Journal: Cell reports

Article Title: Intra- and extra-cellular environments contribute to the fate of HIV-1 infection

doi: 10.1016/j.celrep.2021.109622

Figure Lengend Snippet: (A) HI.fate is based on the chimeric molecular clone HIV-1 NL4-3 , which contains the 5′-long terminal repeat (LTR) from the NY5 isolate and the 3′-LTR from the LAV isolate. Differences between the two LTR sequences decrease the frequency of undesirable recombination events during vector manipulations. Bottom: infection of different cells by HI.fate.E (EF1a-HTLV chimeric internal promoter) viruses leads to the expression of E2-Crimson and ZsGreen fluorescent proteins, which are visualized under a fluorescence microscope. (B) Flow cytometric analysis of different cells infected by HI.fate.E viruses. THP-1 cells were differentiated using 100 nM phorbol myristate acetate (PMA); primary CD4 T cells were isolated from peripheral blood mononuclear cells (PBMCs) by negative selection and activated with anti-CD3/CD28 beads prior to infection. Cells that support HIV-1 gene expression are shown in red and those supporting HIV-1 latency are shown in green. Uninfected cells are colored blue. (C) Increasing amounts of HI.fate.E viruses were used to infect target cells. Left panel shows an example of the readout for 10,000 cells. In the right panel, fluorescence profile of virus titers was plotted for each cell type (mean ± SD of percentage of cells exhibiting a speficied phenotype in a representative experiment). Results represent one of two independent experiments, each performed in duplicate, except for infection of THP-1 cells that were tested in a single experiment performed in duplicate.

Article Snippet: Primary CD4 T cells were isolated from PBMC (1.5 E7 cells, Cat#200-0077, lot# 1608180134 from StemCell Technologies.

Techniques: Plasmid Preparation, Infection, Expressing, Fluorescence, Microscopy, Isolation, Selection

Journal: Cell reports

Article Title: Intra- and extra-cellular environments contribute to the fate of HIV-1 infection

doi: 10.1016/j.celrep.2021.109622

Figure Lengend Snippet: (A) We isolated primary CD4+ T cells from a healthy individual and activated them with anti-CD3/CD28 beads prior to infection with HI.fate. SFFV viruses. We sorted the cells according to their fate but gated on cells with the most prominent phenotype (i.e., 0.3% of highest ZsGreen intensity for latent cells and 3.2% of highest ZsGreen and Crimson intensities for replicating cells; both selections are schematically shown as dashed boxes). (B) We analyzed each sorted cell population by RNA-seq and compared their gene expression. (C) Principal-component analysis evaluates differences between cells supporting different fates. Red unfilled circles represent Crimson + Zs-Green − cells that were analyzed to detect changes related to the loss of Zs-Green expression . Dots were manually resized for visualization. Results represent data of two replicates for each cell population from PBMCs of a single donor, except for control uninfected PBMCs that were analyzed once.

Article Snippet: Primary CD4 T cells were isolated from PBMC (1.5 E7 cells, Cat#200-0077, lot# 1608180134 from StemCell Technologies.

Techniques: Isolation, Infection, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Extracellular vesicle bioactivity and potential clinical utility is determined by mesenchymal stromal cell clonal subtype

doi: 10.1101/2024.09.05.609844

Figure Lengend Snippet: In vitro treatment of activated CD4+ T cells with Y201 and Y202 EVs (n=2, mean events >18,000 counted) A) Proliferative cycles. B) Proliferative index C) Polarisation of activated T cells in the absence and presence of Y201 EVs and Y202 EVs (n=2, mean events >32,000 counted). D/E) Total peritoneal exudate cell (PEC) counts following zymosan (D) or schistosome egg (E) induced inflammation in the absence and presence of Y201 EVs and Y202 EVs. F/G) Examination of TCR+CD4+, naïve and central memory T cells in zymosan or schistosome egg induced inflammation in the absence and presence of Y201 EVs and Y202 EVs (n=3). One-Way ANOVA with Bonferroni post hoc testing,*p<0.05, **p<0.01, ***p<0.001.

Article Snippet: To determine MSC-derived EV immunomodulation for deactivation and suppression of T cell proliferation, suspension cultures of 1.0×10 5 primary human peripheral blood-derived CD4+ T cells (Stem Cell Technologies) were grown in RPMI-1640 with 10% FBS 1% P/S (Gibco, Cat: 10363083) and were pre-treated for 6hrs with 20X EVs isolated from serum-free conditioned medium collected over 24hrs culture from 2.0×10 6 of Y201 or Y202 MSCs.

Techniques: In Vitro

Journal:

Article Title: CD44 MicroBeads Accelerate HIV-1 Infection in T Cells

doi: 10.1016/j.virol.2009.03.022

Figure Lengend Snippet: A, B. Two million CEM cells were exposed to NL-EGFP preparations (see Table 1) at MOI = 0.05 infectious units (IU, P4R5 cell assay) for 4 hr in 1.5 ml total volume. Cells were washed once and and cultured for 7 days. Results from one representative experiment. C, D. Five million primary CD4 cells were infected overnight with MOI = 0.05 IU (P4R5) of NL-EGFP, with or without 1 hour preincubation with CD44 MicroBeads. Three independent experiments. A, C. GFP expression. B. Secreted p24, net values. D. Secreted p24, total values.

Article Snippet: Prescreened, pooled human AB serum for primary CD4 T cell culture was purchased from Omega Scientific (Tarzana, CA).

Techniques: Cell Culture, Infection, Expressing

Journal:

Article Title: CD44 MicroBeads Accelerate HIV-1 Infection in T Cells

doi: 10.1016/j.virol.2009.03.022

Figure Lengend Snippet: Comparison of NL4-3 infections (A, B) of primary CD4 T cells at MOI = 0.01 TCID50 with CD44 MicroBead pretreatment (right panel) and without (left panel). Primary CD4 cells were infected with the R5 clone, JR-CSF (C, E) or clade C, primary X4 isolate 98IN017 (D, F) with an MOI of 0.01 pg p24. Infections were monitored for % infected cells by ICp24 (A,C,D) and soluble p24 (B,E,F). Mean + SEM. n = 3, except for A, left panel (n=4) and B, left panel (n=7).

Article Snippet: Prescreened, pooled human AB serum for primary CD4 T cell culture was purchased from Omega Scientific (Tarzana, CA).

Techniques: Infection

Journal:

Article Title: CD44 MicroBeads Accelerate HIV-1 Infection in T Cells

doi: 10.1016/j.virol.2009.03.022

Figure Lengend Snippet: Primary CD4 cells were infected with the R5 clone, JR-CSF (left panels) or clade C, X4 isolate 98IN017 (right panels) with or without pretreatment with CD44 MicroBeads. At the end of 2 weeks of culture, genomic DNA was isolated for qPCR quantification of HIV integrants per 200 ng input DNA (A) and harvested cells were reactivated with αCD3/αCD28 to determine replication competent infectious units per million (IUPM) by limiting dilution assay (B). Results from three independent cell donors shown (x axis: A, B, C). nd = not done.

Article Snippet: Prescreened, pooled human AB serum for primary CD4 T cell culture was purchased from Omega Scientific (Tarzana, CA).

Techniques: Infection, Isolation, Limiting Dilution Assay

Journal:

Article Title: CD44 MicroBeads Accelerate HIV-1 Infection in T Cells

doi: 10.1016/j.virol.2009.03.022

Figure Lengend Snippet: Primary CD4 T cells were infected with NL4-3 produced from transfection in: 293T (CD44H−), HeLa P4R5 (CD44H+), and CEM (CD44H+) cells, with or without prior incubation with CD44 MicroBeads. The fraction of infected cells was measured by ICp24 with FACS (A) and secreted p24, assayed by ELISA (B). n = 3 except at one time point, as noted. Mean + SEM. Fold change in CD25 (C) and CD69 (D) expression with CD44 MicroBead treatment of virus produced by various cell lines (x axis), assessed one day after infection in unstimulated cells. Mean and range of three experiments.

Article Snippet: Prescreened, pooled human AB serum for primary CD4 T cell culture was purchased from Omega Scientific (Tarzana, CA).

Techniques: Infection, Produced, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Expressing

Journal:

Article Title: CD44 MicroBeads Accelerate HIV-1 Infection in T Cells

doi: 10.1016/j.virol.2009.03.022

Figure Lengend Snippet: Treatments with CD44 MicroBeads, streptavidin-complexed biotinylated αCD44, free αCD44 and CD45 MicroBeads were compared in spreading CEM infections with NL-EGFP (A, B). The fraction of CEM cells expressing GFP was analyzed by FACS (A) and soluble secreted p24 was assayed by ELISA (B). Mean + SEM of three experiments. C. CD69 expression in unstimulated primary CD4 cells, measured one day after exposure to αCD44 or αCD45 reagents alone, or in conjunction with NL4-3 infection. Mean and range of three experiments.

Article Snippet: Prescreened, pooled human AB serum for primary CD4 T cell culture was purchased from Omega Scientific (Tarzana, CA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Infection

Journal:

Article Title: CD44 MicroBeads Accelerate HIV-1 Infection in T Cells

doi: 10.1016/j.virol.2009.03.022

Figure Lengend Snippet: Binding of CD44 MicroBeads to virus particles may accelerate sedimentation onto target cells (1). Simultaneous binding of MicroBeads to virions and cell membrane CD44 may stabilize virion-CD4 interactions and promote coreceptor binding. CD44 MicroBead attachment to cellular CD44 may initiate intracellular signaling that promotes infection (2). Also, virus may spread more efficiently due to cell clustering induced by activated adhesion molecules or by cross-linking of cells directly through MicroBead binding (3). Illustration not to scale.

Article Snippet: Prescreened, pooled human AB serum for primary CD4 T cell culture was purchased from Omega Scientific (Tarzana, CA).

Techniques: Binding Assay, Sedimentation, Infection