Journal:

Article Title: Dendritic Cell-T-Cell Interactions Support Coreceptor-Independent Human Immunodeficiency Virus Type 1 Transmission in the Human Genital Tract

doi:

Figure Lengend Snippet: Expression of HIV-1 coreceptors CCR5 and CXCR4 by individual MMC subpopulations. (A and B) Surface expression was detected by flow cytometry after simultaneous labeling of MMC with MAbs conjugated to three distinct fluorochromes recognizing CD3; either CCR5 or CXCR4; and either CD14, CD83, or CD1a. Clusters of T cells with DC or macrophages were excluded from the analysis. The percentages of mononuclear cells are shown which express CCR5 (A) and CXCR4 (B). Cells examined include MMC and peripheral blood CD3+ T cells from all donors studied (each symbol represents one donor), in comparison to controls, peripheral blood dendritic cells in three donors (PBDC) (A), and native and CCR5-transduced (A) or CXCR4-transduced (B) U373 cells. (C) Detection of CCR5 mRNA by RT-PCR in MMC from one donor sorted into >98% pure T cells (T), macrophages (M), and DC as described in Fig. ​Fig.1G1G to I. Equal amounts of cDNA corresponding to 104 cells in each subpopulation were amplified for CCR5, and the 251-bp reaction products are demonstrated after ethidium bromide staining. cDNAs corresponding to 2 × 104 CCR5-transduced (J CCR5+) and nontransduced (J CCR5−) Jurkat cells were included as positive and negative controls, respectively. cDNAs corresponding to 4 × 103, 6 × 103, 2.5 × 103, and 2 × 104 (T, M, DC, and J, respectively) were also amplified by PCR for the HPRT gene as a control to demonstrate the presence of cDNA in all samples. Control reactions in which RNA was not reverse transcribed (No RT) and in which no cDNA was added are shown. (D) Serial dilutions of CCR5-transduced Jurkat cells containing 1-2 CCR5 copies per cell were individually subjected to RT-PCR for CCR5 and HPRT to demonstrate the sensitivity of detecting these mRNAs by the PCR assay. The approximate copy number is indicated above the lanes, and control reactions omitting RT (No RT) and cDNA are included.

Article Snippet: For CCR5 detection, anti-CCR5 MAb 2D7 (National Institutes of Health AIDS Research and Reference Reagent Program) recognizing the second extracellular domain and anti-CCR5 MAb 227R (ICOS Corp., Seattle, Wash.) recognizing the N-terminal portion was used.

Techniques: Expressing, Flow Cytometry, Labeling, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining

Journal: Arthritis Research & Therapy

Article Title: Therapeutic effects of antibodies to tumor necrosis factor-α, interleukin-6 and cytotoxic T-lymphocyte antigen 4 immunoglobulin in mice with glucose-6-phosphate isomerase induced arthritis

doi: 10.1186/ar2437

Figure Lengend Snippet: In vitro inhibition assay of GPI-induced TNF-α and IFN-γ production using anti-cytokine mAbs or anti-co-stimulators. High amounts of tumor necrosis factor (TNF)-α and IFN-γ were produced by splenocytes cultured with glucose-6-phosphate isomerase (GPI). Thus, we used anti-TNF-α mAb (10 μg/ml), anti-IFN-γ mAb (1 μg/ml), and anti-IL-12 mAb (0.3 μg/ml) to neutralize these cytokines in the in vitro cytometric bead array system. Inhibition study was conducted by adding the above concentrations at commencement of culture. These concentrations were calculated to produce more than 80% blockade of these cytokines. The percentage inhibition rate is calculated by cytokine production with this system: 100 – ([cytokine mAb – control antibody]/control antibody). The inhibition rate of (a) TNFα and (b) IFN-γ are shown. Cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig; 1 μg/ml), anti-inducible co-stimulator (ICOS) mAb (0.5 μg/ml), and anti-CD40L mAb (1 μg/ml) were also used to block co-stimulatory pathways, and the inhibition rate of (c) TNF-α and (d) IFN-γ are shown. Three independent experiments were performed. Data are expressed as mean ± standard error of the mean.

Article Snippet: CTLA-4Ig (BD; 1 μg/ml), anti-inducible co-stimulator (ICOS) mAb (BD; 0.5 μg/ml), and anti-CD40L mAb (BD; 1 μg/ml) were used to block co-stimulatory pathways.

Techniques: In Vitro, Inhibition, Produced, Cell Culture, Blocking Assay

Journal:

Article Title: CCR4-bearing T cells participate in autoimmune diabetes

doi: 10.1172/JCI15547

Figure Lengend Snippet: Functional responsiveness of CCR4+ T cells to the murine CCR4 ligand MDC. (a) Chemotactic response of CCR4+CD4+ T cells to murine chemokines. The migration of FACS-sorted CCR4-negative and -positive BDC2.5 CD4+ T cells to murine MDC (15 ng/ml) was studied in a Transwell assay. The assay was also carried out in the presence of anti-human CCR4 and isotype control mAb’s (left). In addition, the chemotactic function of the various murine chemokines MDC, TARC, SLC, and MCP-1 was evaluated on CCR4-expressing BDC2.5 CD4+ T cells (right). Data representing the percentage of migrating sorted cells (left) or CCR4+CD4+ T cells are expressed as the mean of triplicates ± SEM. Left graph: black bar, CCR4–; white bar, CCR4+; light gray bar, CCR4+/IgG1; dark gray bar, CCR4+/anti-CCR4 mAb. Right graph: black bar, murine MCP-1; white bar, murine MDC; light gray bar, murine TARC; dark gray bar, murine SLC. These data indicate that the human CCR4 mAb cross-reacts with murine CCR4. (b) Expression of CCR4 and CCR5 and activation molecules CD25 and CD69 in leukocyte subsets 8-week-old NOD/shi mice and their modulation through the activation or polarization of BDC2.5 CD4+ T cells. (c) Cytokine profile of polarized Th subsets. To assess the polarization of Th1 and Th2 cells in vitro, we have examined the double intracellular cytokine staining of the polarized Th effectors generated by short-term culture of highly purified naive BDC2.5 T cell populations. We demonstrated that Th1 and Th2 cells were well polarized and differentiated (n = 5).

Article Snippet: Frozen sections were stained with hamster anti-mouse TARC mAb (ICOS Corp.).

Techniques: Functional Assay, Migration, Transwell Assay, Expressing, Activation Assay, In Vitro, Staining, Generated, Purification