Journal: Scientific Reports

Article Title: The colonic mucosa-associated microbiome in SIV infection: shift towards Bacteroidetes coincides with mucosal CD4 + T cell depletion and enterocyte damage

doi: 10.1038/s41598-020-67843-4

Figure Lengend Snippet: Mucosal SIV copy numbers and CD4 + T cells in the colon of SIV-infected animals. ( A ) Cell-associated SIV DNA and RNA copies were quantified longitudinally in colonic tissue before and after i.v. infection with SIVmac251. ( B ) CD4 + T cells in the colonic lamina propria were quantified by immunohistochemical analysis and the proportion CD45RA - memory phenotype CD4 + cells within mucosal CD3 + T cells was determined by flow cytometry before and at different stages of SIV infection. ( C ) CD4 + T cell counts in the peripheral blood were measured in parallel. Mucosal peak viral load was observed on day 7 (black symbols) or on day 14 after infection (grey symbols). Day 7-samples of the three animals, in which mucosal viral load peaked on day 14, represent ‘before peak viral load’ data. Equivalent data samples were not available from animals in which mucosal viral load peaked already on day 7. The chronic phase was analyzed on day 49 after infection. Horizontal solid lines indicate mean values + /− standard deviations. The dashed horizontal line indicates the assay limit of detection. * P < 0.05, *** P < 0.001, **** P < 0.0001. VL viral load.

Article Snippet: The primary antibodies was mouse anti-CD4 (clone 1F6; Novocastra, Newcastle, UK).

Techniques: Infection, Immunohistochemical staining, Flow Cytometry

Journal: Scientific Reports

Article Title: The colonic mucosa-associated microbiome in SIV infection: shift towards Bacteroidetes coincides with mucosal CD4 + T cell depletion and enterocyte damage

doi: 10.1038/s41598-020-67843-4

Figure Lengend Snippet: Associations of SIV infection-related shift towards Bacteroidetes with mucosal CD4 + T cell counts and cytokine secretion. Correlations of abundances of Bacteroidetes at the time mucosal peak viral load with ( A ) initial SIV infection-associated changes of frequencies of CCR5 + CD4 + T cells (determined by flow cytometric analysis) and ( B ) total CD4 + T cell numbers in the colonic mucosa (quantified by in situ immunostaining) and ( C ) with the levels of mucosal secretion of IL-2 (black symbols) and IL-4 (open symbols). Values, depicted as percent change in immune parameters, were calculated for each animal as change between pre-infection levels and those at the time of mucosal peak viral load. ( D ) Correlations between the changes in mucosal CCR5 + CD4 + T cell frequencies and the changes in abundances of Bacteroidetes during the chronic phase of SIV infection. Percent changes were calculated between levels at peak viremia and levels at the chronic stage of infection.

Article Snippet: The primary antibodies was mouse anti-CD4 (clone 1F6; Novocastra, Newcastle, UK).

Techniques: Infection, In Situ, Immunostaining

Journal: Frontiers in Immunology

Article Title: Human CD8 + CD28 − T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner

doi: 10.3389/fimmu.2018.01442

Figure Lengend Snippet: Representative human leukocyte antigen (HLA) typing of donors used for experiments.

Article Snippet: The following antibodies were incubated as the manufacturer’s instructions: rabbit anti-human CD8 Ab (ab93278, abcam) and mouse anti-human CD4 (T Helper/Inducer) monoclonal antibody (mAb) (ZM-0418, ZSGB-BIO) in a humidified chamber overnight at 4°C.

Techniques: Immunopeptidomics

Journal: Frontiers in Immunology

Article Title: Human CD8 + CD28 − T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner

doi: 10.3389/fimmu.2018.01442

Figure Lengend Snippet: CD8 + CD28 − T cells expanded by IL-15 inhibit the proliferation of CD4 + T cells in an allospecific manner in vitro . 5 × 10 4 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled responder CD4 + T cells (R) from donor A were stimulated with 5 × 10 4 antigen-presenting cells (APCs) from donor B (B -APCs ) or indifferent donor I [I -APCs , human leukocyte antigen (HLA)-A, -B, and -DR fully mismatched with donor B and donor A] in the presence of absence of 5 × 10 4 CD8 + CD28 − T cells (A-Ts) in triplicates in 96-well plates for 7 and 11 days. (A) The proliferation of CD4 + T cells was measured by CFSE dilution. (B) The percentages of proliferating CD4 + T cells were compared. The bar graphs indicate the means ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were incubated as the manufacturer’s instructions: rabbit anti-human CD8 Ab (ab93278, abcam) and mouse anti-human CD4 (T Helper/Inducer) monoclonal antibody (mAb) (ZM-0418, ZSGB-BIO) in a humidified chamber overnight at 4°C.

Techniques: In Vitro, Labeling

Journal: Frontiers in Immunology

Article Title: Human CD8 + CD28 − T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner

doi: 10.3389/fimmu.2018.01442

Figure Lengend Snippet: CD8 + CD28 − T cells expanded by IL-15 inhibit allospecific CD4 + T cell proliferation in vivo . (A) 4 × 10 6 purified responder CD4 + T cells (R) from donor A and 4 × 10 6 antigen-presenting cells (APCs) from donor B (B -APCs ) or from an indifferent donor I [I -APCs , human leukocyte antigen (HLA)-A, -B, and -DR fully mismatched with donor B] were adoptive transferred into NOG mice via i.p., the in vitro expanded CD8 + CD28 − T cells were added as putative suppressors (S) at S:R ratios of 0.5:1. (B) The mice were sacrificed to get splenic suspension on day 11, human CD45 and CD4 were as gates in flow cytometry to assess CD4 + T cells number. (C) The bar graphs indicate human CD4 + T cell number in mice splenic suspension, which were average value for three independent experiments. (D) Immunohistochemical staining in NOG mouse spleen tissues (magnification, 400×) showed human CD4 + T cell (A-CD4 + T cells) and CD8 + T cells (human CD8 + CD28 − T cells, A-Ts). Representative data of three mice in three independent experiments. The bar graphs indicate the means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were incubated as the manufacturer’s instructions: rabbit anti-human CD8 Ab (ab93278, abcam) and mouse anti-human CD4 (T Helper/Inducer) monoclonal antibody (mAb) (ZM-0418, ZSGB-BIO) in a humidified chamber overnight at 4°C.

Techniques: In Vivo, Purification, In Vitro, Suspension, Flow Cytometry, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: Human CD8 + CD28 − T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner

doi: 10.3389/fimmu.2018.01442

Figure Lengend Snippet: Suppression by the in vitro expanded CD8 + CD28 − T cells is contact dependent. Transwell assays: the lower chambers of 96-well transwell plates were plated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled responder cells (5 × 10 4 of CD4 + T cells, R) and stimulator cells (5 × 10 4 of B -APCs ), the in vitro expanded CD8 + CD28 − T cells (2.5 × 10 4 , S) were added either in the lower chamber to allow cell–cell contact or in the upper chamber to prevent cell–cell contact. The proliferation of the CD4 + T cells was measured by CFSE dilution assay. Data shown were representative of three independent experiments.

Article Snippet: The following antibodies were incubated as the manufacturer’s instructions: rabbit anti-human CD8 Ab (ab93278, abcam) and mouse anti-human CD4 (T Helper/Inducer) monoclonal antibody (mAb) (ZM-0418, ZSGB-BIO) in a humidified chamber overnight at 4°C.

Techniques: In Vitro, Labeling, Dilution Assay

Journal: Frontiers in Immunology

Article Title: Human CD8 + CD28 − T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner

doi: 10.3389/fimmu.2018.01442

Figure Lengend Snippet: Programmed death-ligand 1 (PD-1):programmed death-ligand 1 (PD-L1) signaling plays a critical role in the suppressive function of in vitro expanded CD8 + CD28 − Ts cells Suppression assays were set up as described in Figure A at an S:R ratio of 0.5:1. The proliferation of the CD4 + T cells was measured by carboxyfluorescein diacetate succinimidyl ester dilution. (A) The percentages of proliferating CD4 + T cells in the presence of anti-human PD-1, anti-human PD-L1 or isotype control antibody at concentrations of 10, 50, or 100 µg/ml were shown. (B) The average percentages of proliferating CD4 + T cells in different antibody concentration groups. The bar graphs indicate the means ± SD, n = 3. * and ** indicate significant difference within group and between different groups. * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were incubated as the manufacturer’s instructions: rabbit anti-human CD8 Ab (ab93278, abcam) and mouse anti-human CD4 (T Helper/Inducer) monoclonal antibody (mAb) (ZM-0418, ZSGB-BIO) in a humidified chamber overnight at 4°C.

Techniques: In Vitro, Control, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display

doi: 10.3389/fmicb.2015.00340

Figure Lengend Snippet: (A) The presence of inserted FLAG sequence can be validated by ELISA on wells coated by anti-FLAG antibody. Phage was detected using HRP-conjugated anti-M13 antibody and Ultra-TMB substrate for horseradish peroxidase (HRP). In control experiments, we coated wells with BSA or used M13KE phage without a FLAG insert. (B) ELISA signal in optimal conditions with oriented immobilization of anti-FLAG IgG on protein A-coated wells yielded signal detectible at 600 femtomolar concentration of phage (3.6 × 10 8 PFU/mL). In this experiment, we used wells coated by non-specific IgG (anti-CD4) as negative control. (C) Comparison of phage displaying FLAG at the N-terminus (NT) and in intra-domain (ID) fashion using setup analogous to (B) . ID-display provides statistically significantly higher signal at equivalent titers. (D) Scheme of selection of ID-FLAG or NT-FLAG from a 1:10,000 mixture with insert-free phage. Enrichment is monitored as increase of blue (LacZ+) plaques on agar overlay containing X-gal. (E) Results of selection of ID-FLAG or NT-FLAG on wells presenting anti-FLAG or anti-CD8 (negative control). ID-display provides minor yet detectable advantage. Input to output ratios for insert-containing clones are displayed below the plot. In all experiments, data represents average from three to four independent wells; error bar is one standard deviation.

Article Snippet: In the case of ID1-mZ (Figure ), the coating antibody was mouse anti-human CD4 IgG (BD Biosciences, San Jose, CA, USA) at a concentration of 4 μg/mL.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control, Selection, Clone Assay, Standard Deviation