CFSE low (proliferated) cells within the CD31 + and CD31 − fraction of naive CD4 + T cells relative to different stimulatory conditions. Cells were treated with vehicle (DMSO) or NF-κB inhibitor (“NFκBinh” 6-amino-4–4-phenoxyphenylethylamino-quinazoline) for each stimulation condition ( S3 Data ). Full Stim: αCD3/αCD28/IL-2. (G) Schematic summary of the dynamics of CD4 + naive T-cell homeostasis in adult humans. A.U., arbitrary units; CFSE, carboxyfluorescein succinimidyl ester; DREVI, conditional-density rescaled visualization; IL, interleukin; nd, no difference; NF-κB, nuclear factor κB; p-ERK1/2, phosphorylation of extracellular signal–regulated kinase 1/2; p-NF-κB, phosphorylation of NF-κB; p-SLP-76, phosphorylation of SH2 domain-containing leukocyte protein of 76 kilodaltons; p-STAT5, phosphorylation of signal transducer and activator of transcription 5; stim, stimulation; TNF, tumor necrosis factor; tSNE, t-stochastic neighborhood embedding.

    Techniques Used: Mass Cytometry, Purification

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    Incubation:

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    Recombinant:

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    Article Title: WSX1 Expression in Tumors Induces Immune Tolerance via Suppression of Effector Immune Cells
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    Article Title: ?? T Cells in Immunity Induced by Mycobacterium bovis Bacillus Calmette-Gu?rin Vaccination
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    Article Title: Focal adhesion kinase negatively regulates Lck function downstream of the T cell antigen receptor
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    Cell Culture:

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    R&D Systems human il 2
    Calcitriol response of TAGAP and IL2RA in CD4+ T cells is not influenced by MS or MS-risk genotype. CD4+ T cells from 8 RRMS patients (MS) and 10 healthy controls (HC) genotyped for MS-risk SNPs in TAGAP (rs1738074) and IL2RA (rs7090512) were cultivated for 7–8 days with αCD3/CD28 and <t>IL-2,</t> treated as described in Materials and Methods before harvesting after 6, 24 and 48 h, with 1,25(OH) 2 D 3 or vehicle control. The box plots with whiskers defining minimum and maximum show the log2 values of relative expression of indicted genes (relative to TBP ) in 1,25(OH) 2 D 3 -treated cells divided by relative expression in vehicle-treated cells, that is, log2 of fold induction by 1,25(OH) 2 D 3 at each time point for ( a ) MS patients compared with HCs and ( b ) the same samples sorted on IL2RA and TAGAP genotype, that is, samples carrying the minor allele ( IL2RA : N =10, MAF (G)=0.3480; TAGAP : N =11, MAF (G)=0.46563) compared with samples homozygous for major allele ( N =8 ( IL2RA ); N =7 ( TAGAP )). The horizontal lines within the boxes represent the median of the groups, and Mann–Whitney U -test was performed to compare the groups (MS vs HC, AA vs AG/GG). The horizontal line at 0 indicates no induction by 1,25(OH) 2 D 3 .
    Human Il 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Calcitriol response of TAGAP and IL2RA in CD4+ T cells is not influenced by MS or MS-risk genotype. CD4+ T cells from 8 RRMS patients (MS) and 10 healthy controls (HC) genotyped for MS-risk SNPs in TAGAP (rs1738074) and IL2RA (rs7090512) were cultivated for 7–8 days with αCD3/CD28 and IL-2, treated as described in Materials and Methods before harvesting after 6, 24 and 48 h, with 1,25(OH) 2 D 3 or vehicle control. The box plots with whiskers defining minimum and maximum show the log2 values of relative expression of indicted genes (relative to TBP ) in 1,25(OH) 2 D 3 -treated cells divided by relative expression in vehicle-treated cells, that is, log2 of fold induction by 1,25(OH) 2 D 3 at each time point for ( a ) MS patients compared with HCs and ( b ) the same samples sorted on IL2RA and TAGAP genotype, that is, samples carrying the minor allele ( IL2RA : N =10, MAF (G)=0.3480; TAGAP : N =11, MAF (G)=0.46563) compared with samples homozygous for major allele ( N =8 ( IL2RA ); N =7 ( TAGAP )). The horizontal lines within the boxes represent the median of the groups, and Mann–Whitney U -test was performed to compare the groups (MS vs HC, AA vs AG/GG). The horizontal line at 0 indicates no induction by 1,25(OH) 2 D 3 .

    Journal: Genes and Immunity

    Article Title: The multiple sclerosis susceptibility genes TAGAP and IL2RA are regulated by vitamin D in CD4+ T cells

    doi: 10.1038/gene.2015.61

    Figure Lengend Snippet: Calcitriol response of TAGAP and IL2RA in CD4+ T cells is not influenced by MS or MS-risk genotype. CD4+ T cells from 8 RRMS patients (MS) and 10 healthy controls (HC) genotyped for MS-risk SNPs in TAGAP (rs1738074) and IL2RA (rs7090512) were cultivated for 7–8 days with αCD3/CD28 and IL-2, treated as described in Materials and Methods before harvesting after 6, 24 and 48 h, with 1,25(OH) 2 D 3 or vehicle control. The box plots with whiskers defining minimum and maximum show the log2 values of relative expression of indicted genes (relative to TBP ) in 1,25(OH) 2 D 3 -treated cells divided by relative expression in vehicle-treated cells, that is, log2 of fold induction by 1,25(OH) 2 D 3 at each time point for ( a ) MS patients compared with HCs and ( b ) the same samples sorted on IL2RA and TAGAP genotype, that is, samples carrying the minor allele ( IL2RA : N =10, MAF (G)=0.3480; TAGAP : N =11, MAF (G)=0.46563) compared with samples homozygous for major allele ( N =8 ( IL2RA ); N =7 ( TAGAP )). The horizontal lines within the boxes represent the median of the groups, and Mann–Whitney U -test was performed to compare the groups (MS vs HC, AA vs AG/GG). The horizontal line at 0 indicates no induction by 1,25(OH) 2 D 3 .

    Article Snippet: Freshly purified CD4+ T cells were either frozen on liquid nitrogen for later use or resuspended in X-VIVO medium (Lonza) with human IL-2 (R & D Systems, Minneapolis, MS, USA) and αCD3/CD28 beads (1:1) (Human T-activator CD3/CD28 Dynabeads for cell expansion and activation, Life Technologies) for activation and 1,25(OH)2 D3 treatment.

    Techniques: Mass Spectrometry, Expressing, MANN-WHITNEY

    Requirements for type 1 cytokine polarization and the cellular origin of IFNγ. Panel A: Requirements for type 1 cytokine polarization in day 5 ATOC. Type 1 cytokine polarization was measured by IFNγ concentrations in the supernatants by ELISA assay. Results are expressed as the mean percent of IFNγ concentration (±1 SD) present in the cultures performed in the presence of 10 ng/ml IL-12, 10 ng/ml IL-2 and 25 μg/ml anti-IL-4 (‘standard conditions’). Data are from three to four independent trials. ∗IFNγ levels are significantly different from standard conditions ( p

    Journal: Journal of Autoimmunity

    Article Title: Type 1 cytokines polarize thymocytes during T cell development in adult thymus organ cultures

    doi: 10.1016/S0896-8411(02)00091-4

    Figure Lengend Snippet: Requirements for type 1 cytokine polarization and the cellular origin of IFNγ. Panel A: Requirements for type 1 cytokine polarization in day 5 ATOC. Type 1 cytokine polarization was measured by IFNγ concentrations in the supernatants by ELISA assay. Results are expressed as the mean percent of IFNγ concentration (±1 SD) present in the cultures performed in the presence of 10 ng/ml IL-12, 10 ng/ml IL-2 and 25 μg/ml anti-IL-4 (‘standard conditions’). Data are from three to four independent trials. ∗IFNγ levels are significantly different from standard conditions ( p

    Article Snippet: Recombinant mouse IL-12, human IL-2, rat IFNγ, rat TNFα, and rat IL-10 were purchased from R & D Systems (Minneapolis, MN).

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

    Cytokine secretion of peripheral blood mononuclear cells (PBMCs) stimulated by different concentrations of recombinant Staphylococcal enterotoxin A–epidermal growth factor (rSEA-EGF). After 24 hours in culture, the supernatants were harvested and IFN-γ, TNFα, and IL-2 concentration was measured by ELISA assay. The rSEA-EGF-stimulated human PBMCs were compared with the negative control (PBS, * P

    Journal: Technology in Cancer Research & Treatment

    Article Title: Construction, Expression, and Characterization of rSEA-EGF and In Vitro Evaluation of its Antitumor Activity Against Nasopharyngeal Cancer

    doi: 10.1177/1533033818762910

    Figure Lengend Snippet: Cytokine secretion of peripheral blood mononuclear cells (PBMCs) stimulated by different concentrations of recombinant Staphylococcal enterotoxin A–epidermal growth factor (rSEA-EGF). After 24 hours in culture, the supernatants were harvested and IFN-γ, TNFα, and IL-2 concentration was measured by ELISA assay. The rSEA-EGF-stimulated human PBMCs were compared with the negative control (PBS, * P

    Article Snippet: Human IL-2, IFN-γ, and TNF-α enzyme linked immunosorbent assay (ELISA) kits were purchased from R & D Systems (Minneapolis, Minnesota, USA). β-tubulin rabbit mAb and EGFR rabbit mAb were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

    Techniques: Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    Intravenous immunoglobulin (IVIg) interferes with phytohaemagglutinin (PHA) during primary T cell activation. Peripheral blood mononuclear cells (PBMC) were activated with 0·5 µg/ml of PHA in the presence of IVIg, IVIg–PHA (5 mg/ml) or F(ab') 2 (3·33 mg/ml) and cultured for 24 h prior to evaluation of interleukin (IL)-2 secretion by enzyme-linked immunosorbent assay (ELISA). Results shown are the mean [± standard deviation (s.d.)] of three independent experiments. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Neutralization of mitogenic lectins by intravenous immunoglobulin (IVIg) prevents T cell activation: does IVIg really have a direct effect on T cells?

    doi: 10.1111/j.1365-2249.2011.04476.x

    Figure Lengend Snippet: Intravenous immunoglobulin (IVIg) interferes with phytohaemagglutinin (PHA) during primary T cell activation. Peripheral blood mononuclear cells (PBMC) were activated with 0·5 µg/ml of PHA in the presence of IVIg, IVIg–PHA (5 mg/ml) or F(ab') 2 (3·33 mg/ml) and cultured for 24 h prior to evaluation of interleukin (IL)-2 secretion by enzyme-linked immunosorbent assay (ELISA). Results shown are the mean [± standard deviation (s.d.)] of three independent experiments. * P

    Article Snippet: After 24 h of culture, T cell function was assessed by measuring the amount of human interleukin (IL)-2 secreted in the culture supernatants by enzyme-linked immunosorbent assay (ELISA), using matched antibody pairs (R & D systems, Minneapolis, MN, USA).

    Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

    F(ab') 2 fragments are as efficient as intravenous immunoglobulin (IVIg) to interact with phytohaemagglutinin (PHA) and inhibit Jurkat T cell activation. (a) The PHA reactivity of equimolar amounts of F(ab') 2 fragments (33 µg/ml) was compared to that of IVIg (50 µg/ml) in enzyme-linked immunosorbent assay (ELISA). Results [± standard deviation (s.d.)] are representative of four separate experiments. n.s.: not significant (Mann–Whitney U -test). (b) Jurkat T cells were incubated in the presence of PHA and equimolar amounts of IVIg (5 mg/ml) or F(ab') 2 fragments (3·3 mg/ml) for 24 h prior to evaluation of interleukin (IL)-2 secretion by ELISA. Results shown are the mean (± s.d.) of four independent experiments. *** P

    Journal: Clinical and Experimental Immunology

    Article Title: Neutralization of mitogenic lectins by intravenous immunoglobulin (IVIg) prevents T cell activation: does IVIg really have a direct effect on T cells?

    doi: 10.1111/j.1365-2249.2011.04476.x

    Figure Lengend Snippet: F(ab') 2 fragments are as efficient as intravenous immunoglobulin (IVIg) to interact with phytohaemagglutinin (PHA) and inhibit Jurkat T cell activation. (a) The PHA reactivity of equimolar amounts of F(ab') 2 fragments (33 µg/ml) was compared to that of IVIg (50 µg/ml) in enzyme-linked immunosorbent assay (ELISA). Results [± standard deviation (s.d.)] are representative of four separate experiments. n.s.: not significant (Mann–Whitney U -test). (b) Jurkat T cells were incubated in the presence of PHA and equimolar amounts of IVIg (5 mg/ml) or F(ab') 2 fragments (3·3 mg/ml) for 24 h prior to evaluation of interleukin (IL)-2 secretion by ELISA. Results shown are the mean (± s.d.) of four independent experiments. *** P

    Article Snippet: After 24 h of culture, T cell function was assessed by measuring the amount of human interleukin (IL)-2 secreted in the culture supernatants by enzyme-linked immunosorbent assay (ELISA), using matched antibody pairs (R & D systems, Minneapolis, MN, USA).

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY, Incubation

    Intravenous immunoglobulin (IVIg)-treated Jurkat T cells are responsive to phytohaemagglutinin (PHA) activation. Jurkat T cells were incubated for 4 h in the presence of IVIg (10 mg/ml) (step 1), followed by three washes and PHA activation (0·5 µg/ml) in the presence or absence of 10 mg/ml of IVIg (step 2). Cells were incubated to 24 h prior to interleukin (IL)-2 determination by enzyme-linked immunosorbent assay (ELISA). Results [± standard deviation (s.d.)] are representative of five independent experiments. n.s.: not significant, ** P

    Journal: Clinical and Experimental Immunology

    Article Title: Neutralization of mitogenic lectins by intravenous immunoglobulin (IVIg) prevents T cell activation: does IVIg really have a direct effect on T cells?

    doi: 10.1111/j.1365-2249.2011.04476.x

    Figure Lengend Snippet: Intravenous immunoglobulin (IVIg)-treated Jurkat T cells are responsive to phytohaemagglutinin (PHA) activation. Jurkat T cells were incubated for 4 h in the presence of IVIg (10 mg/ml) (step 1), followed by three washes and PHA activation (0·5 µg/ml) in the presence or absence of 10 mg/ml of IVIg (step 2). Cells were incubated to 24 h prior to interleukin (IL)-2 determination by enzyme-linked immunosorbent assay (ELISA). Results [± standard deviation (s.d.)] are representative of five independent experiments. n.s.: not significant, ** P

    Article Snippet: After 24 h of culture, T cell function was assessed by measuring the amount of human interleukin (IL)-2 secreted in the culture supernatants by enzyme-linked immunosorbent assay (ELISA), using matched antibody pairs (R & D systems, Minneapolis, MN, USA).

    Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation