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: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A) CD4+ T cells were activated with anti-CD3/CD28 over 72 h. IRF5 expression by western blot. IRF5−/−CD4+ T cells served as a negative control (representative of 2 independent experiments).

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: Expressing, Western Blot, Negative Control

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A and B) IRF5+/+, IRF5+/−, or IRF5−/−CD4+ T cells were activated in vitro with anti-CD3/CD28.

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: In Vitro

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A–C) IRF5+/+, IRF5+/−, or IRF5−/−CD4+ T cells were activated with anti-CD3/CD28.

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques:

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A–C) CD4+ T cells from IRF5+/+ and IRF5−/−mice were stimulated in vitro with anti-CD3/CD28 at the indicated times and phosphorylated proteins were assessed by (A and B) western blot. Representative of 3–4 independent experiments. (C) Flow cytometry. Top: representative flow cytometry with MFI indicated. Bottom: summary phospho-proteins (3 replicates/group).

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: In Vitro, Western Blot, Flow Cytometry

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: CD45RA+ CD4+ T cells were isolated from rs2004640/rs2280714 TT/TT or GG/CC carriers (n = 14/genotype). (A and B) Cells were treated with anti-CD3/CD28 for 72 h.

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: Isolation

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Sonication, DNA Library Preparation, Purification, Activation Assay, cDNA Synthesis, Software

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder

doi: 10.7554/elife.72559

Figure Lengend Snippet: Figure 4. Patient T cell responses to T cell receptor stimulation. (A) Representative flow plots showing expression of IFN-γ in human PBMCs from a healthy donor and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr and cultured for further 4 days in the presence of IL-2 before re-stimulation with PMA plus ionomycin for 5 hr for cytokine analysis. Cells were gated for CD4+ T cells. (B) Quantitative mRNA expression analysis (± s.d.m.) of indicated cytokines from human PBMCs (cultured as mentioned above) with or without stimulation with PMA plus ionomycin for 5 hr. (C) Levels of IL-2 and TNF in human PBMCs from culture supernatants of cells stimulated as described above (B) were determined by ELISA. (D) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after transient stimulation with anti-CD3 antibody cross-linking, or ionomycin (0.5 µM) in the presence of external solution containing 2 mM Ca2+ (left) as indicated. Bar graphs show baseline subtracted ratio values for anti-CD3 antibody cross-linking or ionomycin (average± SEM) from six independent experiments (right). (E) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after store-depletion with thapsigargin (1 µM) stimulation in Ca2+-free Ringer’s solution. SOCE was measured by addition of 2 mM Ca2+-containing Ringer’s solution as indicated (left). Bar graph shows baseline subtracted ratio values at the peak of SOCE (average± SEM) from four independent experiments (right). (F) Phosphorylated JNK levels in CD4+ T cells from healthy control and patient PBMCs (cultured as indicated in A), stimulated with anti-CD3 antibody for indicated times. Bar graphs show average± SEM from three independent experiments. ** p < 0.005, *** p < 0.0001.

Article Snippet: PBMCs from healthy control and patient were activated for 48 hr on a plate coated with 10 μg/ml of anti- CD3 antibody (OKT3, Bio X Cell) and cultured in T cell media (DMEM containing 20% fetal bovine serum and 1% Pen- Strep) supplemented with 5 μg/ml of anti- CD28 antibody (Bio X cell), and 20 U/ml IL- 2 (Peprotech) for ThN differentiation.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder

doi: 10.7554/elife.72559

Figure Lengend Snippet: Figure 5. Cytokine expression profiles in T cells expressing individual allelic mutations in CRACR2A. (A) Representative flow plots showing expression of IL-2 in control or CRACR2A KO Jurkat T cells stably expressing FLAG-tagged WT CRACR2A, CRACR2AE278D (E278D), or CRACR2AR144G/E300* (DM) after stimulation with PMA plus ionomycin for 16 hr (left). Bar graph shows means ± s.e.m. of pooled technical replicates from two independent experiments (right). (B) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells transduced with lentiviruses encoding CRACR2A- targeting sgRNA and those encoding cDNAs of WT or indicated mutants of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. (C) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells purified from healthy donors and transduced with lentiviruses encoding cDNAs for WT or indicated mutant of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. * p < 0.05, *** p < 0.0001.

Article Snippet: PBMCs from healthy control and patient were activated for 48 hr on a plate coated with 10 μg/ml of anti- CD3 antibody (OKT3, Bio X Cell) and cultured in T cell media (DMEM containing 20% fetal bovine serum and 1% Pen- Strep) supplemented with 5 μg/ml of anti- CD28 antibody (Bio X cell), and 20 U/ml IL- 2 (Peprotech) for ThN differentiation.

Techniques: Expressing, Control, Stable Transfection, Transduction, Purification, Mutagenesis

Journal: Journal of Cell Communication and Signaling

Article Title: Platelet-lymphocyte co-culture serves as an ex vivo platform of dynamic heterotypic cross-talk

doi: 10.1007/s12079-022-00676-0

Figure Lengend Snippet: CD4+ and CD8+ population when PBMCs are co-cultured alone or with platelets. PBMCs were cultured without stimuli (unstimulated) or stimulated with anti-CD3/CD28 or PHA for 5 days. PBMCs were cultured alone (A and C) or co-cultured with platelets (B and D). Cell populations positive to CD4 (A and B) and CD8 (C and D) were determined using flow cytometry every day from day 0 to 5. Data are expressed as mean of the fraction of cells among PBMC (%) ± S.E.M. (*P < 0.05, n = 4)

Article Snippet: Anti-human CD3 and anti-human CD28 were purchased from TONBO bioscience (San Diego, CA).

Techniques: Cell Culture, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Platelet-lymphocyte co-culture serves as an ex vivo platform of dynamic heterotypic cross-talk

doi: 10.1007/s12079-022-00676-0

Figure Lengend Snippet: Platelets surface expression of p-selectin and the ability to shed soluble p-selectin are both maintained throughout the culture. Platelets were cultured alone (A) or co-cultured (B) with PBMC in unstimulated environment or with anti-CD3/CD28 or PHA for 5 days. P-selectin expression on platelet surface was detected using flow cytometry every day from day 0 to 5. A P-selectin surface expression was analyzed in platelets co-cultured with PBMCs. B P-selectin surface expression was analyzed in platelets cultured alone and activated with 2Mes-ADP exposure (100 nM – 5 min). Data are expressed as the mean of the fraction of positive cells among platelets (%) ± S.E.M. (*P < 0.05, n = 4). Platelets were co-cultured alone or with PBMC in unstimulated environment (C) or with anti-CD3/CD28 (D) or PHA (E) for 5 days. Soluble P-selectin concentration in the supernatant was analyzed using an ELISA kit every day from day 1 to 5. Platelets alone (patter bars), platelets cultured with PBMC (white bars) and platelets activated with 2Mes-ADP (gray bars, 100 nM – 5 min) were analyzed. Soluble P-selectin concentration by ADP-activated platelets after isolation (day 0) was 168,370 ± 13,342. Data are expressed as pg/mL ± S.E.M. (*P < 0.05, n = 4)

Article Snippet: Anti-human CD3 and anti-human CD28 were purchased from TONBO bioscience (San Diego, CA).

Techniques: Expressing, Cell Culture, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Journal of Cell Communication and Signaling

Article Title: Platelet-lymphocyte co-culture serves as an ex vivo platform of dynamic heterotypic cross-talk

doi: 10.1007/s12079-022-00676-0

Figure Lengend Snippet: Platelet secretion is maintained throughout the co-culture. Platelets were co-cultured alone or with PBMC in unstimulated environment (A) or with anti-CD3/CD28 (B) or PHA (C) for 5 days. PF-4 concentration in the supernatant was analyzed using an ELISA kit every day from day 1 to 5. Platelets alone (patter bars), platelets cultured with PBMC (white bars) and platelets activated with 2Mes-ADP (gray bars, 100 nM – 5 min) were analyzed. PF-4 concentration by ADP-activated platelets immediately after isolation (day 0) was 22,498.6 ± 1128.5. Data are expressed as pg/mL ± S.E.M. (*P < 0.05, n = 4)

Article Snippet: Anti-human CD3 and anti-human CD28 were purchased from TONBO bioscience (San Diego, CA).

Techniques: Co-Culture Assay, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Journal of Cell Communication and Signaling

Article Title: Platelet-lymphocyte co-culture serves as an ex vivo platform of dynamic heterotypic cross-talk

doi: 10.1007/s12079-022-00676-0

Figure Lengend Snippet: Platelet and T lymphocytes aggregate in a time- and stimuli-dependent manner. Platelets and PBMCs were co-cultured in unstimulated environment (A) or with anti-CD3/CD28 (B) or PHA (C) for 5 days. Platelets-T cells aggregate formation was determined using flow cytometry. T cells were gated based on either CD4 or CD8 expression and cell shape, and data were analyzed based on the percentage of aggregates that express both CD41 and CD4 or CD41 and CD8. Aggregates were measured every day from day 1 to 5. Data are expressed as mean of the fraction of cells among T cells (%) ± S.E.M. (*P < 0.05, n = 4)

Article Snippet: Anti-human CD3 and anti-human CD28 were purchased from TONBO bioscience (San Diego, CA).

Techniques: Cell Culture, Flow Cytometry, Expressing