Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CYTOMEGALOVIRUS COINFECTION IS ASSOCIATED WITH INCREASED VASCULAR-HOMING CD57+ CD4 T CELLS IN HIV INFECTION

doi: 10.4049/jimmunol.1900734

Figure Lengend Snippet: IL-15 and TNF enhance the chemoattraction of CD57+ CD4 Tmem toward CX3CL1-expressing endothelial cells. (A) Percentage of CD57− and CD57+ CD4 Tmem from CMV-seronegative (n = 8) and CMV-seropositive (n = 12) donors that express CX3CR1. Significance determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (B) MFI of CX3CR1 staining on CD57− or CD57+ CD4 Tmem from HIV-uninfected control donors (n = 12) after 48h of stimulation with medium control or IL-15 (20ng/ml). Significance determined by Wilcoxon signed rank test. Differences in fold change (FC) determined by Mann-Whitney test. *P ≤ 0.05. (C) Schematic diagram of the transwell assay system. Confluent monolayers of human aortic endothelial cells (HAoECs) were cultured in the presence or absence of TNF (10ng/ml) for 7 days in wells of a 24-well plate. Purified T cells from CMV-seropositive PLWH (n = 11) were exposed to IL-15 (20ng/ml) or medium control for 2 days prior to placement in the upper chamber of a 5μm transwell situated onto the HAoEC monolayer. In some cases, T cells were treated with AZD8797 (500nM) for 1 hour prior to co-culture. After 3 hours, T cells were separately harvested from the upper and lower chambers and analyzed by flow cytometry. (D) Representative dotplots showing CX3CR1 and CD57 expression on CD4 T cells in transwell assay from upper and lower chambers in indicated conditions. (E) Percentage of CD4 T cells recovered from the upper and lower chambers expressing CD57 after 3 hours. Significance determined by Wilcoxon signed rank test. (F) Absolute number of CD57+ CD4 T cells in the lower chamber expressed as a percent of total (upper chamber + lower chamber) CD57+ CD4 T cells (“Percent migrated”) in indicated conditions. Significance determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (G) The proportion of CD57+ CD4 Tmem that expresses CX3CR1 predicts the percent migrated of CD57+ CD4 T cells in the assay after IL-15 and TNF co-treatment (minus no treatment baseline). Significance determined by simple linear regression. (H) (Left) Enrichment in the percentage of CD57+ CD4 Tmem (lower chamber minus upper chamber) versus the percentage change in percent migrated following AZD8797 treatment of CD57+ CD4 Tmem in the IL-15 and TNF co-treatment condition. Significance determined by Spearman analysis. (Right) The percentage change in percent migrated following AZD8797 treatment of CD57+ CD4 Tmem in the IL-15 and TNF co-treatment condition for donors whose CD57+ CD4 Tmem enrichment was at the median value or below and those who enrichment was greater than the median value. Significance determined by Mann-Whitney test.

Article Snippet: Primary HAoECs (PromoCell) were treated for 7 days with recombinant human TNF (R&D Systems).

Techniques: Expressing, Staining, Control, MANN-WHITNEY, Transwell Assay, Cell Culture, Purification, Co-Culture Assay, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis.

doi: 10.4049/jimmunol.179.10.6973

Figure Lengend Snippet: FIGURE 2. TNF- release and the role of TNF- in MTb-mediated macrophage apoptosis. A, MTb-mediated macrophage apoptosis is dependent on TNF-. Differentiated U937 macrophages were incubated with MTb isolates in the presence and absence of specific neutralizing Ab to TNF- (anti-TNF; mAb 210, 5 g/ml) or isotype control (isotype), and after 24 h, apoptosis was determined by ELISA. Data are expressed as percent change in apoptosis compared with unstimulated macrophages (n 3). B, Reduced MTb-mediated TNF- release by HIV human macrophages. Differentiated human U937 and HIV U1 macrophages were incubated with individual MTb isolates at a multiplicity of 10:1 for 24 h, in the presence and absence of a general cellular phosphatase inhibitor, okadaic acid (OA; 50 ng/ml). Cell-free culture supernatants were assayed for TNF- by ELISA. C, Reduced TNF- mRNA transcripts in HIV macrophages. Differentiated U937 and HIV U1 macrophages were incubated for 6 h in the presence or absence of irradiated virulent MTb (iMTbRv). Specific TNF- mRNA was detected by RT-PCR, with -actin as an internal control for mRNA loading. Representative gel of three experiments providing identical results. D, Recovery of apoptosis in HIV macrophages by exogenous TNF- or endogenous macrophage TNF- release. Differentiated HIV U1 macrophages were incubated with irradiated virulent H37Rv MTb (iMTbRv) in the presence or absence of exogenous recombinant human TNF- (100 ng/ml), okadaic acid (OA; 50 ng/ml), or neutralizing anti-TNF Ab (mAb 210) for 24 h at 37°C. Apoptosis was determined by Cell Death ELISA. Data is shown as percentage change in OD from unstimulated condition. All quantitative data values represent mean SEM. , p 0.05 compared with iMTbRv alone; , p 0.05 compared with iMTbRv OA.

Article Snippet: Staurosporine (protein kinase C inhibitor; 5 M) was used as a positive control for apoptosis, and for select experiments, TNF- neutralizing Abs or isotype controls (mAb 210, R&D Systems) were added to culture supernatants to neutralize TNF- bioactivity before addition of MTb.

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Irradiation, Reverse Transcription Polymerase Chain Reaction, Recombinant