Journal: International Journal of Molecular Sciences

Article Title: Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson’s Disease with CMV Infection Background

doi: 10.3390/ijms222313119

Figure Lengend Snippet: The proportion of T cells (CD3 + ), including CD4 + and CD8 + subsets, and NK cells in PD patients, HD group and CMV-seropositive HD individuals.

Article Snippet: The following mouse anti-human fluorescent-labeled antibodies were used for surface cell staining: CD56-APC (clone N901, Beckman Coulter, Miami, FL, USA), CD4-FITC (clone RPA-T4, Sony Biotechnology, San Jose, CA, USA), CD57-PE (clone HCD57, Sony Biotechnology, San Jose, CA, USA), NKG2C-PE (clone 134591, R&D Systems, Minneapolis, MN, USA), CD3-PerCP (clone HIT3a, Sony Biotechnology, San Jose, CA, USA), CD3-FITC (clone FIT3a, Sony Biotechnology, San Jose, CA, USA), CD3-APC (clone F OKT3, Sony Biotechnology, San Jose, CA, USA) CD8-PerCP (clone SK1, Sony Biotechnology, San Jose, CA, USA), CD45RA-APC (clone HI100, Sony Biotechnology, San Jose, CA, USA), CD197-FITC (clone G043H7, Sony Biotechnology, San Jose, CA, USA), CD56-APC-Vio770 (clone REA196, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: (A) IFN-γ spots, each representing a CD8 T cell responding with IFN-γ production, formed upon sensitization of NLV peptide-specific murine (mCD8-NLV; left panel) and human (hCD8-NLV; right panel) cytolytic CD8 T-cell lines (CTLL) by NLV-peptide-pulsed NSG/HHD MEF at an effector-to-stimulator cell ratio of 0.1:1. MEF were pretreated with murine IFN-γ for 48h (filled bars) or left untreated (open bars) and were exogenously loaded with synthetic NLV- or non-cognate gp100 280-288 -peptide at the indicated concentrations. Data are shown as mean of duplicates from one experiment representative of two performed. Error bars represent the range. (B) Structural avidity of antigen binding to mCD8-NLV and hCD8-NLV CTLL was quantified by dose-dependent HLA-A2.1/NLV tetramer binding in flow cytometry. The respective dissociation constant (K D ) was calculated from half-maximal tetramer binding obtained by Scatchard plot analysis. Dotted curves border the 95% confidence regions of the log-linear regression lines. MFI, mean fluorescence intensity. (C) HLA-A2.1 restricted presentation of NLV epitope by NSG/HHD MEF pretreated with murine IFN-γ for 48h (filled bars) or left untreated (open bars) and infected at an MOI of 4 with the indicated viruses for a total time of 22h until the end of the assay. Peptide presentation on the infected MEF during that period was detected in an IFN-γ ELISpot assay with mCD8-NLV (left panel) and hCD8-NLV (right panel) CTLL at an effector-to-stimulator cell ratio of 0.1:1. Data are shown as mean of duplicates from one experiment representative of two performed. Error bars represent the range.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Infection, Enzyme-linked Immunospot

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: (A) Experimental strategy and schedule of adoptive transfer of NLV-specific CD8 T cells into infected NSG/HHD recipients that were preconditioned by total-body γ-irradiation with a dose of 2 Gy. Intraplantar infection was performed throughout with 1x10 5 PFU of chimeric mCMV. Recipients (n = 4–5 per group) were infected with mCMV-NLV (filled symbols) or mCMV-NLV Ala (open symbols), followed by i.v. transfer of graded numbers of (B) murine or (C) human NLV-specific CD8 T cells (mCD8-NLV or hCD8-NLV CTLL, respectively). Infectivity was quantified at d11 post-transfer in spleen and lungs by standard plaque (plaque-forming unit, PFU, assay). In the livers of the same mice, infected cells (filled and open circles for mCMV-NLV and mCMV-NLV Ala , respectively) and tissue-infiltrating CD8 T cells (filled and open squares, correspondingly) were stained by 2C-IHC and counted in representative 10-mm 2 areas of tissue sections. Symbols represent individual mice and horizontal bars mark median values. Statistical analysis for group differences of most interest (bracketed) was performed after log-transformation using Student’s t-test (unpaired, two-sided; p<0.05 considered significant) with Welch’s correction.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Adoptive Transfer Assay, Infection, Irradiation, Staining, Transformation Assay

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: Corresponding to , 2C-IHC of liver tissue sections taken on d11 after transfer of (A) mCD8-NLV CTLL or (B) hCD8-NLV CTLL show mCMV-NLV or mCMV-NLV Ala infected hepatocytes (iHc, red staining of intranuclear IE1 protein) with typical intranuclear inclusion bodies, and infiltrating CD8 T cells (CD8-T, black cytoplasmic and membrane staining of CD3ε), forming nodular inflammatory foci (NIF) or foci of infection in absence of CD8 T cells (IF), respectively. Upper row images give overviews (A,B; a1, b1), higher-magnification lower row images (A, B; a2, b2) reveal details. Bar markers represent 50 μm throughout.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Infection, Staining

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: Groups of 10 γ-irradiated (2 Gy) NSG/HHD mice were infected (A) with 1x10 5 PFU or (B) with 1x10 3 PFU of mCMV-NLV, and in (A; B,b) they received the 1:4 mixtures (see the legend to ) of TCR NLV -transduced (solid graphs) or mock-transduced (dashed graphs) human CD4 and CD8 T cells on the day of infection as a pre-emptive therapy. (B,a) As a reference for comparison, recipients received 1x10 7 cells of CTLL hCD8-NLV (solid graph) or were left with no T cell transfer (dashed graph, w/o T cells). Survival rates over time are displayed as Kaplan Meier survival plots. Statistical significance of differences in survival was calculated using the log-rank test and the Gehan-Wilcoxon test. In the most efficient therapy with the mixture of TCR NLV -transduced CD4 and CD8 T cells (B,b), the median survival time was 26d (range: 21-27d) compared to 21d (range 16-22d) in the mock-transfected control group.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Irradiation, Infection, Transfection

Journal: Cell reports

Article Title: Pro-resolving lipid mediator lipoxin A 4 attenuates neuro-inflammation by modulating T cell responses and modifies the spinal cord lipidome

doi: 10.1016/j.celrep.2021.109201

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-mouse CD8 PerCP-eFluor 710 , BD Biosciences , Cat# 53–6.7; RRID: AB_1603266.

Techniques: Recombinant, Activation Assay, Software

Journal: Physiological Reports

Article Title: Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

doi: 10.14814/phy2.14059

Figure Lengend Snippet: Impact of cyclophosphamide (CYC) on circulating immune cells. Flow cytometry was performed on cryopreserved peripheral blood leukocytes to determine the impact of CYC treatment on lymphoid and myeloid cell populations. (A) Representative scatter plot of peripheral blood leukocytes. (B) Data suggest that CYC treatment reduced CD45R + B cells compared to vehicle treatment in systemic lupus erythematosus (SLE) mice (15.87 ± 8.49% vs. 26.96 ± 4.72%, P = 0.06). (C) There was no significant difference in the percentage of circulating CD3 + CD4 + T cells between groups or (D) the percentage of CD3 + CD8 + T cells.(E) The percentage of circulating neutrophils was significantly increased in CYC‐treated SLE mice compared to vehicle‐treated mice (39.26 ± 4.92 vs. 20.58 ± 6.01, * P < 0.05). (F) There was no significant different in the percentage of circulating monocytes between groups. ○ SLE Vehicle ( n = 8), and ● SLE CYC ( n = 10).

Article Snippet: The following antibodies were diluted with wash buffer (50 μL/sample; 1:100 dilution), and placed in a single tube‐containing sample to measure the relative percentages of circulating lymphocyte populations: CD3e‐PE‐Cy7 (clone 145‐2C11), CD4‐FITC (clone GK1.5), CD8‐PerCP‐Cy (clone 53‐6.7), and CD45R‐Alexa Fluor (clone RA3‐6B2) (BD Biosciences).

Techniques: Flow Cytometry

Journal: Physiological Reports

Article Title: Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

doi: 10.14814/phy2.14059

Figure Lengend Snippet: Impact of cyclophosphamide (CYC) on renal lymphocyte infiltration. (A) Renal CD45R + B cells were significantly increased in systemic lupus erythematosus (SLE) vehicle‐treated mice compared to all other treatment groups (* P < 0.05) (B) Renal CD3 + CD4 + T cells were significantly increased in SLE vehicle‐treated mice compared to all other treatment groups (* P < 0.05). (C) Renal CD3 + CD8 + T cells were not significantly different in response to CYC treatment in control or SLE mice. □ Control Vehicle ( n = 5) ■ Control CYC ( n = 5), ○ SLE Vehicle ( n = 5), and ● SLE CYC ( n = 5).

Article Snippet: The following antibodies were diluted with wash buffer (50 μL/sample; 1:100 dilution), and placed in a single tube‐containing sample to measure the relative percentages of circulating lymphocyte populations: CD3e‐PE‐Cy7 (clone 145‐2C11), CD4‐FITC (clone GK1.5), CD8‐PerCP‐Cy (clone 53‐6.7), and CD45R‐Alexa Fluor (clone RA3‐6B2) (BD Biosciences).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation

doi: 10.3390/ijms25115722

Figure Lengend Snippet: Total T cells and CD4 + , CD8 + subsets quantified by flow cytometry in peripheral blood from patients diagnosed with simple silicosis (SS) or pulmonary massive fibrosis (PMF) and healthy controls (HC). ( A ) Percentage of total T cells (CD3 + ). ( B ) Percentage of T helper cells (CD3 + CD4 + ). ( C ) Percentage of cytotoxic T cells (CD3 + CD8 + ). ( D ) CD4 + /CD8 + ratio. ° indicate outliers. * indicate significance at p < 0.05.

Article Snippet: Surface immunostaining of the cell population was performed by incubating 150 μL of whole peripheral blood for 15 min in the dark with the following antibodies in different combinations ( ): CD45-V500, CD3-APC-H7, CD4-V450, CD56-APC, CD19-PerCP-CY7, CD27-PE, CD16-FITC, CD45RA-FITC, and CD45RO-PE from BD (Becton Dickinson; San Jose, CA, USA); CD19-APC, CD38-FITC, and CD8-PerCP from Immunotools (Friesoythe, Germany); and CD127-PerCP-CY5.5 from BioLegend (San Diego, CA, USA).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation

doi: 10.3390/ijms25115722

Figure Lengend Snippet: Analysis of the surface markers of the lymphocyte subsets.

Article Snippet: Surface immunostaining of the cell population was performed by incubating 150 μL of whole peripheral blood for 15 min in the dark with the following antibodies in different combinations ( ): CD45-V500, CD3-APC-H7, CD4-V450, CD56-APC, CD19-PerCP-CY7, CD27-PE, CD16-FITC, CD45RA-FITC, and CD45RO-PE from BD (Becton Dickinson; San Jose, CA, USA); CD19-APC, CD38-FITC, and CD8-PerCP from Immunotools (Friesoythe, Germany); and CD127-PerCP-CY5.5 from BioLegend (San Diego, CA, USA).

Techniques: