Journal:

Article Title: Borrelia burgdorferi and Interleukin-1 Promote the Transendothelial Migration of Monocytes In Vitro by Different Mechanisms

doi:

Figure Lengend Snippet: CD4+ T cells migrate across B. burgdorferi-stimulated HUVEC in an MCP-1-dependent manner. CD4+ T cells were incubated for 2 h with HUVEC-amnion cultures that had been pretreated with either control medium (Unstim), 5 U of IL-1 per ml, or B. burgdorferi at a ratio of 10 Bb/EC (A). CD4+ T cells were incubated for 2 h with HUVEC-amnion cultures that had been pretreated with either control medium (Unstim) or B. burgdorferi at a ratio of 10 Bb/EC in the presence of no MAb, an isotype-matched control MAb (MOPC-21), or 20 μg of neutralizing anti-MCP-1 MAb per ml (B). Transendothelial migration was assessed as described in Materials and Methods. The total height of each bar represents the number of T cells associated with each culture as a percentage of the total number added. The lower portion of each bar represents the percentage that migrated beneath the endothelium; the upper portion represents the percentage adherent to the apical surface. Bars represent the means ± SD of four to five replicate samples.

Article Snippet: CD4 + T cells, purified using Accu-Prep lymphocytes (Accurate) followed by positive selection with Dynabeads (product M-450 CD4; Dynal, Lake Success, N.Y.) were at least 98% pure, as determined by fluorescence-activated cell sorting (FACS) analysis using fluorescently labeled MAbs against both CD4 and CD3 (Simultest CD3/CD4; Becton Dickinson, San Jose, Calif.).

Techniques: Incubation, Migration

Journal:

Article Title: An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

doi: 10.1038/sj.emboj.7601113

Figure Lengend Snippet: Identification and RNAi knockdown of candidate membrane-associated AKAPs. (A) RII overlays and immunoblotting identify AKAP79, AKAP149 and gravin (AKAP250) as the major membrane-associated AKAPs in HEK293 cells. W: whole-cell extract; M: membrane; C: cytosol. (B) Quantification of AKAP79, AKAP149 and gravin silencing in CD4+ cells at 3 days post-transfection. Lysates were immunoblotted for AKAP79, AKAP149 or gravin (upper blot) and tubulin (loading control, lower blot). AKAP expression levels were normalized to tubulin levels and expressed as a percentage of the amount of the AKAP in control cells. Amalgamated data from three independent experiments are presented as mean±s.e.m.

Article Snippet: To assess the efficacy of RNAi knockdown by immunoblotting, shRNA-positive cells were selected with anti-CD4 magnetic beads (Dynal) for 30 min at 37°C and lysed in 1% (v/v) Triton X-100, 10 mM Na 2 PO 4 , pH 7.4, 150 mM NaCl, 5 mM EDTA and 5 mM EGTA supplemented with protease inhibitors (1 mM benzamidine, 1 mM AEBSF, 0.2 μg/ml leupeptin/pepstatin).

Techniques: Western Blot, Transfection, Expressing

Journal:

Article Title: Protection against Experimental Autoimmune Myocarditis Is Mediated by Interleukin-10-Producing T Cells that Are Controlled by Dendritic Cells

doi:

Figure Lengend Snippet: Polarized CD4+ T-cell response after culture with DCs of S2-16 and adjuvant-treated rats: S2-16:IFA DCs promoted CD4+ T cells to produce more IL-10, whereas S2-16:CFA DCs promoted IFN-γ production from CD4+ T cells. Lewis rats were treated with S2-16:IFA or S2-16:CFA. Fourteen days later, CD4+ T cells and DCs were isolated from spleens of treated rats or untreated naïve rats, and co-cultured together with S2-16 peptide. Proliferation of cells was measured by 3H-thymidine incorporation after 96 hours. Cytokine production in cell culture supernatants was measured by ELISA. A: Proliferative response, IL-10, and IFN-γ production by CD4+ T cells from S2-16:CFA-treated rats after co-culture (5 × 105 CD4+ T cells) with DCs from three groups of rats at various concentrations. B: Proliferative response and IL-10 and IFN-γ production of CD4+ T cells from S2-16:IFA-treated rats after co-culture (5 × 105 CD4+ T cells) with DCs at various concentrations. C: Proliferative response and IFN-γ production of CD4+ T cells from naive rats after co-culture (5 × 105 CD4+ T cells) with DCs at various concentrations. Error bars represent SEMs. Mann-Whitney test was used to determine the difference between S2-16:IFA DC or S2-16:CFA DC versus naïve DC groups (proliferation and IFN-γ), and S2-16:IFA DC versus S2-16:CFA DC groups (IL-10). *P ≤ 0.05, **P ≤ 0.005. D: Intracellular staining suggested that S2-16:IFA DCs induced higher level IL-10 production from CD4+ T cells than S2-16:CFA DCs. DCs were purified from spleens of S2-16:CFA- or S2-16:IFA-treated rats, and cultured with purified CD4+ T cells from these groups of rats, together with S2-16 peptide. Cells were removed from co-culture on day 4 and stained for CD4+ cells and intracellular IL-10-positive cells. Numbers denote percentage of cells in each quadrant. The data are representative of two to three independent experiments with similar results.

Article Snippet: For in vitro stimulation assay of CD4 + T cells, splenocytes were incubated with a saturating concentration of magnetic anti-rat CD4 (OX-38) microbeads (Miltenyi Biotec, Auburn, CA) at 4°C for 20 minutes.

Techniques: Adjuvant, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, MANN-WHITNEY, Staining, Purification

Journal:

Article Title: Direct Binding of Hepatitis C Virus Core to gC1qR on CD4 + and CD8 + T Cells Leads to Impaired Activation of Lck and Akt

doi: 10.1128/JVI.78.12.6409-6419.2004

Figure Lengend Snippet: The HCV core specifically binds to gC1qR displayed on the cell surface of T lymphocytes. (A) Dose-dependent core binding on lymphocytes. A total of 106 PBMC were incubated with various concentrations (0.5, 1, 2, 4, and 8 μg/ml) of the HCV core at 37°C for 2 h. After washing of the residual core protein, core binding was examined by FACS analysis with an anti-core MAb followed by FITC-labeled goat anti-mouse IgG. N/A, not applicable. (B) Anti-gC1qR antibody (a-gC1qR) blocks core binding on lymphocytes. Cells were treated with 2 μg of HCV core/ml in the presence of either 1:10 (vol/vol) anti-gC1qR PAb or prebleed serum at 37°C for 2 h. Core binding was measured as described above. (C) Soluble gC1qR prevents core binding on lymphocytes. Cells were incubated at 37°C for 2 h with 2 μg of HCV core/ml in the presence of various concentrations (2, 4, and 8 μg/ml) of soluble gC1qR (or DHFR) equivalent to core-gC1qR molar ratios of 1:2, 1:4, and 1:8, respectively. Core binding was determined as described above. The results were reproducible in two independent experiments. M1, gated on lymphocyte populations based on isotype control.

Article Snippet: CD4 + and CD8 + T cells were purified from human PBMC by incubation with FITC-anti-CD4 and FITC-anti-CD8 conjugate, followed by positive isolation with anti-FITC magnetic beads (Miltenyi Biotec, Auburn, Calif.) in accordance with the manufacturer's instructions.

Techniques: Binding Assay, Incubation, Labeling, Control

Journal:

Article Title: Direct Binding of Hepatitis C Virus Core to gC1qR on CD4 + and CD8 + T Cells Leads to Impaired Activation of Lck and Akt

doi: 10.1128/JVI.78.12.6409-6419.2004

Figure Lengend Snippet: The HCV core, which binds gC1qR with an affinity similar to that of C1q, elicits a stronger inhibitory signal on T cells. (A) Kinetics of the HCV core-gC1qR interaction and its dissociation constant (Kd) determined by BIAcore analysis. Various concentrations of the HCV core or DHFR were passed over gC1qR (50 μg/ml) covalently coupled to a dextran chip at a flow rate of 50 μl/min. The Kd for the HCV core-gC1qR interaction, as determined by using the BIA analysis computer program, is shown. Results are presented as RU/s, where 1 RU = 1 pg/mm2. (B) Dose-dependent inhibition of T-cell proliferation by the HCV core or C1q. A total of 2 × 105 ConA-stimulated PBMC were incubated with various concentrations of either the HCV core or C1q for 5 days at 37°C. [3H]thymidine was added to the cultures 18 h prior to harvesting, and incorporation was determined by using a MicroBeta liquid scintillation counter. Error bars indicate standard deviations. The results were reproducible in three independent experiments.

Article Snippet: CD4 + and CD8 + T cells were purified from human PBMC by incubation with FITC-anti-CD4 and FITC-anti-CD8 conjugate, followed by positive isolation with anti-FITC magnetic beads (Miltenyi Biotec, Auburn, Calif.) in accordance with the manufacturer's instructions.

Techniques: Inhibition, Incubation

Journal:

Article Title: Direct Binding of Hepatitis C Virus Core to gC1qR on CD4 + and CD8 + T Cells Leads to Impaired Activation of Lck and Akt

doi: 10.1128/JVI.78.12.6409-6419.2004

Figure Lengend Snippet: gC1qR expression and core-induced inhibitory effects are more profound on CD8+ than on CD4+ T cells. (A) gC1qR expression on CD4+ and CD8+ T cells. A total of 106 human PBMC were doubly stained with either FITC-conjugated anti-CD8 or anti-CD4 and anti-gC1qR antibody, followed by PE-conjugated anti-mouse IgG antibody. Cells subjected to FACS analysis were gated on the lymphocyte population. The percentages of CD4+ cells (upper panel) and CD8+ cells (lower panel) that were positive for gC1qR are shown. (B) Proliferation of CD4+ and CD8+ T cells in the presence of the core. Purified CD4+ and CD8+ T cells were activated by ConA in the presence of the HCV core (2 μg/ml) or DHFR (2 μg/ml) for 5 days at 37°C in a 5% CO2 atmosphere. [3H]thymidine was added to the cultures 18 h prior to harvesting. The uptake of [3H]thymidine was measured by using a liquid scintillation counter. P values determined by the Student t test are shown. Error bars indicate standard deviations. The results were reproducible in three independent experiments. (C) CD69 expression on activated CD4+ and CD8+ T cells in the presence of the core. PBMC were activated with ConA in the presence of either the HCV core (right panels) or DHFR (left panels) at 37°C for 24 h in a 5% CO2 atmosphere. CD69 expression on CD4+ T cells (upper panels) and CD8+ T cells (lower panels) was determined by FACS analysis with PE-conjugated anti-CD69 antibody and either FITC-conjugated anti-CD4 or FITC-conjugated anti-CD8 MAb. The percentages of CD4+ and CD8+ T lymphocytes that were positive for CD69 are shown. The results were reproducible in three independent experiments.

Article Snippet: CD4 + and CD8 + T cells were purified from human PBMC by incubation with FITC-anti-CD4 and FITC-anti-CD8 conjugate, followed by positive isolation with anti-FITC magnetic beads (Miltenyi Biotec, Auburn, Calif.) in accordance with the manufacturer's instructions.

Techniques: Expressing, Staining, Purification