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STEMCELL Technologies Inc cd34 cell expansion media
Comparing the efficiency of reprogramming between PBMCs and <t>CD34+</t> cells. PBMCs and CD34+ cells were isolated from single tubes of blood provided from 10 different donors. The efficiency of reprogramming following transfection with DNA Combination Set 2 was determined for each donor and each method. “Total colonies” refers to all iPS colonies derived from either CD34+ or PBMC populations that stain positively for Tra1-60 and “iPS-like” colonies are those that stain positively for Tra1-60, exhibit clear iPS morphology, and are large enough to pick for expansion. Input cells refer to the number of CD34+ cells or PBMCs were used for transfection. The efficiencies across all donors from both methods were compared using the Wilcoxon signed rank test (two-sided), p = 0.007.
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1) Product Images from "Generation of Induced Pluripotent Stem Cells from CD34+ Cells across Blood Drawn from Multiple Donors with Non-Integrating Episomal Vectors"

Article Title: Generation of Induced Pluripotent Stem Cells from CD34+ Cells across Blood Drawn from Multiple Donors with Non-Integrating Episomal Vectors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027956

Comparing the efficiency of reprogramming between PBMCs and CD34+ cells. PBMCs and CD34+ cells were isolated from single tubes of blood provided from 10 different donors. The efficiency of reprogramming following transfection with DNA Combination Set 2 was determined for each donor and each method. “Total colonies” refers to all iPS colonies derived from either CD34+ or PBMC populations that stain positively for Tra1-60 and “iPS-like” colonies are those that stain positively for Tra1-60, exhibit clear iPS morphology, and are large enough to pick for expansion. Input cells refer to the number of CD34+ cells or PBMCs were used for transfection. The efficiencies across all donors from both methods were compared using the Wilcoxon signed rank test (two-sided), p = 0.007.
Figure Legend Snippet: Comparing the efficiency of reprogramming between PBMCs and CD34+ cells. PBMCs and CD34+ cells were isolated from single tubes of blood provided from 10 different donors. The efficiency of reprogramming following transfection with DNA Combination Set 2 was determined for each donor and each method. “Total colonies” refers to all iPS colonies derived from either CD34+ or PBMC populations that stain positively for Tra1-60 and “iPS-like” colonies are those that stain positively for Tra1-60, exhibit clear iPS morphology, and are large enough to pick for expansion. Input cells refer to the number of CD34+ cells or PBMCs were used for transfection. The efficiencies across all donors from both methods were compared using the Wilcoxon signed rank test (two-sided), p = 0.007.

Techniques Used: Isolation, Transfection, Derivative Assay, Staining

Characterization of iPSCs derived from CD34+ blood cells. A subset of iPSC clones were characterized for pluripotency. The experiments demonstrated in this figure provide representative examples of the types of results observed for characterization studies using iPS clones 4 and/or 5 derived from donors 2939 and 3389. A. Cytogenetic analysis on G-banded metaphase cells from iPS clone 4 exhibiting a normal karyotype. B and C. RT-PCR confirms the endogenous expression of classic pluripotency genes and the absence of expression from transgenes. A standard in-house iPS line served as the positive control k. D. Clones were deemed free of episomal (E) DNA and genomic integration (G) by PCR. E. PCR was used to track the loss of oriP/EBNA1-based plasmids at multiple passages using primers that amplify EBNA1. A control plasmid at 1 and 20 copies per genome was used to establish the sensitivity of the PCR at 1 copy per 3,000 cells. F. PCR screen using primers specific for the joining region and all three of the conserved framework regions (FR1, FR2 and FR3) to amplify immunoglobulin heavy chain (IgH) gene rearrangements and two assays with primers specific to the T cell receptor (TCR) gamma gene rearrangement. G. Representative image of donor 2939 clone 5 differentiated in vitro into neurons (i). Clone 5 also demonstrated differentiation into all three germ layers: ii) epithelium iii) endoderm iv) mesoderm v) ectoderm vi) endoderm from teratomas formed when iPSCs were injected into immunodeficient, SCID mice.
Figure Legend Snippet: Characterization of iPSCs derived from CD34+ blood cells. A subset of iPSC clones were characterized for pluripotency. The experiments demonstrated in this figure provide representative examples of the types of results observed for characterization studies using iPS clones 4 and/or 5 derived from donors 2939 and 3389. A. Cytogenetic analysis on G-banded metaphase cells from iPS clone 4 exhibiting a normal karyotype. B and C. RT-PCR confirms the endogenous expression of classic pluripotency genes and the absence of expression from transgenes. A standard in-house iPS line served as the positive control k. D. Clones were deemed free of episomal (E) DNA and genomic integration (G) by PCR. E. PCR was used to track the loss of oriP/EBNA1-based plasmids at multiple passages using primers that amplify EBNA1. A control plasmid at 1 and 20 copies per genome was used to establish the sensitivity of the PCR at 1 copy per 3,000 cells. F. PCR screen using primers specific for the joining region and all three of the conserved framework regions (FR1, FR2 and FR3) to amplify immunoglobulin heavy chain (IgH) gene rearrangements and two assays with primers specific to the T cell receptor (TCR) gamma gene rearrangement. G. Representative image of donor 2939 clone 5 differentiated in vitro into neurons (i). Clone 5 also demonstrated differentiation into all three germ layers: ii) epithelium iii) endoderm iv) mesoderm v) ectoderm vi) endoderm from teratomas formed when iPSCs were injected into immunodeficient, SCID mice.

Techniques Used: Derivative Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Polymerase Chain Reaction, Plasmid Preparation, In Vitro, Injection, Mouse Assay

Plasmid transfections to optimize reprogramming efficiency. A. Representative reprogramming trial from freshly drawn blood (donor 3002) using combination plasmid Set 2 for transfection. A single well is shown from a 6-well plate that contains colonies staining positively for AP activity (i). The white arrowhead highlights the colony magnified in panel ii that also stained positively for Tra-1-81 expression (green), panel iii. B. Schematic of the plasmid sets successfully used for reprogramming trials. Set 1 contains a combination of two plasmids for transfection whereby a 20 kb plasmid that either contains C- or L-myc is depicted. Set 2 includes a three plasmid combination for transfection. C. CD34+ cells purified from four different donors were expanded for 6 days and transfected using the plasmid combination that expresses either Set 1 or Set 2 plasmid sets to compare the total number of resulting iPSCs. D. Reprogramming trials were performed using plasmid Set 2 to transfect a range of cell numbers expanded for 6 days (donor GG, n = 6).
Figure Legend Snippet: Plasmid transfections to optimize reprogramming efficiency. A. Representative reprogramming trial from freshly drawn blood (donor 3002) using combination plasmid Set 2 for transfection. A single well is shown from a 6-well plate that contains colonies staining positively for AP activity (i). The white arrowhead highlights the colony magnified in panel ii that also stained positively for Tra-1-81 expression (green), panel iii. B. Schematic of the plasmid sets successfully used for reprogramming trials. Set 1 contains a combination of two plasmids for transfection whereby a 20 kb plasmid that either contains C- or L-myc is depicted. Set 2 includes a three plasmid combination for transfection. C. CD34+ cells purified from four different donors were expanded for 6 days and transfected using the plasmid combination that expresses either Set 1 or Set 2 plasmid sets to compare the total number of resulting iPSCs. D. Reprogramming trials were performed using plasmid Set 2 to transfect a range of cell numbers expanded for 6 days (donor GG, n = 6).

Techniques Used: Plasmid Preparation, Transfection, Staining, Activity Assay, Expressing, Purification

The generation of iPSCs from blood using completely defined conditions. A. Fold expansion of CD34+ cells pooled from multiple blood donors in standard (n = 13) and completely defined conditions (n = 2) after 6 days of expansion. Fold expansion was calculated from the total number of cells at day 6 divided by the number of cells the day after purification. Percentages indicate the fraction of cells expressing CD34 in the total population as assessed by flow cytometry. B. Reprogramming trials were performed on CD34+ cells obtained by leukapheresis from donors GG and A2389 with and without the B27 supplement.
Figure Legend Snippet: The generation of iPSCs from blood using completely defined conditions. A. Fold expansion of CD34+ cells pooled from multiple blood donors in standard (n = 13) and completely defined conditions (n = 2) after 6 days of expansion. Fold expansion was calculated from the total number of cells at day 6 divided by the number of cells the day after purification. Percentages indicate the fraction of cells expressing CD34 in the total population as assessed by flow cytometry. B. Reprogramming trials were performed on CD34+ cells obtained by leukapheresis from donors GG and A2389 with and without the B27 supplement.

Techniques Used: Purification, Expressing, Flow Cytometry, Cytometry

Hematopoietic cells enriched for CD34 expression are expandable. A. Graphs depict the expansion of purified cells from either two peripheral (PB.1 and PB.2) or cord (CB.1 or CB.2) blood donors over time (lefthand panel) along with the percentages of the total population that are CD34+ (righthand panel). B. Representative profile of a purified population of cells after 6 days of expansion by flow cytometry on cells isolated from Donor 3002. Flow cytometry plots from control staining using IgG antibodies (upper plots) are compared to plots with antibodies specific to lineage markers (lower plots). C. The graph represents an extended analysis by flow cytometry of the characteristic profile of PB.2 cells after 10 days of expansion. The % positive indicates the fraction of the population expressing the cell surface markers on the x-axis. D. The total number of CD34+ cells across 16 different donors was assessed beginning at 0 and 6 days of expansion (left side y-axis). The fold expansion (right side y-axis, orange squares) was determined by dividing the total number of cells at day 6 divided by the number of cells at day 0 after purification. The average percent of CD34 expression across all 16 donors was 48+/−19%.
Figure Legend Snippet: Hematopoietic cells enriched for CD34 expression are expandable. A. Graphs depict the expansion of purified cells from either two peripheral (PB.1 and PB.2) or cord (CB.1 or CB.2) blood donors over time (lefthand panel) along with the percentages of the total population that are CD34+ (righthand panel). B. Representative profile of a purified population of cells after 6 days of expansion by flow cytometry on cells isolated from Donor 3002. Flow cytometry plots from control staining using IgG antibodies (upper plots) are compared to plots with antibodies specific to lineage markers (lower plots). C. The graph represents an extended analysis by flow cytometry of the characteristic profile of PB.2 cells after 10 days of expansion. The % positive indicates the fraction of the population expressing the cell surface markers on the x-axis. D. The total number of CD34+ cells across 16 different donors was assessed beginning at 0 and 6 days of expansion (left side y-axis). The fold expansion (right side y-axis, orange squares) was determined by dividing the total number of cells at day 6 divided by the number of cells at day 0 after purification. The average percent of CD34 expression across all 16 donors was 48+/−19%.

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

Identifying optimal transfection conditions for CD34+ cells. A. PBMCs (donor GG) were isolated and purified for CD34-expression and expanded for 6 days. A range of cell numbers were transfected with a control, oriP/EBNA1-based plasmid expressing GFP. Transfection efficiency was determined by calculating the percentage of viable cells expressing GFP detectable by flow cytometry (n = 6). B. PBMCs (donor A2389) were isolated, purified for CD34-expression and expanded for 3 or 6 days. 6×10 4 to 1×10 5 cells were transfected with the control, GFP-expressing plasmid. The graph depicts the percent of the total population that is GFP-positive along with the absolute number of total cells (n = 3). C. The graph represents the fraction of cells in B that co-express GFP and CD34 when transfected at 3 or 6 days of expansion (n = 3).
Figure Legend Snippet: Identifying optimal transfection conditions for CD34+ cells. A. PBMCs (donor GG) were isolated and purified for CD34-expression and expanded for 6 days. A range of cell numbers were transfected with a control, oriP/EBNA1-based plasmid expressing GFP. Transfection efficiency was determined by calculating the percentage of viable cells expressing GFP detectable by flow cytometry (n = 6). B. PBMCs (donor A2389) were isolated, purified for CD34-expression and expanded for 3 or 6 days. 6×10 4 to 1×10 5 cells were transfected with the control, GFP-expressing plasmid. The graph depicts the percent of the total population that is GFP-positive along with the absolute number of total cells (n = 3). C. The graph represents the fraction of cells in B that co-express GFP and CD34 when transfected at 3 or 6 days of expansion (n = 3).

Techniques Used: Transfection, Isolation, Purification, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

The presence of CD34+ cells correlates with reprogramming efficiency. A. CD34+ cells from four different blood donors were expanded for 3, 6, 9, or 13 days. A large volume of blood was collected from donors 3096, 2849, and 3389 to ensure sufficient cell numbers to perform these studies. Expanding CD34+ cells using plasmid DNA combination Set 2. The efficiency of reprogramming was calculated as the total number of iPSCs exhibiting morphological features characteristic of an ES cell and an ability to stain positively for Tra-1-81 divided by the total number of cells used for transfection. Black Squares depict the percentage of the population expressing CD34 at the indicated days of expansion. B. Representative reprogramming trial whereby both the positive (i) and negative (ii) fraction following purification were used for reprogramming. Panel (i) shows one well of a 6-well plate that contains successfully reprogrammed colonies from donor 2939 based on their ability to demonstrate AP activity. The CD34-depleted fraction from donor 2939 was unable to form colonies as indicated by the lack of AP staining when performed in parallel with the purified population panel, ii. Panels iii and iv magnify the colony in panel (i) marked by a white arrowhead and demonstrates expression of Tra-1-81 (green), panel iv.
Figure Legend Snippet: The presence of CD34+ cells correlates with reprogramming efficiency. A. CD34+ cells from four different blood donors were expanded for 3, 6, 9, or 13 days. A large volume of blood was collected from donors 3096, 2849, and 3389 to ensure sufficient cell numbers to perform these studies. Expanding CD34+ cells using plasmid DNA combination Set 2. The efficiency of reprogramming was calculated as the total number of iPSCs exhibiting morphological features characteristic of an ES cell and an ability to stain positively for Tra-1-81 divided by the total number of cells used for transfection. Black Squares depict the percentage of the population expressing CD34 at the indicated days of expansion. B. Representative reprogramming trial whereby both the positive (i) and negative (ii) fraction following purification were used for reprogramming. Panel (i) shows one well of a 6-well plate that contains successfully reprogrammed colonies from donor 2939 based on their ability to demonstrate AP activity. The CD34-depleted fraction from donor 2939 was unable to form colonies as indicated by the lack of AP staining when performed in parallel with the purified population panel, ii. Panels iii and iv magnify the colony in panel (i) marked by a white arrowhead and demonstrates expression of Tra-1-81 (green), panel iv.

Techniques Used: Plasmid Preparation, Staining, Transfection, Expressing, Purification, Activity Assay

2) Product Images from "Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood"

Article Title: Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood

Journal: Blood Advances

doi: 10.1182/bloodadvances.2019000292

Expression of IRF4 by activated human blood CD8 + T cells. Healthy donor PBMCs were stimulated with 1 μg/mL SEB and analyzed for the expression of surface and intracellular molecules at the indicated time points by flow cytometry. (A) IRF4 and CD69 expression by CD8 + T cells after stimulation with SEB. (B) IRF4 and CD69 expression by CD8 + T cells in unstimulated PBMCs (left panel) and PBMCs stimulated with SEB for 18 hours (right panel). (C) IRF4 and CD69 expression (left panel) and CD25 and CD137 expression (right panel) by CD8 + T cells after 18 hours of SEB stimulation. (D) Frequency of IRF4 + CD69 + , CD137 + , and CD25 + CD137 + cells in CD8 + T cells from healthy donor PBMCs after 18 hours of SEB stimulation (n = 24). Each point represents an individual donor. (E) IRF4 and CD69 expression in CD8 + T CM cells (CD45RA − CCR7 + ), T EM cells (CD45RA − CCR7 − ), naive T cells (CD45RA + CCR7 − ), and T EMRA cells (CD45RA + CCR7 + ) in SEB-stimulated PBMCs. (F) Mean fluorescence intensity (MFI) of IRF4 of baseline CD8 + T CM , T EM , naive T, and T EMRA cells (left panel) and after 18 hours of SEB stimulation (right panel) in healthy donor PBMCs (n = 3). Horizontal lines indicate the mean (± standard deviation). Each symbol represents an individual donor. * P
Figure Legend Snippet: Expression of IRF4 by activated human blood CD8 + T cells. Healthy donor PBMCs were stimulated with 1 μg/mL SEB and analyzed for the expression of surface and intracellular molecules at the indicated time points by flow cytometry. (A) IRF4 and CD69 expression by CD8 + T cells after stimulation with SEB. (B) IRF4 and CD69 expression by CD8 + T cells in unstimulated PBMCs (left panel) and PBMCs stimulated with SEB for 18 hours (right panel). (C) IRF4 and CD69 expression (left panel) and CD25 and CD137 expression (right panel) by CD8 + T cells after 18 hours of SEB stimulation. (D) Frequency of IRF4 + CD69 + , CD137 + , and CD25 + CD137 + cells in CD8 + T cells from healthy donor PBMCs after 18 hours of SEB stimulation (n = 24). Each point represents an individual donor. (E) IRF4 and CD69 expression in CD8 + T CM cells (CD45RA − CCR7 + ), T EM cells (CD45RA − CCR7 − ), naive T cells (CD45RA + CCR7 − ), and T EMRA cells (CD45RA + CCR7 + ) in SEB-stimulated PBMCs. (F) Mean fluorescence intensity (MFI) of IRF4 of baseline CD8 + T CM , T EM , naive T, and T EMRA cells (left panel) and after 18 hours of SEB stimulation (right panel) in healthy donor PBMCs (n = 3). Horizontal lines indicate the mean (± standard deviation). Each symbol represents an individual donor. * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation

IRF4 hi CD8 + T cells highly express CD25. Healthy donor PBMCs were stimulated with CEF peptides for 18 hours. (A) Mean fluorescence intensity of CD25, CD69, and CD137 in IRF4 neg , IRF4 lo , IRF4 int , and IRF4 hi CD137 + populations. (B) A representative viSNE analysis of CEF-specific CD8 + T cells (gated on CD8 + CD69 + cells). Each point represents a single cell, and different colors represent the intensity of the expression of the molecule. CEF-specific CD8 + T cells (IRF4 + CD137 + ) are indicated by the oval.
Figure Legend Snippet: IRF4 hi CD8 + T cells highly express CD25. Healthy donor PBMCs were stimulated with CEF peptides for 18 hours. (A) Mean fluorescence intensity of CD25, CD69, and CD137 in IRF4 neg , IRF4 lo , IRF4 int , and IRF4 hi CD137 + populations. (B) A representative viSNE analysis of CEF-specific CD8 + T cells (gated on CD8 + CD69 + cells). Each point represents a single cell, and different colors represent the intensity of the expression of the molecule. CEF-specific CD8 + T cells (IRF4 + CD137 + ) are indicated by the oval.

Techniques Used: Fluorescence, Expressing

The IRF4-CD137 assay detects Ag-specific CD8 + T cells with low background. PBMCs were cultured with CEF peptides for 18 hours and analyzed for the expression of the indicated molecules. Frequency of IRF4 + CD69 + (top panels), IRF4 + CD137 + (middle panels), and CD25 + CD137 + cells (bottom panels) in CD8 + T cells. Two representative results are shown.
Figure Legend Snippet: The IRF4-CD137 assay detects Ag-specific CD8 + T cells with low background. PBMCs were cultured with CEF peptides for 18 hours and analyzed for the expression of the indicated molecules. Frequency of IRF4 + CD69 + (top panels), IRF4 + CD137 + (middle panels), and CD25 + CD137 + cells (bottom panels) in CD8 + T cells. Two representative results are shown.

Techniques Used: Cell Culture, Expressing

IRF4 expression reflects TCR signal strength. (A) Isolated blood CD8 + T cells were stimulated with increasing concentrations of plate-bound anti-CD3 mAb (0.1-1 μg/mL) for 18 hours. Mean fluorescence intensity of IRF4 in IRF4 + CD137 + cells. A representative result from 3 individual experiments is shown. (B) PBMCs were treated for 30 minutes with increasing concentrations of anti-human pan MHC class I mAb (0-100 μg/mL) and then cultured with CEF peptides for 18 hours. Expression of IRF4, CD69, CD25, and CD137 by activated CD8 + T cells was analyzed. A representative result from 6 individual experiments is shown.
Figure Legend Snippet: IRF4 expression reflects TCR signal strength. (A) Isolated blood CD8 + T cells were stimulated with increasing concentrations of plate-bound anti-CD3 mAb (0.1-1 μg/mL) for 18 hours. Mean fluorescence intensity of IRF4 in IRF4 + CD137 + cells. A representative result from 3 individual experiments is shown. (B) PBMCs were treated for 30 minutes with increasing concentrations of anti-human pan MHC class I mAb (0-100 μg/mL) and then cultured with CEF peptides for 18 hours. Expression of IRF4, CD69, CD25, and CD137 by activated CD8 + T cells was analyzed. A representative result from 6 individual experiments is shown.

Techniques Used: Expressing, Isolation, Fluorescence, Cell Culture

IRF4 expression by tetramer + CD8 + T cells. PBMCs from HLA-A2 + healthy donors were prestained with Flu-M1–A2 tetramer and cultured with Flu-M1 GILGFVFTL peptide for 18 hours. (A) Frequency of tetramer + cells in CD8 + T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. (B) Frequency of IRF4/CD69 (left panels), CD25/CD137 (middle panels), and IRF4/CD137 (right panels) of tetramer + CD8 + T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. A representative result from 5 individual experiments is shown.
Figure Legend Snippet: IRF4 expression by tetramer + CD8 + T cells. PBMCs from HLA-A2 + healthy donors were prestained with Flu-M1–A2 tetramer and cultured with Flu-M1 GILGFVFTL peptide for 18 hours. (A) Frequency of tetramer + cells in CD8 + T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. (B) Frequency of IRF4/CD69 (left panels), CD25/CD137 (middle panels), and IRF4/CD137 (right panels) of tetramer + CD8 + T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. A representative result from 5 individual experiments is shown.

Techniques Used: Expressing, Cell Culture

Low IRF4 expression by HIV-specific CD8 + T cells was independent of PD-1 signals. (A) PD-1 and CD69 expression by IRF4 + CD137 + CD8 + T cells in unstimulated, CEF-stimulated, or HIV-stimulated PBMCs. A representative of 7 samples (left panel). PD-1 MFI by CEF- and HIV-specific CD8 + T cells (middle panels). Gated on IRF4 + CD137 + CD8 + T cells (n = 7). One donor showed high PD-1 by HIV-specific CD8 + T cells (black symbols). (B) PBMCs from HIV + subjects were treated for 30 minutes with anti–PD-L1 antibody and stimulated with HIV peptides for 18 hours. IRF4/CD137 expression with or without pretreatment with anti–PD-L1. Gated on CD8 + T cells. A representative result from 5 experiments is shown. (C) MFI of IRF4 in HIV-specific CD8 + T cells treated or not with anti–PD-L1. Gated on IRF4 + CD137 + CD8 + T cells (n = 5). One donor showed high PD-1 by HIV-specific CD8 + T cells (black symbols). NS, not significant (paired Student t test).
Figure Legend Snippet: Low IRF4 expression by HIV-specific CD8 + T cells was independent of PD-1 signals. (A) PD-1 and CD69 expression by IRF4 + CD137 + CD8 + T cells in unstimulated, CEF-stimulated, or HIV-stimulated PBMCs. A representative of 7 samples (left panel). PD-1 MFI by CEF- and HIV-specific CD8 + T cells (middle panels). Gated on IRF4 + CD137 + CD8 + T cells (n = 7). One donor showed high PD-1 by HIV-specific CD8 + T cells (black symbols). (B) PBMCs from HIV + subjects were treated for 30 minutes with anti–PD-L1 antibody and stimulated with HIV peptides for 18 hours. IRF4/CD137 expression with or without pretreatment with anti–PD-L1. Gated on CD8 + T cells. A representative result from 5 experiments is shown. (C) MFI of IRF4 in HIV-specific CD8 + T cells treated or not with anti–PD-L1. Gated on IRF4 + CD137 + CD8 + T cells (n = 5). One donor showed high PD-1 by HIV-specific CD8 + T cells (black symbols). NS, not significant (paired Student t test).

Techniques Used: Expressing

HIV-specific CD8 + T cells express low IRF4. PBMCs from HIV + subjects were stimulated with 1 μg/mL CEF peptides or HIV peptides for 18 hours. (A) Expression of IRF4 and CD137 by CD8 + T cells. (B) MFI of IRF4 and CD137 expression by CD8 + T cells specific for CEF and HIV peptides (n = 13). (C) viSNE analysis showing differential marker expression by CD8 + T cells specific for CEF peptides (upper panels) or HIV peptides (lower panels). A representative result of 13 samples (gated on CD8 + CD69 + T cells) is shown. Red arrows indicate a cell population uniquely observed in the HIV-specific CD8 + T cells. Blue arrows indicate a cell population shared between CEF- and HIV-specific CD8 + T cells. (D) CD27 and CD25 expression by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides. A representative result of 11 samples is shown. (E) MFI of CD25 (left panel) and percentage of CD25 + (right) by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). (F) MFI of CD27 (left panel) and percentage of CD27 + (right panel) by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). * P
Figure Legend Snippet: HIV-specific CD8 + T cells express low IRF4. PBMCs from HIV + subjects were stimulated with 1 μg/mL CEF peptides or HIV peptides for 18 hours. (A) Expression of IRF4 and CD137 by CD8 + T cells. (B) MFI of IRF4 and CD137 expression by CD8 + T cells specific for CEF and HIV peptides (n = 13). (C) viSNE analysis showing differential marker expression by CD8 + T cells specific for CEF peptides (upper panels) or HIV peptides (lower panels). A representative result of 13 samples (gated on CD8 + CD69 + T cells) is shown. Red arrows indicate a cell population uniquely observed in the HIV-specific CD8 + T cells. Blue arrows indicate a cell population shared between CEF- and HIV-specific CD8 + T cells. (D) CD27 and CD25 expression by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides. A representative result of 11 samples is shown. (E) MFI of CD25 (left panel) and percentage of CD25 + (right) by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). (F) MFI of CD27 (left panel) and percentage of CD27 + (right panel) by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). * P

Techniques Used: Expressing, Marker

3) Product Images from "Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation"

Article Title: Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation

Journal: Nature immunology

doi: 10.1038/s41590-018-0085-3

Antigen-loaded macrophages accumulate more immunological synapses with effector cells compared to antigen-loaded CD4 +  T cells (a)  Assessment of target-effector doublets. Peptide-loaded, FarRed-labeled targets were co-cultured for the indicated times with Violet-labeled expanded CTLs followed by fixation, actin staining, and analysis via imaging flow cytometry. Shown are representative plots (one of two independent experiments) of effector-target co-culture samples and the gating strategy used to quantitate total peptide-loaded targets (red box) and target-effector doublets (blue box).  (b)  Immunological synapses formed between target-effector pairs. Data points within the doublet gate were assessed for concentrated actin (yellow) at the interface between the effectors and targets 32 . As in (a), shown is a representation of one of two independent experiments. White scale bars denote 10μm.  (c)  Summary of immunological synapse accumulation over 60 minutes (n=6 distinct samples from two independent experiments). Frequencies of immunological synapses were calculated as described in the Methods. Shown are means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p=0.001, **p=0.0001, ***p
Figure Legend Snippet: Antigen-loaded macrophages accumulate more immunological synapses with effector cells compared to antigen-loaded CD4 + T cells (a) Assessment of target-effector doublets. Peptide-loaded, FarRed-labeled targets were co-cultured for the indicated times with Violet-labeled expanded CTLs followed by fixation, actin staining, and analysis via imaging flow cytometry. Shown are representative plots (one of two independent experiments) of effector-target co-culture samples and the gating strategy used to quantitate total peptide-loaded targets (red box) and target-effector doublets (blue box). (b) Immunological synapses formed between target-effector pairs. Data points within the doublet gate were assessed for concentrated actin (yellow) at the interface between the effectors and targets 32 . As in (a), shown is a representation of one of two independent experiments. White scale bars denote 10μm. (c) Summary of immunological synapse accumulation over 60 minutes (n=6 distinct samples from two independent experiments). Frequencies of immunological synapses were calculated as described in the Methods. Shown are means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p=0.001, **p=0.0001, ***p

Techniques Used: Labeling, Cell Culture, Staining, Imaging, Flow Cytometry, Cytometry, Co-Culture Assay

HIV-infected macrophages are less susceptible to CTL-mediated killing compared to HIV-infected CD4 +  T cells (a)  HIV-infected target elimination assay. HIV-infected CD4 +  T cells and macrophages were co-cultured with CTL effectors for four hours, followed by quantitation of infected cells by flow cytometry. Live infected cells are depicted in the gate outlining positive Gag-p24 intracellular staining with down-modulation of surface CD4, representing one of four independent experiments. See also  Supplementary Figs. 1  and  2 .  (b) Summary of elimination assays using ex vivo CTL (n=16 distinct samples from four independent experiments) and  (c)  HIV peptide-expanded CTL from HIV-infected donors (n=16 distinct samples from four independent experiments) and CTL from HIV −  healthy donors (n=3 distinct samples from two independent experiments) as a negative control. Shown are the means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p
Figure Legend Snippet: HIV-infected macrophages are less susceptible to CTL-mediated killing compared to HIV-infected CD4 + T cells (a) HIV-infected target elimination assay. HIV-infected CD4 + T cells and macrophages were co-cultured with CTL effectors for four hours, followed by quantitation of infected cells by flow cytometry. Live infected cells are depicted in the gate outlining positive Gag-p24 intracellular staining with down-modulation of surface CD4, representing one of four independent experiments. See also Supplementary Figs. 1 and 2 . (b) Summary of elimination assays using ex vivo CTL (n=16 distinct samples from four independent experiments) and (c) HIV peptide-expanded CTL from HIV-infected donors (n=16 distinct samples from four independent experiments) and CTL from HIV − healthy donors (n=3 distinct samples from two independent experiments) as a negative control. Shown are the means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p

Techniques Used: Infection, CTL Assay, Cell Culture, Quantitation Assay, Flow Cytometry, Cytometry, Staining, Ex Vivo, Negative Control

CTL induce delayed, caspase-3 dependent apoptosis of macrophages resulting in less efficient control of HIV infection (a)  Target killing time course. Peptide-loaded targets were incubated with ex vivo CTL from HIV +  donors (n=4 distinct samples from two independent experiments) for the indicated times, followed by analysis for live FarRed staining via flow cytometry. Shown are the means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p=0.0201, **p=0.0165, ***p=0.0156. See also  Supplementary Fig. 4 . (b)  Viral inhibition assay. Ex vivo CTL from HIV +  donors (n=8 distinct samples from three independent experiments) were co-cultured with HIV-infected CD4 +  T cells or macrophages for seven days, followed by measurement of culture supernatant Gag-p24 antigen. Statistical analysis: two-sided unpaired t-test, * p=0.0005, **p
Figure Legend Snippet: CTL induce delayed, caspase-3 dependent apoptosis of macrophages resulting in less efficient control of HIV infection (a) Target killing time course. Peptide-loaded targets were incubated with ex vivo CTL from HIV + donors (n=4 distinct samples from two independent experiments) for the indicated times, followed by analysis for live FarRed staining via flow cytometry. Shown are the means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p=0.0201, **p=0.0165, ***p=0.0156. See also Supplementary Fig. 4 . (b) Viral inhibition assay. Ex vivo CTL from HIV + donors (n=8 distinct samples from three independent experiments) were co-cultured with HIV-infected CD4 + T cells or macrophages for seven days, followed by measurement of culture supernatant Gag-p24 antigen. Statistical analysis: two-sided unpaired t-test, * p=0.0005, **p

Techniques Used: CTL Assay, Infection, Incubation, Ex Vivo, Staining, Flow Cytometry, Cytometry, Inhibition, Cell Culture

Killing of target cells is MHC-I and perforin-dependent, but granzyme B is dispensable for CD4 +  T cell killing (a and b)  Inhibiting HIV-infected target cell elimination. HIV-infected CD4 +  T cells (a) or macrophages (b) were incubated with HIV-peptide expanded CTL in the presence of an MHC-I blocking antibody, concanamycin A (CMA – an indirect perforin inhibitor), a granzyme B inhibitor, a FAS neutralizing antibody, or recombinant human TRAIL-R1-Fc for four hours. Target cells were analyzed for live cell Gag-p24 intracellular staining via flow cytometry. Shown is the elimination assay summary using HIV +  patients (n = 8 distinct samples from four independent experiments). Statistical analysis: two-sided paired t-test, *p=0.0153, **p=0.0097, *** p=0.0036, ****p
Figure Legend Snippet: Killing of target cells is MHC-I and perforin-dependent, but granzyme B is dispensable for CD4 + T cell killing (a and b) Inhibiting HIV-infected target cell elimination. HIV-infected CD4 + T cells (a) or macrophages (b) were incubated with HIV-peptide expanded CTL in the presence of an MHC-I blocking antibody, concanamycin A (CMA – an indirect perforin inhibitor), a granzyme B inhibitor, a FAS neutralizing antibody, or recombinant human TRAIL-R1-Fc for four hours. Target cells were analyzed for live cell Gag-p24 intracellular staining via flow cytometry. Shown is the elimination assay summary using HIV + patients (n = 8 distinct samples from four independent experiments). Statistical analysis: two-sided paired t-test, *p=0.0153, **p=0.0097, *** p=0.0036, ****p

Techniques Used: Infection, Incubation, CTL Assay, Blocking Assay, Recombinant, Staining, Flow Cytometry, Cytometry

Ex vivo HIV-specific CTL exhibit low perforin + granzyme +  expression compared to expanded CTL (a)  Perforin and granzyme staining of ex vivo HIV tetramer +  CD8 +  T cells. Shown is a representative plot of A02-SL9 (HIV) tetramer staining from two independent experiments. Tetramer +  cells were phenotyped for perforin, and granzyme A, B, H, K, and M staining. Staining controls are shown in  Supplementary Fig. 5a and b . Also see  Supplementary Fig. 5c  for CMV-specific CTL perforin and granzyme expression as a comparison. Shown is a representation of one experiment from two independent experiments.  (b)  Summary of total granzyme expression on HIV +  controller ex vivo HIV-specific CTL (n=7 distinct samples from two independent experiments). Box elements, center line, limits and whiskers are the median, 25 th -75 th  percentiles and min-max, respectively.  (c)  Summary of dual perforin and granzyme expression on ex vivo HIV-specific CD8 +  T cells (n=7 distinct samples from two independent experiments). Box elements, center line, limits and whiskers are the median, 25 th -75 th percentiles and min-max, respectively.  (d)  Cytolytic capacity of HIV-specific ex vivo and expanded CTL. Ex vivo or expanded CTLs were incubated with HIV-infected CD4 +  T cells for six hours followed by flow cytometry analysis of degranulation (surface CD107a expression), perforin and granzyme expression. Shown is a representative analysis of CTL perforin and granzyme phenotyping of degranulated cells from two independent experiments. Supplementary Fig. 5d  shows staining controls.  (e)  Summary of the perforin and granzyme co-expression of degranulated ex vivo and expanded CTL for HIV +  patients (n=8 distinct samples from two independent experiments). Box elements, center line, limits and whiskers are the median, 25 th -75 th  percentiles and min-max, respectively. Statistical analysis: two-tailed unpaired t-test, *p=0.0156, **p=0.0115, ***p=0.005, ****p
Figure Legend Snippet: Ex vivo HIV-specific CTL exhibit low perforin + granzyme + expression compared to expanded CTL (a) Perforin and granzyme staining of ex vivo HIV tetramer + CD8 + T cells. Shown is a representative plot of A02-SL9 (HIV) tetramer staining from two independent experiments. Tetramer + cells were phenotyped for perforin, and granzyme A, B, H, K, and M staining. Staining controls are shown in Supplementary Fig. 5a and b . Also see Supplementary Fig. 5c for CMV-specific CTL perforin and granzyme expression as a comparison. Shown is a representation of one experiment from two independent experiments. (b) Summary of total granzyme expression on HIV + controller ex vivo HIV-specific CTL (n=7 distinct samples from two independent experiments). Box elements, center line, limits and whiskers are the median, 25 th -75 th percentiles and min-max, respectively. (c) Summary of dual perforin and granzyme expression on ex vivo HIV-specific CD8 + T cells (n=7 distinct samples from two independent experiments). Box elements, center line, limits and whiskers are the median, 25 th -75 th percentiles and min-max, respectively. (d) Cytolytic capacity of HIV-specific ex vivo and expanded CTL. Ex vivo or expanded CTLs were incubated with HIV-infected CD4 + T cells for six hours followed by flow cytometry analysis of degranulation (surface CD107a expression), perforin and granzyme expression. Shown is a representative analysis of CTL perforin and granzyme phenotyping of degranulated cells from two independent experiments. Supplementary Fig. 5d shows staining controls. (e) Summary of the perforin and granzyme co-expression of degranulated ex vivo and expanded CTL for HIV + patients (n=8 distinct samples from two independent experiments). Box elements, center line, limits and whiskers are the median, 25 th -75 th percentiles and min-max, respectively. Statistical analysis: two-tailed unpaired t-test, *p=0.0156, **p=0.0115, ***p=0.005, ****p

Techniques Used: Ex Vivo, CTL Assay, Expressing, Staining, Incubation, Infection, Flow Cytometry, Cytometry, Two Tailed Test

HIV-infected macrophages induce stronger CTL cytokine responses than infected CD4 +  T cells (a)  MHC-I surface density. Imaging flow cytometry was used to calculate relative surface densities of MHC-I on CD4 +  T cells and macrophages (see  Supplementary Fig. 6a and b ) (n=7 distinct samples from three independent experiments). Box elements, median and 25 th -75 th  percentiles. Statistical analysis, two-sided Mann-Whitney test. p=0.2209.  (b)  CTL recognition assay. Ex vivo and expanded CTL were incubated with mock/uninfected or HIV-infected CD4 +  T cells or macrophages for six hours followed by analysis of degranulation (CD107a expression). See also  Supplementary Fig. 6c . Shown is one of five independent experiments.  (c)  Comparison of CTL responses to CD4 +  T cells and macrophages. Data shown are the ratios of CTL degranulation in response to macrophages over CD4 +  T cells for ex vivo (n=14 distinct samples from five independent experiments) and expanded CTL (n=16 distinct samples from five independent experiments). Responses against CEF peptide-loaded targets were also assessed (n=6 distinct samples from three independent experiments). Shown are the means +/- SEM. Statistical analysis: two-sided one sample t-test, *p=0.0036, and two-sided Wilcoxon signed rank test, **p
Figure Legend Snippet: HIV-infected macrophages induce stronger CTL cytokine responses than infected CD4 + T cells (a) MHC-I surface density. Imaging flow cytometry was used to calculate relative surface densities of MHC-I on CD4 + T cells and macrophages (see Supplementary Fig. 6a and b ) (n=7 distinct samples from three independent experiments). Box elements, median and 25 th -75 th percentiles. Statistical analysis, two-sided Mann-Whitney test. p=0.2209. (b) CTL recognition assay. Ex vivo and expanded CTL were incubated with mock/uninfected or HIV-infected CD4 + T cells or macrophages for six hours followed by analysis of degranulation (CD107a expression). See also Supplementary Fig. 6c . Shown is one of five independent experiments. (c) Comparison of CTL responses to CD4 + T cells and macrophages. Data shown are the ratios of CTL degranulation in response to macrophages over CD4 + T cells for ex vivo (n=14 distinct samples from five independent experiments) and expanded CTL (n=16 distinct samples from five independent experiments). Responses against CEF peptide-loaded targets were also assessed (n=6 distinct samples from three independent experiments). Shown are the means +/- SEM. Statistical analysis: two-sided one sample t-test, *p=0.0036, and two-sided Wilcoxon signed rank test, **p

Techniques Used: Infection, CTL Assay, Imaging, Flow Cytometry, Cytometry, MANN-WHITNEY, Ex Vivo, Incubation, Expressing

4) Product Images from "Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6+ CD4+ T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection"

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6+ CD4+ T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

Journal: Journal of Virology

doi: 10.1128/JVI.01402-16

TLR2 agonist augments intracellular levels of phosphorylated p65 in CD3/CD28-costimulated CCR6 +  CD4 +  T cells. Purified primary human CD4 +  T cells were subjected to a CD3/CD28 costimulation in the absence or presence of Pam3CSK4. Intracellular expression of phosphorylated p65 was then analyzed by flow cytometry in both CCR6 −  CD4 +  and CCR6 +  CD4 +  T cell subpopulations. The data shown were obtained from CD4 +  T cell preparations isolated from the peripheral blood of five healthy donors. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired  t  test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (*,  P  ≤ 0.05; **,  P  ≤ 0.01).
Figure Legend Snippet: TLR2 agonist augments intracellular levels of phosphorylated p65 in CD3/CD28-costimulated CCR6 + CD4 + T cells. Purified primary human CD4 + T cells were subjected to a CD3/CD28 costimulation in the absence or presence of Pam3CSK4. Intracellular expression of phosphorylated p65 was then analyzed by flow cytometry in both CCR6 − CD4 + and CCR6 + CD4 + T cell subpopulations. The data shown were obtained from CD4 + T cell preparations isolated from the peripheral blood of five healthy donors. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01).

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

TLR2 triggering enhances HIV-1 replication in CD3/CD28-costimulated CD4 +  T cells. Purified primary human CD4 +  T cells were subjected to CD3/CD28 costimulation either in the absence or presence of the TLR2 agonist Pam3CSK4. (A) Cells were incubated with NL4.3 Balenv, and virus production was estimated at the indicated time points by measuring the p24 content in cell-free supernatants. (B) Cells were inoculated with NL4.3 Bal-IRES-HSA reporter virus, and the percentages of cells productively infected with HIV-1 (i.e., HSA + ) were evaluated by flow cytometry. Data shown in panel A represent the means ± standard deviations (SD) of duplicates for a representative donor out of four. Each symbol shown in panel B represents a different donor, with the horizontal line depicting the means for all donors tested. Statistical analyses were made using ratio paired  t  test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (**,  P  ≤ 0.01).
Figure Legend Snippet: TLR2 triggering enhances HIV-1 replication in CD3/CD28-costimulated CD4 + T cells. Purified primary human CD4 + T cells were subjected to CD3/CD28 costimulation either in the absence or presence of the TLR2 agonist Pam3CSK4. (A) Cells were incubated with NL4.3 Balenv, and virus production was estimated at the indicated time points by measuring the p24 content in cell-free supernatants. (B) Cells were inoculated with NL4.3 Bal-IRES-HSA reporter virus, and the percentages of cells productively infected with HIV-1 (i.e., HSA + ) were evaluated by flow cytometry. Data shown in panel A represent the means ± standard deviations (SD) of duplicates for a representative donor out of four. Each symbol shown in panel B represents a different donor, with the horizontal line depicting the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (**, P ≤ 0.01).

Techniques Used: Purification, Incubation, Infection, Flow Cytometry, Cytometry

5) Product Images from "Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy"

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

Journal: Science Advances

doi: 10.1126/sciadv.aaz8521

MAPKi polarizes MΦs toward an alternatively activated, HGF- and GAS6-producing phenotype. ( A ) Schematic of M2-MΦ conditioned medium experiments (left) and data showing ES2 cell count following 72-hour trametinib ± conditioned medium from M2-MΦ that were pretreated with 24-hour trametinib (right) ( n = 3). ( B ) OVCA viability was measured by propidium iodide and annexin V staining for three cell lines treated for 48 hours with 100 nM trametinib ± transwell coculture with M2-MΦ (averaged across five donors with n = 3 reps per donor, two-tailed t test). ( C ) Heat map of supernatant and lysate proteins from PBMC-derived M1-MΦ and M2-MΦ. Analyte levels of MΦs treated with 24-hour trametinib are plotted relative to their respective no-treatment controls ( n = 3, * P
Figure Legend Snippet: MAPKi polarizes MΦs toward an alternatively activated, HGF- and GAS6-producing phenotype. ( A ) Schematic of M2-MΦ conditioned medium experiments (left) and data showing ES2 cell count following 72-hour trametinib ± conditioned medium from M2-MΦ that were pretreated with 24-hour trametinib (right) ( n = 3). ( B ) OVCA viability was measured by propidium iodide and annexin V staining for three cell lines treated for 48 hours with 100 nM trametinib ± transwell coculture with M2-MΦ (averaged across five donors with n = 3 reps per donor, two-tailed t test). ( C ) Heat map of supernatant and lysate proteins from PBMC-derived M1-MΦ and M2-MΦ. Analyte levels of MΦs treated with 24-hour trametinib are plotted relative to their respective no-treatment controls ( n = 3, * P

Techniques Used: Cell Counting, Staining, Two Tailed Test, Derivative Assay

6) Product Images from "Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells"

Article Title: Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

Journal: Scientific Reports

doi: 10.1038/srep15262

CTLs expanded by DCs derived from minigene-modified hPSCs are immunocompetent. ( a ) Expansion of MART-1-specific CD8+ T cells by H1.ME-DCs in bulk culture. HLA-A2+ PBLs were primed and then restimulated twice with H1.ME-DCs. MART-1-specific T cell expansion during this process was monitored by flow cytometry at the indicated time points. The percentages of pentamer+ CD8+ cells in total T cells are shown in the representative contour plots. ( b ) Phenotype of MART-1-specific T cells expanded by H1.ME-DCs. ( c ) GrB secretion by MART-1-specific T cells expanded by H1.ME-DCs as measured by ELISPOT. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3). ( d ) Specific cytotoxicity of MART-1-specific T cells expanded by H1.ME-DCs. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3, *p
Figure Legend Snippet: CTLs expanded by DCs derived from minigene-modified hPSCs are immunocompetent. ( a ) Expansion of MART-1-specific CD8+ T cells by H1.ME-DCs in bulk culture. HLA-A2+ PBLs were primed and then restimulated twice with H1.ME-DCs. MART-1-specific T cell expansion during this process was monitored by flow cytometry at the indicated time points. The percentages of pentamer+ CD8+ cells in total T cells are shown in the representative contour plots. ( b ) Phenotype of MART-1-specific T cells expanded by H1.ME-DCs. ( c ) GrB secretion by MART-1-specific T cells expanded by H1.ME-DCs as measured by ELISPOT. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3). ( d ) Specific cytotoxicity of MART-1-specific T cells expanded by H1.ME-DCs. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3, *p

Techniques Used: Derivative Assay, Modification, Flow Cytometry, Cytometry, Enzyme-linked Immunospot

7) Product Images from "Epigenetic Metabolite Acetate Inhibits Class I/II Histone Deacetylases, Promotes Histone Acetylation, and Increases HIV-1 Integration in CD4+ T Cells"

Article Title: Epigenetic Metabolite Acetate Inhibits Class I/II Histone Deacetylases, Promotes Histone Acetylation, and Increases HIV-1 Integration in CD4+ T Cells

Journal: Journal of Virology

doi: 10.1128/JVI.01943-16

Acetate does not affect cell viability but induces a dose-dependent increase in HIV-1 replication. Purified primary human CD4 +  T cells were costimulated with anti-CD3 and anti-CD28 MAbs in the absence or presence of increasing concentrations of acetate. (A) Cell viability was monitored by flow cytometry at day 6 following acetate treatment. (B) Purified primary human CD4 +  T cells were first treated as described for panel A and then incubated with the NL4.3Bal-IRES-HSA reporter virus for 3 days before quantifying the percentages of HSA +  cells by flow cytometry. Each symbol represents a different donor, with the horizontal lines depicting the means of five donors tested. Statistical analyses were done using ratio-paired Student's  t  tests. The asterisks denote statistically significant data (*,  P  ≤ 0.05; **,  P  ≤ 0.01; ***,  P  ≤ 0.001).
Figure Legend Snippet: Acetate does not affect cell viability but induces a dose-dependent increase in HIV-1 replication. Purified primary human CD4 + T cells were costimulated with anti-CD3 and anti-CD28 MAbs in the absence or presence of increasing concentrations of acetate. (A) Cell viability was monitored by flow cytometry at day 6 following acetate treatment. (B) Purified primary human CD4 + T cells were first treated as described for panel A and then incubated with the NL4.3Bal-IRES-HSA reporter virus for 3 days before quantifying the percentages of HSA + cells by flow cytometry. Each symbol represents a different donor, with the horizontal lines depicting the means of five donors tested. Statistical analyses were done using ratio-paired Student's t tests. The asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Techniques Used: Purification, Flow Cytometry, Cytometry, Incubation

Acetate exerts a more potent effect on HIV-1 integration events. Primary human CD4 +  T cells from 6 distinct donors were first subjected to CD3/CD28 costimulation for 72 h in the absence or presence of acetate. Next, the cells were infected for 24 h with NL4.3Balenv viruses. The cells were processed to determine the number of copies of completed reverse transcripts (A) and proviral DNA (B) by a quantitative real-time PCR test. Each symbol represents a different donor, and the horizontal lines depict the means of 6 donors tested. Statistical analyses were done using ratio-paired Student's  t  tests. The asterisks denote statistically significant data (*,  P  ≤ 0.05; **,  P  ≤ 0.01).
Figure Legend Snippet: Acetate exerts a more potent effect on HIV-1 integration events. Primary human CD4 + T cells from 6 distinct donors were first subjected to CD3/CD28 costimulation for 72 h in the absence or presence of acetate. Next, the cells were infected for 24 h with NL4.3Balenv viruses. The cells were processed to determine the number of copies of completed reverse transcripts (A) and proviral DNA (B) by a quantitative real-time PCR test. Each symbol represents a different donor, and the horizontal lines depict the means of 6 donors tested. Statistical analyses were done using ratio-paired Student's t tests. The asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01).

Techniques Used: Infection, Real-time Polymerase Chain Reaction

HIV-1 replication in CD3/CD28-costimulated CD4 +  T cells is augmented by acetate. Purified primary human CD4 +  T cells were costimulated with anti-CD3 and anti-CD28 MAbs in the absence or presence of acetate (20 mM). (A) The cells were next inoculated with replication-competent NL4.3Balenv viruses, and virus production was estimated either at 6 dpi (left) (6 donors are shown) or at the indicated time points postinfection (right) (a representative donor is shown) by measuring the p24 contents in cell-free supernatants. (B) In some experiments, untreated and acetate-treated cells were incubated with the NL4.3Bal-IRES-HSA reporter virus for 3 days before assessing HSA expression by flow cytometry. Percentages of HSA +  cells are depicted on the left, while the MFI is shown on the right. Each symbol represents a different donor, with the horizontal lines depicting the means of six donors tested. Statistical analyses were done using ratio-paired Student's  t  test. The asterisks denote statistically significant data (**,  P  ≤ 0.01; ***,  P  ≤ 0.001). (C) The gating strategy used in flow cytometry analyses to estimate the percentage of cells productively infected with HIV-1 (HSA +  as defined with an allophycocyanin [APC]-conjugated anti-HSA MAb) for each experimental condition consisted of live lymphocyte gating based on size and complexity on a forward scatter (FSC)/side scatter (SSC) plot (left), followed by doublet discrimination on an FSC-height (H)/FSC-width (W) plot (center), to finally gate HSA +  cells on an FSC-H/APC plot (right). Mock-infected cells were used as negative controls for HSA staining. The number in the plots indicates the percentage of cells within the gate.
Figure Legend Snippet: HIV-1 replication in CD3/CD28-costimulated CD4 + T cells is augmented by acetate. Purified primary human CD4 + T cells were costimulated with anti-CD3 and anti-CD28 MAbs in the absence or presence of acetate (20 mM). (A) The cells were next inoculated with replication-competent NL4.3Balenv viruses, and virus production was estimated either at 6 dpi (left) (6 donors are shown) or at the indicated time points postinfection (right) (a representative donor is shown) by measuring the p24 contents in cell-free supernatants. (B) In some experiments, untreated and acetate-treated cells were incubated with the NL4.3Bal-IRES-HSA reporter virus for 3 days before assessing HSA expression by flow cytometry. Percentages of HSA + cells are depicted on the left, while the MFI is shown on the right. Each symbol represents a different donor, with the horizontal lines depicting the means of six donors tested. Statistical analyses were done using ratio-paired Student's t test. The asterisks denote statistically significant data (**, P ≤ 0.01; ***, P ≤ 0.001). (C) The gating strategy used in flow cytometry analyses to estimate the percentage of cells productively infected with HIV-1 (HSA + as defined with an allophycocyanin [APC]-conjugated anti-HSA MAb) for each experimental condition consisted of live lymphocyte gating based on size and complexity on a forward scatter (FSC)/side scatter (SSC) plot (left), followed by doublet discrimination on an FSC-height (H)/FSC-width (W) plot (center), to finally gate HSA + cells on an FSC-H/APC plot (right). Mock-infected cells were used as negative controls for HSA staining. The number in the plots indicates the percentage of cells within the gate.

Techniques Used: Purification, Incubation, Expressing, Flow Cytometry, Cytometry, Infection, Staining

Acetate mediates histone H3 and H4 acetylation. Primary human CD4 +  T cells were first subjected to CD3/CD28 costimulation for 72 h in the absence or presence of acetate. Control cells were further stimulated for 4 h with the HDAC inhibitor TSA. Histone acetylation was detected by flow cytometry using MAbs specific for H3K9 (A) and H4 K5/8/12/16 (B). Each bar represents the mean percentages and standard deviations (SD) of positive cells from 5 distinct donors. Flow cytometry data obtained with cells costimulated with CD3 and CD28 MAbs were set at 100%. Statistical analyses were performed using ratio-paired  t  tests. The asterisks denote statistically significant data (*,  P  ≤ 0.05; **,  P  ≤ 0.01).
Figure Legend Snippet: Acetate mediates histone H3 and H4 acetylation. Primary human CD4 + T cells were first subjected to CD3/CD28 costimulation for 72 h in the absence or presence of acetate. Control cells were further stimulated for 4 h with the HDAC inhibitor TSA. Histone acetylation was detected by flow cytometry using MAbs specific for H3K9 (A) and H4 K5/8/12/16 (B). Each bar represents the mean percentages and standard deviations (SD) of positive cells from 5 distinct donors. Flow cytometry data obtained with cells costimulated with CD3 and CD28 MAbs were set at 100%. Statistical analyses were performed using ratio-paired t tests. The asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01).

Techniques Used: Flow Cytometry, Cytometry

Acetate decreases virus entry into CD3/CD28-costimulated CD4 +  T cells. Purified primary human CD4 +  T cells were subjected to CD3/CD28 costimulation in the absence or presence of acetate. Virus entry was estimated by quantifying the intracellular p24 contents by an enzyme-linked immunosorbent assay (ELISA) in the total cell population as described in Materials and Methods. The data shown represent the means and standard errors of the mean (SEM) of triplicate samples. Statistical analysis was performed using ratio-paired  t  tests (*,  P
Figure Legend Snippet: Acetate decreases virus entry into CD3/CD28-costimulated CD4 + T cells. Purified primary human CD4 + T cells were subjected to CD3/CD28 costimulation in the absence or presence of acetate. Virus entry was estimated by quantifying the intracellular p24 contents by an enzyme-linked immunosorbent assay (ELISA) in the total cell population as described in Materials and Methods. The data shown represent the means and standard errors of the mean (SEM) of triplicate samples. Statistical analysis was performed using ratio-paired t tests (*, P

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay

8) Product Images from "Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood"

Article Title: Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood

Journal: Blood Advances

doi: 10.1182/bloodadvances.2019000292

Expression of IRF4 by activated human blood CD8 + T cells.  Healthy donor PBMCs were stimulated with 1 μg/mL SEB and analyzed for the expression of surface and intracellular molecules at the indicated time points by flow cytometry. (A) IRF4 and CD69 expression by CD8 +  T cells after stimulation with SEB. (B) IRF4 and CD69 expression by CD8 +  T cells in unstimulated PBMCs (left panel) and PBMCs stimulated with SEB for 18 hours (right panel). (C) IRF4 and CD69 expression (left panel) and CD25 and CD137 expression (right panel) by CD8 +  T cells after 18 hours of SEB stimulation. (D) Frequency of IRF4 + CD69 + , CD137 + , and CD25 + CD137 +  cells in CD8 +  T cells from healthy donor PBMCs after 18 hours of SEB stimulation (n = 24). Each point represents an individual donor. (E) IRF4 and CD69 expression in CD8 +  T CM  cells (CD45RA − CCR7 + ), T EM  cells (CD45RA − CCR7 − ), naive T cells (CD45RA + CCR7 − ), and T EMRA  cells (CD45RA + CCR7 + ) in SEB-stimulated PBMCs. (F) Mean fluorescence intensity (MFI) of IRF4 of baseline CD8 +  T CM , T EM , naive T, and T EMRA  cells (left panel) and after 18 hours of SEB stimulation (right panel) in healthy donor PBMCs (n = 3). Horizontal lines indicate the mean (± standard deviation). Each symbol represents an individual donor. * P
Figure Legend Snippet: Expression of IRF4 by activated human blood CD8 + T cells. Healthy donor PBMCs were stimulated with 1 μg/mL SEB and analyzed for the expression of surface and intracellular molecules at the indicated time points by flow cytometry. (A) IRF4 and CD69 expression by CD8 + T cells after stimulation with SEB. (B) IRF4 and CD69 expression by CD8 + T cells in unstimulated PBMCs (left panel) and PBMCs stimulated with SEB for 18 hours (right panel). (C) IRF4 and CD69 expression (left panel) and CD25 and CD137 expression (right panel) by CD8 + T cells after 18 hours of SEB stimulation. (D) Frequency of IRF4 + CD69 + , CD137 + , and CD25 + CD137 + cells in CD8 + T cells from healthy donor PBMCs after 18 hours of SEB stimulation (n = 24). Each point represents an individual donor. (E) IRF4 and CD69 expression in CD8 + T CM cells (CD45RA − CCR7 + ), T EM cells (CD45RA − CCR7 − ), naive T cells (CD45RA + CCR7 − ), and T EMRA cells (CD45RA + CCR7 + ) in SEB-stimulated PBMCs. (F) Mean fluorescence intensity (MFI) of IRF4 of baseline CD8 + T CM , T EM , naive T, and T EMRA cells (left panel) and after 18 hours of SEB stimulation (right panel) in healthy donor PBMCs (n = 3). Horizontal lines indicate the mean (± standard deviation). Each symbol represents an individual donor. * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation

IRF4 hi CD8 + T cells highly express CD25.  Healthy donor PBMCs were stimulated with CEF peptides for 18 hours. (A) Mean fluorescence intensity of CD25, CD69, and CD137 in IRF4 neg , IRF4 lo , IRF4 int , and IRF4 hi  CD137 +  populations. (B) A representative viSNE analysis of CEF-specific CD8 +  T cells (gated on CD8 + CD69 +  cells). Each point represents a single cell, and different colors represent the intensity of the expression of the molecule. CEF-specific CD8 +  T cells (IRF4 + CD137 + ) are indicated by the oval.
Figure Legend Snippet: IRF4 hi CD8 + T cells highly express CD25. Healthy donor PBMCs were stimulated with CEF peptides for 18 hours. (A) Mean fluorescence intensity of CD25, CD69, and CD137 in IRF4 neg , IRF4 lo , IRF4 int , and IRF4 hi CD137 + populations. (B) A representative viSNE analysis of CEF-specific CD8 + T cells (gated on CD8 + CD69 + cells). Each point represents a single cell, and different colors represent the intensity of the expression of the molecule. CEF-specific CD8 + T cells (IRF4 + CD137 + ) are indicated by the oval.

Techniques Used: Fluorescence, Expressing

The IRF4-CD137 assay detects Ag-specific CD8 + T cells with low background.  PBMCs were cultured with CEF peptides for 18 hours and analyzed for the expression of the indicated molecules. Frequency of IRF4 + CD69 +  (top panels), IRF4 + CD137 +  (middle panels), and CD25 + CD137 +  cells (bottom panels) in CD8 +  T cells. Two representative results are shown.
Figure Legend Snippet: The IRF4-CD137 assay detects Ag-specific CD8 + T cells with low background. PBMCs were cultured with CEF peptides for 18 hours and analyzed for the expression of the indicated molecules. Frequency of IRF4 + CD69 + (top panels), IRF4 + CD137 + (middle panels), and CD25 + CD137 + cells (bottom panels) in CD8 + T cells. Two representative results are shown.

Techniques Used: Cell Culture, Expressing

IRF4 expression reflects TCR signal strength.  (A) Isolated blood CD8 +  T cells were stimulated with increasing concentrations of plate-bound anti-CD3 mAb (0.1-1 μg/mL) for 18 hours. Mean fluorescence intensity of IRF4 in IRF4 + CD137 +  cells. A representative result from 3 individual experiments is shown. (B) PBMCs were treated for 30 minutes with increasing concentrations of anti-human pan MHC class I mAb (0-100 μg/mL) and then cultured with CEF peptides for 18 hours. Expression of IRF4, CD69, CD25, and CD137 by activated CD8 +  T cells was analyzed. A representative result from 6 individual experiments is shown.
Figure Legend Snippet: IRF4 expression reflects TCR signal strength. (A) Isolated blood CD8 + T cells were stimulated with increasing concentrations of plate-bound anti-CD3 mAb (0.1-1 μg/mL) for 18 hours. Mean fluorescence intensity of IRF4 in IRF4 + CD137 + cells. A representative result from 3 individual experiments is shown. (B) PBMCs were treated for 30 minutes with increasing concentrations of anti-human pan MHC class I mAb (0-100 μg/mL) and then cultured with CEF peptides for 18 hours. Expression of IRF4, CD69, CD25, and CD137 by activated CD8 + T cells was analyzed. A representative result from 6 individual experiments is shown.

Techniques Used: Expressing, Isolation, Fluorescence, Cell Culture

IRF4 expression by tetramer + CD8 + T cells.  PBMCs from HLA-A2 +  healthy donors were prestained with Flu-M1–A2 tetramer and cultured with Flu-M1 GILGFVFTL peptide for 18 hours. (A) Frequency of tetramer +  cells in CD8 +  T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. (B) Frequency of IRF4/CD69 (left panels), CD25/CD137 (middle panels), and IRF4/CD137 (right panels) of tetramer +  CD8 +  T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. A representative result from 5 individual experiments is shown.
Figure Legend Snippet: IRF4 expression by tetramer + CD8 + T cells. PBMCs from HLA-A2 + healthy donors were prestained with Flu-M1–A2 tetramer and cultured with Flu-M1 GILGFVFTL peptide for 18 hours. (A) Frequency of tetramer + cells in CD8 + T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. (B) Frequency of IRF4/CD69 (left panels), CD25/CD137 (middle panels), and IRF4/CD137 (right panels) of tetramer + CD8 + T cells in unstimulated, DMSO-treated, and peptide-stimulated PBMCs. A representative result from 5 individual experiments is shown.

Techniques Used: Expressing, Cell Culture

Low IRF4 expression by HIV-specific CD8 + T cells was independent of PD-1 signals.  (A) PD-1 and CD69 expression by IRF4 + CD137 + CD8 +  T cells in unstimulated, CEF-stimulated, or HIV-stimulated PBMCs. A representative of 7 samples (left panel). PD-1 MFI by CEF- and HIV-specific CD8 +  T cells (middle panels). Gated on IRF4 + CD137 + CD8 +  T cells (n = 7). One donor showed high PD-1 by HIV-specific CD8 +  T cells (black symbols). (B) PBMCs from HIV +  subjects were treated for 30 minutes with anti–PD-L1 antibody and stimulated with HIV peptides for 18 hours. IRF4/CD137 expression with or without pretreatment with anti–PD-L1. Gated on CD8 +  T cells. A representative result from 5 experiments is shown. (C) MFI of IRF4 in HIV-specific CD8 +  T cells treated or not with anti–PD-L1. Gated on IRF4 + CD137 + CD8 +  T cells (n = 5). One donor showed high PD-1 by HIV-specific CD8 +  T cells (black symbols). NS, not significant (paired Student  t  test).
Figure Legend Snippet: Low IRF4 expression by HIV-specific CD8 + T cells was independent of PD-1 signals. (A) PD-1 and CD69 expression by IRF4 + CD137 + CD8 + T cells in unstimulated, CEF-stimulated, or HIV-stimulated PBMCs. A representative of 7 samples (left panel). PD-1 MFI by CEF- and HIV-specific CD8 + T cells (middle panels). Gated on IRF4 + CD137 + CD8 + T cells (n = 7). One donor showed high PD-1 by HIV-specific CD8 + T cells (black symbols). (B) PBMCs from HIV + subjects were treated for 30 minutes with anti–PD-L1 antibody and stimulated with HIV peptides for 18 hours. IRF4/CD137 expression with or without pretreatment with anti–PD-L1. Gated on CD8 + T cells. A representative result from 5 experiments is shown. (C) MFI of IRF4 in HIV-specific CD8 + T cells treated or not with anti–PD-L1. Gated on IRF4 + CD137 + CD8 + T cells (n = 5). One donor showed high PD-1 by HIV-specific CD8 + T cells (black symbols). NS, not significant (paired Student t test).

Techniques Used: Expressing

HIV-specific CD8 + T cells express low IRF4.  PBMCs from HIV +  subjects were stimulated with 1 μg/mL CEF peptides or HIV peptides for 18 hours. (A) Expression of IRF4 and CD137 by CD8 +  T cells. (B) MFI of IRF4 and CD137 expression by CD8 +  T cells specific for CEF and HIV peptides (n = 13). (C) viSNE analysis showing differential marker expression by CD8 +  T cells specific for CEF peptides (upper panels) or HIV peptides (lower panels). A representative result of 13 samples (gated on CD8 + CD69 +  T cells) is shown. Red arrows indicate a cell population uniquely observed in the HIV-specific CD8 +  T cells. Blue arrows indicate a cell population shared between CEF- and HIV-specific CD8 +  T cells. (D) CD27 and CD25 expression by IRF4 + CD137 +  CD8 +  T cells in PBMCs stimulated with CEF or HIV peptides. A representative result of 11 samples is shown. (E) MFI of CD25 (left panel) and percentage of CD25 +  (right) by IRF4 + CD137 +  CD8 +  T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). (F) MFI of CD27 (left panel) and percentage of CD27 +  (right panel) by IRF4 + CD137 +  CD8 +  T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). * P
Figure Legend Snippet: HIV-specific CD8 + T cells express low IRF4. PBMCs from HIV + subjects were stimulated with 1 μg/mL CEF peptides or HIV peptides for 18 hours. (A) Expression of IRF4 and CD137 by CD8 + T cells. (B) MFI of IRF4 and CD137 expression by CD8 + T cells specific for CEF and HIV peptides (n = 13). (C) viSNE analysis showing differential marker expression by CD8 + T cells specific for CEF peptides (upper panels) or HIV peptides (lower panels). A representative result of 13 samples (gated on CD8 + CD69 + T cells) is shown. Red arrows indicate a cell population uniquely observed in the HIV-specific CD8 + T cells. Blue arrows indicate a cell population shared between CEF- and HIV-specific CD8 + T cells. (D) CD27 and CD25 expression by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides. A representative result of 11 samples is shown. (E) MFI of CD25 (left panel) and percentage of CD25 + (right) by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). (F) MFI of CD27 (left panel) and percentage of CD27 + (right panel) by IRF4 + CD137 + CD8 + T cells in PBMCs stimulated with CEF or HIV peptides (n = 11). * P

Techniques Used: Expressing, Marker

9) Product Images from "Genetic associations of the interleukin locus at 1q32.1 with clinical outcomes of cutaneous melanoma"

Article Title: Genetic associations of the interleukin locus at 1q32.1 with clinical outcomes of cutaneous melanoma

Journal: Journal of medical genetics

doi: 10.1136/jmedgenet-2014-102832

Analysis of secretion of IL10 in CD4+ T cell stratified by the genotype of rs3024493. The secretion of IL10 was measured in isolated CD4+ T cells from a subset of 75 melanoma patients genotyped in the current study. Y axis represents the absolute concentration
Figure Legend Snippet: Analysis of secretion of IL10 in CD4+ T cell stratified by the genotype of rs3024493. The secretion of IL10 was measured in isolated CD4+ T cells from a subset of 75 melanoma patients genotyped in the current study. Y axis represents the absolute concentration

Techniques Used: Isolation, Concentration Assay

10) Product Images from "Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice"

Article Title: Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice

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

doi: 10.1073/pnas.1711233114

Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of CD4 + T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels
Figure Legend Snippet: Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of CD4 + T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels

Techniques Used: Isolation

11) Product Images from "Increased expression of intrinsic antiviral genes in HLA-B*57-positive individuals"

Article Title: Increased expression of intrinsic antiviral genes in HLA-B*57-positive individuals

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.0313150

CD4 +  T cell activation levels strongly correlate with host restriction factor expression in HLA-B*57-positive individuals.
Figure Legend Snippet: CD4 + T cell activation levels strongly correlate with host restriction factor expression in HLA-B*57-positive individuals.

Techniques Used: Activation Assay, Expressing

12) Product Images from "An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality"

Article Title: An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality

Journal: Genes & Development

doi: 10.1101/gad.307330.117

Homozygous D63H human iPSCs fail to differentiate into functional cardiomyocytes and NPCs. ( A , top panel) Schematic showing the differentiation from iPSCs into cardiomyocytes. ( Bottom panels) Flow cytometry analysis of day 16 cardiomyocytes stained for cTnT-FITC. ( B ) Day 3 ( top panel) and day 7 ( bottom panel) iPSC-derived cardiomyocyte expression for core pluripotent genes and cardiac sarcomeric genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived cardiomyocytes from a heterozygous mother. ( C ) Immunofluorescence staining of day 9 iPSC-derived NPCs for Nestin, Sox2, and DAPI (10×). Images are representative of two clones for each genotype. ( D ) Day 9 ( top panel) and day 15 ( bottom panel) iPSC-derived NPC expression for core pluripotent genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived NPCs from a heterozygous mother.
Figure Legend Snippet: Homozygous D63H human iPSCs fail to differentiate into functional cardiomyocytes and NPCs. ( A , top panel) Schematic showing the differentiation from iPSCs into cardiomyocytes. ( Bottom panels) Flow cytometry analysis of day 16 cardiomyocytes stained for cTnT-FITC. ( B ) Day 3 ( top panel) and day 7 ( bottom panel) iPSC-derived cardiomyocyte expression for core pluripotent genes and cardiac sarcomeric genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived cardiomyocytes from a heterozygous mother. ( C ) Immunofluorescence staining of day 9 iPSC-derived NPCs for Nestin, Sox2, and DAPI (10×). Images are representative of two clones for each genotype. ( D ) Day 9 ( top panel) and day 15 ( bottom panel) iPSC-derived NPC expression for core pluripotent genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived NPCs from a heterozygous mother.

Techniques Used: Functional Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Clone Assay

Homozygous D63H human iPSCs fail to form EBs in vitro and retain pluripotent gene expression. ( A ) EBs derived from heterozygous (mother) and homozygous (fetuses #1 and #3) SIRT6 D63H iPSCs (two clones each; 10×). ( B ) iPSC-derived EB core pluripotent gene expression assessed by qRT–PCR analysis (average of two clones). Data are expressed relative to iPSC-derived EBs from a heterozygous mother. ( C ) Western blot analysis on bulk chromatin for Sox2 and Ac-H3K56 in iPSC-derived EBs. ( D ) EBs derived from iPSCs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 (4×). ( Right panel) Quantified average EB size and number. ( E ) Core pluripotent gene expression in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived EBs from a heterozygous mother infected with pRetro empty vector. ( F ) Western blot analysis on bulk chromatin for Oct4 and Ac-H3K56 in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6.
Figure Legend Snippet: Homozygous D63H human iPSCs fail to form EBs in vitro and retain pluripotent gene expression. ( A ) EBs derived from heterozygous (mother) and homozygous (fetuses #1 and #3) SIRT6 D63H iPSCs (two clones each; 10×). ( B ) iPSC-derived EB core pluripotent gene expression assessed by qRT–PCR analysis (average of two clones). Data are expressed relative to iPSC-derived EBs from a heterozygous mother. ( C ) Western blot analysis on bulk chromatin for Sox2 and Ac-H3K56 in iPSC-derived EBs. ( D ) EBs derived from iPSCs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 (4×). ( Right panel) Quantified average EB size and number. ( E ) Core pluripotent gene expression in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived EBs from a heterozygous mother infected with pRetro empty vector. ( F ) Western blot analysis on bulk chromatin for Oct4 and Ac-H3K56 in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6.

Techniques Used: In Vitro, Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Over Expression, Plasmid Preparation, Infection

13) Product Images from "Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy"

Article Title: Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy

Journal: Science Advances

doi: 10.1126/sciadv.aaz8521

MAPKi polarizes MΦs toward an alternatively activated, HGF- and GAS6-producing phenotype. ( A ) Schematic of M2-MΦ conditioned medium experiments (left) and data showing ES2 cell count following 72-hour trametinib ± conditioned medium from M2-MΦ that were pretreated with 24-hour trametinib (right) ( n = 3). ( B ) OVCA viability was measured by propidium iodide and annexin V staining for three cell lines treated for 48 hours with 100 nM trametinib ± transwell coculture with M2-MΦ (averaged across five donors with n = 3 reps per donor, two-tailed t test). ( C ) Heat map of supernatant and lysate proteins from PBMC-derived M1-MΦ and M2-MΦ. Analyte levels of MΦs treated with 24-hour trametinib are plotted relative to their respective no-treatment controls ( n = 3, * P
Figure Legend Snippet: MAPKi polarizes MΦs toward an alternatively activated, HGF- and GAS6-producing phenotype. ( A ) Schematic of M2-MΦ conditioned medium experiments (left) and data showing ES2 cell count following 72-hour trametinib ± conditioned medium from M2-MΦ that were pretreated with 24-hour trametinib (right) ( n = 3). ( B ) OVCA viability was measured by propidium iodide and annexin V staining for three cell lines treated for 48 hours with 100 nM trametinib ± transwell coculture with M2-MΦ (averaged across five donors with n = 3 reps per donor, two-tailed t test). ( C ) Heat map of supernatant and lysate proteins from PBMC-derived M1-MΦ and M2-MΦ. Analyte levels of MΦs treated with 24-hour trametinib are plotted relative to their respective no-treatment controls ( n = 3, * P

Techniques Used: Cell Counting, Staining, Two Tailed Test, Derivative Assay

14) Product Images from "Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction"

Article Title: Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction

Journal: Nature Communications

doi: 10.1038/ncomms13895

ALPS patient T cells are defective in Fas-induced differentiation and apoptosis. ( a ) Representative data with PBMCs from two unrelated ALPS patients with Fas death domain mutations and a healthy donor control, activated for 7 days with or without FasL-LZ as described in the ‘Methods' section. Differentiation of CD8 + T cells was examined by staining for surface CD27 and CCR7. Cells were gated CD3 + CD4 − CD8 + before analysis of CD27/CCR7 status. ( b ) Summary of fold increase in effector memory T cells by FasL-LZ, in seven ALPS patients ( N =7) and three normal donor controls ( N =3) from two independent experiments. Cumulative data are represented as mean±s.e.m. ( c ) Cumulative specific cell death induced by FasL-LZ in cell cultures shown in b from two independent experiments. P -values are from an unpaired t -test, with data represented as mean±s.e.m. ( d , e ) Intracellular staining for pS6 was performed on isolated CD8 + T cells from three healthy donors ( N =3) and three ALPS patients ( N =3), with representative FACS plots ( d ) shown. Summary of data are shown in e , with fold increase calculated as pS6 MFI vehicle/pS6 MFI FasL-LZ at 20 ng ml −1 .
Figure Legend Snippet: ALPS patient T cells are defective in Fas-induced differentiation and apoptosis. ( a ) Representative data with PBMCs from two unrelated ALPS patients with Fas death domain mutations and a healthy donor control, activated for 7 days with or without FasL-LZ as described in the ‘Methods' section. Differentiation of CD8 + T cells was examined by staining for surface CD27 and CCR7. Cells were gated CD3 + CD4 − CD8 + before analysis of CD27/CCR7 status. ( b ) Summary of fold increase in effector memory T cells by FasL-LZ, in seven ALPS patients ( N =7) and three normal donor controls ( N =3) from two independent experiments. Cumulative data are represented as mean±s.e.m. ( c ) Cumulative specific cell death induced by FasL-LZ in cell cultures shown in b from two independent experiments. P -values are from an unpaired t -test, with data represented as mean±s.e.m. ( d , e ) Intracellular staining for pS6 was performed on isolated CD8 + T cells from three healthy donors ( N =3) and three ALPS patients ( N =3), with representative FACS plots ( d ) shown. Summary of data are shown in e , with fold increase calculated as pS6 MFI vehicle/pS6 MFI FasL-LZ at 20 ng ml −1 .

Techniques Used: Staining, Isolation, FACS

15) Product Images from "An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality"

Article Title: An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality

Journal: Genes & Development

doi: 10.1101/gad.307330.117

Homozygous D63H human iPSCs fail to differentiate into functional cardiomyocytes and NPCs. ( A , top panel) Schematic showing the differentiation from iPSCs into cardiomyocytes. ( Bottom panels) Flow cytometry analysis of day 16 cardiomyocytes stained for cTnT-FITC. ( B ) Day 3 ( top panel) and day 7 ( bottom panel) iPSC-derived cardiomyocyte expression for core pluripotent genes and cardiac sarcomeric genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived cardiomyocytes from a heterozygous mother. ( C ) Immunofluorescence staining of day 9 iPSC-derived NPCs for Nestin, Sox2, and DAPI (10×). Images are representative of two clones for each genotype. ( D ) Day 9 ( top panel) and day 15 ( bottom panel) iPSC-derived NPC expression for core pluripotent genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived NPCs from a heterozygous mother.
Figure Legend Snippet: Homozygous D63H human iPSCs fail to differentiate into functional cardiomyocytes and NPCs. ( A , top panel) Schematic showing the differentiation from iPSCs into cardiomyocytes. ( Bottom panels) Flow cytometry analysis of day 16 cardiomyocytes stained for cTnT-FITC. ( B ) Day 3 ( top panel) and day 7 ( bottom panel) iPSC-derived cardiomyocyte expression for core pluripotent genes and cardiac sarcomeric genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived cardiomyocytes from a heterozygous mother. ( C ) Immunofluorescence staining of day 9 iPSC-derived NPCs for Nestin, Sox2, and DAPI (10×). Images are representative of two clones for each genotype. ( D ) Day 9 ( top panel) and day 15 ( bottom panel) iPSC-derived NPC expression for core pluripotent genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived NPCs from a heterozygous mother.

Techniques Used: Functional Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Clone Assay

Homozygous D63H human iPSCs fail to form EBs in vitro and retain pluripotent gene expression. ( A ) EBs derived from heterozygous (mother) and homozygous (fetuses #1 and #3) SIRT6 D63H iPSCs (two clones each; 10×). ( B ) iPSC-derived EB core pluripotent gene expression assessed by qRT–PCR analysis (average of two clones). Data are expressed relative to iPSC-derived EBs from a heterozygous mother. ( C ) Western blot analysis on bulk chromatin for Sox2 and Ac-H3K56 in iPSC-derived EBs. ( D ) EBs derived from iPSCs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 (4×). ( Right panel) Quantified average EB size and number. ( E ) Core pluripotent gene expression in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived EBs from a heterozygous mother infected with pRetro empty vector. ( F ) Western blot analysis on bulk chromatin for Oct4 and Ac-H3K56 in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6.
Figure Legend Snippet: Homozygous D63H human iPSCs fail to form EBs in vitro and retain pluripotent gene expression. ( A ) EBs derived from heterozygous (mother) and homozygous (fetuses #1 and #3) SIRT6 D63H iPSCs (two clones each; 10×). ( B ) iPSC-derived EB core pluripotent gene expression assessed by qRT–PCR analysis (average of two clones). Data are expressed relative to iPSC-derived EBs from a heterozygous mother. ( C ) Western blot analysis on bulk chromatin for Sox2 and Ac-H3K56 in iPSC-derived EBs. ( D ) EBs derived from iPSCs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 (4×). ( Right panel) Quantified average EB size and number. ( E ) Core pluripotent gene expression in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6 assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived EBs from a heterozygous mother infected with pRetro empty vector. ( F ) Western blot analysis on bulk chromatin for Oct4 and Ac-H3K56 in EBs with doxycycline-inducible overexpression of pRetro empty vector and wild-type SIRT6.

Techniques Used: In Vitro, Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Over Expression, Plasmid Preparation, Infection

16) Product Images from "Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells"

Article Title: Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

Journal: Scientific Reports

doi: 10.1038/srep15262

CTLs expanded by DCs derived from minigene-modified hPSCs are immunocompetent. ( a ) Expansion of MART-1-specific CD8+ T cells by H1.ME-DCs in bulk culture. HLA-A2+ PBLs were primed and then restimulated twice with H1.ME-DCs. MART-1-specific T cell expansion during this process was monitored by flow cytometry at the indicated time points. The percentages of pentamer+ CD8+ cells in total T cells are shown in the representative contour plots. ( b ) Phenotype of MART-1-specific T cells expanded by H1.ME-DCs. ( c ) GrB secretion by MART-1-specific T cells expanded by H1.ME-DCs as measured by ELISPOT. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3). ( d ) Specific cytotoxicity of MART-1-specific T cells expanded by H1.ME-DCs. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3, *p
Figure Legend Snippet: CTLs expanded by DCs derived from minigene-modified hPSCs are immunocompetent. ( a ) Expansion of MART-1-specific CD8+ T cells by H1.ME-DCs in bulk culture. HLA-A2+ PBLs were primed and then restimulated twice with H1.ME-DCs. MART-1-specific T cell expansion during this process was monitored by flow cytometry at the indicated time points. The percentages of pentamer+ CD8+ cells in total T cells are shown in the representative contour plots. ( b ) Phenotype of MART-1-specific T cells expanded by H1.ME-DCs. ( c ) GrB secretion by MART-1-specific T cells expanded by H1.ME-DCs as measured by ELISPOT. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3). ( d ) Specific cytotoxicity of MART-1-specific T cells expanded by H1.ME-DCs. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3, *p

Techniques Used: Derivative Assay, Modification, Flow Cytometry, Cytometry, Enzyme-linked Immunospot

17) Product Images from "Failure to clear intra-monocyte HIV infection linked to persistent neuropsychological testing impairment after first-line combined antiretroviral therapy"

Article Title: Failure to clear intra-monocyte HIV infection linked to persistent neuropsychological testing impairment after first-line combined antiretroviral therapy

Journal: Journal of Neurovirology

doi: 10.1007/s13365-011-0068-8

HIV DNA (log 10 median with inter-quartile range) in peripheral mononuclear cells (PBMC, top ; monocytes, middle ; and monocyte-depleted PBMC, bottom ) comparing dementia cases ( black circle ) to non-dementia cases ( white circle ). Some CART-naïve individuals
Figure Legend Snippet: HIV DNA (log 10 median with inter-quartile range) in peripheral mononuclear cells (PBMC, top ; monocytes, middle ; and monocyte-depleted PBMC, bottom ) comparing dementia cases ( black circle ) to non-dementia cases ( white circle ). Some CART-naïve individuals

Techniques Used:

18) Product Images from "Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation"

Article Title: Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation

Journal: Nature immunology

doi: 10.1038/s41590-018-0085-3

Antigen-loaded macrophages accumulate more immunological synapses with effector cells compared to antigen-loaded CD4 +  T cells (a)  Assessment of target-effector doublets. Peptide-loaded, FarRed-labeled targets were co-cultured for the indicated times with Violet-labeled expanded CTLs followed by fixation, actin staining, and analysis via imaging flow cytometry. Shown are representative plots (one of two independent experiments) of effector-target co-culture samples and the gating strategy used to quantitate total peptide-loaded targets (red box) and target-effector doublets (blue box).  (b)  . As in (a), shown is a representation of one of two independent experiments. White scale bars denote 10μm.  (c)  Summary of immunological synapse accumulation over 60 minutes (n=6 distinct samples from two independent experiments). Frequencies of immunological synapses were calculated as described in the Methods. Shown are means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p=0.001, **p=0.0001, ***p
Figure Legend Snippet: Antigen-loaded macrophages accumulate more immunological synapses with effector cells compared to antigen-loaded CD4 + T cells (a) Assessment of target-effector doublets. Peptide-loaded, FarRed-labeled targets were co-cultured for the indicated times with Violet-labeled expanded CTLs followed by fixation, actin staining, and analysis via imaging flow cytometry. Shown are representative plots (one of two independent experiments) of effector-target co-culture samples and the gating strategy used to quantitate total peptide-loaded targets (red box) and target-effector doublets (blue box). (b) . As in (a), shown is a representation of one of two independent experiments. White scale bars denote 10μm. (c) Summary of immunological synapse accumulation over 60 minutes (n=6 distinct samples from two independent experiments). Frequencies of immunological synapses were calculated as described in the Methods. Shown are means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p=0.001, **p=0.0001, ***p

Techniques Used: Labeling, Cell Culture, Staining, Imaging, Flow Cytometry, Cytometry, Co-Culture Assay

HIV-infected macrophages are less susceptible to CTL-mediated killing compared to HIV-infected CD4 +  T cells (a)  HIV-infected target elimination assay. HIV-infected CD4 + .  (b) Summary of elimination assays using ex vivo CTL (n=16 distinct samples from four independent experiments) and  (c)  HIV peptide-expanded CTL from HIV-infected donors (n=16 distinct samples from four independent experiments) and CTL from HIV −  healthy donors (n=3 distinct samples from two independent experiments) as a negative control. Shown are the means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p
Figure Legend Snippet: HIV-infected macrophages are less susceptible to CTL-mediated killing compared to HIV-infected CD4 + T cells (a) HIV-infected target elimination assay. HIV-infected CD4 + . (b) Summary of elimination assays using ex vivo CTL (n=16 distinct samples from four independent experiments) and (c) HIV peptide-expanded CTL from HIV-infected donors (n=16 distinct samples from four independent experiments) and CTL from HIV − healthy donors (n=3 distinct samples from two independent experiments) as a negative control. Shown are the means +/- SEM. Statistical analysis: two-sided unpaired t-test, *p

Techniques Used: Infection, CTL Assay, Ex Vivo, Negative Control

Killing of target cells is MHC-I and perforin-dependent, but granzyme B is dispensable for CD4 +  T cell killing (a and b)  Inhibiting HIV-infected target cell elimination. HIV-infected CD4 +  T cells (a) or macrophages (b) were incubated with HIV-peptide expanded CTL in the presence of an MHC-I blocking antibody, concanamycin A (CMA – an indirect perforin inhibitor), a granzyme B inhibitor, a FAS neutralizing antibody, or recombinant human TRAIL-R1-Fc for four hours. Target cells were analyzed for live cell Gag-p24 intracellular staining via flow cytometry. Shown is the elimination assay summary using HIV +  patients (n = 8 distinct samples from four independent experiments). Statistical analysis: two-sided paired t-test, *p=0.0153, **p=0.0097, *** p=0.0036, ****p
Figure Legend Snippet: Killing of target cells is MHC-I and perforin-dependent, but granzyme B is dispensable for CD4 + T cell killing (a and b) Inhibiting HIV-infected target cell elimination. HIV-infected CD4 + T cells (a) or macrophages (b) were incubated with HIV-peptide expanded CTL in the presence of an MHC-I blocking antibody, concanamycin A (CMA – an indirect perforin inhibitor), a granzyme B inhibitor, a FAS neutralizing antibody, or recombinant human TRAIL-R1-Fc for four hours. Target cells were analyzed for live cell Gag-p24 intracellular staining via flow cytometry. Shown is the elimination assay summary using HIV + patients (n = 8 distinct samples from four independent experiments). Statistical analysis: two-sided paired t-test, *p=0.0153, **p=0.0097, *** p=0.0036, ****p

Techniques Used: Infection, Incubation, CTL Assay, Blocking Assay, Recombinant, Staining, Flow Cytometry, Cytometry

HIV-infected macrophages induce stronger CTL cytokine responses than infected CD4 +  T cells (a)  MHC-I surface density. Imaging flow cytometry was used to calculate relative surface densities of MHC-I on CD4 + ) (n=7 distinct samples from three independent experiments). Box elements, median and 25 th -75 th  percentiles. Statistical analysis, two-sided Mann-Whitney test. p=0.2209.  (b)  CTL recognition assay. Ex vivo and expanded CTL were incubated with mock/uninfected or HIV-infected CD4 + . Shown is one of five independent experiments.  (c)  Comparison of CTL responses to CD4 +  T cells and macrophages. Data shown are the ratios of CTL degranulation in response to macrophages over CD4 +  T cells for ex vivo (n=14 distinct samples from five independent experiments) and expanded CTL (n=16 distinct samples from five independent experiments). Responses against CEF peptide-loaded targets were also assessed (n=6 distinct samples from three independent experiments). Shown are the means +/- SEM. Statistical analysis: two-sided one sample t-test, *p=0.0036, and two-sided Wilcoxon signed rank test, **p
Figure Legend Snippet: HIV-infected macrophages induce stronger CTL cytokine responses than infected CD4 + T cells (a) MHC-I surface density. Imaging flow cytometry was used to calculate relative surface densities of MHC-I on CD4 + ) (n=7 distinct samples from three independent experiments). Box elements, median and 25 th -75 th percentiles. Statistical analysis, two-sided Mann-Whitney test. p=0.2209. (b) CTL recognition assay. Ex vivo and expanded CTL were incubated with mock/uninfected or HIV-infected CD4 + . Shown is one of five independent experiments. (c) Comparison of CTL responses to CD4 + T cells and macrophages. Data shown are the ratios of CTL degranulation in response to macrophages over CD4 + T cells for ex vivo (n=14 distinct samples from five independent experiments) and expanded CTL (n=16 distinct samples from five independent experiments). Responses against CEF peptide-loaded targets were also assessed (n=6 distinct samples from three independent experiments). Shown are the means +/- SEM. Statistical analysis: two-sided one sample t-test, *p=0.0036, and two-sided Wilcoxon signed rank test, **p

Techniques Used: Infection, CTL Assay, Imaging, Flow Cytometry, Cytometry, MANN-WHITNEY, Ex Vivo, Incubation

19) Product Images from "C reactive protein impairs adaptive immunity in immune cells of patients with melanoma"

Article Title: C reactive protein impairs adaptive immunity in immune cells of patients with melanoma

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2019-000234

C reactive protein (CRP) inhibits the expansion of antigen-specific T cells. (A) MART-1-specific CD8 T-cell expansion. CD8+ T cells isolated from HLA-A*0201 patients with melanoma were stimulated by γ-irradiated, MART-1 26-35  peptide-pulsed peripheral blood mononuclear cells (PBMCs) (antigen-presenting cells (APCs)) for 7 days in presence of different CRP concentration (0, 10, 40 µg/mL). (B and C) Ki67 expression and proliferation by CFSE-labeled Melan-A-specific CD8+ T cells and MART-1 tetramer-positive T cells stimulated by γ-irradiated MART-1 26-35  peptide pulsed APC. (D) Scheme for assessing whether CRP-impaired generation of Melan-A-positive CD8+ T cells was due to its effect on T cells and/or on mature dendritic cells (mDCs). (E) Purified CD8+ T cells and mDCs separately before co-culture were treated with CRP for 48 hours, and co-cultured with MART-1 peptide-pulsed mDCs. Results shown are representative of three patients assessed over three independent experiments. (F) Volcano plot for the differential expression of total genes. The y-axis corresponds to negative log10 transformed adjusted p value, and the x-axis displays the log2 fold change gene expression value. The red and blue dots represent the significantly differential expressed transcripts (adjusted p value
Figure Legend Snippet: C reactive protein (CRP) inhibits the expansion of antigen-specific T cells. (A) MART-1-specific CD8 T-cell expansion. CD8+ T cells isolated from HLA-A*0201 patients with melanoma were stimulated by γ-irradiated, MART-1 26-35 peptide-pulsed peripheral blood mononuclear cells (PBMCs) (antigen-presenting cells (APCs)) for 7 days in presence of different CRP concentration (0, 10, 40 µg/mL). (B and C) Ki67 expression and proliferation by CFSE-labeled Melan-A-specific CD8+ T cells and MART-1 tetramer-positive T cells stimulated by γ-irradiated MART-1 26-35 peptide pulsed APC. (D) Scheme for assessing whether CRP-impaired generation of Melan-A-positive CD8+ T cells was due to its effect on T cells and/or on mature dendritic cells (mDCs). (E) Purified CD8+ T cells and mDCs separately before co-culture were treated with CRP for 48 hours, and co-cultured with MART-1 peptide-pulsed mDCs. Results shown are representative of three patients assessed over three independent experiments. (F) Volcano plot for the differential expression of total genes. The y-axis corresponds to negative log10 transformed adjusted p value, and the x-axis displays the log2 fold change gene expression value. The red and blue dots represent the significantly differential expressed transcripts (adjusted p value

Techniques Used: Isolation, Irradiation, Concentration Assay, Expressing, Labeling, Purification, Co-Culture Assay, Cell Culture, Transformation Assay

Related Articles

Flow Cytometry:

Article Title: Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaques
Article Snippet: .. For polychromatic flow cytometry analysis of memory subsets, cells from uninfected or infected macaques were stained with Pacific Blue-conjugated anti-CD3, PerCP-conjugated anti-CD4, APC-conjugated anti-CD8, Biotin-conjugated anti-PD-1, FITC-conjugated anti-CD38 (clone AT-1) (Stem Cell Technologies, Vancouver, BC), APC-Cy7-conjugated anti-HLA-DR (clone L243) (Pharmingen), PE-Cy7-conjugated anti-CD28 (clone CD28.2) (eBioscience, San Diego, CA), and PE-Cy5-conjugated anti-CD95 (clone DX2) (Pharmingen). .. Cells were washed and then stained with streptavidin-PE.

Cytometry:

Article Title: Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaques
Article Snippet: .. For polychromatic flow cytometry analysis of memory subsets, cells from uninfected or infected macaques were stained with Pacific Blue-conjugated anti-CD3, PerCP-conjugated anti-CD4, APC-conjugated anti-CD8, Biotin-conjugated anti-PD-1, FITC-conjugated anti-CD38 (clone AT-1) (Stem Cell Technologies, Vancouver, BC), APC-Cy7-conjugated anti-HLA-DR (clone L243) (Pharmingen), PE-Cy7-conjugated anti-CD28 (clone CD28.2) (eBioscience, San Diego, CA), and PE-Cy5-conjugated anti-CD95 (clone DX2) (Pharmingen). .. Cells were washed and then stained with streptavidin-PE.

Infection:

Article Title: Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaques
Article Snippet: .. For polychromatic flow cytometry analysis of memory subsets, cells from uninfected or infected macaques were stained with Pacific Blue-conjugated anti-CD3, PerCP-conjugated anti-CD4, APC-conjugated anti-CD8, Biotin-conjugated anti-PD-1, FITC-conjugated anti-CD38 (clone AT-1) (Stem Cell Technologies, Vancouver, BC), APC-Cy7-conjugated anti-HLA-DR (clone L243) (Pharmingen), PE-Cy7-conjugated anti-CD28 (clone CD28.2) (eBioscience, San Diego, CA), and PE-Cy5-conjugated anti-CD95 (clone DX2) (Pharmingen). .. Cells were washed and then stained with streptavidin-PE.

Staining:

Article Title: Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaques
Article Snippet: .. For polychromatic flow cytometry analysis of memory subsets, cells from uninfected or infected macaques were stained with Pacific Blue-conjugated anti-CD3, PerCP-conjugated anti-CD4, APC-conjugated anti-CD8, Biotin-conjugated anti-PD-1, FITC-conjugated anti-CD38 (clone AT-1) (Stem Cell Technologies, Vancouver, BC), APC-Cy7-conjugated anti-HLA-DR (clone L243) (Pharmingen), PE-Cy7-conjugated anti-CD28 (clone CD28.2) (eBioscience, San Diego, CA), and PE-Cy5-conjugated anti-CD95 (clone DX2) (Pharmingen). .. Cells were washed and then stained with streptavidin-PE.

Article Title: Mucosal-associated invariant T (MAIT) cells provide B-cell help in vaccinated and subsequently SIV-infected Rhesus Macaques
Article Snippet: .. The cells were analyzed by flowcytometry following staining with Live/Dead aqua dye, Alexa flour 700 anti-CD3 (SP34-2), BV711 anti-CD69 (FN50), BV605 anti-CD21 (B-ly4) from BD Biosciences, San Jose, CA; BV650 anti-CD20 (2H7), APC anti-CD27 (O323) from Invitrogen Life Tec hnologies, Carlsbad, CA; FITC anti-CD38 (AT-1) from Stemcell Technologies, Vancouver, Canada; and Goat Anti-Human IgD-TXRD from Southernbiotech, Birmingham, AL. Acquisition and analysis followed the same protocol as for MAIT cells. .. The fold change of the different cellular markers was calculated based on marker expression by B cells cultured alone.

Article Title: Hypo-osmolar Formulation of Tenofovir (TFV) Enema Promotes Uptake and Metabolism of TFV in Tissues, Leading to Prevention of SHIV/SIV Infection
Article Snippet: .. Heparinized whole-blood samples collected at 0, 24, and 72 h following enema administration were stained with Live/Dead marker (Invitrogen) and the following antibodies: CD3-Alexa 700 (SP34-2; BD Pharmingen), CD4-V500 (L200; BD Horizon), CD8-V450 (RPA-T8; BD Horizon), CD20-APC-Cy7 (L27; BD Biosciences), CD28-PE-Texas Red (CD28.2; Beckman Coulter), CD95-PE-Cy5 (DX2; BD Pharmingen), CD159a (NKG2a)-APC (Z199; Beckman Coulter), CD38-FITC (AT-1, Stemcell Technologies), HLA-DR-PE-Cy7 (G46-6; BD Pharmingen), and Ki67-PE (B56; BD Pharmingen). .. Stained cells were fixed in 1% paraformaldehyde and acquired on an LSRII instrument (BD).

Article Title: Immunization expands HIV-1 V3-glycan specific B-cells in mice and macaques
Article Snippet: .. Macaque cells were stained with anti-CD16 APC-eFluor780 (Invitrogen, #47–0168-41), anti-CD8a APC-eFluor780 (Invitrogen, #47–0086-42), anti-CD3 APC-eFluor780 (Invitrogen, #47–0037-41), anti-CD14 APC-eFluor780 (eBiosciences, #47–0149-41), anti-CD20 PeCy7 (BD, #335793), anti-CD38 FITC (Stem Cell technologies, #60131FI), anti-IgG BV421 (BD Biosciences, #562581), anti-IgM PerCP-Cy5.5 (BD Biosciences, #561285) at a 1:200 dilution and the live/dead marker Zombie NIR at a 1:400 dilution in FACS buffer. .. Zombie NIR- /CD4- /CD8- /F4/80- /NK1.1- /CD11b- /CD11c- /B220+ /GL7+ /CD95+ /RC1+ /RC1-glycanKO- single cells were isolated from the mouse cell homogenates and Zombie NIR- /CD16- /CD8a- /CD3- /CD14- /CD20+ /CD38+ /IgG+/− /double RC1+ RC1-glycanKO- single cells were isolated from the macaque cell homogenates using a FACS Aria III (Becton Dickinson).

Marker:

Article Title: Hypo-osmolar Formulation of Tenofovir (TFV) Enema Promotes Uptake and Metabolism of TFV in Tissues, Leading to Prevention of SHIV/SIV Infection
Article Snippet: .. Heparinized whole-blood samples collected at 0, 24, and 72 h following enema administration were stained with Live/Dead marker (Invitrogen) and the following antibodies: CD3-Alexa 700 (SP34-2; BD Pharmingen), CD4-V500 (L200; BD Horizon), CD8-V450 (RPA-T8; BD Horizon), CD20-APC-Cy7 (L27; BD Biosciences), CD28-PE-Texas Red (CD28.2; Beckman Coulter), CD95-PE-Cy5 (DX2; BD Pharmingen), CD159a (NKG2a)-APC (Z199; Beckman Coulter), CD38-FITC (AT-1, Stemcell Technologies), HLA-DR-PE-Cy7 (G46-6; BD Pharmingen), and Ki67-PE (B56; BD Pharmingen). .. Stained cells were fixed in 1% paraformaldehyde and acquired on an LSRII instrument (BD).

Article Title: Immunization expands HIV-1 V3-glycan specific B-cells in mice and macaques
Article Snippet: .. Macaque cells were stained with anti-CD16 APC-eFluor780 (Invitrogen, #47–0168-41), anti-CD8a APC-eFluor780 (Invitrogen, #47–0086-42), anti-CD3 APC-eFluor780 (Invitrogen, #47–0037-41), anti-CD14 APC-eFluor780 (eBiosciences, #47–0149-41), anti-CD20 PeCy7 (BD, #335793), anti-CD38 FITC (Stem Cell technologies, #60131FI), anti-IgG BV421 (BD Biosciences, #562581), anti-IgM PerCP-Cy5.5 (BD Biosciences, #561285) at a 1:200 dilution and the live/dead marker Zombie NIR at a 1:400 dilution in FACS buffer. .. Zombie NIR- /CD4- /CD8- /F4/80- /NK1.1- /CD11b- /CD11c- /B220+ /GL7+ /CD95+ /RC1+ /RC1-glycanKO- single cells were isolated from the mouse cell homogenates and Zombie NIR- /CD16- /CD8a- /CD3- /CD14- /CD20+ /CD38+ /IgG+/− /double RC1+ RC1-glycanKO- single cells were isolated from the macaque cell homogenates using a FACS Aria III (Becton Dickinson).

FACS:

Article Title: Immunization expands HIV-1 V3-glycan specific B-cells in mice and macaques
Article Snippet: .. Macaque cells were stained with anti-CD16 APC-eFluor780 (Invitrogen, #47–0168-41), anti-CD8a APC-eFluor780 (Invitrogen, #47–0086-42), anti-CD3 APC-eFluor780 (Invitrogen, #47–0037-41), anti-CD14 APC-eFluor780 (eBiosciences, #47–0149-41), anti-CD20 PeCy7 (BD, #335793), anti-CD38 FITC (Stem Cell technologies, #60131FI), anti-IgG BV421 (BD Biosciences, #562581), anti-IgM PerCP-Cy5.5 (BD Biosciences, #561285) at a 1:200 dilution and the live/dead marker Zombie NIR at a 1:400 dilution in FACS buffer. .. Zombie NIR- /CD4- /CD8- /F4/80- /NK1.1- /CD11b- /CD11c- /B220+ /GL7+ /CD95+ /RC1+ /RC1-glycanKO- single cells were isolated from the mouse cell homogenates and Zombie NIR- /CD16- /CD8a- /CD3- /CD14- /CD20+ /CD38+ /IgG+/− /double RC1+ RC1-glycanKO- single cells were isolated from the macaque cell homogenates using a FACS Aria III (Becton Dickinson).

Recombinase Polymerase Amplification:

Article Title: Safety and Immunological Evaluation of Interleukin-21 Plus Anti-α4β7 mAb Combination Therapy in Rhesus Macaques
Article Snippet: .. The following Abs were used: anti–CD4-APCCy7 (clone OKT4), anti–HLA-DR-BV711 (clone L243), and anti-CD20 PerCpCy5.5 (clone 2H7) all from Biolegend, San Diego, CA, USA; anti–CD95-CF594 (clone DX2), anti–beta7-PECy5 (clone FIB504), anti–CCR7-PECy7 (clone 3D12), anti–Ki67-Alexa700 (clone B56), anti–CD3-BUV395 (clone SP34-2), anti–CD8-BUV496 (clone RPA-T8), anti–CD56-BV605 (clone B159), and anti–CD16-BV650 (clone 3G8) all from Becton–Dickinson, BD Biosciences, San Jose, CA, USA; anti–NKG2A-APC (clone Z199), from Beckman Coulter, Brea, CA, USA; Aqua Live/Dead amine dye-AmCyan from ThermoFisher Scientific, Invitrogen, Waltham, MA, USA; anti–CD38-FITC (clone AT-1) from STEMCELL Technologies, Vancouver, British Columbia, Canada; and anti–α4β7-PE (clone Act-1) obtained from the NIH Non-human Primate Reagent Resource, University of Massachusetts Medical School. .. Flow cytometric acquisition was performed on at least 100,000 CD3+ T cells on a BD LSRII Flow Cytometer driven by BD FACSDiva software.

Article Title: Hypo-osmolar Formulation of Tenofovir (TFV) Enema Promotes Uptake and Metabolism of TFV in Tissues, Leading to Prevention of SHIV/SIV Infection
Article Snippet: .. Heparinized whole-blood samples collected at 0, 24, and 72 h following enema administration were stained with Live/Dead marker (Invitrogen) and the following antibodies: CD3-Alexa 700 (SP34-2; BD Pharmingen), CD4-V500 (L200; BD Horizon), CD8-V450 (RPA-T8; BD Horizon), CD20-APC-Cy7 (L27; BD Biosciences), CD28-PE-Texas Red (CD28.2; Beckman Coulter), CD95-PE-Cy5 (DX2; BD Pharmingen), CD159a (NKG2a)-APC (Z199; Beckman Coulter), CD38-FITC (AT-1, Stemcell Technologies), HLA-DR-PE-Cy7 (G46-6; BD Pharmingen), and Ki67-PE (B56; BD Pharmingen). .. Stained cells were fixed in 1% paraformaldehyde and acquired on an LSRII instrument (BD).

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    STEMCELL Technologies Inc frozen pbmc specimens
    Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of <t>CD4</t> + T cells isolated from <t>PBMCs</t> of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels
    Frozen Pbmc Specimens, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    STEMCELL Technologies Inc peripheral blood mononuclear cells pbmcs
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    Peripheral Blood Mononuclear Cells Pbmcs, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 93/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    STEMCELL Technologies Inc human frozen pbmc
    Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of <t>CD4</t> + T cells isolated from <t>PBMCs</t> of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels
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    STEMCELL Technologies Inc human pbmc
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    Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of CD4 + T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels

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

    Article Title: Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice

    doi: 10.1073/pnas.1711233114

    Figure Lengend Snippet: Gut bacteria from healthy twins trigger an antiinflammatory T cell response. ( A ) IL-10 production of CD4 + T cells isolated from PBMCs of selected twin pairs. T cells were stimulated for 96 h with 1 µg/mL anti-CD3 and anti-CD28 antibodies. Levels

    Article Snippet: Frozen PBMC specimens from these pairs were thawed, and CD4+ T cells were isolated using the RosetteSep Kit (Stemcell Technologies).

    Techniques: Isolation