Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Panel (a) of this figure depicts the expression of the T cell activation marker GITR on CD25+, CD25 bright and CD25 verybright cells from healthy controls (white bars, n = 24) and SSc patients having limited cutaneous SSc (light gray bars, n = 18), late diffuse SSc (dark gray bars, n = 22) and early diffuse SSc (black bars, n = 22) patients. In panel (b) the expression of CD62L on Tregs is investigated. CD25+ and CD25 bright cells from SSc and healthy controls express similar levels of CD62L, whereas CD25 verybright from SSc patient subsets exhibit lower levels of CD62L compared to those from healthy controls. Panel (c) reflects the expression of CD69 on Tregs from healthy donors and SSc patients. CD25+, CD25 bright and CD25 verybright cells from SSc patients express significant lower levels of CD69 than those from healthy donors. CD69 expression on CD25 bright and CD25 verybright cells from edSSc patients was significantly lower then that from ldSSc patients, and ldSSc expressed CD69 significantly lower than those from lSSc. In panel (d) the expression on CD3+ cells is shown for all investigated groups. In contrast with that observed on Tregs from SSc patients, CD69 expression on CD4+ cells was significantly higher in all SSc patient groups. Panel (e) reflects the potential association between CD69 expression on Tregs and disease duration. CD69 expression on T regs from patients with lSSc correlated with disease duration, whereas this was not the case either with ldSSc nor edSSc. In all figures the white bars represent healthy controls, whereas lSSc, ldSSc and edSSc patients are represented by light gray, dark gray and black bars, respectively.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Expressing, Activation Assay, Marker

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Unsorted CD3+ (MACS bead isolated) were stimulated with PHA (5 µg/ml) and consecutively incubated with CD25 high CD127 - or CD25 low CD127 high cells for 5 days. Thereafter, CD3+ cells were incubated with 3 H -thymidine for 24 more hours after which 3 H -thymidine incorporation was measured. Panel (a) reflects the suppressive capacity of Tregs from healthy donors and SSc patients. Proliferation of CD3+ effector cells was effectively inhibited by T regulatory cells from healthy controls, whereas a clearly diminished suppressive activity was observed in the experiments with Tregs from SSc patients. Suppressive effect of Treg (CD25 high CD127 - ) and non-Tregs (CD25 low CD127 high ) is presented in black and white bars, respectively. Results are the mean and SEM of 6 separate experiments using cells from healthy donors (n = 9), lSSc (n = 7), ldSSc (n = 9) and edSSc (n = 7). Panel (b) represents the correlation of CD69 expression and Treg suppressive capacity in Tregs from the various groups under investigation. The percentage of CD69 positive regulatory T cells (CD25 high CD127 - ) correlates well with the percentage of inhibition of CD3+ cells in healthy controls (triangles), lSSc (diamonds), ldSSc (circles) and edSSc (squares). Panel (c) reflects the expression of intracellular TGFβ in Tregs from healthy controls and SSc patients as measured using intracellular flow cytometry. CD25 high CD127 - cells from all SSc patients express lower TGFβ levels compared to controls. Left panel reflects an representative individual from each group whereas the right panel displays the mean of each group comprising 6 individuals (per group) coming forth from 4 independent experiments.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Isolation, Incubation, Activity Assay, Expressing, Inhibition, Flow Cytometry

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: (a) During the co-cultures of unsorted CD3+ cells with either Tregs (CD25 high CD127 - ) or non-Tregs (CD25 low CD127 high ) 10 or 25% plasma from an edSSc patient or healthy control was added to the culture. The graph represents data from 3 independent experiments using 3 healthy control cells, and plasma derived from two edSSc patients and two control individuals. (b) The effect of SSc plasma was evaluated by adding 10% to CD3+ cells for 24 hrs stimulated with PHA or unstimulated. As a control, CD69 expression was measured on CD3+ cells stimulated with PHA only. CD4 and CD25 high /FoxP3 high cells were gated based on the expression of these markers using flow cytometry. (c) CD69 expression and induction upon PHA mediated stimulation of CD4+ and CD25 high /FoxP3 high obtained from healthy donors, lSSc, ldSSc and edSSc patients was investigated using flow cytometry. One representative patient from each group is shown.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Clinical Proteomics, Control, Derivative Assay, Expressing, Flow Cytometry

Journal: BMC complementary medicine and therapies

Article Title: Effect and underlying mechanism of Huangjing Qianshi decoction in pre-diabetes mouse model.

doi: 10.1186/s12906-025-04893-z

Figure Lengend Snippet: Fig. 5 NR3C2 protein level in the tissues of pre-diabetic mice was reduced, whereas protein inhibitor of activated STAT1 (PIAS1) protein level was in creased after Huangjing Qianshi decoction (HJQST) and MET treatment. PIAS1 (A) and NR3C2 (B) proteins were measured using immunohistochemistry. Magnification ×100

Article Snippet: Antibodies against GLUT-4 (1:100 dilution, 66846-1-Ig), INS (1:1000 dilution, 66198-1-Ig), NR3C2 (1:2000 dilution for western blot and 1:100 dilution for IHC, 21854-1-AP), PIAS1 (1:2000 dilution for western blot and 1:100 dilution for IHC, 23395-1-AP), STAT1 (1:2000 dilution, 66545-1- Ig), p-STAT1 (1:2000 dilution, 28977-1-AP), PGC-1a (1:5000 dilution, 66369-1-Ig), and GAPDH (1:20000 dilution, 60004-1-Ig) were purchased from the Proteintech (Wuhan Sanying, Wuhan, China).

Techniques: Immunohistochemistry

Journal: BMC complementary medicine and therapies

Article Title: Effect and underlying mechanism of Huangjing Qianshi decoction in pre-diabetes mouse model.

doi: 10.1186/s12906-025-04893-z

Figure Lengend Snippet: Fig. 6 NR3C2, protein inhibitor of activated STAT1 (PIAS1), p-STAT1, and PGC-1α protein level was affected in the tissues of pre-diabetic mice after Huangjing Qianshi decoction (HJQST) and MET treatment. Proteins in the liver tissue and skeletal muscle tissues of pre-diabetic mice after Huangjing Qianshi decoction (HJQST) and MET treatment were measured using western blot. Full-length blots are presented in Supplementary Fig. 1. *p < 0.05, **p < 0.01, and ***p < 0.001

Article Snippet: Antibodies against GLUT-4 (1:100 dilution, 66846-1-Ig), INS (1:1000 dilution, 66198-1-Ig), NR3C2 (1:2000 dilution for western blot and 1:100 dilution for IHC, 21854-1-AP), PIAS1 (1:2000 dilution for western blot and 1:100 dilution for IHC, 23395-1-AP), STAT1 (1:2000 dilution, 66545-1- Ig), p-STAT1 (1:2000 dilution, 28977-1-AP), PGC-1a (1:5000 dilution, 66369-1-Ig), and GAPDH (1:20000 dilution, 60004-1-Ig) were purchased from the Proteintech (Wuhan Sanying, Wuhan, China).

Techniques: Western Blot

Journal: Immunity

Article Title: Low-Avidity CD4 + T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19

doi: 10.1016/j.immuni.2020.11.016

Figure Lengend Snippet:

Article Snippet: CD69-PE (REA824) , Miltenyi Biotec , Cat#130-112-613; RRID: AB_2659065.

Techniques: Functional Assay, Recombinant, Staining, Sequencing, Gene Expression, Software