Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Antagonism of the A 2A R enhances TCR-stimulated IL-2 mRNA increases in primary human CD4 + T cells and Jurkat T cells. (A) Box plots (top) and difference plots (bottom) show data from naïve and memory CD4 + T cells isolated from the peripheral blood of 20 healthy donors, stimulated with plate-bound anti-CD3 and soluble anti-CD28, and grown in conditions promoting TH1 or TH2 differentiation for three days in the presence or absence of ZM-241385 (ZM). IL-2 mRNA levels were determined by qPCR. In the box plots (top), the height of the box plots equals the interquartile range (IQR) and the horizontal line within the box indicates the median value. The whiskers extend to the lowest and highest data points within 1.5 X IQR and the open circles indicate the outliers, which lie above or below the whiskers. In the difference plots (bottom), open circles show pairwise differences in IL-2 mRNA for each sample when treated with ZM-241385 (ZM) or not (Con). To the right of the open circles are the median values (closed circles) and 95% confidence intervals. (B) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of ZM-241385 (ZM) for three days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05; *** , p < 0.001; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Isolation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: A dominant negative Gα s construct, Gα s DN3, which blocks signaling from G s -coupled receptors, enhances TCR-stimulated IL-2 mRNA increases. Jurkat cells were nucleofected with Gα s DN3 or empty vector (pcDNAI/Amp) and then stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Dominant Negative Mutation, Construct, Plasmid Preparation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA and adenylyl cyclase inhibition with ddA decrease TCR-stimulated IL-2 mRNA levels. Jurkat cells were nucleofected with Gα s siRNA or NT siRNA as described in Methods (A-C) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days (A, C). Gα s siRNA significantly decreased levels of Gα s mRNA (A), Gα s protein (B), and IL-2 mRNA (C). Data for (A) and (C) represent the mean ± SE from 8 experiments. (B) Left, each immunoblot is representative of three immunoblots. Right, quantification of protein expression levels in the presence of Gα s siRNA relative to NT siRNA. Data represent mean ± SE from 3 experiments. (D) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence or absence of ddA. Data represent the mean ± SE from 17 experiments. mRNA levels were determined by qPCR. * , p < 0.05; ** , p < 0.01; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Inhibition, Western Blot, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Inhibiting cAMP production decreases activity of the IL-2 promoter without affecting IL-2 mRNA stability. (A) ddA does not decrease stability of IL-2 mRNA. After 3 days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA, Jurkat cells were incubated for the indicated times with Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 4 experiments. (B) ddA decreases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA for 3 days following nucleofection with the indicated plasmids. (B) Data represent means ± SD from triplicate determinations from a single assay representative of 6 assays. (C) Data represent the means ± SE of values from stimulated cells expressing IL2/pGL3 from the 6 assays. ** , p < 0.01.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Activity Assay, Incubation, Luciferase, Reporter Assay, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA, but not Gα s DN3, decreases TCR-stimulated cAMP. Jurkat cells were nucleofected with the indicated siRNA or plasmids and then assayed for cAMP accumulation as described in Methods. The TCR was stimulated with 2.5 µg/ml plate-bound anti-CD3 and 2.5 µg/ml soluble anti-CD28 (A and B), and the A 2A R was stimulated with 300 µM CGS-21680 (C). Data in (A) represent the mean ± SE from 3 experiments and data in (B and C) represent the mean ± SE from 9 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Evidence for an inhibitory effect of cAMP on TCR-stimulated IL-2 mRNA levels after at least 2 days of TCR stimulation. (A) The potentiating effect of A 2A R antagonism was only observed after at least two days of TCR stimulation. IL-2 levels peaked within 24 hours of TCR stimulation and then decreased over the next 48 hours. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of ZM-241385 (ZM) and IL-2 mRNA levels were determined by qPCR at the indicated times. Data represent the means ± SD from a single experiment that is representative of three such experiments. (B) Stimulation of the TCR for three days followed by one hour of ddA treatment leads to potentiation of TCR-stimulated IL-2 mRNA levels by ddA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28, Jurkat cells were treated with ddA for one hour before determination of IL-2 mRNA levels by qPCR. Data represent the mean ± SE from 14 experiments. *** , p < 0.001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Model of how the source and context of activated Gα s and cAMP may determine whether they enhance or inhibit TCR-stimulated IL-2 transcription. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 co-stimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production . ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including PLC-γ, leading to Ca 2+ increases and activation of a variety of downstream pathways including translocation of NFAT to the nucleus and activation of IL-2 transcription (black and white pathway). Gα s stimulated by a mechanism that does not involve G s PCRs, but which could potentially involve the TCR, enhances TCR-stimulated IL-2 transcription by a mechanism that may involve binding of pCREB to the CRE site of the IL-2 promoter [ ] during the initial stages of TCR stimulation (green pathway, Stimulatory Step 1). In contrast G s PCRs decrease TCR-stimulated IL-2 transcription, potentially by utilizing both Gα s and Gβγ signaling in cells that have been exposed to at least two days of TCR stimulation (red pathway, Inhibitory Step 2). The inhibitory G s PCR/Gα s /cAMP pathway may involve binding of CREM, which gradually replaces pCREB, to the CRE site of the IL-2 promoter or the formation of NFAT/ICER complexes on NFAT/AP-1 composite sites in the IL-2 promoter , leading to repression of transcription (Inhibitory Step 2). Previous studies suggest that cAMP increases stimulated by the TCR are smaller and more transient than those stimulated by G s PCRs, as depicted by the relative sizes of the cAMP symbols, and this may contribute to the opposite effects on IL-2 transcription. Simultaneously, Gβγ may inhibit TCR-stimulated IL-2 transcription by decreasing TCR-stimulated Ca 2+ increases through Ca v 1 channels (Inhibitory Step 2), which are activated by the TCR by an unknown mechanism . Ca 2+ -calmodulin-activated calcineurin dephosphorylates NFAT, exposing a nuclear localization sequence (NLS) and leading to nuclear translocation.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Immunopeptidomics, Phospho-proteomics, Activation Assay, Translocation Assay, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Reversible CD28 checkpoint modulation by cyclic peptides outperforms biologic blockade under exposure-limited conditions

doi: 10.64898/2026.04.09.717469

Figure Lengend Snippet: (A,B) CP8 suppresses CD28-mediated cytokine production in primary human PBMCs with efficacy comparable to clinically advanced CD28-targeting biologics. PBMCs from independent donors (n = 10) were stimulated with anti-CD3/CD28 antibodies in the presence of increasing concentrations (0.1, 0.5, 1 µM) of CP8 or the biologic inhibitors FR104, Acazicolcept, and Lulizumab. CP8 reduced (A) IL-2 and (B) IFN-γ levels in a concentration-dependent manner, reaching levels comparable to biologic-mediated inhibition across donors. (C,D) CP8 does not induce cytokine production in the absence of stimulation. Unstimulated PBMCs treated with CP8 or biologic inhibitors across the same concentration range did not exhibit increased (C) IL-2 or (D) IFN-γ secretion relative to vehicle controls, indicating absence of intrinsic agonist activity. (E-G) CP8 enables rapid restoration of T-cell function following inhibitor removal. PBMCs were preincubated with inhibitors, subjected to washout, and restimulated. CP8-treated cells showed robust recovery of (E) IL-2 and (F) IFN-γ production, as well as (G) CD69 expression, approaching stimulated control levels. In contrast, cells treated with biologic inhibitors remained functionally suppressed despite washout, indicating persistent post-exposure inhibition. Data are presented as mean ± SEM with individual donor values shown.

Article Snippet: To assess the reversibility of CD28 inhibition, primary human PBMCs were preincubated with 500 nM of CP8, FR104 (MedChemExpress, Catalog# HY-P990587), Acazicolcept (MedChemExpress, Catalog# HY-P99420), or Lulizumab (MedChemExpress, Catalog# HY-P99302) for 60 min at 37 °C prior to stimulation.

Techniques: Concentration Assay, Inhibition, Activity Assay, Cell Function Assay, Expressing, Control

Journal: bioRxiv

Article Title: Reversible CD28 checkpoint modulation by cyclic peptides outperforms biologic blockade under exposure-limited conditions

doi: 10.64898/2026.04.09.717469

Figure Lengend Snippet: (A,B) CP8 suppresses cytokine production in peripheral blood mononuclear cells (PBMCs) derived from patients with ulcerative colitis (n = 5). Upon anti-CD3/CD28 stimulation, CP8 reduced (A) IL-2 and (B) IFN-γ secretion in a concentration-dependent manner, with inhibition approaching a plateau at submicromolar to micromolar concentrations, consistent with effective engagement of the CD28 co-stimulatory pathway under inflammatory conditions. (C-E) CP8 preserves the ability of patient-derived T cells to re-engage activation following transient exposure. PBMCs were initially stimulated in the presence of inhibitor, subjected to washout, and subsequently re-stimulated under identical conditions. CP8-treated cells exhibited robust recovery of (C) IL-2 and (D) IFN-γ production, as well as (E) CD69 expression, approaching stimulated control levels upon re-challenge. In contrast, cells exposed to FR104, Acazicolcept, and Lulizumab remained functionally impaired, indicating persistent post-exposure suppression of T-cell responsiveness. Data are presented as individual donor values with mean ± SEM.

Article Snippet: To assess the reversibility of CD28 inhibition, primary human PBMCs were preincubated with 500 nM of CP8, FR104 (MedChemExpress, Catalog# HY-P990587), Acazicolcept (MedChemExpress, Catalog# HY-P99420), or Lulizumab (MedChemExpress, Catalog# HY-P99302) for 60 min at 37 °C prior to stimulation.

Techniques: Derivative Assay, Concentration Assay, Inhibition, Activation Assay, Expressing, Control

Journal: Oncotarget

Article Title: Cell death and restoration of TRAIL-sensitivity by ciglitazone in resistant cervical cancer cells

doi: 10.18632/oncotarget.22632

Figure Lengend Snippet: ( A ) Following a one hour-pre-incubation with or without monoclonal antibodies blocking DR4 and DR5 receptors (5 μg/ml), Ca Ski cells were stimulated by 40 μM ciglitazone and RT4 cells by 50 ng/ml TRAIL ( insert ) for 12 h. The percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis. Data are means ± SEM of three independent experiments performed in triplicates. * P < 0.05 compared to untreated cells, # P < 0.05 compared to ciglitazone-treated Ca Ski cells or TRAIL-treated RT4 cells. Right , after treatment, whole cell lysates were prepared and total protein extracts were subjected to immunoblotting for detection of procaspase 8, 9 and 3 processing. ( B ) Cells were treated for 12 h with ciglitazone at 10 and 40 μM. Cellular proteins were isolated and subjected to immunoblotting for detection of DR4 and DR5. Values of densitometric analyses are indicated. ( C ) Ciglitazone-treated cells were stained with anti-DR4-PE or anti-DR5-PE and analysed by flow cytometry. ( D ) Whole cell lysates prepared from ciglitazone-treated cells were assayed for TRAIL expression by western blotting analysis. Values of densitometric analyses are indicated. ( E ) Ca Ski cells were stained with anti-TRAIL-PE and analysed by flow cytometry. ( F ) Conditioned media were collected from ciglitazone-stimulated cells and the concentration of soluble TRAIL was measured by ELISA. Data are means ± SEM of two independent experiments performed in quadruplicates. * P < 0.05 compared to untreated cells. β-actin was used as an internal loading control.

Article Snippet: Cells (10 6 ) were resuspended in 50 μl of PBS and stained with 10 μl of phycoerythrin (PE)-conjugated mouse monoclonal anti-human DR4 (clone B-N36), DR5 (clone B-K29) or TRAIL (clone B-S23) provided by Diaclone (Besançon, France), for 30 min at 4°C in the dark.

Techniques: Incubation, Bioprocessing, Blocking Assay, Flow Cytometry, Western Blot, Isolation, Staining, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Oncotarget

Article Title: Cell death and restoration of TRAIL-sensitivity by ciglitazone in resistant cervical cancer cells

doi: 10.18632/oncotarget.22632

Figure Lengend Snippet: ( A ) Cells were treated with TRAIL at the indicated concentrations. The percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis. ( B ) Ca Ski cells were treated or not with ciglitazone (30 or 40 μM) or human recombinant TRAIL (50 ng/ml) for 12 h or cotreated with ciglitazone and TRAIL. In this case, the duration of the treatment is 24 h (12 h with ciglitazone plus 12 h with TRAIL). The percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis. ( C ) Cells were pre-incubated for 1 h with 50 μM caspase 8 (Z-IETD-FMK) or 9 (Z-LEHD-FMK) specific inhibitor before indicated treatment; top , the percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis; bottom , cells were treated as indicated. Whole cell lysates were prepared and total protein extracts were subjected to immunoblotting for detection of caspase 8, 9, 3 and Bid processing. ( D ) Cells were pre-incubated with or without monoclonal antibodies blocking DR4 and DR5 receptors for 1 h and stimulated as indicated; top , the percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis; bottom , caspase 8, 9, and 3 cleavage was assayed by western blotting analysis. Data are means ± SEM of three independent experiments performed in triplicates. * P < 0.05 compared to untreated cells. † P < 0.05 compared to 30 μM ciglitazone-treated cells. # P < 0.05 compared to ciglitazone and TRAIL cotreated cells. β-actin was used as an internal loading control.

Article Snippet: Cells (10 6 ) were resuspended in 50 μl of PBS and stained with 10 μl of phycoerythrin (PE)-conjugated mouse monoclonal anti-human DR4 (clone B-N36), DR5 (clone B-K29) or TRAIL (clone B-S23) provided by Diaclone (Besançon, France), for 30 min at 4°C in the dark.

Techniques: Flow Cytometry, Recombinant, Incubation, Western Blot, Bioprocessing, Blocking Assay, Control

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a) Representative flow cytometry density plots with CD62L-CD45RA co-expression profiles in CD4 T cells before and after the stimulation with S-peptides. Quadrants were established with unstained controls. Percentages of the corresponding populations are shown within the quadrants. b , c) CD4 T cell phenotypic changes in H-V (b) and H-CoV-V (c) donors. (-) and (+S), non-stimulated and S-peptide stimulation. N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L- CD45RA-) and effector (CD62L-CD45RA+) phenotypes. d) Phenotypic changes in CD4 T cells within O-CoV-V before and after stimulation with S-peptides. e , f) Effects of previous CoV infection in vaccinated H donors and O patients over T cell phenotypes after stimulation with S-peptides. b-f) Relevant statistical comparisons are indicated by ANOVA followed by pair-wise comparisons with Tukey’s test. g , h) Relative percentages of CD4 T cell differentiation phenotypes in H-V and O-V (g) and in H-CoV-V and O-CoV-V (h) CD27+ CD28+, CD27-CD28+ and CD27+ CD28+ indicate poorly differentiated, intermediate differentiated and highly differentiated T cell phenotypes. U of Mann-Whitney was used to test for significance.*, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing, Infection, Cell Differentiation, MANN-WHITNEY

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a, b, c) Percentage of S-specific CD8 T cells in PBMCs stimulated with S-peptides in H donors and O patients as indicated. d, e, f) Percentage of S-specific CD8 T cells in PBMCs stimulated with M-peptides in H donors and O patients. g, h, i) Percentage of S-specific CD8 T cells in PBMCs stimulated with N-peptides in H donors and O patients. a-i) Significance was tested with Krustal-Wallis followed by Dunn’s test. j) Percentage of activated CD8 T cells after stimulation with S or M specific peptides in O-CoV donors. U of Mann-Whitney was used to test for significance. k) Relative percentages of CD8 T cell differentiation phenotypes in the indicated groups of H and O donors. Means and error bars (standard deviations) are shown. N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L-CD45RA-) and effector (CD62L- CD45RA+) phenotypes. Relevant statistical differences are detailed in . j) Relative percentages of CD8 T cell differentiation phenotypes in the indicated groups of H and O donors. CD27+ CD28+, CD27-CD28+ and CD27+ CD28+ indicate poorly differentiated, intermediate differentiated and highly differentiated T cell phenotypes.. Relevant statistical differences are detailed in . *, ** and *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences. NST, technical control of non-stimulated (NST) PMBCs.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: MANN-WHITNEY, Cell Differentiation, Control

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a) Representative flow cytometry density plots with CD62L-CD45RA co-expression profiles in CD8 T cells before and after the stimulation with S-peptides. Quadrants were established with unstained controls. Percentages of the corresponding populations are shown within the quadrants. b, c, d) Relative percentages of CD8 T cell differentiation phenotypes from the indicated H donors and O patient cohorts. Means and error bars (standard deviations) are shown. N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L-CD45RA-) and effector (CD62L-CD45RA+) phenotypes. e, f, g) Relative percentages of CD8 T cell differentiation phenotypes from the indicated H and O cohort groups. h , i) Relative percentages of CD8 T cell differentiation phenotypes in H-CoV-V and O-CoV-V groups before and after stimulation with S-peptides. j, k) Relative percentages of CD8 T cell differentiation phenotypes in V-H-CoV, V-O-CoV, H-V and O-V groups as indicated in the graphs. CD27+ CD28+, CD27-CD28+ and CD27+ CD28+ indicate poorly differentiated, intermediate differentiated and highly differentiated T cell phenotypes. b-k) Statistical significance was tested by ANOVA followed by Tukey’s pair-wise comparisons. *, ** and *** indicate, significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing, Cell Differentiation

Journal: Journal for immunotherapy of cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models.

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Figure 1 Successful conformal nanoencapsulation of T cells and preservation of original cell functions. (A) Illustration of T-cell encapsulation progression. (B) Depiction of zeta potential changes in T cells throughout the layer-by-layer (LbL) encapsulation process. (C) Absorption peak plots of both alginate and FITC-alginate at 480 nm are presented, accompanied by FITC fluorescence images of the encapsulated T cell’s outer layer. (D) Representative flow scatter plots, demonstrating the encapsulation efficiency achieved when employing a combination of 0.2% gelatin and 0.25% alginate. (E) Comparative scanning electron microscopy images of non-encapsulated and encapsulated T cells are displayed, supplemented by differential interference contrast images. Quantification of cell diameters was executed using ImageJ software. (F–G) Following 48 hours of purified CD3/CD28 antibody-stimulated proliferation, representative flow peak plots of CFSE for both encapsulated and non-encapsulated T cells are exhibited. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. (H–J) The secretion levels of TNF-α, IL-2, and IFN-γ by T cells at 48 hours and 96 hours post- activation by CD3/CD28 antibody were detected by ELISA. (K–L) Comparison of the Anti-CD3 binding capacity between non- encapsulated and encapsulated T cells. All data are represented as mean values±SE, results of at least three (G–J) or five (B, L) repeat experiments each with three samples. *p<0.05, **p<0.01. APC, antigen-presenting cell; CFSE, carboxyfluorescein succinimidyl ester; DPBS, Dulbecco's phosphate-buffered saline; FITC, fluorescein isothiocyanate; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: APC- conjugated anti- mouse CD28 (130- 111- 973) and Anti- PE MicroBeads (130- 048- 801) were purchased from Miltenyi (Germany).

Techniques: Preserving, Encapsulation, Zeta Potential Analyzer, Fluorescence, Electron Microscopy, Software, Purification, Activation Assay, Enzyme-linked Immunosorbent Assay, Comparison, Binding Assay, Saline

Journal: Journal for immunotherapy of cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models.

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Figure 3 Single-cell nanoencapsulation reduced the expression of co-stimulatory molecules between donor T cells and recipient antigen-presenting cells and affected the formation of immune synapses. Mature DCs were co-cultured with encapsulated or non-encapsulated donor T cells to activate unidirectional mixed lymphocyte responses. (A–B) Proliferation of CFSE-labeled T cells was monitored at 48 hours and 96 hours. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. (C–F) Representative flow cytometry histograms and associated statistical analysis of the co-stimulatory molecules CD28, ICOS, and CD40L on T cells and CD80, ICOSL, and CD40 on DCs. (G–K) DCs were activated, sensitized with OVA antigen, and co-cultured with either encapsulated or non-encapsulated donor T cells for 6 hours. Cells within this co-culture system were then collected for further analysis. (G–H) Imaging flow cytometry results comparing the encapsulated group to the non-encapsulated group. (I) Representative immunofluorescence images of T cells co-cultured with DCs in both the encapsulated and non-encapsulated groups. (J–K) Scanning electron microscopy and TEM images of T cells co-cultured with DCs in both the encapsulated and non-encapsulated groups. Mean value±SEM, results of at least five repeat experiments each with three samples. *p<0.05, **p<0.01, ***p<0.01. CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; DC, dendritic cell; MHC, major histocompatibility complex; TEM, transmission electron microscopy.

Article Snippet: APC- conjugated anti- mouse CD28 (130- 111- 973) and Anti- PE MicroBeads (130- 048- 801) were purchased from Miltenyi (Germany).

Techniques: Expressing, Cell Culture, Labeling, Fluorescence, Flow Cytometry, Co-Culture Assay, Imaging, Immunofluorescence, Electron Microscopy, Immunopeptidomics, Transmission Assay

Journal: Journal for immunotherapy of cancer

Article Title: Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models.

doi: 10.1136/jitc-2023-008663

Figure Lengend Snippet: Figure 4 Transplantation of encapsulated T cells in combination with BMCs inhibits the development of GVHD in recipient mice while preserving the GVL effect. (A) Experimental design diagram, demonstrating the use of T cells encapsulated with BMCs for the inhibition of GVHD progression in female BALB/c recipient mice. Mice (20 g each) were administered intraperitoneal (IP) injections of 0.4 mg busulfan and 2 mg cyclophosphamide 7 days prior to BMT. WEHI-3B cells were subsequently infused via tail vein 1-day pre-transplantation. The transplantation involved injecting BMCs (CD45.1) and splenic T cells (CD45.2) from H2-b C57BL/6 mice into the recipient's tail veins. (B–D) Graphical representation of alterations in body weight, clinical scores, and survival rates of mice across all groups, monitored over a period of 60 days. (E–H) Flow cytometry scatter plots and associated statistical results, indicating the proportions of CD3+, CD4+, and CD8+T cell subsets in mice peripheral blood. A comparison is made between groups receiving encapsulated and non-encapsulated T cells. (I) Flow cytometry quantification of cells derived from CD45. One donor mice in recipient mice peripheral blood reflects donor bone marrow-derived cell engraftment. Splenic lymphocytes from recipient mice were analyzed. (J–K) Representative scatter plots depicting Treg and T helper cell 17 cell subsets. (L) Representative flow cytometry peak plots and statistical analysis of CD28, CD40L, and ICOS expression within the H2kb+CD3+ subset. (M) Representative flow cytometry peak plots and statistical analysis of CD80, CD40, and ICOSL expression within the H2kd+LIN-CD11c+MHC-II+ subpopulation was presented. (N–Q) Bar graphs representing the secretion levels of IL-6, IL-10, IFN-γ, and C-X-C motif chemokine ligand 10 in plasma and peritoneal macrophages across both encapsulated and non-encapsulated groups. (S–V) Statistical plot of pathological scores of H&E- stained images of each target organs from mice. Pooled data from three independent experiments each with seven recipients. Survival (D) Kaplan-Meier curve, clinical score (C) weight (B) from two or three independent experiments, each with seven mice per group, are shown. Mean value±SEM; *p<0.05, ***p<0.001. BMC, bone marrow cell; GVHD, graft-versus-host disease; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; Treg, regulatory T cell.

Article Snippet: APC- conjugated anti- mouse CD28 (130- 111- 973) and Anti- PE MicroBeads (130- 048- 801) were purchased from Miltenyi (Germany).

Techniques: Transplantation Assay, Preserving, Inhibition, Flow Cytometry, Comparison, Derivative Assay, Expressing, Clinical Proteomics, Staining, Fluorescence, Immunopeptidomics