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Intracellular Cytokine Staining Response to the AERAS-402 Vaccine. <t>PBMC</t> and leukapheresis specimens from Study Days −84, −14, 28, 56, and 98 were thawed, rested overnight, and stimulated for 5–7 hours with DMSO (negative control), SEB (positive control), or peptide pools corresponding to the vaccine antigens Ag85A, Ag85B, or TB10.4. Specimens were then stained for viability, phenotypic markers, and intracellular cytokine expression and evaluated by flow cytometry. The gating strategy is shown ( a ). The total DMSO-subtracted cytokine response for CD4 + and <t>CD8</t> + T cells following stimulation with Ag85A ( b,c ), Ag85B ( d,e ), and TB10.4 ( f,g ). Each circle represents the response from a single participant. Bars represent the median response for each group. Data is shown for participants immunized with BCG on Study Day −84 and placebo (black circles) and participants vaccinated with BCG on Study Day −84 followed by vaccination with AERAS-402 on Study Days 0 and 28 (3 × 10 10 vp; red circles).
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1) Product Images from "Adenovirally-Induced Polyfunctional T Cells Do Not Necessarily Recognize the Infected Target: Lessons from a Phase I Trial of the AERAS-402 Vaccine"

Article Title: Adenovirally-Induced Polyfunctional T Cells Do Not Necessarily Recognize the Infected Target: Lessons from a Phase I Trial of the AERAS-402 Vaccine

Journal: Scientific Reports

doi: 10.1038/srep36355

Intracellular Cytokine Staining Response to the AERAS-402 Vaccine. PBMC and leukapheresis specimens from Study Days −84, −14, 28, 56, and 98 were thawed, rested overnight, and stimulated for 5–7 hours with DMSO (negative control), SEB (positive control), or peptide pools corresponding to the vaccine antigens Ag85A, Ag85B, or TB10.4. Specimens were then stained for viability, phenotypic markers, and intracellular cytokine expression and evaluated by flow cytometry. The gating strategy is shown ( a ). The total DMSO-subtracted cytokine response for CD4 + and CD8 + T cells following stimulation with Ag85A ( b,c ), Ag85B ( d,e ), and TB10.4 ( f,g ). Each circle represents the response from a single participant. Bars represent the median response for each group. Data is shown for participants immunized with BCG on Study Day −84 and placebo (black circles) and participants vaccinated with BCG on Study Day −84 followed by vaccination with AERAS-402 on Study Days 0 and 28 (3 × 10 10 vp; red circles).
Figure Legend Snippet: Intracellular Cytokine Staining Response to the AERAS-402 Vaccine. PBMC and leukapheresis specimens from Study Days −84, −14, 28, 56, and 98 were thawed, rested overnight, and stimulated for 5–7 hours with DMSO (negative control), SEB (positive control), or peptide pools corresponding to the vaccine antigens Ag85A, Ag85B, or TB10.4. Specimens were then stained for viability, phenotypic markers, and intracellular cytokine expression and evaluated by flow cytometry. The gating strategy is shown ( a ). The total DMSO-subtracted cytokine response for CD4 + and CD8 + T cells following stimulation with Ag85A ( b,c ), Ag85B ( d,e ), and TB10.4 ( f,g ). Each circle represents the response from a single participant. Bars represent the median response for each group. Data is shown for participants immunized with BCG on Study Day −84 and placebo (black circles) and participants vaccinated with BCG on Study Day −84 followed by vaccination with AERAS-402 on Study Days 0 and 28 (3 × 10 10 vp; red circles).

Techniques Used: Staining, Negative Control, Positive Control, Expressing, Flow Cytometry, Cytometry

Ex Vivo ELISPOT Response to the AERAS-402 Vaccine. IFN-γ ELISPOT was performed on either 250,000 PBMC or 250,000 CD4-depleted PBMC (CD8/others) using single peptide pools consisting of 15-mers overlapping by 11 amino acids (final concentration of each peptide pool is 5μg/ml) spanning each of the three antigens contained in the AERAS-402 vaccine. All determinations are performed in duplicate. Solid line: Ag85A, dashed line: Ag85B, dotted line: TB10.4. For the CD8 ELISPOT assay (CD8/others), CD8 + T cells were negatively selected from PBMC using a combination of CD4 and CD56 magnetic beads. For the PBMC ELISPOT, unfractionated PBMC were used as the source of responding T cells and largely consist of CD4 + T cells. Participant numbers are designated over the graphs with the two participants receiving placebo noted in parentheses. Participant 02309 received only the first dose of AERAS-402 vaccine (day 0), due to an abnormal hematocrit prior to the planned day 28 dose.
Figure Legend Snippet: Ex Vivo ELISPOT Response to the AERAS-402 Vaccine. IFN-γ ELISPOT was performed on either 250,000 PBMC or 250,000 CD4-depleted PBMC (CD8/others) using single peptide pools consisting of 15-mers overlapping by 11 amino acids (final concentration of each peptide pool is 5μg/ml) spanning each of the three antigens contained in the AERAS-402 vaccine. All determinations are performed in duplicate. Solid line: Ag85A, dashed line: Ag85B, dotted line: TB10.4. For the CD8 ELISPOT assay (CD8/others), CD8 + T cells were negatively selected from PBMC using a combination of CD4 and CD56 magnetic beads. For the PBMC ELISPOT, unfractionated PBMC were used as the source of responding T cells and largely consist of CD4 + T cells. Participant numbers are designated over the graphs with the two participants receiving placebo noted in parentheses. Participant 02309 received only the first dose of AERAS-402 vaccine (day 0), due to an abnormal hematocrit prior to the planned day 28 dose.

Techniques Used: Ex Vivo, Enzyme-linked Immunospot, Concentration Assay, Magnetic Beads

2) Product Images from "Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects"

Article Title: Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-8-34

Colony Forming Unit Endothelium Assay : PBMC were plated on 24-well fibronectin-coated plates at a concentration of 10(6) cells per well. After 5 days of culture cells were Giemsa stained and clusters of > 50 cells were quantified as colonies.
Figure Legend Snippet: Colony Forming Unit Endothelium Assay : PBMC were plated on 24-well fibronectin-coated plates at a concentration of 10(6) cells per well. After 5 days of culture cells were Giemsa stained and clusters of > 50 cells were quantified as colonies.

Techniques Used: Concentration Assay, Staining

3) Product Images from "Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus"

Article Title: Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus

Journal: Annals of the Rheumatic Diseases

doi: 10.1136/annrheumdis-2017-212916

Effect of B cell stimulation and iberdomide (CC-220) on Ikaros and Aiolos protein levels over time. (A) CD19+ B cells were isolated from the peripheral blood mononuclear cells of normal donors and stimulated in the presence of CC-220 or a Syk inhibitor. (B, C) Ikaros and Aiolos were measured by Western blot analysis. Representative blots are shown; graphs represent the mean and SE of the mean (n=3).
Figure Legend Snippet: Effect of B cell stimulation and iberdomide (CC-220) on Ikaros and Aiolos protein levels over time. (A) CD19+ B cells were isolated from the peripheral blood mononuclear cells of normal donors and stimulated in the presence of CC-220 or a Syk inhibitor. (B, C) Ikaros and Aiolos were measured by Western blot analysis. Representative blots are shown; graphs represent the mean and SE of the mean (n=3).

Techniques Used: Cell Stimulation, Isolation, Western Blot

4) Product Images from "Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation"

Article Title: Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation

Journal: Immunobiology

doi: 10.1016/j.imbio.2018.08.007

Impact of Stimulation on Activation of PBMC vs Purified B cells from 5 control vs 5 viremic HIV-1-infected subjects.
Figure Legend Snippet: Impact of Stimulation on Activation of PBMC vs Purified B cells from 5 control vs 5 viremic HIV-1-infected subjects.

Techniques Used: Activation Assay, Purification, Infection

AID expression in PBMC by qRT-PCR in 14 control and 21 HIV-1-infected subjects.
Figure Legend Snippet: AID expression in PBMC by qRT-PCR in 14 control and 21 HIV-1-infected subjects.

Techniques Used: Expressing, Quantitative RT-PCR, Infection

5) Product Images from "The CD8+ T-Cell Response to an Epstein-Barr Virus-Related Gammaherpesvirus Infecting Rhesus Macaques Provides Evidence for Immune Evasion by the EBNA-1 Homologue"

Article Title: The CD8+ T-Cell Response to an Epstein-Barr Virus-Related Gammaherpesvirus Infecting Rhesus Macaques Provides Evidence for Immune Evasion by the EBNA-1 Homologue

Journal:

doi: 10.1128/JVI.79.20.12681-12691.2005

rhEBNA-1 peptide-specific IFN-γ ELISPOT responses in PBMC, CD4 + and CD8 + T-lymphocyte-enriched populations. Peptide 67 and peptide 82 were identified as potential rhEBNA-1 epitope-containing peptides for Mm141-97 (peptide 67) and
Figure Legend Snippet: rhEBNA-1 peptide-specific IFN-γ ELISPOT responses in PBMC, CD4 + and CD8 + T-lymphocyte-enriched populations. Peptide 67 and peptide 82 were identified as potential rhEBNA-1 epitope-containing peptides for Mm141-97 (peptide 67) and

Techniques Used: Enzyme-linked Immunospot

CD8 + T-lymphocyte-restricted IFN-γ ELISPOT responses to rhLCV latent infection proteins by ex vivo analysis of PBMC. Unfractionated, CD4 + or CD8 + T-lymphocyte-depleted PBMC from three rhesus macaques (Mm141-97, Mm144-97,
Figure Legend Snippet: CD8 + T-lymphocyte-restricted IFN-γ ELISPOT responses to rhLCV latent infection proteins by ex vivo analysis of PBMC. Unfractionated, CD4 + or CD8 + T-lymphocyte-depleted PBMC from three rhesus macaques (Mm141-97, Mm144-97,

Techniques Used: Enzyme-linked Immunospot, Infection, Ex Vivo

6) Product Images from "A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells"

Article Title: A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

Journal: mAbs

doi: 10.1080/19420862.2015.1029216

Cytokine release by AFM11-His is strictly dependent on the presence of CD19 + target cells. ( A ) Cytokine release in cultures of PBMC, B cell-depleted PBMC, and enriched human T cells. Unfractionated human PBMC, B cell-depleted PBMC, and enriched T cells were cultured in the presence of: i) 10 µg/mL AFM11-His or OKT3, ii) 100 µg/mL PHA, or iii) without antibodies (w/o). After 48 h incubation IL-2, IL-6, IL-10, TNF, and IFN-γ in the cell-free culture supernatant were quantified by multiplexing and the results of representative experiments were plotted. ( B ) TNF, ( C ) IL-2, ( D ) IL-6, and ( E ) IFN-γ levels in culture supernatants of PBMC from 7 individual donors. Cytokine levels were determined by Luminex upon stimulation with antibodies in solution. PBMC were either stimulated with AFM11 (open circles) or OKT3 (closed triangles) at the indicated concentrations. IL-2 and TNF levels were measured after 24 h, IL-6 and IFN-γ levels were measured after 48 h. Background (asterisks) was measured in cultures supplemented with vehicle (formulation buffer) corresponding to the volume of highest AFM11 concentration used. Horizontal bars indicate mean values across the 7 donors.
Figure Legend Snippet: Cytokine release by AFM11-His is strictly dependent on the presence of CD19 + target cells. ( A ) Cytokine release in cultures of PBMC, B cell-depleted PBMC, and enriched human T cells. Unfractionated human PBMC, B cell-depleted PBMC, and enriched T cells were cultured in the presence of: i) 10 µg/mL AFM11-His or OKT3, ii) 100 µg/mL PHA, or iii) without antibodies (w/o). After 48 h incubation IL-2, IL-6, IL-10, TNF, and IFN-γ in the cell-free culture supernatant were quantified by multiplexing and the results of representative experiments were plotted. ( B ) TNF, ( C ) IL-2, ( D ) IL-6, and ( E ) IFN-γ levels in culture supernatants of PBMC from 7 individual donors. Cytokine levels were determined by Luminex upon stimulation with antibodies in solution. PBMC were either stimulated with AFM11 (open circles) or OKT3 (closed triangles) at the indicated concentrations. IL-2 and TNF levels were measured after 24 h, IL-6 and IFN-γ levels were measured after 48 h. Background (asterisks) was measured in cultures supplemented with vehicle (formulation buffer) corresponding to the volume of highest AFM11 concentration used. Horizontal bars indicate mean values across the 7 donors.

Techniques Used: Cell Culture, Incubation, Multiplexing, Luminex, Concentration Assay

AFM11 does not facilitate activation of human T cells in the absence of CD19 + target cells. Dose-responsive induction of CD25 by AFM11-His ( A ) and CD69 ( B ) expression on human T cells was assayed in cultures of human PBMC, B cell-depleted PBMC, and enriched T cells after 48 h incubation. Unfractionated PBMC contained 3.8% CD19 + cells. B cell-depleted cultures possessed 0.6% CD19 + cells, and the enriched T cell cultures contained 0.1% CD19 + cells. The kinetics of CD25 ( C ) and CD69 ( D ) expression induced by AFM11-His (10 ng/mL) were determined in human PBMC cultures containing 3.5% CD19 + cells and in B cell-depleted PBMC cultures containing
Figure Legend Snippet: AFM11 does not facilitate activation of human T cells in the absence of CD19 + target cells. Dose-responsive induction of CD25 by AFM11-His ( A ) and CD69 ( B ) expression on human T cells was assayed in cultures of human PBMC, B cell-depleted PBMC, and enriched T cells after 48 h incubation. Unfractionated PBMC contained 3.8% CD19 + cells. B cell-depleted cultures possessed 0.6% CD19 + cells, and the enriched T cell cultures contained 0.1% CD19 + cells. The kinetics of CD25 ( C ) and CD69 ( D ) expression induced by AFM11-His (10 ng/mL) were determined in human PBMC cultures containing 3.5% CD19 + cells and in B cell-depleted PBMC cultures containing

Techniques Used: Activation Assay, Expressing, Incubation

7) Product Images from "Human Articular Chondrocytes Regulate Immune Response by Affecting Directly T Cell Proliferation and Indirectly Inhibiting Monocyte Differentiation to Professional Antigen-Presenting Cells"

Article Title: Human Articular Chondrocytes Regulate Immune Response by Affecting Directly T Cell Proliferation and Indirectly Inhibiting Monocyte Differentiation to Professional Antigen-Presenting Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00415

Human AC do not stimulate allo-response and inhibit MLR as a third party . (A) Allogeneic hAC were used as stimulator of PBMC of three donors labeled with CFSE at the PBMC:hAC ratio of 10:1. Proliferation was evaluated at day 7 and it was assessed as decrement of CFSE content in CD3 + T cells (identified by indirect immunofluorescence with anti-CD3 mAb UCHT-1 followed by goat APC-conjugated anti-mouse isotype specific antiserum). Results are expressed as % of CD3 + T cells with decrement of CFSE content compared to PBMC cultured alone. MLR between the three different PBMC donors was shown for comparison to define whether PBMC can indeed proliferate upon allo-recognition. (B) Regulation of MLR with hAC as a third party. MLR were set up in the presence of hAC (as a third party) at the responder PBMC:hAC ratio of 10:1 and proliferation evaluated as in (A) as decrement of CFSE content in CD3 + responder T cells. Ctr: level of proliferation of CD3 + T cell in the MLR. Results are expressed as % of CD3 + T cells with decrement of CFSE content. Responder PBMC cultured alone showed a proliferation of 3–7%.
Figure Legend Snippet: Human AC do not stimulate allo-response and inhibit MLR as a third party . (A) Allogeneic hAC were used as stimulator of PBMC of three donors labeled with CFSE at the PBMC:hAC ratio of 10:1. Proliferation was evaluated at day 7 and it was assessed as decrement of CFSE content in CD3 + T cells (identified by indirect immunofluorescence with anti-CD3 mAb UCHT-1 followed by goat APC-conjugated anti-mouse isotype specific antiserum). Results are expressed as % of CD3 + T cells with decrement of CFSE content compared to PBMC cultured alone. MLR between the three different PBMC donors was shown for comparison to define whether PBMC can indeed proliferate upon allo-recognition. (B) Regulation of MLR with hAC as a third party. MLR were set up in the presence of hAC (as a third party) at the responder PBMC:hAC ratio of 10:1 and proliferation evaluated as in (A) as decrement of CFSE content in CD3 + responder T cells. Ctr: level of proliferation of CD3 + T cell in the MLR. Results are expressed as % of CD3 + T cells with decrement of CFSE content. Responder PBMC cultured alone showed a proliferation of 3–7%.

Techniques Used: HAC Assay, Labeling, Immunofluorescence, Cell Culture

Human articular condrocyte (hAC) regulate T cell proliferation . (A) T cell proliferation of CD3 + T cells with the indicated stimuli at different PBMC:hAC ratio (20:1, 10:1, 5:1) or PBMC alone (medium). (B) T cell proliferation of PBMC on hAC at the PBMC:hAC ratio of 5:1 in contact or separated by a Millicell transwell chamber (TW, PBMC in TW) or by PBMC in TW separated by a co-cultures of PBMC and hAC (TW-contact). Results are the mean ± SD of eight different experiments. (C) Schematic representation of the different culture conditions: contact (A,B) , TW (B) , and TW-Contact (B) . * p
Figure Legend Snippet: Human articular condrocyte (hAC) regulate T cell proliferation . (A) T cell proliferation of CD3 + T cells with the indicated stimuli at different PBMC:hAC ratio (20:1, 10:1, 5:1) or PBMC alone (medium). (B) T cell proliferation of PBMC on hAC at the PBMC:hAC ratio of 5:1 in contact or separated by a Millicell transwell chamber (TW, PBMC in TW) or by PBMC in TW separated by a co-cultures of PBMC and hAC (TW-contact). Results are the mean ± SD of eight different experiments. (C) Schematic representation of the different culture conditions: contact (A,B) , TW (B) , and TW-Contact (B) . * p

Techniques Used: HAC Assay

Effect of PGE 2 , kynurenine, and adenosine on differentiation of Mo to iDC and presence of PGE 2 in conditioned media of hAC–Mo co-cultures . To determine the effect on the expression of exogeneous PGE 2 (A) or kynurenine (B) or adenosine (C) , Mo from PBMC were induced to differentiate to iDC with GM-CSF and IL-4 and incubated starting at the onset of culture with different doses (as indicated) of these three drugs. The expression of CD1a and CD14 antigens were assessed with direct immunofluorescence using CD1a–FITC and CD14PE–Cy7 mAbs on day 6 of culture. Medium indicates the expression of CD1a and CD14 in cultures without the addition of any drug and it represents the typical differentiation of Mo to iDC. In (C) , lined bar represents the expression of CD1a and CD14 in co-cultures with hAC showing the impairment in Mo differentiation to iDC. Results are shown as mean of fluorescence intensity of each marker and are the mean ± SD of three independent experiments for each drug. (D) The conditioned media (CM) from Mo co-cultured with hAC with GM-CSF and IL-4 was harvested on day 4 of culture and analyzed for the presence of PGE 2 using a specific ELISA. The amount of PGE 2 in CM from hAC or Mo is shown for comparison. Results are the mean ± SD of determination of three independent experiments. * p
Figure Legend Snippet: Effect of PGE 2 , kynurenine, and adenosine on differentiation of Mo to iDC and presence of PGE 2 in conditioned media of hAC–Mo co-cultures . To determine the effect on the expression of exogeneous PGE 2 (A) or kynurenine (B) or adenosine (C) , Mo from PBMC were induced to differentiate to iDC with GM-CSF and IL-4 and incubated starting at the onset of culture with different doses (as indicated) of these three drugs. The expression of CD1a and CD14 antigens were assessed with direct immunofluorescence using CD1a–FITC and CD14PE–Cy7 mAbs on day 6 of culture. Medium indicates the expression of CD1a and CD14 in cultures without the addition of any drug and it represents the typical differentiation of Mo to iDC. In (C) , lined bar represents the expression of CD1a and CD14 in co-cultures with hAC showing the impairment in Mo differentiation to iDC. Results are shown as mean of fluorescence intensity of each marker and are the mean ± SD of three independent experiments for each drug. (D) The conditioned media (CM) from Mo co-cultured with hAC with GM-CSF and IL-4 was harvested on day 4 of culture and analyzed for the presence of PGE 2 using a specific ELISA. The amount of PGE 2 in CM from hAC or Mo is shown for comparison. Results are the mean ± SD of determination of three independent experiments. * p

Techniques Used: HAC Assay, Expressing, Incubation, Immunofluorescence, Fluorescence, Marker, Cell Culture, Enzyme-linked Immunosorbent Assay

8) Product Images from "Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection"

Article Title: Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006888

ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Infection, In Vitro, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Expressing, Flow Cytometry, Cytometry, Infection

ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.
Figure Legend Snippet: ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.

Techniques Used: Expressing, Activation Assay, Labeling, Plasmid Preparation, Real-time Polymerase Chain Reaction, Infection

ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure Legend Snippet: ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

Phenotypic characterization of ALVAC- and Ad5 vector-specific CD4 T cells. PBMC from RV144 or HVTN204 vaccine recipients were stained with CFSE and re-stimulated with vector for 6 days. Phenotypes and cytokine profile of CFSE-low, vector-specific CD4 T cells were measured by flow cytometry. (A) Comparison for percent of central memory (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45R)+) subsets in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (B) Comparison for α4β7+% in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (C) Representative flow cytometric plots for cytokine expression (IFN-γ, IL-2, and IL-17) in CFSE-low, ALVAC-specific (top) or Ad5 vector-specific (bottom) CD4 T cells; (D) Comparison for cytokine expression in CFSE-low, vector-specific CD4 T cells (% cytokine+ CFSE-low) between ALVAC and Ad5 vector from multiple vaccine recipients (n = 11). n.s.: not significant, *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: Phenotypic characterization of ALVAC- and Ad5 vector-specific CD4 T cells. PBMC from RV144 or HVTN204 vaccine recipients were stained with CFSE and re-stimulated with vector for 6 days. Phenotypes and cytokine profile of CFSE-low, vector-specific CD4 T cells were measured by flow cytometry. (A) Comparison for percent of central memory (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45R)+) subsets in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (B) Comparison for α4β7+% in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (C) Representative flow cytometric plots for cytokine expression (IFN-γ, IL-2, and IL-17) in CFSE-low, ALVAC-specific (top) or Ad5 vector-specific (bottom) CD4 T cells; (D) Comparison for cytokine expression in CFSE-low, vector-specific CD4 T cells (% cytokine+ CFSE-low) between ALVAC and Ad5 vector from multiple vaccine recipients (n = 11). n.s.: not significant, *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Flow Cytometry, Cytometry, Infection, Plasmid Preparation, Staining, Expressing

ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Magnetic Beads, Staining, Flow Cytometry, Cytometry, Expressing

9) Product Images from "Regulatory T cells exhibit distinct features in human breast cancer"

Article Title: Regulatory T cells exhibit distinct features in human breast cancer

Journal: Immunity

doi: 10.1016/j.immuni.2016.10.032

Treg cells densely populate human breast tumors Freshly isolated human primary breast tumor, NBP, and peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry. (A) Representative flow cytometric analysis of lymphocytes from tumor, NBP, and PBMC isolated from a patient with primary breast cancer. (Data are representative of findings in samples analyzed from 105 patients). (B) Scatter plots indicating the cumulative frequency of Treg cells among all T cells and the CD8 + :Treg cell ratio. (C) Ratio of CD4 + to CD8 + T cells in tumor, NBP, and PBMC. Data represent the analysis of over 100 individual patients; error bars represent SEM; *p
Figure Legend Snippet: Treg cells densely populate human breast tumors Freshly isolated human primary breast tumor, NBP, and peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry. (A) Representative flow cytometric analysis of lymphocytes from tumor, NBP, and PBMC isolated from a patient with primary breast cancer. (Data are representative of findings in samples analyzed from 105 patients). (B) Scatter plots indicating the cumulative frequency of Treg cells among all T cells and the CD8 + :Treg cell ratio. (C) Ratio of CD4 + to CD8 + T cells in tumor, NBP, and PBMC. Data represent the analysis of over 100 individual patients; error bars represent SEM; *p

Techniques Used: Isolation, Flow Cytometry, Cytometry

10) Product Images from "Blood Biomarkers of Expressed and Inducible HIV-1"

Article Title: Blood Biomarkers of Expressed and Inducible HIV-1

Journal: AIDS (London, England)

doi: 10.1097/QAD.0000000000001748

Unstimulated virus release from cultured PBMC and resting CD4+T-cells correlates with persistent viremia on ART (a) Unstimulated virus release from 15×10 6 PBMC following 5 days of culture correlated with levels of plasma viremia (HIV-1 RNA) measured by large-volume iSCA (rho=0.50, p=0.098). (b) Similarly, unstimulated virus release from 5×10 6 resting CD4+T-cells after 7 days of culture correlated with the levels of plasma viremia by large-volume iSCA (rho = 0.67, p=0.0016). In both (a) and (b), open symbols denote samples that had undetectable HIV-1 RNA by large-volume iSCA (interpolated at 50% of the limit of detection based on plasma volume assayed) or CAP/CTM qRT-PCR of culture supernatant (interpolated at 50% of the limit of detection, or 10 copies per milliliter of culture supernatant assayed).
Figure Legend Snippet: Unstimulated virus release from cultured PBMC and resting CD4+T-cells correlates with persistent viremia on ART (a) Unstimulated virus release from 15×10 6 PBMC following 5 days of culture correlated with levels of plasma viremia (HIV-1 RNA) measured by large-volume iSCA (rho=0.50, p=0.098). (b) Similarly, unstimulated virus release from 5×10 6 resting CD4+T-cells after 7 days of culture correlated with the levels of plasma viremia by large-volume iSCA (rho = 0.67, p=0.0016). In both (a) and (b), open symbols denote samples that had undetectable HIV-1 RNA by large-volume iSCA (interpolated at 50% of the limit of detection based on plasma volume assayed) or CAP/CTM qRT-PCR of culture supernatant (interpolated at 50% of the limit of detection, or 10 copies per milliliter of culture supernatant assayed).

Techniques Used: Cell Culture, Quantitative RT-PCR

Spontaneous virion release is correlated with the frequency of infected cells and their transcriptional activity in PBMC (a) The frequency of infected cells (cellular HIV-1 DNA in PBMC) is correlated with the spontaneous release of virions from unstimulated cultured PBMC (rho=0.55, p=0.010). (b) The level of unspliced cellular HIV-1 RNA transcription in PBMC is correlated with spontaneous virion release from PBMC (rho=0.64, p=0.0016). (c) The frequency of infected cells in PBMC is correlated with spontaneous virion release from resting CD4+T-cells (rho=0.69, p
Figure Legend Snippet: Spontaneous virion release is correlated with the frequency of infected cells and their transcriptional activity in PBMC (a) The frequency of infected cells (cellular HIV-1 DNA in PBMC) is correlated with the spontaneous release of virions from unstimulated cultured PBMC (rho=0.55, p=0.010). (b) The level of unspliced cellular HIV-1 RNA transcription in PBMC is correlated with spontaneous virion release from PBMC (rho=0.64, p=0.0016). (c) The frequency of infected cells in PBMC is correlated with spontaneous virion release from resting CD4+T-cells (rho=0.69, p

Techniques Used: Infection, Activity Assay, Cell Culture

The frequency of infected cells and their transcriptional activity in PBMC are correlated with the level of inducible virion release from PBMC and resting CD4+T-cells (a) The frequency of infected cells in PBMC is correlated with the level of inducible virion release from cultured PBMC treated with PMA/iono for 5 days (rho=0.64, p=0.0017). (b) The level of cellular unspliced HIV-1 RNA transcription is correlated with the level of inducible virion release from cultured PBMC (rho=0.77, p
Figure Legend Snippet: The frequency of infected cells and their transcriptional activity in PBMC are correlated with the level of inducible virion release from PBMC and resting CD4+T-cells (a) The frequency of infected cells in PBMC is correlated with the level of inducible virion release from cultured PBMC treated with PMA/iono for 5 days (rho=0.64, p=0.0017). (b) The level of cellular unspliced HIV-1 RNA transcription is correlated with the level of inducible virion release from cultured PBMC (rho=0.77, p

Techniques Used: Infection, Activity Assay, Cell Culture

11) Product Images from "Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection"

Article Title: Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006888

ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Infection, In Vitro, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Expressing, Flow Cytometry, Cytometry, Infection

ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.
Figure Legend Snippet: ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.

Techniques Used: Expressing, Activation Assay, Labeling, Plasmid Preparation, Real-time Polymerase Chain Reaction, Infection

ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure Legend Snippet: ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Flow Cytometry, Cytometry, Infection, Plasmid Preparation, Staining, Expressing

ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Magnetic Beads, Staining, Flow Cytometry, Cytometry, Expressing

12) Product Images from "Decreased expression of microRNA-21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis"

Article Title: Decreased expression of microRNA-21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12353

Increased circulating CD3 + CD4 + IL-17 + Th17 cells and decreased CD4 + CD25 + Foxp3 + Treg cells in rheumatoid arthritis (RA) patients. ( A ) Peripheral blood mononuclear cells (PBMC) from RA patients ( n = 25) and healthy controls ( n = 20) were stimulated with PMA, ionomycin and BFA for 5 hrs, and then stained with fluorescently labelled anti-human antibodies. FCM analysis for CD3, CD4 and IL-17 was performed. The representative flow cytometric results are shown, and values indicate the percentage of events in the indicated quadrant. ( B ) Collective analysis of Th17 cells from RA patients and healthy controls; data are expressed as box plots. Each box represents the interquartile range (IQR). Lines inside the boxed represent the median. Whiskers represent the highest and lowest values. * P
Figure Legend Snippet: Increased circulating CD3 + CD4 + IL-17 + Th17 cells and decreased CD4 + CD25 + Foxp3 + Treg cells in rheumatoid arthritis (RA) patients. ( A ) Peripheral blood mononuclear cells (PBMC) from RA patients ( n = 25) and healthy controls ( n = 20) were stimulated with PMA, ionomycin and BFA for 5 hrs, and then stained with fluorescently labelled anti-human antibodies. FCM analysis for CD3, CD4 and IL-17 was performed. The representative flow cytometric results are shown, and values indicate the percentage of events in the indicated quadrant. ( B ) Collective analysis of Th17 cells from RA patients and healthy controls; data are expressed as box plots. Each box represents the interquartile range (IQR). Lines inside the boxed represent the median. Whiskers represent the highest and lowest values. * P

Techniques Used: Staining, Flow Cytometry

MiR-21 is decreased in peripheral blood mononuclear cells (PBMCs) and CD4 + T cells from rheumatoid arthritis (RA) patients. ( A ) PBMCs (3 × 10 6 ) were isolated from RA patients ( n = 25) and healthy control ( n = 20), and miR-21 was quantified by real-time PCR. ( B ) Expression of miR-21 in sorted CD4 + T cells from RA patients ( n = 5) and healthy control ( n = 5) by real-time PCR. The expression of miR-21 in patients with RA is shown as relative levels compared with healthy controls. Data are expressed as the mean ± SEM. * P
Figure Legend Snippet: MiR-21 is decreased in peripheral blood mononuclear cells (PBMCs) and CD4 + T cells from rheumatoid arthritis (RA) patients. ( A ) PBMCs (3 × 10 6 ) were isolated from RA patients ( n = 25) and healthy control ( n = 20), and miR-21 was quantified by real-time PCR. ( B ) Expression of miR-21 in sorted CD4 + T cells from RA patients ( n = 5) and healthy control ( n = 5) by real-time PCR. The expression of miR-21 in patients with RA is shown as relative levels compared with healthy controls. Data are expressed as the mean ± SEM. * P

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

13) Product Images from "Telomerase-Based Pharmacologic Enhancement of Antiviral Function of Human CD8+ T Lymphocytes 1"

Article Title: Telomerase-Based Pharmacologic Enhancement of Antiviral Function of Human CD8+ T Lymphocytes 1

Journal:

doi:

Telomerase activity in PBMC and T lymphocytes treated with TAT2. a , An 8% acrylamide gel showing TRAP products from PHA (12 μ g/ml)-stimulated PBMC of an HIV+ donor. Cells were treated with TAT2 (1 μ M) or DMSO (0.1%) every 48 h for 12 days,
Figure Legend Snippet: Telomerase activity in PBMC and T lymphocytes treated with TAT2. a , An 8% acrylamide gel showing TRAP products from PHA (12 μ g/ml)-stimulated PBMC of an HIV+ donor. Cells were treated with TAT2 (1 μ M) or DMSO (0.1%) every 48 h for 12 days,

Techniques Used: Activity Assay, Acrylamide Gel Assay

14) Product Images from "Buprenorphine decreases CCL2-mediated migration of CD14+CD16+ monocytes"

Article Title: Buprenorphine decreases CCL2-mediated migration of CD14+CD16+ monocytes

Journal: Journal of leukocyte biology

doi: 10.1002/JLB.3HI0118-015R

Mature monocytes express surface MOR and KOR. CD14 + monocytes were isolated from PBMC using magnetic beads and cultured non-adherently for three days with MCSF-1. After culture, more than 70% of the monocytes were CD14 + CD16 + . Surface MOR and KOR on mature
Figure Legend Snippet: Mature monocytes express surface MOR and KOR. CD14 + monocytes were isolated from PBMC using magnetic beads and cultured non-adherently for three days with MCSF-1. After culture, more than 70% of the monocytes were CD14 + CD16 + . Surface MOR and KOR on mature

Techniques Used: Isolation, Magnetic Beads, Cell Culture

CD14 + CD16 + monocytes have higher expression of MOR and KOR as compared to CD14 + CD16 − monocytes in PBMC from uninfected people. PBMC were isolated from peripheral blood by Ficoll gradient centrifugation. Cells were analyzed by flow cytometry for
Figure Legend Snippet: CD14 + CD16 + monocytes have higher expression of MOR and KOR as compared to CD14 + CD16 − monocytes in PBMC from uninfected people. PBMC were isolated from peripheral blood by Ficoll gradient centrifugation. Cells were analyzed by flow cytometry for

Techniques Used: Expressing, Isolation, Gradient Centrifugation, Flow Cytometry, Cytometry

15) Product Images from "Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection"

Article Title: Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006888

ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Infection, In Vitro, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Expressing, Flow Cytometry, Cytometry, Infection

ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.
Figure Legend Snippet: ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.

Techniques Used: Expressing, Activation Assay, Labeling, Plasmid Preparation, Real-time Polymerase Chain Reaction, Infection

ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure Legend Snippet: ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

Phenotypic characterization of ALVAC- and Ad5 vector-specific CD4 T cells. PBMC from RV144 or HVTN204 vaccine recipients were stained with CFSE and re-stimulated with vector for 6 days. Phenotypes and cytokine profile of CFSE-low, vector-specific CD4 T cells were measured by flow cytometry. (A) Comparison for percent of central memory (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45R)+) subsets in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (B) Comparison for α4β7+% in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (C) Representative flow cytometric plots for cytokine expression (IFN-γ, IL-2, and IL-17) in CFSE-low, ALVAC-specific (top) or Ad5 vector-specific (bottom) CD4 T cells; (D) Comparison for cytokine expression in CFSE-low, vector-specific CD4 T cells (% cytokine+ CFSE-low) between ALVAC and Ad5 vector from multiple vaccine recipients (n = 11). n.s.: not significant, *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: Phenotypic characterization of ALVAC- and Ad5 vector-specific CD4 T cells. PBMC from RV144 or HVTN204 vaccine recipients were stained with CFSE and re-stimulated with vector for 6 days. Phenotypes and cytokine profile of CFSE-low, vector-specific CD4 T cells were measured by flow cytometry. (A) Comparison for percent of central memory (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45R)+) subsets in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (B) Comparison for α4β7+% in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (C) Representative flow cytometric plots for cytokine expression (IFN-γ, IL-2, and IL-17) in CFSE-low, ALVAC-specific (top) or Ad5 vector-specific (bottom) CD4 T cells; (D) Comparison for cytokine expression in CFSE-low, vector-specific CD4 T cells (% cytokine+ CFSE-low) between ALVAC and Ad5 vector from multiple vaccine recipients (n = 11). n.s.: not significant, *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Flow Cytometry, Cytometry, Infection, Plasmid Preparation, Staining, Expressing

ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Magnetic Beads, Staining, Flow Cytometry, Cytometry, Expressing

16) Product Images from "Naturally occurring subclinical endotoxemia in humans alters adaptive and innate immune functions through reduced MAPK and increased STAT1 phosphorylation"

Article Title: Naturally occurring subclinical endotoxemia in humans alters adaptive and innate immune functions through reduced MAPK and increased STAT1 phosphorylation

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

doi: 10.4049/jimmunol.1501888

LPS affects CD4 + and CD8 + T cell proliferative capacity Cryopreserved PBMCs were thawed, washed and re-suspended at 10 6 cells/ml in R10 supplemented with rIL-2 (50 U/ml). Cells were stained with 0.5μM CellTrace ™ Violet dye and subsequently stimulated with R10 (vehicle) or anti-CD3/CD28 Dynabeads ® for 3, 4, or 5 days. Proliferation was assessed by flow cytometry and division cycles calculated and adjusted for cell number per division at each time point as previously described (see methods). Division cycles are depicted as
Figure Legend Snippet: LPS affects CD4 + and CD8 + T cell proliferative capacity Cryopreserved PBMCs were thawed, washed and re-suspended at 10 6 cells/ml in R10 supplemented with rIL-2 (50 U/ml). Cells were stained with 0.5μM CellTrace ™ Violet dye and subsequently stimulated with R10 (vehicle) or anti-CD3/CD28 Dynabeads ® for 3, 4, or 5 days. Proliferation was assessed by flow cytometry and division cycles calculated and adjusted for cell number per division at each time point as previously described (see methods). Division cycles are depicted as

Techniques Used: Staining, Flow Cytometry, Cytometry

17) Product Images from "Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection"

Article Title: Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005201

Expression of CD4, CCR5 and activation markers on Vδ2 cells. PBMC from HIV-uninfected donors were cultured and treated with IL-2 alone or IPP and IL-2 for six days. The surface expression of CD4, CCR5, and activation markers (HLA-DR, CD38 and CD25) on Vδ2 cells were analyzed by flow cytometry on days 0 and 6. A) Majority of peripheral Vδ2 cells do not express the CD4 receptor but CD4 is significantly upregulated after six days in culture with IL-2 or with IPP and IL-2 (*p
Figure Legend Snippet: Expression of CD4, CCR5 and activation markers on Vδ2 cells. PBMC from HIV-uninfected donors were cultured and treated with IL-2 alone or IPP and IL-2 for six days. The surface expression of CD4, CCR5, and activation markers (HLA-DR, CD38 and CD25) on Vδ2 cells were analyzed by flow cytometry on days 0 and 6. A) Majority of peripheral Vδ2 cells do not express the CD4 receptor but CD4 is significantly upregulated after six days in culture with IL-2 or with IPP and IL-2 (*p

Techniques Used: Expressing, Activation Assay, Cell Culture, Flow Cytometry, Cytometry

Quantification of total HIV DNA levels. A) Total pol HIV copies were quantified by ddPCR within Vδ2 cells (n = 12), total resting CD4 + T cells (r-CD4) (n = 8) and unfractionated PBMC (n = 12) from HIV-1 suppressed patients treated in the acute HIV infection (AHI) or in the chronic HIV infection (CHI). Limit of quantitation (LOQ) was 50.6 copies/ 10 6 Vδ2 cells and 5.1 copies/ 10 6 r-CD4 cells and PBMC, and is depicted with a dotted line. Each color represents one patient. B) Pie charts reflecting the contribution of Vδ2 cells (purple) and r-CD4 cells (red) to the total HIV DNA + PBMC in CHI patients (left pie) and AHI patients (right pie).
Figure Legend Snippet: Quantification of total HIV DNA levels. A) Total pol HIV copies were quantified by ddPCR within Vδ2 cells (n = 12), total resting CD4 + T cells (r-CD4) (n = 8) and unfractionated PBMC (n = 12) from HIV-1 suppressed patients treated in the acute HIV infection (AHI) or in the chronic HIV infection (CHI). Limit of quantitation (LOQ) was 50.6 copies/ 10 6 Vδ2 cells and 5.1 copies/ 10 6 r-CD4 cells and PBMC, and is depicted with a dotted line. Each color represents one patient. B) Pie charts reflecting the contribution of Vδ2 cells (purple) and r-CD4 cells (red) to the total HIV DNA + PBMC in CHI patients (left pie) and AHI patients (right pie).

Techniques Used: Infection, Quantitation Assay

18) Product Images from "Antibody and TLR7 Agonist Delay Viral Rebound in SHIV-Infected Monkeys"

Article Title: Antibody and TLR7 Agonist Delay Viral Rebound in SHIV-Infected Monkeys

Journal: Nature

doi: 10.1038/s41586-018-0600-6

In vitro killing studies. ( a ) GS-9620 treatment led to CD69 upregulation of CD56+ NK cells and CD3+ T cells in vitro following incubation of human PBMC with 1,000 nM GS-9620 for 5 days (N=9). ( b ) GS-9620 treatment augmented PGT121-mediated killing of PGT121 in vitro . Percent p24 reduction in CD4+ T cells (N=6) using an antibody-mediated killing assay (see Methods). Percent killing was calculated as the percent reduction of p24 in CD4+ T cells with PGT121 compared with no PGT121. P-values reflect 2-sided paired Student’s t tests.
Figure Legend Snippet: In vitro killing studies. ( a ) GS-9620 treatment led to CD69 upregulation of CD56+ NK cells and CD3+ T cells in vitro following incubation of human PBMC with 1,000 nM GS-9620 for 5 days (N=9). ( b ) GS-9620 treatment augmented PGT121-mediated killing of PGT121 in vitro . Percent p24 reduction in CD4+ T cells (N=6) using an antibody-mediated killing assay (see Methods). Percent killing was calculated as the percent reduction of p24 in CD4+ T cells with PGT121 compared with no PGT121. P-values reflect 2-sided paired Student’s t tests.

Techniques Used: In Vitro, Incubation

Viral DNA prior to ART discontinuation. Log viral DNA copies/10 6 CD4+ T cells are shown (limit of detection 3 DNA copies/10 6 cells) in PBMC and lymph nodes prior to the interventions ( a ) at week 96 prior to the interventions and ( b ) at week 120 following the interventions (N=11 animals/group). Red horizontal bars indicate median values. P-values reflect 2-sided Mann-Whitney tests.
Figure Legend Snippet: Viral DNA prior to ART discontinuation. Log viral DNA copies/10 6 CD4+ T cells are shown (limit of detection 3 DNA copies/10 6 cells) in PBMC and lymph nodes prior to the interventions ( a ) at week 96 prior to the interventions and ( b ) at week 120 following the interventions (N=11 animals/group). Red horizontal bars indicate median values. P-values reflect 2-sided Mann-Whitney tests.

Techniques Used: MANN-WHITNEY

19) Product Images from "Increased Frequency of Regulatory T Cells Accompanies Increased Immune Activation in Rectal Mucosae of HIV-Positive Noncontrollers ▿"

Article Title: Increased Frequency of Regulatory T Cells Accompanies Increased Immune Activation in Rectal Mucosae of HIV-Positive Noncontrollers ▿

Journal: Journal of Virology

doi: 10.1128/JVI.05608-11

Peripheral and mucosal Treg of noncontrollers, controllers, and seronegative subjects are suppressive ex vivo . CD4 + CD25 − non-Treg and CD4 + CD25 + Treg were separated from PBMC and RMNC using magnetic beads as described in Materials and Methods.
Figure Legend Snippet: Peripheral and mucosal Treg of noncontrollers, controllers, and seronegative subjects are suppressive ex vivo . CD4 + CD25 − non-Treg and CD4 + CD25 + Treg were separated from PBMC and RMNC using magnetic beads as described in Materials and Methods.

Techniques Used: Ex Vivo, Magnetic Beads

Numbers of CD4 + FOXP3 + CD25 + Treg are not significantly different between patient groups despite a decline in total CD4 + T cells of noncontrollers and controllers. (A and B) Treg frequencies in PBMC (PB-; solid symbols) and RMNC (R-; open symbols) of
Figure Legend Snippet: Numbers of CD4 + FOXP3 + CD25 + Treg are not significantly different between patient groups despite a decline in total CD4 + T cells of noncontrollers and controllers. (A and B) Treg frequencies in PBMC (PB-; solid symbols) and RMNC (R-; open symbols) of

Techniques Used:

Treg frequency is increased in the rectal mucosae of noncontrollers regardless of the gating strategy used to identify Treg. (A) Representative flow cytometry plots depicting four different approaches for identifying CD4 + Treg in human PBMC (top panels)
Figure Legend Snippet: Treg frequency is increased in the rectal mucosae of noncontrollers regardless of the gating strategy used to identify Treg. (A) Representative flow cytometry plots depicting four different approaches for identifying CD4 + Treg in human PBMC (top panels)

Techniques Used: Flow Cytometry, Cytometry

Markers of T cell activation are elevated in total CD8 + , CD4 + , and Treg populations in the mucosae of noncontrollers. (A and B) Treg frequencies in PBMC (solid symbols) and RMNC (open symbols) of noncontrollers (triangles), controllers (circles), and
Figure Legend Snippet: Markers of T cell activation are elevated in total CD8 + , CD4 + , and Treg populations in the mucosae of noncontrollers. (A and B) Treg frequencies in PBMC (solid symbols) and RMNC (open symbols) of noncontrollers (triangles), controllers (circles), and

Techniques Used: Activation Assay

20) Product Images from "T cell recognition of Mycobacterium tuberculosis peptides presented by HLA-E derived from infected human cells"

Article Title: T cell recognition of Mycobacterium tuberculosis peptides presented by HLA-E derived from infected human cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0188288

The CD8 + T cell response to Rv0634A 19-29 is restricted by HLA-E. A) A549 cells were pulsed with the Rv0634 19-29 peptide and used as antigen presenting cells in an ELISPOT assay with IFN-γ production by CD8 + T cells from mismatched donors as a readout. B-D) D481 CFSE-labeled CD8+ T cells were isolated and cultured with Rv0634A 19-29 peptide-pulsed autologous macrophages and DC. CFSE-dim cells were sorted and stained with the Rv0634A 19-29 HLA-E tetramer and antibodies against CD3 and CD8. As a control, the D481 line was also stained with a non-HLA-E tetramer (C), or with anti-NKG2A (D) do demonstrate that the tetramer staining is specific. Whole PBMC were stained with the anti-NKG2A antibody as a positive control. E) The mismatched A549 cell line was pulsed with the Rv0634A 19-29 peptide and used as antigen presenting cells with D481 CD8 + T cell line.
Figure Legend Snippet: The CD8 + T cell response to Rv0634A 19-29 is restricted by HLA-E. A) A549 cells were pulsed with the Rv0634 19-29 peptide and used as antigen presenting cells in an ELISPOT assay with IFN-γ production by CD8 + T cells from mismatched donors as a readout. B-D) D481 CFSE-labeled CD8+ T cells were isolated and cultured with Rv0634A 19-29 peptide-pulsed autologous macrophages and DC. CFSE-dim cells were sorted and stained with the Rv0634A 19-29 HLA-E tetramer and antibodies against CD3 and CD8. As a control, the D481 line was also stained with a non-HLA-E tetramer (C), or with anti-NKG2A (D) do demonstrate that the tetramer staining is specific. Whole PBMC were stained with the anti-NKG2A antibody as a positive control. E) The mismatched A549 cell line was pulsed with the Rv0634A 19-29 peptide and used as antigen presenting cells with D481 CD8 + T cell line.

Techniques Used: Enzyme-linked Immunospot, Labeling, Isolation, Cell Culture, Staining, Positive Control

21) Product Images from "Fractalkine Expression Induces Endothelial Progenitor Cell Lysis by Natural Killer Cells"

Article Title: Fractalkine Expression Induces Endothelial Progenitor Cell Lysis by Natural Killer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026663

FKN expression is induced on CD34 + circulating progenitors by inflammatory cytokines and KTR sera. FKN expression was assessed by flow cytometry after gating of CD34 + cells within PBMC isolated from healthy donors after 20 h treatment with ( A ) sera from transplant patients with high % of CD34 + FKN + cells or with ( B ) TNF-α (20 ng/ml) and IFN-γ (50 ng/ml). Serum from healthy blood donors or control medium served as controls. ( C ) FKN expression was also induced on CD34 + cells gated within CD133-purified progenitors after a 20 h treatment with sera from transplant patients.
Figure Legend Snippet: FKN expression is induced on CD34 + circulating progenitors by inflammatory cytokines and KTR sera. FKN expression was assessed by flow cytometry after gating of CD34 + cells within PBMC isolated from healthy donors after 20 h treatment with ( A ) sera from transplant patients with high % of CD34 + FKN + cells or with ( B ) TNF-α (20 ng/ml) and IFN-γ (50 ng/ml). Serum from healthy blood donors or control medium served as controls. ( C ) FKN expression was also induced on CD34 + cells gated within CD133-purified progenitors after a 20 h treatment with sera from transplant patients.

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

Membrane FKN targets CD34 + -derived ECFC lysis by NK cells. ( A ) Cytotoxicity assay performed using PBMC effector cells isolated from 7 independent blood donors as described in Methods . Each independent assay was performed in triplicate (n = 7) and analysed using an effector: target ratio = 100∶1. ( B ) Representative illustration of ECFC lysis. CMFDA labeled target cells were analyzed by epi-fluorescence microscopy on an inverted microscope Nikon Eclipse TE 2000-U with a Plan Fluor 4x /0.13 objective, images were acquired using NIS elements AR software. ( C ) FKN enhances ECFC lysis by purified NK cells. CMFDA cytotoxicity assay performed using purified NK cells as effectors, effector: target ratio = 10∶1, (n = 3). ( D ) Addition of FKN neutralizing antibody reduces ECFC lysis. FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody before addition of PBMC (n = 3). ( E ) Lysis of FKN expressing ECFC resulted in increased microparticles release after 24 h of incubation with PBMC.ECFC were labeled with the lipophilic DiD tracer and transfected with pCDNA3.1 vector coding for membrane FKN (grey bars) or with pCDNA 3.1 empty vector (white bars) and cultured for 24 h. PBMC were added at an effector: target ratio of 50∶1 and co-cultured with target cells for 4 h and 24 h, (n = 5). DiD positive microparticles released by target cells were enumerated by calculation of
Figure Legend Snippet: Membrane FKN targets CD34 + -derived ECFC lysis by NK cells. ( A ) Cytotoxicity assay performed using PBMC effector cells isolated from 7 independent blood donors as described in Methods . Each independent assay was performed in triplicate (n = 7) and analysed using an effector: target ratio = 100∶1. ( B ) Representative illustration of ECFC lysis. CMFDA labeled target cells were analyzed by epi-fluorescence microscopy on an inverted microscope Nikon Eclipse TE 2000-U with a Plan Fluor 4x /0.13 objective, images were acquired using NIS elements AR software. ( C ) FKN enhances ECFC lysis by purified NK cells. CMFDA cytotoxicity assay performed using purified NK cells as effectors, effector: target ratio = 10∶1, (n = 3). ( D ) Addition of FKN neutralizing antibody reduces ECFC lysis. FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody before addition of PBMC (n = 3). ( E ) Lysis of FKN expressing ECFC resulted in increased microparticles release after 24 h of incubation with PBMC.ECFC were labeled with the lipophilic DiD tracer and transfected with pCDNA3.1 vector coding for membrane FKN (grey bars) or with pCDNA 3.1 empty vector (white bars) and cultured for 24 h. PBMC were added at an effector: target ratio of 50∶1 and co-cultured with target cells for 4 h and 24 h, (n = 5). DiD positive microparticles released by target cells were enumerated by calculation of

Techniques Used: Derivative Assay, Lysis, Cytotoxicity Assay, Isolation, Labeling, Fluorescence, Microscopy, Inverted Microscopy, Software, Purification, Transfection, Expressing, Incubation, Plasmid Preparation, Cell Culture

22) Product Images from "Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify"

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

Journal: Cells

doi: 10.3390/cells7050045

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.
Figure Legend 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.

Techniques Used:

23) Product Images from "Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models"

Article Title: Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models

Journal: Journal of Virology

doi: 10.1128/JVI.00242-19

Reduction of CCR5 expression on nonconventional T cells following seminal plasma exposure. (A) Gating strategy used to identify and compare CCR5 expression on conventional and nonconventional T cell populations. Lymphocytes were gated based on FSC-A and FSC-H to identify single cells, followed by gating on viable CD3 + T cells. Vδ2 + gamma delta T cells were identified based on Vδ2 TCR expression. MAIT cells were identified as Vδ2 − cells with a Vα7.2 + CD161 ++ phenotype. Vδ2 − non-MAIT conventional T cells were gated for CD4 and CD8 coreceptor expression. (B) Comparison of CCR5 expression levels among Vδ2 + , MAIT, CD4, and CD8 T cell populations. Gates for CCR5 expression were determined based on fluorescence-minus-one (FMO) controls. The proportion of CCR5 + cells within each population and the median fluorescent intensity (MFI) of CCR5 + cells is expressed in the lower left corner of each plot. (C) Representative histograms of Vδ2 + and MAIT cell CCR5 expression following 5 h or 16 h of exposure to 1% pooled SP in whole PBMC culture compared to an untreated control. (D) The MFI of CCR5 on CCR5 + Vδ2 + cells or MAIT cells following 1% SP treatment was determined as a percentage of the MFI of the untreated control at each time point ( n = 5). Differences compared to the untreated control were assessed by Friedman test with Dunn’s posttest. *, P
Figure Legend Snippet: Reduction of CCR5 expression on nonconventional T cells following seminal plasma exposure. (A) Gating strategy used to identify and compare CCR5 expression on conventional and nonconventional T cell populations. Lymphocytes were gated based on FSC-A and FSC-H to identify single cells, followed by gating on viable CD3 + T cells. Vδ2 + gamma delta T cells were identified based on Vδ2 TCR expression. MAIT cells were identified as Vδ2 − cells with a Vα7.2 + CD161 ++ phenotype. Vδ2 − non-MAIT conventional T cells were gated for CD4 and CD8 coreceptor expression. (B) Comparison of CCR5 expression levels among Vδ2 + , MAIT, CD4, and CD8 T cell populations. Gates for CCR5 expression were determined based on fluorescence-minus-one (FMO) controls. The proportion of CCR5 + cells within each population and the median fluorescent intensity (MFI) of CCR5 + cells is expressed in the lower left corner of each plot. (C) Representative histograms of Vδ2 + and MAIT cell CCR5 expression following 5 h or 16 h of exposure to 1% pooled SP in whole PBMC culture compared to an untreated control. (D) The MFI of CCR5 on CCR5 + Vδ2 + cells or MAIT cells following 1% SP treatment was determined as a percentage of the MFI of the untreated control at each time point ( n = 5). Differences compared to the untreated control were assessed by Friedman test with Dunn’s posttest. *, P

Techniques Used: Expressing, Fluorescence

Inhibition of NK cell function following seminal plasma treatment. (A) Representative plot of intracellular calcium flux in response to CD16 triggering. Cells were gated on live lymphocytes and then CD3 − CD56 dim cells. Red arrows indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). (B) Representative plot of Fluo4-AM MFI over time in CD3 − CD19 − CD56 dim NK cells. Dashed lines indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). Calcium flux in cells that were cultured for 5 h in the absence of SP (untreated) was compared to the flux elicited in cells that were cultured for 5 h but exposed to an individual SP sample for 3 or 5 h. The plot is representative of cells exposed to either individual SP samples or the SP pool. (C) The stimulation-induced AUC of the SP-treated samples is expressed as a percentage of the untreated control for four PBMC donors (median with IQR). Differences were assessed by Friedman test with Dunn’s multiple-comparison posttest. (D) Gating strategy to assess NK cell function following stimulation with gp120-coated CEM.NKr cells and HIVIG. Lymphocytes were gated on FSC-A versus FSC-H to identify singlets, and NK cells were defined as CD3 − CD56 dim cells. Incubation of PBMC with gp120-coated CEM.NKr cells resulted in minimal background expression of IFN-γ, CD107a, and MIP-1α/MIP-1β. Addition of HIVIG resulted in NK cell recognition of CEM.NKr target cells and induced expression of IFN-γ, CD107a, and MIP-1α/MIP-1β, which is inhibited in the presence of seminal plasma (SP). (E) Addition of 1% SP to the PBMC/CEM.NKr/antibody coculture results in a significant inhibition of IFN-γ, CD107a, and MIP-1α/MIP-1β expression by NK cells ( n = 10 PBMC donors). (F) The SP-induced percent inhibition of IFN-γ and CD107a expression is significantly greater than the inhibition observed for MIP-1α/MIP-1β ( n = 10 PBMC donors). Differences were assessed by Wilcoxon test or Friedman test with Dunn’s multiple-comparison posttest.
Figure Legend Snippet: Inhibition of NK cell function following seminal plasma treatment. (A) Representative plot of intracellular calcium flux in response to CD16 triggering. Cells were gated on live lymphocytes and then CD3 − CD56 dim cells. Red arrows indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). (B) Representative plot of Fluo4-AM MFI over time in CD3 − CD19 − CD56 dim NK cells. Dashed lines indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). Calcium flux in cells that were cultured for 5 h in the absence of SP (untreated) was compared to the flux elicited in cells that were cultured for 5 h but exposed to an individual SP sample for 3 or 5 h. The plot is representative of cells exposed to either individual SP samples or the SP pool. (C) The stimulation-induced AUC of the SP-treated samples is expressed as a percentage of the untreated control for four PBMC donors (median with IQR). Differences were assessed by Friedman test with Dunn’s multiple-comparison posttest. (D) Gating strategy to assess NK cell function following stimulation with gp120-coated CEM.NKr cells and HIVIG. Lymphocytes were gated on FSC-A versus FSC-H to identify singlets, and NK cells were defined as CD3 − CD56 dim cells. Incubation of PBMC with gp120-coated CEM.NKr cells resulted in minimal background expression of IFN-γ, CD107a, and MIP-1α/MIP-1β. Addition of HIVIG resulted in NK cell recognition of CEM.NKr target cells and induced expression of IFN-γ, CD107a, and MIP-1α/MIP-1β, which is inhibited in the presence of seminal plasma (SP). (E) Addition of 1% SP to the PBMC/CEM.NKr/antibody coculture results in a significant inhibition of IFN-γ, CD107a, and MIP-1α/MIP-1β expression by NK cells ( n = 10 PBMC donors). (F) The SP-induced percent inhibition of IFN-γ and CD107a expression is significantly greater than the inhibition observed for MIP-1α/MIP-1β ( n = 10 PBMC donors). Differences were assessed by Wilcoxon test or Friedman test with Dunn’s multiple-comparison posttest.

Techniques Used: Inhibition, Cell Function Assay, Cell Culture, Incubation, Expressing

Impact of SP on lymphocyte viability during cell culture. Cells were cultured with 1% (final concentration) SP for 5 or 16 h in RF10 medium. (A) Representative plots of bulk PBMC forward scatter (FSC)/side scatter (SSC) characteristics and viability after 16 h of culture in either RF10 alone or RF10 plus 1% SP. Cells were gated based on FSC area versus SSC area to identify lymphocytes. Singlets were identified by FSC area versus height. CD3 + viability dye − (live) cells were gated for further downstream analysis. (B) Quantification of T cell viability in bulk PBMC cultures after 5 or 16 h. After gating on lymphocytes and singlets, total CD3 + cells were gated, and the proportion of viability dye − (live) cells was quantified. (C) PBMC were cultured for 16 h in the presence or absence of SP pool and the frequencies of CD3 + Vδ2 TCR + , CD3 + Vδ2 TCR − Va7.2 + CD161 ++ (MAIT), CD4 + , and CD8 + T cells were quantified. (D) Representative viability dye staining of isolated T cell cultures. CD3 + cells were gated among the lymphocyte population and subsequently gated as viability dye + (dead) or viability dye − (live). (E) Quantification of the proportion of live cells after 16 h of culture of isolated T cells in RF10 medium alone or RF10 plus 1% SP.
Figure Legend Snippet: Impact of SP on lymphocyte viability during cell culture. Cells were cultured with 1% (final concentration) SP for 5 or 16 h in RF10 medium. (A) Representative plots of bulk PBMC forward scatter (FSC)/side scatter (SSC) characteristics and viability after 16 h of culture in either RF10 alone or RF10 plus 1% SP. Cells were gated based on FSC area versus SSC area to identify lymphocytes. Singlets were identified by FSC area versus height. CD3 + viability dye − (live) cells were gated for further downstream analysis. (B) Quantification of T cell viability in bulk PBMC cultures after 5 or 16 h. After gating on lymphocytes and singlets, total CD3 + cells were gated, and the proportion of viability dye − (live) cells was quantified. (C) PBMC were cultured for 16 h in the presence or absence of SP pool and the frequencies of CD3 + Vδ2 TCR + , CD3 + Vδ2 TCR − Va7.2 + CD161 ++ (MAIT), CD4 + , and CD8 + T cells were quantified. (D) Representative viability dye staining of isolated T cell cultures. CD3 + cells were gated among the lymphocyte population and subsequently gated as viability dye + (dead) or viability dye − (live). (E) Quantification of the proportion of live cells after 16 h of culture of isolated T cells in RF10 medium alone or RF10 plus 1% SP.

Techniques Used: Cell Culture, Concentration Assay, Staining, Isolation

Response of macaque T cells to SP exposure in vitro and in vivo . (A) Representative staining of CD4 + T cell CCR5 expression and CCR5/CCR6 coexpression following exposure of macaque PBMC to 1% SP 3 for 16 h. (B) Quantification of CCR5 MFI on CCR5 + CD4 + T cells and proportion of CCR5 dim CD4 + T cells following 16 h of SP exposure ( n = 5). (C) Vaginal biopsy specimens were collected from five female macaques at baseline. Two weeks later, animals were vaginally exposed to 2.5 ml of seminal plasma, and a second biopsy specimen was collected 24 h later. CD4 + T cells (identified as live, CD45 + EpCam − CD20 − CD14 − CD3 + CD4 + lymphocytes) were assessed for expression of CCR5 and CCR5 surface density (MFI). Plots are representative of results from two animals with low baseline CCR5 expression.
Figure Legend Snippet: Response of macaque T cells to SP exposure in vitro and in vivo . (A) Representative staining of CD4 + T cell CCR5 expression and CCR5/CCR6 coexpression following exposure of macaque PBMC to 1% SP 3 for 16 h. (B) Quantification of CCR5 MFI on CCR5 + CD4 + T cells and proportion of CCR5 dim CD4 + T cells following 16 h of SP exposure ( n = 5). (C) Vaginal biopsy specimens were collected from five female macaques at baseline. Two weeks later, animals were vaginally exposed to 2.5 ml of seminal plasma, and a second biopsy specimen was collected 24 h later. CD4 + T cells (identified as live, CD45 + EpCam − CD20 − CD14 − CD3 + CD4 + lymphocytes) were assessed for expression of CCR5 and CCR5 surface density (MFI). Plots are representative of results from two animals with low baseline CCR5 expression.

Techniques Used: In Vitro, In Vivo, Staining, Expressing

24) Product Images from "Decreased Suppression and Increased Phosphorylated STAT3 in Regulatory T-cells are Associated with Benefit from Adjuvant PD-1 Blockade in Resected Metastatic Melanoma"

Article Title: Decreased Suppression and Increased Phosphorylated STAT3 in Regulatory T-cells are Associated with Benefit from Adjuvant PD-1 Blockade in Resected Metastatic Melanoma

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-18-1100

PD-1 Blockade Increases Expression of Phosphorylated STAT3 in T-cells. (A) Paired PBMC samples (baseline and week 13 post-nivolumab treatment initiation) from patients treated with adjuvant nivolumab were assessed by flow cytometry and the geometric mean fluorescent intensity (gMFI) of pSTAT3 S727 determined in Tregs (CD4+CD127 -/low CD25+FOXP3+), CD4+ Tcons and CD8+ T-cells. Blue lines connect paired NED patient samples and red, dotted lines connect relapsed patient samples. (B) PBMC samples from active metastatic disease patients treated with nivolumab were likewise assessed. (C) Changes in pSTAT3 intensity of PBMC Tregs (red circles), CD4+ Tcons (blue squares) and CD8+ T-cells (purple triangles) in metastatic disease patients are plotted in relation to overall patient survival. (D) CD3+ T-cells were isolated from baseline patient PBMC and cultured for 48 hours in the presence of IgG (black line histogram) or PD-1 blocking antibodies (blue line histogram), then assessed by flow cytometry for expression of pSTAT3 S727 in CD4+CD127 -/low CD25+ Tregs, CD4+ Tcons and CD8+ T-cells. Fluorescence minus one (FMO) histograms are shown in solid gray.
Figure Legend Snippet: PD-1 Blockade Increases Expression of Phosphorylated STAT3 in T-cells. (A) Paired PBMC samples (baseline and week 13 post-nivolumab treatment initiation) from patients treated with adjuvant nivolumab were assessed by flow cytometry and the geometric mean fluorescent intensity (gMFI) of pSTAT3 S727 determined in Tregs (CD4+CD127 -/low CD25+FOXP3+), CD4+ Tcons and CD8+ T-cells. Blue lines connect paired NED patient samples and red, dotted lines connect relapsed patient samples. (B) PBMC samples from active metastatic disease patients treated with nivolumab were likewise assessed. (C) Changes in pSTAT3 intensity of PBMC Tregs (red circles), CD4+ Tcons (blue squares) and CD8+ T-cells (purple triangles) in metastatic disease patients are plotted in relation to overall patient survival. (D) CD3+ T-cells were isolated from baseline patient PBMC and cultured for 48 hours in the presence of IgG (black line histogram) or PD-1 blocking antibodies (blue line histogram), then assessed by flow cytometry for expression of pSTAT3 S727 in CD4+CD127 -/low CD25+ Tregs, CD4+ Tcons and CD8+ T-cells. Fluorescence minus one (FMO) histograms are shown in solid gray.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Cell Culture, Blocking Assay, Fluorescence

Nivolumab Therapy Reduces CD4+CD127 low/− CD25+ T-cell Suppressive Function in Non-Relapsing Patients. (A) Patient baseline PBMC samples were assessed by flow cytometry for Tregs (CD4+CD127 -/low CD25+FOXP3+) as a percentage of total CD3+ T-cells. (B) PBMC samples from patients with active disease treated with nivolumab were assessed as in (A). Error bars show mean with 95% confidence intervals. (C-D) Post-treatment samples from (C) resected disease patients and (D) active disease patients were likewise assessed. Paired patient samples are plotted with connecting lines. NED or responding patients are represented with blue lines and relapsing or non-responding patients with red, dotted lines. (E) Baseline (black bars) and after 13 weeks of nivolumab therapy (blue bars) patient PBMC CD4+CD127 low/− CD25+ T-cells were isolated and co-cultured with allogeneic CD8+ T-cells and irradiated PBMC at indicated ratios. After 5 days, tritiated thymidine was added to cultures for a further 18 hours and cultures assessed for radioactive incorporation. Suppression was normalized against control cultures containing no CD4+CD127 low/− CD25+ T-cells (100% proliferation). *p
Figure Legend Snippet: Nivolumab Therapy Reduces CD4+CD127 low/− CD25+ T-cell Suppressive Function in Non-Relapsing Patients. (A) Patient baseline PBMC samples were assessed by flow cytometry for Tregs (CD4+CD127 -/low CD25+FOXP3+) as a percentage of total CD3+ T-cells. (B) PBMC samples from patients with active disease treated with nivolumab were assessed as in (A). Error bars show mean with 95% confidence intervals. (C-D) Post-treatment samples from (C) resected disease patients and (D) active disease patients were likewise assessed. Paired patient samples are plotted with connecting lines. NED or responding patients are represented with blue lines and relapsing or non-responding patients with red, dotted lines. (E) Baseline (black bars) and after 13 weeks of nivolumab therapy (blue bars) patient PBMC CD4+CD127 low/− CD25+ T-cells were isolated and co-cultured with allogeneic CD8+ T-cells and irradiated PBMC at indicated ratios. After 5 days, tritiated thymidine was added to cultures for a further 18 hours and cultures assessed for radioactive incorporation. Suppression was normalized against control cultures containing no CD4+CD127 low/− CD25+ T-cells (100% proliferation). *p

Techniques Used: Flow Cytometry, Cytometry, Isolation, Cell Culture, Irradiation

25) Product Images from "Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins"

Article Title: Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002906

Activation markers on CD4+ and CD8+ T cell subsets upon T. cruzi and ribosomal protein activation. PBMC isolated from patients with chronic Chagas' disease Cardiomyopathy (CCC; n = 27) and non-infected individuals (NI; n = 20) were seeded at 2.5×10 6 cells/well and stimulated with T. cruzi lysate, P2β or CP0 proteins (10 µg/ml) or medium alone for 6 days. PBMC were stained with CD3-APC, CD4-PE-Cy5 or CD8-PE-Cy5 and activation marker-specific labeled antibodies (CD25-FITC and HLA-DR-PE) prior to flow cytometry analysis. 10,000–15,000 events in the lymphocyte gate (R1 gate) were acquired using a FACSAria flow cytometer (Becton Dickinson); dead cells were excluded by forward vs side-scatter (FSC/SSC) gating. A) Gate-pathway used to determine the activation expression in the populations graphed in B. B) Results were expressed as the percentage of CD25+ or HLA-DR+ cells in CD3+CD4+ (R2 gate) or CD3+CD8+ (R3 gate) lymphocytes. Horizontal lines represent the median and percentiles 25–75th, vertical lines represent percentiles 5–95th. Statistical analysis was performed using the Mann-Whitney U Test, *** P
Figure Legend Snippet: Activation markers on CD4+ and CD8+ T cell subsets upon T. cruzi and ribosomal protein activation. PBMC isolated from patients with chronic Chagas' disease Cardiomyopathy (CCC; n = 27) and non-infected individuals (NI; n = 20) were seeded at 2.5×10 6 cells/well and stimulated with T. cruzi lysate, P2β or CP0 proteins (10 µg/ml) or medium alone for 6 days. PBMC were stained with CD3-APC, CD4-PE-Cy5 or CD8-PE-Cy5 and activation marker-specific labeled antibodies (CD25-FITC and HLA-DR-PE) prior to flow cytometry analysis. 10,000–15,000 events in the lymphocyte gate (R1 gate) were acquired using a FACSAria flow cytometer (Becton Dickinson); dead cells were excluded by forward vs side-scatter (FSC/SSC) gating. A) Gate-pathway used to determine the activation expression in the populations graphed in B. B) Results were expressed as the percentage of CD25+ or HLA-DR+ cells in CD3+CD4+ (R2 gate) or CD3+CD8+ (R3 gate) lymphocytes. Horizontal lines represent the median and percentiles 25–75th, vertical lines represent percentiles 5–95th. Statistical analysis was performed using the Mann-Whitney U Test, *** P

Techniques Used: Activation Assay, Isolation, Countercurrent Chromatography, Infection, Staining, Marker, Labeling, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

Cytokine production profile by CD4+ and CD8+ T cells derived from chagasic patients. CD4+ and CD8+ T cells from three chronic Chagas' disease Cardiomyopathy patients, called RM11, RM12 and RM14, were isolated as described under Methods . Enriched CD4+ or CD8+ T cells were co-cultured with the autologous CD3 (neg) fraction as antigen presenting cells and the indicated stimulus. Supernatants were collected on day 6 and cytokines quantified by multiplex technology. For comparison, results from whole PBMC from each of the patients are represented in the left panel. Symbol (+) indicates a fold induction between 3 to 5 and (++) a fold induction > 5. The fold induction was calculated as: [(cytokine in stimulated culture) - (cytokine in NS culture)]/(cytokine in NS culture), where NS denotes non-stimulated cells.
Figure Legend Snippet: Cytokine production profile by CD4+ and CD8+ T cells derived from chagasic patients. CD4+ and CD8+ T cells from three chronic Chagas' disease Cardiomyopathy patients, called RM11, RM12 and RM14, were isolated as described under Methods . Enriched CD4+ or CD8+ T cells were co-cultured with the autologous CD3 (neg) fraction as antigen presenting cells and the indicated stimulus. Supernatants were collected on day 6 and cytokines quantified by multiplex technology. For comparison, results from whole PBMC from each of the patients are represented in the left panel. Symbol (+) indicates a fold induction between 3 to 5 and (++) a fold induction > 5. The fold induction was calculated as: [(cytokine in stimulated culture) - (cytokine in NS culture)]/(cytokine in NS culture), where NS denotes non-stimulated cells.

Techniques Used: Derivative Assay, Isolation, Cell Culture, Multiplex Assay

26) Product Images from "Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus"

Article Title: Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus

Journal: Annals of the Rheumatic Diseases

doi: 10.1136/annrheumdis-2017-212916

Effect of B cell stimulation and iberdomide (CC-220) on Ikaros and Aiolos protein levels over time. (A) CD19+ B cells were isolated from the peripheral blood mononuclear cells of normal donors and stimulated in the presence of CC-220 or a Syk inhibitor. (B, C) Ikaros and Aiolos were measured by Western blot analysis. Representative blots are shown; graphs represent the mean and SE of the mean (n=3).
Figure Legend Snippet: Effect of B cell stimulation and iberdomide (CC-220) on Ikaros and Aiolos protein levels over time. (A) CD19+ B cells were isolated from the peripheral blood mononuclear cells of normal donors and stimulated in the presence of CC-220 or a Syk inhibitor. (B, C) Ikaros and Aiolos were measured by Western blot analysis. Representative blots are shown; graphs represent the mean and SE of the mean (n=3).

Techniques Used: Cell Stimulation, Isolation, Western Blot

27) Product Images from "Multiple NF-?B Sites in HIV-1 Subtype C Long Terminal Repeat Confer Superior Magnitude of Transcription and Thereby the Enhanced Viral Predominance *"

Article Title: Multiple NF-?B Sites in HIV-1 Subtype C Long Terminal Repeat Confer Superior Magnitude of Transcription and Thereby the Enhanced Viral Predominance *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.397158

4-κB viruses out-compete the 3-κB strains in T-cells A, schematic representation of the paired isogenic viral constructs used in the competition assays. The 22-bp sequence consisting of the F-κB site was engineered into the 3′-LTR of the Indie-C1 molecular clone (HHC) to generate the 4-κB LTR (FHHC). Replication profiles of the viral strains in vitro using CEM-CCR5 T-cells ( B ) or the CD8-depleted, mitogen-activated PBMC ( C ) from a representative subject. The cells were infected with 500 infectious units of FHHC or HHC viruses, and the secretion of p24 into the medium was monitored for several weeks as indicated. The data are presented as the mean of triplicate wells ± 1 S.D. and are representative of three independent experiments. Pairwise competition between the HHC and FHHC isogenic viruses in CEM-CCR5 cells ( D ) or activated PBMC ( E . Genomic DNA was extracted on day 10 following the viral infection and subjected to the HTA analysis. The heteroduplex band intensities are plotted as relative values compared with the monoinfections (see formula ).
Figure Legend Snippet: 4-κB viruses out-compete the 3-κB strains in T-cells A, schematic representation of the paired isogenic viral constructs used in the competition assays. The 22-bp sequence consisting of the F-κB site was engineered into the 3′-LTR of the Indie-C1 molecular clone (HHC) to generate the 4-κB LTR (FHHC). Replication profiles of the viral strains in vitro using CEM-CCR5 T-cells ( B ) or the CD8-depleted, mitogen-activated PBMC ( C ) from a representative subject. The cells were infected with 500 infectious units of FHHC or HHC viruses, and the secretion of p24 into the medium was monitored for several weeks as indicated. The data are presented as the mean of triplicate wells ± 1 S.D. and are representative of three independent experiments. Pairwise competition between the HHC and FHHC isogenic viruses in CEM-CCR5 cells ( D ) or activated PBMC ( E . Genomic DNA was extracted on day 10 following the viral infection and subjected to the HTA analysis. The heteroduplex band intensities are plotted as relative values compared with the monoinfections (see formula ).

Techniques Used: Construct, Sequencing, In Vitro, Infection

28) Product Images from "Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models"

Article Title: Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models

Journal: Journal of Virology

doi: 10.1128/JVI.00242-19

Reduction of CCR5 expression on nonconventional T cells following seminal plasma exposure. (A) Gating strategy used to identify and compare CCR5 expression on conventional and nonconventional T cell populations. Lymphocytes were gated based on FSC-A and FSC-H to identify single cells, followed by gating on viable CD3 + T cells. Vδ2 + gamma delta T cells were identified based on Vδ2 TCR expression. MAIT cells were identified as Vδ2 − cells with a Vα7.2 + CD161 ++ phenotype. Vδ2 − non-MAIT conventional T cells were gated for CD4 and CD8 coreceptor expression. (B) Comparison of CCR5 expression levels among Vδ2 + , MAIT, CD4, and CD8 T cell populations. Gates for CCR5 expression were determined based on fluorescence-minus-one (FMO) controls. The proportion of CCR5 + cells within each population and the median fluorescent intensity (MFI) of CCR5 + cells is expressed in the lower left corner of each plot. (C) Representative histograms of Vδ2 + and MAIT cell CCR5 expression following 5 h or 16 h of exposure to 1% pooled SP in whole PBMC culture compared to an untreated control. (D) The MFI of CCR5 on CCR5 + Vδ2 + cells or MAIT cells following 1% SP treatment was determined as a percentage of the MFI of the untreated control at each time point ( n  = 5). Differences compared to the untreated control were assessed by Friedman test with Dunn’s posttest. *, P
Figure Legend Snippet: Reduction of CCR5 expression on nonconventional T cells following seminal plasma exposure. (A) Gating strategy used to identify and compare CCR5 expression on conventional and nonconventional T cell populations. Lymphocytes were gated based on FSC-A and FSC-H to identify single cells, followed by gating on viable CD3 + T cells. Vδ2 + gamma delta T cells were identified based on Vδ2 TCR expression. MAIT cells were identified as Vδ2 − cells with a Vα7.2 + CD161 ++ phenotype. Vδ2 − non-MAIT conventional T cells were gated for CD4 and CD8 coreceptor expression. (B) Comparison of CCR5 expression levels among Vδ2 + , MAIT, CD4, and CD8 T cell populations. Gates for CCR5 expression were determined based on fluorescence-minus-one (FMO) controls. The proportion of CCR5 + cells within each population and the median fluorescent intensity (MFI) of CCR5 + cells is expressed in the lower left corner of each plot. (C) Representative histograms of Vδ2 + and MAIT cell CCR5 expression following 5 h or 16 h of exposure to 1% pooled SP in whole PBMC culture compared to an untreated control. (D) The MFI of CCR5 on CCR5 + Vδ2 + cells or MAIT cells following 1% SP treatment was determined as a percentage of the MFI of the untreated control at each time point ( n  = 5). Differences compared to the untreated control were assessed by Friedman test with Dunn’s posttest. *, P

Techniques Used: Expressing, Fluorescence

Inhibition of NK cell function following seminal plasma treatment. (A) Representative plot of intracellular calcium flux in response to CD16 triggering. Cells were gated on live lymphocytes and then CD3 − CD56 dim cells. Red arrows indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). (B) Representative plot of Fluo4-AM MFI over time in CD3 − CD19 − CD56 dim NK cells. Dashed lines indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). Calcium flux in cells that were cultured for 5 h in the absence of SP (untreated) was compared to the flux elicited in cells that were cultured for 5 h but exposed to an individual SP sample for 3 or 5 h. The plot is representative of cells exposed to either individual SP samples or the SP pool. (C) The stimulation-induced AUC of the SP-treated samples is expressed as a percentage of the untreated control for four PBMC donors (median with IQR). Differences were assessed by Friedman test with Dunn’s multiple-comparison posttest. (D) Gating strategy to assess NK cell function following stimulation with gp120-coated CEM.NKr cells and HIVIG. Lymphocytes were gated on FSC-A versus FSC-H to identify singlets, and NK cells were defined as CD3 − CD56 dim cells. Incubation of PBMC with gp120-coated CEM.NKr cells resulted in minimal background expression of IFN-γ, CD107a, and MIP-1α/MIP-1β. Addition of HIVIG resulted in NK cell recognition of CEM.NKr target cells and induced expression of IFN-γ, CD107a, and MIP-1α/MIP-1β, which is inhibited in the presence of seminal plasma (SP). (E) Addition of 1% SP to the PBMC/CEM.NKr/antibody coculture results in a significant inhibition of IFN-γ, CD107a, and MIP-1α/MIP-1β expression by NK cells ( n  = 10 PBMC donors). (F) The SP-induced percent inhibition of IFN-γ and CD107a expression is significantly greater than the inhibition observed for MIP-1α/MIP-1β ( n  = 10 PBMC donors). Differences were assessed by Wilcoxon test or Friedman test with Dunn’s multiple-comparison posttest.
Figure Legend Snippet: Inhibition of NK cell function following seminal plasma treatment. (A) Representative plot of intracellular calcium flux in response to CD16 triggering. Cells were gated on live lymphocytes and then CD3 − CD56 dim cells. Red arrows indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). (B) Representative plot of Fluo4-AM MFI over time in CD3 − CD19 − CD56 dim NK cells. Dashed lines indicate the time of 3G8 or cross-linking F(ab′) 2 additions (at 30 and 60 s). Calcium flux in cells that were cultured for 5 h in the absence of SP (untreated) was compared to the flux elicited in cells that were cultured for 5 h but exposed to an individual SP sample for 3 or 5 h. The plot is representative of cells exposed to either individual SP samples or the SP pool. (C) The stimulation-induced AUC of the SP-treated samples is expressed as a percentage of the untreated control for four PBMC donors (median with IQR). Differences were assessed by Friedman test with Dunn’s multiple-comparison posttest. (D) Gating strategy to assess NK cell function following stimulation with gp120-coated CEM.NKr cells and HIVIG. Lymphocytes were gated on FSC-A versus FSC-H to identify singlets, and NK cells were defined as CD3 − CD56 dim cells. Incubation of PBMC with gp120-coated CEM.NKr cells resulted in minimal background expression of IFN-γ, CD107a, and MIP-1α/MIP-1β. Addition of HIVIG resulted in NK cell recognition of CEM.NKr target cells and induced expression of IFN-γ, CD107a, and MIP-1α/MIP-1β, which is inhibited in the presence of seminal plasma (SP). (E) Addition of 1% SP to the PBMC/CEM.NKr/antibody coculture results in a significant inhibition of IFN-γ, CD107a, and MIP-1α/MIP-1β expression by NK cells ( n  = 10 PBMC donors). (F) The SP-induced percent inhibition of IFN-γ and CD107a expression is significantly greater than the inhibition observed for MIP-1α/MIP-1β ( n  = 10 PBMC donors). Differences were assessed by Wilcoxon test or Friedman test with Dunn’s multiple-comparison posttest.

Techniques Used: Inhibition, Cell Function Assay, Cell Culture, Incubation, Expressing

Impact of SP on lymphocyte viability during cell culture. Cells were cultured with 1% (final concentration) SP for 5 or 16 h in RF10 medium. (A) Representative plots of bulk PBMC forward scatter (FSC)/side scatter (SSC) characteristics and viability after 16 h of culture in either RF10 alone or RF10 plus 1% SP. Cells were gated based on FSC area versus SSC area to identify lymphocytes. Singlets were identified by FSC area versus height. CD3 + viability dye − (live) cells were gated for further downstream analysis. (B) Quantification of T cell viability in bulk PBMC cultures after 5 or 16 h. After gating on lymphocytes and singlets, total CD3 + cells were gated, and the proportion of viability dye − (live) cells was quantified. (C) PBMC were cultured for 16 h in the presence or absence of SP pool and the frequencies of CD3 + Vδ2 TCR + , CD3 + Vδ2 TCR − Va7.2 + CD161 ++ (MAIT), CD4 + , and CD8 + T cells were quantified. (D) Representative viability dye staining of isolated T cell cultures. CD3 + cells were gated among the lymphocyte population and subsequently gated as viability dye + (dead) or viability dye − (live). (E) Quantification of the proportion of live cells after 16 h of culture of isolated T cells in RF10 medium alone or RF10 plus 1% SP.
Figure Legend Snippet: Impact of SP on lymphocyte viability during cell culture. Cells were cultured with 1% (final concentration) SP for 5 or 16 h in RF10 medium. (A) Representative plots of bulk PBMC forward scatter (FSC)/side scatter (SSC) characteristics and viability after 16 h of culture in either RF10 alone or RF10 plus 1% SP. Cells were gated based on FSC area versus SSC area to identify lymphocytes. Singlets were identified by FSC area versus height. CD3 + viability dye − (live) cells were gated for further downstream analysis. (B) Quantification of T cell viability in bulk PBMC cultures after 5 or 16 h. After gating on lymphocytes and singlets, total CD3 + cells were gated, and the proportion of viability dye − (live) cells was quantified. (C) PBMC were cultured for 16 h in the presence or absence of SP pool and the frequencies of CD3 + Vδ2 TCR + , CD3 + Vδ2 TCR − Va7.2 + CD161 ++ (MAIT), CD4 + , and CD8 + T cells were quantified. (D) Representative viability dye staining of isolated T cell cultures. CD3 + cells were gated among the lymphocyte population and subsequently gated as viability dye + (dead) or viability dye − (live). (E) Quantification of the proportion of live cells after 16 h of culture of isolated T cells in RF10 medium alone or RF10 plus 1% SP.

Techniques Used: Cell Culture, Concentration Assay, Staining, Isolation

Response of macaque T cells to SP exposure in vitro and in vivo . (A) Representative staining of CD4 + T cell CCR5 expression and CCR5/CCR6 coexpression following exposure of macaque PBMC to 1% SP 3 for 16 h. (B) Quantification of CCR5 MFI on CCR5 + CD4 + T cells and proportion of CCR5 dim CD4 + T cells following 16 h of SP exposure ( n  = 5). (C) Vaginal biopsy specimens were collected from five female macaques at baseline. Two weeks later, animals were vaginally exposed to 2.5 ml of seminal plasma, and a second biopsy specimen was collected 24 h later. CD4 + T cells (identified as live, CD45 + EpCam − CD20 − CD14 − CD3 + CD4 + lymphocytes) were assessed for expression of CCR5 and CCR5 surface density (MFI). Plots are representative of results from two animals with low baseline CCR5 expression.
Figure Legend Snippet: Response of macaque T cells to SP exposure in vitro and in vivo . (A) Representative staining of CD4 + T cell CCR5 expression and CCR5/CCR6 coexpression following exposure of macaque PBMC to 1% SP 3 for 16 h. (B) Quantification of CCR5 MFI on CCR5 + CD4 + T cells and proportion of CCR5 dim CD4 + T cells following 16 h of SP exposure ( n  = 5). (C) Vaginal biopsy specimens were collected from five female macaques at baseline. Two weeks later, animals were vaginally exposed to 2.5 ml of seminal plasma, and a second biopsy specimen was collected 24 h later. CD4 + T cells (identified as live, CD45 + EpCam − CD20 − CD14 − CD3 + CD4 + lymphocytes) were assessed for expression of CCR5 and CCR5 surface density (MFI). Plots are representative of results from two animals with low baseline CCR5 expression.

Techniques Used: In Vitro, In Vivo, Staining, Expressing

29) Product Images from "Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors"

Article Title: Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006740

Ruxolitinib does not inhibit normal TCR function and signaling that is independent of HIV-1 infection. (A) Mean CD3 zeta and SLP76 phosphorylation (MFI in CD4 cells) as quantified by flow cytometry in CD4 + cells isolated from HIV negative donors and stimulated with anti-CD3/CD28 in the presence of increasing concentrations of Ruxoltinib versus DMSO treated control cells (n = 3). Statistical significance was determined by an upaired t-test corrected for multiple comparisons using the Holm-Sidak method. (B) Mean cytokine production (% of IL-2 + , TNF-α + and IFN-γ + triple positive cells) in CD3+CD8- cells or CD3+CD8+ cells as measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 6 hr with aCD3/CD28, Brefeldin A (5 μg/ml) and increasing concentrations of Ruxoltinib versus DMSO treated cells (n = 3). Statistical significance for (B) determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p
Figure Legend Snippet: Ruxolitinib does not inhibit normal TCR function and signaling that is independent of HIV-1 infection. (A) Mean CD3 zeta and SLP76 phosphorylation (MFI in CD4 cells) as quantified by flow cytometry in CD4 + cells isolated from HIV negative donors and stimulated with anti-CD3/CD28 in the presence of increasing concentrations of Ruxoltinib versus DMSO treated control cells (n = 3). Statistical significance was determined by an upaired t-test corrected for multiple comparisons using the Holm-Sidak method. (B) Mean cytokine production (% of IL-2 + , TNF-α + and IFN-γ + triple positive cells) in CD3+CD8- cells or CD3+CD8+ cells as measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 6 hr with aCD3/CD28, Brefeldin A (5 μg/ml) and increasing concentrations of Ruxoltinib versus DMSO treated cells (n = 3). Statistical significance for (B) determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p

Techniques Used: Infection, Flow Cytometry, Cytometry, Isolation

Jak inhibitors block cytokine-induced STAT5 phosphorylation and Bcl-2 expression. STAT5 phosphorylation (% in CD4+ T cells) or Bcl-2 expression (MFI in CD4+ T cells) was measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 15 min (pSTAT studies; A, C) or 6 days (Bcl-2 studies; B, D) with IL-2 (left panels), IL-7 (middle panels) and IL-15 (right panels) (n = 3) and increasing concentrations (0.01, 0.1, and 1.0 μM) of ruxolitinib or tofacitinib (A-D). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. (-) indicates no cytokine was added. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: **p
Figure Legend Snippet: Jak inhibitors block cytokine-induced STAT5 phosphorylation and Bcl-2 expression. STAT5 phosphorylation (% in CD4+ T cells) or Bcl-2 expression (MFI in CD4+ T cells) was measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 15 min (pSTAT studies; A, C) or 6 days (Bcl-2 studies; B, D) with IL-2 (left panels), IL-7 (middle panels) and IL-15 (right panels) (n = 3) and increasing concentrations (0.01, 0.1, and 1.0 μM) of ruxolitinib or tofacitinib (A-D). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. (-) indicates no cytokine was added. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: **p

Techniques Used: Blocking Assay, Expressing, Flow Cytometry, Cytometry, Isolation

30) Product Images from "PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C"

Article Title: PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.815829

Replication profile of the single- and double-PTAP viral molecular strains of the Indie-C1 molecular clone. A ). The viral strains of the panel are genetically identical except for the differences shown in the PTAP motifs. Both the PTAP motif sequences are compared with the HIV-1C consensus sequence in the sequence alignment. The dashes represent sequence deletion, and dots represent sequence identity. B , replication profile of VT1 ( top panels , CEM-CCR5 T-cells or CD8-depleted and activated PBMC of a healthy subject) and VT2 ( bottom panel , CEM-CCR5 T-cells) strains was compared with that of WT. The cells were infected with 100 TCID 50 units of single- or double-PTAP viral strain in duplicate wells, and the secretion of p24 into the medium was monitored up to 21–28 days. The data are presented as the mean of quadruplicate wells ± S.D. and representative of two independent experiments. A two-way ANOVA test was performed to determine the significance of the difference in proliferation between the viral strains. Comparable results were obtained with the PBMC of several other healthy donors. C, pairwise growth competition between single- and double-PTAP viral strains in HTA. The left panel shows the schematic representation of the ratios of the paired viruses used in the assay. Genomic DNA was extracted from the infected cells every week following infection and used in the assay as described under “Materials and methods.” The relative fitness values (depicted above the bars ) of the competing viral strains WT versus VT1 ( middle panel , days 7, 14, and 21) and WT versus VT2 ( right panel, days 7 and 14) in CEM-CCR5 cells are presented. A two-way ANOVA test was performed to determine the statistical significance. Data are representative of two independent experiments.
Figure Legend Snippet: Replication profile of the single- and double-PTAP viral molecular strains of the Indie-C1 molecular clone. A ). The viral strains of the panel are genetically identical except for the differences shown in the PTAP motifs. Both the PTAP motif sequences are compared with the HIV-1C consensus sequence in the sequence alignment. The dashes represent sequence deletion, and dots represent sequence identity. B , replication profile of VT1 ( top panels , CEM-CCR5 T-cells or CD8-depleted and activated PBMC of a healthy subject) and VT2 ( bottom panel , CEM-CCR5 T-cells) strains was compared with that of WT. The cells were infected with 100 TCID 50 units of single- or double-PTAP viral strain in duplicate wells, and the secretion of p24 into the medium was monitored up to 21–28 days. The data are presented as the mean of quadruplicate wells ± S.D. and representative of two independent experiments. A two-way ANOVA test was performed to determine the significance of the difference in proliferation between the viral strains. Comparable results were obtained with the PBMC of several other healthy donors. C, pairwise growth competition between single- and double-PTAP viral strains in HTA. The left panel shows the schematic representation of the ratios of the paired viruses used in the assay. Genomic DNA was extracted from the infected cells every week following infection and used in the assay as described under “Materials and methods.” The relative fitness values (depicted above the bars ) of the competing viral strains WT versus VT1 ( middle panel , days 7, 14, and 21) and WT versus VT2 ( right panel, days 7 and 14) in CEM-CCR5 cells are presented. A two-way ANOVA test was performed to determine the statistical significance. Data are representative of two independent experiments.

Techniques Used: Sequencing, Infection

31) Product Images from "Both HLA-B*57 and Plasma HIV RNA Levels Contribute to the HIV-Specific CD8+ T Cell Response in HIV Controllers"

Article Title: Both HLA-B*57 and Plasma HIV RNA Levels Contribute to the HIV-Specific CD8+ T Cell Response in HIV Controllers

Journal: Journal of Virology

doi: 10.1128/JVI.02098-13

CD8 + T cell responses were measured by IFN-γ ELISpot assay in HLA-B*57 + (black circles) and HLA-B*57 − (clear circles) subjects and are expressed as SFC/10 6 PBMC. (A) Total HIV-specific response. (B) Response directed against Env, Gag,
Figure Legend Snippet: CD8 + T cell responses were measured by IFN-γ ELISpot assay in HLA-B*57 + (black circles) and HLA-B*57 − (clear circles) subjects and are expressed as SFC/10 6 PBMC. (A) Total HIV-specific response. (B) Response directed against Env, Gag,

Techniques Used: Enzyme-linked Immunospot

32) Product Images from "The majority of the in vitro erythroid expansion potential resides in CD34− cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples"

Article Title: The majority of the in vitro erythroid expansion potential resides in CD34− cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples

Journal: Haematologica

doi: 10.3324/haematol.2009.019828

Differentiation of erythroblasts derived from total PBMC or CD34 + cells is comparable. (A) Proliferation and cell cycle arrest during differentiation of erythroblasts from 1x10 8 total PBMC (black squares) or 0.2x10 6 CD34 + cells purified from 1x10 8 total
Figure Legend Snippet: Differentiation of erythroblasts derived from total PBMC or CD34 + cells is comparable. (A) Proliferation and cell cycle arrest during differentiation of erythroblasts from 1x10 8 total PBMC (black squares) or 0.2x10 6 CD34 + cells purified from 1x10 8 total

Techniques Used: Derivative Assay, Purification

Expansion of erythroblasts from PBMC outperforms expansion from CD34 + cells purified from PBMC. (A and B) Dot plots showing forward and side scatter plots before (A, total PBMC) and after (B) CD34 + isolation. Gate-1 shows area where CD34 + cells reside
Figure Legend Snippet: Expansion of erythroblasts from PBMC outperforms expansion from CD34 + cells purified from PBMC. (A and B) Dot plots showing forward and side scatter plots before (A, total PBMC) and after (B) CD34 + isolation. Gate-1 shows area where CD34 + cells reside

Techniques Used: Purification, Isolation

33) Product Images from "Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection"

Article Title: Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006888

ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Infection, In Vitro, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Expressing, Flow Cytometry, Cytometry, Infection

ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.
Figure Legend Snippet: ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.

Techniques Used: Expressing, Activation Assay, Labeling, Plasmid Preparation, Real-time Polymerase Chain Reaction, Infection

ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure Legend Snippet: ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry

CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Flow Cytometry, Cytometry, Infection, Plasmid Preparation, Staining, Expressing

ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Magnetic Beads, Staining, Flow Cytometry, Cytometry, Expressing

34) Product Images from "Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins"

Article Title: Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01106

The functional responses of Treg cells are quantitatively sensitive to attenuated IL-2 signal. (A) Proliferation of Treg cells in response to select muteins from each class was measured in human PBMC assay and shown as percent Ki67-positive cells in CD25+ Foxp3+ CD4-gated Treg cells. Shown are data representative of four donors. (B) Proliferative responses measured as percent Ki67 positives of Treg, CD25+ Tconv, NK (CD56+ CD3–), and CD8 T cell gated subpopulations to increasing concentrations of wildtype or IL-2 muteins are compared in a total PBMC assay, for select muteins from each class. Shown are representative of data from four donors. (C) IL-2 mutein activity on induction of Foxp3 and CTLA4 in Treg cells are shown, represented as MFI of Foxp3 or CTLA4 in Foxp3-positive and CTLA4-positive gated Treg cell populations, respectively. (D) Treg phenotype before and after stimulation. Purified human Treg cells were stimulated with anti-CD3 and wildtype or IL-2 mutein at 66.7 nM for 3 days and analyzed for Foxp3 and CD25 expression. Day 0 unstimulated Treg cells analyzed at the same time to show baseline expression of these markers. Each color represents the mutein class or day 0: wildtype (black closed circles), class A (blue), class B (green), class C (red), and day 0 (black open circles). Shown are combined data from three different donors. ** represents p -value of 0.005 from a one-way ANOVA analysis. (E) Treg suppression assay. Purified human Treg cells that were pre-stimulated with anti-CD3 and IL-2 mutein were co-cultured with purified CD8 T cells from an unmatched donor at various ratios. CD8 T cells were stimulated with CD3+CD28 activation beads and Treg-mediated suppression was measured by activation marker induction on CD8 T cells on day 1. These values were converted to percent suppression, each point representing the average of values from two donors. Error bars indicate standard deviation.
Figure Legend Snippet: The functional responses of Treg cells are quantitatively sensitive to attenuated IL-2 signal. (A) Proliferation of Treg cells in response to select muteins from each class was measured in human PBMC assay and shown as percent Ki67-positive cells in CD25+ Foxp3+ CD4-gated Treg cells. Shown are data representative of four donors. (B) Proliferative responses measured as percent Ki67 positives of Treg, CD25+ Tconv, NK (CD56+ CD3–), and CD8 T cell gated subpopulations to increasing concentrations of wildtype or IL-2 muteins are compared in a total PBMC assay, for select muteins from each class. Shown are representative of data from four donors. (C) IL-2 mutein activity on induction of Foxp3 and CTLA4 in Treg cells are shown, represented as MFI of Foxp3 or CTLA4 in Foxp3-positive and CTLA4-positive gated Treg cell populations, respectively. (D) Treg phenotype before and after stimulation. Purified human Treg cells were stimulated with anti-CD3 and wildtype or IL-2 mutein at 66.7 nM for 3 days and analyzed for Foxp3 and CD25 expression. Day 0 unstimulated Treg cells analyzed at the same time to show baseline expression of these markers. Each color represents the mutein class or day 0: wildtype (black closed circles), class A (blue), class B (green), class C (red), and day 0 (black open circles). Shown are combined data from three different donors. ** represents p -value of 0.005 from a one-way ANOVA analysis. (E) Treg suppression assay. Purified human Treg cells that were pre-stimulated with anti-CD3 and IL-2 mutein were co-cultured with purified CD8 T cells from an unmatched donor at various ratios. CD8 T cells were stimulated with CD3+CD28 activation beads and Treg-mediated suppression was measured by activation marker induction on CD8 T cells on day 1. These values were converted to percent suppression, each point representing the average of values from two donors. Error bars indicate standard deviation.

Techniques Used: Functional Assay, PBMC Assay, Activity Assay, Purification, Expressing, Suppression Assay, Cell Culture, Activation Assay, Marker, Standard Deviation

35) Product Images from "Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify"

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

Journal: Cells

doi: 10.3390/cells7050045

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.
Figure Legend 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.

Techniques Used:

36) Product Images from "Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation"

Article Title: Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation

Journal: Immunobiology

doi: 10.1016/j.imbio.2018.08.007

Impact of Stimulation on Activation of PBMC vs Purified B cells from 5 control vs 5 viremic HIV-1-infected subjects.
Figure Legend Snippet: Impact of Stimulation on Activation of PBMC vs Purified B cells from 5 control vs 5 viremic HIV-1-infected subjects.

Techniques Used: Activation Assay, Purification, Infection

AID expression in PBMC by qRT-PCR in 14 control and 21 HIV-1-infected subjects.
Figure Legend Snippet: AID expression in PBMC by qRT-PCR in 14 control and 21 HIV-1-infected subjects.

Techniques Used: Expressing, Quantitative RT-PCR, Infection

37) Product Images from "Malaria-induced interferon-γ drives the expansion of Tbethi atypical memory B cells"

Article Title: Malaria-induced interferon-γ drives the expansion of Tbethi atypical memory B cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006576

Tfh-1 and Th1 cells are superior inducers of T-bet expression in naïve B cells. PBMCs of U.S. adults (n = 15) were FACs-sorted into naïve B cells (CD19+CD21+IgD+), cTfh cells (CD4+PD-1+CXCR5+CXCR3-), Tfh-1 cells (CD4+PD-1+CXCR5+CXCR3+) and Th-1 cells (CD4+PD-1+CXCR5-CXCR3+). Autologous naïve B cells were cultured for 2 days with each T cell subset in the presence of SEB, or with SEB alone. ( A ) T-bet MFI in B cells at 2 days of co-culture with T cell subsets or SEB alone. Representative histogram on right. ( B ) Percentage of T-bet hi B cells at 2 days of co-culture with T cell subsets or SEB alone. p values were determined by paired Student’s t test with Bonferroni adjustments. **** P
Figure Legend Snippet: Tfh-1 and Th1 cells are superior inducers of T-bet expression in naïve B cells. PBMCs of U.S. adults (n = 15) were FACs-sorted into naïve B cells (CD19+CD21+IgD+), cTfh cells (CD4+PD-1+CXCR5+CXCR3-), Tfh-1 cells (CD4+PD-1+CXCR5+CXCR3+) and Th-1 cells (CD4+PD-1+CXCR5-CXCR3+). Autologous naïve B cells were cultured for 2 days with each T cell subset in the presence of SEB, or with SEB alone. ( A ) T-bet MFI in B cells at 2 days of co-culture with T cell subsets or SEB alone. Representative histogram on right. ( B ) Percentage of T-bet hi B cells at 2 days of co-culture with T cell subsets or SEB alone. p values were determined by paired Student’s t test with Bonferroni adjustments. **** P

Techniques Used: Expressing, FACS, Cell Culture, Co-Culture Assay

Secreted cytokine profiles of T cell subset/naïve B cell co-cultures. PBMCs of U.S. adults (n = 5) were FACs-sorted into naïve B cells (CD19+CD21+IgD+), cTfh cells (CD4+PD-1+CXCR5+CXCR3-), Tfh-1 cells (CD4+PD-1+CXCR5+CXCR3+) and Th-1 cells (CD4+PD-1+CXCR5-CXCR3+). Autologous naïve B cells were cultured for 2 days with each T cell subset in the presence of SEB, or with SEB alone. Shown are cytokine concentrations in supernatants at 2 days. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P
Figure Legend Snippet: Secreted cytokine profiles of T cell subset/naïve B cell co-cultures. PBMCs of U.S. adults (n = 5) were FACs-sorted into naïve B cells (CD19+CD21+IgD+), cTfh cells (CD4+PD-1+CXCR5+CXCR3-), Tfh-1 cells (CD4+PD-1+CXCR5+CXCR3+) and Th-1 cells (CD4+PD-1+CXCR5-CXCR3+). Autologous naïve B cells were cultured for 2 days with each T cell subset in the presence of SEB, or with SEB alone. Shown are cytokine concentrations in supernatants at 2 days. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P

Techniques Used: FACS, Cell Culture

Blockade of P . falciparum -induced IFN-γ reduces T-bet expression in naïve B cells. T-bet and CD69 expression in naïve B cells (n = 5 U.S. adults) incubated with supernatants of iRBC-stimulated PBMCs plus anti-IgM in the presence of increasing concentrations of IFN-γ neutralizing antibodies ( A ) or IFN-γ receptor blocking antibodies ( B ) (representative histograms, right). ( C ) T-bet and CD69 expression in naïve B cells (n = 5 U.S. adults) incubated with supernatants of iRBC-stimulated PBMCs plus anti-IgM in the presence of IFN-γ neutralizing antibodies and IFN-γ receptor blocking antibodies separately or combined. Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P
Figure Legend Snippet: Blockade of P . falciparum -induced IFN-γ reduces T-bet expression in naïve B cells. T-bet and CD69 expression in naïve B cells (n = 5 U.S. adults) incubated with supernatants of iRBC-stimulated PBMCs plus anti-IgM in the presence of increasing concentrations of IFN-γ neutralizing antibodies ( A ) or IFN-γ receptor blocking antibodies ( B ) (representative histograms, right). ( C ) T-bet and CD69 expression in naïve B cells (n = 5 U.S. adults) incubated with supernatants of iRBC-stimulated PBMCs plus anti-IgM in the presence of IFN-γ neutralizing antibodies and IFN-γ receptor blocking antibodies separately or combined. Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P

Techniques Used: Expressing, Incubation, Blocking Assay

T-bet expression in naïve B cells in the presence of supernatant of iRBC-stimulated PBMCs or increasing concentrations of IFN-γ with or without BCR crosslinking. ( A ) T-bet expression in naïve B cells (n = 5 U.S. adults) with or without BCR crosslinking in the presence of supernatant of iRBC-stimulated PBMCs, or in the presence of increasing concentrations of recombinant human IFN-γ (right, representative histograms). ( B ) Intermediate and high T-bet expression in naïve B cells (n = 5 U.S. adults) after BCR cross-linking in the presence of iRBC-stimulated PBMC supernatant or rhIFN-γ (right, representative histograms). Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P
Figure Legend Snippet: T-bet expression in naïve B cells in the presence of supernatant of iRBC-stimulated PBMCs or increasing concentrations of IFN-γ with or without BCR crosslinking. ( A ) T-bet expression in naïve B cells (n = 5 U.S. adults) with or without BCR crosslinking in the presence of supernatant of iRBC-stimulated PBMCs, or in the presence of increasing concentrations of recombinant human IFN-γ (right, representative histograms). ( B ) Intermediate and high T-bet expression in naïve B cells (n = 5 U.S. adults) after BCR cross-linking in the presence of iRBC-stimulated PBMC supernatant or rhIFN-γ (right, representative histograms). Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P

Techniques Used: Expressing, Recombinant

Supernatants of PBMCs stimulated with P . falciparum -infected RBCs plus BCR cross-linking drive T-bet expression in B cells. ( A-C ) PBMCs of healthy U.S. adults (n = 5) were stimulated in vitro with P . falciparum -infected red blood cell (iRBC) lysate or uninfected red blood cell (uRBC) lysate for 3 days. The resulting supernatants or the iRBC lysate alone were transferred to PBMCs from the same U.S. adults (n = 5) in the presence of media alone, anti-IgM, anti-CD40, or both, followed by staining for T-bet, CD10, CD19 and IgD. ( A ) Fold change in T-bet MFI in stimulated naïve B cells relative to unstimulated naïve B cells (left, representative histograms). Fold change in percentage of T-bet intermediate (T-bet int ) and T-bet high (T-bet hi ) ( B ) naïve B cells and ( C ) memory B cells after BCR cross-linking with anti-IgM/G/A in the presence of media alone, uRBC/PBMC supernatant or iRBC/PBMC supernatant, relative to unstimulated cells. Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P
Figure Legend Snippet: Supernatants of PBMCs stimulated with P . falciparum -infected RBCs plus BCR cross-linking drive T-bet expression in B cells. ( A-C ) PBMCs of healthy U.S. adults (n = 5) were stimulated in vitro with P . falciparum -infected red blood cell (iRBC) lysate or uninfected red blood cell (uRBC) lysate for 3 days. The resulting supernatants or the iRBC lysate alone were transferred to PBMCs from the same U.S. adults (n = 5) in the presence of media alone, anti-IgM, anti-CD40, or both, followed by staining for T-bet, CD10, CD19 and IgD. ( A ) Fold change in T-bet MFI in stimulated naïve B cells relative to unstimulated naïve B cells (left, representative histograms). Fold change in percentage of T-bet intermediate (T-bet int ) and T-bet high (T-bet hi ) ( B ) naïve B cells and ( C ) memory B cells after BCR cross-linking with anti-IgM/G/A in the presence of media alone, uRBC/PBMC supernatant or iRBC/PBMC supernatant, relative to unstimulated cells. Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s t test with Bonferroni adjustments where appropriate. **** P

Techniques Used: Infection, Expressing, In Vitro, Staining

P . falciparum -induced IFN-γ correlates with T-bet expression in naïve B cells. PBMCs of healthy U.S. adults (n = 8) were stimulated in vitro with iRBC lysate or uRBC lysate for 3 days. Cytokine concentrations in the resulting supernatants were determined by a multiplex assay. Supernatants were transferred to PBMCs from the same U.S. adults (n = 8) in the presence of anti-IgM, followed by staining for T-bet, CD10, CD19 and IgD. Shown are correlations between fold changes in cytokine concentrations in supernatants of iRBC-stimulated vs. uRBC-stimulated PBMCs, and fold changes in T-bet expression in naïve B cells stimulated with supernatants of iRBC-stimulated vs. uRBC-stimulated PBMCs. Pearson correlation were used for correlative analyses. **** P
Figure Legend Snippet: P . falciparum -induced IFN-γ correlates with T-bet expression in naïve B cells. PBMCs of healthy U.S. adults (n = 8) were stimulated in vitro with iRBC lysate or uRBC lysate for 3 days. Cytokine concentrations in the resulting supernatants were determined by a multiplex assay. Supernatants were transferred to PBMCs from the same U.S. adults (n = 8) in the presence of anti-IgM, followed by staining for T-bet, CD10, CD19 and IgD. Shown are correlations between fold changes in cytokine concentrations in supernatants of iRBC-stimulated vs. uRBC-stimulated PBMCs, and fold changes in T-bet expression in naïve B cells stimulated with supernatants of iRBC-stimulated vs. uRBC-stimulated PBMCs. Pearson correlation were used for correlative analyses. **** P

Techniques Used: Expressing, In Vitro, Multiplex Assay, Staining

38) Product Images from "A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool"

Article Title: A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

Journal: Cells

doi: 10.3390/cells6040047

Th1, Th2 and Th17 lineage of CPI-reactive CD4 cells. Seventeen healthy donors were tested at 400,000 PBMC/well in IL-2, IL-4, IL-5, and IL-17 ELISPOT assays for the CPI-induced recall response. The percentage of PBMC donors displaying a positive cytokine recall response is shown.
Figure Legend Snippet: Th1, Th2 and Th17 lineage of CPI-reactive CD4 cells. Seventeen healthy donors were tested at 400,000 PBMC/well in IL-2, IL-4, IL-5, and IL-17 ELISPOT assays for the CPI-induced recall response. The percentage of PBMC donors displaying a positive cytokine recall response is shown.

Techniques Used: Enzyme-linked Immunospot

CPI (inactivated cytomegalo-, parainfluenza-, and influenza virions) recalls CD4 cells, CEF, (peptides of Cytomegalo-, Epstein-Barr and Flu-virus) recalls CD8 cells, respectively. Unseparated, as well as CD4- and CD8 cell-depleted PBMC of the same donor were tested for the CEF and CPI recall response, as specified. Representative well images are shown in ( A ). In ( B ) the percentage of response in the depleted PBMC fractions is shown relative to the unseparated PBMC.
Figure Legend Snippet: CPI (inactivated cytomegalo-, parainfluenza-, and influenza virions) recalls CD4 cells, CEF, (peptides of Cytomegalo-, Epstein-Barr and Flu-virus) recalls CD8 cells, respectively. Unseparated, as well as CD4- and CD8 cell-depleted PBMC of the same donor were tested for the CEF and CPI recall response, as specified. Representative well images are shown in ( A ). In ( B ) the percentage of response in the depleted PBMC fractions is shown relative to the unseparated PBMC.

Techniques Used:

Establishing the CD4/CD8 lineage of responding PBMC. Unseparated PBMC (open bars), PBMC of the same subject that have been depleted of CD8 cells (blue bars), or of CD4 cells (light blue bars) were tested at the same cell number (250,000) per well. The numbers of IFN-γ producing cells were measured by ELISPOT after the addition of the antigens specified on the x-axis. Percent response in the depleted PBMC fractions is shown relative to the unseparated PBMC. Note, for varicella, parainfluenza, mumps and influenza the light blue bars are barely/non visible due to the close to complete abrogation of Spot Forming Units (SFU) formation in CD4 cell-depleted PBMC.
Figure Legend Snippet: Establishing the CD4/CD8 lineage of responding PBMC. Unseparated PBMC (open bars), PBMC of the same subject that have been depleted of CD8 cells (blue bars), or of CD4 cells (light blue bars) were tested at the same cell number (250,000) per well. The numbers of IFN-γ producing cells were measured by ELISPOT after the addition of the antigens specified on the x-axis. Percent response in the depleted PBMC fractions is shown relative to the unseparated PBMC. Note, for varicella, parainfluenza, mumps and influenza the light blue bars are barely/non visible due to the close to complete abrogation of Spot Forming Units (SFU) formation in CD4 cell-depleted PBMC.

Techniques Used: Enzyme-linked Immunospot

39) Product Images from "Enriched Cd141+ DCs in the joint are transcriptionally distinct, activated, and contribute to joint pathogenesis"

Article Title: Enriched Cd141+ DCs in the joint are transcriptionally distinct, activated, and contribute to joint pathogenesis

Journal: JCI Insight

doi: 10.1172/jci.insight.95228

DC are enriched at the site of inflammation, where they reside in a semimature state. ( A ) Frequency of mDC and pDC in the peripheral blood of healthy control (HC) donors ( n = 7) and inflammatory arthritis (IA) patients ( n = 5). Frequency was calculated as percentage of mDC and pDC in the Lin – HLADR + population. mDC were gated as Lin – HLADR + CD11c + cells, and pDC were gated as Lin – HLADR + CD123 + cells. ( B ) The percentage of CD141 + mDC and CD1c + mDC was analyzed in HC and IA patients as a percentage of Lin – HLADR + CD11c + cells (mDC) ( n = 7). mDC were gated as described above, and CD141 + DC were gated on CD1c – CD141 + Clec9A + cells. CD1c + mDC were gated based on the expression of CD1c in the mDC population. ( C ) The median fluorescence intensity (MFI) of CD40 on mDC and pDC in HC and IA. ( D ) Identification of DC within the inflamed synovium. Dissociated IA synovial tissue (ST) and matched whole blood (WB) ( n = 6) were stained with a panel of fluorochrome-conjugated antibodies to identify DC. Cells were gated as CD45 + , HLA-DR + , and CD11c + , and the MFI of CD80 and CD40 was assessed. Percentage of mDC in ST and WB of IA patients as a percentage of CD45 + cells. ( E ) Representative histogram of CD80 and CD40 expression in WB (shaded area) and ST (clear line) and bar chart representing MFI. ( F ) Frequency and activation of mDC in synovial fluid and matched peripheral blood of IA patients ( n = 5). DC were gated as described in A . ( G ) The percentage of mDC in IA PBMC and IA synovial fluid mononuclear cells (SFMC) is presented as a percentage of Lin – HLA-DR + cells. ( H ) The MFI of CD40 on mDC is represented in IA PBMC and SFMC. Data were analyzed using Mann-Whitney U test. * P
Figure Legend Snippet: DC are enriched at the site of inflammation, where they reside in a semimature state. ( A ) Frequency of mDC and pDC in the peripheral blood of healthy control (HC) donors ( n = 7) and inflammatory arthritis (IA) patients ( n = 5). Frequency was calculated as percentage of mDC and pDC in the Lin – HLADR + population. mDC were gated as Lin – HLADR + CD11c + cells, and pDC were gated as Lin – HLADR + CD123 + cells. ( B ) The percentage of CD141 + mDC and CD1c + mDC was analyzed in HC and IA patients as a percentage of Lin – HLADR + CD11c + cells (mDC) ( n = 7). mDC were gated as described above, and CD141 + DC were gated on CD1c – CD141 + Clec9A + cells. CD1c + mDC were gated based on the expression of CD1c in the mDC population. ( C ) The median fluorescence intensity (MFI) of CD40 on mDC and pDC in HC and IA. ( D ) Identification of DC within the inflamed synovium. Dissociated IA synovial tissue (ST) and matched whole blood (WB) ( n = 6) were stained with a panel of fluorochrome-conjugated antibodies to identify DC. Cells were gated as CD45 + , HLA-DR + , and CD11c + , and the MFI of CD80 and CD40 was assessed. Percentage of mDC in ST and WB of IA patients as a percentage of CD45 + cells. ( E ) Representative histogram of CD80 and CD40 expression in WB (shaded area) and ST (clear line) and bar chart representing MFI. ( F ) Frequency and activation of mDC in synovial fluid and matched peripheral blood of IA patients ( n = 5). DC were gated as described in A . ( G ) The percentage of mDC in IA PBMC and IA synovial fluid mononuclear cells (SFMC) is presented as a percentage of Lin – HLA-DR + cells. ( H ) The MFI of CD40 on mDC is represented in IA PBMC and SFMC. Data were analyzed using Mann-Whitney U test. * P

Techniques Used: IA, Expressing, Fluorescence, Western Blot, Staining, Activation Assay, MANN-WHITNEY

40) Product Images from "Buprenorphine decreases CCL2-mediated migration of CD14+CD16+ monocytes"

Article Title: Buprenorphine decreases CCL2-mediated migration of CD14+CD16+ monocytes

Journal: Journal of leukocyte biology

doi: 10.1002/JLB.3HI0118-015R

Mature monocytes express surface MOR and KOR. CD14 + monocytes were isolated from PBMC using magnetic beads and cultured non-adherently for three days with MCSF-1. After culture, more than 70% of the monocytes were CD14 + CD16 + . Surface MOR and KOR on mature
Figure Legend Snippet: Mature monocytes express surface MOR and KOR. CD14 + monocytes were isolated from PBMC using magnetic beads and cultured non-adherently for three days with MCSF-1. After culture, more than 70% of the monocytes were CD14 + CD16 + . Surface MOR and KOR on mature

Techniques Used: Isolation, Magnetic Beads, Cell Culture

CD14 + CD16 + monocytes have higher expression of MOR and KOR as compared to CD14 + CD16 − monocytes in PBMC from uninfected people. PBMC were isolated from peripheral blood by Ficoll gradient centrifugation. Cells were analyzed by flow cytometry for
Figure Legend Snippet: CD14 + CD16 + monocytes have higher expression of MOR and KOR as compared to CD14 + CD16 − monocytes in PBMC from uninfected people. PBMC were isolated from peripheral blood by Ficoll gradient centrifugation. Cells were analyzed by flow cytometry for

Techniques Used: Expressing, Isolation, Gradient Centrifugation, Flow Cytometry, Cytometry

Related Articles

Selection:

Article Title: The CD8+ T-Cell Response to an Epstein-Barr Virus-Related Gammaherpesvirus Infecting Rhesus Macaques Provides Evidence for Immune Evasion by the EBNA-1 Homologue
Article Snippet: .. CD4+ or CD8+ T lymphocytes were selectively depleted from PBMC by immunomagnetic selection (StemCell Technologies). .. Briefly, 107 PBMC in 1 ml of phosphate-buffered saline were incubated at room temperature for 15 min with 20 μl of either anti-dextran/anti-CD4 or anti-dextran/anti-CD8 tetrameric antibody cocktail followed by a 15-min incubation at room temperature with 60 μl of dextran coated magnetic nanoparticle colloid.

Article Title: Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation
Article Snippet: .. To determine the ability of stimuli to activate B cells and induce AID production in the absence of T and accessory cell help, we purified B cells from approximately 50 million PBMC each from 5 additional viremic HIV-1-infected subjects (age 45–48 years; median 248 CD4+ T cells/μL (range 90–435); plasma HIV-1 RNA 136,000–946,000 copies/mL) and 5 age- and gender-matched HIV-1-seronegative control subjects with a negative selection kit (STEMCELL Technologies, Vancouver, BC). .. The median yield of B cells was 2.5 × 106 (range 1.85–3.1 × 106 ) and the median purity was 96.4% (range 92.4–98.5%) as measured by flow cytometry ( ).

Article Title: Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses
Article Snippet: .. Briefly, PBMCs were obtained by leukapheresis, and CD4+ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions and were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin 2 (rIL-2) (100 U/ml). .. Proviral constructs.

Magnetic Beads:

Article Title: Adenovirally-Induced Polyfunctional T Cells Do Not Necessarily Recognize the Infected Target: Lessons from a Phase I Trial of the AERAS-402 Vaccine
Article Snippet: .. Second, CD8+ T cells were positively selected from PBMC using magnetic beads (Stemcell Technologies) such that > 97% of the cell population were CD8+ T cells. .. These CD8+ T cells were used as a source of responder T cells and tested in duplicate at a cell concentration of 250,000 cells per well.

Isolation:

Article Title: Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus
Article Snippet: .. PBMCs were generated from human buffy coats and CD19+ B cells were isolated from PBMC using the EasySep Human B Cell Enrichment Cocktail protocol (STEMCELL Technologies, Vancouver, British Columbia, Canada). .. The human primary B cell differentiation protocol was adapted from previously described methods.

Article Title: Plasma and Immunoglobulin G Galactosylation Associate with HIV Persistence During Antiretroviral Therapy
Article Snippet: .. CD4+ T cells were isolated from PBMCS using EasySep Human CD4+ T cell enrichment kit (Stemcell Technologies, Vancouver, British Columbia, Canada). .. Cellular RNA and DNA from total unfractionated PBMCs and isolated CD4+ T cells were purified using the AllPrep DNA/RNA kit (Qiagen, Ventura CA) as specified by the manufacturer, quantified using a Nanodrop (ND-1000) spectrophotometer and normalized to cell equivalents by qPCR using human genomic TERT for DNA and RPLP0 expression for RNA (Life Technologies, Grand Island NY).

Purification:

Article Title: Effective B cell Activation in vitro during Viremic HIV-1 Infection with Surrogate T cell Stimulation
Article Snippet: .. To determine the ability of stimuli to activate B cells and induce AID production in the absence of T and accessory cell help, we purified B cells from approximately 50 million PBMC each from 5 additional viremic HIV-1-infected subjects (age 45–48 years; median 248 CD4+ T cells/μL (range 90–435); plasma HIV-1 RNA 136,000–946,000 copies/mL) and 5 age- and gender-matched HIV-1-seronegative control subjects with a negative selection kit (STEMCELL Technologies, Vancouver, BC). .. The median yield of B cells was 2.5 × 106 (range 1.85–3.1 × 106 ) and the median purity was 96.4% (range 92.4–98.5%) as measured by flow cytometry ( ).

Article Title: Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses
Article Snippet: .. Briefly, PBMCs were obtained by leukapheresis, and CD4+ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions and were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin 2 (rIL-2) (100 U/ml). .. Proviral constructs.

Generated:

Article Title: Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus
Article Snippet: .. PBMCs were generated from human buffy coats and CD19+ B cells were isolated from PBMC using the EasySep Human B Cell Enrichment Cocktail protocol (STEMCELL Technologies, Vancouver, British Columbia, Canada). .. The human primary B cell differentiation protocol was adapted from previously described methods.

Recombinant:

Article Title: Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses
Article Snippet: .. Briefly, PBMCs were obtained by leukapheresis, and CD4+ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions and were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin 2 (rIL-2) (100 U/ml). .. Proviral constructs.

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    STEMCELL Technologies Inc pbmcs
    Susceptibility of cells infected with different parental and reporter IMCs to ADCC mediated by A32 and HIV + sera. Primary <t>CD4</t> + T cells infected with IMCs expressing different Env strains (CH58, CH40, CH77, CH505, YU2, SF162, and BaL) and expressing or not the LucR reporter gene were used as target cells, and autologous <t>PBMCs</t> were used as effector cells in a well-established FACS-based ADCC assay ( 1 , 9 , 10 , 60 , 84 ). (A and C) Percentages of ADCC-mediated killing obtained with A32 or HIV + sera from 5 HIV-1-infected individuals obtained in at least 5 independent experiments. (B and D) Correlations between levels of cell surface CD4 and ADCC responses mediated by A32 or HIV + sera, using the same panel of IMC constructs, detected with the FACS-based ADCC assay. Statistical significance was tested using an unpaired t test (A and C), a Pearson correlation test (B), or a Spearman rank correlation test (D) (*, P
    Pbmcs, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 94/100, based on 511 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmcs/product/STEMCELL Technologies Inc
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    STEMCELL Technologies Inc human b lymphocytes peripheral blood mononuclear cells pbmcs
    Endogenous expression of C3 is very low in <t>human</t> B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, <t>PBMCs,</t> and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.
    Human B Lymphocytes Peripheral Blood Mononuclear Cells Pbmcs, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    STEMCELL Technologies Inc monocyte derived macrophages pbmcs
    SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with VSV-G pseudotyped HIV-1 and SIV vectors. a Schematic showing experimental timeline. Briefly, <t>CD14+</t> monocytes were isolated from human donor <t>PBMCs</t> using positive selection with magnetic beads. Monocytes were differentiated into MDMs and on day 7 were electroporated with siRNA and plated at 1.5 × 10 4 MDMs per well in a 48 well plate. After 96 h, protein knockdown was confirmed by immunoblot and cells were infected with 100 μl (corresponding to 164 ng p24 HIV-1 and 234 ng p27 SIV viral vectors) of the indicated virus. Luciferase and cell viability measurements were determined at 24 h post-infection. b Representative immunoblot showing SULT1A1 knockdown 96 h post transfection with SULT1A1 siRNAs (1–3) and AllStars Negative siRNA Control (ASN), Qiagen. SULT1A1 expression is compared to endogenous Ku86 used as a loading control. Results from one representative donor are shown. SULT1A1 expression was generally decreased by 70–80 % with siRNA treatment compared to the control ASN siRNA (as shown in Fig. 2c, middle panel). c HIV-1 luciferase reporter expression ( left panel ), SULT1A1 protein expression ( middle panel ), and cell viability of MDMs ( right panel ) were measured 24 h after infection with the VSV-G pseudotyped NL43-Luc HIV-1 vector. Results shown are from 6 donors assayed twice. All values were compared to ASN control. d SIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression ( middle panel ), and cell viability ( right panel ) for MDMs 24 h post infection with VSV-G-pseudotyped SIVagm-Luc. Mean and SD shown, *** p
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    Susceptibility of cells infected with different parental and reporter IMCs to ADCC mediated by A32 and HIV + sera. Primary CD4 + T cells infected with IMCs expressing different Env strains (CH58, CH40, CH77, CH505, YU2, SF162, and BaL) and expressing or not the LucR reporter gene were used as target cells, and autologous PBMCs were used as effector cells in a well-established FACS-based ADCC assay ( 1 , 9 , 10 , 60 , 84 ). (A and C) Percentages of ADCC-mediated killing obtained with A32 or HIV + sera from 5 HIV-1-infected individuals obtained in at least 5 independent experiments. (B and D) Correlations between levels of cell surface CD4 and ADCC responses mediated by A32 or HIV + sera, using the same panel of IMC constructs, detected with the FACS-based ADCC assay. Statistical significance was tested using an unpaired t test (A and C), a Pearson correlation test (B), or a Spearman rank correlation test (D) (*, P

    Journal: Journal of Virology

    Article Title: Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses

    doi: 10.1128/JVI.00484-18

    Figure Lengend Snippet: Susceptibility of cells infected with different parental and reporter IMCs to ADCC mediated by A32 and HIV + sera. Primary CD4 + T cells infected with IMCs expressing different Env strains (CH58, CH40, CH77, CH505, YU2, SF162, and BaL) and expressing or not the LucR reporter gene were used as target cells, and autologous PBMCs were used as effector cells in a well-established FACS-based ADCC assay ( 1 , 9 , 10 , 60 , 84 ). (A and C) Percentages of ADCC-mediated killing obtained with A32 or HIV + sera from 5 HIV-1-infected individuals obtained in at least 5 independent experiments. (B and D) Correlations between levels of cell surface CD4 and ADCC responses mediated by A32 or HIV + sera, using the same panel of IMC constructs, detected with the FACS-based ADCC assay. Statistical significance was tested using an unpaired t test (A and C), a Pearson correlation test (B), or a Spearman rank correlation test (D) (*, P

    Article Snippet: Briefly, PBMCs were obtained by leukapheresis, and CD4+ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions and were activated with phytohemagglutinin-L (10 μg/ml) for 48 h and then maintained in RPMI 1640 complete medium supplemented with recombinant interleukin 2 (rIL-2) (100 U/ml).

    Techniques: Infection, Expressing, FACS, ADCC Assay, Construct

    Endogenous expression of C3 is very low in human B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, PBMCs, and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

    doi: 10.3389/fimmu.2019.00493

    Figure Lengend Snippet: Endogenous expression of C3 is very low in human B cells. (A) RNA was isolated from malignant B cell lines and blood-derived B cells, reverse-transcribed and analyzed for C3 expression by qPCR. As positive control, blood-derived T cells, PBMCs, and total, liver tissue RNA were used. Data were normalized to the housekeeping HPRT gene and are shown as mean 2-dCt values with SD of three independent experiments. (B) The presence of full length C3 mRNA was confirmed by primer pairs, covering the whole region of human C3 coding sequence. As positive control, liver tissue RNA was used. Data shown are representative of three independent experiments. Numbers indicate DNA length in base pair (bp). The start positions of forward (Fw) and reverse (Rv) primers are shown under the gel picture. (C) Western blot results analyzing endogenous C3 expression of human B cells. Lysates prepared from the human B cell lines, Raji and Namalwa and blood-derived B cells and PBMCs were analyzed for the presence of C3 by Western blot with the goat polyclonal anti-C3 antibody from Quidel under non reducing and reducing conditions. As positive control, lysate of Raji cells incubated with 10% NHS in EDTA-GVB buffer was used. Results shown are representative of five independent experiments.

    Article Snippet: Isolation of Human B Lymphocytes Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Stemcell Technologies) density gradient centrifugation from superfluous buffy coat obtained from the Medical Service (Clinical Immunology and Transfusion Medicine, Lund) according to standard procedures ( ) and permit granted by the local ethics committee of Lund.

    Techniques: Expressing, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Positive Control, Sequencing, Western Blot, Incubation

    SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with VSV-G pseudotyped HIV-1 and SIV vectors. a Schematic showing experimental timeline. Briefly, CD14+ monocytes were isolated from human donor PBMCs using positive selection with magnetic beads. Monocytes were differentiated into MDMs and on day 7 were electroporated with siRNA and plated at 1.5 × 10 4 MDMs per well in a 48 well plate. After 96 h, protein knockdown was confirmed by immunoblot and cells were infected with 100 μl (corresponding to 164 ng p24 HIV-1 and 234 ng p27 SIV viral vectors) of the indicated virus. Luciferase and cell viability measurements were determined at 24 h post-infection. b Representative immunoblot showing SULT1A1 knockdown 96 h post transfection with SULT1A1 siRNAs (1–3) and AllStars Negative siRNA Control (ASN), Qiagen. SULT1A1 expression is compared to endogenous Ku86 used as a loading control. Results from one representative donor are shown. SULT1A1 expression was generally decreased by 70–80 % with siRNA treatment compared to the control ASN siRNA (as shown in Fig. 2c, middle panel). c HIV-1 luciferase reporter expression ( left panel ), SULT1A1 protein expression ( middle panel ), and cell viability of MDMs ( right panel ) were measured 24 h after infection with the VSV-G pseudotyped NL43-Luc HIV-1 vector. Results shown are from 6 donors assayed twice. All values were compared to ASN control. d SIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression ( middle panel ), and cell viability ( right panel ) for MDMs 24 h post infection with VSV-G-pseudotyped SIVagm-Luc. Mean and SD shown, *** p

    Journal: Virology Journal

    Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

    doi: 10.1186/s12985-016-0491-9

    Figure Lengend Snippet: SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with VSV-G pseudotyped HIV-1 and SIV vectors. a Schematic showing experimental timeline. Briefly, CD14+ monocytes were isolated from human donor PBMCs using positive selection with magnetic beads. Monocytes were differentiated into MDMs and on day 7 were electroporated with siRNA and plated at 1.5 × 10 4 MDMs per well in a 48 well plate. After 96 h, protein knockdown was confirmed by immunoblot and cells were infected with 100 μl (corresponding to 164 ng p24 HIV-1 and 234 ng p27 SIV viral vectors) of the indicated virus. Luciferase and cell viability measurements were determined at 24 h post-infection. b Representative immunoblot showing SULT1A1 knockdown 96 h post transfection with SULT1A1 siRNAs (1–3) and AllStars Negative siRNA Control (ASN), Qiagen. SULT1A1 expression is compared to endogenous Ku86 used as a loading control. Results from one representative donor are shown. SULT1A1 expression was generally decreased by 70–80 % with siRNA treatment compared to the control ASN siRNA (as shown in Fig. 2c, middle panel). c HIV-1 luciferase reporter expression ( left panel ), SULT1A1 protein expression ( middle panel ), and cell viability of MDMs ( right panel ) were measured 24 h after infection with the VSV-G pseudotyped NL43-Luc HIV-1 vector. Results shown are from 6 donors assayed twice. All values were compared to ASN control. d SIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression ( middle panel ), and cell viability ( right panel ) for MDMs 24 h post infection with VSV-G-pseudotyped SIVagm-Luc. Mean and SD shown, *** p

    Article Snippet: Monocyte-derived macrophages PBMCs were thawed in RPMI, washed, and CD14+ monocytes were isolated using CD14 positive selection magnetic cell sorting according to the manufacturer’s instructions (Easy Sep Human CD14 Positive Selection kit, StemCell Technologies).

    Techniques: Expressing, Infection, Isolation, Selection, Magnetic Beads, Luciferase, Transfection, Plasmid Preparation

    SULT1A1 is highly expressed in primary human monocyte-derived macrophages (MDMs). a The cellular sulfonation pathway. The first step of the cellular sulfonation pathway involves import through a sulfate transporter of a sulfate ion that is then used as a substrate by either 3′-phosphoadenosine-5′phosphosulfate (PAPS) synthetase enzymes PAPSS1 or PAPSS2. These proteins catalyze two enzymatic steps to generate PAPS, the high-energy universal sulfonate-donor from sulfate and two molcules of ATP. PAPS can be transported across the Golgi membrane and used by the Golgi sulfotransferases to generate sulfonated proteins, glycoproteins, glycoproteins, glycolipids, and proteoglycans. Alternatively, PAPS can be used by cytosolic sulfotransferases (SULTS) to sulfonate small molecules such as hormones, neurotransmitters, and xenobiotics. b Human CD4+ T cells and CD14+ monocytes were isolated from donor PBMCs by magnetic bead isolation. Resting CD4+ T cells were lysed directly after separation, and the remaining CD4+ T cells were activated using CD3/CD28 beads for three days. Monocytes were cultured for 7 days in the presence of 20 ng/ml M-CSF, were lysed, subjected to gel electrophoresis, and immunoblotting was performed to detect SULT1A1 or the loading control Ku86 protein

    Journal: Virology Journal

    Article Title: Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

    doi: 10.1186/s12985-016-0491-9

    Figure Lengend Snippet: SULT1A1 is highly expressed in primary human monocyte-derived macrophages (MDMs). a The cellular sulfonation pathway. The first step of the cellular sulfonation pathway involves import through a sulfate transporter of a sulfate ion that is then used as a substrate by either 3′-phosphoadenosine-5′phosphosulfate (PAPS) synthetase enzymes PAPSS1 or PAPSS2. These proteins catalyze two enzymatic steps to generate PAPS, the high-energy universal sulfonate-donor from sulfate and two molcules of ATP. PAPS can be transported across the Golgi membrane and used by the Golgi sulfotransferases to generate sulfonated proteins, glycoproteins, glycoproteins, glycolipids, and proteoglycans. Alternatively, PAPS can be used by cytosolic sulfotransferases (SULTS) to sulfonate small molecules such as hormones, neurotransmitters, and xenobiotics. b Human CD4+ T cells and CD14+ monocytes were isolated from donor PBMCs by magnetic bead isolation. Resting CD4+ T cells were lysed directly after separation, and the remaining CD4+ T cells were activated using CD3/CD28 beads for three days. Monocytes were cultured for 7 days in the presence of 20 ng/ml M-CSF, were lysed, subjected to gel electrophoresis, and immunoblotting was performed to detect SULT1A1 or the loading control Ku86 protein

    Article Snippet: Monocyte-derived macrophages PBMCs were thawed in RPMI, washed, and CD14+ monocytes were isolated using CD14 positive selection magnetic cell sorting according to the manufacturer’s instructions (Easy Sep Human CD14 Positive Selection kit, StemCell Technologies).

    Techniques: Derivative Assay, Papanicolaou Stain, Isolation, Cell Culture, Nucleic Acid Electrophoresis