Structured Review

Enzo Biochem cd40l
Intracellular calcium mobilization and CysLTR 1 antagonism. (Left panel) DCs were loaded with FluoForte and intracellular calcium release was determined using a Tecan Infinite F200 Pro fluorometer. <t>CD40L</t> was added at t-d and LTC 4 or LTD 4 were injected
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1) Product Images from "Leukotriene C4 induces migration of human monocyte\u2013derived dendritic cells without loss of immunostimulatory function"

Article Title: Leukotriene C4 induces migration of human monocyte\u2013derived dendritic cells without loss of immunostimulatory function

Journal: Blood

doi: 10.1182/blood-2011-10-385930

Intracellular calcium mobilization and CysLTR 1 antagonism. (Left panel) DCs were loaded with FluoForte and intracellular calcium release was determined using a Tecan Infinite F200 Pro fluorometer. CD40L was added at t-d and LTC 4 or LTD 4 were injected
Figure Legend Snippet: Intracellular calcium mobilization and CysLTR 1 antagonism. (Left panel) DCs were loaded with FluoForte and intracellular calcium release was determined using a Tecan Infinite F200 Pro fluorometer. CD40L was added at t-d and LTC 4 or LTD 4 were injected

Techniques Used: Injection

Cytokine secretion and IDO activity of mature DCs. (Left panel) DCs were matured for 24 hours with CC or CD40L in the presence or absence of LTC 4 or PGE 2 . Supernatants were harvested and secretion of IL-10, IL-12p70, and IL-12p40 were analyzed by ELISA.
Figure Legend Snippet: Cytokine secretion and IDO activity of mature DCs. (Left panel) DCs were matured for 24 hours with CC or CD40L in the presence or absence of LTC 4 or PGE 2 . Supernatants were harvested and secretion of IL-10, IL-12p70, and IL-12p40 were analyzed by ELISA.

Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay

2) Product Images from "Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation"

Article Title: Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aax0904

T H 1 and T FH cytokines modulate expression of T-bet and migration receptors. ( A ) Conventional flow cytometry performed on LN GCBC and CD19 hi MBC depicting the expression of Bcl6, T-bet, and CXCR5 expression of an HIV-infected individual. Red pattern and numbers identify Bcl6 frequencies within each quadrant. ( B ) Conventional flow cytometry performed on tonsillar B cells isolated from HIV-negative donors depicting Bcl6 and T-bet expression before and after 48 hours of stimulation of IgD + cells with anti-human BCR and CD40L with or without IFN-γ or IL-21. ( C ) Histograms and comparison ( n = 10) of T-bet and CXCR5 protein expression under conditions in (B). ( D ) Comparison ( n = 6) of gene expression for TBX21 , S1PR2 , and BACH2 under conditions in (B). * P
Figure Legend Snippet: T H 1 and T FH cytokines modulate expression of T-bet and migration receptors. ( A ) Conventional flow cytometry performed on LN GCBC and CD19 hi MBC depicting the expression of Bcl6, T-bet, and CXCR5 expression of an HIV-infected individual. Red pattern and numbers identify Bcl6 frequencies within each quadrant. ( B ) Conventional flow cytometry performed on tonsillar B cells isolated from HIV-negative donors depicting Bcl6 and T-bet expression before and after 48 hours of stimulation of IgD + cells with anti-human BCR and CD40L with or without IFN-γ or IL-21. ( C ) Histograms and comparison ( n = 10) of T-bet and CXCR5 protein expression under conditions in (B). ( D ) Comparison ( n = 6) of gene expression for TBX21 , S1PR2 , and BACH2 under conditions in (B). * P

Techniques Used: Expressing, Migration, Flow Cytometry, Infection, Isolation

3) Product Images from "Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency"

Article Title: Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency

Journal: Breast Cancer Research

doi: 10.1186/bcr1001

Maturation of dendritic cells (DCs) from healthy donors or from breast cancer patients. Data are expressed as the percentage of the cells (with standard error) expressing the CD80, CD83 and CD86 surface markers after treatment of immature DCs with a combination of maturating agents: (a) tumour necrosis factor (TNF)-α/lipopolysaccharide (LPS)/CD40L ( n = 3); (b) IL-1β/IL-6/TNF-α/prostaglandin (PG)E 2 ( n = 4–5); and (c) Ribomunyl ® /Imukin ® ( n = 3). a Different from corresponding donors in each individual assay.
Figure Legend Snippet: Maturation of dendritic cells (DCs) from healthy donors or from breast cancer patients. Data are expressed as the percentage of the cells (with standard error) expressing the CD80, CD83 and CD86 surface markers after treatment of immature DCs with a combination of maturating agents: (a) tumour necrosis factor (TNF)-α/lipopolysaccharide (LPS)/CD40L ( n = 3); (b) IL-1β/IL-6/TNF-α/prostaglandin (PG)E 2 ( n = 4–5); and (c) Ribomunyl ® /Imukin ® ( n = 3). a Different from corresponding donors in each individual assay.

Techniques Used: Expressing

4) Product Images from "Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response"

Article Title: Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

doi: 10.1002/cyto.a.23737

Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3 − CD19 + CD20 ++ CD38 + and non-GC B cells as CD3 − CD19 + CD38 − . The non-GC population was further gated to enrich for IgD + naïve B cells (possibly containing unswitched memory B cells) and IgD − memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p
Figure Legend Snippet: Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3 − CD19 + CD20 ++ CD38 + and non-GC B cells as CD3 − CD19 + CD38 − . The non-GC population was further gated to enrich for IgD + naïve B cells (possibly containing unswitched memory B cells) and IgD − memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p

Techniques Used: Expressing, Mass Cytometry

CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils. CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20 hi CD38 + , non-GC (CD20 + CD38 − ) were split into CD27 − IgD + naïve and CD27 + IgD − memory B cells. * p
Figure Legend Snippet: CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils. CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20 hi CD38 + , non-GC (CD20 + CD38 − ) were split into CD27 − IgD + naïve and CD27 + IgD − memory B cells. * p

Techniques Used: Fluorescence, Flow Cytometry

CD40L-induced IκBα degradation is not proportional to level of CD40L binding CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20 + CD38 − and GC B cells as CD20 hi CD38 + . (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p
Figure Legend Snippet: CD40L-induced IκBα degradation is not proportional to level of CD40L binding CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20 + CD38 − and GC B cells as CD20 hi CD38 + . (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p

Techniques Used: Binding Assay, Fluorescence, Flow Cytometry

5) Product Images from "Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses"

Article Title: Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses

Journal: bioRxiv

doi: 10.1101/2020.06.01.127514

LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws PBMC were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hours, in the abence/presence of Bafilomycin A1 for the last 2h. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. ( a ) Monocytes gated on CD14+ treated with IFNg or LPS, ( b ) B cells gated on CD19+ treated with anti-CD40L and anti-IgM, ( c ) CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28, ( d ) CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28.
Figure Legend Snippet: LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws PBMC were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hours, in the abence/presence of Bafilomycin A1 for the last 2h. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. ( a ) Monocytes gated on CD14+ treated with IFNg or LPS, ( b ) B cells gated on CD19+ treated with anti-CD40L and anti-IgM, ( c ) CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28, ( d ) CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28.

Techniques Used: Flow Cytometry, Generated, Cell Culture, Fluorescence

6) Product Images from "Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation"

Article Title: Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt886

Comparison of histone modification changes during transformation of RBLs with mutant forms of EBV and IL-4/CD40L-stimulated cells. ( A ) Comparison of activation and proliferation levels between B cells infected with wild type EBV (WT EBV), the LMP-1 and EBNA-2 deficient forms of EBV (LMP1 KO and EBNA2 KO), and cells stimulated with IL-4/CD40L (ACT). The left panel shows the proportion of activated cells (left; determined by FACS analysis with CD86) and the right panel the proportion of dividing cells (determined with tritiated thymidine). ( B ) Quantitation of levels of infection with LMP1-expressing lentivirus measured by GFP fluorescence and FACS analysis. ( C ) Analysis by western blot of a time course experiment with antibodies against H3K4me3, H3K9me3, H3K27me3, total H3, H4K20me3 and total H4 of RBLs infected with wild type EBV, EBV deficient for LMP-1, EBV deficient for EBNA-2, IL-4/CD40L-stimulated B cells and IL-4/CD40L-stimulated B cells infected with LMP-1. ( D ) Densitometric quantitation of western blot data. The average of three independent experiments is represented. Data are presented relative to levels in RBL. ( E ) Top panel, XIC of isobaric H3 peptides from different B-cell populations. Differentially modified peptides were chromatographically separated and individual peaks identified by tandem MS spectra. Relative levels of individual isoforms were quantified using the Genesis peak detection module of the Xcalibur software package (Thermo Fisher Scientific). X -axis represents retention time, y -axis the intensity of ion currents in the Orbitrap mass spectrometer. Bottom panel, bar chart of H3K27me3 levels relative to the two other modification states of this peptide. Error bars represent the SD of two independent biological replicates. Abbreviations : unmod = unmodified peptide, me1 = single mono-methylated lysine. ( F ) Quantitative ChIP assays showing changes in H4K20me3, H3K27me3 and H3K9me3 between RBLs and B cells infected wild type EBV, EBV deficient for LMP-1, EBV deficient for EBNA-2, IL-4/CD40L-stimulated B cells and IL-4/CD40L-stimulated B cells infected with LMP-1 (LMP1 LV). The sequences correspond to promoter gene sequences from the ChIP-seq analysis. GAPDH was used as a control sequence.
Figure Legend Snippet: Comparison of histone modification changes during transformation of RBLs with mutant forms of EBV and IL-4/CD40L-stimulated cells. ( A ) Comparison of activation and proliferation levels between B cells infected with wild type EBV (WT EBV), the LMP-1 and EBNA-2 deficient forms of EBV (LMP1 KO and EBNA2 KO), and cells stimulated with IL-4/CD40L (ACT). The left panel shows the proportion of activated cells (left; determined by FACS analysis with CD86) and the right panel the proportion of dividing cells (determined with tritiated thymidine). ( B ) Quantitation of levels of infection with LMP1-expressing lentivirus measured by GFP fluorescence and FACS analysis. ( C ) Analysis by western blot of a time course experiment with antibodies against H3K4me3, H3K9me3, H3K27me3, total H3, H4K20me3 and total H4 of RBLs infected with wild type EBV, EBV deficient for LMP-1, EBV deficient for EBNA-2, IL-4/CD40L-stimulated B cells and IL-4/CD40L-stimulated B cells infected with LMP-1. ( D ) Densitometric quantitation of western blot data. The average of three independent experiments is represented. Data are presented relative to levels in RBL. ( E ) Top panel, XIC of isobaric H3 peptides from different B-cell populations. Differentially modified peptides were chromatographically separated and individual peaks identified by tandem MS spectra. Relative levels of individual isoforms were quantified using the Genesis peak detection module of the Xcalibur software package (Thermo Fisher Scientific). X -axis represents retention time, y -axis the intensity of ion currents in the Orbitrap mass spectrometer. Bottom panel, bar chart of H3K27me3 levels relative to the two other modification states of this peptide. Error bars represent the SD of two independent biological replicates. Abbreviations : unmod = unmodified peptide, me1 = single mono-methylated lysine. ( F ) Quantitative ChIP assays showing changes in H4K20me3, H3K27me3 and H3K9me3 between RBLs and B cells infected wild type EBV, EBV deficient for LMP-1, EBV deficient for EBNA-2, IL-4/CD40L-stimulated B cells and IL-4/CD40L-stimulated B cells infected with LMP-1 (LMP1 LV). The sequences correspond to promoter gene sequences from the ChIP-seq analysis. GAPDH was used as a control sequence.

Techniques Used: Modification, Transformation Assay, Mutagenesis, Activation Assay, Infection, Activated Clotting Time Assay, FACS, Quantitation Assay, Expressing, Fluorescence, Western Blot, Mass Spectrometry, Software, Methylation, Chromatin Immunoprecipitation, Sequencing

7) Product Images from "Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2"

Article Title: Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2

Journal: Immunology

doi: 10.1111/j.1365-2567.2003.01764.x

PGE 2 up-regulates C5aR on DC and prevents its down-regulation upon maturation. Immature DC were incubated with CD40L or LPS, respectively, with or without the presence of PGE 2 for 48 hr. The expression of C5aR and CD83 was analysed by fluorescence cytometry. Presence of PGE 2 led to up-regulation of C5aR, while LPS and CD40L led to down-regulation. (a) One representative experiment (numbers are percentage of cells in the corresponding quadrants), (b) mean fluorescence intensity ± SEM (of all cells acquired) of five independent experiments (*, significant difference, P
Figure Legend Snippet: PGE 2 up-regulates C5aR on DC and prevents its down-regulation upon maturation. Immature DC were incubated with CD40L or LPS, respectively, with or without the presence of PGE 2 for 48 hr. The expression of C5aR and CD83 was analysed by fluorescence cytometry. Presence of PGE 2 led to up-regulation of C5aR, while LPS and CD40L led to down-regulation. (a) One representative experiment (numbers are percentage of cells in the corresponding quadrants), (b) mean fluorescence intensity ± SEM (of all cells acquired) of five independent experiments (*, significant difference, P

Techniques Used: Incubation, Expressing, Fluorescence, Cytometry

8) Product Images from "Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment"

Article Title: Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment

Journal: Oncotarget

doi: 10.18632/oncotarget.14588

Cerdulatinib effectively blocks BCR and JAK-STAT signaling pathways A. Immunoblots for early BCR components SYK and BTK following cerdulatinib treatment. Freshly isolated CLL cells ( N = 6) were treated with increasing doses of cerdulatinib in the presence of combined stimulation with soluble anti-IgM, CD40L and CpG. p-SYK, total SYK, p-BTK and total BTK were blotted in whole cell extracts. GAPDH served as a loading control. B. Left panel, phospho-flow assay of p-PLCγ2 (Tyr759) in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. Middle panel, Mean fluorescence intensity (MFI) of p-PLCγ2 under indicated conditions ( N = 43). Data represents mean+SE. **** P
Figure Legend Snippet: Cerdulatinib effectively blocks BCR and JAK-STAT signaling pathways A. Immunoblots for early BCR components SYK and BTK following cerdulatinib treatment. Freshly isolated CLL cells ( N = 6) were treated with increasing doses of cerdulatinib in the presence of combined stimulation with soluble anti-IgM, CD40L and CpG. p-SYK, total SYK, p-BTK and total BTK were blotted in whole cell extracts. GAPDH served as a loading control. B. Left panel, phospho-flow assay of p-PLCγ2 (Tyr759) in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. Middle panel, Mean fluorescence intensity (MFI) of p-PLCγ2 under indicated conditions ( N = 43). Data represents mean+SE. **** P

Techniques Used: Western Blot, Isolation, Fluorescence

Cerdulatinib inhibits the activity of NF-κB pathway A. Phosphorylation of IκBα following cerdulatinib treatment. CLL cells were stimulated with either plate-bound αIgM alone or combined IL4+CD40L+plate-bound αIgM stimuli, and treated with 2 µM cerdulatnib for 24 hrs. Whole Cell lysates were immunoblotted for p-IκBα and GAPDH as the loading control. Four representative blots are shown. B. Reduction in p-IκBα is dose-dependent on cerdulatinib. CLL cells were stimulated with combined IL4+CD40L+plate-bound αIgM stimuli. C. Cerdulatinib decreased nuclear P65. Nuclear extracts were prepared from each sample and were immunoblotted for P65 and lamin B, a nuclear marker. D. DNA binding activity of NFκB subunit p50. Nuclear extracts were prepared from each sample. P50 activity was measured by ELISA plates coated with oligonucleotides containing the NF-kB consensus sequence (59-GGGACTTTCC-39). Concentration of p50 was determined by comparing samples with a standard curve of purified p50 protein, results represent mean±SE of 6 CLL samples. **** P
Figure Legend Snippet: Cerdulatinib inhibits the activity of NF-κB pathway A. Phosphorylation of IκBα following cerdulatinib treatment. CLL cells were stimulated with either plate-bound αIgM alone or combined IL4+CD40L+plate-bound αIgM stimuli, and treated with 2 µM cerdulatnib for 24 hrs. Whole Cell lysates were immunoblotted for p-IκBα and GAPDH as the loading control. Four representative blots are shown. B. Reduction in p-IκBα is dose-dependent on cerdulatinib. CLL cells were stimulated with combined IL4+CD40L+plate-bound αIgM stimuli. C. Cerdulatinib decreased nuclear P65. Nuclear extracts were prepared from each sample and were immunoblotted for P65 and lamin B, a nuclear marker. D. DNA binding activity of NFκB subunit p50. Nuclear extracts were prepared from each sample. P50 activity was measured by ELISA plates coated with oligonucleotides containing the NF-kB consensus sequence (59-GGGACTTTCC-39). Concentration of p50 was determined by comparing samples with a standard curve of purified p50 protein, results represent mean±SE of 6 CLL samples. **** P

Techniques Used: Activity Assay, Marker, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Concentration Assay, Purification

Cerdulatinib, but not ibrutinib, is able to overcome the support of the microenvironment and induces CLL cell death A. Time course of cell viability following 2 µM cerdulatinb treatment in the presence or absence of stromal NKTert cells ( N = 14) (Left panel) or stromal HS-5 cells ( N = 31) (Right panel). B. Dose-titration of cerdulatinib treatment (72 hrs) in the presence of stromal NKTert cells ( N = 14) or HS-5 cells ( N = 31). C. Effect of 2 µM cerdulatinib or 0.5 µM ibrutinib on CLL viability in the presence of 10 ng/mL IL4, 1µg/mLl CD40L, and 10µg/mL plate-bound αIgM. Data in A-C were analyzed by ANOVA test. **, P
Figure Legend Snippet: Cerdulatinib, but not ibrutinib, is able to overcome the support of the microenvironment and induces CLL cell death A. Time course of cell viability following 2 µM cerdulatinb treatment in the presence or absence of stromal NKTert cells ( N = 14) (Left panel) or stromal HS-5 cells ( N = 31) (Right panel). B. Dose-titration of cerdulatinib treatment (72 hrs) in the presence of stromal NKTert cells ( N = 14) or HS-5 cells ( N = 31). C. Effect of 2 µM cerdulatinib or 0.5 µM ibrutinib on CLL viability in the presence of 10 ng/mL IL4, 1µg/mLl CD40L, and 10µg/mL plate-bound αIgM. Data in A-C were analyzed by ANOVA test. **, P

Techniques Used: Titration

Activity of cerdulatinib is transduced to inhibition of AKT and ERK A. Phospho-flow assays of p-AKT (Ser473) and p-ERK (Thr202/Tyr204), in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with soluble anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. B. Top panels, Mean fluorescence intensity (MFI) of p-AKT or p-ERK under indicated conditions ( N = 43). Data represents mean+SE. **** P
Figure Legend Snippet: Activity of cerdulatinib is transduced to inhibition of AKT and ERK A. Phospho-flow assays of p-AKT (Ser473) and p-ERK (Thr202/Tyr204), in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with soluble anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. B. Top panels, Mean fluorescence intensity (MFI) of p-AKT or p-ERK under indicated conditions ( N = 43). Data represents mean+SE. **** P

Techniques Used: Activity Assay, Inhibition, Fluorescence

9) Product Images from "Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation"

Article Title: Overexpression of T-bet in HIV infection is associated with accumulation of B cells outside germinal centers and poor affinity maturation

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aax0904

T H 1 and T FH cytokines modulate expression of T-bet and migration receptors. ( A ) Conventional flow cytometry performed on LN GCBC and CD19 hi MBC depicting the expression of Bcl6, T-bet, and CXCR5 expression of an HIV-infected individual. Red pattern and numbers identify Bcl6 frequencies within each quadrant. ( B ) Conventional flow cytometry performed on tonsillar B cells isolated from HIV-negative donors depicting Bcl6 and T-bet expression before and after 48 hours of stimulation of IgD + cells with anti-human BCR and CD40L with or without IFN-γ or IL-21. ( C ) Histograms and comparison ( n = 10) of T-bet and CXCR5 protein expression under conditions in (B). ( D ) Comparison ( n = 6) of gene expression for TBX21 , S1PR2 , and BACH2 under conditions in (B). * P
Figure Legend Snippet: T H 1 and T FH cytokines modulate expression of T-bet and migration receptors. ( A ) Conventional flow cytometry performed on LN GCBC and CD19 hi MBC depicting the expression of Bcl6, T-bet, and CXCR5 expression of an HIV-infected individual. Red pattern and numbers identify Bcl6 frequencies within each quadrant. ( B ) Conventional flow cytometry performed on tonsillar B cells isolated from HIV-negative donors depicting Bcl6 and T-bet expression before and after 48 hours of stimulation of IgD + cells with anti-human BCR and CD40L with or without IFN-γ or IL-21. ( C ) Histograms and comparison ( n = 10) of T-bet and CXCR5 protein expression under conditions in (B). ( D ) Comparison ( n = 6) of gene expression for TBX21 , S1PR2 , and BACH2 under conditions in (B). * P

Techniques Used: Expressing, Migration, Flow Cytometry, Infection, Isolation

10) Product Images from "Thyrotropin and CD40L Stimulate Interleukin-12 Expression in Fibrocytes: Implications for Pathogenesis of Thyroid-Associated Ophthalmopathy"

Article Title: Thyrotropin and CD40L Stimulate Interleukin-12 Expression in Fibrocytes: Implications for Pathogenesis of Thyroid-Associated Ophthalmopathy

Journal: Thyroid

doi: 10.1089/thy.2016.0243

TSH did not induce IL-12 promoter activity ( A ). The vector containing the promoter was transfected into fibrocytes and cells were incubated with TSH for 2 h. TSH stabilized IL-12p40 mRNA ( B , left panel), whereas CD40L did not affect mRNA stability ( B , right panel). IL-12 mRNA production was stimulated for 12 h with TSH. Cells were then washed and treated with 50 μM of DRB alone, DRB and TSH, or DRB and CD40L (time zero). Degradation of existing mRNA was slower in the presence of TSH and DRB (●) than in the presence of DRB alone (◯), whereas mRNA degradation proceeded at similar rate in the absence (□) and presence (■) of CD40L. TSH stimulation of IL-12p40 mRNA depended on de novo protein production ( C , left panel). Adding cycloheximide (10 μg/mL) 1 h before TSH stimulation significantly lowered the TSH-induced IL-12p40 mRNA expression. Adding cycloheximide did not affect CD40L stimulation of IL-12p40 mRNA ( C , right panel), indicating that CD40L does not depend on de novo protein synthesis.
Figure Legend Snippet: TSH did not induce IL-12 promoter activity ( A ). The vector containing the promoter was transfected into fibrocytes and cells were incubated with TSH for 2 h. TSH stabilized IL-12p40 mRNA ( B , left panel), whereas CD40L did not affect mRNA stability ( B , right panel). IL-12 mRNA production was stimulated for 12 h with TSH. Cells were then washed and treated with 50 μM of DRB alone, DRB and TSH, or DRB and CD40L (time zero). Degradation of existing mRNA was slower in the presence of TSH and DRB (●) than in the presence of DRB alone (◯), whereas mRNA degradation proceeded at similar rate in the absence (□) and presence (■) of CD40L. TSH stimulation of IL-12p40 mRNA depended on de novo protein production ( C , left panel). Adding cycloheximide (10 μg/mL) 1 h before TSH stimulation significantly lowered the TSH-induced IL-12p40 mRNA expression. Adding cycloheximide did not affect CD40L stimulation of IL-12p40 mRNA ( C , right panel), indicating that CD40L does not depend on de novo protein synthesis.

Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Incubation, Expressing

CD40 signaling of IL-12 production proceeded through TRAF6 in fibrocytes. Two doses of 50 μM control peptide or TRAF6 or TRAF2 inhibitor peptide were added to cells 24 h and 1 h before CD40L stimulation. Six hours after CD40L stimulation, RNA was isolated, and IL-12p40 mRNA levels were measured by real-time PCR. TRAF6 inhibitor peptide significantly inhibited the action of CD40L, whereas the TRAF2 peptide had no effect.
Figure Legend Snippet: CD40 signaling of IL-12 production proceeded through TRAF6 in fibrocytes. Two doses of 50 μM control peptide or TRAF6 or TRAF2 inhibitor peptide were added to cells 24 h and 1 h before CD40L stimulation. Six hours after CD40L stimulation, RNA was isolated, and IL-12p40 mRNA levels were measured by real-time PCR. TRAF6 inhibitor peptide significantly inhibited the action of CD40L, whereas the TRAF2 peptide had no effect.

Techniques Used: Isolation, Real-time Polymerase Chain Reaction

TSH and CD40L induced IL-12 at the pre-translational level. Steady-state IL-12p40 mRNA expression stimulated with TSH (▲) or CD40L (■) was followed by real-time polymerase chain reaction (PCR) in cultured fibrocytes for 24 h ( A ). IL-12p40 mRNA production increased responding to TSH, M22, and CD40L stimulation in fibrocytes from healthy controls ( B , left panel) and from patients with GD ( B , right panel).
Figure Legend Snippet: TSH and CD40L induced IL-12 at the pre-translational level. Steady-state IL-12p40 mRNA expression stimulated with TSH (▲) or CD40L (■) was followed by real-time polymerase chain reaction (PCR) in cultured fibrocytes for 24 h ( A ). IL-12p40 mRNA production increased responding to TSH, M22, and CD40L stimulation in fibrocytes from healthy controls ( B , left panel) and from patients with GD ( B , right panel).

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture

Thyrotropin (TSH) and CD40L induced interleukin-12 (IL-12) production in circulating fibrocytes ( A ) and in cultured fibrocytes ( B ). ( A ) Freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 18 h. Flow cytometry showed that TSH and CD40L induced IL-12p40/p70 (the combined p40 monomer and p70 heterodimer) in circulating fibrocytes, resulting in MFI values that were 1.54- and 1.25-fold higher, respectively, than in untreated cells. The MFIs of IL-12 p70 (heterodimer) were 2.43- and 2.33-fold higher after TSH or CD40L was added (right panels). ( B ) The fibrocytes in cultures were treated with TSH and CD40L for 48 h. The time course of extracellular IL-12p40/p70 concentrations shows that TSH (▲) and CD40L (■) stimulated IL-12 production, whereas unstimulated cells (◯) produced negligible amounts. Peak production was reached at 24 h ( p
Figure Legend Snippet: Thyrotropin (TSH) and CD40L induced interleukin-12 (IL-12) production in circulating fibrocytes ( A ) and in cultured fibrocytes ( B ). ( A ) Freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 18 h. Flow cytometry showed that TSH and CD40L induced IL-12p40/p70 (the combined p40 monomer and p70 heterodimer) in circulating fibrocytes, resulting in MFI values that were 1.54- and 1.25-fold higher, respectively, than in untreated cells. The MFIs of IL-12 p70 (heterodimer) were 2.43- and 2.33-fold higher after TSH or CD40L was added (right panels). ( B ) The fibrocytes in cultures were treated with TSH and CD40L for 48 h. The time course of extracellular IL-12p40/p70 concentrations shows that TSH (▲) and CD40L (■) stimulated IL-12 production, whereas unstimulated cells (◯) produced negligible amounts. Peak production was reached at 24 h ( p

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

TSH and CD40L induced IL-12p40 mRNA expression through Akt ( A ) and NF-κB ( B ) signaling in cultured fibrocytes. Akt inhibitor (AKTi) and NF-κB inhibitor (MG132) were added 1 h before TSH or CD40L stimulation. RNA was isolated after 6 h of induction. AKTi and MG132 blocked IL-12p40 mRNA expression induced by TSH ( p
Figure Legend Snippet: TSH and CD40L induced IL-12p40 mRNA expression through Akt ( A ) and NF-κB ( B ) signaling in cultured fibrocytes. Akt inhibitor (AKTi) and NF-κB inhibitor (MG132) were added 1 h before TSH or CD40L stimulation. RNA was isolated after 6 h of induction. AKTi and MG132 blocked IL-12p40 mRNA expression induced by TSH ( p

Techniques Used: Expressing, Cell Culture, Isolation

11) Product Images from "MicroRNA-155 influences B-cell function through PU.1 in rheumatoid arthritis"

Article Title: MicroRNA-155 influences B-cell function through PU.1 in rheumatoid arthritis

Journal: Nature Communications

doi: 10.1038/ncomms12970

Rationale for the use of antagomiR-155 in the treatment of RA synovitis. ( a ) miR-155 control of SHIP-1 and PU.1 is crucial for the regulation of inflammatory response in macrophages and for antibody production and terminal differentiation of B cells; ( b ) In RA synovial compartment, macrophages and B cells are characterized by higher expression of miR-155 due to endogenous TLR ligands 6 and other soluble factors, for example, CD40L, IL21, CpG, IFN-alpha, IL-6 and BAFF. This leads to the downregulation of SHIP-1 and PU.1 expression and thus to the amplification of the inflammatory cascade by macrophages and increase in antibody production and B-cell maturation; ( c ) Thus, the administration of antagomiR-155 could restore the normal expression of SHIP-1 and PU.1 acting both on macrophages and B cells and facilitate the resolution of autoimmunity and inflammation in RA. SHIP-1, inositol polyphosphate-5-phosphatase; PU.1, Spi-1 proto-oncogene; TLR, Toll like receptor BAFF, B-cell activating factor; CD40L, CD40 ligand, BCR, B-cell receptor.
Figure Legend Snippet: Rationale for the use of antagomiR-155 in the treatment of RA synovitis. ( a ) miR-155 control of SHIP-1 and PU.1 is crucial for the regulation of inflammatory response in macrophages and for antibody production and terminal differentiation of B cells; ( b ) In RA synovial compartment, macrophages and B cells are characterized by higher expression of miR-155 due to endogenous TLR ligands 6 and other soluble factors, for example, CD40L, IL21, CpG, IFN-alpha, IL-6 and BAFF. This leads to the downregulation of SHIP-1 and PU.1 expression and thus to the amplification of the inflammatory cascade by macrophages and increase in antibody production and B-cell maturation; ( c ) Thus, the administration of antagomiR-155 could restore the normal expression of SHIP-1 and PU.1 acting both on macrophages and B cells and facilitate the resolution of autoimmunity and inflammation in RA. SHIP-1, inositol polyphosphate-5-phosphatase; PU.1, Spi-1 proto-oncogene; TLR, Toll like receptor BAFF, B-cell activating factor; CD40L, CD40 ligand, BCR, B-cell receptor.

Techniques Used: Expressing, Amplification

PU.1 expression in CD19 + cells of RA patients. ( a ) Expression of PU.1 in CD19 + cells isolated from PB and SF of RA patients compared to HC, ** P =0.002 SF-derived CD19 + cells from ACPA + RA versus HC; * P =0.001 SF-derived CD19 + cells from ACPA - RA versus HC; and § P =0.001 SF-derived CD19 + cells from ACPA + RA versus PB-derived CD19 + cells from ACPA + RA; data are shown as mean±s.d., 25–75 percentiles; Mann-Whitney U -test. ( b ) Expression of PU.1 in CD19 + cells in paired RA PB and SF samples; * P =0.002; Wilcoxon test; ( c ) Inverse correlation between miR-155 and PU.1 expression in SF derived CD19 + cells in RA patients [ r = −0.65; P =0.04; 95%CI (−0.38 to −0.88)]; Spearman Rank Correlation coefficient; ( d ) PU.1 expression in PB CD19 + cells isolated from HC ( n =5) after in vitro stimulation with CD40L (2 μg/ml), IL-21 (50 ng/ml), anti-IgM (10 μg/ml), CpG (1 μg/ml), Interferon alpha (400 IU/ml), IL-6 (30 ng/ml) and BAFF (20 ng/ml) for 24–72 h; * P
Figure Legend Snippet: PU.1 expression in CD19 + cells of RA patients. ( a ) Expression of PU.1 in CD19 + cells isolated from PB and SF of RA patients compared to HC, ** P =0.002 SF-derived CD19 + cells from ACPA + RA versus HC; * P =0.001 SF-derived CD19 + cells from ACPA - RA versus HC; and § P =0.001 SF-derived CD19 + cells from ACPA + RA versus PB-derived CD19 + cells from ACPA + RA; data are shown as mean±s.d., 25–75 percentiles; Mann-Whitney U -test. ( b ) Expression of PU.1 in CD19 + cells in paired RA PB and SF samples; * P =0.002; Wilcoxon test; ( c ) Inverse correlation between miR-155 and PU.1 expression in SF derived CD19 + cells in RA patients [ r = −0.65; P =0.04; 95%CI (−0.38 to −0.88)]; Spearman Rank Correlation coefficient; ( d ) PU.1 expression in PB CD19 + cells isolated from HC ( n =5) after in vitro stimulation with CD40L (2 μg/ml), IL-21 (50 ng/ml), anti-IgM (10 μg/ml), CpG (1 μg/ml), Interferon alpha (400 IU/ml), IL-6 (30 ng/ml) and BAFF (20 ng/ml) for 24–72 h; * P

Techniques Used: Expressing, Isolation, Derivative Assay, MANN-WHITNEY, In Vitro

miR-155 in CD19 + cells is induced by inflammatory and maturation factors. miR-155 expression in PB CD19 + cells isolated from HC ( n =5) after in vitro stimulation with CD40L (2 μg/ml), IL-21 (50 ng/ml), anti-IgM (10 μg/ml), CpG (1 μg/ml), Interferon alpha (400 IU/ml), IL-6 (30 ng/ml) and BAFF (20 ng/ml) for 24–72 h; * P
Figure Legend Snippet: miR-155 in CD19 + cells is induced by inflammatory and maturation factors. miR-155 expression in PB CD19 + cells isolated from HC ( n =5) after in vitro stimulation with CD40L (2 μg/ml), IL-21 (50 ng/ml), anti-IgM (10 μg/ml), CpG (1 μg/ml), Interferon alpha (400 IU/ml), IL-6 (30 ng/ml) and BAFF (20 ng/ml) for 24–72 h; * P

Techniques Used: Expressing, Isolation, In Vitro

12) Product Images from "NF-κB-inducing kinase is a key regulator of inflammation-induced and tumour-associated angiogenesis"

Article Title: NF-κB-inducing kinase is a key regulator of inflammation-induced and tumour-associated angiogenesis

Journal: The Journal of Pathology

doi: 10.1002/path.4403

Non-canonical NF-κB signalling induces in vitro sprouting of endothelial cells. (A) Representative pictures of tube formation by HMVECs under basal conditions or after LT, LIGHT, and CD40L stimulation (scale bar for all panels = 500 µm). (B) Quantification of LT-, LIGHT-, and CD40L-enhanced tube formation. n = 7 per group, three independent experiments. (C–F) HMVECs were transfected by the indicated siRNAs. Scrambled non-targeting siRNA (=siC) was used as a control. (C) Basal tube formation; (D) LT-enhanced tube formation; (E) LIGHT-enhanced tube formation; (F) CD40L-enhanced tube formation. n = 6 per group, three independent experiments. All panels: mean ± SEM. * p
Figure Legend Snippet: Non-canonical NF-κB signalling induces in vitro sprouting of endothelial cells. (A) Representative pictures of tube formation by HMVECs under basal conditions or after LT, LIGHT, and CD40L stimulation (scale bar for all panels = 500 µm). (B) Quantification of LT-, LIGHT-, and CD40L-enhanced tube formation. n = 7 per group, three independent experiments. (C–F) HMVECs were transfected by the indicated siRNAs. Scrambled non-targeting siRNA (=siC) was used as a control. (C) Basal tube formation; (D) LT-enhanced tube formation; (E) LIGHT-enhanced tube formation; (F) CD40L-enhanced tube formation. n = 6 per group, three independent experiments. All panels: mean ± SEM. * p

Techniques Used: In Vitro, Transfection

13) Product Images from "Epistatic interactions between mutations of TACI (TNFRSF13B) and TCF3 result in a severe primary immunodeficiency disorder and systemic lupus erythematosus"

Article Title: Epistatic interactions between mutations of TACI (TNFRSF13B) and TCF3 result in a severe primary immunodeficiency disorder and systemic lupus erythematosus

Journal: Clinical & Translational Immunology

doi: 10.1038/cti.2017.41

Severe defect in in vitro antibody production in proband demonstrates epistasis. ( a ) Immunoglobulin production from supernatants collected from in vitro cultures of naïve B cells isolated from PBMCs of each family member, stimulated as indicated with CD40L (100 ng ml –1 ), IL-4 (50 ng ml –1 ), IL-21 (50 ng ml –1 ), CpG (1 μg ml –1 ) and APRIL (500 ng ml –1 ). Supernatants were assessed for secretion of IgG, IgA and IgM as indicated. ( b ) Representative Cell Trace Violet (CTV) plots and IgG isotype switched cells following in vitro stimulation of naïve B cells with CD40L+IL-4+IL-21 for 6 days (representative from two independent experiments). Cells were isolated, labelled with CTV, stimulated and collected after 6 days of culture and the division profiles and proportions of IgG expressing cells determined. FMO, fluorochrome minus one.
Figure Legend Snippet: Severe defect in in vitro antibody production in proband demonstrates epistasis. ( a ) Immunoglobulin production from supernatants collected from in vitro cultures of naïve B cells isolated from PBMCs of each family member, stimulated as indicated with CD40L (100 ng ml –1 ), IL-4 (50 ng ml –1 ), IL-21 (50 ng ml –1 ), CpG (1 μg ml –1 ) and APRIL (500 ng ml –1 ). Supernatants were assessed for secretion of IgG, IgA and IgM as indicated. ( b ) Representative Cell Trace Violet (CTV) plots and IgG isotype switched cells following in vitro stimulation of naïve B cells with CD40L+IL-4+IL-21 for 6 days (representative from two independent experiments). Cells were isolated, labelled with CTV, stimulated and collected after 6 days of culture and the division profiles and proportions of IgG expressing cells determined. FMO, fluorochrome minus one.

Techniques Used: In Vitro, Isolation, Expressing

Severe defect in generation of antibody secreting cells in E2A/TACI-deficient cells. ( a – c ) Summary graphs from in vitro proliferation of naïve B cells stimulated as indicated with CD40L (100 ng ml −1 ), IL-4 (50 ng ml −1 ), IL-21 (50 ng ml −1 ), CpG (1 μg ml −1 ) and APRIL (500 ng ml −1 ) as indicated. Isolated cells were collected after 5 days of culture, cell surface stained and analysed by flow cytometry for the ( a ) proportion of antibody secreting cells (ASC, CD27 hi CD38 + ) and ( b ) total number of ASC and ( c ) total lymphocyte number. Cell counts and proportion of ASC are shown for the proband, with both TNFRSF13B /TACI and TCF3 mutations in white; her son (III.1), expressing TCF3 T168fsX191 mutant B cells only (blue); TACI-deficient individuals (II.3, II.4, gray); and wild-type (III.2 and HD, black). Summary graphs of the proportions and total number of differentiated cells for all family members and healthy donors (HD, n =4) was performed in two independent experiments.
Figure Legend Snippet: Severe defect in generation of antibody secreting cells in E2A/TACI-deficient cells. ( a – c ) Summary graphs from in vitro proliferation of naïve B cells stimulated as indicated with CD40L (100 ng ml −1 ), IL-4 (50 ng ml −1 ), IL-21 (50 ng ml −1 ), CpG (1 μg ml −1 ) and APRIL (500 ng ml −1 ) as indicated. Isolated cells were collected after 5 days of culture, cell surface stained and analysed by flow cytometry for the ( a ) proportion of antibody secreting cells (ASC, CD27 hi CD38 + ) and ( b ) total number of ASC and ( c ) total lymphocyte number. Cell counts and proportion of ASC are shown for the proband, with both TNFRSF13B /TACI and TCF3 mutations in white; her son (III.1), expressing TCF3 T168fsX191 mutant B cells only (blue); TACI-deficient individuals (II.3, II.4, gray); and wild-type (III.2 and HD, black). Summary graphs of the proportions and total number of differentiated cells for all family members and healthy donors (HD, n =4) was performed in two independent experiments.

Techniques Used: In Vitro, Isolation, Staining, Flow Cytometry, Cytometry, Expressing, Mutagenesis

14) Product Images from "Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses"

Article Title: Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses

Journal: bioRxiv

doi: 10.1101/2020.06.01.127514

LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws PBMC were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hours, in the abence/presence of Bafilomycin A1 for the last 2h. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. ( a ) Monocytes gated on CD14+ treated with IFNg or LPS, ( b ) B cells gated on CD19+ treated with anti-CD40L and anti-IgM, ( c ) CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28, ( d ) CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28.
Figure Legend Snippet: LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws PBMC were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hours, in the abence/presence of Bafilomycin A1 for the last 2h. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. ( a ) Monocytes gated on CD14+ treated with IFNg or LPS, ( b ) B cells gated on CD19+ treated with anti-CD40L and anti-IgM, ( c ) CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28, ( d ) CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28.

Techniques Used: Flow Cytometry, Generated, Cell Culture, Fluorescence

15) Product Images from "B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients"

Article Title: B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients

Journal: The Journal of allergy and clinical immunology

doi: 10.1016/j.jaci.2013.12.1043

Impaired B-cell proliferation after BCR/TLR triggering in patients undergoing ERT is corrected after HSC-GT. A, Percentage of CFSE-diluting B cells in patients undergoing HSC-GT (n = 4 for CpG stimulation and n = 6 when either immunoglobulin or CD40L
Figure Legend Snippet: Impaired B-cell proliferation after BCR/TLR triggering in patients undergoing ERT is corrected after HSC-GT. A, Percentage of CFSE-diluting B cells in patients undergoing HSC-GT (n = 4 for CpG stimulation and n = 6 when either immunoglobulin or CD40L

Techniques Used:

16) Product Images from "Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response"

Article Title: Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

doi: 10.1002/cyto.a.23737

Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3 − CD19 + CD20 ++ CD38 + and non-GC B cells as CD3 − CD19 + CD38 − . The non-GC population was further gated to enrich for IgD + naïve B cells (possibly containing unswitched memory B cells) and IgD − memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p
Figure Legend Snippet: Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3 − CD19 + CD20 ++ CD38 + and non-GC B cells as CD3 − CD19 + CD38 − . The non-GC population was further gated to enrich for IgD + naïve B cells (possibly containing unswitched memory B cells) and IgD − memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p

Techniques Used: Expressing, Mass Cytometry

CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils. CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20 hi CD38 + , non-GC (CD20 + CD38 − ) were split into CD27 − IgD + naïve and CD27 + IgD − memory B cells. * p
Figure Legend Snippet: CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils. CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20 hi CD38 + , non-GC (CD20 + CD38 − ) were split into CD27 − IgD + naïve and CD27 + IgD − memory B cells. * p

Techniques Used: Fluorescence, Flow Cytometry

CD40L-induced IκBα degradation is not proportional to level of CD40L binding CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20 + CD38 − and GC B cells as CD20 hi CD38 + . (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p
Figure Legend Snippet: CD40L-induced IκBα degradation is not proportional to level of CD40L binding CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20 + CD38 − and GC B cells as CD20 hi CD38 + . (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p

Techniques Used: Binding Assay, Fluorescence, Flow Cytometry

17) Product Images from "Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment"

Article Title: Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment

Journal: Oncotarget

doi: 10.18632/oncotarget.14588

Cerdulatinib effectively blocks BCR and JAK-STAT signaling pathways A. Immunoblots for early BCR components SYK and BTK following cerdulatinib treatment. Freshly isolated CLL cells ( N = 6) were treated with increasing doses of cerdulatinib in the presence of combined stimulation with soluble anti-IgM, CD40L and CpG. p-SYK, total SYK, p-BTK and total BTK were blotted in whole cell extracts. GAPDH served as a loading control. B. Left panel, phospho-flow assay of p-PLCγ2 (Tyr759) in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. Middle panel, Mean fluorescence intensity (MFI) of p-PLCγ2 under indicated conditions ( N = 43). Data represents mean+SE. **** P
Figure Legend Snippet: Cerdulatinib effectively blocks BCR and JAK-STAT signaling pathways A. Immunoblots for early BCR components SYK and BTK following cerdulatinib treatment. Freshly isolated CLL cells ( N = 6) were treated with increasing doses of cerdulatinib in the presence of combined stimulation with soluble anti-IgM, CD40L and CpG. p-SYK, total SYK, p-BTK and total BTK were blotted in whole cell extracts. GAPDH served as a loading control. B. Left panel, phospho-flow assay of p-PLCγ2 (Tyr759) in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. Middle panel, Mean fluorescence intensity (MFI) of p-PLCγ2 under indicated conditions ( N = 43). Data represents mean+SE. **** P

Techniques Used: Western Blot, Isolation, Flow Cytometry, Fluorescence

Cerdulatinib inhibits the activity of NF-κB pathway A. Phosphorylation of IκBα following cerdulatinib treatment. CLL cells were stimulated with either plate-bound αIgM alone or combined IL4+CD40L+plate-bound αIgM stimuli, and treated with 2 µM cerdulatnib for 24 hrs. Whole Cell lysates were immunoblotted for p-IκBα and GAPDH as the loading control. Four representative blots are shown. B. Reduction in p-IκBα is dose-dependent on cerdulatinib. CLL cells were stimulated with combined IL4+CD40L+plate-bound αIgM stimuli. C. Cerdulatinib decreased nuclear P65. Nuclear extracts were prepared from each sample and were immunoblotted for P65 and lamin B, a nuclear marker. D. DNA binding activity of NFκB subunit p50. Nuclear extracts were prepared from each sample. P50 activity was measured by ELISA plates coated with oligonucleotides containing the NF-kB consensus sequence (59-GGGACTTTCC-39). Concentration of p50 was determined by comparing samples with a standard curve of purified p50 protein, results represent mean±SE of 6 CLL samples. **** P
Figure Legend Snippet: Cerdulatinib inhibits the activity of NF-κB pathway A. Phosphorylation of IκBα following cerdulatinib treatment. CLL cells were stimulated with either plate-bound αIgM alone or combined IL4+CD40L+plate-bound αIgM stimuli, and treated with 2 µM cerdulatnib for 24 hrs. Whole Cell lysates were immunoblotted for p-IκBα and GAPDH as the loading control. Four representative blots are shown. B. Reduction in p-IκBα is dose-dependent on cerdulatinib. CLL cells were stimulated with combined IL4+CD40L+plate-bound αIgM stimuli. C. Cerdulatinib decreased nuclear P65. Nuclear extracts were prepared from each sample and were immunoblotted for P65 and lamin B, a nuclear marker. D. DNA binding activity of NFκB subunit p50. Nuclear extracts were prepared from each sample. P50 activity was measured by ELISA plates coated with oligonucleotides containing the NF-kB consensus sequence (59-GGGACTTTCC-39). Concentration of p50 was determined by comparing samples with a standard curve of purified p50 protein, results represent mean±SE of 6 CLL samples. **** P

Techniques Used: Activity Assay, Marker, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Concentration Assay, Purification

Cerdulatinib, but not ibrutinib, is able to overcome the support of the microenvironment and induces CLL cell death A. Time course of cell viability following 2 µM cerdulatinb treatment in the presence or absence of stromal NKTert cells ( N = 14) (Left panel) or stromal HS-5 cells ( N = 31) (Right panel). B. Dose-titration of cerdulatinib treatment (72 hrs) in the presence of stromal NKTert cells ( N = 14) or HS-5 cells ( N = 31). C. Effect of 2 µM cerdulatinib or 0.5 µM ibrutinib on CLL viability in the presence of 10 ng/mL IL4, 1µg/mLl CD40L, and 10µg/mL plate-bound αIgM. Data in A-C were analyzed by ANOVA test. **, P
Figure Legend Snippet: Cerdulatinib, but not ibrutinib, is able to overcome the support of the microenvironment and induces CLL cell death A. Time course of cell viability following 2 µM cerdulatinb treatment in the presence or absence of stromal NKTert cells ( N = 14) (Left panel) or stromal HS-5 cells ( N = 31) (Right panel). B. Dose-titration of cerdulatinib treatment (72 hrs) in the presence of stromal NKTert cells ( N = 14) or HS-5 cells ( N = 31). C. Effect of 2 µM cerdulatinib or 0.5 µM ibrutinib on CLL viability in the presence of 10 ng/mL IL4, 1µg/mLl CD40L, and 10µg/mL plate-bound αIgM. Data in A-C were analyzed by ANOVA test. **, P

Techniques Used: Titration

Activity of cerdulatinib is transduced to inhibition of AKT and ERK A. Phospho-flow assays of p-AKT (Ser473) and p-ERK (Thr202/Tyr204), in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with soluble anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. B. Top panels, Mean fluorescence intensity (MFI) of p-AKT or p-ERK under indicated conditions ( N = 43). Data represents mean+SE. **** P
Figure Legend Snippet: Activity of cerdulatinib is transduced to inhibition of AKT and ERK A. Phospho-flow assays of p-AKT (Ser473) and p-ERK (Thr202/Tyr204), in 4 representative cases under different conditions. CLL cells were treated with 2 µM of cerdulatinib for 1 hr before stimulation with soluble anti-IgM, IL-4 and CD40L. Unsti, unstimulated cells. Sti, cells stimulated with combined stimuli. Sti+Cerd, stimulated cells with exposure to 2 µM cerdulatinib. B. Top panels, Mean fluorescence intensity (MFI) of p-AKT or p-ERK under indicated conditions ( N = 43). Data represents mean+SE. **** P

Techniques Used: Activity Assay, Inhibition, Flow Cytometry, Fluorescence

18) Product Images from "Clinical Adjuvant Combinations Stimulate Potent B-Cell Responses In Vitro by Activating Dermal Dendritic Cells"

Article Title: Clinical Adjuvant Combinations Stimulate Potent B-Cell Responses In Vitro by Activating Dermal Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063785

CD14 + DDCs stimulated with two TLR ligands have an increased capacity to induce naïve B-cells to proliferate and differentiate into Ig-secreting B-cells. (A) Proliferation of B-cells after stimulation with TLR ligands in the presence or absence of CD14 + DDCs, as measured by BrdU incorporation. The following concentrations of TLR ligands were used: Hiltonol (2.5 µg/ml), GLA (125 ng/ml) and R-848 (0.6 µg/ml). Naïve B-cells were isolated and cultured for 5 days with TLR ligands in the presence of CD40L and IL-2, and with or without CD14 + DDCs (DC: B-cell ratio of 1∶5). The bars represent mean O.D. values (± SEM) derived from triplicate samples from each of three donors. ‡ Wilcoxon matched-pairs test. (B) Phenotypic analysis of B-cells after 7 days of stimulation with TLR ligands in the presence or absence of CD14 + DDCs (plus CD40L and IL-2). The numbers denote the percentage of CD38 + CD27 + B-cells. (C) Ig secretion in B-cell cultures stimulated for 14 days with the indicated TLR ligands, and CD40L plus IL-2, in the presence or absence of CD14 + DDCs. Ig secretion was quantified by ELISA. Data are presented as means ± SEM of duplicates and are representative of three independent experiments.
Figure Legend Snippet: CD14 + DDCs stimulated with two TLR ligands have an increased capacity to induce naïve B-cells to proliferate and differentiate into Ig-secreting B-cells. (A) Proliferation of B-cells after stimulation with TLR ligands in the presence or absence of CD14 + DDCs, as measured by BrdU incorporation. The following concentrations of TLR ligands were used: Hiltonol (2.5 µg/ml), GLA (125 ng/ml) and R-848 (0.6 µg/ml). Naïve B-cells were isolated and cultured for 5 days with TLR ligands in the presence of CD40L and IL-2, and with or without CD14 + DDCs (DC: B-cell ratio of 1∶5). The bars represent mean O.D. values (± SEM) derived from triplicate samples from each of three donors. ‡ Wilcoxon matched-pairs test. (B) Phenotypic analysis of B-cells after 7 days of stimulation with TLR ligands in the presence or absence of CD14 + DDCs (plus CD40L and IL-2). The numbers denote the percentage of CD38 + CD27 + B-cells. (C) Ig secretion in B-cell cultures stimulated for 14 days with the indicated TLR ligands, and CD40L plus IL-2, in the presence or absence of CD14 + DDCs. Ig secretion was quantified by ELISA. Data are presented as means ± SEM of duplicates and are representative of three independent experiments.

Techniques Used: BrdU Incorporation Assay, Isolation, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay

19) Product Images from "CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes"

Article Title: CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200208083

Binding of Hsp70 complex to CD40-expressing HEK293T cells induces signaling via p38 and causes peptide uptake. (A) HEK293T cells, stably transfected with cDNA encoding human CD40 or the unrelated membrane protein (MCAT) were incubated for 20 min at 37°C with CD40L or with Hsp70, N70, C70, or bacterial DnaK in the presence of peptide C and ADP or AMPPNP, or buffer alone. Thereafter, cells were washed, solubilized in SDS-sample buffer, and analyzed by immunoblotting with antibodies directed against phosphorylated (active) p38. Blots were also developed with an antibody against tubulin in order to control for equal loading. (B) Cells were incubated at 0°C for 30 min with recombinant human Hsp70, N70, or C70, all in the presence FITC-labeled peptide, or with the FITC-labeled peptide alone. Thereafter, cells were washed, incubated for 15 min at 37°C, and processed for fluorescence microscopy. (1) Hsp70, (2) FITC-labeled peptide alone, (3) C70, and (4) N70. Only cells stably transfected with CD40 are shown. Cell boundaries are emphasized with broken lines. MCAT-expressing control cells did not show fluorescence above background.
Figure Legend Snippet: Binding of Hsp70 complex to CD40-expressing HEK293T cells induces signaling via p38 and causes peptide uptake. (A) HEK293T cells, stably transfected with cDNA encoding human CD40 or the unrelated membrane protein (MCAT) were incubated for 20 min at 37°C with CD40L or with Hsp70, N70, C70, or bacterial DnaK in the presence of peptide C and ADP or AMPPNP, or buffer alone. Thereafter, cells were washed, solubilized in SDS-sample buffer, and analyzed by immunoblotting with antibodies directed against phosphorylated (active) p38. Blots were also developed with an antibody against tubulin in order to control for equal loading. (B) Cells were incubated at 0°C for 30 min with recombinant human Hsp70, N70, or C70, all in the presence FITC-labeled peptide, or with the FITC-labeled peptide alone. Thereafter, cells were washed, incubated for 15 min at 37°C, and processed for fluorescence microscopy. (1) Hsp70, (2) FITC-labeled peptide alone, (3) C70, and (4) N70. Only cells stably transfected with CD40 are shown. Cell boundaries are emphasized with broken lines. MCAT-expressing control cells did not show fluorescence above background.

Techniques Used: Binding Assay, Expressing, Stable Transfection, Transfection, Incubation, Recombinant, Labeling, Fluorescence, Microscopy

20) Product Images from "Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response"

Article Title: Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

doi: 10.1002/cyto.a.23737

Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3 − CD19 + CD20 ++ CD38 + and non-GC B cells as CD3 − CD19 + CD38 − . The non-GC population was further gated to enrich for IgD + naïve B cells (possibly containing unswitched memory B cells) and IgD − memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p
Figure Legend Snippet: Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3 − CD19 + CD20 ++ CD38 + and non-GC B cells as CD3 − CD19 + CD38 − . The non-GC population was further gated to enrich for IgD + naïve B cells (possibly containing unswitched memory B cells) and IgD − memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p

Techniques Used: Expressing, Mass Cytometry

CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils. CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20 hi CD38 + , non-GC (CD20 + CD38 − ) were split into CD27 − IgD + naïve and CD27 + IgD − memory B cells. * p
Figure Legend Snippet: CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils. CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20 hi CD38 + , non-GC (CD20 + CD38 − ) were split into CD27 − IgD + naïve and CD27 + IgD − memory B cells. * p

Techniques Used: Fluorescence, Flow Cytometry

CD40L-induced IκBα degradation is not proportional to level of CD40L binding CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20 + CD38 − and GC B cells as CD20 hi CD38 + . (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p
Figure Legend Snippet: CD40L-induced IκBα degradation is not proportional to level of CD40L binding CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20 + CD38 − and GC B cells as CD20 hi CD38 + . (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p

Techniques Used: Binding Assay, Fluorescence, Flow Cytometry

21) Product Images from "Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence"

Article Title: Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence

Journal: Molecular Cell

doi: 10.1016/j.molcel.2019.08.005

Spermidine Induces TFEB Expression and Improves the Function of Old Human B Cells (A) The protein levels of TFEB, hypusinated eIF5A, and overall eIF5A of PBMCs from healthy human donors of indicated ages were assessed using western blot. A representative plot (left) and quantifications (right) are shown. n = 8–15 donors. (B) TFEB mRNA of human PBMCs was measured using qPCR with GAPDH as the reference gene. n = 7–9 donors. (C) Sorted B cells from young human donors were cultured with anti-IgM/CD40L and treated with DFMO with or without 10 μM spermidine for 7 days. n = 5 donors. (D) Sorted B cells from human donors were cultured as in (C) with spermidine and/or GC7 as indicated. The expression of hypusinated (Hyp) or non-hypusinated (AcLys or Lys) eIF5A was distinguished by two-dimensional western blot of eIF5A. Black arrow, hypusinated Lys 50 , pH 5.2; red arrow, unmodified Lys 50 , pH 5.1; blue arrow, acetylated Lys 47 with unmodified Lys 50 , pH 5.0. The non-hypusination ratio was calculated as eIF5A dot densitometric intensity (AcLys + Lys)/(AcLys + Lys + Hyp). n = 4 donors. (E–G) Sorted B cells from old human donors (age 77.5 ± 6.3 years) were cultured as in (C) together with spermidine and GC7. (E) The protein levels of TFEB and eIF5A hypusination were measured using western blot. (F) Autophagic flux was determined by flow cytometry as in Figure 1 A. (G) Supernatant IgG was assessed using ELISA. n = 4–14 donors. Data are represented as mean ± SEM. Unpaired two-tailed Student’s t test (A and D, comparison of young versus old). Welch’s t test (B). Paired one-way ANOVA with post hoc Tukey’s test (C). Paired one-tailed t test (D, comparisons of old versus old + Spd, old + Spd versus old + Spd + GC7 comparison, E–G). ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .
Figure Legend Snippet: Spermidine Induces TFEB Expression and Improves the Function of Old Human B Cells (A) The protein levels of TFEB, hypusinated eIF5A, and overall eIF5A of PBMCs from healthy human donors of indicated ages were assessed using western blot. A representative plot (left) and quantifications (right) are shown. n = 8–15 donors. (B) TFEB mRNA of human PBMCs was measured using qPCR with GAPDH as the reference gene. n = 7–9 donors. (C) Sorted B cells from young human donors were cultured with anti-IgM/CD40L and treated with DFMO with or without 10 μM spermidine for 7 days. n = 5 donors. (D) Sorted B cells from human donors were cultured as in (C) with spermidine and/or GC7 as indicated. The expression of hypusinated (Hyp) or non-hypusinated (AcLys or Lys) eIF5A was distinguished by two-dimensional western blot of eIF5A. Black arrow, hypusinated Lys 50 , pH 5.2; red arrow, unmodified Lys 50 , pH 5.1; blue arrow, acetylated Lys 47 with unmodified Lys 50 , pH 5.0. The non-hypusination ratio was calculated as eIF5A dot densitometric intensity (AcLys + Lys)/(AcLys + Lys + Hyp). n = 4 donors. (E–G) Sorted B cells from old human donors (age 77.5 ± 6.3 years) were cultured as in (C) together with spermidine and GC7. (E) The protein levels of TFEB and eIF5A hypusination were measured using western blot. (F) Autophagic flux was determined by flow cytometry as in Figure 1 A. (G) Supernatant IgG was assessed using ELISA. n = 4–14 donors. Data are represented as mean ± SEM. Unpaired two-tailed Student’s t test (A and D, comparison of young versus old). Welch’s t test (B). Paired one-way ANOVA with post hoc Tukey’s test (C). Paired one-tailed t test (D, comparisons of old versus old + Spd, old + Spd versus old + Spd + GC7 comparison, E–G). ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test, One-tailed Test

22) Product Images from "PD-1highCXCR5–CD4+ Peripheral Helper T (Tph) cells Promote Tissue-Homing Plasmablasts in COVID-19"

Article Title: PD-1highCXCR5–CD4+ Peripheral Helper T (Tph) cells Promote Tissue-Homing Plasmablasts in COVID-19

Journal: medRxiv

doi: 10.1101/2021.03.13.21253527

The functions of PD-1 high CXCR5 − Tph cells PD-1 high CXCR5 − Tph cells promote B cell differentiation and produce IFNγ much higher than cTfh cells, which affects the expression levels of tissue-homing receptors on plasmablasts. a-b , Each T cell subset and autologous CD20 + CD27 + B cells were sorted and co-cultured with SEB and LPS for 7 days (n=5, COVID-19 patients). Representative flow data of CD27 high CD138 + plasma cells after co-culture ( a ). IgG concentrations in supernatants of co-cultures were evaluated by One-way ANOVA with Dunn’ s multiple comparisons tests ( b ). c , Sorted T cells (n=4, COVID-19 patients) were stimulated with anti-CD3/28 (each 1 μg/ml) for 48 hrs, then cytokine production levels were measured (IFNγ, IL-17A, IL-10). d-f , Sorted CD20 + CD27 + memory B cells (n=5, healthy donors) were cultured with CD40L (0.05 ng/ml), IL-21 (20ng/ml), and IL-10 (10 ng/ml) or different concentration of IFNγ (0, 3, 10, 30 ng/ml) for 7 days (n=5, healthy controls). Representative histogram of flow data for CXCR3 expression on plasma cells ( d , left), CXCR3 gMFI was evaluated by One-way ANOVA with Tukey’ s multiple comparisons tests ( d , right). After 7 days in culture, CD19 + CD27 + CD138 + plasma cells were sorted and gene expression measured relative to B2M by qPCR ( e, f ). The expression levels were evaluated by One-way ANOVA with Tukey’ s multiple comparisons tests ( c-f ).
Figure Legend Snippet: The functions of PD-1 high CXCR5 − Tph cells PD-1 high CXCR5 − Tph cells promote B cell differentiation and produce IFNγ much higher than cTfh cells, which affects the expression levels of tissue-homing receptors on plasmablasts. a-b , Each T cell subset and autologous CD20 + CD27 + B cells were sorted and co-cultured with SEB and LPS for 7 days (n=5, COVID-19 patients). Representative flow data of CD27 high CD138 + plasma cells after co-culture ( a ). IgG concentrations in supernatants of co-cultures were evaluated by One-way ANOVA with Dunn’ s multiple comparisons tests ( b ). c , Sorted T cells (n=4, COVID-19 patients) were stimulated with anti-CD3/28 (each 1 μg/ml) for 48 hrs, then cytokine production levels were measured (IFNγ, IL-17A, IL-10). d-f , Sorted CD20 + CD27 + memory B cells (n=5, healthy donors) were cultured with CD40L (0.05 ng/ml), IL-21 (20ng/ml), and IL-10 (10 ng/ml) or different concentration of IFNγ (0, 3, 10, 30 ng/ml) for 7 days (n=5, healthy controls). Representative histogram of flow data for CXCR3 expression on plasma cells ( d , left), CXCR3 gMFI was evaluated by One-way ANOVA with Tukey’ s multiple comparisons tests ( d , right). After 7 days in culture, CD19 + CD27 + CD138 + plasma cells were sorted and gene expression measured relative to B2M by qPCR ( e, f ). The expression levels were evaluated by One-way ANOVA with Tukey’ s multiple comparisons tests ( c-f ).

Techniques Used: Cell Differentiation, Expressing, Cell Culture, Co-Culture Assay, Concentration Assay, Real-time Polymerase Chain Reaction

23) Product Images from "Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses"

Article Title: Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses

Journal: eLife

doi: 10.7554/eLife.57950

LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws. PBMCs were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hr, in the abence/presence of Bafilomycin A1 for the last 2 hr. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. Monocytes gated on CD14+ treated with IFNγ or LPS ( a ), B cells gated on CD19+ treated with anti-CD40L and anti-IgM ( b ), CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28 ( c ), CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28 ( d ). LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.
Figure Legend Snippet: LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws. PBMCs were generated from blood taken at 3 weeks intervals (samples 1, 2, 3) from young human donors and were cultured for 24 hr, in the abence/presence of Bafilomycin A1 for the last 2 hr. Here basal autophagic flux was calculated as LC3-II mean fluorescence intensity (treatment-basal)/basal. Monocytes gated on CD14+ treated with IFNγ or LPS ( a ), B cells gated on CD19+ treated with anti-CD40L and anti-IgM ( b ), CD4+ T cells gated on CD3+CD4+ treated with anti-CD3/CD28 ( c ), CD8+ T cells gated on CD3+ CD8+ treated with anti-CD3/CD28 ( d ). LC3-II detection by flow cytometry is a reliable and reproducible technique in immune cells over several blood draws.

Techniques Used: Flow Cytometry, Generated, Cell Culture, Fluorescence

24) Product Images from "A Nonsense N –Terminus NFKB2 Mutation Leading to Haploinsufficiency in a Patient with a Predominantly Antibody Deficiency"

Article Title: A Nonsense N –Terminus NFKB2 Mutation Leading to Haploinsufficiency in a Patient with a Predominantly Antibody Deficiency

Journal: Journal of Clinical Immunology

doi: 10.1007/s10875-020-00842-2

Proliferation of T and B cells from patients and healthy donor controls. (a ) Total PBMCs were stained with CellTrace Violet and stimulated with anti-CD3 and anti-CD28 for 3 days. Data are representative of three experiments. ( b ) For B cell proliferation, cells were stimulated with indicated agonists (anti-IgM, CD40L, IL-4, CpG, IL-21) for 4–5 days. Numbers indicate the percentage of cells having undergone at least one cellular division assessed by CellTrace Violet dye dilution. Data are representative of two experiments
Figure Legend Snippet: Proliferation of T and B cells from patients and healthy donor controls. (a ) Total PBMCs were stained with CellTrace Violet and stimulated with anti-CD3 and anti-CD28 for 3 days. Data are representative of three experiments. ( b ) For B cell proliferation, cells were stimulated with indicated agonists (anti-IgM, CD40L, IL-4, CpG, IL-21) for 4–5 days. Numbers indicate the percentage of cells having undergone at least one cellular division assessed by CellTrace Violet dye dilution. Data are representative of two experiments

Techniques Used: Staining

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    Enzo Biochem cd40l
    Intracellular calcium mobilization and CysLTR 1 antagonism. (Left panel) DCs were loaded with FluoForte and intracellular calcium release was determined using a Tecan Infinite F200 Pro fluorometer. <t>CD40L</t> was added at t-d and LTC 4 or LTD 4 were injected
    Cd40l, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem trimeric cd40l
    IgA Increases Systemic BCAAs Capable of Enhancing IgG Production (A) Heat map of significantly differentially abundant plasma metabolites from 9 HCs, 4 IGAD-R patients and 3 IGAD-NR patients. Data represent standardized peak areas and have been row mean-centered and row-normalized. Metabolites highlighted in bold are discussed in the text. SPA, standardized peak area. The positioning of the methyl group on methyl-2-oxvaleric acid was unable to be differentiated between attachment to either the third or fourth carbon of the molecule. (B) Plasma concentration of leucine, isoleucine and valine in donors described in A. Standardized peak area compared to internal standard and quantitative estimation ( μ M) was performed by normalizing relative peak areas and comparing to standard curve. (C) ELISA of IgG secreted by B cells from 7-10 HCs upon exposure of peripheral blood mononuclear cells to medium alone (ctrl) or T cell-associated stimuli <t>CD40L</t> and IL-21 for 6 days in the presence of decreasing amounts of BCAAs. From left to right: complete BCAA-sufficient media, (120 mg/L BCAAs at a 2:5:5 ratio of L-valine, L-leucine, and L-isoleucine), BCAA-depleted media with one tenth as much BCAAs (12 mg/L BCAAs at the same ratio as above), and BCAA-deficient media with no BCAAs. (D) ELISA of IgM secreted by B cells from 10 HCs upon exposure of peripheral blood mononuclear cells for 6 days to medium alone (ctrl) or the TI ligand CpG-DNA in the presence of decreasing amounts of BCAAs as in (C). Metabolomics (A, B) were from one experiment. In vitro BCAA experiments (C, D) summarize two independent experiments involving peripheral blood mononuclear cells from 5 HCs each. Data are presented with mean and significance was determined through Kruskal-Wallis test with Dunn’s correction for multiple comparisons. *p
    Trimeric Cd40l, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem trimeric cd40l reagent
    <t>CD40L:CD28</t> CSP surface expression kinetic in PBLs after retroviral transduction. Human primary T cells were retrovirally transduced with pMP71 encoding the CD40L:CD28 sequences and CSP surface expression was measured by flow cytometry at 3, 6, 10 and 13 days after transduction using CD40L antibody. Percentage of CD40L-positive cells within gated live, single, CD3 + populations are displayed as histograms. Mock-transduced T cells were used as negative control (red line), T cells transduced with the native CD40L (nCD40L) protein were used as expression reference and are depicted in blue, CD40L:IgGFc:CD28 CSP is depicted in orange, CD40L:Fil3:CD28 CSP is depicted in green and CD40L:CD28i CSP is depicted in purple. Shown is one representative experiment of at least 5 repeats. A summary graph of 5 experiments is shown in Supplementary Figure 1 .
    Trimeric Cd40l Reagent, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Intracellular calcium mobilization and CysLTR 1 antagonism. (Left panel) DCs were loaded with FluoForte and intracellular calcium release was determined using a Tecan Infinite F200 Pro fluorometer. CD40L was added at t-d and LTC 4 or LTD 4 were injected

    Journal: Blood

    Article Title: Leukotriene C4 induces migration of human monocyte\u2013derived dendritic cells without loss of immunostimulatory function

    doi: 10.1182/blood-2011-10-385930

    Figure Lengend Snippet: Intracellular calcium mobilization and CysLTR 1 antagonism. (Left panel) DCs were loaded with FluoForte and intracellular calcium release was determined using a Tecan Infinite F200 Pro fluorometer. CD40L was added at t-d and LTC 4 or LTD 4 were injected

    Article Snippet: CD40L and Enhancer were obtained from Alexis Biochemicals.

    Techniques: Injection

    Cytokine secretion and IDO activity of mature DCs. (Left panel) DCs were matured for 24 hours with CC or CD40L in the presence or absence of LTC 4 or PGE 2 . Supernatants were harvested and secretion of IL-10, IL-12p70, and IL-12p40 were analyzed by ELISA.

    Journal: Blood

    Article Title: Leukotriene C4 induces migration of human monocyte\u2013derived dendritic cells without loss of immunostimulatory function

    doi: 10.1182/blood-2011-10-385930

    Figure Lengend Snippet: Cytokine secretion and IDO activity of mature DCs. (Left panel) DCs were matured for 24 hours with CC or CD40L in the presence or absence of LTC 4 or PGE 2 . Supernatants were harvested and secretion of IL-10, IL-12p70, and IL-12p40 were analyzed by ELISA.

    Article Snippet: CD40L and Enhancer were obtained from Alexis Biochemicals.

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

    IgA Increases Systemic BCAAs Capable of Enhancing IgG Production (A) Heat map of significantly differentially abundant plasma metabolites from 9 HCs, 4 IGAD-R patients and 3 IGAD-NR patients. Data represent standardized peak areas and have been row mean-centered and row-normalized. Metabolites highlighted in bold are discussed in the text. SPA, standardized peak area. The positioning of the methyl group on methyl-2-oxvaleric acid was unable to be differentiated between attachment to either the third or fourth carbon of the molecule. (B) Plasma concentration of leucine, isoleucine and valine in donors described in A. Standardized peak area compared to internal standard and quantitative estimation ( μ M) was performed by normalizing relative peak areas and comparing to standard curve. (C) ELISA of IgG secreted by B cells from 7-10 HCs upon exposure of peripheral blood mononuclear cells to medium alone (ctrl) or T cell-associated stimuli CD40L and IL-21 for 6 days in the presence of decreasing amounts of BCAAs. From left to right: complete BCAA-sufficient media, (120 mg/L BCAAs at a 2:5:5 ratio of L-valine, L-leucine, and L-isoleucine), BCAA-depleted media with one tenth as much BCAAs (12 mg/L BCAAs at the same ratio as above), and BCAA-deficient media with no BCAAs. (D) ELISA of IgM secreted by B cells from 10 HCs upon exposure of peripheral blood mononuclear cells for 6 days to medium alone (ctrl) or the TI ligand CpG-DNA in the presence of decreasing amounts of BCAAs as in (C). Metabolomics (A, B) were from one experiment. In vitro BCAA experiments (C, D) summarize two independent experiments involving peripheral blood mononuclear cells from 5 HCs each. Data are presented with mean and significance was determined through Kruskal-Wallis test with Dunn’s correction for multiple comparisons. *p

    Journal: bioRxiv

    Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

    doi: 10.1101/2021.04.29.439534

    Figure Lengend Snippet: IgA Increases Systemic BCAAs Capable of Enhancing IgG Production (A) Heat map of significantly differentially abundant plasma metabolites from 9 HCs, 4 IGAD-R patients and 3 IGAD-NR patients. Data represent standardized peak areas and have been row mean-centered and row-normalized. Metabolites highlighted in bold are discussed in the text. SPA, standardized peak area. The positioning of the methyl group on methyl-2-oxvaleric acid was unable to be differentiated between attachment to either the third or fourth carbon of the molecule. (B) Plasma concentration of leucine, isoleucine and valine in donors described in A. Standardized peak area compared to internal standard and quantitative estimation ( μ M) was performed by normalizing relative peak areas and comparing to standard curve. (C) ELISA of IgG secreted by B cells from 7-10 HCs upon exposure of peripheral blood mononuclear cells to medium alone (ctrl) or T cell-associated stimuli CD40L and IL-21 for 6 days in the presence of decreasing amounts of BCAAs. From left to right: complete BCAA-sufficient media, (120 mg/L BCAAs at a 2:5:5 ratio of L-valine, L-leucine, and L-isoleucine), BCAA-depleted media with one tenth as much BCAAs (12 mg/L BCAAs at the same ratio as above), and BCAA-deficient media with no BCAAs. (D) ELISA of IgM secreted by B cells from 10 HCs upon exposure of peripheral blood mononuclear cells for 6 days to medium alone (ctrl) or the TI ligand CpG-DNA in the presence of decreasing amounts of BCAAs as in (C). Metabolomics (A, B) were from one experiment. In vitro BCAA experiments (C, D) summarize two independent experiments involving peripheral blood mononuclear cells from 5 HCs each. Data are presented with mean and significance was determined through Kruskal-Wallis test with Dunn’s correction for multiple comparisons. *p

    Article Snippet: Human B Cell Cultures 3 × 105 PBMCs (200 μl/well) were cultured in 96-well round bottom plates and stimulated with 500 ng/ml trimeric CD40L (Enzo Life Sciences) and 100 ng/ml IL-21 (Peprotech), or 500 ng/ml CpG-DNA (Invivogen) for 7 days.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, In Vitro

    Additive effect of TLR9 activation on CLL cells after [anti-IgM + CD40L + IL-4 + IL-21] stimulation. ( A) CLL cells (n = 39) were stimulated either by CpG-ODN2006, or IgM + CD40L + IL-4 + IL-21 or the combination of CpG-ODN2006 and anti-IgM + CD40L + IL-4 + IL-21. After initial CFSE staining (day 0), the percentage of cell proliferation was measured at day 6 in soluble medium and ( B ) in semi-solid medium. (C) Determination of the number of cell generations for responding CLL cells after anti-IgM + CD40L + IL-4 + IL-21 stimulation with or without CpG-ODN2006 in soluble medium and (D) in semi-solid medium. (E) Comparison of CLL cells proliferation after anti-IgM + CD40L + IL-4 + IL-21 stimulation with or without CpG-ODN2006 in soluble medium and (F) in semi-solid medium. Symbols represent CLL cells sub-types (circle: UM ZAP+; triangle: M ZAP+; square: M ZAP−; diamond: UM ZAP−). *p

    Journal: Scientific Reports

    Article Title: BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

    doi: 10.1038/s41598-018-36853-8

    Figure Lengend Snippet: Additive effect of TLR9 activation on CLL cells after [anti-IgM + CD40L + IL-4 + IL-21] stimulation. ( A) CLL cells (n = 39) were stimulated either by CpG-ODN2006, or IgM + CD40L + IL-4 + IL-21 or the combination of CpG-ODN2006 and anti-IgM + CD40L + IL-4 + IL-21. After initial CFSE staining (day 0), the percentage of cell proliferation was measured at day 6 in soluble medium and ( B ) in semi-solid medium. (C) Determination of the number of cell generations for responding CLL cells after anti-IgM + CD40L + IL-4 + IL-21 stimulation with or without CpG-ODN2006 in soluble medium and (D) in semi-solid medium. (E) Comparison of CLL cells proliferation after anti-IgM + CD40L + IL-4 + IL-21 stimulation with or without CpG-ODN2006 in soluble medium and (F) in semi-solid medium. Symbols represent CLL cells sub-types (circle: UM ZAP+; triangle: M ZAP+; square: M ZAP−; diamond: UM ZAP−). *p

    Article Snippet: B cells at a density of 106 cells/ml were stimulated in the absence or presence of 10 µg/ml of soluble F(ab’)2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA), 100 ng/ml of trimeric CD40L (Enzo Life Science, Villeurbanne, France), 10 ng/ml of IL-4 (R & D Systems-Bio-Techne, Lille, France) and 25 ng/ml of IL-21 (Invitrogen, Maryland, USA).

    Techniques: Activation Assay, Staining

    Cell proliferation of CLL and healthy B cells after soluble [anti-IgM + CD40L + IL-4 + IL-21] stimulation. ( A ) CLL cells isolated from a cohort of 59 CLL patients and healthy donors (naïve B cells, n = 16 or total B cells, n = 20) were stimulated ex vivo with anti-IgM, CD40L, IL-4 and IL-21. After initial CFSE staining (day 0), the percentage of cell proliferation was measured at day 6 for CLL cells and at day 4 for healthy B cells in stimulated cells (S) and control unstimulated cells (US). A threshold of dividing cells greater than 25% among living cells (dashed line) and the presence of at least one generation of daughter cells defines proliferation. 95% confidence interval for median is shown in each group. *p

    Journal: Scientific Reports

    Article Title: BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

    doi: 10.1038/s41598-018-36853-8

    Figure Lengend Snippet: Cell proliferation of CLL and healthy B cells after soluble [anti-IgM + CD40L + IL-4 + IL-21] stimulation. ( A ) CLL cells isolated from a cohort of 59 CLL patients and healthy donors (naïve B cells, n = 16 or total B cells, n = 20) were stimulated ex vivo with anti-IgM, CD40L, IL-4 and IL-21. After initial CFSE staining (day 0), the percentage of cell proliferation was measured at day 6 for CLL cells and at day 4 for healthy B cells in stimulated cells (S) and control unstimulated cells (US). A threshold of dividing cells greater than 25% among living cells (dashed line) and the presence of at least one generation of daughter cells defines proliferation. 95% confidence interval for median is shown in each group. *p

    Article Snippet: B cells at a density of 106 cells/ml were stimulated in the absence or presence of 10 µg/ml of soluble F(ab’)2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA), 100 ng/ml of trimeric CD40L (Enzo Life Science, Villeurbanne, France), 10 ng/ml of IL-4 (R & D Systems-Bio-Techne, Lille, France) and 25 ng/ml of IL-21 (Invitrogen, Maryland, USA).

    Techniques: Isolation, Ex Vivo, Staining

    Cell proliferation of CLL and healthy B cells after [anti-IgM + CD40L + IL-4 + IL-21] stimulation on a semi-solid medium. ( A ) CLL cells isolated from a cohort of 38 CLL patients and naïve and total B cells (n = 11) isolated from healthy donors have been stimulated ex vivo with anti-IgM, CD40L, IL-4 and IL-21 on a semi-solid culture medium. After initial CFSE staining (day 0), the percentage of cell proliferation was measured at day 6 for CLL cells and at day 4 for healthy B cells in stimulated cells (S) and control unstimulated cells (US). *p

    Journal: Scientific Reports

    Article Title: BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

    doi: 10.1038/s41598-018-36853-8

    Figure Lengend Snippet: Cell proliferation of CLL and healthy B cells after [anti-IgM + CD40L + IL-4 + IL-21] stimulation on a semi-solid medium. ( A ) CLL cells isolated from a cohort of 38 CLL patients and naïve and total B cells (n = 11) isolated from healthy donors have been stimulated ex vivo with anti-IgM, CD40L, IL-4 and IL-21 on a semi-solid culture medium. After initial CFSE staining (day 0), the percentage of cell proliferation was measured at day 6 for CLL cells and at day 4 for healthy B cells in stimulated cells (S) and control unstimulated cells (US). *p

    Article Snippet: B cells at a density of 106 cells/ml were stimulated in the absence or presence of 10 µg/ml of soluble F(ab’)2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA), 100 ng/ml of trimeric CD40L (Enzo Life Science, Villeurbanne, France), 10 ng/ml of IL-4 (R & D Systems-Bio-Techne, Lille, France) and 25 ng/ml of IL-21 (Invitrogen, Maryland, USA).

    Techniques: Isolation, Ex Vivo, Staining

    Signaling pathways activated by [anti-IgM + CD40L + IL-4 + IL-21] stimulation of responding and non-responding UM ZAP70+ CLL cells. ( A ) Normalized protein expression of ZAP70, phospho-ZAP70 Tyr319 /SYK Tyr352 (pZAP70/pSYK), phospho-SYK Tyr323 (pSYK), phospho-AKT Thr308 (pAKT), phospho-ERK1/2 Tyr204 (pERK), phospho-IκB Ser32/36 (pIκB) and phospho-STAT6 Tyr641 (pSTAT6), based on immunoblot results of proliferating (Responders, n = 4) and non-proliferating (Non Responders, n = 3) UM-CLL ZAP+ CLL cells following stimulation (S) or in unstimulated (US) conditions. Protein expression corresponds to the value of a signal (determined with ImageJ) normalized to that of GAPDH. ( B ) Role of IL-4 co-stimulation on CLL cells proliferation. CLL cells isolated from 6 CLL patients were stimulated ex vivo with anti-IgM, CD40L, IL-4 and IL-21, with or without a specific JAK3 inhibitor (PF-956980), or with anti-IgM, CD40L and IL-21. After initial CFSE staining (at day 0), the percentage of cell proliferation was measured at day 6. Symbols (circle) represent UM ZAP+ CLL cells. 95% confidence interval for median is shown in each graph. *p

    Journal: Scientific Reports

    Article Title: BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

    doi: 10.1038/s41598-018-36853-8

    Figure Lengend Snippet: Signaling pathways activated by [anti-IgM + CD40L + IL-4 + IL-21] stimulation of responding and non-responding UM ZAP70+ CLL cells. ( A ) Normalized protein expression of ZAP70, phospho-ZAP70 Tyr319 /SYK Tyr352 (pZAP70/pSYK), phospho-SYK Tyr323 (pSYK), phospho-AKT Thr308 (pAKT), phospho-ERK1/2 Tyr204 (pERK), phospho-IκB Ser32/36 (pIκB) and phospho-STAT6 Tyr641 (pSTAT6), based on immunoblot results of proliferating (Responders, n = 4) and non-proliferating (Non Responders, n = 3) UM-CLL ZAP+ CLL cells following stimulation (S) or in unstimulated (US) conditions. Protein expression corresponds to the value of a signal (determined with ImageJ) normalized to that of GAPDH. ( B ) Role of IL-4 co-stimulation on CLL cells proliferation. CLL cells isolated from 6 CLL patients were stimulated ex vivo with anti-IgM, CD40L, IL-4 and IL-21, with or without a specific JAK3 inhibitor (PF-956980), or with anti-IgM, CD40L and IL-21. After initial CFSE staining (at day 0), the percentage of cell proliferation was measured at day 6. Symbols (circle) represent UM ZAP+ CLL cells. 95% confidence interval for median is shown in each graph. *p

    Article Snippet: B cells at a density of 106 cells/ml were stimulated in the absence or presence of 10 µg/ml of soluble F(ab’)2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA), 100 ng/ml of trimeric CD40L (Enzo Life Science, Villeurbanne, France), 10 ng/ml of IL-4 (R & D Systems-Bio-Techne, Lille, France) and 25 ng/ml of IL-21 (Invitrogen, Maryland, USA).

    Techniques: Expressing, Isolation, Ex Vivo, Staining

    Proliferative response according to the IGHV mutational status and ZAP70 protein expression. Recapitulation of CLL cells proliferation for UM ZAP+ (circle), M ZAP70+ (triangle) and M ZAP70− (square) CLL cells after anti-IgM + CD40L + IL-4 + IL-21 stimulation in soluble medium, semi-solid medium, soluble medium + CpG-ODN2006 and semi-solid medium + CpG-ODN2006. *p

    Journal: Scientific Reports

    Article Title: BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

    doi: 10.1038/s41598-018-36853-8

    Figure Lengend Snippet: Proliferative response according to the IGHV mutational status and ZAP70 protein expression. Recapitulation of CLL cells proliferation for UM ZAP+ (circle), M ZAP70+ (triangle) and M ZAP70− (square) CLL cells after anti-IgM + CD40L + IL-4 + IL-21 stimulation in soluble medium, semi-solid medium, soluble medium + CpG-ODN2006 and semi-solid medium + CpG-ODN2006. *p

    Article Snippet: B cells at a density of 106 cells/ml were stimulated in the absence or presence of 10 µg/ml of soluble F(ab’)2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA), 100 ng/ml of trimeric CD40L (Enzo Life Science, Villeurbanne, France), 10 ng/ml of IL-4 (R & D Systems-Bio-Techne, Lille, France) and 25 ng/ml of IL-21 (Invitrogen, Maryland, USA).

    Techniques: Expressing

    CD40L:CD28 CSP surface expression kinetic in PBLs after retroviral transduction. Human primary T cells were retrovirally transduced with pMP71 encoding the CD40L:CD28 sequences and CSP surface expression was measured by flow cytometry at 3, 6, 10 and 13 days after transduction using CD40L antibody. Percentage of CD40L-positive cells within gated live, single, CD3 + populations are displayed as histograms. Mock-transduced T cells were used as negative control (red line), T cells transduced with the native CD40L (nCD40L) protein were used as expression reference and are depicted in blue, CD40L:IgGFc:CD28 CSP is depicted in orange, CD40L:Fil3:CD28 CSP is depicted in green and CD40L:CD28i CSP is depicted in purple. Shown is one representative experiment of at least 5 repeats. A summary graph of 5 experiments is shown in Supplementary Figure 1 .

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: CD40L:CD28 CSP surface expression kinetic in PBLs after retroviral transduction. Human primary T cells were retrovirally transduced with pMP71 encoding the CD40L:CD28 sequences and CSP surface expression was measured by flow cytometry at 3, 6, 10 and 13 days after transduction using CD40L antibody. Percentage of CD40L-positive cells within gated live, single, CD3 + populations are displayed as histograms. Mock-transduced T cells were used as negative control (red line), T cells transduced with the native CD40L (nCD40L) protein were used as expression reference and are depicted in blue, CD40L:IgGFc:CD28 CSP is depicted in orange, CD40L:Fil3:CD28 CSP is depicted in green and CD40L:CD28i CSP is depicted in purple. Shown is one representative experiment of at least 5 repeats. A summary graph of 5 experiments is shown in Supplementary Figure 1 .

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Expressing, Transduction, Flow Cytometry, Negative Control

    T cells expressing the CD40L:CD28 CSPs can activate dendritic cells and induce a pro-inflammatory secretome in tumor-conditioned ercDCs through CD40/CD40L interaction. (A) Schematic drawing of the DC maturation assay. TCR-T58 T cells expressing the different CSPs were thawed and re-activated to induce CSP expression as described in Figure 7 . Co-cultures used immature dendritic cells (iDCs) or ercDCs. Negative control consisted of iDCs without T cells. Positive control consisted of mature DCs (mDCs). Density plots depicting the CD40L:CD28 CSP expression on the transduced T cells after re-activation. (B) Graphs depict the fold change of mean fluorescence intensity (MFI) relative to control of CD83, CCR7, PD-L1, HLA-DR and CD80 surface expression on DCs after overnight co-culture with CSP-expressing T58 T cells. Controls correspond to the first two bars distinguished by light colors, respectively. Bars are the mean values of 5 independent experiments. Error bars are the standard deviation of the 5 experiments. (C) Chemokine and cytokine secretion of iDCs and ercDCs after co-culture with T cells (measured using 45Plex bead array, representing thousands of individual bead measurements). Bars are the fold change in secretion relative to DC without T cell co-culture. Shaded area indicates the effect induced by Mock-T cells without CSP. Black bars indicate secretion by iDC, grey bars depict the secretion by ercDCs. ercDCs are tumor-conditioned DCs.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: T cells expressing the CD40L:CD28 CSPs can activate dendritic cells and induce a pro-inflammatory secretome in tumor-conditioned ercDCs through CD40/CD40L interaction. (A) Schematic drawing of the DC maturation assay. TCR-T58 T cells expressing the different CSPs were thawed and re-activated to induce CSP expression as described in Figure 7 . Co-cultures used immature dendritic cells (iDCs) or ercDCs. Negative control consisted of iDCs without T cells. Positive control consisted of mature DCs (mDCs). Density plots depicting the CD40L:CD28 CSP expression on the transduced T cells after re-activation. (B) Graphs depict the fold change of mean fluorescence intensity (MFI) relative to control of CD83, CCR7, PD-L1, HLA-DR and CD80 surface expression on DCs after overnight co-culture with CSP-expressing T58 T cells. Controls correspond to the first two bars distinguished by light colors, respectively. Bars are the mean values of 5 independent experiments. Error bars are the standard deviation of the 5 experiments. (C) Chemokine and cytokine secretion of iDCs and ercDCs after co-culture with T cells (measured using 45Plex bead array, representing thousands of individual bead measurements). Bars are the fold change in secretion relative to DC without T cell co-culture. Shaded area indicates the effect induced by Mock-T cells without CSP. Black bars indicate secretion by iDC, grey bars depict the secretion by ercDCs. ercDCs are tumor-conditioned DCs.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Expressing, Negative Control, Positive Control, Activation Assay, Fluorescence, Co-Culture Assay, Standard Deviation

    CD40L:CD28 CSPs improve cytolytic activity of antigen-specific T cells. TCR-D115 tyrosinase-specific T cells and TCR53 RCC-specific T cells expressing the CD40L:CD28 CSPs were thawed and used immediately without re-activation in a 4h chromium release assay at indicated T cell to target cell ratio with tyrosinase-positive target cells SK-Mel23 and HEK293/Tyr/CD40 for TCR-D115 T cells, and RCC-26 and RCC-53 RCC tumor cell lines for TCR53 T cells. Shown are mean values of duplicates from one representative experiment. Error bars show the standard deviation. All target cells expressed CD40 and HLA-A2 (see histograms Figures 6 , 10 ).

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: CD40L:CD28 CSPs improve cytolytic activity of antigen-specific T cells. TCR-D115 tyrosinase-specific T cells and TCR53 RCC-specific T cells expressing the CD40L:CD28 CSPs were thawed and used immediately without re-activation in a 4h chromium release assay at indicated T cell to target cell ratio with tyrosinase-positive target cells SK-Mel23 and HEK293/Tyr/CD40 for TCR-D115 T cells, and RCC-26 and RCC-53 RCC tumor cell lines for TCR53 T cells. Shown are mean values of duplicates from one representative experiment. Error bars show the standard deviation. All target cells expressed CD40 and HLA-A2 (see histograms Figures 6 , 10 ).

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Activity Assay, Expressing, Activation Assay, Release Assay, Standard Deviation

    Biological activity of the CD28 ICD within the CSPs by measuring the phosphorylation of AKT, mTOR and RPS6. CD40L:CD28 CSP-expressing TCR-T58 T cells were thawed and re-activated to induce CSP expression as described in Figure 7 . On day 6, they were stimulated with HEK293/Tyr/CD40 for 30 min. Unstimulated T cells were used as a negative control. (A) Graphs depict the percentage of phosphorylated p-AKT, p-mTOR and p-RPS6 in T cells after 30 minutes co-culture, grey bars correspond to the unstimulated controls and black bars correspond to the stimulated T cells. Bars are the mean values of the percentages of three independent experiments. The error bars are the standard deviation. (B) Histograms depicting the fluorescence intensi ty of each phosphorylated protein in T cells expressing the different CSPs; unstimulated (grey histogram) and after stimulation (colored histograms). (C) Density plots showing the correlation between p-AKT with p-mTOR and p-mTOR with p-RPS6 in T cells expressing the different CSPs. Mock-transduced T cells (red) and T cells transduced to express the native CD40L (blue) were used as references. T cells transduced with CD40L:IgGFc:CD28 are depicted in orange, those with CD40L:Fil3:CD28 CSP are depicted in green and T cells with CD40L:CD28i CSP are depicted in purple.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: Biological activity of the CD28 ICD within the CSPs by measuring the phosphorylation of AKT, mTOR and RPS6. CD40L:CD28 CSP-expressing TCR-T58 T cells were thawed and re-activated to induce CSP expression as described in Figure 7 . On day 6, they were stimulated with HEK293/Tyr/CD40 for 30 min. Unstimulated T cells were used as a negative control. (A) Graphs depict the percentage of phosphorylated p-AKT, p-mTOR and p-RPS6 in T cells after 30 minutes co-culture, grey bars correspond to the unstimulated controls and black bars correspond to the stimulated T cells. Bars are the mean values of the percentages of three independent experiments. The error bars are the standard deviation. (B) Histograms depicting the fluorescence intensi ty of each phosphorylated protein in T cells expressing the different CSPs; unstimulated (grey histogram) and after stimulation (colored histograms). (C) Density plots showing the correlation between p-AKT with p-mTOR and p-mTOR with p-RPS6 in T cells expressing the different CSPs. Mock-transduced T cells (red) and T cells transduced to express the native CD40L (blue) were used as references. T cells transduced with CD40L:IgGFc:CD28 are depicted in orange, those with CD40L:Fil3:CD28 CSP are depicted in green and T cells with CD40L:CD28i CSP are depicted in purple.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Activity Assay, Expressing, Negative Control, Co-Culture Assay, Standard Deviation, Fluorescence, Transduction

    Design of CD40L:CD28 CSPs. (A) CD28 and CD40L are depicted according to their classification as type I and type II membrane proteins, respectively. C-terminal amino acid (C) and N-terminal amino acid (N) determines the membrane orientation. (B) Schematic representation of the three different approaches for the structure of the CD40L:CD28 CSPs, AA length and origin of protein fragments are specified next to each molecule. (C) Domain representation of the CSPs including the signal peptide (SP) from the PD-1 protein for type I membrane protein approaches. SP, signal peptide from PD-1 (20 AA); ICD, intracellular domain; TMD, Transmembrane domain; ECD, extracellular domain.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: Design of CD40L:CD28 CSPs. (A) CD28 and CD40L are depicted according to their classification as type I and type II membrane proteins, respectively. C-terminal amino acid (C) and N-terminal amino acid (N) determines the membrane orientation. (B) Schematic representation of the three different approaches for the structure of the CD40L:CD28 CSPs, AA length and origin of protein fragments are specified next to each molecule. (C) Domain representation of the CSPs including the signal peptide (SP) from the PD-1 protein for type I membrane protein approaches. SP, signal peptide from PD-1 (20 AA); ICD, intracellular domain; TMD, Transmembrane domain; ECD, extracellular domain.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques:

    CD40L:CD28 CSP surface expression is regulated by anti-CD3/CD28 T cell activation. (A) Timeline depicting the re-activation of transduced T cells, which were frozen on day 12 after CSP transduction. T cells were thawed and re-activated using anti-CD3 plus anti-CD28 antibodies and 100 U/ml IL-2 for 3 days, then transferred to a new plate without antibodies in fresh medium plus 100 U/ml IL-2 for 3 additional days to allow downregulation of endogenous CD40L expression on Mock-T cells. (B) Histograms showing CD40L surface expression (identifying the CD40L:CD28 CSPs, the transduced native CD40L protein and the endogenously expressed CD40L) by flow cytometry after thawing and every third day after re-activation. (C) Gated CD40L-positive CD4 and CD8 cells within the live, single, CD3 + population are shown in dot plots as the blue population superimposed on the CD40L-negative T cells shown in red. Mock-transduced T cells were used as negative control and T cells transduced with the native CD40L (nCD40L) sequence are the reference against the CD40L:CD28 CSPs. (D) Percentage of live, single, CD3 positive cells are shown in dot plots measured 6 days after re-activation of CD40L:CD28 CSP-transduced T cells. This stimulation was performed for each B cell and DC assay, as well as CD28 signaling assay, thus at least 10 times. The exact number of repeats is listed with each of these processes.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: CD40L:CD28 CSP surface expression is regulated by anti-CD3/CD28 T cell activation. (A) Timeline depicting the re-activation of transduced T cells, which were frozen on day 12 after CSP transduction. T cells were thawed and re-activated using anti-CD3 plus anti-CD28 antibodies and 100 U/ml IL-2 for 3 days, then transferred to a new plate without antibodies in fresh medium plus 100 U/ml IL-2 for 3 additional days to allow downregulation of endogenous CD40L expression on Mock-T cells. (B) Histograms showing CD40L surface expression (identifying the CD40L:CD28 CSPs, the transduced native CD40L protein and the endogenously expressed CD40L) by flow cytometry after thawing and every third day after re-activation. (C) Gated CD40L-positive CD4 and CD8 cells within the live, single, CD3 + population are shown in dot plots as the blue population superimposed on the CD40L-negative T cells shown in red. Mock-transduced T cells were used as negative control and T cells transduced with the native CD40L (nCD40L) sequence are the reference against the CD40L:CD28 CSPs. (D) Percentage of live, single, CD3 positive cells are shown in dot plots measured 6 days after re-activation of CD40L:CD28 CSP-transduced T cells. This stimulation was performed for each B cell and DC assay, as well as CD28 signaling assay, thus at least 10 times. The exact number of repeats is listed with each of these processes.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Expressing, Activation Assay, Transduction, Flow Cytometry, Negative Control, Sequencing

    CD40L:CD28 CSP expression kinetic in T cells is modulated by medium replacement. Human primary T cells were retrovirally transduced with the native CD40L sequence or the CSP constructs and expanded for 12 days. Cells were transferred to a new flask or plate with fresh medium/100 U/ml IL-2 on day 6 and 12. (A) Timeline describing the transduction steps and expansion of human primary T cells. (B) CSP surface expression was measured by flow cytometry (FCM) at day 3, 6, 10, 12 and 13 after transduction using CD40L antibody. The CD4 and CD8 cells within the live, single, CD3 + population are depicted in the dot plots and the CD40L-positive (blue dots) and CD40L-negative (red dots) are graphically superimposed. Mock-transduced T cells were used as negative control and T cells transduced with the native CD40L (nCD40L) sequence were used as positive reference. (C) Percentage of CD40L-positive T cells at the different time points after transduction. Summary of 2 independent experiments, plus SEM. Mock-transduced T cells (red line) and T cells transduced to express the native CD40L (blue line) were used as reference. T cells transduced with CD40L:IgGFc:CD28 are depicted in orange, those with CD40L:Fil3:CD28 CSP are depicted in green and T cells with CD40L:CD28i CSP are depicted in purple.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: CD40L:CD28 CSP expression kinetic in T cells is modulated by medium replacement. Human primary T cells were retrovirally transduced with the native CD40L sequence or the CSP constructs and expanded for 12 days. Cells were transferred to a new flask or plate with fresh medium/100 U/ml IL-2 on day 6 and 12. (A) Timeline describing the transduction steps and expansion of human primary T cells. (B) CSP surface expression was measured by flow cytometry (FCM) at day 3, 6, 10, 12 and 13 after transduction using CD40L antibody. The CD4 and CD8 cells within the live, single, CD3 + population are depicted in the dot plots and the CD40L-positive (blue dots) and CD40L-negative (red dots) are graphically superimposed. Mock-transduced T cells were used as negative control and T cells transduced with the native CD40L (nCD40L) sequence were used as positive reference. (C) Percentage of CD40L-positive T cells at the different time points after transduction. Summary of 2 independent experiments, plus SEM. Mock-transduced T cells (red line) and T cells transduced to express the native CD40L (blue line) were used as reference. T cells transduced with CD40L:IgGFc:CD28 are depicted in orange, those with CD40L:Fil3:CD28 CSP are depicted in green and T cells with CD40L:CD28i CSP are depicted in purple.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Expressing, Transduction, Sequencing, Construct, Flow Cytometry, Negative Control

    CD40L:CD28 CSP expression kinetic on T cells after recognition of peptide/MHC ligands on target cells in the presence or absence of target cell-expressed CD40 receptor. Human TCR-T58 T cells (tyrosinase-specific, HLA-A2 restricted) expressing the CD40L:CD28 CSPs were frozen on day 12 after CSP transduction. After thawing, T cells were used in co-cultures at a T cell to target cell ratio of 1:10. CSP surface expression on TCR-T58 T cells was analyzed by flow cytometry and is expressed as percentage of gated single, live, CD8+ CD40L+ cells. Mock-transduced TCR-T58 T cells (red line) and TCR-T58 T cells transduced to express the native CD40L (blue line) were used as reference. TCR-T58 T cells transduced with CD40L:IgGFc:CD28 are depicted in orange, those with CD40L:Fil3:CD28 CSP are depicted in green and T cells with CD40L:CD28i CSP are depicted in purple. (A) Target cells were SK-Mel23 cells or SK-Mel23 that had been pre-treated with anti-CD40 antibody in a 1:11 concentration (clone HB14 pure functional grade, Miltenyi) before setting the co-culture to block the endogenous CD40 receptor. Histograms of CD40 expression on SK-Mel23 cells, line histogram depicts the specific staining for CD40 and the grey filled histogram is the unstained control. (B) Target cells were HEK293/Tyr/mock cells that had very low endogenous CD40 expression or were transduced to strongly express CD40 (HEK293/Tyr/CD40). Line histogram depicts the specific staining for CD40 and the grey filled histogram is the unstained control. This is one representative experiment of two.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: CD40L:CD28 CSP expression kinetic on T cells after recognition of peptide/MHC ligands on target cells in the presence or absence of target cell-expressed CD40 receptor. Human TCR-T58 T cells (tyrosinase-specific, HLA-A2 restricted) expressing the CD40L:CD28 CSPs were frozen on day 12 after CSP transduction. After thawing, T cells were used in co-cultures at a T cell to target cell ratio of 1:10. CSP surface expression on TCR-T58 T cells was analyzed by flow cytometry and is expressed as percentage of gated single, live, CD8+ CD40L+ cells. Mock-transduced TCR-T58 T cells (red line) and TCR-T58 T cells transduced to express the native CD40L (blue line) were used as reference. TCR-T58 T cells transduced with CD40L:IgGFc:CD28 are depicted in orange, those with CD40L:Fil3:CD28 CSP are depicted in green and T cells with CD40L:CD28i CSP are depicted in purple. (A) Target cells were SK-Mel23 cells or SK-Mel23 that had been pre-treated with anti-CD40 antibody in a 1:11 concentration (clone HB14 pure functional grade, Miltenyi) before setting the co-culture to block the endogenous CD40 receptor. Histograms of CD40 expression on SK-Mel23 cells, line histogram depicts the specific staining for CD40 and the grey filled histogram is the unstained control. (B) Target cells were HEK293/Tyr/mock cells that had very low endogenous CD40 expression or were transduced to strongly express CD40 (HEK293/Tyr/CD40). Line histogram depicts the specific staining for CD40 and the grey filled histogram is the unstained control. This is one representative experiment of two.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Expressing, Transduction, Flow Cytometry, Concentration Assay, Functional Assay, Co-Culture Assay, Blocking Assay, Staining

    Postulated effector mechanisms for the CD40L:CD28 chimeric co-stimulatory switch proteins (CSPs). Depicted are the 4 potential axes of effector mechanisms of T cells engineered with the CD40L:CD28 CSPs when interacting with different components of the tumor microenvironment. 1) Strengthening the MHC/peptide-triggered TCR signaling through eliciting an intracellular co-stimulatory signaling cascade allowing the T cells to overcome inhibition of effector function in the tumor milieu. 2) Activation of tumor-resident CD40-expressing DCs. Interaction of CD40 on DCs with CD40L expressed on the engineered T cells could induce signals in DCs leading to their maturation with gain in de novo priming capability. 3) Targeting tumor endothelium. CD40 is expressed, amongst others, on neovascular endothelium and CD40 stimulation has been shown to activate human endothelial cells including proliferation and the upregulation of adhesion molecules, enabling T cell attachment and infiltration. Targeting this stromal compartment could potentially enhance the immunotherapy effect by depriving the tumor bed of live supporting surroundings and enhancing T cell infiltration. 4) Apoptotic effects on tumor cells. It is reported that tumor cells aberrantly express CD40 and that CD40 signals induce apoptotic cell death independent of MHC/peptide-specific targeting. Thus, CD40L:CD28-engineered T cells may kill tumor cells expressing CD40 even if they do not present the cognate TCR-MHC ligand.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: Postulated effector mechanisms for the CD40L:CD28 chimeric co-stimulatory switch proteins (CSPs). Depicted are the 4 potential axes of effector mechanisms of T cells engineered with the CD40L:CD28 CSPs when interacting with different components of the tumor microenvironment. 1) Strengthening the MHC/peptide-triggered TCR signaling through eliciting an intracellular co-stimulatory signaling cascade allowing the T cells to overcome inhibition of effector function in the tumor milieu. 2) Activation of tumor-resident CD40-expressing DCs. Interaction of CD40 on DCs with CD40L expressed on the engineered T cells could induce signals in DCs leading to their maturation with gain in de novo priming capability. 3) Targeting tumor endothelium. CD40 is expressed, amongst others, on neovascular endothelium and CD40 stimulation has been shown to activate human endothelial cells including proliferation and the upregulation of adhesion molecules, enabling T cell attachment and infiltration. Targeting this stromal compartment could potentially enhance the immunotherapy effect by depriving the tumor bed of live supporting surroundings and enhancing T cell infiltration. 4) Apoptotic effects on tumor cells. It is reported that tumor cells aberrantly express CD40 and that CD40 signals induce apoptotic cell death independent of MHC/peptide-specific targeting. Thus, CD40L:CD28-engineered T cells may kill tumor cells expressing CD40 even if they do not present the cognate TCR-MHC ligand.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Inhibition, Activation Assay, Expressing, Cell Attachment Assay

    CD40L:CD28 CSPs improve T cell cytokine secretion. TCR-T58 tyrosinase-specific T cells and TCR53 RCC-specific T cells expressing the CD40L:CD28 CSPs were thawed and used immediately without re-activation in co-cultures at a T cell to target cell ratios of 1:10 with tyrosinase-positive target cells SK-Mel23 for TCR-T58 T cells (A) , and RCC-26 and RCC-53 RCC tumor cell lines for TCR53 T cells (B, C) . All target cells expressed CD40 and HLA-A2 endogenously (shown as histograms). IFN-ү was measured in supernatants of co-cultures by ELISA. Shown are mean values of duplicates from one representative experiment repeated 2 times. Error bars are the standard deviation.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: CD40L:CD28 CSPs improve T cell cytokine secretion. TCR-T58 tyrosinase-specific T cells and TCR53 RCC-specific T cells expressing the CD40L:CD28 CSPs were thawed and used immediately without re-activation in co-cultures at a T cell to target cell ratios of 1:10 with tyrosinase-positive target cells SK-Mel23 for TCR-T58 T cells (A) , and RCC-26 and RCC-53 RCC tumor cell lines for TCR53 T cells (B, C) . All target cells expressed CD40 and HLA-A2 endogenously (shown as histograms). IFN-ү was measured in supernatants of co-cultures by ELISA. Shown are mean values of duplicates from one representative experiment repeated 2 times. Error bars are the standard deviation.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Biologic activity of the CD40L ECD within the CSPs using a B cell stimulation assay. Primary human T cells transduced with the CSPs were re-activated, collected after 6 days when endogenous CD40L expression on Mock-T cells was absent, and co-cultured with freshly isolated B cells at a 1:1 ratio. Co-cultures were harvested and analyzed by flow cytometry for B cell-specific surface activation markers CD83, CD86 and Fas. Three controls were included: i) a negative control consisting of B cells without T cells (unstimulated control), ii) a positive control using B cells stimulated with a soluble enhanced trimeric CD40L, and iii) B cells co-cultured with CD40L-expressing HEK293 cells. (A) Schematic diagram of the stimulation assay. (B) Histograms depicting the CD40L:CD28 CSP expression on the transduced T cells after thawing and anti-CD3/CD28 re-activation and culture for 6 days. (C) Graphs depicting the fold change of median fluorescence intensity (MFI) relative to control of CD83, CD86 and Fas on the surface of B cells after overnight co-culture with T cells transduced with CD40L:IgGFc:CD28 depicted in orange, CD40L:Fil3:CD28 CSP depicted in green and T cells with CD40L:CD28i CSP depicted in purple. Mock-transduced T cells (red) and T cells transduced to express the native CD40L (blue) were used as references. Controls are the first three bars distinguished by light grey color. Bars are the mean values of indicated number of independent experiments. Error bars are the standard deviation of the corresponding number of experiments n.

    Journal: Frontiers in Immunology

    Article Title: Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

    doi: 10.3389/fimmu.2021.750478

    Figure Lengend Snippet: Biologic activity of the CD40L ECD within the CSPs using a B cell stimulation assay. Primary human T cells transduced with the CSPs were re-activated, collected after 6 days when endogenous CD40L expression on Mock-T cells was absent, and co-cultured with freshly isolated B cells at a 1:1 ratio. Co-cultures were harvested and analyzed by flow cytometry for B cell-specific surface activation markers CD83, CD86 and Fas. Three controls were included: i) a negative control consisting of B cells without T cells (unstimulated control), ii) a positive control using B cells stimulated with a soluble enhanced trimeric CD40L, and iii) B cells co-cultured with CD40L-expressing HEK293 cells. (A) Schematic diagram of the stimulation assay. (B) Histograms depicting the CD40L:CD28 CSP expression on the transduced T cells after thawing and anti-CD3/CD28 re-activation and culture for 6 days. (C) Graphs depicting the fold change of median fluorescence intensity (MFI) relative to control of CD83, CD86 and Fas on the surface of B cells after overnight co-culture with T cells transduced with CD40L:IgGFc:CD28 depicted in orange, CD40L:Fil3:CD28 CSP depicted in green and T cells with CD40L:CD28i CSP depicted in purple. Mock-transduced T cells (red) and T cells transduced to express the native CD40L (blue) were used as references. Controls are the first three bars distinguished by light grey color. Bars are the mean values of indicated number of independent experiments. Error bars are the standard deviation of the corresponding number of experiments n.

    Article Snippet: Positive controls were B cells activated using soluble enhanced trimeric CD40L reagent (Enzo Life Science) (1:10 dilution) and B cells activated using HEK293/CD40L cells.

    Techniques: Activity Assay, Cell Stimulation, Transduction, Expressing, Cell Culture, Isolation, Flow Cytometry, Activation Assay, Negative Control, Positive Control, Fluorescence, Co-Culture Assay, Standard Deviation