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    Veri Cells CD4 Low PBMC
    Description:
    Veri Cells CD4 Low PBMC Apps FC ICFC Size 25 tests
    Catalog Number:
    425601
    Price:
    185
    Category:
    Buffer Solution Chemical
    Applications:
    FC, ICFC
    Size:
    25 tests
    Quantity:
    1
    Preparation:
    The Veri Cells CD4 Low PBMC Kit is prepared from lyophilized human peripheral blood mononuclear cells and a vial of reconstitution buffer
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    BioLegend veri cells cd4 low pbmc
    Veri Cells CD4 Low PBMC
    Veri Cells CD4 Low PBMC Apps FC ICFC Size 25 tests
    https://www.bioz.com/result/veri cells cd4 low pbmc/product/BioLegend
    Average 96 stars, based on 1 article reviews
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    veri cells cd4 low pbmc - by Bioz Stars, 2021-03
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    1) Product Images from "Inhibition of ITK differentiates GVT and GVHD in allo-HSCT"

    Article Title: Inhibition of ITK differentiates GVT and GVHD in allo-HSCT

    Journal: bioRxiv

    doi: 10.1101/2020.07.15.204693

    Novel peptide SLP76pTYR specifically targets ITK signaling and enhances Treg cell development. (A) Total T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for CD4 + and FoxP3 + . n=3 one representative experiment shown. (B) Quantification of three experiments as in (A). (C) Cell lysates from T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for phosphorylation of ITK, PLCγ1 and ERK. Lysates were also examined for phosphorylation of PI3K, mTOR, P13K, and AKT. n=3 one representative experiment shown. ( D ) Western blots were normalized to β−Actin and quantitative data from 3 independent experiments presented. ( E ) Human GVHD patients’ samples were cells stimulated in the presence of SLP76pTYR or vehicle only. T cell lysates were used in western blots for analysis of pPLCγ, pERK, pAKT, pMTOR, and (H) three experiments were quantitated and normalized to β−Actin. n=3, one representative experiment shown. ( G ) Primary human T cells purified from PBMC were stimulated with CD3 and CD28 for 5 hours in the presence of vehicle alone or SLP76pTYR in the presence BFA. Intracellular IFN-γ and TNF-α expression by CD8 + and CD4 + T cells was determined by flow cytometry. For statistical analysis we used two-way ANOVA and Student’s t test. P values are presented.
    Figure Legend Snippet: Novel peptide SLP76pTYR specifically targets ITK signaling and enhances Treg cell development. (A) Total T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for CD4 + and FoxP3 + . n=3 one representative experiment shown. (B) Quantification of three experiments as in (A). (C) Cell lysates from T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for phosphorylation of ITK, PLCγ1 and ERK. Lysates were also examined for phosphorylation of PI3K, mTOR, P13K, and AKT. n=3 one representative experiment shown. ( D ) Western blots were normalized to β−Actin and quantitative data from 3 independent experiments presented. ( E ) Human GVHD patients’ samples were cells stimulated in the presence of SLP76pTYR or vehicle only. T cell lysates were used in western blots for analysis of pPLCγ, pERK, pAKT, pMTOR, and (H) three experiments were quantitated and normalized to β−Actin. n=3, one representative experiment shown. ( G ) Primary human T cells purified from PBMC were stimulated with CD3 and CD28 for 5 hours in the presence of vehicle alone or SLP76pTYR in the presence BFA. Intracellular IFN-γ and TNF-α expression by CD8 + and CD4 + T cells was determined by flow cytometry. For statistical analysis we used two-way ANOVA and Student’s t test. P values are presented.

    Techniques Used: Western Blot, Purification, Expressing, Flow Cytometry

    2) Product Images from "Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth"

    Article Title: Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth

    Journal: Vaccines

    doi: 10.3390/vaccines8030360

    Functionality of mRNA-elicited CD4 + and CD8 + T cells. Groups of BALB/cJ mice were immunized with monovalent R1, R2, RIII and RIV or multivalent R1 + 2, R1 + R2 + RIII and R1 + R2 + RIII + RIV vaccines at weeks 0 and 4. Each mRNA was administered intramuscularly at a 3-µg dose resulting in a total of 6-, 9- and 12-µg amounts for the combined vaccines, respectively. The frequencies of responding PBMCs and/or splenocytes were determined using combined pool of 28 peptides corresponding to 7 defined H-2 d class I epitopes and their 4 variants present in the vaccine ( Table 1 ). Multicolor flow cytometry analysis was performed to assess the functional phenotypes of vaccine-elicited CD8 + and CD4 + T cells in terms of CD107a, IFN-γ, IL-2 and TNF-α expression at 1 ( a ) and 5 ( b ) weeks after the homologous boost. The same cytokine legend as in (a) applies to (b). Specific T-cell frequencies are shown as group median (column) and individual animal values ( n = 6). ( c ) Group median ( n = 6) proportions of CD8 + and CD4 + T cells expressing 1 (black), 2 (light grey), 3 (dark grey) or 4 (white) functions are depicted using pie charts. See Figure S2 for the gating strategy. (a and b) Groups were compared using Kruskal–Wallis test with Dunn’s multiple comparison correction and significant two-tailed p values are indicated by an asterisk: * p
    Figure Legend Snippet: Functionality of mRNA-elicited CD4 + and CD8 + T cells. Groups of BALB/cJ mice were immunized with monovalent R1, R2, RIII and RIV or multivalent R1 + 2, R1 + R2 + RIII and R1 + R2 + RIII + RIV vaccines at weeks 0 and 4. Each mRNA was administered intramuscularly at a 3-µg dose resulting in a total of 6-, 9- and 12-µg amounts for the combined vaccines, respectively. The frequencies of responding PBMCs and/or splenocytes were determined using combined pool of 28 peptides corresponding to 7 defined H-2 d class I epitopes and their 4 variants present in the vaccine ( Table 1 ). Multicolor flow cytometry analysis was performed to assess the functional phenotypes of vaccine-elicited CD8 + and CD4 + T cells in terms of CD107a, IFN-γ, IL-2 and TNF-α expression at 1 ( a ) and 5 ( b ) weeks after the homologous boost. The same cytokine legend as in (a) applies to (b). Specific T-cell frequencies are shown as group median (column) and individual animal values ( n = 6). ( c ) Group median ( n = 6) proportions of CD8 + and CD4 + T cells expressing 1 (black), 2 (light grey), 3 (dark grey) or 4 (white) functions are depicted using pie charts. See Figure S2 for the gating strategy. (a and b) Groups were compared using Kruskal–Wallis test with Dunn’s multiple comparison correction and significant two-tailed p values are indicated by an asterisk: * p

    Techniques Used: Mouse Assay, Flow Cytometry, Functional Assay, Expressing, Two Tailed Test

    3) Product Images from "A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)"

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-01856-8

    GMCSF-MOG was superior to GMCSF-NFM for eliciting persistence of a CD44 high Tcon phenotype. a – d 2D2-FIG mice were vaccinated with 4 nmol of GMCSF-MOG ( n = 5), 4 nmol of GMCSF-NFM ( n = 5), or with saline ( n = 5). PBMCs were analyzed on days 5, 12, and 19 for CD3, CD4, CD44, and FOXP3 expression. a – c The day 0 time point was derived from the CD44 expression of CD3 + CD4 + T cells from naïve 2D2-FIG mice ( n = 23). Shown are percentages of a CD44 high T cells, b CD44 high Tcons, and c CD44 high Tregs of gated CD3 + CD4 + T cells on days 0, 5, 12, and 19. Shown in d are the percentage of CD62L low T cells of gated CD44 high CD3 + CD4 + T cells on day 5, 12, and 19. Shown in e are representative dot plots of CD44 ( y -axis) and FOXP3 ( x -axis) expression of CD4 + CD3 + T cells on day 5. These data are representative of three independent experiments. Statistical significance was analyzed using a one-way ANOVA. Shown in a , b and d are significant differences between the groups GMCSF-MOG, GMCSF-NFM, and saline, and c statistical differences comparing GMCSF-MOG treatment to both GMCSF-NFM and saline. ( *p
    Figure Legend Snippet: GMCSF-MOG was superior to GMCSF-NFM for eliciting persistence of a CD44 high Tcon phenotype. a – d 2D2-FIG mice were vaccinated with 4 nmol of GMCSF-MOG ( n = 5), 4 nmol of GMCSF-NFM ( n = 5), or with saline ( n = 5). PBMCs were analyzed on days 5, 12, and 19 for CD3, CD4, CD44, and FOXP3 expression. a – c The day 0 time point was derived from the CD44 expression of CD3 + CD4 + T cells from naïve 2D2-FIG mice ( n = 23). Shown are percentages of a CD44 high T cells, b CD44 high Tcons, and c CD44 high Tregs of gated CD3 + CD4 + T cells on days 0, 5, 12, and 19. Shown in d are the percentage of CD62L low T cells of gated CD44 high CD3 + CD4 + T cells on day 5, 12, and 19. Shown in e are representative dot plots of CD44 ( y -axis) and FOXP3 ( x -axis) expression of CD4 + CD3 + T cells on day 5. These data are representative of three independent experiments. Statistical significance was analyzed using a one-way ANOVA. Shown in a , b and d are significant differences between the groups GMCSF-MOG, GMCSF-NFM, and saline, and c statistical differences comparing GMCSF-MOG treatment to both GMCSF-NFM and saline. ( *p

    Techniques Used: Mouse Assay, Expressing, Derivative Assay

    GMCSF-MOG preferentially expanded Tregs from a memory T cell pool. On day − 1, a CD45.1-2D2-FIG mixed Treg and Tcon line (40% Tregs and 60% Tcons) was injected intravenously (1.25 × 10 6 cells) into CD45.2 2D2-FIG- Rag1 −/− mice ( n = 3). On day 0, 2D2-FIG- Rag1 −/− recipient mice were vaccinated with 4 nmol of GMCSF-MOG or 4 nmol GM-CSF + 4 nmol MOG 35–55 . PBMCs were analyzed on day 4 for CD3, CD4, CD45.1, CD44, and FOXP3. Shown are analyses of donor CD45.1 CD3 + CD4 + T cells including a representative dot plots of GMCSF-MOG and “GM-CSF + MOG”-treated mice for CD44 ( y -axis) and FOXP3 expression ( x -axis) together with ( b , d , f ) cell numbers per microliter of blood and ( c , e , g ) percentages of designated cell populations. Statistical significance was analyzed by use of a two-tailed t test. ( *p
    Figure Legend Snippet: GMCSF-MOG preferentially expanded Tregs from a memory T cell pool. On day − 1, a CD45.1-2D2-FIG mixed Treg and Tcon line (40% Tregs and 60% Tcons) was injected intravenously (1.25 × 10 6 cells) into CD45.2 2D2-FIG- Rag1 −/− mice ( n = 3). On day 0, 2D2-FIG- Rag1 −/− recipient mice were vaccinated with 4 nmol of GMCSF-MOG or 4 nmol GM-CSF + 4 nmol MOG 35–55 . PBMCs were analyzed on day 4 for CD3, CD4, CD45.1, CD44, and FOXP3. Shown are analyses of donor CD45.1 CD3 + CD4 + T cells including a representative dot plots of GMCSF-MOG and “GM-CSF + MOG”-treated mice for CD44 ( y -axis) and FOXP3 expression ( x -axis) together with ( b , d , f ) cell numbers per microliter of blood and ( c , e , g ) percentages of designated cell populations. Statistical significance was analyzed by use of a two-tailed t test. ( *p

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

    The high-efficiency GMCSF-NFM vaccine induced memory Tcon responses in 2D2-FIG mice. On day 0, 2D2-FIG mice were SC injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. On day 8, splenocytes were harvested from vaccinated mice, and CD4 + T cells were purified and analyzed for a Vβ11 ( y -axis), b CD44 ( y -axis), and a , b FOXP3 expression ( x -axis). To assess generality of these findings ( c , d ), data from seven controlled experiments (analysis of PBMC ranging from day 4 to day 7) were pooled to assess 2D2-FIG mice vaccinated SC with saline ( n = 24), 4 nmol of GMCSF-MOG ( n = 24), or 4 nmol of GMCSF-NFM ( n = 25) for total Treg percentages ( c ) and CD44 + Treg percentages ( d ) among CD4 + T cells. a , b , e , f Purified 2D2-FIG splenic T cells (25,000/well) from each vaccinated mouse were cultured in duplicate with 200,000 irradiated naïve splenocytes (C57BL/6) and designated concentrations (x-axis) of e MOG 35–55 and f NFM 13–37 . Cultures were pulsed with 1 μCi of [ 3 H]thymidine during the last 24 h of a 3-day culture. These data are representative of two independent experiments. Statistical significance was analyzed by use of a one-way ANOVA. ( *p
    Figure Legend Snippet: The high-efficiency GMCSF-NFM vaccine induced memory Tcon responses in 2D2-FIG mice. On day 0, 2D2-FIG mice were SC injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. On day 8, splenocytes were harvested from vaccinated mice, and CD4 + T cells were purified and analyzed for a Vβ11 ( y -axis), b CD44 ( y -axis), and a , b FOXP3 expression ( x -axis). To assess generality of these findings ( c , d ), data from seven controlled experiments (analysis of PBMC ranging from day 4 to day 7) were pooled to assess 2D2-FIG mice vaccinated SC with saline ( n = 24), 4 nmol of GMCSF-MOG ( n = 24), or 4 nmol of GMCSF-NFM ( n = 25) for total Treg percentages ( c ) and CD44 + Treg percentages ( d ) among CD4 + T cells. a , b , e , f Purified 2D2-FIG splenic T cells (25,000/well) from each vaccinated mouse were cultured in duplicate with 200,000 irradiated naïve splenocytes (C57BL/6) and designated concentrations (x-axis) of e MOG 35–55 and f NFM 13–37 . Cultures were pulsed with 1 μCi of [ 3 H]thymidine during the last 24 h of a 3-day culture. These data are representative of two independent experiments. Statistical significance was analyzed by use of a one-way ANOVA. ( *p

    Techniques Used: Mouse Assay, Injection, Purification, Expressing, Cell Culture, Irradiation

    GMCSF-MOG induced robust Treg responses even when mixed with an immunogenic vaccine. a – n On day 0, 2D2-FIG ( n = 3–5) mice were injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. Separate groups were also injected with either 2 nmol of GMCSF-MOG + 2 nmol of GMCSF-NFM ( a , b ) or 4 nmol of GMCSF-MOG + 4 nmol GMCSF-NFM ( c–n ). PBMCs were assayed for CD3, CD4, FOXP3, Vβ11 (2D2 TCRβ), CD44, and CD62L expression. Shown are a , c percentages and b , d numbers (per microliter of blood) of FOXP3 + Tregs for CD3 + CD4 + T cells collected on days 0, 5, 12, and 19 ( a , b ) or days 0, 7, and 15 ( c , d ). Also shown for day 7 are e representative dot plots of CD3 + CD4 + T cells analyzed for Vβ11 ( y -axis) and FOXP3 expression ( x -axis); f the total number of CD3 + T cells per μl of blood; representative histograms for g Vβ11, i CD3, k CD44, and m CD62L expression of CD3 + CD4 + T cells; and the mean florescence intensity (MFI) of h Vβ11, j CD3, l CD44, and n CD62L. a , b Statistical significance was analyzed by use of a two-way repeated measures ANOVA. Means for groups G-MOG, “G-MOG + G-NFM”, and G-NFM represented statistically significant differences compared to each other ( *p
    Figure Legend Snippet: GMCSF-MOG induced robust Treg responses even when mixed with an immunogenic vaccine. a – n On day 0, 2D2-FIG ( n = 3–5) mice were injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. Separate groups were also injected with either 2 nmol of GMCSF-MOG + 2 nmol of GMCSF-NFM ( a , b ) or 4 nmol of GMCSF-MOG + 4 nmol GMCSF-NFM ( c–n ). PBMCs were assayed for CD3, CD4, FOXP3, Vβ11 (2D2 TCRβ), CD44, and CD62L expression. Shown are a , c percentages and b , d numbers (per microliter of blood) of FOXP3 + Tregs for CD3 + CD4 + T cells collected on days 0, 5, 12, and 19 ( a , b ) or days 0, 7, and 15 ( c , d ). Also shown for day 7 are e representative dot plots of CD3 + CD4 + T cells analyzed for Vβ11 ( y -axis) and FOXP3 expression ( x -axis); f the total number of CD3 + T cells per μl of blood; representative histograms for g Vβ11, i CD3, k CD44, and m CD62L expression of CD3 + CD4 + T cells; and the mean florescence intensity (MFI) of h Vβ11, j CD3, l CD44, and n CD62L. a , b Statistical significance was analyzed by use of a two-way repeated measures ANOVA. Means for groups G-MOG, “G-MOG + G-NFM”, and G-NFM represented statistically significant differences compared to each other ( *p

    Techniques Used: Mouse Assay, Injection, Expressing

    Administration of a CD40 agonist in vivo inhibited the Treg-inductive activity of GMCSF-MOG. On day − 2 and 0, 2D2-FIG mice ( n = 7/group) were injected i.p. with 100 μg of an anti-CD40 mAb (clone FGK4.5, rat anti-mouse CD40, IgG2a) or control mAb (clone 2A2, rat anti-trinitrophenol, IgG2a) in 500 μl saline. All mice were injected with 4 nmol of GMCSF-MOG on day 0. PBMC were analyzed on day − 8 before vaccination and day 3 post-vaccination for side-scatter (SSC), CD3, and FOXP3. Lymph nodes were harvested and analyzed on day 4 for CD4, CD11b, MHCII, and FOXP3. Shown are a representative histograms analyzed for MHCII expression ( x -axis), b MHCII median fluorescence intensity, and c numbers of CD4 − CD11b − MHCII + cells (B cells) and CD11b + CD4 − cells (myeloid APC) from lymph nodes on day 4. Also shown are d representative dot plots of CD3 + T cells analyzed for SSC ( y -axis) and FOXP3 ( x -axis) from blood on day 3 together with percentages e and numbers f of Tregs (per μl of blood) on days − 8 and 3. Statistical significance was analyzed by use of a one-tailed t test. ( *p
    Figure Legend Snippet: Administration of a CD40 agonist in vivo inhibited the Treg-inductive activity of GMCSF-MOG. On day − 2 and 0, 2D2-FIG mice ( n = 7/group) were injected i.p. with 100 μg of an anti-CD40 mAb (clone FGK4.5, rat anti-mouse CD40, IgG2a) or control mAb (clone 2A2, rat anti-trinitrophenol, IgG2a) in 500 μl saline. All mice were injected with 4 nmol of GMCSF-MOG on day 0. PBMC were analyzed on day − 8 before vaccination and day 3 post-vaccination for side-scatter (SSC), CD3, and FOXP3. Lymph nodes were harvested and analyzed on day 4 for CD4, CD11b, MHCII, and FOXP3. Shown are a representative histograms analyzed for MHCII expression ( x -axis), b MHCII median fluorescence intensity, and c numbers of CD4 − CD11b − MHCII + cells (B cells) and CD11b + CD4 − cells (myeloid APC) from lymph nodes on day 4. Also shown are d representative dot plots of CD3 + T cells analyzed for SSC ( y -axis) and FOXP3 ( x -axis) from blood on day 3 together with percentages e and numbers f of Tregs (per μl of blood) on days − 8 and 3. Statistical significance was analyzed by use of a one-tailed t test. ( *p

    Techniques Used: In Vivo, Activity Assay, Mouse Assay, Injection, Expressing, Fluorescence, One-tailed Test

    Related Articles

    Mouse Assay:

    Article Title: Use of a Humanized Mouse Model System in the Validation of Human Radiation Biodosimetry Standards
    Article Snippet: Human T-cell viability was analyzed using Annexin-V apoptosis detection kit (Biolegend) according to manufacturer instructions. .. For this assay, human peripheral blood mononuclear cells (PBMC) from human donors and humanized mice were purified, as described above. .. Human T cells were identified by staining with APC-labeled anti-human CD3 antibody (clone UCHT1; Biolegend) allowing minimal interference with the FITC-labeled Annexin-V during flow cytometry.

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)
    Article Snippet: The PC61-5.3 anti-CD25 rat IgG1 (λ) hybridoma was obtained from ATCC, and the PC61 mAb was purified as previously described [ ]. .. Flow cytometric analyses of leukocytes For vaccination studies and ex vivo/in vitro analyses, mice were vaccinated or cells were stimulated with designated antigens (e.g., GMCSF-MOG, GMCSF-NFM, MOG35–55 , NFM13–37 ) and peripheral blood mononuclear cells (PBMC) were collected via the submandibular vein and were diluted into sodium citrate (130 mM). .. Alternatively, lymph nodes and spleens were dissected from mice and pressed through 70 μm cell strainer (Corning, NY) to obtain a single cell suspension.

    Purification:

    Article Title: Use of a Humanized Mouse Model System in the Validation of Human Radiation Biodosimetry Standards
    Article Snippet: Human T-cell viability was analyzed using Annexin-V apoptosis detection kit (Biolegend) according to manufacturer instructions. .. For this assay, human peripheral blood mononuclear cells (PBMC) from human donors and humanized mice were purified, as described above. .. Human T cells were identified by staining with APC-labeled anti-human CD3 antibody (clone UCHT1; Biolegend) allowing minimal interference with the FITC-labeled Annexin-V during flow cytometry.

    Isolation:

    Article Title: Inhibition of ITK differentiates GVT and GVHD in allo-HSCT
    Article Snippet: .. Human Patient Samples T cells were isolated from Peripheral Blood Mononuclear Cells (PBMC) of GVHD patients or normal healthy donors as previously described . .. Briefly, mononuclear cells were isolated from GVHD patient samples by Ficoll-Hypaque density centrifugation and separated by positive selection with CD8 and CD4 MACS Microbeads.

    Article Title: Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain
    Article Snippet: .. Peripheral blood mononuclear cells were isolated by density centrifugation with Lymphoprep. .. CD4+ T cells were obtained using CD4 MicroBeads from Miltenyi and cultivated in RPMI-1640 medium supplemented with 10% FCS (Biowest), 1% penicillin/streptomycin, and IL-2 (100 U/mL).

    Staining:

    Article Title: Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth
    Article Snippet: .. Intracellular Cytokine Staining (ICS) AssaySplenocytes or peripheral blood mononuclear cells (PBMC) were stimulated with peptide at 2 µg/mL; ionomycin and phorbol myristate acetate (PMA) at 2.0 mg/mL and 0.5 mg/mL, respectively, as positive assay controls; tissue culture medium with 1% DMSO was used as a negative control and processed as previously described [ ]. .. The following mAb reagents were used: anti-CD107a phycoerythrin (PE)-conjugated mAb, anti-CD3 PerCP-eFluor710, anti-CD8a eFluor 450, anti-IFN-γ PE-Cy7, anti-IL-2 APC, and anti-tumor necrosis factor (TNF)-α fluorescein isothiocyanate (FITC) (all from eBioscience, San Diego, CA, USA) and anti-CD4 allophycocyanin (APC)/Cy7 (BioLegend, San Diego, CA, USA).

    Article Title: Activation of bone marrow adaptive immunity in type 2 diabetes: rescue by co-stimulation modulator Abatacept
    Article Snippet: After centrifugation, plasma was removed, and the pellet incubated with 1X RBC Lysis Buffer on ice for 10 min. Mononuclear cells were then resuspended at 2 x 106 cells/mL in PBS. .. Flow cytometry analyses Immunofluorescence surface staining was performed by adding a panel of directly conjugated mAb to freshly prepared BM mononuclear cells, splenocytes and peripheral blood mononuclear cells. .. Before staining, cell viability was assessed using Zombie NIR™ Fixable Viability Kit (#423106) for 30 min a RT.

    Negative Control:

    Article Title: Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth
    Article Snippet: .. Intracellular Cytokine Staining (ICS) AssaySplenocytes or peripheral blood mononuclear cells (PBMC) were stimulated with peptide at 2 µg/mL; ionomycin and phorbol myristate acetate (PMA) at 2.0 mg/mL and 0.5 mg/mL, respectively, as positive assay controls; tissue culture medium with 1% DMSO was used as a negative control and processed as previously described [ ]. .. The following mAb reagents were used: anti-CD107a phycoerythrin (PE)-conjugated mAb, anti-CD3 PerCP-eFluor710, anti-CD8a eFluor 450, anti-IFN-γ PE-Cy7, anti-IL-2 APC, and anti-tumor necrosis factor (TNF)-α fluorescein isothiocyanate (FITC) (all from eBioscience, San Diego, CA, USA) and anti-CD4 allophycocyanin (APC)/Cy7 (BioLegend, San Diego, CA, USA).

    Centrifugation:

    Article Title: Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain
    Article Snippet: .. Peripheral blood mononuclear cells were isolated by density centrifugation with Lymphoprep. .. CD4+ T cells were obtained using CD4 MicroBeads from Miltenyi and cultivated in RPMI-1640 medium supplemented with 10% FCS (Biowest), 1% penicillin/streptomycin, and IL-2 (100 U/mL).

    Flow Cytometry:

    Article Title: Activation of bone marrow adaptive immunity in type 2 diabetes: rescue by co-stimulation modulator Abatacept
    Article Snippet: After centrifugation, plasma was removed, and the pellet incubated with 1X RBC Lysis Buffer on ice for 10 min. Mononuclear cells were then resuspended at 2 x 106 cells/mL in PBS. .. Flow cytometry analyses Immunofluorescence surface staining was performed by adding a panel of directly conjugated mAb to freshly prepared BM mononuclear cells, splenocytes and peripheral blood mononuclear cells. .. Before staining, cell viability was assessed using Zombie NIR™ Fixable Viability Kit (#423106) for 30 min a RT.

    Article Title: Safety and immunogenicity of VGX-3100 formulations in a healthy young adult population
    Article Snippet: .. Flow cytometry was used to assess the ability of T-cells to synthesize lytic proteins following long-term exposure to an antigen (i.e. Lytic Granule Loading assay): Peripheral blood mononuclear cells (PBMC) were recovered from cryopreservation overnight in cell culture medium and spun, washed, and resuspended the following day. .. After counting, 106 PBMCs were plated into a 96-well plate in R10 medium.

    Immunofluorescence:

    Article Title: Activation of bone marrow adaptive immunity in type 2 diabetes: rescue by co-stimulation modulator Abatacept
    Article Snippet: After centrifugation, plasma was removed, and the pellet incubated with 1X RBC Lysis Buffer on ice for 10 min. Mononuclear cells were then resuspended at 2 x 106 cells/mL in PBS. .. Flow cytometry analyses Immunofluorescence surface staining was performed by adding a panel of directly conjugated mAb to freshly prepared BM mononuclear cells, splenocytes and peripheral blood mononuclear cells. .. Before staining, cell viability was assessed using Zombie NIR™ Fixable Viability Kit (#423106) for 30 min a RT.

    Cell Culture:

    Article Title: Safety and immunogenicity of VGX-3100 formulations in a healthy young adult population
    Article Snippet: .. Flow cytometry was used to assess the ability of T-cells to synthesize lytic proteins following long-term exposure to an antigen (i.e. Lytic Granule Loading assay): Peripheral blood mononuclear cells (PBMC) were recovered from cryopreservation overnight in cell culture medium and spun, washed, and resuspended the following day. .. After counting, 106 PBMCs were plated into a 96-well plate in R10 medium.

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    BioLegend veri cells cd4 low pbmc
    Functionality of mRNA-elicited <t>CD4</t> + and CD8 + T cells. Groups of BALB/cJ mice were immunized with monovalent R1, R2, RIII and RIV or multivalent R1 + 2, R1 + R2 + RIII and R1 + R2 + RIII + RIV vaccines at weeks 0 and 4. Each mRNA was administered intramuscularly at a 3-µg dose resulting in a total of 6-, 9- and 12-µg amounts for the combined vaccines, respectively. The frequencies of responding <t>PBMCs</t> and/or splenocytes were determined using combined pool of 28 peptides corresponding to 7 defined H-2 d class I epitopes and their 4 variants present in the vaccine ( Table 1 ). Multicolor flow cytometry analysis was performed to assess the functional phenotypes of vaccine-elicited CD8 + and CD4 + T cells in terms of CD107a, IFN-γ, IL-2 and TNF-α expression at 1 ( a ) and 5 ( b ) weeks after the homologous boost. The same cytokine legend as in (a) applies to (b). Specific T-cell frequencies are shown as group median (column) and individual animal values ( n = 6). ( c ) Group median ( n = 6) proportions of CD8 + and CD4 + T cells expressing 1 (black), 2 (light grey), 3 (dark grey) or 4 (white) functions are depicted using pie charts. See Figure S2 for the gating strategy. (a and b) Groups were compared using Kruskal–Wallis test with Dunn’s multiple comparison correction and significant two-tailed p values are indicated by an asterisk: * p
    Veri Cells Cd4 Low Pbmc, supplied by BioLegend, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Functionality of mRNA-elicited CD4 + and CD8 + T cells. Groups of BALB/cJ mice were immunized with monovalent R1, R2, RIII and RIV or multivalent R1 + 2, R1 + R2 + RIII and R1 + R2 + RIII + RIV vaccines at weeks 0 and 4. Each mRNA was administered intramuscularly at a 3-µg dose resulting in a total of 6-, 9- and 12-µg amounts for the combined vaccines, respectively. The frequencies of responding PBMCs and/or splenocytes were determined using combined pool of 28 peptides corresponding to 7 defined H-2 d class I epitopes and their 4 variants present in the vaccine ( Table 1 ). Multicolor flow cytometry analysis was performed to assess the functional phenotypes of vaccine-elicited CD8 + and CD4 + T cells in terms of CD107a, IFN-γ, IL-2 and TNF-α expression at 1 ( a ) and 5 ( b ) weeks after the homologous boost. The same cytokine legend as in (a) applies to (b). Specific T-cell frequencies are shown as group median (column) and individual animal values ( n = 6). ( c ) Group median ( n = 6) proportions of CD8 + and CD4 + T cells expressing 1 (black), 2 (light grey), 3 (dark grey) or 4 (white) functions are depicted using pie charts. See Figure S2 for the gating strategy. (a and b) Groups were compared using Kruskal–Wallis test with Dunn’s multiple comparison correction and significant two-tailed p values are indicated by an asterisk: * p

    Journal: Vaccines

    Article Title: Tetravalent Immunogen Assembled from Conserved Regions of HIV-1 and Delivered as mRNA Demonstrates Potent Preclinical T-Cell Immunogenicity and Breadth

    doi: 10.3390/vaccines8030360

    Figure Lengend Snippet: Functionality of mRNA-elicited CD4 + and CD8 + T cells. Groups of BALB/cJ mice were immunized with monovalent R1, R2, RIII and RIV or multivalent R1 + 2, R1 + R2 + RIII and R1 + R2 + RIII + RIV vaccines at weeks 0 and 4. Each mRNA was administered intramuscularly at a 3-µg dose resulting in a total of 6-, 9- and 12-µg amounts for the combined vaccines, respectively. The frequencies of responding PBMCs and/or splenocytes were determined using combined pool of 28 peptides corresponding to 7 defined H-2 d class I epitopes and their 4 variants present in the vaccine ( Table 1 ). Multicolor flow cytometry analysis was performed to assess the functional phenotypes of vaccine-elicited CD8 + and CD4 + T cells in terms of CD107a, IFN-γ, IL-2 and TNF-α expression at 1 ( a ) and 5 ( b ) weeks after the homologous boost. The same cytokine legend as in (a) applies to (b). Specific T-cell frequencies are shown as group median (column) and individual animal values ( n = 6). ( c ) Group median ( n = 6) proportions of CD8 + and CD4 + T cells expressing 1 (black), 2 (light grey), 3 (dark grey) or 4 (white) functions are depicted using pie charts. See Figure S2 for the gating strategy. (a and b) Groups were compared using Kruskal–Wallis test with Dunn’s multiple comparison correction and significant two-tailed p values are indicated by an asterisk: * p

    Article Snippet: Intracellular Cytokine Staining (ICS) AssaySplenocytes or peripheral blood mononuclear cells (PBMC) were stimulated with peptide at 2 µg/mL; ionomycin and phorbol myristate acetate (PMA) at 2.0 mg/mL and 0.5 mg/mL, respectively, as positive assay controls; tissue culture medium with 1% DMSO was used as a negative control and processed as previously described [ ].

    Techniques: Mouse Assay, Flow Cytometry, Functional Assay, Expressing, Two Tailed Test

    GMCSF-MOG was superior to GMCSF-NFM for eliciting persistence of a CD44 high Tcon phenotype. a – d 2D2-FIG mice were vaccinated with 4 nmol of GMCSF-MOG ( n = 5), 4 nmol of GMCSF-NFM ( n = 5), or with saline ( n = 5). PBMCs were analyzed on days 5, 12, and 19 for CD3, CD4, CD44, and FOXP3 expression. a – c The day 0 time point was derived from the CD44 expression of CD3 + CD4 + T cells from naïve 2D2-FIG mice ( n = 23). Shown are percentages of a CD44 high T cells, b CD44 high Tcons, and c CD44 high Tregs of gated CD3 + CD4 + T cells on days 0, 5, 12, and 19. Shown in d are the percentage of CD62L low T cells of gated CD44 high CD3 + CD4 + T cells on day 5, 12, and 19. Shown in e are representative dot plots of CD44 ( y -axis) and FOXP3 ( x -axis) expression of CD4 + CD3 + T cells on day 5. These data are representative of three independent experiments. Statistical significance was analyzed using a one-way ANOVA. Shown in a , b and d are significant differences between the groups GMCSF-MOG, GMCSF-NFM, and saline, and c statistical differences comparing GMCSF-MOG treatment to both GMCSF-NFM and saline. ( *p

    Journal: Journal of Neuroinflammation

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

    doi: 10.1186/s12974-020-01856-8

    Figure Lengend Snippet: GMCSF-MOG was superior to GMCSF-NFM for eliciting persistence of a CD44 high Tcon phenotype. a – d 2D2-FIG mice were vaccinated with 4 nmol of GMCSF-MOG ( n = 5), 4 nmol of GMCSF-NFM ( n = 5), or with saline ( n = 5). PBMCs were analyzed on days 5, 12, and 19 for CD3, CD4, CD44, and FOXP3 expression. a – c The day 0 time point was derived from the CD44 expression of CD3 + CD4 + T cells from naïve 2D2-FIG mice ( n = 23). Shown are percentages of a CD44 high T cells, b CD44 high Tcons, and c CD44 high Tregs of gated CD3 + CD4 + T cells on days 0, 5, 12, and 19. Shown in d are the percentage of CD62L low T cells of gated CD44 high CD3 + CD4 + T cells on day 5, 12, and 19. Shown in e are representative dot plots of CD44 ( y -axis) and FOXP3 ( x -axis) expression of CD4 + CD3 + T cells on day 5. These data are representative of three independent experiments. Statistical significance was analyzed using a one-way ANOVA. Shown in a , b and d are significant differences between the groups GMCSF-MOG, GMCSF-NFM, and saline, and c statistical differences comparing GMCSF-MOG treatment to both GMCSF-NFM and saline. ( *p

    Article Snippet: Flow cytometric analyses of leukocytes For vaccination studies and ex vivo/in vitro analyses, mice were vaccinated or cells were stimulated with designated antigens (e.g., GMCSF-MOG, GMCSF-NFM, MOG35–55 , NFM13–37 ) and peripheral blood mononuclear cells (PBMC) were collected via the submandibular vein and were diluted into sodium citrate (130 mM).

    Techniques: Mouse Assay, Expressing, Derivative Assay

    GMCSF-MOG preferentially expanded Tregs from a memory T cell pool. On day − 1, a CD45.1-2D2-FIG mixed Treg and Tcon line (40% Tregs and 60% Tcons) was injected intravenously (1.25 × 10 6 cells) into CD45.2 2D2-FIG- Rag1 −/− mice ( n = 3). On day 0, 2D2-FIG- Rag1 −/− recipient mice were vaccinated with 4 nmol of GMCSF-MOG or 4 nmol GM-CSF + 4 nmol MOG 35–55 . PBMCs were analyzed on day 4 for CD3, CD4, CD45.1, CD44, and FOXP3. Shown are analyses of donor CD45.1 CD3 + CD4 + T cells including a representative dot plots of GMCSF-MOG and “GM-CSF + MOG”-treated mice for CD44 ( y -axis) and FOXP3 expression ( x -axis) together with ( b , d , f ) cell numbers per microliter of blood and ( c , e , g ) percentages of designated cell populations. Statistical significance was analyzed by use of a two-tailed t test. ( *p

    Journal: Journal of Neuroinflammation

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

    doi: 10.1186/s12974-020-01856-8

    Figure Lengend Snippet: GMCSF-MOG preferentially expanded Tregs from a memory T cell pool. On day − 1, a CD45.1-2D2-FIG mixed Treg and Tcon line (40% Tregs and 60% Tcons) was injected intravenously (1.25 × 10 6 cells) into CD45.2 2D2-FIG- Rag1 −/− mice ( n = 3). On day 0, 2D2-FIG- Rag1 −/− recipient mice were vaccinated with 4 nmol of GMCSF-MOG or 4 nmol GM-CSF + 4 nmol MOG 35–55 . PBMCs were analyzed on day 4 for CD3, CD4, CD45.1, CD44, and FOXP3. Shown are analyses of donor CD45.1 CD3 + CD4 + T cells including a representative dot plots of GMCSF-MOG and “GM-CSF + MOG”-treated mice for CD44 ( y -axis) and FOXP3 expression ( x -axis) together with ( b , d , f ) cell numbers per microliter of blood and ( c , e , g ) percentages of designated cell populations. Statistical significance was analyzed by use of a two-tailed t test. ( *p

    Article Snippet: Flow cytometric analyses of leukocytes For vaccination studies and ex vivo/in vitro analyses, mice were vaccinated or cells were stimulated with designated antigens (e.g., GMCSF-MOG, GMCSF-NFM, MOG35–55 , NFM13–37 ) and peripheral blood mononuclear cells (PBMC) were collected via the submandibular vein and were diluted into sodium citrate (130 mM).

    Techniques: Injection, Mouse Assay, Expressing, Two Tailed Test

    The high-efficiency GMCSF-NFM vaccine induced memory Tcon responses in 2D2-FIG mice. On day 0, 2D2-FIG mice were SC injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. On day 8, splenocytes were harvested from vaccinated mice, and CD4 + T cells were purified and analyzed for a Vβ11 ( y -axis), b CD44 ( y -axis), and a , b FOXP3 expression ( x -axis). To assess generality of these findings ( c , d ), data from seven controlled experiments (analysis of PBMC ranging from day 4 to day 7) were pooled to assess 2D2-FIG mice vaccinated SC with saline ( n = 24), 4 nmol of GMCSF-MOG ( n = 24), or 4 nmol of GMCSF-NFM ( n = 25) for total Treg percentages ( c ) and CD44 + Treg percentages ( d ) among CD4 + T cells. a , b , e , f Purified 2D2-FIG splenic T cells (25,000/well) from each vaccinated mouse were cultured in duplicate with 200,000 irradiated naïve splenocytes (C57BL/6) and designated concentrations (x-axis) of e MOG 35–55 and f NFM 13–37 . Cultures were pulsed with 1 μCi of [ 3 H]thymidine during the last 24 h of a 3-day culture. These data are representative of two independent experiments. Statistical significance was analyzed by use of a one-way ANOVA. ( *p

    Journal: Journal of Neuroinflammation

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

    doi: 10.1186/s12974-020-01856-8

    Figure Lengend Snippet: The high-efficiency GMCSF-NFM vaccine induced memory Tcon responses in 2D2-FIG mice. On day 0, 2D2-FIG mice were SC injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. On day 8, splenocytes were harvested from vaccinated mice, and CD4 + T cells were purified and analyzed for a Vβ11 ( y -axis), b CD44 ( y -axis), and a , b FOXP3 expression ( x -axis). To assess generality of these findings ( c , d ), data from seven controlled experiments (analysis of PBMC ranging from day 4 to day 7) were pooled to assess 2D2-FIG mice vaccinated SC with saline ( n = 24), 4 nmol of GMCSF-MOG ( n = 24), or 4 nmol of GMCSF-NFM ( n = 25) for total Treg percentages ( c ) and CD44 + Treg percentages ( d ) among CD4 + T cells. a , b , e , f Purified 2D2-FIG splenic T cells (25,000/well) from each vaccinated mouse were cultured in duplicate with 200,000 irradiated naïve splenocytes (C57BL/6) and designated concentrations (x-axis) of e MOG 35–55 and f NFM 13–37 . Cultures were pulsed with 1 μCi of [ 3 H]thymidine during the last 24 h of a 3-day culture. These data are representative of two independent experiments. Statistical significance was analyzed by use of a one-way ANOVA. ( *p

    Article Snippet: Flow cytometric analyses of leukocytes For vaccination studies and ex vivo/in vitro analyses, mice were vaccinated or cells were stimulated with designated antigens (e.g., GMCSF-MOG, GMCSF-NFM, MOG35–55 , NFM13–37 ) and peripheral blood mononuclear cells (PBMC) were collected via the submandibular vein and were diluted into sodium citrate (130 mM).

    Techniques: Mouse Assay, Injection, Purification, Expressing, Cell Culture, Irradiation

    GMCSF-MOG induced robust Treg responses even when mixed with an immunogenic vaccine. a – n On day 0, 2D2-FIG ( n = 3–5) mice were injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. Separate groups were also injected with either 2 nmol of GMCSF-MOG + 2 nmol of GMCSF-NFM ( a , b ) or 4 nmol of GMCSF-MOG + 4 nmol GMCSF-NFM ( c–n ). PBMCs were assayed for CD3, CD4, FOXP3, Vβ11 (2D2 TCRβ), CD44, and CD62L expression. Shown are a , c percentages and b , d numbers (per microliter of blood) of FOXP3 + Tregs for CD3 + CD4 + T cells collected on days 0, 5, 12, and 19 ( a , b ) or days 0, 7, and 15 ( c , d ). Also shown for day 7 are e representative dot plots of CD3 + CD4 + T cells analyzed for Vβ11 ( y -axis) and FOXP3 expression ( x -axis); f the total number of CD3 + T cells per μl of blood; representative histograms for g Vβ11, i CD3, k CD44, and m CD62L expression of CD3 + CD4 + T cells; and the mean florescence intensity (MFI) of h Vβ11, j CD3, l CD44, and n CD62L. a , b Statistical significance was analyzed by use of a two-way repeated measures ANOVA. Means for groups G-MOG, “G-MOG + G-NFM”, and G-NFM represented statistically significant differences compared to each other ( *p

    Journal: Journal of Neuroinflammation

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

    doi: 10.1186/s12974-020-01856-8

    Figure Lengend Snippet: GMCSF-MOG induced robust Treg responses even when mixed with an immunogenic vaccine. a – n On day 0, 2D2-FIG ( n = 3–5) mice were injected with 4 nmol of GMCSF-MOG, 4 nmol of GMCSF-NFM, or saline. Separate groups were also injected with either 2 nmol of GMCSF-MOG + 2 nmol of GMCSF-NFM ( a , b ) or 4 nmol of GMCSF-MOG + 4 nmol GMCSF-NFM ( c–n ). PBMCs were assayed for CD3, CD4, FOXP3, Vβ11 (2D2 TCRβ), CD44, and CD62L expression. Shown are a , c percentages and b , d numbers (per microliter of blood) of FOXP3 + Tregs for CD3 + CD4 + T cells collected on days 0, 5, 12, and 19 ( a , b ) or days 0, 7, and 15 ( c , d ). Also shown for day 7 are e representative dot plots of CD3 + CD4 + T cells analyzed for Vβ11 ( y -axis) and FOXP3 expression ( x -axis); f the total number of CD3 + T cells per μl of blood; representative histograms for g Vβ11, i CD3, k CD44, and m CD62L expression of CD3 + CD4 + T cells; and the mean florescence intensity (MFI) of h Vβ11, j CD3, l CD44, and n CD62L. a , b Statistical significance was analyzed by use of a two-way repeated measures ANOVA. Means for groups G-MOG, “G-MOG + G-NFM”, and G-NFM represented statistically significant differences compared to each other ( *p

    Article Snippet: Flow cytometric analyses of leukocytes For vaccination studies and ex vivo/in vitro analyses, mice were vaccinated or cells were stimulated with designated antigens (e.g., GMCSF-MOG, GMCSF-NFM, MOG35–55 , NFM13–37 ) and peripheral blood mononuclear cells (PBMC) were collected via the submandibular vein and were diluted into sodium citrate (130 mM).

    Techniques: Mouse Assay, Injection, Expressing

    Administration of a CD40 agonist in vivo inhibited the Treg-inductive activity of GMCSF-MOG. On day − 2 and 0, 2D2-FIG mice ( n = 7/group) were injected i.p. with 100 μg of an anti-CD40 mAb (clone FGK4.5, rat anti-mouse CD40, IgG2a) or control mAb (clone 2A2, rat anti-trinitrophenol, IgG2a) in 500 μl saline. All mice were injected with 4 nmol of GMCSF-MOG on day 0. PBMC were analyzed on day − 8 before vaccination and day 3 post-vaccination for side-scatter (SSC), CD3, and FOXP3. Lymph nodes were harvested and analyzed on day 4 for CD4, CD11b, MHCII, and FOXP3. Shown are a representative histograms analyzed for MHCII expression ( x -axis), b MHCII median fluorescence intensity, and c numbers of CD4 − CD11b − MHCII + cells (B cells) and CD11b + CD4 − cells (myeloid APC) from lymph nodes on day 4. Also shown are d representative dot plots of CD3 + T cells analyzed for SSC ( y -axis) and FOXP3 ( x -axis) from blood on day 3 together with percentages e and numbers f of Tregs (per μl of blood) on days − 8 and 3. Statistical significance was analyzed by use of a one-tailed t test. ( *p

    Journal: Journal of Neuroinflammation

    Article Title: A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE)

    doi: 10.1186/s12974-020-01856-8

    Figure Lengend Snippet: Administration of a CD40 agonist in vivo inhibited the Treg-inductive activity of GMCSF-MOG. On day − 2 and 0, 2D2-FIG mice ( n = 7/group) were injected i.p. with 100 μg of an anti-CD40 mAb (clone FGK4.5, rat anti-mouse CD40, IgG2a) or control mAb (clone 2A2, rat anti-trinitrophenol, IgG2a) in 500 μl saline. All mice were injected with 4 nmol of GMCSF-MOG on day 0. PBMC were analyzed on day − 8 before vaccination and day 3 post-vaccination for side-scatter (SSC), CD3, and FOXP3. Lymph nodes were harvested and analyzed on day 4 for CD4, CD11b, MHCII, and FOXP3. Shown are a representative histograms analyzed for MHCII expression ( x -axis), b MHCII median fluorescence intensity, and c numbers of CD4 − CD11b − MHCII + cells (B cells) and CD11b + CD4 − cells (myeloid APC) from lymph nodes on day 4. Also shown are d representative dot plots of CD3 + T cells analyzed for SSC ( y -axis) and FOXP3 ( x -axis) from blood on day 3 together with percentages e and numbers f of Tregs (per μl of blood) on days − 8 and 3. Statistical significance was analyzed by use of a one-tailed t test. ( *p

    Article Snippet: Flow cytometric analyses of leukocytes For vaccination studies and ex vivo/in vitro analyses, mice were vaccinated or cells were stimulated with designated antigens (e.g., GMCSF-MOG, GMCSF-NFM, MOG35–55 , NFM13–37 ) and peripheral blood mononuclear cells (PBMC) were collected via the submandibular vein and were diluted into sodium citrate (130 mM).

    Techniques: In Vivo, Activity Assay, Mouse Assay, Injection, Expressing, Fluorescence, One-tailed Test

    Novel peptide SLP76pTYR specifically targets ITK signaling and enhances Treg cell development. (A) Total T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for CD4 + and FoxP3 + . n=3 one representative experiment shown. (B) Quantification of three experiments as in (A). (C) Cell lysates from T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for phosphorylation of ITK, PLCγ1 and ERK. Lysates were also examined for phosphorylation of PI3K, mTOR, P13K, and AKT. n=3 one representative experiment shown. ( D ) Western blots were normalized to β−Actin and quantitative data from 3 independent experiments presented. ( E ) Human GVHD patients’ samples were cells stimulated in the presence of SLP76pTYR or vehicle only. T cell lysates were used in western blots for analysis of pPLCγ, pERK, pAKT, pMTOR, and (H) three experiments were quantitated and normalized to β−Actin. n=3, one representative experiment shown. ( G ) Primary human T cells purified from PBMC were stimulated with CD3 and CD28 for 5 hours in the presence of vehicle alone or SLP76pTYR in the presence BFA. Intracellular IFN-γ and TNF-α expression by CD8 + and CD4 + T cells was determined by flow cytometry. For statistical analysis we used two-way ANOVA and Student’s t test. P values are presented.

    Journal: bioRxiv

    Article Title: Inhibition of ITK differentiates GVT and GVHD in allo-HSCT

    doi: 10.1101/2020.07.15.204693

    Figure Lengend Snippet: Novel peptide SLP76pTYR specifically targets ITK signaling and enhances Treg cell development. (A) Total T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for CD4 + and FoxP3 + . n=3 one representative experiment shown. (B) Quantification of three experiments as in (A). (C) Cell lysates from T cells stimulated in the presence of SLP76pTYR or vehicle alone were examined for phosphorylation of ITK, PLCγ1 and ERK. Lysates were also examined for phosphorylation of PI3K, mTOR, P13K, and AKT. n=3 one representative experiment shown. ( D ) Western blots were normalized to β−Actin and quantitative data from 3 independent experiments presented. ( E ) Human GVHD patients’ samples were cells stimulated in the presence of SLP76pTYR or vehicle only. T cell lysates were used in western blots for analysis of pPLCγ, pERK, pAKT, pMTOR, and (H) three experiments were quantitated and normalized to β−Actin. n=3, one representative experiment shown. ( G ) Primary human T cells purified from PBMC were stimulated with CD3 and CD28 for 5 hours in the presence of vehicle alone or SLP76pTYR in the presence BFA. Intracellular IFN-γ and TNF-α expression by CD8 + and CD4 + T cells was determined by flow cytometry. For statistical analysis we used two-way ANOVA and Student’s t test. P values are presented.

    Article Snippet: Human Patient Samples T cells were isolated from Peripheral Blood Mononuclear Cells (PBMC) of GVHD patients or normal healthy donors as previously described .

    Techniques: Western Blot, Purification, Expressing, Flow Cytometry