Structured Review

BioLegend annexin v staining
Apoptosis of leukocytes and monocytes after different 8-MOP/UVA treatments. Leukocytes were stained for the pan leukocyte marker CD45 and for <t>annexin</t> V as apoptosis marker and analyzed by flow cytometry (A). Monocytes were detected by CD14 staining (B). Percentages of annexin V positive cells out of all CD45 or CD14 cells are shown in the bar chart. Treatment conditions for Figs. 1 - 3 : Without treatment (ctrl.), with 30 minutes’ 8-MOP pre-incubation and subsequent washing step for 8-MOP removal before culturing (30 min pre-incubation + wash), with 8-MOP addition immediately before UVA irradiation and subsequent 8-MOP removal before culturing (no pre-incubation + wash) and with 8-MOP addition immediately before UVA irradiation without 8-MOP removal before culturing (no pre-incubation w/o wash). Apoptosis of untreated ctrl. samples after 1, 2 and 3 days in culture was always significantly lower ( p
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1) Product Images from "In vitro effects of different 8-methoxypsoralen treatment protocols for extracorporeal photopheresis on mononuclear cells"

Article Title: In vitro effects of different 8-methoxypsoralen treatment protocols for extracorporeal photopheresis on mononuclear cells

Journal: Central-European Journal of Immunology

doi: 10.5114/ceji.2017.67312

Apoptosis of leukocytes and monocytes after different 8-MOP/UVA treatments. Leukocytes were stained for the pan leukocyte marker CD45 and for annexin V as apoptosis marker and analyzed by flow cytometry (A). Monocytes were detected by CD14 staining (B). Percentages of annexin V positive cells out of all CD45 or CD14 cells are shown in the bar chart. Treatment conditions for Figs. 1 - 3 : Without treatment (ctrl.), with 30 minutes’ 8-MOP pre-incubation and subsequent washing step for 8-MOP removal before culturing (30 min pre-incubation + wash), with 8-MOP addition immediately before UVA irradiation and subsequent 8-MOP removal before culturing (no pre-incubation + wash) and with 8-MOP addition immediately before UVA irradiation without 8-MOP removal before culturing (no pre-incubation w/o wash). Apoptosis of untreated ctrl. samples after 1, 2 and 3 days in culture was always significantly lower ( p
Figure Legend Snippet: Apoptosis of leukocytes and monocytes after different 8-MOP/UVA treatments. Leukocytes were stained for the pan leukocyte marker CD45 and for annexin V as apoptosis marker and analyzed by flow cytometry (A). Monocytes were detected by CD14 staining (B). Percentages of annexin V positive cells out of all CD45 or CD14 cells are shown in the bar chart. Treatment conditions for Figs. 1 - 3 : Without treatment (ctrl.), with 30 minutes’ 8-MOP pre-incubation and subsequent washing step for 8-MOP removal before culturing (30 min pre-incubation + wash), with 8-MOP addition immediately before UVA irradiation and subsequent 8-MOP removal before culturing (no pre-incubation + wash) and with 8-MOP addition immediately before UVA irradiation without 8-MOP removal before culturing (no pre-incubation w/o wash). Apoptosis of untreated ctrl. samples after 1, 2 and 3 days in culture was always significantly lower ( p

Techniques Used: Staining, Marker, Flow Cytometry, Cytometry, Incubation, Irradiation

Apoptosis of B cells, NK cells and NKT cells after different 8-MOP/UVA treatments. B cells were identified with marker CD19 (A). NK cells were defined as CD56 positive and CD3 negative cells (B) and NKT cells as CD56 CD3 double-positive cells (C). Percentages of annexin V positive cells out of all cells from the corresponding cell type are shown in the bar charts. For treatment conditions, see legend of Fig. 1
Figure Legend Snippet: Apoptosis of B cells, NK cells and NKT cells after different 8-MOP/UVA treatments. B cells were identified with marker CD19 (A). NK cells were defined as CD56 positive and CD3 negative cells (B) and NKT cells as CD56 CD3 double-positive cells (C). Percentages of annexin V positive cells out of all cells from the corresponding cell type are shown in the bar charts. For treatment conditions, see legend of Fig. 1

Techniques Used: Marker

Apoptosis of T cells after different 8-MOP/UVA treatments. Leukocytes were stained with CD3 as pan T cell marker (A), CD4 as T helper cell marker (B), CD8 as cytotoxic T cell marker (C) and with CD4 CD25 co-staining as regulatory T cell marker (D). Percentages of annexin V positive cells out of all cells from the corresponding cell type are shown in the bar charts. For treatment conditions, see legend of Fig. 1
Figure Legend Snippet: Apoptosis of T cells after different 8-MOP/UVA treatments. Leukocytes were stained with CD3 as pan T cell marker (A), CD4 as T helper cell marker (B), CD8 as cytotoxic T cell marker (C) and with CD4 CD25 co-staining as regulatory T cell marker (D). Percentages of annexin V positive cells out of all cells from the corresponding cell type are shown in the bar charts. For treatment conditions, see legend of Fig. 1

Techniques Used: Staining, Marker

2) Product Images from "Distinct Differences on Neointima Formation in Immunodeficient and Humanized Mice after Carotid or Femoral Arterial Injury"

Article Title: Distinct Differences on Neointima Formation in Immunodeficient and Humanized Mice after Carotid or Femoral Arterial Injury

Journal: Scientific Reports

doi: 10.1038/srep35387

Presence of human PBMC-derived cells within injured carotid ( A–D ) and femoral ( E–H ) arteries in NSG mice 4 weeks after reconstitution with 5 × 10 6  human PBMCs. Sections were immunofluorescently stained with αSMA (in red) and specific antibodies (in green) recognizing human CD45 ( A,E ), human CD4 ( B,F ), human CD8 ( C,G ) and human CD68 ( D,H ). Representative images of arterial staining from 3 different mice per group. Specificity controls for the antibodies used are shown in  Figs S6 and S7A . Nuclei were stained with DAPI (in blue). Dotted line indicates the internal elastic lamina. Arrows indicate positively stained human leukocytes. Original magnification: 400x. Confocal microscopy ortho images hCD45/αSMA (I) or hCD8/αSMA ( J ) on wire-injured femoral arteries. Abbreviations:  a : adventitia;  m : media;  ni : neointima.
Figure Legend Snippet: Presence of human PBMC-derived cells within injured carotid ( A–D ) and femoral ( E–H ) arteries in NSG mice 4 weeks after reconstitution with 5 × 10 6 human PBMCs. Sections were immunofluorescently stained with αSMA (in red) and specific antibodies (in green) recognizing human CD45 ( A,E ), human CD4 ( B,F ), human CD8 ( C,G ) and human CD68 ( D,H ). Representative images of arterial staining from 3 different mice per group. Specificity controls for the antibodies used are shown in Figs S6 and S7A . Nuclei were stained with DAPI (in blue). Dotted line indicates the internal elastic lamina. Arrows indicate positively stained human leukocytes. Original magnification: 400x. Confocal microscopy ortho images hCD45/αSMA (I) or hCD8/αSMA ( J ) on wire-injured femoral arteries. Abbreviations: a : adventitia; m : media; ni : neointima.

Techniques Used: Derivative Assay, Mouse Assay, Staining, Confocal Microscopy

3) Product Images from "Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization"

Article Title: Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization

Journal: Scientific Reports

doi: 10.1038/srep24779

Impact of porcine liver matrices on T cell and B cell subpopulation proliferation and Th1/Th2 cytokine secretion in co-cultures in the presence of OKT3. ( a – f ) The proliferation patterns of T cells, B cells and their subsets were analyzed using anti-human CD3, CD8, CD4, HLA-DR and CD19 antibodies. ( g – k ) Th1 and Th2 cytokine levels for TNF-α, IFN-γ, IL-5, IL-6 and IL-10 are shown for co-cultures of PBMCs alone or in combination with protein extracts of the porcine matrix. Negative controls without any proteins from the liver materials in OKT3-coated wells are included. Na = native liver; De = decellularized; GP = genipin; GA = glutaraldehyde. *p
Figure Legend Snippet: Impact of porcine liver matrices on T cell and B cell subpopulation proliferation and Th1/Th2 cytokine secretion in co-cultures in the presence of OKT3. ( a – f ) The proliferation patterns of T cells, B cells and their subsets were analyzed using anti-human CD3, CD8, CD4, HLA-DR and CD19 antibodies. ( g – k ) Th1 and Th2 cytokine levels for TNF-α, IFN-γ, IL-5, IL-6 and IL-10 are shown for co-cultures of PBMCs alone or in combination with protein extracts of the porcine matrix. Negative controls without any proteins from the liver materials in OKT3-coated wells are included. Na = native liver; De = decellularized; GP = genipin; GA = glutaraldehyde. *p

Techniques Used:

4) Product Images from "Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis"

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis

Journal: Immunity

doi: 10.1016/j.immuni.2018.04.009

GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Quantitative RT-PCR, Staining, Injection

5) Product Images from "T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis"

Article Title: T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00944

Kinetics of T follicular helper (Tfh)-like cell in the secondary lymphoid organs (SLOs) during the different phases of experimental autoimmune encephalomyelitis (EAE). (A) The clinical course of MOG 35–55 -induced EAE in C57BL/6J mice. Mean clinical score is shown ( n = 8). (B) Representative flow cytometry results of CD3 + CD4 + CXCR5 + PD-1 + Tfh-like cells in the spleens of EAE mice during different clinical phases. Gates were set on CD3 + CD4 + cells. Numbers in the upright corner illustrated the percentage of CXCR5 + PD-1 + cells in CD4 + T cells. Comparisons of the frequency of Tfh cells in spleens [ (C) left] and draining lymph nodes [ (D) left] at different phases of EAE mice ( n = 8/time point). The correlation analysis between the EAE score and the frequency of Tfh-like cells in spleens [ (C) right] or draining lymph nodes [ (D) right]. (E) The serum level of IL-21 at different phases of EAE mice (left, n = 8/time point) and its correlation with EAE score (right). (F) Representative blots band of Bcl-6, CXCR5, and IL-21 in splenocytes of EAE mice at different phases. (G) Statistical data of the relative protein level of Bcl-6, CXCR5, and IL-21 in splenocytes of EAE mice at different phases. Values are mean ± SEM. For (C–E) , each data point represents an individual subject and the horizontal lines represent the means ( n = 8). Results are representative of three independent experiments. Abbreviations: Pre, pre-clinical; P, peak; R, remission; C, chronic. * P
Figure Legend Snippet: Kinetics of T follicular helper (Tfh)-like cell in the secondary lymphoid organs (SLOs) during the different phases of experimental autoimmune encephalomyelitis (EAE). (A) The clinical course of MOG 35–55 -induced EAE in C57BL/6J mice. Mean clinical score is shown ( n = 8). (B) Representative flow cytometry results of CD3 + CD4 + CXCR5 + PD-1 + Tfh-like cells in the spleens of EAE mice during different clinical phases. Gates were set on CD3 + CD4 + cells. Numbers in the upright corner illustrated the percentage of CXCR5 + PD-1 + cells in CD4 + T cells. Comparisons of the frequency of Tfh cells in spleens [ (C) left] and draining lymph nodes [ (D) left] at different phases of EAE mice ( n = 8/time point). The correlation analysis between the EAE score and the frequency of Tfh-like cells in spleens [ (C) right] or draining lymph nodes [ (D) right]. (E) The serum level of IL-21 at different phases of EAE mice (left, n = 8/time point) and its correlation with EAE score (right). (F) Representative blots band of Bcl-6, CXCR5, and IL-21 in splenocytes of EAE mice at different phases. (G) Statistical data of the relative protein level of Bcl-6, CXCR5, and IL-21 in splenocytes of EAE mice at different phases. Values are mean ± SEM. For (C–E) , each data point represents an individual subject and the horizontal lines represent the means ( n = 8). Results are representative of three independent experiments. Abbreviations: Pre, pre-clinical; P, peak; R, remission; C, chronic. * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

Increases in the percentages of circulating T follicular helper (Tfh)-like and B cells in relapsing-remitting MS (RR-MS) patients during the relapsing phase of the disease. Representative flow cytometry results of CD3 + CD4 + CXCR5 + PD-1 + Tfh-like cells (A) and B cells (B) in healthy controls (HCs), relapsing and remitting patients. Numbers in the upright corner illustrated the percentage of CXCR5 + PD-1 + cells in CD4 + T cells and the percentage of CD19 + B cells in lymphocytes. Comparisons of the frequencies of Tfh-like cells (C) and B cells (D) among HCs, relapsing patients and remitting patients. (E) Comparison of serum IL-21 level among HCs, relapsing patients, and remitting patients. (F) The correlation analysis between the frequency of Tfh-like cells and the frequency of B cells in patients with RR-MS. (G) The correlation analysis between the frequency of Tfh-like cells and serum IL-21 level in patients with RR-MS. (H) The correlation analysis between the frequency of B cells and serum IL-21 level in patients with RR-MS. Each data point represents an individual subject. The horizontal lines represent the mean values. HCs: n = 20; relapsing patients, n = 13; remitting patients, n = 15. * P
Figure Legend Snippet: Increases in the percentages of circulating T follicular helper (Tfh)-like and B cells in relapsing-remitting MS (RR-MS) patients during the relapsing phase of the disease. Representative flow cytometry results of CD3 + CD4 + CXCR5 + PD-1 + Tfh-like cells (A) and B cells (B) in healthy controls (HCs), relapsing and remitting patients. Numbers in the upright corner illustrated the percentage of CXCR5 + PD-1 + cells in CD4 + T cells and the percentage of CD19 + B cells in lymphocytes. Comparisons of the frequencies of Tfh-like cells (C) and B cells (D) among HCs, relapsing patients and remitting patients. (E) Comparison of serum IL-21 level among HCs, relapsing patients, and remitting patients. (F) The correlation analysis between the frequency of Tfh-like cells and the frequency of B cells in patients with RR-MS. (G) The correlation analysis between the frequency of Tfh-like cells and serum IL-21 level in patients with RR-MS. (H) The correlation analysis between the frequency of B cells and serum IL-21 level in patients with RR-MS. Each data point represents an individual subject. The horizontal lines represent the mean values. HCs: n = 20; relapsing patients, n = 13; remitting patients, n = 15. * P

Techniques Used: Mass Spectrometry, Flow Cytometry, Cytometry

6) Product Images from "LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation"

Article Title: LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1502462

CD161 is expressed on FDCs and on a subset of tonsillar T cells. ( A ) CD161 expression negatively correlated with CXCR5 expression on CD45RA − T cells (CD3 + CD4+) assessed by FACS ( n = 5, three independent experiments) and real-time PCR ( n = 3, two
Figure Legend Snippet: CD161 is expressed on FDCs and on a subset of tonsillar T cells. ( A ) CD161 expression negatively correlated with CXCR5 expression on CD45RA − T cells (CD3 + CD4+) assessed by FACS ( n = 5, three independent experiments) and real-time PCR ( n = 3, two

Techniques Used: Expressing, FACS, Real-time Polymerase Chain Reaction

7) Product Images from "Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice"

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

Journal: Diabetologia

doi: 10.1007/s00125-019-04974-y

Anti-insulin B cells repopulate pancreatic islets more rapidly than insulin-negative B cells after anti-CD20 treatment. Groups of VH125.hCD20/NOD mice, aged 6–8 weeks old, were injected with 2H7 anti-CD20 or IgG isotype control. Spleen, PLNs and pancreatic islets were analysed for insulin-positive and insulin-negative B cells at 8 weeks and 12 weeks post depletion by flow cytometry. ( a – f ) No. of cells from IgG control-treated (black circles) and 2H7-treated (grey squares) mice for insulin-negative B cells ( a – c ) and insulin-positive B cells ( d – f ) from spleen ( a , d ) PLNs ( b , e ) and islets ( c , f ). ( g – i ) Percentage of B cells repopulated at 8 and 12 weeks after treatment from spleen ( g ), PLNs ( h ) and islets ( i ) of mice shown in ( a – f ). Percentages were calculated as individual numbers from each 2H7-treated mouse / mean number from all IgG control antibody-treated mice. Horizontal lines represent the median value. Data represent three independent experiments. At 8 weeks, n = 7 (spleen), n = 7 (PLNs) and n = 9 (islets) for control IgG-treated mice and n = 10 (spleen), n = 9 (PLNs) and n = 12 (islets) for 2H7-treated mice. At 12 weeks, n = 8 (spleen), n = 6 (PLNs) and n = 7 (islets) for control IgG and n = 11 (spleen), n = 7 (PLNs) and n = 11 (islets) for 2H7-treated mice. * p
Figure Legend Snippet: Anti-insulin B cells repopulate pancreatic islets more rapidly than insulin-negative B cells after anti-CD20 treatment. Groups of VH125.hCD20/NOD mice, aged 6–8 weeks old, were injected with 2H7 anti-CD20 or IgG isotype control. Spleen, PLNs and pancreatic islets were analysed for insulin-positive and insulin-negative B cells at 8 weeks and 12 weeks post depletion by flow cytometry. ( a – f ) No. of cells from IgG control-treated (black circles) and 2H7-treated (grey squares) mice for insulin-negative B cells ( a – c ) and insulin-positive B cells ( d – f ) from spleen ( a , d ) PLNs ( b , e ) and islets ( c , f ). ( g – i ) Percentage of B cells repopulated at 8 and 12 weeks after treatment from spleen ( g ), PLNs ( h ) and islets ( i ) of mice shown in ( a – f ). Percentages were calculated as individual numbers from each 2H7-treated mouse / mean number from all IgG control antibody-treated mice. Horizontal lines represent the median value. Data represent three independent experiments. At 8 weeks, n = 7 (spleen), n = 7 (PLNs) and n = 9 (islets) for control IgG-treated mice and n = 10 (spleen), n = 9 (PLNs) and n = 12 (islets) for 2H7-treated mice. At 12 weeks, n = 8 (spleen), n = 6 (PLNs) and n = 7 (islets) for control IgG and n = 11 (spleen), n = 7 (PLNs) and n = 11 (islets) for 2H7-treated mice. * p

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry

CD138 int anti-insulin B cells are enriched in pancreatic islets after anti-CD20 treatment. Groups of 6- to 8-week-old VH125.hCD20/NOD mice were injected with 2H7 anti-CD20 or IgG isotype control. Groups of mice ( n = 2 or 3 per group) were pooled and insulin + B cells from pancreatic islets were analysed for four different populations based on CD138 expression: CD138 − (blue); CD138 int IgM + (orange); CD138 int IgM lo (grey) and CD138 hi IgM lo (red). ( a , b ) Representative flow plots showing gating on live CD3 − CD11b − CD11c − ( a ) and graph showing the overall percentages of the four different populations ( b ). ( c , d ) Representative flow plots showing insulin − CD19 + , insulin + CD19 + and insulin + CD19 − cells ( c ) and graph showing the overall percentages of these cells ( d ); 2H7 (black circles), IgG (grey circles). ( e ) Representative flow plots showing CD138 and IgM expression in insulin + CD19 + and insulin + CD19 − cells. ( f , g ) Graphs showing CD138 and IgM populations on insulin + CD19 + ( f ) and insulin + CD19 − cells ( g ) ( n = 5 groups for control IgG treatment; n = 4 groups for 2H7 treatment). Horizontal lines represent the median values. Data represent two independent experiments. * p
Figure Legend Snippet: CD138 int anti-insulin B cells are enriched in pancreatic islets after anti-CD20 treatment. Groups of 6- to 8-week-old VH125.hCD20/NOD mice were injected with 2H7 anti-CD20 or IgG isotype control. Groups of mice ( n = 2 or 3 per group) were pooled and insulin + B cells from pancreatic islets were analysed for four different populations based on CD138 expression: CD138 − (blue); CD138 int IgM + (orange); CD138 int IgM lo (grey) and CD138 hi IgM lo (red). ( a , b ) Representative flow plots showing gating on live CD3 − CD11b − CD11c − ( a ) and graph showing the overall percentages of the four different populations ( b ). ( c , d ) Representative flow plots showing insulin − CD19 + , insulin + CD19 + and insulin + CD19 − cells ( c ) and graph showing the overall percentages of these cells ( d ); 2H7 (black circles), IgG (grey circles). ( e ) Representative flow plots showing CD138 and IgM expression in insulin + CD19 + and insulin + CD19 − cells. ( f , g ) Graphs showing CD138 and IgM populations on insulin + CD19 + ( f ) and insulin + CD19 − cells ( g ) ( n = 5 groups for control IgG treatment; n = 4 groups for 2H7 treatment). Horizontal lines represent the median values. Data represent two independent experiments. * p

Techniques Used: Mouse Assay, Injection, Expressing, Flow Cytometry

Anti-insulin B cells successfully present antigen to insulin-specific CD8 T cells. ( a , b ) Anti-insulin B cells from spleens of hCD20/NOD (hCD20), VH125, VH125.hCD20/NOD (VH125.hCD20) and 125Tg mice analysed by flow cytometry using human insulin conjugated to FITC fluorochrome. Representative flow cytometric plots ( a ) and quantification of total frequency of insulin-positive B cells ( b ) in each transgenic strain are shown. n = 6 (hCD20/NOD); n = 13 (VH125); n = 15 (VH125.hCD20/NOD); n = 18 (125Tg). Cells were gated on live CD3 − CD19 + cells. ( c – e ) VH125.hCD20/NOD splenic B cells were labelled for compartments (follicular zone [CD23 hi CD21 lo ], marginal zone [CD23 lo CD21 hi ], transitional 2 cells [CD23 hi CD21 hi ]) and counterstained with insulin–FITC ( n = 8 mice). Representative flow cytometric plots of insulin + B cells in each compartment ( c ), quantification of the percentage of B cells ( d ) and quantification of absolute insulin + B cell numbers ( e ) are shown. ( f – i ) Anti-insulin B cells from spleens of groups ( n = 3–5 pooled) of VH125.hCD20/NOD mice were enriched using insulin–FITC labelling and FITC microbeads and stimulated with 5 mg/ml anti-CD40 for 24 h before co-culture with purified G9 Cα −/− insulin-specific CD8 T cells. CD8 T cells were then analysed for activation markers after 24 h. Positive insulin fractions after enrichment ranged from 25% to 45% ( n = 4 groups). Representative plots showing enrichment of insulin-binding B cells by anti-FITC microbeads, labelled with B220 (as B220-based enrichment) and insulin ( f ) and the percentage of CD69 ( g ) and IFN-γ ( h ) expression on live CD8 T cells after culture with insulin-positive and -negative fractions are shown. ( i ) Fold change in GMFI of co-stimulation markers CD80 and CD86 and MHC I on insulin-positive and -negative B cells (live CD19 + ) after co-culture. All data shown are representative of at least three independent experiments. Horizontal lines represent median values. * p
Figure Legend Snippet: Anti-insulin B cells successfully present antigen to insulin-specific CD8 T cells. ( a , b ) Anti-insulin B cells from spleens of hCD20/NOD (hCD20), VH125, VH125.hCD20/NOD (VH125.hCD20) and 125Tg mice analysed by flow cytometry using human insulin conjugated to FITC fluorochrome. Representative flow cytometric plots ( a ) and quantification of total frequency of insulin-positive B cells ( b ) in each transgenic strain are shown. n = 6 (hCD20/NOD); n = 13 (VH125); n = 15 (VH125.hCD20/NOD); n = 18 (125Tg). Cells were gated on live CD3 − CD19 + cells. ( c – e ) VH125.hCD20/NOD splenic B cells were labelled for compartments (follicular zone [CD23 hi CD21 lo ], marginal zone [CD23 lo CD21 hi ], transitional 2 cells [CD23 hi CD21 hi ]) and counterstained with insulin–FITC ( n = 8 mice). Representative flow cytometric plots of insulin + B cells in each compartment ( c ), quantification of the percentage of B cells ( d ) and quantification of absolute insulin + B cell numbers ( e ) are shown. ( f – i ) Anti-insulin B cells from spleens of groups ( n = 3–5 pooled) of VH125.hCD20/NOD mice were enriched using insulin–FITC labelling and FITC microbeads and stimulated with 5 mg/ml anti-CD40 for 24 h before co-culture with purified G9 Cα −/− insulin-specific CD8 T cells. CD8 T cells were then analysed for activation markers after 24 h. Positive insulin fractions after enrichment ranged from 25% to 45% ( n = 4 groups). Representative plots showing enrichment of insulin-binding B cells by anti-FITC microbeads, labelled with B220 (as B220-based enrichment) and insulin ( f ) and the percentage of CD69 ( g ) and IFN-γ ( h ) expression on live CD8 T cells after culture with insulin-positive and -negative fractions are shown. ( i ) Fold change in GMFI of co-stimulation markers CD80 and CD86 and MHC I on insulin-positive and -negative B cells (live CD19 + ) after co-culture. All data shown are representative of at least three independent experiments. Horizontal lines represent median values. * p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Transgenic Assay, Co-Culture Assay, Purification, Activation Assay, Binding Assay, Expressing

Anti-insulin B cells are selectively recruited to the pancreatic islets. PLNs and pancreatic islets were analysed for the frequency of insulin-positive B cells in VH125.hCD20/NOD mice aged 6–8 weeks ( n = 5 mice) and 18–20 weeks ( n = 8 mice [PLNs]; n = 12 mice [islets]). Cells were gated on live CD3 − CD19 + populations. ( a ) Flow cytometric plots showing insulin staining in spleen, PLNs and pancreatic islets and competitive binding assay with excess (20×) unlabelled insulin. ( b ) Numbers of insulin-positive B cells from mice aged 6–8 weeks and 18–20 weeks from both PLNs and pancreatic islets. ( c ) Ratio of insulin-positive to insulin-negative B cells in PLNs and pancreatic islets in mice of different ages, compared with spleen (pooled ages, n = 17 mice). Horizontal lines represent median values. Data are representative of at least two independent experiments. * p
Figure Legend Snippet: Anti-insulin B cells are selectively recruited to the pancreatic islets. PLNs and pancreatic islets were analysed for the frequency of insulin-positive B cells in VH125.hCD20/NOD mice aged 6–8 weeks ( n = 5 mice) and 18–20 weeks ( n = 8 mice [PLNs]; n = 12 mice [islets]). Cells were gated on live CD3 − CD19 + populations. ( a ) Flow cytometric plots showing insulin staining in spleen, PLNs and pancreatic islets and competitive binding assay with excess (20×) unlabelled insulin. ( b ) Numbers of insulin-positive B cells from mice aged 6–8 weeks and 18–20 weeks from both PLNs and pancreatic islets. ( c ) Ratio of insulin-positive to insulin-negative B cells in PLNs and pancreatic islets in mice of different ages, compared with spleen (pooled ages, n = 17 mice). Horizontal lines represent median values. Data are representative of at least two independent experiments. * p

Techniques Used: Mouse Assay, Flow Cytometry, Staining, Competitive Binding Assay

Anti-insulin islet B cells are enriched in the CD138 int subset. Pancreatic islets from groups ( n = 2 or 3) of NOD and VH125.hCD20/NOD (VH125.hCD20) mice were pooled and insulin + B cells were analysed by flow cytometry. ( a ) Representative flow plots showing insulin-positive B cells in spleen and islets, either stained at 4°C or 37°C, against CD19 expression in NOD and VH125.hCD20/NOD mice. ( b , c ) Line graphs represent percentages of insulin-positive B cells from spleens and islet that were detected by staining at 37°C in NOD ( b ) and VH125.hCD20/NOD mice ( c ) ( n = 4 groups). ( d ) Four different populations express different combinations of CD138 and IgD in NOD mice and IgM in VH125.hCD20/NOD mice: CD138 − IgD + /IgM + (blue); CD138 int IgD + /IgM + (orange); CD138 int IgD lo /IgM lo (grey) and CD138 hi IgD lo /IgM lo (red). ( e , f ) Percentage of insulin + CD19 + and insulin + CD19 − enrichment categorised by CD138 and IgM in VH125.hCD20 mice ( e ) and IgD subsets in NOD mice ( f ) ( n = 3 groups). * p
Figure Legend Snippet: Anti-insulin islet B cells are enriched in the CD138 int subset. Pancreatic islets from groups ( n = 2 or 3) of NOD and VH125.hCD20/NOD (VH125.hCD20) mice were pooled and insulin + B cells were analysed by flow cytometry. ( a ) Representative flow plots showing insulin-positive B cells in spleen and islets, either stained at 4°C or 37°C, against CD19 expression in NOD and VH125.hCD20/NOD mice. ( b , c ) Line graphs represent percentages of insulin-positive B cells from spleens and islet that were detected by staining at 37°C in NOD ( b ) and VH125.hCD20/NOD mice ( c ) ( n = 4 groups). ( d ) Four different populations express different combinations of CD138 and IgD in NOD mice and IgM in VH125.hCD20/NOD mice: CD138 − IgD + /IgM + (blue); CD138 int IgD + /IgM + (orange); CD138 int IgD lo /IgM lo (grey) and CD138 hi IgD lo /IgM lo (red). ( e , f ) Percentage of insulin + CD19 + and insulin + CD19 − enrichment categorised by CD138 and IgM in VH125.hCD20 mice ( e ) and IgD subsets in NOD mice ( f ) ( n = 3 groups). * p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Staining, Expressing

Established pancreatic islet B cell infiltration is enriched for CD138 but few of the cells express Blimp-1. Pancreatic islets from groups ( n = 2 or 3) of NOD and VH125.hCD20/NOD (VH125.hCD20) mice were pooled, and B cells were analysed by flow cytometry. ( a ) Representative flow plots to show islet B cells expressing IgD, IgM, CD11b and CD5 from NOD mice (black) and VH125.hCD20/NOD mice (grey), gated on live CD3 − CD19 + . ( b – d ) Gating on live CD3 − CD11b − CD11c − revealed four different populations based on CD138 and IgD/M expression: CD138 − (blue gate); CD138 int IgD/M + (orange gate); CD138 int IgD/M lo (grey gate) and CD138 hi IgD/M lo (red gate). Representative flow cytometry plots on splenocytes ( b ) and pancreatic islets ( c ) from NOD and VH125.hCD20/NOD mice; histograms reveal various surface markers and Blimp-1 transcription factor expression on each population. Overall percentages of different CD138 and IgD/M populations in NOD and VH125.hCD20/NOD mice are shown in ( d ). * p
Figure Legend Snippet: Established pancreatic islet B cell infiltration is enriched for CD138 but few of the cells express Blimp-1. Pancreatic islets from groups ( n = 2 or 3) of NOD and VH125.hCD20/NOD (VH125.hCD20) mice were pooled, and B cells were analysed by flow cytometry. ( a ) Representative flow plots to show islet B cells expressing IgD, IgM, CD11b and CD5 from NOD mice (black) and VH125.hCD20/NOD mice (grey), gated on live CD3 − CD19 + . ( b – d ) Gating on live CD3 − CD11b − CD11c − revealed four different populations based on CD138 and IgD/M expression: CD138 − (blue gate); CD138 int IgD/M + (orange gate); CD138 int IgD/M lo (grey gate) and CD138 hi IgD/M lo (red gate). Representative flow cytometry plots on splenocytes ( b ) and pancreatic islets ( c ) from NOD and VH125.hCD20/NOD mice; histograms reveal various surface markers and Blimp-1 transcription factor expression on each population. Overall percentages of different CD138 and IgD/M populations in NOD and VH125.hCD20/NOD mice are shown in ( d ). * p

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

8) Product Images from "Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis"

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis

Journal: Immunity

doi: 10.1016/j.immuni.2018.04.009

GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Quantitative RT-PCR, Staining, Injection

GM-CSF from Non-T Cells Is Crucial for the Initiation of Autoimmune Arthritis (A) Experimental design of adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− or Csf2 −/− Rag2 −/− mice. Arthritis scores of four groups (a–d) of mice were assessed 3 months after transfer of 1 × 10 6 CD4 + T cells. (B) Arthritis scores of the four groups mice (n = 15 or 16 each) shown in (A). Horizontal bars indicate the means. (C) Representative joint histology of the groups shown in (A). Scale bars indicate 200 μm. (D) Flow cytometry of splenic CD4 + T cells stained for intracellular IL-17 and GM-CSF or IFN-γ. (E) Proportion of IL-17-producing CD4 + T cells from individual mice as shown in (D). Vertical bars mean SD (n = 3). ∗∗ p
Figure Legend Snippet: GM-CSF from Non-T Cells Is Crucial for the Initiation of Autoimmune Arthritis (A) Experimental design of adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− or Csf2 −/− Rag2 −/− mice. Arthritis scores of four groups (a–d) of mice were assessed 3 months after transfer of 1 × 10 6 CD4 + T cells. (B) Arthritis scores of the four groups mice (n = 15 or 16 each) shown in (A). Horizontal bars indicate the means. (C) Representative joint histology of the groups shown in (A). Scale bars indicate 200 μm. (D) Flow cytometry of splenic CD4 + T cells stained for intracellular IL-17 and GM-CSF or IFN-γ. (E) Proportion of IL-17-producing CD4 + T cells from individual mice as shown in (D). Vertical bars mean SD (n = 3). ∗∗ p

Techniques Used: Adoptive Transfer Assay, Mouse Assay, Flow Cytometry, Cytometry, Staining

GM-CSF-Producing ILCs in Synovial Fluid of RA Patients (A) The presence of ILCs (defined as CD45 + CD3 − CD4 − CD8 − CD11b − CD11c − CD19 − CD56 − ) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right). (B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD. (C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom). (D) Gating strategies for GM-CSF + CD45 + lineage markers-negative (ILCs), GM-CSF + CD45 + CD3 − CD11b + (myeloid cells), and GM-CSF + CD45 + CD11b − CD3 + cells (T cells). (E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Synovial Fluid of RA Patients (A) The presence of ILCs (defined as CD45 + CD3 − CD4 − CD8 − CD11b − CD11c − CD19 − CD56 − ) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right). (B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD. (C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom). (D) Gating strategies for GM-CSF + CD45 + lineage markers-negative (ILCs), GM-CSF + CD45 + CD3 − CD11b + (myeloid cells), and GM-CSF + CD45 + CD11b − CD3 + cells (T cells). (E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing

Induction of GM-CSF in FLSs Stimulated with IL-17 (A) Quantitative RT-PCR analysis for the expression of designated genes in IL-17-stimulated FLSs. FLSs (2.5 × 10 4 ) were stimulated with 50 ng/mL rmIL-17 and harvested at the indicated time points. mRNA expression is presented relative to the expression of Hprt1 . (B) Quantitative RT-PCR analysis for the expression of designated genes in synoviocytes from Rag2 −/− mice with CD4 + T cell transfer. CD45 − Podoplanin + synoviocytes (3 × 10 4 ) were sorted from inflamed joints of Rag2 −/− mice 4 weeks after transfer of 1 × 10 6 SKG or Il17a −/− SKG CD4 + T cells. Vertical bars mean SD (n = 3). Data are representative of two independent experiments.
Figure Legend Snippet: Induction of GM-CSF in FLSs Stimulated with IL-17 (A) Quantitative RT-PCR analysis for the expression of designated genes in IL-17-stimulated FLSs. FLSs (2.5 × 10 4 ) were stimulated with 50 ng/mL rmIL-17 and harvested at the indicated time points. mRNA expression is presented relative to the expression of Hprt1 . (B) Quantitative RT-PCR analysis for the expression of designated genes in synoviocytes from Rag2 −/− mice with CD4 + T cell transfer. CD45 − Podoplanin + synoviocytes (3 × 10 4 ) were sorted from inflamed joints of Rag2 −/− mice 4 weeks after transfer of 1 × 10 6 SKG or Il17a −/− SKG CD4 + T cells. Vertical bars mean SD (n = 3). Data are representative of two independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

GM-CSF-Producing T Helper Cells Are Dispensable for GM-CSF-Dependent Autoimmune Arthritis Development (A) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from popliteal LNs or inflamed joints. (B) Proportion of cytokine-producing cells in CD4 + T cells from individual mice as shown in (A). Vertical bars mean SD (n = 3). (C) Arthritis scores assessed in individual SKG, Csf2 −/− SKG, or Il17a −/− SKG mice (n = 20 each) 3 months after single i.p. injection of 20 mg mannan. (D) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from inflamed joints of Il17a Cre R26R eYFP SKG mice. (E) Arthritis development after adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− mice (n = 17 each, SEM). The severity of arthritis was monitored every week after transfer of 1 × 10 6 CD4 + T cells. (F) Intracellular IL-17 and GM-CSF staining of CD4 + T cells from spleens and inflamed joints of Rag2 −/− mice with CD4 + T cells transfer as shown in (E). ∗ p
Figure Legend Snippet: GM-CSF-Producing T Helper Cells Are Dispensable for GM-CSF-Dependent Autoimmune Arthritis Development (A) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from popliteal LNs or inflamed joints. (B) Proportion of cytokine-producing cells in CD4 + T cells from individual mice as shown in (A). Vertical bars mean SD (n = 3). (C) Arthritis scores assessed in individual SKG, Csf2 −/− SKG, or Il17a −/− SKG mice (n = 20 each) 3 months after single i.p. injection of 20 mg mannan. (D) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from inflamed joints of Il17a Cre R26R eYFP SKG mice. (E) Arthritis development after adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− mice (n = 17 each, SEM). The severity of arthritis was monitored every week after transfer of 1 × 10 6 CD4 + T cells. (F) Intracellular IL-17 and GM-CSF staining of CD4 + T cells from spleens and inflamed joints of Rag2 −/− mice with CD4 + T cells transfer as shown in (E). ∗ p

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

9) Product Images from "Effect of Age on NK Cell Compartment in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors"

Article Title: Effect of Age on NK Cell Compartment in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02587

Expression of Natural Cytotoxicity Receptors (NCRs) on CD56 bright and CD56 dim NK cells. (A) Representative histograms for NKp30, NKp46, and NKp80 are shown (the shaded area of light gray, the CD56 dim cells, the shaded one of gray, the CD56 bright cells). Results were expressed as Median Fluorescence Intensity (MFI), measured in the total of cells. (B) Expression of activating receptors NKp30, NKp46, and NKp80 on CD56 bright and CD56 dim NK subsets from healthy individuals and TKI-treated CML patients, stratified according to age (middle-aged 35–65 years and old > 65 years). Number of donors: NKp30 and NKp80 middle-aged healthy n = 19, middle-aged CML n = 11, old healthy n = 23, and old CML n = 9; NKp46 middle-aged healthy n = 19, middle-aged CML n = 16, old healthy n = 23 and old CML n = 10. The results were expressed as median with interquartile range. P- values were determined comparing middle age with old and healthy with TKI-treated CML patients and were considered significant at p
Figure Legend Snippet: Expression of Natural Cytotoxicity Receptors (NCRs) on CD56 bright and CD56 dim NK cells. (A) Representative histograms for NKp30, NKp46, and NKp80 are shown (the shaded area of light gray, the CD56 dim cells, the shaded one of gray, the CD56 bright cells). Results were expressed as Median Fluorescence Intensity (MFI), measured in the total of cells. (B) Expression of activating receptors NKp30, NKp46, and NKp80 on CD56 bright and CD56 dim NK subsets from healthy individuals and TKI-treated CML patients, stratified according to age (middle-aged 35–65 years and old > 65 years). Number of donors: NKp30 and NKp80 middle-aged healthy n = 19, middle-aged CML n = 11, old healthy n = 23, and old CML n = 9; NKp46 middle-aged healthy n = 19, middle-aged CML n = 16, old healthy n = 23 and old CML n = 10. The results were expressed as median with interquartile range. P- values were determined comparing middle age with old and healthy with TKI-treated CML patients and were considered significant at p

Techniques Used: Expressing, Fluorescence

10) Product Images from "Late-stage tumors induce anemia and immunosuppressive extramedullary erythroid progenitor cells"

Article Title: Late-stage tumors induce anemia and immunosuppressive extramedullary erythroid progenitor cells

Journal: Nature medicine

doi: 10.1038/s41591-018-0205-5

CD45 + CD71 + TER119 + erythroid progenitor cells accumulate in tumor-bearing mice and exert immunosuppressive effects on CD8 + T cells a, The number (left) and frequency (right) of MDSCs (CD11b + Gr1 + ), erythroid progenitor cells (CD71 + TER119 + ), stromal cells (CD45 − TER119 − ), macrophages (F4/80 + ), CD4 + T, CD8 + T and B (B220 + ) cells in the spleens of C57BL/6 mice were quantified at different time points (0, 7, 14, 21 and 28 days) after LLC cell inoculation (n=4). b, The frequencies of MDSCs, CD71 + TER119 + cells and Tregs in the spleen 28 days after LLC inoculation were determined (n=4). c , Representative flow cytometry (left) and cumulative composite data (right) showing the proliferation of CFSE-labeled CD8 + T cells after co-culture with CD71 + TER119 + erythroid progenitor cells isolated from the spleens of tumor-bearing mice at different CD8 + T cell: erythroid progenitor cell ratios (n=4). d, Mature red blood cell (RBC) counts were measured in tumor-bearing mice at different time points (n=4 or 5) after LLC or B16F10 inoculation. Each point represents data from an individual mouse, and the data are representative of at least three independent experiments. e, The correlation between the total number of CD71 + TER119 + erythroid progenitor cells in the spleens of tumor-bearing mice and hemoglobin (HGB) concentration was analyzed by Pearson’s correlation coefficient. (n=16). f, Gating strategy: after excluding doublets or larger aggregates, DAPI-positive cells, which are likely membrane-permeable apoptotic cells, were excluded from further analysis. Next, very small events, likely nuclei or debris, were excluded. Finally, we selected TER119 + cells for further analysis. Representative flow cytometry and cumulative composite data show the frequency of CD45 + CD71 + cells within the TER119 + population in the spleens of tumor-bearing (21d), anemic(4d) and neonatal mice. Right panel: cumulative composite data show the total number of CD45 + CD71 + TER119 + cells in the spleens of tumor-bearing mice at the indicated days after tumor inoculation (n=5). g, Cumulative composite data show the frequencies of CD45 + CD71 + TER119 + EPCs, MDSCs and Tregs in the spleen of mice with advanced tumors (28 days after LLC inoculation)(left). The ratio of immunosuppressive cells, CD45 + CD71 + TER119 + EPCs, MDSCs or Treg cells, against CD8 + (middle) or CD4 + (right) T cells in the spleen of mice with advanced tumors (28 days after LLC inoculation) (n=4). h, CD45 + CD71 + TER119 + or CD45 − CD71 + TER119 + erythroid progenitor cells isolated from the spleens of tumor-bearing mice were co-cultured with sorted CD8 + T cells and the T cell ex vivo killing efficiency was determined after 6 h (n=5). i, 2×10 5 CFSE-labeled P14 CD8 + T cells were mixed with 2×10 6 sorted CD45 + CD71 + TER119 + or CD45 − CD71 + TER119 + cells. The mixed cells were then adoptively transferred into naïve B6 mice immediately (n=8), which were subsequently infected with LCMV-Armstrong. These mice were sacrificed on day 3 post infection and CFSE high CD8 + T cells were analyzed by flow cytometry. j–k, A total of 2×10 5 B16F10-Ova melanoma cells were subcutaneously injected into C57BL/6 mice on day 0. Next, 2×10 6 CD45 + CD71 + TER119 + or CD45 − CD71 + TER119 + cells were intravenously injected on days 0 and 5. j , Tumor growth was monitored every 2 or 3 days ( left ). Mice were sacrificed on day 22and tumors were collected and weighed ( right ). Each point represents data from an individual mouse (CD45 + CD71 + TER119 + group n=3, CD45 − CD71 + TER119 + group n=5), and data were analyzed by two-tailed unpaired t-test. k, Tumor infiltrating leukocytes were enriched and loaded with the OVA 257–264 (SIINFEKL) peptide in vitro for 24-hour restimulation. Frequencies of IFN-γ and TNF-α producing T cells were analyzed by intracellular cytokine staining. Each point represents data from an individual mouse (n=3), and data were analyzed by two-tailed unpaired t-test. l–n , A total of 1×10 6 Lewis lung cancer cells were subcutaneously injected into C57BL/6 mice (PBS was used as control). Anti-CD71 antibody (1 mg/mouse) was intravenously injected at day 21 after tumor cell inoculation (IgG was used as control, 1 mg/mouse). To attenuate the anti-CD71 antibody, anti-IgG2a antibody (3 mg/mouse) was intravenously injected 24 h later. Finally, we adoptively transferred P14 CD8 + T cells (CD90.1, 2×10 6 cells/mouse) into mice and infected with LCMV cl13 simultaneously 36 h after administration of anti-CD71 antibody. All mice were sacrificed at day 2 after LCMV infection (l) . Representative flow cytometry ( m, left ) and cumulative composite data ( m, middle ) show the frequency of Ki67 + cells among P14 CD8 + T cells. Cumulative composite data show the Ki67 MFI in P14 CD8 + T cell ( m, right ). Cumulative composite data show the total number of CD90.1 + CD8 + P14 cells in the spleen (n). o–q, The hemoglobin (HGB)concentration ( o ) and number of CD45 + CD71 + TER119 + cells (p) in the peripheral blood of MMTV-PyMT female mice which developed palpable mammary tumors at 12 weeks old were determined at the indicated weeks. The proliferative capacity of CFSE-labeled CD8 + T cells in response to anti-CD3 and anti-CD28 was analyzed after co-culture with CD45 + CD71 + TER119 + EPCs isolated from the spleens of 20 week old MMTV-PyMT female mice at a CD8 + T cell/EPC ratio of 1:2 ( q ); CD45 + CD71 + TER119 + EPCs isolated from spleens of 20 week old MMTV-PyMT females mice were co-cultured with sorted CD8 + T cells and the ex vivo T cell killing efficiency was determined after 6 h ( r ). s, The proliferative capacity of CFSE-labeled CD8+ T cells in response to anti-CD3 and anti-CD28 was analyzed after co-culture with CD45 + CD71 + TER119 + erythroid progenitor cells isolated from the spleens of tumor-bearing, anemic or neonatal mice at the indicated CD8 + T cell:EPC ratios (n=5). Each point in (b–e) and (h–m) represents data from an individual mouse. Data are representative of three independent experiments and were analyzed by two-tailed unpaired t -test. Two-tailed p-values were reported. Bar graphs denote mean values with SEM.
Figure Legend Snippet: CD45 + CD71 + TER119 + erythroid progenitor cells accumulate in tumor-bearing mice and exert immunosuppressive effects on CD8 + T cells a, The number (left) and frequency (right) of MDSCs (CD11b + Gr1 + ), erythroid progenitor cells (CD71 + TER119 + ), stromal cells (CD45 − TER119 − ), macrophages (F4/80 + ), CD4 + T, CD8 + T and B (B220 + ) cells in the spleens of C57BL/6 mice were quantified at different time points (0, 7, 14, 21 and 28 days) after LLC cell inoculation (n=4). b, The frequencies of MDSCs, CD71 + TER119 + cells and Tregs in the spleen 28 days after LLC inoculation were determined (n=4). c , Representative flow cytometry (left) and cumulative composite data (right) showing the proliferation of CFSE-labeled CD8 + T cells after co-culture with CD71 + TER119 + erythroid progenitor cells isolated from the spleens of tumor-bearing mice at different CD8 + T cell: erythroid progenitor cell ratios (n=4). d, Mature red blood cell (RBC) counts were measured in tumor-bearing mice at different time points (n=4 or 5) after LLC or B16F10 inoculation. Each point represents data from an individual mouse, and the data are representative of at least three independent experiments. e, The correlation between the total number of CD71 + TER119 + erythroid progenitor cells in the spleens of tumor-bearing mice and hemoglobin (HGB) concentration was analyzed by Pearson’s correlation coefficient. (n=16). f, Gating strategy: after excluding doublets or larger aggregates, DAPI-positive cells, which are likely membrane-permeable apoptotic cells, were excluded from further analysis. Next, very small events, likely nuclei or debris, were excluded. Finally, we selected TER119 + cells for further analysis. Representative flow cytometry and cumulative composite data show the frequency of CD45 + CD71 + cells within the TER119 + population in the spleens of tumor-bearing (21d), anemic(4d) and neonatal mice. Right panel: cumulative composite data show the total number of CD45 + CD71 + TER119 + cells in the spleens of tumor-bearing mice at the indicated days after tumor inoculation (n=5). g, Cumulative composite data show the frequencies of CD45 + CD71 + TER119 + EPCs, MDSCs and Tregs in the spleen of mice with advanced tumors (28 days after LLC inoculation)(left). The ratio of immunosuppressive cells, CD45 + CD71 + TER119 + EPCs, MDSCs or Treg cells, against CD8 + (middle) or CD4 + (right) T cells in the spleen of mice with advanced tumors (28 days after LLC inoculation) (n=4). h, CD45 + CD71 + TER119 + or CD45 − CD71 + TER119 + erythroid progenitor cells isolated from the spleens of tumor-bearing mice were co-cultured with sorted CD8 + T cells and the T cell ex vivo killing efficiency was determined after 6 h (n=5). i, 2×10 5 CFSE-labeled P14 CD8 + T cells were mixed with 2×10 6 sorted CD45 + CD71 + TER119 + or CD45 − CD71 + TER119 + cells. The mixed cells were then adoptively transferred into naïve B6 mice immediately (n=8), which were subsequently infected with LCMV-Armstrong. These mice were sacrificed on day 3 post infection and CFSE high CD8 + T cells were analyzed by flow cytometry. j–k, A total of 2×10 5 B16F10-Ova melanoma cells were subcutaneously injected into C57BL/6 mice on day 0. Next, 2×10 6 CD45 + CD71 + TER119 + or CD45 − CD71 + TER119 + cells were intravenously injected on days 0 and 5. j , Tumor growth was monitored every 2 or 3 days ( left ). Mice were sacrificed on day 22and tumors were collected and weighed ( right ). Each point represents data from an individual mouse (CD45 + CD71 + TER119 + group n=3, CD45 − CD71 + TER119 + group n=5), and data were analyzed by two-tailed unpaired t-test. k, Tumor infiltrating leukocytes were enriched and loaded with the OVA 257–264 (SIINFEKL) peptide in vitro for 24-hour restimulation. Frequencies of IFN-γ and TNF-α producing T cells were analyzed by intracellular cytokine staining. Each point represents data from an individual mouse (n=3), and data were analyzed by two-tailed unpaired t-test. l–n , A total of 1×10 6 Lewis lung cancer cells were subcutaneously injected into C57BL/6 mice (PBS was used as control). Anti-CD71 antibody (1 mg/mouse) was intravenously injected at day 21 after tumor cell inoculation (IgG was used as control, 1 mg/mouse). To attenuate the anti-CD71 antibody, anti-IgG2a antibody (3 mg/mouse) was intravenously injected 24 h later. Finally, we adoptively transferred P14 CD8 + T cells (CD90.1, 2×10 6 cells/mouse) into mice and infected with LCMV cl13 simultaneously 36 h after administration of anti-CD71 antibody. All mice were sacrificed at day 2 after LCMV infection (l) . Representative flow cytometry ( m, left ) and cumulative composite data ( m, middle ) show the frequency of Ki67 + cells among P14 CD8 + T cells. Cumulative composite data show the Ki67 MFI in P14 CD8 + T cell ( m, right ). Cumulative composite data show the total number of CD90.1 + CD8 + P14 cells in the spleen (n). o–q, The hemoglobin (HGB)concentration ( o ) and number of CD45 + CD71 + TER119 + cells (p) in the peripheral blood of MMTV-PyMT female mice which developed palpable mammary tumors at 12 weeks old were determined at the indicated weeks. The proliferative capacity of CFSE-labeled CD8 + T cells in response to anti-CD3 and anti-CD28 was analyzed after co-culture with CD45 + CD71 + TER119 + EPCs isolated from the spleens of 20 week old MMTV-PyMT female mice at a CD8 + T cell/EPC ratio of 1:2 ( q ); CD45 + CD71 + TER119 + EPCs isolated from spleens of 20 week old MMTV-PyMT females mice were co-cultured with sorted CD8 + T cells and the ex vivo T cell killing efficiency was determined after 6 h ( r ). s, The proliferative capacity of CFSE-labeled CD8+ T cells in response to anti-CD3 and anti-CD28 was analyzed after co-culture with CD45 + CD71 + TER119 + erythroid progenitor cells isolated from the spleens of tumor-bearing, anemic or neonatal mice at the indicated CD8 + T cell:EPC ratios (n=5). Each point in (b–e) and (h–m) represents data from an individual mouse. Data are representative of three independent experiments and were analyzed by two-tailed unpaired t -test. Two-tailed p-values were reported. Bar graphs denote mean values with SEM.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Labeling, Co-Culture Assay, Isolation, Concentration Assay, Cell Culture, Ex Vivo, Infection, Injection, Two Tailed Test, In Vitro, Staining

11) Product Images from "The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR"

Article Title: The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR

Journal: Nature immunology

doi: 10.1038/s41590-018-0137-8

Itch interacts with WWP2 through its third WW domain. a , Immunoblot analysis of HEK293T cells transfected with expression vectors for Xpress-tagged Itch and/or Myc-tagged WWP2 or not (above lanes), assessed after immunoprecipitation (IP) with anti-Xpress (α-Xpress) or no immunoprecipitation (Total lysate) (left margin); right margin, molecular size in kilodaltons (throughout). b , Immunoblot analysis of the interaction of endogenous Itch with WWP2 in Jurkat T cells, assessed after immunoprecipitation with anti-Itch or the control antibody IgG (above lanes; left) or no immunoprecipitation (right). c , Confocal microscopy of the intracellular localization of Xpress-tagged Itch and Myc-tagged WWP2 in NIH3T3 cells transfected to express those molecules. Scale bars, 20 μm. d , Full-length (WT) Itch and its deletion mutants lacking functional domains (left; ranges (left margin) indicate amino acids present in construct), and immunoblot analysis of the interaction of Itch and WWP2 in HEK293T cells co-transfected (above lanes) to express Myc-tagged WWP2 plus empty vector (EV) or Xpress-tagged full-length Itch or various deletion mutants (middle and right), assessed after immunoprecipitation with anti-Myc (middle) or no immunoprecipitation (right). Data are representative of two to four independent experiments.
Figure Legend Snippet: Itch interacts with WWP2 through its third WW domain. a , Immunoblot analysis of HEK293T cells transfected with expression vectors for Xpress-tagged Itch and/or Myc-tagged WWP2 or not (above lanes), assessed after immunoprecipitation (IP) with anti-Xpress (α-Xpress) or no immunoprecipitation (Total lysate) (left margin); right margin, molecular size in kilodaltons (throughout). b , Immunoblot analysis of the interaction of endogenous Itch with WWP2 in Jurkat T cells, assessed after immunoprecipitation with anti-Itch or the control antibody IgG (above lanes; left) or no immunoprecipitation (right). c , Confocal microscopy of the intracellular localization of Xpress-tagged Itch and Myc-tagged WWP2 in NIH3T3 cells transfected to express those molecules. Scale bars, 20 μm. d , Full-length (WT) Itch and its deletion mutants lacking functional domains (left; ranges (left margin) indicate amino acids present in construct), and immunoblot analysis of the interaction of Itch and WWP2 in HEK293T cells co-transfected (above lanes) to express Myc-tagged WWP2 plus empty vector (EV) or Xpress-tagged full-length Itch or various deletion mutants (middle and right), assessed after immunoprecipitation with anti-Myc (middle) or no immunoprecipitation (right). Data are representative of two to four independent experiments.

Techniques Used: Transfection, Expressing, Immunoprecipitation, Confocal Microscopy, Functional Assay, Construct, Plasmid Preparation

Accelerated autoimmunity and inflammation in DKO mice. a , Body weight of 6-week-old male wild-type (WT) mice, Wwp2 −/− , Itch f/f Cd4 -Cre and Itch f/f Cd4 -Cre Wwp2 −/− (DKO) mice (key; n = 8 per group). b , ELISA of IL-6 in the serum of 6- to 8-week-old mice as in a (key; n = 9 per group). c , ELISA of antibodies to double-stranded DNA (Anti-dsDNA) in the serum of 6-week-old mice as in a (key; n = 7 per group), presented as optical density at 450 nm (OD 450 ). d , Lung sections from 6-week-old mice as in a (above images), stained with hematoxylin and eosin. Scale bars, 100 μm. e , Absolute number of cells in the lungs (left) or bronchoalveolar lavage fluid (BALF) (right) of wild-type or DKObm mice (key; n = 8 per group) at 6-8 weeks after cell transplantation. f , Frequency (left) and number (right) of eosinophils, macrophages, dendritic cells and neutrophils (horizontal axis) in the bronchoalveolar lavage fluid of mice as in e (key; n = 8 per group (eosinophils, macrophages and neutrophils) or n = 5 per group (dendritic cells)). g , ELISA of the immunoglobulin subclasses IgE, IgG1 and IgA in serum from 6-week-old wild-type mice ( n = 5), Wwp2 −/− mice ( n = 6), Itch f/f Cd4 -Cre mice ( n = 6) or DKO mice ( n = 6) as in a (key). Each symbol ( a - c , e - g ) represents an individual mouse; small horizontal lines ( c , e , g ) indicate the mean (±s.d.). NS, not significant ( P > 0.05); * P
Figure Legend Snippet: Accelerated autoimmunity and inflammation in DKO mice. a , Body weight of 6-week-old male wild-type (WT) mice, Wwp2 −/− , Itch f/f Cd4 -Cre and Itch f/f Cd4 -Cre Wwp2 −/− (DKO) mice (key; n = 8 per group). b , ELISA of IL-6 in the serum of 6- to 8-week-old mice as in a (key; n = 9 per group). c , ELISA of antibodies to double-stranded DNA (Anti-dsDNA) in the serum of 6-week-old mice as in a (key; n = 7 per group), presented as optical density at 450 nm (OD 450 ). d , Lung sections from 6-week-old mice as in a (above images), stained with hematoxylin and eosin. Scale bars, 100 μm. e , Absolute number of cells in the lungs (left) or bronchoalveolar lavage fluid (BALF) (right) of wild-type or DKObm mice (key; n = 8 per group) at 6-8 weeks after cell transplantation. f , Frequency (left) and number (right) of eosinophils, macrophages, dendritic cells and neutrophils (horizontal axis) in the bronchoalveolar lavage fluid of mice as in e (key; n = 8 per group (eosinophils, macrophages and neutrophils) or n = 5 per group (dendritic cells)). g , ELISA of the immunoglobulin subclasses IgE, IgG1 and IgA in serum from 6-week-old wild-type mice ( n = 5), Wwp2 −/− mice ( n = 6), Itch f/f Cd4 -Cre mice ( n = 6) or DKO mice ( n = 6) as in a (key). Each symbol ( a - c , e - g ) represents an individual mouse; small horizontal lines ( c , e , g ) indicate the mean (±s.d.). NS, not significant ( P > 0.05); * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Transplantation Assay

SHP-1 ubiquitination regulates the function of Lck. a , Immunoblot analysis of SHP-1 and actin (loading control) in lysates of wild-type and DKO Jurkat T cells (above blots) treated for 0, 1, 3 or 6 h (above lanes) with 50 μg/ml of cycloheximide (CHX). b , Immunoblot analysis of lysates of Jurkat T cells expressing wild-type SHP-1 or 3KR–SHP-1 (above blots) and treated with cycloheximide as in a . c , Protein tyrosine-phosphatase (PTP) activity of 3KR–SHP-1 among proteins immunoprecipitated, with the control antibody IgG or anti-Flag (below plot), from lysates of Jurkat T cells transfected with empty vector or expression vector for FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (key), presented relative to the activity of wild-type-SHP-1 (bottom), and immunoblot analysis of such immunoprecipitates (above lanes) (top). d , Immunoblot analysis of the interaction of SHP-1 and Lck in lysates of HEK293T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 plus either Lck (top group) or ZAP70 (bottom group) (above lanes), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). e , Immunoblot analysis of the interaction of SHP-1 and Lck in Jurkat T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 (above lanes) and left unstimulated or stimulated for 5 min with anti-CD3 (above blots), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). f , Immunoblot analysis of Lck phosphorylated at Tyr397 (p-Lck(Y397)) or Tyr505 (p-Lck(Y505)) and total Lck in CD4 + T cells sorted from wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre or DKO mice (above blots) and stimulated for 0, 5 or 15 min (above lanes) with anti-CD3 plus anti-CD28; numbers below lanes indicate the ratio of phosphorylated protein to total protein. g , Immunoblot analysis of phosphorylated and total signaling molecules (left margin), and FLAG (loading control), in lysates of Jurkat T cells expressing short hairpin RNA targeting SHP-1 (SHP-1 shRNA) together with FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (above blots) and stimulated for 0, 2 or 15 min (above lanes) with anti-CD3 (numbers below lanes, as in f ) (right blots), and immunoblot analysis of SHP-1 in Jurkat T cells expressing control (non-targeting) or SHP-1-targeting short hairpin RNA (above lanes) (left). h , ELISA of IL-4 in supernatants of GFP + (transduced) CD4 + T cells sorted from bone marrow chimeras reconstituted with bone marrow cells transduced with retroviral vector encoding green fluorescent protein (GFP) and either wild-type SHP-1 or 3KR–SHP-1 (horizontal access), then stimulated for 48 h with anti-CD3 plus anti-CD28. i , ELISA of IL-4 in supernatants of naive CD4 + T cells transduced to express wild-type SHP-1 or 3KR–SHP-1 (horizontal axis) under neutral conditions (top) or T H 2 conditions (bottom) and then allowed to ‘rest’ for 3 d, followed by sorting of GFP + (transduced) cells and re-stimulation for 24 h with anti-CD3 plus anti-CD28. Each symbol ( c , h , i ) represents an individual technical replicate ( c , i ) or mouse ( h ). * P
Figure Legend Snippet: SHP-1 ubiquitination regulates the function of Lck. a , Immunoblot analysis of SHP-1 and actin (loading control) in lysates of wild-type and DKO Jurkat T cells (above blots) treated for 0, 1, 3 or 6 h (above lanes) with 50 μg/ml of cycloheximide (CHX). b , Immunoblot analysis of lysates of Jurkat T cells expressing wild-type SHP-1 or 3KR–SHP-1 (above blots) and treated with cycloheximide as in a . c , Protein tyrosine-phosphatase (PTP) activity of 3KR–SHP-1 among proteins immunoprecipitated, with the control antibody IgG or anti-Flag (below plot), from lysates of Jurkat T cells transfected with empty vector or expression vector for FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (key), presented relative to the activity of wild-type-SHP-1 (bottom), and immunoblot analysis of such immunoprecipitates (above lanes) (top). d , Immunoblot analysis of the interaction of SHP-1 and Lck in lysates of HEK293T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 plus either Lck (top group) or ZAP70 (bottom group) (above lanes), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). e , Immunoblot analysis of the interaction of SHP-1 and Lck in Jurkat T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 (above lanes) and left unstimulated or stimulated for 5 min with anti-CD3 (above blots), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). f , Immunoblot analysis of Lck phosphorylated at Tyr397 (p-Lck(Y397)) or Tyr505 (p-Lck(Y505)) and total Lck in CD4 + T cells sorted from wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre or DKO mice (above blots) and stimulated for 0, 5 or 15 min (above lanes) with anti-CD3 plus anti-CD28; numbers below lanes indicate the ratio of phosphorylated protein to total protein. g , Immunoblot analysis of phosphorylated and total signaling molecules (left margin), and FLAG (loading control), in lysates of Jurkat T cells expressing short hairpin RNA targeting SHP-1 (SHP-1 shRNA) together with FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (above blots) and stimulated for 0, 2 or 15 min (above lanes) with anti-CD3 (numbers below lanes, as in f ) (right blots), and immunoblot analysis of SHP-1 in Jurkat T cells expressing control (non-targeting) or SHP-1-targeting short hairpin RNA (above lanes) (left). h , ELISA of IL-4 in supernatants of GFP + (transduced) CD4 + T cells sorted from bone marrow chimeras reconstituted with bone marrow cells transduced with retroviral vector encoding green fluorescent protein (GFP) and either wild-type SHP-1 or 3KR–SHP-1 (horizontal access), then stimulated for 48 h with anti-CD3 plus anti-CD28. i , ELISA of IL-4 in supernatants of naive CD4 + T cells transduced to express wild-type SHP-1 or 3KR–SHP-1 (horizontal axis) under neutral conditions (top) or T H 2 conditions (bottom) and then allowed to ‘rest’ for 3 d, followed by sorting of GFP + (transduced) cells and re-stimulation for 24 h with anti-CD3 plus anti-CD28. Each symbol ( c , h , i ) represents an individual technical replicate ( c , i ) or mouse ( h ). * P

Techniques Used: Expressing, Activity Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Mouse Assay, shRNA, Enzyme-linked Immunosorbent Assay, Transduction

Itch and WWP2 promote the polyubiquitination of SHP-1. a , b , Immunoblot analysis of lysates of Jurkat T cells transfected to express FLAG-tagged Itch ( a ) or WWP2 ( b ), assessed after immunoprecipitation with anti-FLAG or the control antibody IgG (above lanes, left) or without immunoprecipitation (right). c , d , Immunoblot analysis of an in vivo ubiquitination assay of SHP-1 in HEK293T cells transfected with various combinations (above lanes) of empty vector and expression vectors for Xpress-tagged Itch, Myc-tagged WWP2, FLAG-tagged SHP-1 and hemagglutinin-tagged ubiquitin (HA-Ub) ( c ) or with expression vectors for Xpress-tagged Itch or FLAG-tagged WWP2 (each wild-type (WT) or catalytically inactive (with the active-site cysteine replaced with alanine (CA)), and hemagglutinin-tagged ubiquitin and Myc-tagged SHP-1 ( d ), then lysed under denaturing conditions and immunoprecipitated with anti-FLAG ( c ) or anti-Myc ( d ) or assessed without immunoprecipitation (left margin). e , Immunoblot analysis of the ubiquitination of SHP-1 in lysates of wild-type and DKO Jurkat T cells (above blots) transfected to express FLAG-tagged SHP-1 together with HA-tagged ubiquitin, stimulated for 0, 5 or 15 min (above lanes) with anti-CD3, assessed after immunoprecipitation with anti-FLAG (left margin). Data are representative of three to four independent experiments.
Figure Legend Snippet: Itch and WWP2 promote the polyubiquitination of SHP-1. a , b , Immunoblot analysis of lysates of Jurkat T cells transfected to express FLAG-tagged Itch ( a ) or WWP2 ( b ), assessed after immunoprecipitation with anti-FLAG or the control antibody IgG (above lanes, left) or without immunoprecipitation (right). c , d , Immunoblot analysis of an in vivo ubiquitination assay of SHP-1 in HEK293T cells transfected with various combinations (above lanes) of empty vector and expression vectors for Xpress-tagged Itch, Myc-tagged WWP2, FLAG-tagged SHP-1 and hemagglutinin-tagged ubiquitin (HA-Ub) ( c ) or with expression vectors for Xpress-tagged Itch or FLAG-tagged WWP2 (each wild-type (WT) or catalytically inactive (with the active-site cysteine replaced with alanine (CA)), and hemagglutinin-tagged ubiquitin and Myc-tagged SHP-1 ( d ), then lysed under denaturing conditions and immunoprecipitated with anti-FLAG ( c ) or anti-Myc ( d ) or assessed without immunoprecipitation (left margin). e , Immunoblot analysis of the ubiquitination of SHP-1 in lysates of wild-type and DKO Jurkat T cells (above blots) transfected to express FLAG-tagged SHP-1 together with HA-tagged ubiquitin, stimulated for 0, 5 or 15 min (above lanes) with anti-CD3, assessed after immunoprecipitation with anti-FLAG (left margin). Data are representative of three to four independent experiments.

Techniques Used: Transfection, Immunoprecipitation, In Vivo, Ubiquitin Assay, Plasmid Preparation, Expressing

12) Product Images from "Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis"

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis

Journal: Immunity

doi: 10.1016/j.immuni.2018.04.009

Induction of GM-CSF in FLSs Stimulated with IL-17 (A) Quantitative RT-PCR analysis for the expression of designated genes in IL-17-stimulated FLSs. FLSs (2.5 × 10 4 ) were stimulated with 50 ng/mL rmIL-17 and harvested at the indicated time points. mRNA expression is presented relative to the expression of Hprt1 . (B) Quantitative RT-PCR analysis for the expression of designated genes in synoviocytes from Rag2 −/− mice with CD4 + T cell transfer. CD45 − Podoplanin + synoviocytes (3 × 10 4 ) were sorted from inflamed joints of Rag2 −/− mice 4 weeks after transfer of 1 × 10 6 SKG or Il17a −/− SKG CD4 + T cells. Vertical bars mean SD (n = 3). Data are representative of two independent experiments.
Figure Legend Snippet: Induction of GM-CSF in FLSs Stimulated with IL-17 (A) Quantitative RT-PCR analysis for the expression of designated genes in IL-17-stimulated FLSs. FLSs (2.5 × 10 4 ) were stimulated with 50 ng/mL rmIL-17 and harvested at the indicated time points. mRNA expression is presented relative to the expression of Hprt1 . (B) Quantitative RT-PCR analysis for the expression of designated genes in synoviocytes from Rag2 −/− mice with CD4 + T cell transfer. CD45 − Podoplanin + synoviocytes (3 × 10 4 ) were sorted from inflamed joints of Rag2 −/− mice 4 weeks after transfer of 1 × 10 6 SKG or Il17a −/− SKG CD4 + T cells. Vertical bars mean SD (n = 3). Data are representative of two independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

13) Product Images from "Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis"

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis

Journal: Immunity

doi: 10.1016/j.immuni.2018.04.009

GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Quantitative RT-PCR, Staining, Injection

GM-CSF from ILCs and Radio-Resistant Stromal Cells Is Crucial for Autoimmune Arthritis (A) Preparation of experimental groups for assessing arthritogenic effects of GM-CSF from ILCs or radio-resistant stromal cells. Rag2 −/− or Csf2 −/− Rag2 −/− mice were x-irradiated (X- Rag2 −/− mice) and transferred with BM cells from Csf2 −/− or WT Rag2 −/− mice. The resulting four groups of BM chimeras were transferred with CD4 + T cells from Csf2 −/− SKG mice 6 weeks after BM reconstitution and assessed for arthritis development 12 weeks later. (B) Arthritis scores of four groups of mice shown in (A). (C) Proportion of total synovial ILCs from x-irradiated Rag2 −/− mice reconstituted with Csf2 −/− or WT Rag2 −/− BM cells. (D) Flow cytometry of synovial ILCs for the expression of GM-CSF and IL-13 in arthritic joints of BM chimeras shown in (C). (E) Proportion of GM-CSF + IL-13 − , GM-CSF + IL-13 + , and GM-CSF − IL-13 + synovial ILCs (n = 6 each) as shown in (D). (F and G) The effects of ILC depletion on arthritis development. Thy1.1 + Rag2 −/− mice were x-irradiated and transferred with BM cells from Thy1.1 + Rag2 −/− and Thy1.2 + Csf2 −/− SKG mice. The resulting BM chimeras were i.p. injected with 20 mg mannan 6 weeks later, followed by i.v. injection with 500 μg anti-Thy1.1 mAb or control Rat IgG every week. Flow cytometry of synovial ILCs for GM-CSF and IL-13 expression (F). Arthritis scores in each group of mice monitored every week (G). Vertical bars mean SD (n = 8 each). ∗ p
Figure Legend Snippet: GM-CSF from ILCs and Radio-Resistant Stromal Cells Is Crucial for Autoimmune Arthritis (A) Preparation of experimental groups for assessing arthritogenic effects of GM-CSF from ILCs or radio-resistant stromal cells. Rag2 −/− or Csf2 −/− Rag2 −/− mice were x-irradiated (X- Rag2 −/− mice) and transferred with BM cells from Csf2 −/− or WT Rag2 −/− mice. The resulting four groups of BM chimeras were transferred with CD4 + T cells from Csf2 −/− SKG mice 6 weeks after BM reconstitution and assessed for arthritis development 12 weeks later. (B) Arthritis scores of four groups of mice shown in (A). (C) Proportion of total synovial ILCs from x-irradiated Rag2 −/− mice reconstituted with Csf2 −/− or WT Rag2 −/− BM cells. (D) Flow cytometry of synovial ILCs for the expression of GM-CSF and IL-13 in arthritic joints of BM chimeras shown in (C). (E) Proportion of GM-CSF + IL-13 − , GM-CSF + IL-13 + , and GM-CSF − IL-13 + synovial ILCs (n = 6 each) as shown in (D). (F and G) The effects of ILC depletion on arthritis development. Thy1.1 + Rag2 −/− mice were x-irradiated and transferred with BM cells from Thy1.1 + Rag2 −/− and Thy1.2 + Csf2 −/− SKG mice. The resulting BM chimeras were i.p. injected with 20 mg mannan 6 weeks later, followed by i.v. injection with 500 μg anti-Thy1.1 mAb or control Rat IgG every week. Flow cytometry of synovial ILCs for GM-CSF and IL-13 expression (F). Arthritis scores in each group of mice monitored every week (G). Vertical bars mean SD (n = 8 each). ∗ p

Techniques Used: Mouse Assay, Irradiation, Flow Cytometry, Cytometry, Expressing, Injection

GM-CSF from Non-T Cells Is Crucial for the Initiation of Autoimmune Arthritis (A) Experimental design of adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− or Csf2 −/− Rag2 −/− mice. Arthritis scores of four groups (a–d) of mice were assessed 3 months after transfer of 1 × 10 6 CD4 + T cells. (B) Arthritis scores of the four groups mice (n = 15 or 16 each) shown in (A). Horizontal bars indicate the means. (C) Representative joint histology of the groups shown in (A). Scale bars indicate 200 μm. (D) Flow cytometry of splenic CD4 + T cells stained for intracellular IL-17 and GM-CSF or IFN-γ. (E) Proportion of IL-17-producing CD4 + T cells from individual mice as shown in (D). Vertical bars mean SD (n = 3). ∗∗ p
Figure Legend Snippet: GM-CSF from Non-T Cells Is Crucial for the Initiation of Autoimmune Arthritis (A) Experimental design of adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− or Csf2 −/− Rag2 −/− mice. Arthritis scores of four groups (a–d) of mice were assessed 3 months after transfer of 1 × 10 6 CD4 + T cells. (B) Arthritis scores of the four groups mice (n = 15 or 16 each) shown in (A). Horizontal bars indicate the means. (C) Representative joint histology of the groups shown in (A). Scale bars indicate 200 μm. (D) Flow cytometry of splenic CD4 + T cells stained for intracellular IL-17 and GM-CSF or IFN-γ. (E) Proportion of IL-17-producing CD4 + T cells from individual mice as shown in (D). Vertical bars mean SD (n = 3). ∗∗ p

Techniques Used: Adoptive Transfer Assay, Mouse Assay, Flow Cytometry, Cytometry, Staining

GM-CSF-Producing ILCs in Synovial Fluid of RA Patients (A) The presence of ILCs (defined as CD45 + CD3 − CD4 − CD8 − CD11b − CD11c − CD19 − CD56 − ) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right). (B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD. (C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom). (D) Gating strategies for GM-CSF + CD45 + lineage markers-negative (ILCs), GM-CSF + CD45 + CD3 − CD11b + (myeloid cells), and GM-CSF + CD45 + CD11b − CD3 + cells (T cells). (E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Synovial Fluid of RA Patients (A) The presence of ILCs (defined as CD45 + CD3 − CD4 − CD8 − CD11b − CD11c − CD19 − CD56 − ) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right). (B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD. (C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom). (D) Gating strategies for GM-CSF + CD45 + lineage markers-negative (ILCs), GM-CSF + CD45 + CD3 − CD11b + (myeloid cells), and GM-CSF + CD45 + CD11b − CD3 + cells (T cells). (E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing

Induction of GM-CSF in FLSs Stimulated with IL-17 (A) Quantitative RT-PCR analysis for the expression of designated genes in IL-17-stimulated FLSs. FLSs (2.5 × 10 4 ) were stimulated with 50 ng/mL rmIL-17 and harvested at the indicated time points. mRNA expression is presented relative to the expression of Hprt1 . (B) Quantitative RT-PCR analysis for the expression of designated genes in synoviocytes from Rag2 −/− mice with CD4 + T cell transfer. CD45 − Podoplanin + synoviocytes (3 × 10 4 ) were sorted from inflamed joints of Rag2 −/− mice 4 weeks after transfer of 1 × 10 6 SKG or Il17a −/− SKG CD4 + T cells. Vertical bars mean SD (n = 3). Data are representative of two independent experiments.
Figure Legend Snippet: Induction of GM-CSF in FLSs Stimulated with IL-17 (A) Quantitative RT-PCR analysis for the expression of designated genes in IL-17-stimulated FLSs. FLSs (2.5 × 10 4 ) were stimulated with 50 ng/mL rmIL-17 and harvested at the indicated time points. mRNA expression is presented relative to the expression of Hprt1 . (B) Quantitative RT-PCR analysis for the expression of designated genes in synoviocytes from Rag2 −/− mice with CD4 + T cell transfer. CD45 − Podoplanin + synoviocytes (3 × 10 4 ) were sorted from inflamed joints of Rag2 −/− mice 4 weeks after transfer of 1 × 10 6 SKG or Il17a −/− SKG CD4 + T cells. Vertical bars mean SD (n = 3). Data are representative of two independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

GM-CSF-Producing T Helper Cells Are Dispensable for GM-CSF-Dependent Autoimmune Arthritis Development (A) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from popliteal LNs or inflamed joints. (B) Proportion of cytokine-producing cells in CD4 + T cells from individual mice as shown in (A). Vertical bars mean SD (n = 3). (C) Arthritis scores assessed in individual SKG, Csf2 −/− SKG, or Il17a −/− SKG mice (n = 20 each) 3 months after single i.p. injection of 20 mg mannan. (D) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from inflamed joints of Il17a Cre R26R eYFP SKG mice. (E) Arthritis development after adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− mice (n = 17 each, SEM). The severity of arthritis was monitored every week after transfer of 1 × 10 6 CD4 + T cells. (F) Intracellular IL-17 and GM-CSF staining of CD4 + T cells from spleens and inflamed joints of Rag2 −/− mice with CD4 + T cells transfer as shown in (E). ∗ p
Figure Legend Snippet: GM-CSF-Producing T Helper Cells Are Dispensable for GM-CSF-Dependent Autoimmune Arthritis Development (A) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from popliteal LNs or inflamed joints. (B) Proportion of cytokine-producing cells in CD4 + T cells from individual mice as shown in (A). Vertical bars mean SD (n = 3). (C) Arthritis scores assessed in individual SKG, Csf2 −/− SKG, or Il17a −/− SKG mice (n = 20 each) 3 months after single i.p. injection of 20 mg mannan. (D) Intracellular IL-17, IFN-γ, and GM-CSF staining of CD4 + T cells from inflamed joints of Il17a Cre R26R eYFP SKG mice. (E) Arthritis development after adoptive transfer of CD4 + T cells from WT or Csf2 −/− SKG mice into Rag2 −/− mice (n = 17 each, SEM). The severity of arthritis was monitored every week after transfer of 1 × 10 6 CD4 + T cells. (F) Intracellular IL-17 and GM-CSF staining of CD4 + T cells from spleens and inflamed joints of Rag2 −/− mice with CD4 + T cells transfer as shown in (E). ∗ p

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

14) Product Images from "Targeting the STING pathway in tumor-associated macrophages regulates innate immune sensing of gastric cancer cells"

Article Title: Targeting the STING pathway in tumor-associated macrophages regulates innate immune sensing of gastric cancer cells

Journal: Theranostics

doi: 10.7150/thno.37745

Knocking-down STING and STING activation promote both PBMC-DMs and BM-DMs differentiating into pro-inflammatory subtype. (A) Upper panel, flow cytometric analysis of TAMs for pro-inflammatory (CD45 + CD11b + CD80 + ) and anti-inflammatory (CD45 + CD11b + CD163 + ) subtypes; Lower panel, quantification of pro-inflammatory, anti-inflammatory macrophages and the corresponding ratios (n=8); *, p
Figure Legend Snippet: Knocking-down STING and STING activation promote both PBMC-DMs and BM-DMs differentiating into pro-inflammatory subtype. (A) Upper panel, flow cytometric analysis of TAMs for pro-inflammatory (CD45 + CD11b + CD80 + ) and anti-inflammatory (CD45 + CD11b + CD163 + ) subtypes; Lower panel, quantification of pro-inflammatory, anti-inflammatory macrophages and the corresponding ratios (n=8); *, p

Techniques Used: Activation Assay, Flow Cytometry

15) Product Images from "CD300c is an Activating Receptor Expressed on Human Monocytes"

Article Title: CD300c is an Activating Receptor Expressed on Human Monocytes

Journal: Journal of Innate Immunity

doi: 10.1159/000350523

Expression of CD300c is regulated on monocytes by LPS. a Enriched monocytes from healthy donors were either left untreated (Alone) or treated with LPS for 2 and 24 h. RNA was extracted and the levels of CD300c mRNA were determined by real-time PCR. Graph bars represent the average ± SEM. Data are from 5 independent experiments. b Enriched monocytes were either left untreated or treated with LPS for 2 and 24 h. The cells were harvested and checked for CD300c cell surface expression. The shaded histograms represent the staining with the isotype control (MOPC-21) and the open histograms represent the staining with anti-CD300c (clone TX45) monoclonal antibody. The numbers indicate the values of median fluorescence intensity. Results are representative of 5 healthy donors.
Figure Legend Snippet: Expression of CD300c is regulated on monocytes by LPS. a Enriched monocytes from healthy donors were either left untreated (Alone) or treated with LPS for 2 and 24 h. RNA was extracted and the levels of CD300c mRNA were determined by real-time PCR. Graph bars represent the average ± SEM. Data are from 5 independent experiments. b Enriched monocytes were either left untreated or treated with LPS for 2 and 24 h. The cells were harvested and checked for CD300c cell surface expression. The shaded histograms represent the staining with the isotype control (MOPC-21) and the open histograms represent the staining with anti-CD300c (clone TX45) monoclonal antibody. The numbers indicate the values of median fluorescence intensity. Results are representative of 5 healthy donors.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence

CD300c is expressed on human monocytes and monocyte-derived cells. a Cells were isolated from normal healthy donors and stained with specific antibodies to distinguish different cell subsets. In addition, cells were stained with anti-CD300a/c (clone E59.126) and anti-CD300c (clone TX45) monoclonal antibodies. Shaded histograms represent the staining with isotype-matched controls and empty histograms the staining with anti-CD300a/c (clone E59.126; upper panel) and anti-CD300c (clone TX45; lower panel). Data from a representative out of 6 are shown. b Enriched monocytes were differentiated into mDC and macrophages according to the protocol described in the Materials and Methods. The expression of CD300c was determined using anti-CD300c (clone TX45) monoclonal antibody. The shaded histograms represent the staining with isotype-matched control and the open histograms correspond to the specific staining with anti-CD300c (clone TX45). Results from a representative out of 4 are shown.
Figure Legend Snippet: CD300c is expressed on human monocytes and monocyte-derived cells. a Cells were isolated from normal healthy donors and stained with specific antibodies to distinguish different cell subsets. In addition, cells were stained with anti-CD300a/c (clone E59.126) and anti-CD300c (clone TX45) monoclonal antibodies. Shaded histograms represent the staining with isotype-matched controls and empty histograms the staining with anti-CD300a/c (clone E59.126; upper panel) and anti-CD300c (clone TX45; lower panel). Data from a representative out of 6 are shown. b Enriched monocytes were differentiated into mDC and macrophages according to the protocol described in the Materials and Methods. The expression of CD300c was determined using anti-CD300c (clone TX45) monoclonal antibody. The shaded histograms represent the staining with isotype-matched control and the open histograms correspond to the specific staining with anti-CD300c (clone TX45). Results from a representative out of 4 are shown.

Techniques Used: Derivative Assay, Isolation, Staining, Expressing

Monoclonal antibody clone TX45 binds specifically to CD300c. a 293T cells were transiently transfected with empty vector (upper panels), vectors expressing full-length CD300c (middle panels) or CD300a (lower panels). After 16 h of transfection, cells were stained with the following monoclonal antibodies: anti-CD300c, clone TX45 (left lane); anti-CD300a/c, clone E59.126 (middle lane), and anti-CD300a/c, clone TX49 (right lane). Shaded histograms represent unstained cells and the open histograms represent the staining with the specific antibodies. These data are representative of 3 independent experiments. b YTS cells stably transfected with empty vector (upper panel), vector expressing CD300c (middle panel) and CD300a (lower panel) were stained with anti-CD300c, clone TX45 (left lane) and anti-CD300a/c, clone E59.126 (middle lane). The shaded histograms represent the isotype-matched controls and the open histograms the staining with the specific monoclonal antibodies. Results are representative of 3 independent experiments.
Figure Legend Snippet: Monoclonal antibody clone TX45 binds specifically to CD300c. a 293T cells were transiently transfected with empty vector (upper panels), vectors expressing full-length CD300c (middle panels) or CD300a (lower panels). After 16 h of transfection, cells were stained with the following monoclonal antibodies: anti-CD300c, clone TX45 (left lane); anti-CD300a/c, clone E59.126 (middle lane), and anti-CD300a/c, clone TX49 (right lane). Shaded histograms represent unstained cells and the open histograms represent the staining with the specific antibodies. These data are representative of 3 independent experiments. b YTS cells stably transfected with empty vector (upper panel), vector expressing CD300c (middle panel) and CD300a (lower panel) were stained with anti-CD300c, clone TX45 (left lane) and anti-CD300a/c, clone E59.126 (middle lane). The shaded histograms represent the isotype-matched controls and the open histograms the staining with the specific monoclonal antibodies. Results are representative of 3 independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Stable Transfection

Cross-linking of CD300c induces calcium mobilization and upregulation of CD86. a Enriched monocytes from human healthy donors were loaded with Fluo-4 and Fura-Red. To establish a baseline, monocytes were first acquired using a FACSCalibur for 30 s at which point the primary antibodies, anti-CD300c (clone TX45) or isotype-matched control (clone MOPC-21), were added. Then, at 60 s, the primary antibodies were cross-linked with goat anti-mouse IgG F(ab′) 2 and fluorescence was measured. Ca 2+ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. Results are representative of 3 independent experiments. b Enriched monocytes from human healthy donors were left either nonstimulated (None) or stimulated with plate-bound isotype control antibody, MOPC-21 (Isotype), and anti-CD300c monoclonal antibody, clone TX45 (TX45) for 24 h. Then, cells were harvested, stained and analyzed by flow cytometry. Shaded histograms correspond to unstained cells and open histograms to the specific staining with anti-CD86 monoclonal antibody. Numbers indicate the values of the median fluorescence intensity. These results are representative of 3 independent experiments from 3 different healthy donors.
Figure Legend Snippet: Cross-linking of CD300c induces calcium mobilization and upregulation of CD86. a Enriched monocytes from human healthy donors were loaded with Fluo-4 and Fura-Red. To establish a baseline, monocytes were first acquired using a FACSCalibur for 30 s at which point the primary antibodies, anti-CD300c (clone TX45) or isotype-matched control (clone MOPC-21), were added. Then, at 60 s, the primary antibodies were cross-linked with goat anti-mouse IgG F(ab′) 2 and fluorescence was measured. Ca 2+ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. Results are representative of 3 independent experiments. b Enriched monocytes from human healthy donors were left either nonstimulated (None) or stimulated with plate-bound isotype control antibody, MOPC-21 (Isotype), and anti-CD300c monoclonal antibody, clone TX45 (TX45) for 24 h. Then, cells were harvested, stained and analyzed by flow cytometry. Shaded histograms correspond to unstained cells and open histograms to the specific staining with anti-CD86 monoclonal antibody. Numbers indicate the values of the median fluorescence intensity. These results are representative of 3 independent experiments from 3 different healthy donors.

Techniques Used: Fluorescence, Staining, Flow Cytometry, Cytometry

Cross-linking of CD300c in monocytes induces the secretion of inflammatory cytokines. Freshly isolated monocytes from healthy donors were either stimulated with plate-bound isotype-matched control antibody, MOPC-21 (empty bars), or with anti-CD300c antibody, TX45 (black bars), in the absence (Untreated) or presence of LPS for 24 h. Culture supernatants were harvested and tested for the secretion of human inflammatory cytokines using flow-cytometric bead analysis. The values on the y-axis correspond to the concentrations of cytokines: TNF-α, IL-1β and IL-10 (pg/ml), and IL-8 and IL-6 (ng/ml). Graph bars represent the average ± SEM. Data are from 5 independent experiments.
Figure Legend Snippet: Cross-linking of CD300c in monocytes induces the secretion of inflammatory cytokines. Freshly isolated monocytes from healthy donors were either stimulated with plate-bound isotype-matched control antibody, MOPC-21 (empty bars), or with anti-CD300c antibody, TX45 (black bars), in the absence (Untreated) or presence of LPS for 24 h. Culture supernatants were harvested and tested for the secretion of human inflammatory cytokines using flow-cytometric bead analysis. The values on the y-axis correspond to the concentrations of cytokines: TNF-α, IL-1β and IL-10 (pg/ml), and IL-8 and IL-6 (ng/ml). Graph bars represent the average ± SEM. Data are from 5 independent experiments.

Techniques Used: Isolation, Flow Cytometry

16) Product Images from "T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target"

Article Title: T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

Journal: Theranostics

doi: 10.7150/thno.27275

CD2, CD3, and CD7 are expressed on all T-cell subsets and CD2 as well as CD7 show an upregulation on specifically activated T cells. (A) Analysis of CD2, CD3, and CD7 expression on the surface of different T-cell subsets from freshly isolated peripheral blood mononuclear cells (PBMC). Mean fluorescence intensity (MFI) of PE-labeled anti-CD2, anti-CD3, and anti-CD7 antibodies is shown normalized to PE-isotype control for cells pre-gated on CD4 or CD8. T-cell subpopulations include T CM (central memory T cells; CD45RA - CD62L + ), T EFF (effector T cells; CD45RA + CD62L - ), T EM (effector memory T cells; CD45RA - CD62L - ), T N (naive T cells; CD45RA + CD62L + ), and T Reg (regulatory T cells; CD4 + CD25 + CD127 low ). Analysis of CD2 and CD7 on T-cell subpopulations of two other donors revealed the same pattern for each subset at other MFI values. Flow cytometry data for LSR II was evaluated using FlowJoSoftware7.6.5 and is shown for cells pre-gated on single and 7-AAD - cells. (B) CD2, CD3, and CD7 molecules on the surface of T cells and tumor cells (control). TCR2.5D6iRFP or iRFP T CM (both GFP - ) were co-cultivated for 24 h with ML2-B7 or ML2-B15 (both GFP + ) tumor cells as indicated and subsequently analyzed for surface expression with respective PE-labeled antibodies. For calculation of the number of surface molecules, detected geometric mean (GM) was related to GM of PE quantification beads and the labeling efficiency of antibodies was determined by nanophotometer.
Figure Legend Snippet: CD2, CD3, and CD7 are expressed on all T-cell subsets and CD2 as well as CD7 show an upregulation on specifically activated T cells. (A) Analysis of CD2, CD3, and CD7 expression on the surface of different T-cell subsets from freshly isolated peripheral blood mononuclear cells (PBMC). Mean fluorescence intensity (MFI) of PE-labeled anti-CD2, anti-CD3, and anti-CD7 antibodies is shown normalized to PE-isotype control for cells pre-gated on CD4 or CD8. T-cell subpopulations include T CM (central memory T cells; CD45RA - CD62L + ), T EFF (effector T cells; CD45RA + CD62L - ), T EM (effector memory T cells; CD45RA - CD62L - ), T N (naive T cells; CD45RA + CD62L + ), and T Reg (regulatory T cells; CD4 + CD25 + CD127 low ). Analysis of CD2 and CD7 on T-cell subpopulations of two other donors revealed the same pattern for each subset at other MFI values. Flow cytometry data for LSR II was evaluated using FlowJoSoftware7.6.5 and is shown for cells pre-gated on single and 7-AAD - cells. (B) CD2, CD3, and CD7 molecules on the surface of T cells and tumor cells (control). TCR2.5D6iRFP or iRFP T CM (both GFP - ) were co-cultivated for 24 h with ML2-B7 or ML2-B15 (both GFP + ) tumor cells as indicated and subsequently analyzed for surface expression with respective PE-labeled antibodies. For calculation of the number of surface molecules, detected geometric mean (GM) was related to GM of PE quantification beads and the labeling efficiency of antibodies was determined by nanophotometer.

Techniques Used: Expressing, Isolation, Fluorescence, Labeling, Flow Cytometry, Cytometry

17) Product Images from "N‐methyl‐D‐aspartate receptor antibody production from germinal center reactions: Therapeutic implications"

Article Title: N‐methyl‐D‐aspartate receptor antibody production from germinal center reactions: Therapeutic implications

Journal: Annals of Neurology

doi: 10.1002/ana.25173

NR1‐IgG antibody generation from ovarian teratomas. (A) An ovarian teratoma from patient number 2 (scale bar, 0.2cm), showed dense B cell (CD20) infiltration, with more sparse T cells (CD3) and few plasma cells (CD138, inset × 80 magnification). T and B cells express CD27, whereas CD38 is more restricted to the B cell regions. The NR1 subunit is densely expressed throughout this region. Scale bar, 100 μm. (B) Cystic aspirate from another teratoma (a 28‐year‐old female recruited after patients 1–10) was studied by flow cytometry and found to contain CD19 + B cells, which (C) expressed surface IgM, IgG, and IgD. (D) In addition, some CD19 + cells were CD27 ++ CD38 ++ antibody‐secreting cells (ASCs). (E,F) Culture of these cells with an ASC‐maintenance condition (interleukin‐6), but not B cell proliferative condition (R848, CD40‐ligand, and interleukin‐2), resulted in NR1‐IgG generation (E,F, lower panel). Scale bar, 5 μm. NR1‐IgG was also detected from supernatants of the teratoma explants of patient 2 (F, upper panel) after incubation with B cell proliferative conditions or interleukin‐6. In addition, NR1‐IgG was detected in aspirates directly from the teratoma at higher levels than in the serum from these 2 patients (F). DAPI = 4',6‐diamidino‐2‐phenylindole; EGFP = enhanced green fluorescent protein; Ig = immunoglobulin.
Figure Legend Snippet: NR1‐IgG antibody generation from ovarian teratomas. (A) An ovarian teratoma from patient number 2 (scale bar, 0.2cm), showed dense B cell (CD20) infiltration, with more sparse T cells (CD3) and few plasma cells (CD138, inset × 80 magnification). T and B cells express CD27, whereas CD38 is more restricted to the B cell regions. The NR1 subunit is densely expressed throughout this region. Scale bar, 100 μm. (B) Cystic aspirate from another teratoma (a 28‐year‐old female recruited after patients 1–10) was studied by flow cytometry and found to contain CD19 + B cells, which (C) expressed surface IgM, IgG, and IgD. (D) In addition, some CD19 + cells were CD27 ++ CD38 ++ antibody‐secreting cells (ASCs). (E,F) Culture of these cells with an ASC‐maintenance condition (interleukin‐6), but not B cell proliferative condition (R848, CD40‐ligand, and interleukin‐2), resulted in NR1‐IgG generation (E,F, lower panel). Scale bar, 5 μm. NR1‐IgG was also detected from supernatants of the teratoma explants of patient 2 (F, upper panel) after incubation with B cell proliferative conditions or interleukin‐6. In addition, NR1‐IgG was detected in aspirates directly from the teratoma at higher levels than in the serum from these 2 patients (F). DAPI = 4',6‐diamidino‐2‐phenylindole; EGFP = enhanced green fluorescent protein; Ig = immunoglobulin.

Techniques Used: Flow Cytometry, Cytometry, Incubation

18) Product Images from "Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery–Dreifuss muscular dystrophy"

Article Title: Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery–Dreifuss muscular dystrophy

Journal: Neuromuscular Disorders

doi: 10.1016/j.nmd.2014.09.012

Highly lobulated nuclei in patient blood include non-granulocyte cells. A) Immunofluorescence staining of control blood cells for the following markers: CD3 (T-cells), CD14 (macrophages), CD66b (granulocytes). B) Immunofluorescence staining of patient blood cells for the same markers. The right panels are magnifications of the boxed areas in the merged images.
Figure Legend Snippet: Highly lobulated nuclei in patient blood include non-granulocyte cells. A) Immunofluorescence staining of control blood cells for the following markers: CD3 (T-cells), CD14 (macrophages), CD66b (granulocytes). B) Immunofluorescence staining of patient blood cells for the same markers. The right panels are magnifications of the boxed areas in the merged images.

Techniques Used: Immunofluorescence, Staining

Blood cell distributions and nuclear lobulation in the patient. A) Dapi staining of nuclei from cells isolated from patient blood, 20× magnification. B) Dapi staining of nuclei from cells isolated from patient blood, 100× magnification. C) FACS staining of cells isolated from patient and control blood for following markers: CD3 (T-cells), CD14 (macrophages), CD66b (granulocytes) and CD19 (B-cells).
Figure Legend Snippet: Blood cell distributions and nuclear lobulation in the patient. A) Dapi staining of nuclei from cells isolated from patient blood, 20× magnification. B) Dapi staining of nuclei from cells isolated from patient blood, 100× magnification. C) FACS staining of cells isolated from patient and control blood for following markers: CD3 (T-cells), CD14 (macrophages), CD66b (granulocytes) and CD19 (B-cells).

Techniques Used: Staining, Isolation, FACS

19) Product Images from "Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer"

Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

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

doi: 10.4049/jimmunol.1501853

Particulate β-glucan treatment in vivo reduces tumor burden and impacts the frequency of MDSC in spleens and tumors of LLC and E0771-bearing mice (A) C57BL/6 WT mice (n=7, 8) were injected subcutaneously (s.c) with LLC or E0771 tumor cell lines. Once palpable tumors were formed (day 8), mice were orally administered with particulate β-glucan (800 µg, daily) or PBS with a gavage needle at indicated time. Tumor diameters were measured every three days and tumor volumes were then calculated. (B) On day 32 (LLC model) or day 35 (E0771 model), mice were killed and spleens were excised and weighed. Each point in the data plot represents the spleen weight of each mouse in grams. PBS-treated group was compared to particulate β-glucan treated group (WGP) in both models (C) Tumor tissues were excised and weighted from WGP or PBS-treated mice. (D) Flow cytometry analysis of the frequencies of M-MDSC (Ly6G − Ly6C high ) and PMN-MDSC (Ly6G + Ly6C int ) in the spleens of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD11b + cells. (E) Frequencies of M-MDSC and PMN-MDSC in the tumors of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD45 + CD11b + cells. *p
Figure Legend Snippet: Particulate β-glucan treatment in vivo reduces tumor burden and impacts the frequency of MDSC in spleens and tumors of LLC and E0771-bearing mice (A) C57BL/6 WT mice (n=7, 8) were injected subcutaneously (s.c) with LLC or E0771 tumor cell lines. Once palpable tumors were formed (day 8), mice were orally administered with particulate β-glucan (800 µg, daily) or PBS with a gavage needle at indicated time. Tumor diameters were measured every three days and tumor volumes were then calculated. (B) On day 32 (LLC model) or day 35 (E0771 model), mice were killed and spleens were excised and weighed. Each point in the data plot represents the spleen weight of each mouse in grams. PBS-treated group was compared to particulate β-glucan treated group (WGP) in both models (C) Tumor tissues were excised and weighted from WGP or PBS-treated mice. (D) Flow cytometry analysis of the frequencies of M-MDSC (Ly6G − Ly6C high ) and PMN-MDSC (Ly6G + Ly6C int ) in the spleens of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD11b + cells. (E) Frequencies of M-MDSC and PMN-MDSC in the tumors of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD45 + CD11b + cells. *p

Techniques Used: In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry

20) Product Images from "PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy"

Article Title: PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.112

In vivo roles of Tim-3 and PD-1 during early pregnancy. ( a ) The weight of pregnant CBA/J females treated with PBS, anti-Tim-3 antibody, anti-PD-1 antibody, or both antibodies i.p. at doses of 500, 250, and 250 mg at days 4.5, 6.5, and 8.5, respectively. ( b ) The number of live fetuses per uterus from pregnant CBA/J females following treatment with the indicated blocking antibodies. Pregnant mice were kiled at day 10.5 of pregnancy, the uteri were removed, and the implantation sites and resorbed/live embryos were counted to assess the effect of anti-Tim-3 and anti-PD-1 antibody treatment on pregnancy. ( c – f ) Quantification of flow-cytometric analysis of cytokine production and transcription factor expression by dCD8 + T cells of mice following treatment with the indicated blocking antibodies. Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. Data represent mean±S.E.M. of n =6–8 mice per group and are representative of four independent analyses. * P
Figure Legend Snippet: In vivo roles of Tim-3 and PD-1 during early pregnancy. ( a ) The weight of pregnant CBA/J females treated with PBS, anti-Tim-3 antibody, anti-PD-1 antibody, or both antibodies i.p. at doses of 500, 250, and 250 mg at days 4.5, 6.5, and 8.5, respectively. ( b ) The number of live fetuses per uterus from pregnant CBA/J females following treatment with the indicated blocking antibodies. Pregnant mice were kiled at day 10.5 of pregnancy, the uteri were removed, and the implantation sites and resorbed/live embryos were counted to assess the effect of anti-Tim-3 and anti-PD-1 antibody treatment on pregnancy. ( c – f ) Quantification of flow-cytometric analysis of cytokine production and transcription factor expression by dCD8 + T cells of mice following treatment with the indicated blocking antibodies. Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. Data represent mean±S.E.M. of n =6–8 mice per group and are representative of four independent analyses. * P

Techniques Used: In Vivo, Crocin Bleaching Assay, Blocking Assay, Mouse Assay, Flow Cytometry, Expressing, Isolation, Cytometry

Proliferation and cytokine production in dCD8 + T cells during normal pregnancy. ( a ) Freshly isolated DICs were stained with antibodies against Ki-67 to detect the proliferation of dCD8 + T cells by flow cytometry. A representative flow-cytometry plot (right) and quantification (left) of Ki-67 staining in dCD8 + T cells are shown. n =9. ( b and c ) Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. ( b ) Expression of the Th2-type cytokines IL-4 and IL-10 in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. A representative flow-cytometry plot (right) and quantitation (left) are shown. n =9. ( c ) Quantitation of flow-cytometric analysis of the Th1-type cytokines IFN- γ and TNF- α in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. n =12. Data represent mean±S.E.M. The flow-cytometry plots are representative of three independent experiments. * P
Figure Legend Snippet: Proliferation and cytokine production in dCD8 + T cells during normal pregnancy. ( a ) Freshly isolated DICs were stained with antibodies against Ki-67 to detect the proliferation of dCD8 + T cells by flow cytometry. A representative flow-cytometry plot (right) and quantification (left) of Ki-67 staining in dCD8 + T cells are shown. n =9. ( b and c ) Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested and analyzed by flow cytometry. ( b ) Expression of the Th2-type cytokines IL-4 and IL-10 in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. A representative flow-cytometry plot (right) and quantitation (left) are shown. n =9. ( c ) Quantitation of flow-cytometric analysis of the Th1-type cytokines IFN- γ and TNF- α in Tim-3 + PD-1 + , Tim-3 − PD-1 + , Tim-3 + PD-1 − , and Tim-3 − PD-1 − dCD8 + T cells. n =12. Data represent mean±S.E.M. The flow-cytometry plots are representative of three independent experiments. * P

Techniques Used: Isolation, Staining, Flow Cytometry, Cytometry, Expressing, Quantitation Assay

Decreased number of CD8 + T cells co-expressing Tim-3 and PD-1 with disregulated cytokine production is observed in human early pregnancy loss. ( a ) Frequency of Tim-3 and PD-1 expression on gated CD8 + T cells from DICs and PBMCs from normal pregnancy (NP; n =30) and miscarriage (abnormal pregnancy, AP; n =36) as measured by flow cytometry. ( b ) Flow-cytometric analysis of Ki-67 staining in dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). ( c and d ) Quantification of flow-cytometric analysis of cytokine production by dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested, fixed, permeabilized, and stained with antibodies against IL-4, IL-10, IFN- γ , TNF- α , CD8, PD-1, and Tim-3. Data represent mean±S.E.M. The flow-cytometry plot is representative of six independent experiments. * P
Figure Legend Snippet: Decreased number of CD8 + T cells co-expressing Tim-3 and PD-1 with disregulated cytokine production is observed in human early pregnancy loss. ( a ) Frequency of Tim-3 and PD-1 expression on gated CD8 + T cells from DICs and PBMCs from normal pregnancy (NP; n =30) and miscarriage (abnormal pregnancy, AP; n =36) as measured by flow cytometry. ( b ) Flow-cytometric analysis of Ki-67 staining in dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). ( c and d ) Quantification of flow-cytometric analysis of cytokine production by dCD8 + T cells from normal pregnancy ( n =30) and miscarriage ( n =36). Freshly isolated DICs were treated with brefeldin A (10 μ g/ml), PMA (50 ng/ml), and ionomycin (1 μ g/ml) for 4 h, then the cells were harvested, fixed, permeabilized, and stained with antibodies against IL-4, IL-10, IFN- γ , TNF- α , CD8, PD-1, and Tim-3. Data represent mean±S.E.M. The flow-cytometry plot is representative of six independent experiments. * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Isolation

21) Product Images from "GMP-Compliant, Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo"

Article Title: GMP-Compliant, Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053611

Susceptibility of tumor cells to cytotoxicity of expanded NK cells. (A) Cytotoxicity of expanded NK cells against various tumor cell lines was analyzed by 51 Cr-release assay with the indicated effector∶target (E∶T) ratio in triplicate. Cytotoxicity against normal PBMCs was also analyzed. The assay was performed two times with expanded NK cells from different donors, and representative data was presented. Each graph represents mean±SD. (B) Expression of HLA-class I, ULBP-1, ULBP-2, MIC-A/B, CD112 and CD155 was analyzed by flow cytometry in various tumor cell lines and normal PBMCs (solid lines). Grey histograms represent isotype controls. (C) Expanded NK cells were pre-incubated with blocking antibodies for DNAM-1, NKG2D, NKp30 and/or NKp44, and cytotoxicity was analyzed against SW480 or SNU398 cells by 51 Cr-release assay in triplicate. E∶T ratio was 10∶1. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. The assay was performed two times with expanded NK cells from different donors, and representative data are presented. Each bar graph represents mean+SD.
Figure Legend Snippet: Susceptibility of tumor cells to cytotoxicity of expanded NK cells. (A) Cytotoxicity of expanded NK cells against various tumor cell lines was analyzed by 51 Cr-release assay with the indicated effector∶target (E∶T) ratio in triplicate. Cytotoxicity against normal PBMCs was also analyzed. The assay was performed two times with expanded NK cells from different donors, and representative data was presented. Each graph represents mean±SD. (B) Expression of HLA-class I, ULBP-1, ULBP-2, MIC-A/B, CD112 and CD155 was analyzed by flow cytometry in various tumor cell lines and normal PBMCs (solid lines). Grey histograms represent isotype controls. (C) Expanded NK cells were pre-incubated with blocking antibodies for DNAM-1, NKG2D, NKp30 and/or NKp44, and cytotoxicity was analyzed against SW480 or SNU398 cells by 51 Cr-release assay in triplicate. E∶T ratio was 10∶1. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. The assay was performed two times with expanded NK cells from different donors, and representative data are presented. Each bar graph represents mean+SD.

Techniques Used: Release Assay, Expressing, Flow Cytometry, Cytometry, Incubation, Blocking Assay, Inhibition

22) Product Images from "Reduction of circulating innate lymphoid cell progenitors results in impaired cytokine production by innate lymphoid cells in patients with lupus nephritis"

Article Title: Reduction of circulating innate lymphoid cell progenitors results in impaired cytokine production by innate lymphoid cells in patients with lupus nephritis

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-020-2114-5

Circulating ILCs correlate with clinical parameters that reflect LN disease activity. Correlation between the percentage of ( a ) total ILCs, ( b ) CD117 + ILCs, and ( c ) cytokine-negative ILCs within PBMC, and clinical parameters (WBC counts, UPCR, and eGFR). **P
Figure Legend Snippet: Circulating ILCs correlate with clinical parameters that reflect LN disease activity. Correlation between the percentage of ( a ) total ILCs, ( b ) CD117 + ILCs, and ( c ) cytokine-negative ILCs within PBMC, and clinical parameters (WBC counts, UPCR, and eGFR). **P

Techniques Used: Activity Assay

Patients with stable LN have higher numbers of circulating CD117 + ILCs and cytokine-producing ILCs. a Differences in clinical parameters of those with active and stable LN. b Changes in total ILCs, CD117 + ILCs, CRTH2 + ILCs, and CD117 − /CRTH2 − ILCs are shown as min. to max. Box-and-whisker plots (line represents the median). c , d Changes in IFN-γ + , IL-13 + , IL-17A + , PD-1 + ILCs, and cytokine-negative ILCs in PBMC from those with active LN ( n = 12) or stable LN ( n = 10). * P
Figure Legend Snippet: Patients with stable LN have higher numbers of circulating CD117 + ILCs and cytokine-producing ILCs. a Differences in clinical parameters of those with active and stable LN. b Changes in total ILCs, CD117 + ILCs, CRTH2 + ILCs, and CD117 − /CRTH2 − ILCs are shown as min. to max. Box-and-whisker plots (line represents the median). c , d Changes in IFN-γ + , IL-13 + , IL-17A + , PD-1 + ILCs, and cytokine-negative ILCs in PBMC from those with active LN ( n = 12) or stable LN ( n = 10). * P

Techniques Used: Whisker Assay

Patients with LN show reduced percentages of CD117 + ILCs in the circulation and in renal tissue. a Gating strategy for blood ILCs. b Representative flow cytometry plots of ILC subsets within the PBMC. c Percentage of total ILCs (CD45 + Lin − CD127 + ), CD117 + ILCs, CRTH2 + ILCs, and CD117 − /CRTH2 − ILCs within the PBMC from HC ( n = 8), LN ( n = 12), AAV ( n = 5), IgAN ( n = 8), and MCD/FSGS ( n = 5). d Representative flow cytometry plots of ILC subsets in urine. e Percentage of CD117 + ILCs and CRTH2 + ILCs in urine from patients with LN ( n = 15), AAV ( n = 6), IgAN ( n = 9), and MCD/FSGS ( n = 5). * P
Figure Legend Snippet: Patients with LN show reduced percentages of CD117 + ILCs in the circulation and in renal tissue. a Gating strategy for blood ILCs. b Representative flow cytometry plots of ILC subsets within the PBMC. c Percentage of total ILCs (CD45 + Lin − CD127 + ), CD117 + ILCs, CRTH2 + ILCs, and CD117 − /CRTH2 − ILCs within the PBMC from HC ( n = 8), LN ( n = 12), AAV ( n = 5), IgAN ( n = 8), and MCD/FSGS ( n = 5). d Representative flow cytometry plots of ILC subsets in urine. e Percentage of CD117 + ILCs and CRTH2 + ILCs in urine from patients with LN ( n = 15), AAV ( n = 6), IgAN ( n = 9), and MCD/FSGS ( n = 5). * P

Techniques Used: Flow Cytometry

Differentiation of ILC progenitors is affected by LN microenvironment. a PBMCs from HC were cultured for 24 h with plasma from either HC ( n = 9) or patients with active LN ( n = 12). b Percentages of CD117 + , CRTH2 + , and CD117 − CRTH2 − ILCs in total ILCs were compared under both conditions. c , d Annexin V was stained to compare the apoptosis of ILCs in either conditions ( n = 8 for HC; n = 10 for LN). e FACS-sorted CD117 + ILCs were cultured for 8 days in plasma from HC ( n = 7) or patients with active LN ( n = 11). f Percentages of CD117 + , CRTH2 + , and CD117 − CRTH2 − ILCs in CD45 + immune cells and total ILCs were compared between two culture systems. g The effect of blocking of IL-1 receptor was compared in HC ( n = 9) and LN ( n = 12) plasma-cultured condition as in Fig. 5a. h Serum IL-1β level was quantified for HC and LN, presented in optical density at 450 nm (OD 450nm ) and concentration (pg/mL). * P
Figure Legend Snippet: Differentiation of ILC progenitors is affected by LN microenvironment. a PBMCs from HC were cultured for 24 h with plasma from either HC ( n = 9) or patients with active LN ( n = 12). b Percentages of CD117 + , CRTH2 + , and CD117 − CRTH2 − ILCs in total ILCs were compared under both conditions. c , d Annexin V was stained to compare the apoptosis of ILCs in either conditions ( n = 8 for HC; n = 10 for LN). e FACS-sorted CD117 + ILCs were cultured for 8 days in plasma from HC ( n = 7) or patients with active LN ( n = 11). f Percentages of CD117 + , CRTH2 + , and CD117 − CRTH2 − ILCs in CD45 + immune cells and total ILCs were compared between two culture systems. g The effect of blocking of IL-1 receptor was compared in HC ( n = 9) and LN ( n = 12) plasma-cultured condition as in Fig. 5a. h Serum IL-1β level was quantified for HC and LN, presented in optical density at 450 nm (OD 450nm ) and concentration (pg/mL). * P

Techniques Used: Cell Culture, Staining, FACS, Blocking Assay, Concentration Assay

23) Product Images from "A randomized phase II trial of personalized peptide vaccine with low dose cyclophosphamide in biliary tract cancer"

Article Title: A randomized phase II trial of personalized peptide vaccine with low dose cyclophosphamide in biliary tract cancer

Journal: Cancer Science

doi: 10.1111/cas.13193

Inhibitory immune cells before and after personalized peptide vaccination ( PPV ). Inhibitory immune cells, including Treg cells, granulocytic myeloid‐derived suppressor cells ( MDSC ), and monocytic MDSC , in peripheral blood mononuclear cells ( PBMC ) were examined before and after PPV (6 th and 12 th vaccination). (a) The percentages of Foxp3 + CD 25 + cells in CD 4 + cells were determined before and after PPV . (b) In the cell subset negative for lineage markers ( CD 3, CD 19, CD 56, CD 14) and HLA ‐ DR in the lymphocyte/monocyte gate, granulocytic MDSC were identified as positive for CD 33 and CD 11b. (c) The percentages of CD 11b + CD 33 + cells in PBMC were determined before and after PPV . (d) Monocytic MDSC were identified as positive for CD 14 and low (or negative) for HLA ‐ DR in the monocyte gate. (e) The percentages of HLA ‐ DR low/− cells in CD 14 + cells were determined before and after PPV . Box plots show median and interquartile range ( IQR ). The whiskers (vertical bars) are the lowest value within 1.5× IQR of the lower quartile and the highest value within 1.5× IQR of the upper quartile. Data not included between the whiskers were plotted as an outlier with dots. “X” shows the mean of the data.
Figure Legend Snippet: Inhibitory immune cells before and after personalized peptide vaccination ( PPV ). Inhibitory immune cells, including Treg cells, granulocytic myeloid‐derived suppressor cells ( MDSC ), and monocytic MDSC , in peripheral blood mononuclear cells ( PBMC ) were examined before and after PPV (6 th and 12 th vaccination). (a) The percentages of Foxp3 + CD 25 + cells in CD 4 + cells were determined before and after PPV . (b) In the cell subset negative for lineage markers ( CD 3, CD 19, CD 56, CD 14) and HLA ‐ DR in the lymphocyte/monocyte gate, granulocytic MDSC were identified as positive for CD 33 and CD 11b. (c) The percentages of CD 11b + CD 33 + cells in PBMC were determined before and after PPV . (d) Monocytic MDSC were identified as positive for CD 14 and low (or negative) for HLA ‐ DR in the monocyte gate. (e) The percentages of HLA ‐ DR low/− cells in CD 14 + cells were determined before and after PPV . Box plots show median and interquartile range ( IQR ). The whiskers (vertical bars) are the lowest value within 1.5× IQR of the lower quartile and the highest value within 1.5× IQR of the upper quartile. Data not included between the whiskers were plotted as an outlier with dots. “X” shows the mean of the data.

Techniques Used: Derivative Assay

24) Product Images from "Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor"

Article Title: Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor

Journal: Cell Proliferation

doi: 10.1111/cpr.12858

Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry
Figure Legend Snippet: Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

Techniques Used: Isolation, Labeling, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Flow Cytometry

25) Product Images from "The Act1 D10N Missense Variant Impairs CD40 Signaling in Human B-Cells"

Article Title: The Act1 D10N Missense Variant Impairs CD40 Signaling in Human B-Cells

Journal: Genes and immunity

doi: 10.1038/s41435-017-0007-7

Act1 is expressed in different subsets of PBMCs. PBMCs were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain, PE-Cy5-coupled anti-CD3, PE-coupled anti-CD19, V450-coupled anti-CD14, PE-Cy7-coupled anti-CD4 and V500-coupled anti-CD8 and eFluor 660-coupled anti-Act1 or eFluor 660-coupled isotype control. CD19 + B-cells, CD4 + T-cells, CD8 + T-cells and CD14 + monocytes were identified. ( a ) Cell subset distributions from a representative flow cytometry experiment (one of four experiments). ( b ) Act1 + cells percentages within each PBMC population from a representative flow cytometry experiment (one of four experiments). Mean Act1 + percentages from all experiments (n = 4). Bars represent mean ± SEM. * P
Figure Legend Snippet: Act1 is expressed in different subsets of PBMCs. PBMCs were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain, PE-Cy5-coupled anti-CD3, PE-coupled anti-CD19, V450-coupled anti-CD14, PE-Cy7-coupled anti-CD4 and V500-coupled anti-CD8 and eFluor 660-coupled anti-Act1 or eFluor 660-coupled isotype control. CD19 + B-cells, CD4 + T-cells, CD8 + T-cells and CD14 + monocytes were identified. ( a ) Cell subset distributions from a representative flow cytometry experiment (one of four experiments). ( b ) Act1 + cells percentages within each PBMC population from a representative flow cytometry experiment (one of four experiments). Mean Act1 + percentages from all experiments (n = 4). Bars represent mean ± SEM. * P

Techniques Used: Staining, Flow Cytometry, Cytometry

26) Product Images from "APOBEC3D and APOBEC3F Potently Promote HIV-1 Diversification and Evolution in Humanized Mouse Model"

Article Title: APOBEC3D and APOBEC3F Potently Promote HIV-1 Diversification and Evolution in Humanized Mouse Model

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004453

Dynamics of HIV-1 vif mutant infection in humanized mice. (A and B) The virus solutions containing 5 ng of p24 antigen (WT HIV-1 [n = 7], 4A HIV-1 [n = 11], 5A HIV-1 [n = 12], and 4A5A HIV-1 [n = 8]) or RPMI 1640 (n = 3; for mock infection) were inoculated into humanized mice, and the amount of viral RNA in plasma (A) and the level of peripheral CD4 + T cells (CD45 + CD3 + CD4 + cells) (B) were analyzed at 0, 1, 2, 3, 5, and 6 wpi. The averages are shown in circles with SEMs, and the values from each mouse are shown by line. X-axes, wpi. In panel A, the detection limit of HIV-1 RNA is 800 copies/ml plasma. (C) Area under the curve (AUC). AUCs of the VL of the mice infected with WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), 4A5A HIV-1 (n = 8) were calculated using the trapezoidal rule as described in Materials and Methods . (D) Virus replication rate. Virus replication rates of WT HIV-1 (n = 7), 4A HIV-1 (n = 21), 5A HIV-1 (n = 27), and 4A5A HIV-1 (n = 19) were estimated by using the data of VL and peripheral CD4 + T cell counts as described in Materials and Methods . In panels C and D, horizontal bars represent the averages. Asterisks represent statistically significant differences ( P
Figure Legend Snippet: Dynamics of HIV-1 vif mutant infection in humanized mice. (A and B) The virus solutions containing 5 ng of p24 antigen (WT HIV-1 [n = 7], 4A HIV-1 [n = 11], 5A HIV-1 [n = 12], and 4A5A HIV-1 [n = 8]) or RPMI 1640 (n = 3; for mock infection) were inoculated into humanized mice, and the amount of viral RNA in plasma (A) and the level of peripheral CD4 + T cells (CD45 + CD3 + CD4 + cells) (B) were analyzed at 0, 1, 2, 3, 5, and 6 wpi. The averages are shown in circles with SEMs, and the values from each mouse are shown by line. X-axes, wpi. In panel A, the detection limit of HIV-1 RNA is 800 copies/ml plasma. (C) Area under the curve (AUC). AUCs of the VL of the mice infected with WT HIV-1 (n = 7), 4A HIV-1 (n = 11), 5A HIV-1 (n = 12), 4A5A HIV-1 (n = 8) were calculated using the trapezoidal rule as described in Materials and Methods . (D) Virus replication rate. Virus replication rates of WT HIV-1 (n = 7), 4A HIV-1 (n = 21), 5A HIV-1 (n = 27), and 4A5A HIV-1 (n = 19) were estimated by using the data of VL and peripheral CD4 + T cell counts as described in Materials and Methods . In panels C and D, horizontal bars represent the averages. Asterisks represent statistically significant differences ( P

Techniques Used: Mutagenesis, Infection, Mouse Assay

27) Product Images from "Factors associated with the elevated percentage of CD4CD69 T cells in maintained hemodialysis patients"

Article Title: Factors associated with the elevated percentage of CD4CD69 T cells in maintained hemodialysis patients

Journal: Renal Failure

doi: 10.1080/0886022X.2017.1349672

The distribution of antigen CD4 and CD69 between the MHD group and healthy controls. (A) the CD4 T cells gated on CD3 T cells and the expression of CD69 in two groups; (B)-(C) the expression of CD4 and CD4CD69 on T cells before and after propensity score matching. * p
Figure Legend Snippet: The distribution of antigen CD4 and CD69 between the MHD group and healthy controls. (A) the CD4 T cells gated on CD3 T cells and the expression of CD69 in two groups; (B)-(C) the expression of CD4 and CD4CD69 on T cells before and after propensity score matching. * p

Techniques Used: Expressing

28) Product Images from "Endothelial microparticles delivering microRNA-155 into T lymphocytes are involved in the initiation of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation"

Article Title: Endothelial microparticles delivering microRNA-155 into T lymphocytes are involved in the initiation of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

Journal: Oncotarget

doi: 10.18632/oncotarget.15579

Inhibition of miR-155 in EMPs influences T lymphocytes differentiation in aGVHD mice After being subjected to lethal total body irradiation, BALB/c mice were transplanted intravenously with 1 × 10 7 BM cells and 2 × 10 7 spleen cells isolated from C57BL/6 donors to establish aGVHD model. EMPs (60 μg) from mouse TNF-α-stimulated MAECs, TNF-α-stimulated MAECs protected by 2.5 μmol/L simvastatin, TNF-α-stimulated MAECs transfected by antagomir-155, TNF-α-stimulated MAECs transfected by antagomir-NC were given intravenously to aGVHD mice on day0 and +7d, retrospectively. Antagomir-155 and antagomir-NC of 25 mg/kg was given intravenously on +7d followed by 5 mg/kg twice weekly up to +21d. Peripheral blood T lymphocytes subsets were assessed by flow cytometry analysis on +21d. Shown is the mean ± SD from three combined independent experiments. * P
Figure Legend Snippet: Inhibition of miR-155 in EMPs influences T lymphocytes differentiation in aGVHD mice After being subjected to lethal total body irradiation, BALB/c mice were transplanted intravenously with 1 × 10 7 BM cells and 2 × 10 7 spleen cells isolated from C57BL/6 donors to establish aGVHD model. EMPs (60 μg) from mouse TNF-α-stimulated MAECs, TNF-α-stimulated MAECs protected by 2.5 μmol/L simvastatin, TNF-α-stimulated MAECs transfected by antagomir-155, TNF-α-stimulated MAECs transfected by antagomir-NC were given intravenously to aGVHD mice on day0 and +7d, retrospectively. Antagomir-155 and antagomir-NC of 25 mg/kg was given intravenously on +7d followed by 5 mg/kg twice weekly up to +21d. Peripheral blood T lymphocytes subsets were assessed by flow cytometry analysis on +21d. Shown is the mean ± SD from three combined independent experiments. * P

Techniques Used: Inhibition, Mouse Assay, Irradiation, Isolation, Transfection, Flow Cytometry, Cytometry

miR-155 in EMPs promotes Th1, Th9 and Th17 and inhibit Th2 and Treg cells differentiation Purified T lymphocytes were co-cultured with 10 μg/mL EMPs from EA.hy926, TNF-α-stimulated EA.hy926, TNF-α-stimulated EA.hy926 protected by 2.5 μmol/L simvastatin, TNF-α-stimulated EA.hy926 transinfected by antagomir-155 and TNF-α-stimulated EA.hy926 transinfected by antagomir-NC, respectively. Cytokines were determined using ELISA kits 3 days after co-culture. T lymphocytes subsets were analyzed by flow cytometry. Data represent mean ± SD from three independent experiments. * P
Figure Legend Snippet: miR-155 in EMPs promotes Th1, Th9 and Th17 and inhibit Th2 and Treg cells differentiation Purified T lymphocytes were co-cultured with 10 μg/mL EMPs from EA.hy926, TNF-α-stimulated EA.hy926, TNF-α-stimulated EA.hy926 protected by 2.5 μmol/L simvastatin, TNF-α-stimulated EA.hy926 transinfected by antagomir-155 and TNF-α-stimulated EA.hy926 transinfected by antagomir-NC, respectively. Cytokines were determined using ELISA kits 3 days after co-culture. T lymphocytes subsets were analyzed by flow cytometry. Data represent mean ± SD from three independent experiments. * P

Techniques Used: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Flow Cytometry, Cytometry

miR-155 expressions in MPs, plasma and T lymphocytes of peripheral blood from BM and aGVHD mice before allo-BMT, at +4d, +8d, +12d, +16d and aGVHD point Average values ± SD from three independent experiments were plotted. ( A ) EMPs isolated from the peripheral blood of BM and aGVHD groups were assessed by flow cytometry analysis. * P
Figure Legend Snippet: miR-155 expressions in MPs, plasma and T lymphocytes of peripheral blood from BM and aGVHD mice before allo-BMT, at +4d, +8d, +12d, +16d and aGVHD point Average values ± SD from three independent experiments were plotted. ( A ) EMPs isolated from the peripheral blood of BM and aGVHD groups were assessed by flow cytometry analysis. * P

Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Cytometry

29) Product Images from "HLF Expression Defines the Human Haematopoietic Stem Cell State"

Article Title: HLF Expression Defines the Human Haematopoietic Stem Cell State

Journal: bioRxiv

doi: 10.1101/2020.06.29.177709

HLF-reporter labels repopulating cells in CD34+ cord blood cell cultures. a) FACS plots showing the sorting of HLF-reporter targeted population for transplantation. rAAV6 HLF-ZE : recombinant rAAV6 particle encoding an HLF repair template with ires ZsGreen P2A tEGFR cassette. b) Summary of transplantation layout. Transplantation cohorts and cell doses are represented using the same color-code as in (a). c) Human engraftment summary of transplanted NSGS recipients. Human bone marrow chimerism determined based on human CD45+ cells among total CD45+ (mouse and human) cells at short (week 3), intermediate (week 9) and long-term (week 16) post-transplantation timepoints is plotted using the same color code as in (a) and (b). Each recipient mouse is represented along the x-axis (NSGS-ID). Recipients are arranged by descending average reconstitution across all timepoints. Recipients #25912, #25914 and #25916 were sacrificed at week 10 post-transplantation to be used as donors for secondary transplantation (summarized in Fig. 5 ). d) Lineage proportion of transplanted recipients. Bone marrow biopsies were analyzed and are arranged along timepoints and the individual recipients as in (e). Normalized proportions of B-cells (CD19), myeloid cells (CD33) and T-cells (CD3) within human CD45 + cells for each timepoint and recipient are color-coded as indicated. e) HR allele frequencies in pre-transplanted cell populations. top panel: ddPCR droplets are pre-gated based on FAM-positivity, black droplets represent FAM+/HEX-events indicative of untargeted alleles, red droplets (FAM/HEX double positive) indicate targeted alleles, sub-sampled to 300 droplets per specimen. bottom panel, quantification summary of HR frequencies calculated based on targeted/(untargeted+targeted) droplets. f) ddPCR analysis of bone marrow biopsies at weeks 3, 9 and 16. Specimens are arranged as in (c), ddPCR droplets are represented as in (e), sub-sampled to 50 droplets per specimen and timepoint. g) HR allele tracing summary. Summarized data representation of (e) and (f). Dashed red lines represent allele frequencies at time of transplantation. Bars represent average HR allele frequencies from (e) with standard error bars, color-codes as in (a-c).
Figure Legend Snippet: HLF-reporter labels repopulating cells in CD34+ cord blood cell cultures. a) FACS plots showing the sorting of HLF-reporter targeted population for transplantation. rAAV6 HLF-ZE : recombinant rAAV6 particle encoding an HLF repair template with ires ZsGreen P2A tEGFR cassette. b) Summary of transplantation layout. Transplantation cohorts and cell doses are represented using the same color-code as in (a). c) Human engraftment summary of transplanted NSGS recipients. Human bone marrow chimerism determined based on human CD45+ cells among total CD45+ (mouse and human) cells at short (week 3), intermediate (week 9) and long-term (week 16) post-transplantation timepoints is plotted using the same color code as in (a) and (b). Each recipient mouse is represented along the x-axis (NSGS-ID). Recipients are arranged by descending average reconstitution across all timepoints. Recipients #25912, #25914 and #25916 were sacrificed at week 10 post-transplantation to be used as donors for secondary transplantation (summarized in Fig. 5 ). d) Lineage proportion of transplanted recipients. Bone marrow biopsies were analyzed and are arranged along timepoints and the individual recipients as in (e). Normalized proportions of B-cells (CD19), myeloid cells (CD33) and T-cells (CD3) within human CD45 + cells for each timepoint and recipient are color-coded as indicated. e) HR allele frequencies in pre-transplanted cell populations. top panel: ddPCR droplets are pre-gated based on FAM-positivity, black droplets represent FAM+/HEX-events indicative of untargeted alleles, red droplets (FAM/HEX double positive) indicate targeted alleles, sub-sampled to 300 droplets per specimen. bottom panel, quantification summary of HR frequencies calculated based on targeted/(untargeted+targeted) droplets. f) ddPCR analysis of bone marrow biopsies at weeks 3, 9 and 16. Specimens are arranged as in (c), ddPCR droplets are represented as in (e), sub-sampled to 50 droplets per specimen and timepoint. g) HR allele tracing summary. Summarized data representation of (e) and (f). Dashed red lines represent allele frequencies at time of transplantation. Bars represent average HR allele frequencies from (e) with standard error bars, color-codes as in (a-c).

Techniques Used: FACS, Transplantation Assay, Recombinant

HLF-reporter labels human HSCs with extensive self-renewal capacity. a) FACS plots of CD34+/HLF-ZsG+ population . Representative bone marrow biopsies of reporter-negative ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG − sorted, left) and reporter-positive ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted, right panel) primary recipients, gated on human CD45+. b) Summary of CD34+/HLF-ZsG+ population. Population overview of all primary recipients, pre-gated on human CD45+, recipient mice are arranged according to engraftment levels as in Fig. 4c . c) Strategy for secondary transplantation. Bone marrow of three primary recipients ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted cohort) was pooled and magnetically enriched for human CD34 expression. Reporter-expressing (ZsG+) and non-expressing cells (ZsG-) with comparable levels of CD34 expression were sorted for transplantation. Intra-hepatic transplantation into newborn NSGS recipients as outlined. Corresponding cell doses of HLF-ZsG+ (n=10) and HLF-ZsG- (n=7) were transplanted. d) Human engraftment summary of secondary recipients. Human bone chimerism in indicated tissues was determined based on human CD45-expressing cells among total (mouse and human) CD45+ cells at short (week 5, blood), intermediate (week 9, marrow) and long-term (week 16, marrow and spleen) post-transplantation. Dashed line represents the 0.1% mark used as cut-off for engraftment positivity. Significance was calculated by unpaired, one-sided (alternative = “greater”) Wilcoxon test and is provided as p-value for a given comparison. d) Lineage distribution of engrafted human cells. Positive specimens from (d) are shown and color-coded for B-cells (CD19+), myeloid cells (CD33+) and T-cells (CD3). Normalized for lineage proportions within human CD45+ cells. Samples with less than 0.1% of human chimerism are designated negative (neg.).
Figure Legend Snippet: HLF-reporter labels human HSCs with extensive self-renewal capacity. a) FACS plots of CD34+/HLF-ZsG+ population . Representative bone marrow biopsies of reporter-negative ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG − sorted, left) and reporter-positive ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted, right panel) primary recipients, gated on human CD45+. b) Summary of CD34+/HLF-ZsG+ population. Population overview of all primary recipients, pre-gated on human CD45+, recipient mice are arranged according to engraftment levels as in Fig. 4c . c) Strategy for secondary transplantation. Bone marrow of three primary recipients ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted cohort) was pooled and magnetically enriched for human CD34 expression. Reporter-expressing (ZsG+) and non-expressing cells (ZsG-) with comparable levels of CD34 expression were sorted for transplantation. Intra-hepatic transplantation into newborn NSGS recipients as outlined. Corresponding cell doses of HLF-ZsG+ (n=10) and HLF-ZsG- (n=7) were transplanted. d) Human engraftment summary of secondary recipients. Human bone chimerism in indicated tissues was determined based on human CD45-expressing cells among total (mouse and human) CD45+ cells at short (week 5, blood), intermediate (week 9, marrow) and long-term (week 16, marrow and spleen) post-transplantation. Dashed line represents the 0.1% mark used as cut-off for engraftment positivity. Significance was calculated by unpaired, one-sided (alternative = “greater”) Wilcoxon test and is provided as p-value for a given comparison. d) Lineage distribution of engrafted human cells. Positive specimens from (d) are shown and color-coded for B-cells (CD19+), myeloid cells (CD33+) and T-cells (CD3). Normalized for lineage proportions within human CD45+ cells. Samples with less than 0.1% of human chimerism are designated negative (neg.).

Techniques Used: FACS, Mouse Assay, Transplantation Assay, Expressing

30) Product Images from "ZNF410 represses fetal globin by devoted control of CHD4/NuRD"

Article Title: ZNF410 represses fetal globin by devoted control of CHD4/NuRD

Journal: bioRxiv

doi: 10.1101/2020.08.31.272856

ZNF410 deficient human HSPCs de-repress HbF and retain repopulation potential. (a) Schematic of gene editing and transplant of human CD34+ HSPCs in immunodeficient NBSGW mice. Animals were euthanized 16 weeks post-transplant and bone marrow (BM) was harvested and sorted into various subpopulations by flow cytometry. (b-e) Two independent CD34+ HSPC donors were edited and transplanted into 6 mice for each condition (mock or ZNF410 edited). Each symbol represents one mouse, recipients of donor 1 depicted as circles and donor 2 as triangles. Bars indicate median value. (b) Indel frequency at ZNF410 was quantified in input cells 4 days after electroporation and in total and sorted engrafted BM cells. Percentage of frameshift alleles is represented in gray and the percentage of in-frame alleles is represented in white for each bar. (c) Engraftment of human hematopoietic cells assessed by hCD45+ compared to total CD45+ cells. (d) B-lymphocytes (CD19+), granulocytes (CD33-dim SSC-high) and monocytes (CD33-bright SSC-low) expressed as fraction of hCD45+ cells. HSPCs (CD34+) and T-lymphocytes (CD3+) expressed as fraction of hCD45+ CD19-CD33-cells. (e) Erythroid cells (hCD235a+) expressed as fraction of hCD45-mCD45-cells. (f) CHD4 expression measured by RT-qPCR in human erythroid cells from control (n=4) and ZNF410 edited (n=4) xenografts. (g) HbF measured by HPLC from hemolysates of sorted BM hCD235a+ cells.
Figure Legend Snippet: ZNF410 deficient human HSPCs de-repress HbF and retain repopulation potential. (a) Schematic of gene editing and transplant of human CD34+ HSPCs in immunodeficient NBSGW mice. Animals were euthanized 16 weeks post-transplant and bone marrow (BM) was harvested and sorted into various subpopulations by flow cytometry. (b-e) Two independent CD34+ HSPC donors were edited and transplanted into 6 mice for each condition (mock or ZNF410 edited). Each symbol represents one mouse, recipients of donor 1 depicted as circles and donor 2 as triangles. Bars indicate median value. (b) Indel frequency at ZNF410 was quantified in input cells 4 days after electroporation and in total and sorted engrafted BM cells. Percentage of frameshift alleles is represented in gray and the percentage of in-frame alleles is represented in white for each bar. (c) Engraftment of human hematopoietic cells assessed by hCD45+ compared to total CD45+ cells. (d) B-lymphocytes (CD19+), granulocytes (CD33-dim SSC-high) and monocytes (CD33-bright SSC-low) expressed as fraction of hCD45+ cells. HSPCs (CD34+) and T-lymphocytes (CD3+) expressed as fraction of hCD45+ CD19-CD33-cells. (e) Erythroid cells (hCD235a+) expressed as fraction of hCD45-mCD45-cells. (f) CHD4 expression measured by RT-qPCR in human erythroid cells from control (n=4) and ZNF410 edited (n=4) xenografts. (g) HbF measured by HPLC from hemolysates of sorted BM hCD235a+ cells.

Techniques Used: Mouse Assay, Flow Cytometry, Electroporation, Expressing, Quantitative RT-PCR, High Performance Liquid Chromatography

31) Product Images from "Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer"

Article Title: Yeast-derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-derived Suppressor Cells by Inducing PMN-MDSC Apoptosis and M-MDSC Differentiation to APC in Cancer

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

doi: 10.4049/jimmunol.1501853

Particulate β-glucan treatment induces M-MDSC differentiation in vivo with reduced tumor growth Tumor M-MDSC sorted from C57Bl/6 LLC tumor-bearing mice (CD45.2) were treated with or without WGP for overnight and then intratumorally injected into SJL tumor-bearing mice (CD45.1). Mice were sacrificed after 7 days and single cell suspensions from tumors were stained with anti-CD45.2 or isotype control mAb (A) and CD11c and MHC class II. Cells were gated on CD45.2 + cells (B). The percentage of CD11c + MHC class II + cells was summarized. (C) M-MDSC sorted from LLC tumor-bearing mice were treated with or without WGP and then mixed with LLC cells for injection. LLC alone was used as control. Tumor growth was monitored and recorded. (D) M-MDSC sorted from LLC tumor-bearing wildtype or dectin-1 KO mice were treated with WGP and mixed with LLC cells for injection. Tumor growth was monitored. *p
Figure Legend Snippet: Particulate β-glucan treatment induces M-MDSC differentiation in vivo with reduced tumor growth Tumor M-MDSC sorted from C57Bl/6 LLC tumor-bearing mice (CD45.2) were treated with or without WGP for overnight and then intratumorally injected into SJL tumor-bearing mice (CD45.1). Mice were sacrificed after 7 days and single cell suspensions from tumors were stained with anti-CD45.2 or isotype control mAb (A) and CD11c and MHC class II. Cells were gated on CD45.2 + cells (B). The percentage of CD11c + MHC class II + cells was summarized. (C) M-MDSC sorted from LLC tumor-bearing mice were treated with or without WGP and then mixed with LLC cells for injection. LLC alone was used as control. Tumor growth was monitored and recorded. (D) M-MDSC sorted from LLC tumor-bearing wildtype or dectin-1 KO mice were treated with WGP and mixed with LLC cells for injection. Tumor growth was monitored. *p

Techniques Used: In Vivo, Mouse Assay, Injection, Staining

Particulate β-glucan reduces tumor Gr-1 + CD11b + MDSC-mediated T cell suppression (A) Tumor Gr-1 + CD11b + CD45 + MDSC sorted from LLC-bearing mice were co-cultured with CFSE-labeled OT-II splenocytes at indicated ratios, in the presence of OVA (100 µg/ml) with or without particulate β-glucan (50 µg/ml) for 3–4 days. Data represent the frequency of CFSE diluted cells gated on CD4 + T cells. The experiment was repeated twice with similar results. (B) Same cell cultures as (A) were further stimulated with PMA/Ionomycin for intracellular IFN-γ staining. The experiment was repeated twice with similar results. (C) Splenocytes of OT-I mice were co-cultured with sorted Gr-1 + CD11b + CD45 + tumor MDSC from LLC-bearing mice at indicated ratios, in the presence of OVA (50 µg/ml in M-MDSC cultures and 10 µg/ml in PMN-MDSC cultures) with or without particulate β-glucan (50 µg/ml). Data represent the percentage of IFN-γ + cells and CFSE diluted cells gated on CD8 + T cells. Results are representative of two independent experiments. *p
Figure Legend Snippet: Particulate β-glucan reduces tumor Gr-1 + CD11b + MDSC-mediated T cell suppression (A) Tumor Gr-1 + CD11b + CD45 + MDSC sorted from LLC-bearing mice were co-cultured with CFSE-labeled OT-II splenocytes at indicated ratios, in the presence of OVA (100 µg/ml) with or without particulate β-glucan (50 µg/ml) for 3–4 days. Data represent the frequency of CFSE diluted cells gated on CD4 + T cells. The experiment was repeated twice with similar results. (B) Same cell cultures as (A) were further stimulated with PMA/Ionomycin for intracellular IFN-γ staining. The experiment was repeated twice with similar results. (C) Splenocytes of OT-I mice were co-cultured with sorted Gr-1 + CD11b + CD45 + tumor MDSC from LLC-bearing mice at indicated ratios, in the presence of OVA (50 µg/ml in M-MDSC cultures and 10 µg/ml in PMN-MDSC cultures) with or without particulate β-glucan (50 µg/ml). Data represent the percentage of IFN-γ + cells and CFSE diluted cells gated on CD8 + T cells. Results are representative of two independent experiments. *p

Techniques Used: Mouse Assay, Cell Culture, Labeling, Staining

Particulate β-glucan treatment in vivo reduces tumor burden and impacts the frequency of MDSC in spleens and tumors of LLC and E0771-bearing mice (A) C57BL/6 WT mice (n=7, 8) were injected subcutaneously (s.c) with LLC or E0771 tumor cell lines. Once palpable tumors were formed (day 8), mice were orally administered with particulate β-glucan (800 µg, daily) or PBS with a gavage needle at indicated time. Tumor diameters were measured every three days and tumor volumes were then calculated. (B) On day 32 (LLC model) or day 35 (E0771 model), mice were killed and spleens were excised and weighed. Each point in the data plot represents the spleen weight of each mouse in grams. PBS-treated group was compared to particulate β-glucan treated group (WGP) in both models (C) Tumor tissues were excised and weighted from WGP or PBS-treated mice. (D) Flow cytometry analysis of the frequencies of M-MDSC (Ly6G − Ly6C high ) and PMN-MDSC (Ly6G + Ly6C int ) in the spleens of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD11b + cells. (E) Frequencies of M-MDSC and PMN-MDSC in the tumors of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD45 + CD11b + cells. *p
Figure Legend Snippet: Particulate β-glucan treatment in vivo reduces tumor burden and impacts the frequency of MDSC in spleens and tumors of LLC and E0771-bearing mice (A) C57BL/6 WT mice (n=7, 8) were injected subcutaneously (s.c) with LLC or E0771 tumor cell lines. Once palpable tumors were formed (day 8), mice were orally administered with particulate β-glucan (800 µg, daily) or PBS with a gavage needle at indicated time. Tumor diameters were measured every three days and tumor volumes were then calculated. (B) On day 32 (LLC model) or day 35 (E0771 model), mice were killed and spleens were excised and weighed. Each point in the data plot represents the spleen weight of each mouse in grams. PBS-treated group was compared to particulate β-glucan treated group (WGP) in both models (C) Tumor tissues were excised and weighted from WGP or PBS-treated mice. (D) Flow cytometry analysis of the frequencies of M-MDSC (Ly6G − Ly6C high ) and PMN-MDSC (Ly6G + Ly6C int ) in the spleens of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD11b + cells. (E) Frequencies of M-MDSC and PMN-MDSC in the tumors of LLC and E0771-bearing mice treated with PBS or particulate β-glucan (WGP). Cells were gated on CD45 + CD11b + cells. *p

Techniques Used: In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry

32) Product Images from "LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration"

Article Title: LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration

Journal: Nature

doi: 10.1038/s41586-018-0615-z

LILRB4 promotes infiltration of AML cells. a and c, Examination of LILRB4 expression on mouse AML cells, C1498 ( a ) or WEHI-3 ( c ) that stably express lilrb4. b and d, Forced expression of LILRB4 did not affect cell proliferation of mouse AML cells, C1498 ( b, n=3 biologically independent samples with mean and s.e.m.) or WEHI-3 ( d , n=3 biologically independent samples with mean and s.e.m.). e, Forced expression of human LILRB4 promoted transendothelial migration of mouse AML WEHI-3 cells (n=3 biologically independent samples with mean and s.e.m.). f, NSG mice (n=6 mice) were injected with 1×10 6 THP-1 cells followed immediately by IgG or anti-LILRB4 antibody treatment and were monitored by bioluminescence imaging. g-h, Anti-LILRB4 antibodies decreased AML cells infiltration into internal organs. Mice were sacrificed at 21 days for ex vivo bioluminescence imaging of internal organs after transplantation of 1×10 6 luciferase-expressed THP-1 cells. Images of luminescence flux (radiance) from representative mice are shown ( g ). 1: GI tract; 2: legs; 3: lung; 4: spleen; 5: liver; 6: kidneys; 7: brain; 8: heart. Infiltrated leukemia cells formed tumor nodules in liver ( h ). i-j, Anti-LILRB4 antibodies did not have effect on LILRB4-negative cancer cells. LILRB4 is expressed on THP-1 and MV4–11 human AML cells but not on U937 cells as analyzed by flow cytometry ( i ). Isotype IgG was used as control. NSG mice were injected with U937 human AML cells, which do not express LILRB4, and then treated with anti-LILRB4 antibodies ( j ). IgG served as control antibodies. Mice were sacrificed at day 25 post-transplant for analysis of LV, BM, SP, and PB by flow cytometry. The presence of human AML cells was detected by anti-human CD45 antibody staining (n=4 mice with mean and s.e.m.). k-t, Anti-LILRB4 antibodies decreased infiltration of THP-1 ( k-o ) or MV4–11 ( p-t ) human AML cells. Comparison of transendothelial migration abilities of GFP-expressing THP-1 ( k ) or CFSE-labeled MV4–11 ( p ) cells after treatment with anti-LILRB4 antibody or IgG control in a transwell assay (n=3 biologically independent samples with mean and s.e.m.). Comparison of the homing abilities of CFSE-labeled MV4–11 cells (5×10 6 per mouse) that were injected into NSG mice followed immediately by IgG or anti-LILRB4 antibody treatment at 20 hr post-injection (n=5 mice). Numbers of leukemia cells (GFP + in l or CFSE + in q ) in LV, SP, and BM normalized to that in PB as determined by flow cytometry are shown. NSG mice were injected with 1×10 6 THP-1 or MV4–11 cells followed immediately by IgG or anti-LILRB4 antibody treatment (n=6 mice for THP-1 or 5 mice for MV4–11 xenografts). Shown are percentages of MV4–11 cells (stained with anti-human CD45) as determined by flow cytometry in indicated organs at day 21 post-transplant ( m and r ), overall survival ( n and s ), and body weights as a function of time ( o and t ). u, Targeted immune cell populations were depleted in NSG mice. Representative flow cytometry plots demonstrating successful reduction of NK cell (CD45 + CD49b + ), macrophage (CD11b + F4/80 + ), and neutrophil (CD11b + CD11c - ) frequency in NSG mice depleted of the respective immune cell subtype by treatment with anti-asialo GM1 antibodies, clodronate liposomes, and anti-Ly6G antibodies, respectively, compared to non-depleted (wild-type) NSG mice. v, CFSE-labeled MV4–11 cells (5×10 6 per mouse) were injected into NSG mice in that respective innate immune cells were depleted, followed immediately by IgG or anti-LILRB4-N297A antibody treatment (n=5 mice). Numbers of leukemia cells (CFSE positive) in LV, SP, and BM normalized to that in PB at 20 hr post-injection are shown. ( a, c, i and u ) These experiments were repeated independently three times with similar results. See Methods for definition of box plot elements in l-m, o, q-r, t and v . All p values (except of n and s from long-rank test) were from two-tailed student t -test.
Figure Legend Snippet: LILRB4 promotes infiltration of AML cells. a and c, Examination of LILRB4 expression on mouse AML cells, C1498 ( a ) or WEHI-3 ( c ) that stably express lilrb4. b and d, Forced expression of LILRB4 did not affect cell proliferation of mouse AML cells, C1498 ( b, n=3 biologically independent samples with mean and s.e.m.) or WEHI-3 ( d , n=3 biologically independent samples with mean and s.e.m.). e, Forced expression of human LILRB4 promoted transendothelial migration of mouse AML WEHI-3 cells (n=3 biologically independent samples with mean and s.e.m.). f, NSG mice (n=6 mice) were injected with 1×10 6 THP-1 cells followed immediately by IgG or anti-LILRB4 antibody treatment and were monitored by bioluminescence imaging. g-h, Anti-LILRB4 antibodies decreased AML cells infiltration into internal organs. Mice were sacrificed at 21 days for ex vivo bioluminescence imaging of internal organs after transplantation of 1×10 6 luciferase-expressed THP-1 cells. Images of luminescence flux (radiance) from representative mice are shown ( g ). 1: GI tract; 2: legs; 3: lung; 4: spleen; 5: liver; 6: kidneys; 7: brain; 8: heart. Infiltrated leukemia cells formed tumor nodules in liver ( h ). i-j, Anti-LILRB4 antibodies did not have effect on LILRB4-negative cancer cells. LILRB4 is expressed on THP-1 and MV4–11 human AML cells but not on U937 cells as analyzed by flow cytometry ( i ). Isotype IgG was used as control. NSG mice were injected with U937 human AML cells, which do not express LILRB4, and then treated with anti-LILRB4 antibodies ( j ). IgG served as control antibodies. Mice were sacrificed at day 25 post-transplant for analysis of LV, BM, SP, and PB by flow cytometry. The presence of human AML cells was detected by anti-human CD45 antibody staining (n=4 mice with mean and s.e.m.). k-t, Anti-LILRB4 antibodies decreased infiltration of THP-1 ( k-o ) or MV4–11 ( p-t ) human AML cells. Comparison of transendothelial migration abilities of GFP-expressing THP-1 ( k ) or CFSE-labeled MV4–11 ( p ) cells after treatment with anti-LILRB4 antibody or IgG control in a transwell assay (n=3 biologically independent samples with mean and s.e.m.). Comparison of the homing abilities of CFSE-labeled MV4–11 cells (5×10 6 per mouse) that were injected into NSG mice followed immediately by IgG or anti-LILRB4 antibody treatment at 20 hr post-injection (n=5 mice). Numbers of leukemia cells (GFP + in l or CFSE + in q ) in LV, SP, and BM normalized to that in PB as determined by flow cytometry are shown. NSG mice were injected with 1×10 6 THP-1 or MV4–11 cells followed immediately by IgG or anti-LILRB4 antibody treatment (n=6 mice for THP-1 or 5 mice for MV4–11 xenografts). Shown are percentages of MV4–11 cells (stained with anti-human CD45) as determined by flow cytometry in indicated organs at day 21 post-transplant ( m and r ), overall survival ( n and s ), and body weights as a function of time ( o and t ). u, Targeted immune cell populations were depleted in NSG mice. Representative flow cytometry plots demonstrating successful reduction of NK cell (CD45 + CD49b + ), macrophage (CD11b + F4/80 + ), and neutrophil (CD11b + CD11c - ) frequency in NSG mice depleted of the respective immune cell subtype by treatment with anti-asialo GM1 antibodies, clodronate liposomes, and anti-Ly6G antibodies, respectively, compared to non-depleted (wild-type) NSG mice. v, CFSE-labeled MV4–11 cells (5×10 6 per mouse) were injected into NSG mice in that respective innate immune cells were depleted, followed immediately by IgG or anti-LILRB4-N297A antibody treatment (n=5 mice). Numbers of leukemia cells (CFSE positive) in LV, SP, and BM normalized to that in PB at 20 hr post-injection are shown. ( a, c, i and u ) These experiments were repeated independently three times with similar results. See Methods for definition of box plot elements in l-m, o, q-r, t and v . All p values (except of n and s from long-rank test) were from two-tailed student t -test.

Techniques Used: Expressing, Stable Transfection, Migration, Mouse Assay, Injection, Imaging, Ex Vivo, Transplantation Assay, Luciferase, Flow Cytometry, Cytometry, Staining, Labeling, Transwell Assay, Two Tailed Test

33) Product Images from "Retinoic Acid Exerts Disease Stage-Dependent Effects on Pristane-Induced Lupus"

Article Title: Retinoic Acid Exerts Disease Stage-Dependent Effects on Pristane-Induced Lupus

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00408

Enhancement of systemic inflammation by tRA pre-treatment in pristane-induced lupus. (A,B) The percentages of bone marrow cDC subsets determined by flow cytometry at the experimental endpoint (6 months post induction of lupus) are shown. Expansion of CD11b + (A) and CD11b − (B) cDCs. (C,D) Upregulation of the inflammatory marker Ly6C on CD11b + (C) and CD11b − (D) subsets of cDCs. (E,F) Percentage of MHC-II high cells on CD11b + (E) and CD11b − (F) cDCs. (G) Immunohistochemical stains of splenic sections showing the infiltration of T cells (CD3, purple) and the expression of CD11/CD18 (or LFA1, red) and CD54 (or ICAM1, green). Pictures were captured with a Zeiss LSM 880 confocal microscope (red arrows indicate the interaction between LFA1 and ICAM1). Significant differences were shown as * p
Figure Legend Snippet: Enhancement of systemic inflammation by tRA pre-treatment in pristane-induced lupus. (A,B) The percentages of bone marrow cDC subsets determined by flow cytometry at the experimental endpoint (6 months post induction of lupus) are shown. Expansion of CD11b + (A) and CD11b − (B) cDCs. (C,D) Upregulation of the inflammatory marker Ly6C on CD11b + (C) and CD11b − (D) subsets of cDCs. (E,F) Percentage of MHC-II high cells on CD11b + (E) and CD11b − (F) cDCs. (G) Immunohistochemical stains of splenic sections showing the infiltration of T cells (CD3, purple) and the expression of CD11/CD18 (or LFA1, red) and CD54 (or ICAM1, green). Pictures were captured with a Zeiss LSM 880 confocal microscope (red arrows indicate the interaction between LFA1 and ICAM1). Significant differences were shown as * p

Techniques Used: Flow Cytometry, Marker, Immunohistochemistry, Expressing, Microscopy

34) Product Images from "Increased Frequency of Peripheral B and T Cells Expressing Granulocyte Monocyte Colony-Stimulating Factor in Rheumatoid Arthritis Patients"

Article Title: Increased Frequency of Peripheral B and T Cells Expressing Granulocyte Monocyte Colony-Stimulating Factor in Rheumatoid Arthritis Patients

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.01967

B cell phenotype of rheumatoid arthritis (RA) granulocyte monocyte colony-stimulating factor (GM-CSF+) and − cells. Freshly isolated PBMCs from 15 RA patients were stimulated with PMA and inonomycin and then stained with specific antibodies against CD19 (anti-CD19-FITC), GM-CSF (anti-GM-CSF-PE), CD27 (anti-CD27-APC), and CD38 (anti-CD38-PE/Cy5, as described in Materials and Methods). B cells (CD19+) either GM-CSF+ or − were classified as: transitional (CD38+ CD27−), memory (CD27+ CD38−), plasmablasts (CD27+ CD38+), or naive B cells (CD27− CD38−). The % of each B cell subpopulation was compared between GM-CSF+ (black bars) and − (white bars) cells. The bars represent the mean ± SD (%) of each subtype. The statistically significant p values (
Figure Legend Snippet: B cell phenotype of rheumatoid arthritis (RA) granulocyte monocyte colony-stimulating factor (GM-CSF+) and − cells. Freshly isolated PBMCs from 15 RA patients were stimulated with PMA and inonomycin and then stained with specific antibodies against CD19 (anti-CD19-FITC), GM-CSF (anti-GM-CSF-PE), CD27 (anti-CD27-APC), and CD38 (anti-CD38-PE/Cy5, as described in Materials and Methods). B cells (CD19+) either GM-CSF+ or − were classified as: transitional (CD38+ CD27−), memory (CD27+ CD38−), plasmablasts (CD27+ CD38+), or naive B cells (CD27− CD38−). The % of each B cell subpopulation was compared between GM-CSF+ (black bars) and − (white bars) cells. The bars represent the mean ± SD (%) of each subtype. The statistically significant p values (

Techniques Used: Isolation, Staining

35) Product Images from "In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice"

Article Title: In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice

Journal: Journal of Virology

doi: 10.1128/JVI.00563-15

Effect of in vivo IL-15 superagonist treatment and/or HIV-1 infection on the populations of human NK cells and T cells in hu-spl-PBMC-NSG mouse spleens. NSG mice were intrasplenically injected with activated human PBMCs (∼8 × 10 6 cells). In parallel, some of these mice were also inoculated by intrasplenic injection with HIV-LucR (1 × 10 7 IU). One day later, some of the uninoculated and HIV-LucR-inoculated mice were intravenously treated with the IL-15 superagonist (0.2 mg/kg). Six days later, the spleens were harvested, and after gating on the live human CD45 + population, the fraction of splenocytes that were NK cells (CD3 − CD56 + CD16 + ) or T cells (CD3 + ) (A) and CD8 + T cells or CD4 + T cells (B) was determined by flow cytometry. Mean values ± standard error of the mean for each group of hu-spl-PBMC-NSG mice, which were inoculated with HIV-LucR and/or treated with the IL-15 superagonist, as indicated, are shown.
Figure Legend Snippet: Effect of in vivo IL-15 superagonist treatment and/or HIV-1 infection on the populations of human NK cells and T cells in hu-spl-PBMC-NSG mouse spleens. NSG mice were intrasplenically injected with activated human PBMCs (∼8 × 10 6 cells). In parallel, some of these mice were also inoculated by intrasplenic injection with HIV-LucR (1 × 10 7 IU). One day later, some of the uninoculated and HIV-LucR-inoculated mice were intravenously treated with the IL-15 superagonist (0.2 mg/kg). Six days later, the spleens were harvested, and after gating on the live human CD45 + population, the fraction of splenocytes that were NK cells (CD3 − CD56 + CD16 + ) or T cells (CD3 + ) (A) and CD8 + T cells or CD4 + T cells (B) was determined by flow cytometry. Mean values ± standard error of the mean for each group of hu-spl-PBMC-NSG mice, which were inoculated with HIV-LucR and/or treated with the IL-15 superagonist, as indicated, are shown.

Techniques Used: In Vivo, Infection, Mouse Assay, Injection, Flow Cytometry, Cytometry

In vivo activation of human NK cells by the IL-15 superagonist in spleens of HIV-1-infected hu-spl-PBMC-NSG mice. NSG mice were intrasplenically injected with human PBMCs (∼8 × 10 6 cells) and in parallel inoculated with HIV-LucR (1 × 10 7 IU) and then either untreated or treated with one intravenous dose of the IL-15 superagonist (0.2 mg/kg) 1 day after infection. Two days later, the harvested splenocytes were analyzed by flow cytometry for the expression of human CD3, CD56, CD16, and CD69. After gating on the human CD3 − /CD56 + /CD16 + NK cell population, the fraction of NK cells expressing CD69 was determined. The data points for each mouse are shown with the mean ± standard error for each group. ***, P
Figure Legend Snippet: In vivo activation of human NK cells by the IL-15 superagonist in spleens of HIV-1-infected hu-spl-PBMC-NSG mice. NSG mice were intrasplenically injected with human PBMCs (∼8 × 10 6 cells) and in parallel inoculated with HIV-LucR (1 × 10 7 IU) and then either untreated or treated with one intravenous dose of the IL-15 superagonist (0.2 mg/kg) 1 day after infection. Two days later, the harvested splenocytes were analyzed by flow cytometry for the expression of human CD3, CD56, CD16, and CD69. After gating on the human CD3 − /CD56 + /CD16 + NK cell population, the fraction of NK cells expressing CD69 was determined. The data points for each mouse are shown with the mean ± standard error for each group. ***, P

Techniques Used: In Vivo, Activation Assay, Infection, Mouse Assay, Injection, Flow Cytometry, Cytometry, Expressing

Treatment of human PBMCs with the IL-15 superagonist increases their cytotoxic activity. (A) Schematic structure of the IL-15 superagonist. (B and C) Intracellular perforin expression (B) and intracellular granzyme B expression (C) in CD3 − CD16 + CD56 + cells were determined by flow cytometry after human PBMCs from six seronegative donors were incubated for 2 days with the indicated concentrations of the IL-15 superagonist. Data represent the mean ± standard error of MFI values obtained from the six donors. (D) After human PBMCs were stimulated with the indicated concentrations of the IL-15 superagonist for 16 h, they were coincubated with K562 target cells (5:1 E:T ratio) for 4 h, and CD107a expression by CD3 − CD56 + NK cells was determined by flow cytometry. The results are expressed as mean percentages of CD107a-positive CD3 − CD56 + NK cells from PBMCs isolated from 4 normal donors ± standard errors. (E) Human PBMCs were cultured with K562 cells at the indicated E:T ratios with or without the IL-15 superagonist (10 nM). After 3 days, the fraction of dead K562 cells was quantified by flow cytometry. (F) Human PBMCs were cultured with activated ACH2 cells with or without the indicated doses of the IL-15 superagonist. After 24 h, the percent cell lysis of target cells was measured by using the CytoTox-One homogeneous membrane integrity assay. Data represent results from at least three replicates per group with the mean ± standard error for the group. *, P
Figure Legend Snippet: Treatment of human PBMCs with the IL-15 superagonist increases their cytotoxic activity. (A) Schematic structure of the IL-15 superagonist. (B and C) Intracellular perforin expression (B) and intracellular granzyme B expression (C) in CD3 − CD16 + CD56 + cells were determined by flow cytometry after human PBMCs from six seronegative donors were incubated for 2 days with the indicated concentrations of the IL-15 superagonist. Data represent the mean ± standard error of MFI values obtained from the six donors. (D) After human PBMCs were stimulated with the indicated concentrations of the IL-15 superagonist for 16 h, they were coincubated with K562 target cells (5:1 E:T ratio) for 4 h, and CD107a expression by CD3 − CD56 + NK cells was determined by flow cytometry. The results are expressed as mean percentages of CD107a-positive CD3 − CD56 + NK cells from PBMCs isolated from 4 normal donors ± standard errors. (E) Human PBMCs were cultured with K562 cells at the indicated E:T ratios with or without the IL-15 superagonist (10 nM). After 3 days, the fraction of dead K562 cells was quantified by flow cytometry. (F) Human PBMCs were cultured with activated ACH2 cells with or without the indicated doses of the IL-15 superagonist. After 24 h, the percent cell lysis of target cells was measured by using the CytoTox-One homogeneous membrane integrity assay. Data represent results from at least three replicates per group with the mean ± standard error for the group. *, P

Techniques Used: Activity Assay, Expressing, Flow Cytometry, Cytometry, Incubation, Isolation, Cell Culture, Lysis, Integrity Assay

NK cells mediate IL-15 superagonist inhibition of HIV-1 infection in hu-spl-PBMC-NSG mice. Activated PBMCs that were unfractionated or were depleted of CD8 + T cells or NK cells by immunomagnetic sorting were intrasplenically injected into NSG mice (∼8 × 10 6 cells) in parallel with HIV-LucR (1 × 10 7 IU). One day later, the mice were treated with an intravenous dose of the IL-15 superagonist or PBS. (A) The effectiveness of the depletion of CD8 + T cells or CD56 + NK cells from the PBMCs prior to injection was assessed by flow cytometry after staining with antibodies to human CD3, CD8, and CD56. (B) Six days after intrasplenic injection, HIV-1 infection in the spleens of mice treated as indicated was quantified by measuring LucR activity in the splenic lysates. The dot plot shows the LucR values for each mouse with the mean ± standard error for each group. ****, P
Figure Legend Snippet: NK cells mediate IL-15 superagonist inhibition of HIV-1 infection in hu-spl-PBMC-NSG mice. Activated PBMCs that were unfractionated or were depleted of CD8 + T cells or NK cells by immunomagnetic sorting were intrasplenically injected into NSG mice (∼8 × 10 6 cells) in parallel with HIV-LucR (1 × 10 7 IU). One day later, the mice were treated with an intravenous dose of the IL-15 superagonist or PBS. (A) The effectiveness of the depletion of CD8 + T cells or CD56 + NK cells from the PBMCs prior to injection was assessed by flow cytometry after staining with antibodies to human CD3, CD8, and CD56. (B) Six days after intrasplenic injection, HIV-1 infection in the spleens of mice treated as indicated was quantified by measuring LucR activity in the splenic lysates. The dot plot shows the LucR values for each mouse with the mean ± standard error for each group. ****, P

Techniques Used: Inhibition, Infection, Mouse Assay, Injection, Flow Cytometry, Cytometry, Staining, Activity Assay

36) Product Images from "Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit"

Article Title: Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit

Journal: mAbs

doi: 10.1080/19420862.2019.1654303

Sintilimab showed in vitro and in vivo higher levels of PD-1 receptor occupancy. Human PBMC were stimulated to express PD-1 before incubation with sintilimab, MDX-1106 or MK-3475. Flow cytometry results showing proportions of CD3+ T cells that bind with different anti-PD-1 mAbs (a) and the mean fluorescence intensity of PD-1 (b). Data are expressed as the means ± SE of three independent experiments. (c) The effects of anti-PD-1 mAbs on mixed lymphocyte reaction (MLR) response. CD4+ T cells isolated from human PBMC were co-cultured with mature monocyte-derived dendritic cells at a ratio of 10:1 in the presence of different concentrations of anti-PD-1 mAbs. Twelve hours later, unbound mAbs was removed. Cells were co-cultured for 4 more days and the concentration of IL-2 in cultural supernatant was detected by Cisbio kit. In NOG mice reconstituted with human immune cells, PD-1 receptor occupancy on circulating CD3+ T cells 24 h (d) and 72 h (e) after anti-PD-1 mAbs intraperitoneal injection at doses of 1, 3 and 10 mg/kg ( n ≥ 3 mice/group). (f) Mean (± SE) serum concentration-time profiles following a single IV administration of 10 mg/kg sintilimab, MDX-1106 or MK-3475 to hPD-1 knock-in mice (n = 3 animals per group).
Figure Legend Snippet: Sintilimab showed in vitro and in vivo higher levels of PD-1 receptor occupancy. Human PBMC were stimulated to express PD-1 before incubation with sintilimab, MDX-1106 or MK-3475. Flow cytometry results showing proportions of CD3+ T cells that bind with different anti-PD-1 mAbs (a) and the mean fluorescence intensity of PD-1 (b). Data are expressed as the means ± SE of three independent experiments. (c) The effects of anti-PD-1 mAbs on mixed lymphocyte reaction (MLR) response. CD4+ T cells isolated from human PBMC were co-cultured with mature monocyte-derived dendritic cells at a ratio of 10:1 in the presence of different concentrations of anti-PD-1 mAbs. Twelve hours later, unbound mAbs was removed. Cells were co-cultured for 4 more days and the concentration of IL-2 in cultural supernatant was detected by Cisbio kit. In NOG mice reconstituted with human immune cells, PD-1 receptor occupancy on circulating CD3+ T cells 24 h (d) and 72 h (e) after anti-PD-1 mAbs intraperitoneal injection at doses of 1, 3 and 10 mg/kg ( n ≥ 3 mice/group). (f) Mean (± SE) serum concentration-time profiles following a single IV administration of 10 mg/kg sintilimab, MDX-1106 or MK-3475 to hPD-1 knock-in mice (n = 3 animals per group).

Techniques Used: In Vitro, In Vivo, Incubation, Flow Cytometry, Cytometry, Fluorescence, Isolation, Cell Culture, Derivative Assay, Concentration Assay, Mouse Assay, Injection, Knock-In

37) Product Images from "Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis"

Article Title: Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis

Journal: Nature immunology

doi: 10.1038/s41590-018-0103-5

Cav2 and cJun genes localize to the nuclear periphery and Nup210 is required for the repression of the proapoptoticreceptor Fas ( a ) DNA-FISH analysis of Cav2 and Nup62 in Nup210 +/+ naïve CD4 + T cells. Gene loci are shown in red and the nuclear periphery marker Lamin B1 in green. ( b ) The percentage of nuclei from (a) that show one or both loci associated with the nuclear periphery was quantified from 3D nuclear reconstructions (see Supplementary Fig. 8a ). ( c ) qPCR analysis of Cav1 , Cav2 , and Irf4 mRNA levels in Nup210 +/+ naïve CD4 + T cells unstimulated or activated with soluble anti-CD3. Expression was normalized to Hprt . ( d ) DNA-FISH of cJun in Nup210 +/+ naïve CD4 + T cells. Gene loci (red) and Lamin B1 (green). ( e ) Chromatin immunoprecipitation (ChIP) analysis of STAT3 binding to the Fas promoter in unstimulated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. Binding of STAT3 was quantified by qPCR. ( f ) Flow cytometric viability analysis of Nup210 +/+ or Nup210 −/− naïve CD4 + T cells co-cultured with splenocytes from TCRbeta- TCRdelta- mice and soluble FasL. Viability was determined using annexin V and propidium iodide staining. ( a , d ) representative of three independent experiments; ( b ) mean ± s.d. pooled from three independent experiments, n ≥ 200 cells quantified from n = 4, 5 or 8 individual fields, symbols show individual field quantification; ( c ) mean ± s.e.m, n = 5 mice per time point, representative of one experiment; ( e ) mean ± s.d., each symbol represents a technical replicate, n = 3, representative of two independent experiments, ( f ) mean ± s.e.m, each symbol represents an individual mouse, n = 9 mice per group, data pooled from two independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test). Scale bar: 5 μm.
Figure Legend Snippet: Cav2 and cJun genes localize to the nuclear periphery and Nup210 is required for the repression of the proapoptoticreceptor Fas ( a ) DNA-FISH analysis of Cav2 and Nup62 in Nup210 +/+ naïve CD4 + T cells. Gene loci are shown in red and the nuclear periphery marker Lamin B1 in green. ( b ) The percentage of nuclei from (a) that show one or both loci associated with the nuclear periphery was quantified from 3D nuclear reconstructions (see Supplementary Fig. 8a ). ( c ) qPCR analysis of Cav1 , Cav2 , and Irf4 mRNA levels in Nup210 +/+ naïve CD4 + T cells unstimulated or activated with soluble anti-CD3. Expression was normalized to Hprt . ( d ) DNA-FISH of cJun in Nup210 +/+ naïve CD4 + T cells. Gene loci (red) and Lamin B1 (green). ( e ) Chromatin immunoprecipitation (ChIP) analysis of STAT3 binding to the Fas promoter in unstimulated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. Binding of STAT3 was quantified by qPCR. ( f ) Flow cytometric viability analysis of Nup210 +/+ or Nup210 −/− naïve CD4 + T cells co-cultured with splenocytes from TCRbeta- TCRdelta- mice and soluble FasL. Viability was determined using annexin V and propidium iodide staining. ( a , d ) representative of three independent experiments; ( b ) mean ± s.d. pooled from three independent experiments, n ≥ 200 cells quantified from n = 4, 5 or 8 individual fields, symbols show individual field quantification; ( c ) mean ± s.e.m, n = 5 mice per time point, representative of one experiment; ( e ) mean ± s.d., each symbol represents a technical replicate, n = 3, representative of two independent experiments, ( f ) mean ± s.e.m, each symbol represents an individual mouse, n = 9 mice per group, data pooled from two independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test). Scale bar: 5 μm.

Techniques Used: Fluorescence In Situ Hybridization, Marker, Real-time Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, Flow Cytometry, Cell Culture, Mouse Assay, Staining, Two Tailed Test

Nup210 is required for TCR signaling ( a ) Immunoblot analysis of p-Lck (Y394) and total Lck protein levels in Nup210 +/+ and Nup210 −/− naïve CD4 + T cells. Hsp90 used as loading control. ( b , c ) Flow cytometric analysis of p-Lck (Y505) expression (b) and mean fluorescence intensity (MFI) (c) in resting naïve CD4 + (CD4 + CD62L hi CD44 lo CD25 − ) T cells from Nup210 +/+ and Nup210 −/− mice. Activation with sodium peroxyvanadate used as positive control. Secondary Ab: fluorescently labeled secondary antibody stained sample. MFI is shown relative to that of Nup210 +/+ mice. ( d , e ) Flow cytometric analysis of p-PLC-γ1 (Y783) expression (d) and MFI (e) in resting naïve CD4 + (CD4 + CD62L hi CD44 lo CD25 − ) T cells from Nup210 +/+ and Nup210 −/− mice. ( f ) Flow cytometric analysis of Nur77 expression in Nup210 +/+ or Nup210 −/− CD4 + CD44 lo T cells stimulated for 16 hours with soluble (sol.) anti-CD3 mAb. Values are displayed relative to sol. anti-CD3-stimulated Nup210 +/+ cells. ( g ) Flow cytometric analysis of Nur77 expression in unstimulated (left) or TCR-stimulated (right) Nup210 +/+ and Nup210 −/− CD4 + CD44 lo T cells. TCR stimulation was performed with plate-bound anti-CD3 and anti-CD28 mAbs, and soluble IL-2 for 14 hours. FMO, fluorescence minus one. ( h ) Flow cytometric analysis of Nur77 levels in CD4 + T cells stimulated as in (g) or without IL-2 supplementation. Values shown are relative to control treated cells. ( i ) Flow cytometric analysis of CFSE and CTV dilutions 48 hours after stimulation of CFSE-labeled Nup210 +/+ and CTV-labeled Nup210 −/− CD4 + CD44 lo T cells with plate-bound anti-CD3/ anti-CD28 mAbs and soluble IL-2. The percentage of cells that have undergone proliferation is indicated. ( j ) The percentage of cells from (i) that underwent 1–3 divisions was quantified. ( c , e , h ) mean ± s.e.m., each symbol represents an individual mouse, ( f , j ) mean ± s.d.; each symbol represents a technical replicate; ( a ) biological samples prepared pooling cells from n = 4 or 8 mice; mice per group: ( c ) n = 8 or 9, ( e ) n = 13 or 14, ( h ) n = 4 or 5; ( f ) n = 2 technical replicates of one biological sample from each genotype, each prepared pooling cells from 4 mice; ( j ) n = 3 technical replicates of one biological sample from each genotype, each prepared pooling cells from 5 or 10 mice per group. Data are representative of ( a , f , i , j ) two, ( b , d , g , h ) three independent experiments, or are pooled from ( c , e ) three independent experiments. NS, not significant ( P > 0.05); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test).
Figure Legend Snippet: Nup210 is required for TCR signaling ( a ) Immunoblot analysis of p-Lck (Y394) and total Lck protein levels in Nup210 +/+ and Nup210 −/− naïve CD4 + T cells. Hsp90 used as loading control. ( b , c ) Flow cytometric analysis of p-Lck (Y505) expression (b) and mean fluorescence intensity (MFI) (c) in resting naïve CD4 + (CD4 + CD62L hi CD44 lo CD25 − ) T cells from Nup210 +/+ and Nup210 −/− mice. Activation with sodium peroxyvanadate used as positive control. Secondary Ab: fluorescently labeled secondary antibody stained sample. MFI is shown relative to that of Nup210 +/+ mice. ( d , e ) Flow cytometric analysis of p-PLC-γ1 (Y783) expression (d) and MFI (e) in resting naïve CD4 + (CD4 + CD62L hi CD44 lo CD25 − ) T cells from Nup210 +/+ and Nup210 −/− mice. ( f ) Flow cytometric analysis of Nur77 expression in Nup210 +/+ or Nup210 −/− CD4 + CD44 lo T cells stimulated for 16 hours with soluble (sol.) anti-CD3 mAb. Values are displayed relative to sol. anti-CD3-stimulated Nup210 +/+ cells. ( g ) Flow cytometric analysis of Nur77 expression in unstimulated (left) or TCR-stimulated (right) Nup210 +/+ and Nup210 −/− CD4 + CD44 lo T cells. TCR stimulation was performed with plate-bound anti-CD3 and anti-CD28 mAbs, and soluble IL-2 for 14 hours. FMO, fluorescence minus one. ( h ) Flow cytometric analysis of Nur77 levels in CD4 + T cells stimulated as in (g) or without IL-2 supplementation. Values shown are relative to control treated cells. ( i ) Flow cytometric analysis of CFSE and CTV dilutions 48 hours after stimulation of CFSE-labeled Nup210 +/+ and CTV-labeled Nup210 −/− CD4 + CD44 lo T cells with plate-bound anti-CD3/ anti-CD28 mAbs and soluble IL-2. The percentage of cells that have undergone proliferation is indicated. ( j ) The percentage of cells from (i) that underwent 1–3 divisions was quantified. ( c , e , h ) mean ± s.e.m., each symbol represents an individual mouse, ( f , j ) mean ± s.d.; each symbol represents a technical replicate; ( a ) biological samples prepared pooling cells from n = 4 or 8 mice; mice per group: ( c ) n = 8 or 9, ( e ) n = 13 or 14, ( h ) n = 4 or 5; ( f ) n = 2 technical replicates of one biological sample from each genotype, each prepared pooling cells from 4 mice; ( j ) n = 3 technical replicates of one biological sample from each genotype, each prepared pooling cells from 5 or 10 mice per group. Data are representative of ( a , f , i , j ) two, ( b , d , g , h ) three independent experiments, or are pooled from ( c , e ) three independent experiments. NS, not significant ( P > 0.05); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test).

Techniques Used: Flow Cytometry, Expressing, Fluorescence, Mouse Assay, Activation Assay, Positive Control, Labeling, Staining, Planar Chromatography, Two Tailed Test

Nup210 is required for naïve CD4 + T cell homeostasis Analysis of White Blood Cell (WBC) ( a ) and Red Blood Cell (RBC) ( b ) concentration in blood of Nup210 +/+ and Nup210 −/− mice. Lym: lymphocytes; Mon: monocytes; Gra: granulocytes; HGB: Hemoglobin; HCT: Hematocrit; MCV: Mean Cell Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; RDWc: Red Cell Distribution Width; PLT: Platelet; Pct: Platelet hematocrit; MPV: Mean Platelet Volume; PDWc: Platelet Distribution Width. ( c – i ) Flow cytometric analysis of Nup210 +/+ and Nup210 −/− spleens. (c) Gating strategy used to define splenic CD3 + T cells. (d) Splenic CD3 + T cell numbers. (e) Splenic CD4 + /CD8 + T cell ratios. (f) Gating strategy used to define splenic CD4 + and CD8 + T cell populations. (g) Splenic CD4 + and CD8 + T cell numbers. (h) Gating strategy used to identify naïve (CD62L hi CD44 lo ), effector memory (CD62L lo CD44 hi ) and central memory (CD62L hi CD44 hi ) splenic CD4 + T cells. (i) Splenic naïve and memory CD4 + T cell numbers. ( a , b , d , e , g , i ) mean ± s.e.m; each symbol represents an individual mouse; mice per group: ( a , b ) n = 4, ( d ) n = 28 or 29, ( e ) n = 35 or 39, ( g ) n = 37 or 41, and ( i ) n = 33 or 36. Data are representative of ( a , b ) two, ( c ) eight, ( f ) eleven, and ( h ) ten independent experiments, or are pooled from ( d ) eight, ( e , g ) eleven, and ( i ) ten independent experiments. NS, not significant ( P > 0.05); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test).
Figure Legend Snippet: Nup210 is required for naïve CD4 + T cell homeostasis Analysis of White Blood Cell (WBC) ( a ) and Red Blood Cell (RBC) ( b ) concentration in blood of Nup210 +/+ and Nup210 −/− mice. Lym: lymphocytes; Mon: monocytes; Gra: granulocytes; HGB: Hemoglobin; HCT: Hematocrit; MCV: Mean Cell Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; RDWc: Red Cell Distribution Width; PLT: Platelet; Pct: Platelet hematocrit; MPV: Mean Platelet Volume; PDWc: Platelet Distribution Width. ( c – i ) Flow cytometric analysis of Nup210 +/+ and Nup210 −/− spleens. (c) Gating strategy used to define splenic CD3 + T cells. (d) Splenic CD3 + T cell numbers. (e) Splenic CD4 + /CD8 + T cell ratios. (f) Gating strategy used to define splenic CD4 + and CD8 + T cell populations. (g) Splenic CD4 + and CD8 + T cell numbers. (h) Gating strategy used to identify naïve (CD62L hi CD44 lo ), effector memory (CD62L lo CD44 hi ) and central memory (CD62L hi CD44 hi ) splenic CD4 + T cells. (i) Splenic naïve and memory CD4 + T cell numbers. ( a , b , d , e , g , i ) mean ± s.e.m; each symbol represents an individual mouse; mice per group: ( a , b ) n = 4, ( d ) n = 28 or 29, ( e ) n = 35 or 39, ( g ) n = 37 or 41, and ( i ) n = 33 or 36. Data are representative of ( a , b ) two, ( c ) eight, ( f ) eleven, and ( h ) ten independent experiments, or are pooled from ( d ) eight, ( e , g ) eleven, and ( i ) ten independent experiments. NS, not significant ( P > 0.05); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test).

Techniques Used: Concentration Assay, Mouse Assay, Flow Cytometry, Two Tailed Test

Nup210 is required for the TCR-dependent induction of Cav2 expression ( a ) Immunofluorescence analysis of Nup210 and mAb414 in NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. ( b ) Immunoblot analysis of Nup210 and cFos in resting and soluble anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. Hsp90 was used as loading control ( > exp: higher exposure). ( c ) qPCR analysis of Il2 mRNA levels in resting or soluble anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. Il2 expression was normalized to Hprt1 . ( d ) Immunofluorescence analysis of SLP-76 clustering on the surface of unstimulated or anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. ( e ) The percentage of cells with SLP-76 foci from (d) was quantified. ( f ) Immunofluorescence analysis of Cav1 and Cav2 in unstimulated or anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. ( g ) The percentage of activated (SLP-76 cluster-positive cells) NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells expressing control (empty vector) or Cav2 was quantified after stimulation with soluble anti-CD3. ( h ) Immunoblot analysis of Nup210, Cav2, p-Lck Y394, p-Lck Y505, and total Lck in unstimulated or anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells expressing control or Cav2. Hsp90 was used as loading control. ( a , b , d,f ) representative of three independent experiments; ( c ) mean ± s.d., each symbol represents a technical replicate n = 3, representative of three independent experiments; ( e , g ) mean ± s.e.m, pooled from four independent experiments, n = 4 biological samples, n ≥ 200 cells per group; ( h ) representative of two independent experiments. NS, not significant ( P > 0.05); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test). Scale bar: 5 μm.
Figure Legend Snippet: Nup210 is required for the TCR-dependent induction of Cav2 expression ( a ) Immunofluorescence analysis of Nup210 and mAb414 in NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. ( b ) Immunoblot analysis of Nup210 and cFos in resting and soluble anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. Hsp90 was used as loading control ( > exp: higher exposure). ( c ) qPCR analysis of Il2 mRNA levels in resting or soluble anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. Il2 expression was normalized to Hprt1 . ( d ) Immunofluorescence analysis of SLP-76 clustering on the surface of unstimulated or anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. ( e ) The percentage of cells with SLP-76 foci from (d) was quantified. ( f ) Immunofluorescence analysis of Cav1 and Cav2 in unstimulated or anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells. ( g ) The percentage of activated (SLP-76 cluster-positive cells) NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells expressing control (empty vector) or Cav2 was quantified after stimulation with soluble anti-CD3. ( h ) Immunoblot analysis of Nup210, Cav2, p-Lck Y394, p-Lck Y505, and total Lck in unstimulated or anti-CD3-activated NUP210 +/+ or NUP210 −/− J14 SLP-76-EYFP cells expressing control or Cav2. Hsp90 was used as loading control. ( a , b , d,f ) representative of three independent experiments; ( c ) mean ± s.d., each symbol represents a technical replicate n = 3, representative of three independent experiments; ( e , g ) mean ± s.e.m, pooled from four independent experiments, n = 4 biological samples, n ≥ 200 cells per group; ( h ) representative of two independent experiments. NS, not significant ( P > 0.05); * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (two-tailed unpaired Student’s t -test). Scale bar: 5 μm.

Techniques Used: Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

38) Product Images from "Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells"

Article Title: Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells

Journal: Oncotarget

doi:

AR suppresses anti-tumor activity of CD8 + T cells in vivo A. Proportion of each T subset in the Hepa1–6 xenografts was determined by flow cytometry. B. Histograms of the expression of CTLA-4 and ICOS on intratumoral Tregs determined by flow cytometry. This is a representative of three independent experiments. C. AR expression in the xenografts was determined by Western blotting. This is a representative of two independent experiments. D. Intratumoral Tregs proliferation was determined by ki67 staining. Numbers in the plots were the percentages of ki67 + cells presented as mean ± SD. E. Expression of IFN-γ, TNF-α, perforin and granzyme B in intratumoral CD8 + T cells were analyzed using qRT-PCR. Ctrl, xenografts of non-transfected Hepa1–6 cells; shRNA, xenografts of LV-shRNA-transfected Hepa1–6 cells; Scramble, xenografts of LV-scramble-transfected Hepa1–6 cells. N = 7 per group. * p
Figure Legend Snippet: AR suppresses anti-tumor activity of CD8 + T cells in vivo A. Proportion of each T subset in the Hepa1–6 xenografts was determined by flow cytometry. B. Histograms of the expression of CTLA-4 and ICOS on intratumoral Tregs determined by flow cytometry. This is a representative of three independent experiments. C. AR expression in the xenografts was determined by Western blotting. This is a representative of two independent experiments. D. Intratumoral Tregs proliferation was determined by ki67 staining. Numbers in the plots were the percentages of ki67 + cells presented as mean ± SD. E. Expression of IFN-γ, TNF-α, perforin and granzyme B in intratumoral CD8 + T cells were analyzed using qRT-PCR. Ctrl, xenografts of non-transfected Hepa1–6 cells; shRNA, xenografts of LV-shRNA-transfected Hepa1–6 cells; Scramble, xenografts of LV-scramble-transfected Hepa1–6 cells. N = 7 per group. * p

Techniques Used: Activity Assay, In Vivo, Flow Cytometry, Cytometry, Expressing, Western Blot, Staining, Quantitative RT-PCR, Transfection, shRNA

39) Product Images from "Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis"

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis

Journal: Immunity

doi: 10.1016/j.immuni.2018.04.009

GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Inflamed Joints (A) Flow cytometry analysis of GM-CSF expression by CD4 + T cells and CD4 − cells among CD45 + joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. (B) Quantitative RT-PCR analysis of Csf2 , Il1b , and Tnf in CD11b + Ly-6G − (CD11b + ), CD11b + Ly-6G + (Ly-6G + ), CD4 + T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1 . (C) Flow cytometry of joint infiltrating cells in Rag2 −/− mice transferred with CD4 + T cells from WT or Csf2 −/− SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5). (D) Proportion of ILCs in Rag2 −/− mice transferred with CD4 + T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means. (E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3). (F) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for Ki-67 expression. (G) Flow cytometry of synovial ILCs (CD45.2 + lineage markers-negative Thy1.2 + cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2. (H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Rorγt, and Foxp3. (I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H). (J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13. (K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments. (L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + T cells (naive CD4 + T) and synovial ILCs (n = 3) as shown in (C). (N) The effects of ILC depletion on arthritis development. CD4 + T cells (1 × 10 6 ) from Thy1.1 + SKG mice were adoptively transferred into Thy1.2 + Rag2 −/− mice, which were i.v. injected with 500 μg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Quantitative RT-PCR, Staining, Injection

GM-CSF-Producing ILCs in Synovial Fluid of RA Patients (A) The presence of ILCs (defined as CD45 + CD3 − CD4 − CD8 − CD11b − CD11c − CD19 − CD56 − ) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right). (B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD. (C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom). (D) Gating strategies for GM-CSF + CD45 + lineage markers-negative (ILCs), GM-CSF + CD45 + CD3 − CD11b + (myeloid cells), and GM-CSF + CD45 + CD11b − CD3 + cells (T cells). (E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means. ∗ p
Figure Legend Snippet: GM-CSF-Producing ILCs in Synovial Fluid of RA Patients (A) The presence of ILCs (defined as CD45 + CD3 − CD4 − CD8 − CD11b − CD11c − CD19 − CD56 − ) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right). (B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD. (C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom). (D) Gating strategies for GM-CSF + CD45 + lineage markers-negative (ILCs), GM-CSF + CD45 + CD3 − CD11b + (myeloid cells), and GM-CSF + CD45 + CD11b − CD3 + cells (T cells). (E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means. ∗ p

Techniques Used: Flow Cytometry, Cytometry, Expressing

40) Product Images from "Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy"

Article Title: Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy

Journal: Nature biomedical engineering

doi: 10.1038/s41551-018-0310-2

S–P–aPD-1 induced a durable immune response. a , Flow cytometry analysis of CD44 hi CD62L hi T cells (gated on CD8+ T cells) in saline- and S–P–aPD-1-treated mice ( n = 3). b , Bioluminescence images of native and treated mice re-challenged with 1 × 10 6 C1498 cells at 3 weeks. c , Survival curves for native and treated mice after re-challenge with C1498 cells ( n = 7). *** P
Figure Legend Snippet: S–P–aPD-1 induced a durable immune response. a , Flow cytometry analysis of CD44 hi CD62L hi T cells (gated on CD8+ T cells) in saline- and S–P–aPD-1-treated mice ( n = 3). b , Bioluminescence images of native and treated mice re-challenged with 1 × 10 6 C1498 cells at 3 weeks. c , Survival curves for native and treated mice after re-challenge with C1498 cells ( n = 7). *** P

Techniques Used: Flow Cytometry, Mouse Assay

Related Articles

Flow Cytometry:

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Article Snippet: .. Flow cytometry The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences). .. For intracellular staining for transcription factors, cells were stained using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instruction.

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Article Snippet: .. The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences). .. For intracellular staining for transcription factors, cells were stained using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instruction.

Cytometry:

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Article Snippet: .. Flow cytometry The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences). .. For intracellular staining for transcription factors, cells were stained using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instruction.

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Article Snippet: .. The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences). .. For intracellular staining for transcription factors, cells were stained using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instruction.

Cell Culture:

Article Title: Bone marrow myeloid cells regulate myeloid-biased hematopoietic stem cells via a histamine-dependent feedback loop
Article Snippet: .. 10 ng/ml GM-CSF (BioLegend) was added to the myeloid cell culture. .. LPS (1 μg/ml, LPS-EB Ultrapure, InvivoGen) was added 12 hours after cells were plated.

Article Title: Role of PKCtheta in macrophage-mediated immune response to Salmonella typhimurium infection in mice
Article Snippet: .. CD14+ cells were positively selected with human CD14 MicroBeads (130-050-201; MACS Miltenyi Biotec) and cultured in DMEM (BioWhittaker BE12-707 F, Lonza) supplemented with 10 % FCS, 2 mM L-glutamine, 1 % penicillin plus streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin in 0.9 % NaCl), and 100 ng/ml of human GM-CSF (572903, BioLegend). .. Cells were fed on day 4 and stimulated with 10 ng/ml LPS (lipopolysaccharide from S. typhimurium L6511; Sigma-Aldrich, Vienna, Austria) and 10 ng/ml human IFNgamma (570204, BioLegend) on day 7 for the indicated time periods.

Purification:

Article Title: Regulation of the actin cytoskeleton by Rho kinase controls antigen presentation by CD1d 1
Article Snippet: .. Purified and biotinylated anti-mouse IL-2 (BD Biosciences), purified and biotinylated antihuman IL-4 and GM-CSF (BioLegend, San Diego, CA), and avidin-peroxidase (Sigma-Aldrich), were used to measure cytokine production by ELISA. .. Recombinant murine IL-2, human IL-4 and GM-CSF (Peprotech, Rocky Hill, NJ) were used as standards.

Enzyme-linked Immunosorbent Assay:

Article Title: The immunostimulatory effects and pro‐apoptotic activity of rhCNB against Lewis lung cancer is mediated by Toll‐like receptor 4. The immunostimulatory effects and pro‐apoptotic activity of rhCNB against Lewis lung cancer is mediated by Toll‐like receptor 4
Article Snippet: .. The ELISA kits for mouse TNFα, CCL5, IL‐12p40, IFN‐γ, IL4, and IL10 were from Neobioscience Technology Co., Ltd., and the mouse IFNβ ELISA kit was from Cloud Clone Corp; Recombinant mouse IL‐4 and GM‐CSF were from BioLegend. .. 2.2.1 Isolation of mouse peritoneal macrophages 5 × 105 mouse peritoneal macrophages from C57BL/6 WT and TLR4−/− mice were plated in the wells of sterile 12‐well cell culture plates in 1 mL RPMI 1640 containing 10% FBS, and incubated overnight at 37°C with 5% CO2 .

Article Title: Regulation of the actin cytoskeleton by Rho kinase controls antigen presentation by CD1d 1
Article Snippet: .. Purified and biotinylated anti-mouse IL-2 (BD Biosciences), purified and biotinylated antihuman IL-4 and GM-CSF (BioLegend, San Diego, CA), and avidin-peroxidase (Sigma-Aldrich), were used to measure cytokine production by ELISA. .. Recombinant murine IL-2, human IL-4 and GM-CSF (Peprotech, Rocky Hill, NJ) were used as standards.

Concentration Assay:

Article Title: Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade
Article Snippet: .. Recombinant mouse G-CSF (Shenandoah Biotechnology), SCF (Gemini Bioproducts), GM-CSF (BioLegend), IL6 (R & D Systems) were used at a final concentration of 50 ng ml−1 . .. Mouse studies and MDSCs depletion All mice procedures were conducted in accordance with the Institutional Animal Care and Use Committee at Augusta University.

Avidin-Biotin Assay:

Article Title: Regulation of the actin cytoskeleton by Rho kinase controls antigen presentation by CD1d 1
Article Snippet: .. Purified and biotinylated anti-mouse IL-2 (BD Biosciences), purified and biotinylated antihuman IL-4 and GM-CSF (BioLegend, San Diego, CA), and avidin-peroxidase (Sigma-Aldrich), were used to measure cytokine production by ELISA. .. Recombinant murine IL-2, human IL-4 and GM-CSF (Peprotech, Rocky Hill, NJ) were used as standards.

FACS:

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Article Snippet: .. Flow cytometry The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences). .. For intracellular staining for transcription factors, cells were stained using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instruction.

Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Article Snippet: .. The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences). .. For intracellular staining for transcription factors, cells were stained using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instruction.

Recombinant:

Article Title: Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade
Article Snippet: .. Recombinant mouse G-CSF (Shenandoah Biotechnology), SCF (Gemini Bioproducts), GM-CSF (BioLegend), IL6 (R & D Systems) were used at a final concentration of 50 ng ml−1 . .. Mouse studies and MDSCs depletion All mice procedures were conducted in accordance with the Institutional Animal Care and Use Committee at Augusta University.

Article Title: The immunostimulatory effects and pro‐apoptotic activity of rhCNB against Lewis lung cancer is mediated by Toll‐like receptor 4. The immunostimulatory effects and pro‐apoptotic activity of rhCNB against Lewis lung cancer is mediated by Toll‐like receptor 4
Article Snippet: .. The ELISA kits for mouse TNFα, CCL5, IL‐12p40, IFN‐γ, IL4, and IL10 were from Neobioscience Technology Co., Ltd., and the mouse IFNβ ELISA kit was from Cloud Clone Corp; Recombinant mouse IL‐4 and GM‐CSF were from BioLegend. .. 2.2.1 Isolation of mouse peritoneal macrophages 5 × 105 mouse peritoneal macrophages from C57BL/6 WT and TLR4−/− mice were plated in the wells of sterile 12‐well cell culture plates in 1 mL RPMI 1640 containing 10% FBS, and incubated overnight at 37°C with 5% CO2 .

Magnetic Cell Separation:

Article Title: Role of PKCtheta in macrophage-mediated immune response to Salmonella typhimurium infection in mice
Article Snippet: .. CD14+ cells were positively selected with human CD14 MicroBeads (130-050-201; MACS Miltenyi Biotec) and cultured in DMEM (BioWhittaker BE12-707 F, Lonza) supplemented with 10 % FCS, 2 mM L-glutamine, 1 % penicillin plus streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin in 0.9 % NaCl), and 100 ng/ml of human GM-CSF (572903, BioLegend). .. Cells were fed on day 4 and stimulated with 10 ng/ml LPS (lipopolysaccharide from S. typhimurium L6511; Sigma-Aldrich, Vienna, Austria) and 10 ng/ml human IFNgamma (570204, BioLegend) on day 7 for the indicated time periods.

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    BioLegend flow cytometry analysis mouse anti human antibodies anti cd3
    Enhancement of ZIKV infection in primary monocytes triggered by DENV immune sera. Freshly isolated PBMCs from healthy donors were either uninfected or infected with ZIKV at an MOI of 2 in the presence or absence of serially diluted DENV immune serum samples. Cells were harvested and stained with anti-E antibody in combination with antibodies to <t>CD3,</t> CD14, CD19 and then analyzed by flow <t>cytometry.</t> (A) and (C) Representative flow cytometry analyses of ZIKV-infected (Anti-E) monocytes (CD14+CD3-CD19-), T cells (CD14-CD3+CD19-) and B cells (CD14-CD3-CD19+) in the presence of a DENV immune serum sample D1-E01 (A) and a control serum Naïve-01 (C) were presented. (B) and (D) Verification of enhancement in ZIKV infection by the boosted assay on Vero cells. Culture supernatants from ZIKV-infected PBMCs were harvested and used to infect Vero cells for two days followed by intracellular staining with an anti-E antibody and flow cytometry analysis. Representative results of the boosted infection assay on Vero cells for the DENV immune serum D1-01 (B) and the control serum Naïve-01 (D) were shown.
    Flow Cytometry Analysis Mouse Anti Human Antibodies Anti Cd3, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry analysis mouse anti human antibodies anti cd3/product/BioLegend
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    flow cytometry analysis mouse anti human antibodies anti cd3 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    BioLegend anti human cd3 pe cy7
    Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with <t>anti‐CD3‐PE‐Cy7,</t> anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry
    Anti Human Cd3 Pe Cy7, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human cd3 pe cy7/product/BioLegend
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    anti human cd3 pe cy7 - by Bioz Stars, 2020-09
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    Enhancement of ZIKV infection in primary monocytes triggered by DENV immune sera. Freshly isolated PBMCs from healthy donors were either uninfected or infected with ZIKV at an MOI of 2 in the presence or absence of serially diluted DENV immune serum samples. Cells were harvested and stained with anti-E antibody in combination with antibodies to CD3, CD14, CD19 and then analyzed by flow cytometry. (A) and (C) Representative flow cytometry analyses of ZIKV-infected (Anti-E) monocytes (CD14+CD3-CD19-), T cells (CD14-CD3+CD19-) and B cells (CD14-CD3-CD19+) in the presence of a DENV immune serum sample D1-E01 (A) and a control serum Naïve-01 (C) were presented. (B) and (D) Verification of enhancement in ZIKV infection by the boosted assay on Vero cells. Culture supernatants from ZIKV-infected PBMCs were harvested and used to infect Vero cells for two days followed by intracellular staining with an anti-E antibody and flow cytometry analysis. Representative results of the boosted infection assay on Vero cells for the DENV immune serum D1-01 (B) and the control serum Naïve-01 (D) were shown.

    Journal: PLoS ONE

    Article Title: Dengue immune sera enhance Zika virus infection in human peripheral blood monocytes through Fc gamma receptors

    doi: 10.1371/journal.pone.0200478

    Figure Lengend Snippet: Enhancement of ZIKV infection in primary monocytes triggered by DENV immune sera. Freshly isolated PBMCs from healthy donors were either uninfected or infected with ZIKV at an MOI of 2 in the presence or absence of serially diluted DENV immune serum samples. Cells were harvested and stained with anti-E antibody in combination with antibodies to CD3, CD14, CD19 and then analyzed by flow cytometry. (A) and (C) Representative flow cytometry analyses of ZIKV-infected (Anti-E) monocytes (CD14+CD3-CD19-), T cells (CD14-CD3+CD19-) and B cells (CD14-CD3-CD19+) in the presence of a DENV immune serum sample D1-E01 (A) and a control serum Naïve-01 (C) were presented. (B) and (D) Verification of enhancement in ZIKV infection by the boosted assay on Vero cells. Culture supernatants from ZIKV-infected PBMCs were harvested and used to infect Vero cells for two days followed by intracellular staining with an anti-E antibody and flow cytometry analysis. Representative results of the boosted infection assay on Vero cells for the DENV immune serum D1-01 (B) and the control serum Naïve-01 (D) were shown.

    Article Snippet: Flow cytometry analysis Mouse anti-human antibodies anti-CD3 (PE-Cy7; clone UCHT1; Biolegend), anti-CD4 (BV-785; clone RPA-T4; Biolegend), anti-CD14 (PE; clone M5E2; Biolegend), anti-CD19 (BV-711; clone HIB19; Biolegend), anti-CD19 (APC; clone HIB19; Biolegend), anti-HLA-DR (Percp; clone L243; Biolegend), anti-CD11c (APC; clone 3.9; eBioscience), anti-DC-SIGN (PE-Cy7; clone 9E9A8; Biolegend), anti-CD83 (FITC; clone HB15e; Biolegend) and anti-CD86 (PE; clone IT2.2; eBioscience) were purchased from Biolegend and eBioscience.

    Techniques: Infection, Isolation, Staining, Flow Cytometry, Cytometry

    Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

    Journal: Cell Proliferation

    Article Title: Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor

    doi: 10.1111/cpr.12858

    Figure Lengend Snippet: Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

    Article Snippet: The following reagents were used for this analysis: anti‐human CD3‐PE‐Cy7, anti‐human CD56‐PE, anti‐human CD69‐APC‐Cy7, mouse control PE, mouse control APC‐Cy7, mouse control PE‐Cy7, and Human TruStain FcX™ blocking solution purchased from BioLegend; anti‐EGFR antibody purchased from Cell Signal Technology; and goat anti‐rabbit IgG purchased from Abcam.

    Techniques: Isolation, Labeling, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Flow Cytometry