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A. In vivo persistence of CTL clones as measured by tetramer+ CD8+ T cells from post-infusion, <t>cryopreserved</t> <t>PBMCs.</t> Superimposed are the correlative IFN-γ ELISPOT data. ELISPOT results are presented as the mean number of spot forming cells/10 5 PBMCs. Shown is patient #8 who demonstrated prolonged persistence of Mart-1 27–35 specific CTL. B. In vivo persistence and ELIspot data for patient #7 who received only one infusion of Gp100 154–162 specific CTL clones. The long arrow indicates initiation of high dose dexamethasone (dex).
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1) Product Images from "Fludarabine Modulates Immune Response and Extends In Vivo Survival of Adoptively Transferred CD8 T Cells in Patients with Metastatic Melanoma"

Article Title: Fludarabine Modulates Immune Response and Extends In Vivo Survival of Adoptively Transferred CD8 T Cells in Patients with Metastatic Melanoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004749

A. In vivo persistence of CTL clones as measured by tetramer+ CD8+ T cells from post-infusion, cryopreserved PBMCs. Superimposed are the correlative IFN-γ ELISPOT data. ELISPOT results are presented as the mean number of spot forming cells/10 5 PBMCs. Shown is patient #8 who demonstrated prolonged persistence of Mart-1 27–35 specific CTL. B. In vivo persistence and ELIspot data for patient #7 who received only one infusion of Gp100 154–162 specific CTL clones. The long arrow indicates initiation of high dose dexamethasone (dex).
Figure Legend Snippet: A. In vivo persistence of CTL clones as measured by tetramer+ CD8+ T cells from post-infusion, cryopreserved PBMCs. Superimposed are the correlative IFN-γ ELISPOT data. ELISPOT results are presented as the mean number of spot forming cells/10 5 PBMCs. Shown is patient #8 who demonstrated prolonged persistence of Mart-1 27–35 specific CTL. B. In vivo persistence and ELIspot data for patient #7 who received only one infusion of Gp100 154–162 specific CTL clones. The long arrow indicates initiation of high dose dexamethasone (dex).

Techniques Used: In Vivo, CTL Assay, Clone Assay, Enzyme-linked Immunospot

2) Product Images from "Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists"

Article Title: Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-11-88

TLR9 activation by B lymphocytes, PBMC, and CAL-1 cells . (A) Activation of NF-κB in human B lymphocyte cell lines, Ramos-Blue cells, stably transfected with NF-kB/AP-1-inducible SEAP reporter gene. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006: * P
Figure Legend Snippet: TLR9 activation by B lymphocytes, PBMC, and CAL-1 cells . (A) Activation of NF-κB in human B lymphocyte cell lines, Ramos-Blue cells, stably transfected with NF-kB/AP-1-inducible SEAP reporter gene. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006: * P

Techniques Used: Activation Assay, Stable Transfection, Transfection

3) Product Images from "ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques"

Article Title: ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques

Journal: bioRxiv

doi: 10.1101/2020.05.13.093195

Humoral and cellular immune responses to ChAdOx1 nCoV-19 vaccination in rhesus macaques. a. Study schedule for NHPs. V = vaccination with ChAdOx1 nCoV-19; G = vaccination with ChAdOx1 GFP; E = exam; N = exam and necropsy. b. Virus neutralizing titer in serum. d. Summed IFN-γ ELISpot responses in PBMCs toward peptides spanning the spike protein Vaccinated animals = red circles; control animals = blue squares; dotted line = limit of detection; line – median; SFU = spot-forming units.
Figure Legend Snippet: Humoral and cellular immune responses to ChAdOx1 nCoV-19 vaccination in rhesus macaques. a. Study schedule for NHPs. V = vaccination with ChAdOx1 nCoV-19; G = vaccination with ChAdOx1 GFP; E = exam; N = exam and necropsy. b. Virus neutralizing titer in serum. d. Summed IFN-γ ELISpot responses in PBMCs toward peptides spanning the spike protein Vaccinated animals = red circles; control animals = blue squares; dotted line = limit of detection; line – median; SFU = spot-forming units.

Techniques Used: Enzyme-linked Immunospot

4) Product Images from "Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells"

Article Title: Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells

Journal: Vaccine

doi: 10.1016/j.vaccine.2016.04.034

Human HLA-A*0201 PBMCs recognize HMPV N39 and M39 peptides
Figure Legend Snippet: Human HLA-A*0201 PBMCs recognize HMPV N39 and M39 peptides

Techniques Used:

Frequency of immune T CD8 in human HLA-A*0201 PBMCs recognizing the HMPV M39 epitope
Figure Legend Snippet: Frequency of immune T CD8 in human HLA-A*0201 PBMCs recognizing the HMPV M39 epitope

Techniques Used:

5) Product Images from "Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections"

Article Title: Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections

Journal: JCI Insight

doi: 10.1172/jci.insight.140329

Suppressed innate immune TNF-α response in COVID-19. Representative ELISpot photomicrographs displaying baseline innate immune (monocyte) function with LPS-stimulated TNF-α production in PBMCs. Comparison between different donor types, including ( A ) healthy control volunteers and ( B ) CINS, ( C ) septic, and ( D ) COVID-19 patients. Number of spots demonstrates the number of cytokine-producing monocytes, and counts are presented as corrected number of spots per thousand monocytes plated as fraction of the 2.5 × 10 3 PBMCs plated in each well. COVID-19 patients had suppressed TNF-α production when compared with controls. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.
Figure Legend Snippet: Suppressed innate immune TNF-α response in COVID-19. Representative ELISpot photomicrographs displaying baseline innate immune (monocyte) function with LPS-stimulated TNF-α production in PBMCs. Comparison between different donor types, including ( A ) healthy control volunteers and ( B ) CINS, ( C ) septic, and ( D ) COVID-19 patients. Number of spots demonstrates the number of cytokine-producing monocytes, and counts are presented as corrected number of spots per thousand monocytes plated as fraction of the 2.5 × 10 3 PBMCs plated in each well. COVID-19 patients had suppressed TNF-α production when compared with controls. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.

Techniques Used: Enzyme-linked Immunospot

Adaptive immune suppression in COVID-19 patients. Representative ELISpot photomicrographs displaying IFN-ɣ production following overnight stimulation with anti-CD3/anti-CD28 antibodies for ( A ) healthy volunteers, ( B ) CINS patients, and ( C ) septic non–COVID-19 patients. ( D ) Three representative COVID-19–positive samples. Number of spots demonstrates the number of cytokine-producing T cells. Counts are presented as the corrected number of spots per thousand lymphocytes plated as fraction of the 2.5 × 10 4 PBMCs plated in each well. Note the reduction in IFN-ɣ production in both septic and COVID-19 patients compared with CINS patients. Note also a degree of heterogeneity in IFN-ɣ production in COVID-19 and septic patients. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.
Figure Legend Snippet: Adaptive immune suppression in COVID-19 patients. Representative ELISpot photomicrographs displaying IFN-ɣ production following overnight stimulation with anti-CD3/anti-CD28 antibodies for ( A ) healthy volunteers, ( B ) CINS patients, and ( C ) septic non–COVID-19 patients. ( D ) Three representative COVID-19–positive samples. Number of spots demonstrates the number of cytokine-producing T cells. Counts are presented as the corrected number of spots per thousand lymphocytes plated as fraction of the 2.5 × 10 4 PBMCs plated in each well. Note the reduction in IFN-ɣ production in both septic and COVID-19 patients compared with CINS patients. Note also a degree of heterogeneity in IFN-ɣ production in COVID-19 and septic patients. Each photomicrograph was captured with the same magnification, and each image is to scale. ELISpot assays were performed using the PBMC fraction from freshly drawn whole blood. Each condition was run in duplicate for control samples and triplicate for COVID-19 samples.

Techniques Used: Enzyme-linked Immunospot

6) Product Images from "Evaluation of a DNA Aβ42 vaccine in adult rhesus monkeys (Macaca mulatta): antibody kinetics and immune profile after intradermal immunization with full-length DNA Aβ42 trimer"

Article Title: Evaluation of a DNA Aβ42 vaccine in adult rhesus monkeys (Macaca mulatta): antibody kinetics and immune profile after intradermal immunization with full-length DNA Aβ42 trimer

Journal: Alzheimer's Research & Therapy

doi: 10.1186/s13195-017-0257-7

Interleukin (IL)-17 and IL-4 Enzyme-Linked ImmunoSpot (ELISPOT) assays, as well as interferon (IFN)-γ enzyme-linked immunosorbent assay (ELISA) from amyloid-β peptide 1–42 (Aβ42) peptide-restimulated peripheral blood mononuclear cell cultures of DNA Aβ42-immunized rhesus monkeys after a 3-month rest period. IL-17- and IL-4-secreting cells were analyzed in an ELISPOT assay ( a–c ). IFN-γ secretion was tested with a cytokine ELISA ( d ). The individual rhesus monkeys are indicated with numbers on the x -axis of all graphs. a–c The y -axis shows the number of cytokine-secreting cells (spots) per 10 6 cells. d The y -axis shows the amount of IFN-γ found (expressed in picograms per milliliter of culture supernatant)
Figure Legend Snippet: Interleukin (IL)-17 and IL-4 Enzyme-Linked ImmunoSpot (ELISPOT) assays, as well as interferon (IFN)-γ enzyme-linked immunosorbent assay (ELISA) from amyloid-β peptide 1–42 (Aβ42) peptide-restimulated peripheral blood mononuclear cell cultures of DNA Aβ42-immunized rhesus monkeys after a 3-month rest period. IL-17- and IL-4-secreting cells were analyzed in an ELISPOT assay ( a–c ). IFN-γ secretion was tested with a cytokine ELISA ( d ). The individual rhesus monkeys are indicated with numbers on the x -axis of all graphs. a–c The y -axis shows the number of cytokine-secreting cells (spots) per 10 6 cells. d The y -axis shows the amount of IFN-γ found (expressed in picograms per milliliter of culture supernatant)

Techniques Used: Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Interferon (IFN)-γ, interleukin (IL)-17, and IL-4 Enzyme-Linked ImmunoSpot assays for amyloid-β peptide 1–42 (Aβ42) peptide restimulated peripheral blood mononuclear cell cultures of DNA Aβ42-immunized rhesus monkeys. The first column shows the number of IFN-γ-, IL-17-, and IL-4-secreting cells after three immunization time points (48 h in cell culture, medium controls, and Aβ42 peptide restimulation). The second column provides results from the same analyses after the sixth immunization time point. Individual rhesus monkeys are indicated with numbers on the x -axis of all graphs. The y -axis of all graphs shows the number of cytokine-secreting cells (spots) per 10 6 cells. Increased numbers for all three cytokines were found in the immunized animals as well as in the nontreated control animals. Therefore, the cytokine secretion was considered as nonspecific and not due to the DNA Aβ42 immunization. a IL-17-secreting cells. b IFN-γ-secreting cells. c The number of IL-4-secreting cells per 10 6 splenocytes. * p
Figure Legend Snippet: Interferon (IFN)-γ, interleukin (IL)-17, and IL-4 Enzyme-Linked ImmunoSpot assays for amyloid-β peptide 1–42 (Aβ42) peptide restimulated peripheral blood mononuclear cell cultures of DNA Aβ42-immunized rhesus monkeys. The first column shows the number of IFN-γ-, IL-17-, and IL-4-secreting cells after three immunization time points (48 h in cell culture, medium controls, and Aβ42 peptide restimulation). The second column provides results from the same analyses after the sixth immunization time point. Individual rhesus monkeys are indicated with numbers on the x -axis of all graphs. The y -axis of all graphs shows the number of cytokine-secreting cells (spots) per 10 6 cells. Increased numbers for all three cytokines were found in the immunized animals as well as in the nontreated control animals. Therefore, the cytokine secretion was considered as nonspecific and not due to the DNA Aβ42 immunization. a IL-17-secreting cells. b IFN-γ-secreting cells. c The number of IL-4-secreting cells per 10 6 splenocytes. * p

Techniques Used: Enzyme-linked Immunospot, Cell Culture

7) Product Images from "Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4"

Article Title: Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

Journal: Scientific Reports

doi: 10.1038/srep44485

HIV MDSC control CMV specific IFNγ production from CD4 + CX3CR1 + T cells. HIV MDSC were expanded, sorted and cultured with autologous freshly isolated PBMCs as in Fig. 2 . Cells were sorted and cultured for 48 hrs, Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. (a) Representative flow cytometry histograms are shown. (b) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. (c) Supernatants were collected and the quantity of IFNγ determined by ELISA. (d) Blood obtained from HIV(−) (n = 7) and HIV(+) individuals on ART (HIV+) (n = 12) was stained with anti-CD3,-CD19,-CD11b,-CD33,-CD14, -HLA DR Abs, cells were analyzed as CD3 − CD19 − CD11b + CD33 + CD14 + HLA DR −/lo by flow cytometry. Percentages of MDSCs are shown. (e) Representative dot plot with HLA DR −/lo region is shown. For ( d ) Each dot in the plots depict data of each individual donor, the plots include observations from 25 th to 75 th percentile. The horizontal line represents the median value. For ( b and c ), histograms are presented as mean+/−SD; n = 3 donors; *p
Figure Legend Snippet: HIV MDSC control CMV specific IFNγ production from CD4 + CX3CR1 + T cells. HIV MDSC were expanded, sorted and cultured with autologous freshly isolated PBMCs as in Fig. 2 . Cells were sorted and cultured for 48 hrs, Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. (a) Representative flow cytometry histograms are shown. (b) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. (c) Supernatants were collected and the quantity of IFNγ determined by ELISA. (d) Blood obtained from HIV(−) (n = 7) and HIV(+) individuals on ART (HIV+) (n = 12) was stained with anti-CD3,-CD19,-CD11b,-CD33,-CD14, -HLA DR Abs, cells were analyzed as CD3 − CD19 − CD11b + CD33 + CD14 + HLA DR −/lo by flow cytometry. Percentages of MDSCs are shown. (e) Representative dot plot with HLA DR −/lo region is shown. For ( d ) Each dot in the plots depict data of each individual donor, the plots include observations from 25 th to 75 th percentile. The horizontal line represents the median value. For ( b and c ), histograms are presented as mean+/−SD; n = 3 donors; *p

Techniques Used: Cell Culture, Isolation, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

IL-27 regulates B7-H4 expression on HIV expanded MDSC. (a) To determine if IL-27 regulates B7-H4 expression, PBMCs from HIV(−) donors were cultured with or without rIL-27 (5 ng/ml) for 72 hrs. Cells cultured in the presence of non-infectious HIV BaL (p24, 12 ng/10 7 cells) or rIL-10 (5 ng/ml) served as additional controls. Whole cell lysates of PBMCs were prepared and immunoblotted with Abs to GAPDH as loading control and B7-H4. (ai) Immunoblot shown is representative of 4 donors; cells cultured with rIL10 and rIL-27 were run in duplicates. (aii) The histogram bar graph shows mean+/−SD of the densitometric analysis for B7-H4 (n = 4). (b–d) Effect of IL-6, IL-10 and IL-27 on MDSC and B7-H4 + HIV MDSC was determined. PBMCs of healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) in the presence or absence of neutralizing anti-IL6, -IL-10 or –IL-27 or respective isotype antibodies for 5 days. Cells were stained with anti-CD11b, -CD33, -CD14, -HLA DR and –B7-H4 Abs and analyzed using flow cytometry. (b) %B7-H4 + MDSC (bi) and MFI of B7-H4 on MDSC (bii) is shown in presence of anti-IL-6 or-IL-10 antibodies. (c) %B7-H4 + MDSC (ci) and MFI of B7-H4 on MDSC (cii) is shown in presence of anti-IL-27 antibody. (d) %MDSC is shown in presence of anti-IL-6 and –IL-10 (di) and anti-IL-27 (dii) antibodies. Histograms are presented as mean+/−SD; n = 4 donors. (e) Regulation of immunity in HIV/CMV co-infection: HIV/CMV co-infected individuals on ART with suppressed HIV replication and recovered CD4 + T cell numbers have increased IL-27 and reduced MDSC, IL-27 induces (1) B7-H4 on MDSC which binds to CMV-CD4 + T cells and (2) mediates CD4 + IL-10 + T cells, these collectively regulates T cell activation such that IFNγ is produced that is sufficient to control CMV replication without inducing inflammation (right side of straight line) . HIV/CMV co-infected ART naïve individuals with replicating virus have increased IL-6 that induces MDSC expansion and MDSC contributes to immune suppression (left side of straight line) observed during chronic disease stage. *p
Figure Legend Snippet: IL-27 regulates B7-H4 expression on HIV expanded MDSC. (a) To determine if IL-27 regulates B7-H4 expression, PBMCs from HIV(−) donors were cultured with or without rIL-27 (5 ng/ml) for 72 hrs. Cells cultured in the presence of non-infectious HIV BaL (p24, 12 ng/10 7 cells) or rIL-10 (5 ng/ml) served as additional controls. Whole cell lysates of PBMCs were prepared and immunoblotted with Abs to GAPDH as loading control and B7-H4. (ai) Immunoblot shown is representative of 4 donors; cells cultured with rIL10 and rIL-27 were run in duplicates. (aii) The histogram bar graph shows mean+/−SD of the densitometric analysis for B7-H4 (n = 4). (b–d) Effect of IL-6, IL-10 and IL-27 on MDSC and B7-H4 + HIV MDSC was determined. PBMCs of healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) in the presence or absence of neutralizing anti-IL6, -IL-10 or –IL-27 or respective isotype antibodies for 5 days. Cells were stained with anti-CD11b, -CD33, -CD14, -HLA DR and –B7-H4 Abs and analyzed using flow cytometry. (b) %B7-H4 + MDSC (bi) and MFI of B7-H4 on MDSC (bii) is shown in presence of anti-IL-6 or-IL-10 antibodies. (c) %B7-H4 + MDSC (ci) and MFI of B7-H4 on MDSC (cii) is shown in presence of anti-IL-27 antibody. (d) %MDSC is shown in presence of anti-IL-6 and –IL-10 (di) and anti-IL-27 (dii) antibodies. Histograms are presented as mean+/−SD; n = 4 donors. (e) Regulation of immunity in HIV/CMV co-infection: HIV/CMV co-infected individuals on ART with suppressed HIV replication and recovered CD4 + T cell numbers have increased IL-27 and reduced MDSC, IL-27 induces (1) B7-H4 on MDSC which binds to CMV-CD4 + T cells and (2) mediates CD4 + IL-10 + T cells, these collectively regulates T cell activation such that IFNγ is produced that is sufficient to control CMV replication without inducing inflammation (right side of straight line) . HIV/CMV co-infected ART naïve individuals with replicating virus have increased IL-6 that induces MDSC expansion and MDSC contributes to immune suppression (left side of straight line) observed during chronic disease stage. *p

Techniques Used: Expressing, Cell Culture, Staining, Flow Cytometry, Cytometry, Infection, Activation Assay, Produced

In vitro expanded HIV MDSC are functionally similar to MDSC in HIV-infected individuals and modulate CMV specific T cell response. PBMCs of CMV(+) HIV(−) donors were cultured without (PBMCs) or with inactivated HIV BaL (HIV PBMCs) (p24 Ag, 12 ng/10 7 cells). After 5 days, cells were stained with anti-CD11b, -CD33, CD14, HLA DR. ( a and b ) CD14 + HLA DR −/lo MDSC were depleted from HIV PBMCs. HIV PBMCs, HIV PBMCs and MDSC depleted HIV PBMCs (HIV MDSC dep) were cultured with or without CMVpp65 for 24 and 48 hrs. The amounts of IL-2 ( a ) and IFNγ ( b ) were determined in culture supernatants at 24 and 48 hrs, respectively. Net IL-2 or IFNγ was calculated as CMVpp65 – Control. ( c and d ) PBMCs were cultured and stained as above, DR hi and MDSC were sorted. (ci and cii) Representative dot plots show HLA DR vs CD14 in control cells before sort and after sort (Left panel) and in HIV BaL treated cells before sort and after sort (Right panel) . Cells isolated are > 98% pure. ( d and e ) Isolated HIV DR hi and MDSC (5 × 10 4 ) were cultured overnight with autologous freshly isolated PBMCs (1 × 10 5 ) on ELISPOT plates with or without CMVpp65 to determine frequency of ( d ) IFNγ producing cells and ( e ) IL-10 producing cells. Histograms are presented as mean+/−SD; n = 3 donors; *p
Figure Legend Snippet: In vitro expanded HIV MDSC are functionally similar to MDSC in HIV-infected individuals and modulate CMV specific T cell response. PBMCs of CMV(+) HIV(−) donors were cultured without (PBMCs) or with inactivated HIV BaL (HIV PBMCs) (p24 Ag, 12 ng/10 7 cells). After 5 days, cells were stained with anti-CD11b, -CD33, CD14, HLA DR. ( a and b ) CD14 + HLA DR −/lo MDSC were depleted from HIV PBMCs. HIV PBMCs, HIV PBMCs and MDSC depleted HIV PBMCs (HIV MDSC dep) were cultured with or without CMVpp65 for 24 and 48 hrs. The amounts of IL-2 ( a ) and IFNγ ( b ) were determined in culture supernatants at 24 and 48 hrs, respectively. Net IL-2 or IFNγ was calculated as CMVpp65 – Control. ( c and d ) PBMCs were cultured and stained as above, DR hi and MDSC were sorted. (ci and cii) Representative dot plots show HLA DR vs CD14 in control cells before sort and after sort (Left panel) and in HIV BaL treated cells before sort and after sort (Right panel) . Cells isolated are > 98% pure. ( d and e ) Isolated HIV DR hi and MDSC (5 × 10 4 ) were cultured overnight with autologous freshly isolated PBMCs (1 × 10 5 ) on ELISPOT plates with or without CMVpp65 to determine frequency of ( d ) IFNγ producing cells and ( e ) IL-10 producing cells. Histograms are presented as mean+/−SD; n = 3 donors; *p

Techniques Used: In Vitro, Infection, Cell Culture, Staining, Isolation, Enzyme-linked Immunospot

B7-H4 down regulates pAkt to regulate CMV induced T cell activation. To determine the effect of HIV MDSC on CMVpp65 induced pZap70 or pAkt, PBMCs of CMV(+) HIV(−) donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti -CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. (a–c) Sorted cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 for 15 min. pZap70 was determined as detailed in Methods section. (a) Representative flow cytometry histogram plot is shown, (b) pZap70 expressed as %Net CD3 + CD4 + pZap70 + cells and (c) MFI of pZap70 in CD3 + CD4 + cells was determined. (d–f) Sorted MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen); HIV DR hi cells were cultured in presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, transfected cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 peptide pool for 15–30 min pAkt was determined as above. (d) Representative flow cytometry histogram plot is shown, (e) pAkt expressed as %Net CD3 + CD4 + pAkt + cells and (f) MFI of pAkt in CD3 + CD4 + cells was determined. Histograms are presented as mean+/−SD; n = 4 donors. *p
Figure Legend Snippet: B7-H4 down regulates pAkt to regulate CMV induced T cell activation. To determine the effect of HIV MDSC on CMVpp65 induced pZap70 or pAkt, PBMCs of CMV(+) HIV(−) donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti -CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. (a–c) Sorted cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 for 15 min. pZap70 was determined as detailed in Methods section. (a) Representative flow cytometry histogram plot is shown, (b) pZap70 expressed as %Net CD3 + CD4 + pZap70 + cells and (c) MFI of pZap70 in CD3 + CD4 + cells was determined. (d–f) Sorted MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen); HIV DR hi cells were cultured in presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, transfected cells and freshly isolated autologous PBMCs were cultured in a ratio of 1:2 (1 sorted cell: 2 PBMCs) with or without CMVpp65 peptide pool for 15–30 min pAkt was determined as above. (d) Representative flow cytometry histogram plot is shown, (e) pAkt expressed as %Net CD3 + CD4 + pAkt + cells and (f) MFI of pAkt in CD3 + CD4 + cells was determined. Histograms are presented as mean+/−SD; n = 4 donors. *p

Techniques Used: Activation Assay, Cell Culture, Staining, Isolation, Flow Cytometry, Cytometry, Transfection

HIV MDSC overexpress the inhibitory ligand B7-H4 and regulate CMV T cell activation. PBMCs from healthy donors were cultured in the presence or absence of HIV BaL . After 5 days, cells were stained using anti-CD11b, -CD33, -CD14, -HLA DR and -B7-H1/-B7-H3/-PD-L2/-ICOS-L or –B7-H4. (a and b ) Expression of B7-H1, B7-H3, PD-L2, ICOS-L or B7-H4 was determined by flow cytometry and fold expression of each ligand calculated: MDSC (Control cells - HIV BaL stimulated cells)/DR hi (Control cell - HIV BaL stimulated cells). (a) Mean values ± SD from 5 donors are shown (b) Representative flow cytometry histogram plot is shown. (c) Net Mean Fluorescence Intensity (MFI) of respective inhibitory ligands in DR hi and MDSC was determined: MFI in Control cells – MFI in HIV BaL stimulated. Mean ± SD from 5 donors are shown. ( d and e ) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti- CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen). HIV DR hi cells were cultured in the presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, cell lysates of control and B7-H4 siRNA transfected cells were prepared and immunoblotted using anti-GAPDH and –B7-H4 Ab to determine expression of B7-H4. 2.5 × 10 4 transfected cells were cultured with 5 × 10 4 freshly isolated autologous PBMCs in the presence or absence of CMVpp65. (d) After 18 hrs, IL-2 and (e) after 48 hrs IFNγ was determined in culture supernatants by ELISA; partial knockdown of B7-H4 is shown in d. (f and g) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) and MDSC isolated as above. 0.05 × 10 6 MDSC were cultured with 0.1 × 10 6 autologous PBMCs in presence or absence of CMVpp65 with or without rB7-H4 (10 ng/ml). (f) After 18 hrs, IL-2 and (g) after 48 hrs IFNγ was determined in culture supernatants by ELISA. Mean+/−SD are shown for 3 healthy donors. *p
Figure Legend Snippet: HIV MDSC overexpress the inhibitory ligand B7-H4 and regulate CMV T cell activation. PBMCs from healthy donors were cultured in the presence or absence of HIV BaL . After 5 days, cells were stained using anti-CD11b, -CD33, -CD14, -HLA DR and -B7-H1/-B7-H3/-PD-L2/-ICOS-L or –B7-H4. (a and b ) Expression of B7-H1, B7-H3, PD-L2, ICOS-L or B7-H4 was determined by flow cytometry and fold expression of each ligand calculated: MDSC (Control cells - HIV BaL stimulated cells)/DR hi (Control cell - HIV BaL stimulated cells). (a) Mean values ± SD from 5 donors are shown (b) Representative flow cytometry histogram plot is shown. (c) Net Mean Fluorescence Intensity (MFI) of respective inhibitory ligands in DR hi and MDSC was determined: MFI in Control cells – MFI in HIV BaL stimulated. Mean ± SD from 5 donors are shown. ( d and e ) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells). After 5 days, cells were stained with anti- CD11b, -CD33, CD14, HLA DR; DR hi and MDSC were sorted. MDSC were transfected with control or B7-H4 siRNA (75 nM) using Lipofectamine RNAiMAX reagent (Invitrogen). HIV DR hi cells were cultured in the presence of Lipofectamine RNAiMAX reagent used for transfecting MDSC. After 48–72 hrs, cell lysates of control and B7-H4 siRNA transfected cells were prepared and immunoblotted using anti-GAPDH and –B7-H4 Ab to determine expression of B7-H4. 2.5 × 10 4 transfected cells were cultured with 5 × 10 4 freshly isolated autologous PBMCs in the presence or absence of CMVpp65. (d) After 18 hrs, IL-2 and (e) after 48 hrs IFNγ was determined in culture supernatants by ELISA; partial knockdown of B7-H4 is shown in d. (f and g) PBMCs of CMV(+) HIV(−) healthy donors were cultured with or without non-infectious HIV BaL (p24, 12 ng/10 7 cells) and MDSC isolated as above. 0.05 × 10 6 MDSC were cultured with 0.1 × 10 6 autologous PBMCs in presence or absence of CMVpp65 with or without rB7-H4 (10 ng/ml). (f) After 18 hrs, IL-2 and (g) after 48 hrs IFNγ was determined in culture supernatants by ELISA. Mean+/−SD are shown for 3 healthy donors. *p

Techniques Used: Activation Assay, Cell Culture, Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence, Transfection, Isolation, Enzyme-linked Immunosorbent Assay

MDSC regulate CD4 + CX3CR1 + IFNγ + cells in HIV/CMV co-infected individuals. (a–c) All donors were CMV(+) and either HIV-infected (HIV+) or HIV–uninfected (HIV−). PBMCs of HIV(−) and HIV(+) donors were cultured with or without CMVpp65 for 72 hrs. Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. ( a ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. The plots include observations from 25th to 75th percentile; the horizontal line represents the median value. ( b ) Percentage of IFNγ + cells were determined in CD14 − CD3 + CD4 + CX3CR1 + (open histograms) and CD14 − CD3 + CD4 + CX3CR1 − (shaded histograms) subsets. ( c ) Representative dot plots showing CD4 + IFNγ + cells gated on CD14 − CD3 + CD4 + CX3CR1 + cells are shown from HIV-uninfected (ci HIV-ve) and HIV-infected (cii HIV+ve) individual. (d and e) Freshly isolated PBMCs from CMV(+) HIV-infected individuals on ART and with suppressed viral replication were stained with anti-CD14 and –HLA DR antibodies; CD14 + HLA DR −/lo MDSC were depleted from PBMCs by flow cytometry. Whole PBMC (PBMC) and MDSC depleted PBMC (PBMC-MDSC) were cultured with or without CMVpp65 for 72 hrs as above. Culture supernatant was stored and cells were stained as detailed above. ( d ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. ( e ) Amount of IFNγ in the culture supernatants was determined by ELISA. The histograms in b show mean values+/−SD; n = 8 donors. For all other graphs each dot represents an individual donor; *p
Figure Legend Snippet: MDSC regulate CD4 + CX3CR1 + IFNγ + cells in HIV/CMV co-infected individuals. (a–c) All donors were CMV(+) and either HIV-infected (HIV+) or HIV–uninfected (HIV−). PBMCs of HIV(−) and HIV(+) donors were cultured with or without CMVpp65 for 72 hrs. Brefeldin A was added for the last 5 hrs of culture. Cells were surface stained with anti-CD14, -CD3, -CD4 and –CX3CR1, fixed, permeabilized and stained using anti-IFNγ for intracellular IFNγ. ( a ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. The plots include observations from 25th to 75th percentile; the horizontal line represents the median value. ( b ) Percentage of IFNγ + cells were determined in CD14 − CD3 + CD4 + CX3CR1 + (open histograms) and CD14 − CD3 + CD4 + CX3CR1 − (shaded histograms) subsets. ( c ) Representative dot plots showing CD4 + IFNγ + cells gated on CD14 − CD3 + CD4 + CX3CR1 + cells are shown from HIV-uninfected (ci HIV-ve) and HIV-infected (cii HIV+ve) individual. (d and e) Freshly isolated PBMCs from CMV(+) HIV-infected individuals on ART and with suppressed viral replication were stained with anti-CD14 and –HLA DR antibodies; CD14 + HLA DR −/lo MDSC were depleted from PBMCs by flow cytometry. Whole PBMC (PBMC) and MDSC depleted PBMC (PBMC-MDSC) were cultured with or without CMVpp65 for 72 hrs as above. Culture supernatant was stored and cells were stained as detailed above. ( d ) Percentage of CD14 − CD3 + CD4 + CX3CR1 + IFNγ + cells was determined by flow cytometry. ( e ) Amount of IFNγ in the culture supernatants was determined by ELISA. The histograms in b show mean values+/−SD; n = 8 donors. For all other graphs each dot represents an individual donor; *p

Techniques Used: Infection, Cell Culture, Staining, Flow Cytometry, Cytometry, Isolation, Enzyme-linked Immunosorbent Assay

Related Articles

Quantitation Assay:

Article Title: Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections
Article Snippet: ELISpot quantitation of IFN-ɣ and TNF-α production. .. Quantitation of IFN-ɣ– and TNF-α–producing cells was performed on isolated PBMCs by ELISpot analysis, as per the manufacturer’s instruction (Cellular Technologies Limited [CTL] Immunospot, R & D Systems) and as previously described ( , ). .. Patient PBMCs were harvested from whole blood via Ficoll-Paque, counted using the Vi-Cell counter from Beckman Coulter, and incubated overnight plated in 96 well ELISpot culture plates with CLT media or RPMI 1640 media (Sigma-Aldrich) supplemented with human AB serum, nonessential amino acids, penicillin/streptomycin, and l -glutamine.

Isolation:

Article Title: Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections
Article Snippet: ELISpot quantitation of IFN-ɣ and TNF-α production. .. Quantitation of IFN-ɣ– and TNF-α–producing cells was performed on isolated PBMCs by ELISpot analysis, as per the manufacturer’s instruction (Cellular Technologies Limited [CTL] Immunospot, R & D Systems) and as previously described ( , ). .. Patient PBMCs were harvested from whole blood via Ficoll-Paque, counted using the Vi-Cell counter from Beckman Coulter, and incubated overnight plated in 96 well ELISpot culture plates with CLT media or RPMI 1640 media (Sigma-Aldrich) supplemented with human AB serum, nonessential amino acids, penicillin/streptomycin, and l -glutamine.

Enzyme-linked Immunospot:

Article Title: Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections
Article Snippet: ELISpot quantitation of IFN-ɣ and TNF-α production. .. Quantitation of IFN-ɣ– and TNF-α–producing cells was performed on isolated PBMCs by ELISpot analysis, as per the manufacturer’s instruction (Cellular Technologies Limited [CTL] Immunospot, R & D Systems) and as previously described ( , ). .. Patient PBMCs were harvested from whole blood via Ficoll-Paque, counted using the Vi-Cell counter from Beckman Coulter, and incubated overnight plated in 96 well ELISpot culture plates with CLT media or RPMI 1640 media (Sigma-Aldrich) supplemented with human AB serum, nonessential amino acids, penicillin/streptomycin, and l -glutamine.

Article Title: ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques
Article Snippet: Cytokine positive responses are presented after subtraction of the background response detected in the corresponding unstimulated sample (media containing CD107a and Golgi-plug) of each individual spleen sample. .. NHPs – IFN-γ ELISpot assay of PBMCs was performed using the ImmunoSpot® Human IFN- γ Single-Color Enzymatic ELISpot Assay Kit according to the manufacturer’s protocol (Cellular Technology Limited). .. PBMCs were plated at a concentration of 100,000 cells per well and were stimulated with four contiguous peptide pools spanning the length of the SARS-CoV-2 spike protein sequence at a concentration of 2 μg/mL per peptide (Mimotopes).

Article Title: Immunotherapy of high-risk acute leukemia with a recipient (autologous) vaccine expressing transgenic human CD40L and IL-2 after chemotherapy and allogeneic stem cell transplantation
Article Snippet: .. When adequate numbers of PBMCs and target cells were available, we measured the profile of T cells responding to the immunizing blast cells using granzyme B (GrB), interferon-γ (IFN-γ), and IL-5 enzyme-linked immunospot (ELISPOT; Pharmingen/BD Bioscience, San Diego, CA). .. Frozen PBMCs obtained before and after immunization were thawed and seeded at 3 × 105 to 5 × 105 cells per well in a 96-well plate coated with monoclonal antibodies (mAbs) specific for GrB, IFN-γ, or IL-5 in RPMI 1640 (BioWhittaker) containing 5% heat-inactivated human AB serum (Gemini BioProducts, Woodland, CA) and 1% L-glutamine (BioWhittaker).

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    Cellular Technology Ltd regulatory t cell treg assessment frozen pbmcs
    <t>Regulatory</t> T cells <t>(Treg)</t> in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. a Gating strategy used to identify blood Treg cells within the CD4 + and CD8 + T-cell compartment. b Percentage of CD4 + Treg cells in different patient groups (TKIs group, n = 33; TKIs plus IFN-α group, n = 8) and in healthy controls (n = 20). c Frequency of CD4 + Treg cells in patients with CML receiving imatinib (n = 26) or 2nd generation TKIs (n = 7). d Frequency of CD8 + Treg cells in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. The p values in the figure reflect statistically significant differences among study groups
    Regulatory T Cell Treg Assessment Frozen Pbmcs, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellular Technology Ltd pbmc
    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
    Pbmc, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellular Technology Ltd peripheral blood mononuclear cells pbmc
    Qualification of <t>HCMV-specific</t> T-cell IFN-γ ImmunoSpot® testing of <t>PBMC.</t> ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.
    Peripheral Blood Mononuclear Cells Pbmc, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellular Technology Ltd interferon γ enzyme linked immunospot peripheral blood mononuclear cells
    Cellular immunity to pandemic influenza virus (pH1N1) antigens in donor baseline peripheral blood mononuclear cells. Responses were measured by interferon (IFN)-γ enzyme-linked <t>immunospot</t> (ELISPOT). Stimulation used 25 pH1N1 2009 peptide pools (see Methods and Supplementary Table 1), at 2 μg/mL final concentration of each peptide in the culture. A standard CEF peptide pool and phytohemagglutinin were used as positive controls, and media alone was used as negative control. Spots were counted 24 hours later. Bars show responses for individual donors as IFN-γ spot-forming cells (SFC)/10 6 cells above background. Colored segments of bars indicate antigen recognized. (A) Responses at the baseline time point for 153 donors (not all of whom were evaluable for infection because some lacked follow-up serum samples). (B and C) Responses at the baseline time point in those donors later infected with pH1N1, grouped for symptoms of differing severity. Due to limited availability of cells, certain infected donors were not tested with the 25 peptide pools and thus are not shown. (B) Infected donors with influenza-like illness (ILI) symptoms. (C) Donors with milder or no symptoms. Total IFN-γ ELISPOT response did not differ significantly between the ILI and mild/asymptomatic groups, P = .127 by 2-tailed Student t test.
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    Regulatory T cells (Treg) in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. a Gating strategy used to identify blood Treg cells within the CD4 + and CD8 + T-cell compartment. b Percentage of CD4 + Treg cells in different patient groups (TKIs group, n = 33; TKIs plus IFN-α group, n = 8) and in healthy controls (n = 20). c Frequency of CD4 + Treg cells in patients with CML receiving imatinib (n = 26) or 2nd generation TKIs (n = 7). d Frequency of CD8 + Treg cells in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. The p values in the figure reflect statistically significant differences among study groups

    Journal: Journal of Translational Medicine

    Article Title: Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-α

    doi: 10.1186/s12967-019-02194-x

    Figure Lengend Snippet: Regulatory T cells (Treg) in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. a Gating strategy used to identify blood Treg cells within the CD4 + and CD8 + T-cell compartment. b Percentage of CD4 + Treg cells in different patient groups (TKIs group, n = 33; TKIs plus IFN-α group, n = 8) and in healthy controls (n = 20). c Frequency of CD4 + Treg cells in patients with CML receiving imatinib (n = 26) or 2nd generation TKIs (n = 7). d Frequency of CD8 + Treg cells in patients with CML receiving TKIs, either alone or in combination with IFN-α, and in healthy controls. The p values in the figure reflect statistically significant differences among study groups

    Article Snippet: Regulatory T cell (Treg) assessment Frozen PBMCs were thawed following the Cellular Technology Limited protocol (available online at http://www.immunospot.com ).

    Techniques:

    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques: Ex Vivo, Standard Deviation, Isolation, Enzyme-linked Immunospot

    Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques:

    Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques: Enzyme-linked Immunospot, Standard Deviation

    Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.

    Journal: Cells

    Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

    doi: 10.3390/cells7050045

    Figure Lengend Snippet: Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.

    Article Snippet: Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

    Techniques:

    Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.

    Journal: Cells

    Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

    doi: 10.3390/cells7050045

    Figure Lengend Snippet: Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.

    Article Snippet: Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

    Techniques: Negative Control

    Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

    Journal: Cells

    Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

    doi: 10.3390/cells7050045

    Figure Lengend Snippet: Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

    Article Snippet: Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

    Techniques: Positive Control

    Cellular immunity to pandemic influenza virus (pH1N1) antigens in donor baseline peripheral blood mononuclear cells. Responses were measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT). Stimulation used 25 pH1N1 2009 peptide pools (see Methods and Supplementary Table 1), at 2 μg/mL final concentration of each peptide in the culture. A standard CEF peptide pool and phytohemagglutinin were used as positive controls, and media alone was used as negative control. Spots were counted 24 hours later. Bars show responses for individual donors as IFN-γ spot-forming cells (SFC)/10 6 cells above background. Colored segments of bars indicate antigen recognized. (A) Responses at the baseline time point for 153 donors (not all of whom were evaluable for infection because some lacked follow-up serum samples). (B and C) Responses at the baseline time point in those donors later infected with pH1N1, grouped for symptoms of differing severity. Due to limited availability of cells, certain infected donors were not tested with the 25 peptide pools and thus are not shown. (B) Infected donors with influenza-like illness (ILI) symptoms. (C) Donors with milder or no symptoms. Total IFN-γ ELISPOT response did not differ significantly between the ILI and mild/asymptomatic groups, P = .127 by 2-tailed Student t test.

    Journal: Open Forum Infectious Diseases

    Article Title: Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic

    doi: 10.1093/ofid/ofx023

    Figure Lengend Snippet: Cellular immunity to pandemic influenza virus (pH1N1) antigens in donor baseline peripheral blood mononuclear cells. Responses were measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT). Stimulation used 25 pH1N1 2009 peptide pools (see Methods and Supplementary Table 1), at 2 μg/mL final concentration of each peptide in the culture. A standard CEF peptide pool and phytohemagglutinin were used as positive controls, and media alone was used as negative control. Spots were counted 24 hours later. Bars show responses for individual donors as IFN-γ spot-forming cells (SFC)/10 6 cells above background. Colored segments of bars indicate antigen recognized. (A) Responses at the baseline time point for 153 donors (not all of whom were evaluable for infection because some lacked follow-up serum samples). (B and C) Responses at the baseline time point in those donors later infected with pH1N1, grouped for symptoms of differing severity. Due to limited availability of cells, certain infected donors were not tested with the 25 peptide pools and thus are not shown. (B) Infected donors with influenza-like illness (ILI) symptoms. (C) Donors with milder or no symptoms. Total IFN-γ ELISPOT response did not differ significantly between the ILI and mild/asymptomatic groups, P = .127 by 2-tailed Student t test.

    Article Snippet: Interferon-γ Enzyme-Linked Immunospot Peripheral blood mononuclear cells were cultured in triplicate for 24 hours with stimulating peptide pools containing 2 µg/mL of each peptide, in anti-interferon (IFN)-γ-coated plates.

    Techniques: Enzyme-linked Immunospot, Concentration Assay, Negative Control, Infection