Journal: Cell Reports Methods

Article Title: Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

doi: 10.1016/j.crmeth.2023.100485

Figure Lengend Snippet:

Article Snippet: Per well 1–3X10E5 patient’s cells were plated with addition of 10E4 SupT1 hu R5 in 50% ImmunoCult-XF T cell Expansion Medium, 50% conditioned medium, 100IU IL-2/mL and anti-human CD3/CD28/CD2 Antibody stimulation cocktail (Stem Cell Technologies).

Techniques: Control, Virus, Plasmid Preparation, Derivative Assay, Infection, Recombinant, Cell Culture, Cell Isolation, PCR Cloning, Transfection, Software, Sequencing, Blocking Assay

Journal: bioRxiv

Article Title: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

doi: 10.1101/111260

Figure Lengend Snippet: (A) The construction designs of CART are listed as CD19-GFP, SS1-GFP, CD19-IL18, and SS1-IL18. (B,C) NSG mice (n=5) were inoculated with 2.5e6 AsPC1 expressing CBG-T2A-GFP. 3 weeks later, the mice received 2e6 of different CART as indicated. (B) Tumor growth and body weight. (C) CD8+ and CD4+ T cells in peripheral blood. IL18 levels were assessed by ELISA. (D) Representative FACS plots showing percentages of CAR+/CD8+ in CART cells during expansion in vitro and in the spleens of mice. Each data point represents cells from a single spleen, except that 5 spleens from mice receiving SS1 CART were pooled. (E) The percentage of CAR+/CD4+ or CAR+/CD8+ T cells in spleens from (D). (F) Tumor free NSG mice (n=5) were inoculated with 5e6 SS1 or SS1-IL18 CART cells, and monitored for circulating T cells, serum IL18 concentrations, body weight and survival. (G) H&E staining of tumors treated with different CART cells as indicated. (H) IHC staining for human CD3+ T cells in tumor sections from mice treated with CD19-IL18 or SS1-IL18 CART cells. (I) Representative pictures of liver, lung and pancreas from mice receiving SS1-IL18 CART cells demonstrated T cell infiltration and GVHD (see also Table S1). All data with error bars are presented as mean±SEM.

Article Snippet: In brief, primary donor T cells were activated with anti-CD3/CD28 Dynabeads (LifeTechnologies) at D0, and transduced with lentivector at D1.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

doi: 10.1101/111260

Figure Lengend Snippet: Primary T cells from normal donors were activated with anti-CD3 beads and varying concentrations (1.56 doubled up to 200 ng/ml) of recombinant IL18 (added at D0 and D3). Activated T cells were de-beaded at D5 and counted every day afterwards. (A) changes in T cell size and (B) proliferation kinetics. (C) IL18 dependent expansion of T cells from two different donors. (D) Luminex analysis of secreted IFNG and IL2. Additional cytokine data are shown in Figure 2-figure supplement 1. (E,F) Normal donor T cells expanded with anti-CD3/CD28 beads were selected by MACS columns to purify CD8 and CD4 T cells. Purified CD8, CD4 T cells, or bulk CD8+CD4 T cells were restimulated with anti-CD3 beads and 100ng/ml IL18 (added at D0 and D3). Cell size (E) and proliferation kinetics (F) were monitored after stimulation with anti-CD3 beads and recombinant IL18. (G-M) T cells were transduced with lentivector encoding GFP or GFP-IL18 at D1 during standard T cells expansion and GFP-IL18/CD8 and GFP-IL18/CD4 T cells were selected using MACS columns. NSG mice (n=5) were injected intravenously with 2×10 6 GFP-IL18 T cells, GFP-IL18/CD8, or GFP-IL18/CD4 T cells. GFP T cells were included as a negative control. The data shown is representative of two independent experiments. (G) Circulating CD45+ T cells at d15 and D23 after T cells injection. (H) Spleens from various groups, indicating splenomegaly in mice receiving GFP-IL18/CD4 or bulk T cells. (I) Circulating CD4+CD45+ T cells at D23 following T cell injection. (J) The ratio of CD8 to CD4 T cells. Circulating Serum levels of IL18 (K) and IFNG (L) at D15 and D23. (M) Serum at D23 was analyzed by luminex (duplicate) to assess cytokine profile.

Article Snippet: In brief, primary donor T cells were activated with anti-CD3/CD28 Dynabeads (LifeTechnologies) at D0, and transduced with lentivector at D1.

Techniques: Recombinant, Luminex, Purification, Transduction, Injection, Negative Control

Journal: bioRxiv

Article Title: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

doi: 10.1101/111260

Figure Lengend Snippet: (A) Expansion kinetics and size of TCR-deficient (TCR-) CD19-GFP or CD19-IL18 CART cells following stimulation with anti-CD3/CD28 beads and RNA electroporation. (B) Expansion kinetics and size of TCR+ and TCR-deficient SS1-GFP or SS1-IL18 CART cells following restimulation with irradiated mesothelin-expressing K562 cells in the presence of exogenous IL2 (until day 9 or day 11, as indicated). (C) Expansion kinetics and size of TCR+ and TCR-deficient CD19-GFP or CD19-IL18 CART cells following restimulation with irradiated CD19-expressing K562 cells in the presence of exogenous IL2. (D) TCR-deficient CART cells expanded from (B) and (C) were further restimulated with aAPC in the absence of exogenous IL2. (E) IL18 secretion (duplicate) of indicated CART cells following restimulation with cognate antigen. (F,G) NSG mice (n=5) were injected with 2×10 6 TCR+ or TCR-deficient T cells expressing either GFP or GFP-IL18. After 18 days, the number of GFP+ and GFP-CD4+ and CD8+ cells in blood (F) or spleens (G) was assessed. All data with error bars are presented as mean±SEM.

Article Snippet: In brief, primary donor T cells were activated with anti-CD3/CD28 Dynabeads (LifeTechnologies) at D0, and transduced with lentivector at D1.

Techniques: Electroporation, Irradiation, Expressing, Injection

Journal: bioRxiv

Article Title: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

doi: 10.1101/111260

Figure Lengend Snippet: (A) Cell competition assay design. CD19-IL18 WT, GFP WT, Amcyan IL18R KO, and DsRed TCR KO T cells were mixed together and injected into NSG mouse (n=5) intravenously, to evaluate the expansion of each cell population in response to IL18 secretion. (B) Normal human primary T cells were activated and transduced with lentiviral expression vectors by standard protocols, then electroporated with sgRNA targeting TCRB or IL18RA, and Cas9 RNA as described in Methods. Three days after electroporation, T cells were stained with anti-CD3 or anti-IL18RA to confirm gene knockout. T cells with TCR or IL18R knockout were stained with anti-CD3, or anti-IL18RA microbeads and negatively sorted by MACS. (C) 1×10 6 CD19-IL18 CART cells, 1×10 6 GFP T cells, 1×10 6 IL18R deficient Amcyan T cells, and 1×10 6 TCR-deficient DsRed T cells were mixed together and injected intravenously into NSG mice. (D) Expression levels of IL18RA on GFP WT, CD19 CAR WT, and Amcyan IL18R- cells in blood (left) and spleen (right). (E) Frequencies of CD19-IL18 WT, GFP WT, IL18R-deficient Amcyan, and TCR-deficient DsRed T cells in blood. (F) Total circulating CD45+ cells, indicating robust expansion. (G) The ratios of CD19 CART WT, IL18R-deficient Amcyan, and TCR-deficient DsRed to GFP T cells in blood and spleen. All data with error bars are presented as mean±SEM.

Article Snippet: In brief, primary donor T cells were activated with anti-CD3/CD28 Dynabeads (LifeTechnologies) at D0, and transduced with lentivector at D1.

Techniques: Competitive Binding Assay, Injection, Transduction, Expressing, Electroporation, Staining, Gene Knockout, Knock-Out

Journal: bioRxiv

Article Title: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

doi: 10.1101/111260

Figure Lengend Snippet: NSG mice were injected intravenously with 1×10 6 Nalm6 cells expressing CBG and GFP reporter proteins. At D7 post tumor injection, mice (n=5) were imaged and randomized into groups to receive 1×10 6 TCR-deficient CD19-GFP T cells or CD19-IL18 CART cells. Mice receiving no CART cells or TCR-deficient SS1-IL18 CART cells were included as controls. The data shown is representative of two independent experiments. (A) Live animal imaging of CBG expressing tumors in mice receiving CD19-GFP or CD19-IL18 TCR-deficient CART cells. (B) Left panel: tumor growth curves for mice injected with CD19-GFP CART cells, SS1-IL18 CART cells, or CD19-IL18 CART cells (all TCR-deficient). Untreated control received no T cells. Right panel: tumor growth in mice receiving TCR-deficient CD19-GFP CART cells or TCR-deficient CD19-IL18 CART cells. Mice were monitored for body weight (C) and survival (D) . The number of CD3-CD45+ T cells (E) and IL18 concentration (F) in blood were monitored over time. (G) The percentage of CD3-cells among total CD45+ cells, distribution of CD4+ and CD8+ T cells within CD19 CART cells (H) , and memory phenotype in CD4+ and CD8+ CART cells (I) in mice receiving TCR-deficient CD19-IL18 CART cells. All data with error bars are presented as mean±SEM.

Article Snippet: In brief, primary donor T cells were activated with anti-CD3/CD28 Dynabeads (LifeTechnologies) at D0, and transduced with lentivector at D1.

Techniques: Injection, Expressing, Imaging, Concentration Assay

Journal: bioRxiv

Article Title: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

doi: 10.1101/111260

Figure Lengend Snippet: (A) Upper panel: schematic of lentiviral construct in which the NFAT promoter drives expression of CD19-GFP-IL18. Lower panel: the ex vivo timeline to generate TCR-deficient T cells transduced with NFAT-CD19-GFP-IL18 and electroporated with mRNA CD19 CAR (designated as TCR-/NFAT-CD19-GFP-IL18). (B) TCR-/NFAT-CD19-GFP-IL18 CART cells were stimulated with anti-CD3/28 beads, K562-CD19, or PMA/ionomycin, after which CD45GFP+ cells were estimated by flowcytometry. (C) Summary of GFP+/CD45+ percentages (upper panel) and IL18 concentrations (lower panel) under different conditions as indicated. (D) NSG mice (n=5) were inoculated with 1×10 6 Nalm6-CBG/GFP cells, and treated 1 week later with TCR-deficient untransduced, NFAT-CD19-GFP, or NFAT-CD19-GFP-IL18 CART (all bearing mRNA encoded CD19 CAR). Tumor progression was monitored by live animal imaging. (E) Tumor growth kinetics (Student's t-test: **p=0.0057, *p=0.0359), survival rate after T cells infusion, change of body weight, and IL18 concentrations at D10 and D15 following T cell injection. All data with error bars are presented as mean±SEM.

Article Snippet: In brief, primary donor T cells were activated with anti-CD3/CD28 Dynabeads (LifeTechnologies) at D0, and transduced with lentivector at D1.

Techniques: Construct, Expressing, Ex Vivo, Transduction, Imaging, Injection

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: Establishment of pre- and postactivation latency in primary T cells in vitro. (A) Preactivation latency. Resting CD4+ T cells were isolated from PBMCs of healthy blood donors by negative selection using MACS, pretreated with CCL19 (100 nM) for 24 h, and infected with NL4.3-EGFP at an MOI of 0.5. Further rounds of infection were blocked by the addition of antiretrovirals at 2 days postinfection and maintained during culture. Cells expressing EGFP (EGFP+) were enumerated and removed by FCM sorting at day 5 postinfection. EGFP expression in EGFP− cells was determined by FCM after activation in the presence of the antiretroviral. (B) Postactivation latency. Naive T cells were sorted from total CD4+ T cells by negative selection using CD45RO microbeads. Cells were activated with anti-CD3/anti-CD28 beads with anti-IL-4, anti-IL-12, and TGF-β. Beads were removed at day 3, and cells were expanded in 30 U IL-2 for 4 days. At day 7 postactivation, cells were infected with NL4.3-EGFP. Antiretrovirals were added to the cultures at 2 days postinfection and maintained during culture. At day 7 postinfection, EGFP-expressing cells were enumerated and removed by FCM sorting, and EGFP− cells were activated with the same panel of stimuli used in the preactivation model. EGFP expression was measured at 3 days poststimulation by FCM. Ral, raltegravir; FACS, fluorescence-activated cell sorter.

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: In Vitro, Isolation, Selection, Infection, Expressing, Activation Assay, Fluorescence

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: HIV infection and virus reactivation in pre- and postactivation latency. (A) Flow cytometry gating and analysis used to measure EGFP expression in CD4+ T cells using the in vitro latency models. Live cells were defined by forward scatter (FSC) versus side scatter (SSC). Uninfected T cells were used to detect any background EGFP expression in our system. EGFP expression was measured against the PE channel. Plots show EGFP expression in mock-infected T cells (uninfected resting CD4+) with chemokine treatment (+CCL19) or mock-activated T cells (uninfected activated CD4+) and infected T cells (activated CD4+). (B) Plots representing EGFP expression following activation. Data are shown as percentages of EGFP in each plot in preactivation (+CCL19 or untreated CD4+ T cells) or postactivation latency. Data are representative of results for a matched sample in one experiment. (C) Frequency of productive infection in infected CD4+ T cells measured by EGFP expression at 5 days postinfection in the preactivation models (CCL19 treated [+] [red open circles] and untreated [−] [blue open circles]) and at day 7 in the postactivation model (black open circles). (D to F) Induced EGFP expression was measured following stimulation of sorted EGFP− cells in preactivation latency with CCL19 (D) or without CCL19 (E) and in postactivation latency (F), using monocytes (mono), monocytes and anti-CD3 (20 μg/ml) (mono+aCD3), PHA (10 μg/ml) and PMA (50 ng/ml) (PMA/PHA), or PMA and ionomycin (500 ng/ml) (PMA/Iono) as well as plate-bound anti-CD3 (20 μg/ml) and anti-CD28 (3.6 μg/ml) (aCD3/aCD28) stimulation or culturing with antiretrovirals only (Unstim). EGFP expression was measured 72 h after coculture. Each dot represents data for a single donor, and the box plots show 25th and 75th percentiles, medians, and ranges. *, P ≤ 0.05; **, P ≤ 0.01 (as determined by a Wilcoxon matched-pairs signed-rank test).

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: Infection, Flow Cytometry, Expressing, In Vitro, Activation Assay

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: EGFP expression following the addition of irradiated allogeneic monocytes. EGFP− cells were sorted and cocultured with allogeneic monocytes with or without soluble anti-CD3 (+, anti-CD3 [20 μg/ml]) in the presence of antiretrovirals. Monocytes were irradiated (+irr) (closed symbols) or nonirradiated (−irr) (open symbols) and added to latently infected EGFP− T cells. EGFP expression was measured at 72 h. (A to C) CCL19-treated (A) or untreated (B) cells and cells during postactivation latency (C). Each symbol represents data for a single donor. *, P ≤ 0.05; ns, not significant (as determined by a Wilcoxon matched-pairs signed-rank test). Blue lines indicate the median. (D) Comparison of the frequencies of induced EGFP expression following stimulation with soluble anti-CD3 with or without monocytes and anti-CD3/anti-CD28 beads (with or without monocytes) in the postactivation model. Sorted EGFP− cells were cocultured with soluble anti-CD3 (20 μg/ml) or with anti-CD3/anti-CD28 beads (1:1 ratio) with and without monocytes in the presence of antiretrovirals. Induced EGFP expression was measured at 72 h. Each point represents data for a single donor. *, P ≤ 0.05; **, P ≤ 0.01 (as determined by a Wilcoxon matched-pairs signed-rank test). The median is indicated with a blue line.

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: Expressing, Irradiation, Infection

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: Cellular proliferation and virus expression from sorted EGFP− cells following stimulation. (A) Levels of cell proliferation following treatment with different stimuli. The sorted EGFP− T cells in the preactivation models (CCL19 treated [+] [open red circles] and untreated [−] [open blue circles]) and postactivation models were stained with proliferation dye (eFluor670) and then cocultured with allogeneic monocytes, with and without anti-CD3, plate-bound anti-CD/anti-CD28, or PMA-PHA and PMA-ionomycin, or with an antiretroviral alone (unstim). At 72 h postactivation, cellular proliferation was measured by FCM. Each point represents data for an individual donor; the box plots show 25th and 75th percentiles, medians, and ranges. *, P ≤ 0.05 (as determined by a Wilcoxon matched-pairs signed-rank test). (B) EGFP expression in nonproliferating (eFluor670hi) and proliferating (eFluor670lo) cells under each stimulation condition. Each point represents data for a single donor. *, P ≤ 0.05; ***, P ≤ 0.001 (as determined by a Wilcoxon matched-pairs signed-rank test). The median is shown as a blue line. (C) Correlation between EGFP expression and cell proliferation in pre- and postactivation latency, determined by using Spearman's rank test. (D) Correlation between the frequency of EGFP expression in nonproliferating (eFluor670hi) and proliferating (eFluor670lo) cells in both models. Data from all cultures were pooled, and the level of EGFP expression in nonproliferating (eFlour670hi) cells was plotted against the level of EGFP expression in proliferating (eFluor670lo) cells in both models.

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: Expressing, Staining

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: Postsort spontaneous EGFP expression is associated with higher-level productive infection. (A) The level of spontaneous EGFP expression from infected cells was measured 72 h after coculture of sorted EGFP− cells with antiretrovirals only. Each point represents data for an individual donor; the box plots show 25th and 75th percentiles, medians, and ranges. ****, P ≤ 0.0001 (as determined by a Mann-Whitney test). (B) Correlation between EGFP+ productively infected T cells and EGFP expression from sorted EGFP− cells (spontaneous expression) 72 h after stimulation with monocytes, monocytes–anti-CD3 (20 μg/ml), PHA (10 μg/ml)–PMA (50 ng/ml), PMA–ionomycin (500 ng/ml), plate-coated anti-CD3 (20 μg/ml)/anti-CD28 (3.6 μg/ml), or antiretrovirals only. (C) Comparison of induced EGFP expression after stimulation and spontaneous expression (unstimulated expression). (D) Correlation between EGFP+ productively infected cells and induced EGFP expression. Each point represents data for a single donor; correlations were determined by Spearman's rank test.

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: Expressing, Infection, MANN-WHITNEY

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: Coculturing of APCs with activated infected T cells during establishment of latency inhibits subsequent inducible EGFP expression. (A) Infected T cells in postactivation latency were cocultured with APCs directly or in the bottom reservoir of a transwell system from day 2 postinfection. Antiretrovirals were added at the same time, and cells were cultured for 5 days. At day 7 postinfection, the EGFP− cells were sorted and cultured with or without stimulation. EGFP expression was measured by FCM. (B) Numbers of productively infected EGFP-expressing cells from APC-treated cultures with transwells (open squares) or without transwells (closed squares) and activation-induced expression compared to the number of productively expressing cells among T cells alone (open circles) and PMA-ionomycin-treated T cells (closed circles). The median is shown as a blue line. *, P ≤ 0.05 (as determined by a Wilcoxon matched-pairs signed-rank test). Each dot represents data for an individual donor. APC cocultures during establishment of latency are shown by gray shading. (C) Numbers of spontaneous EGFP-expressing cells from T cells alone, APC cocultures, and mitogen-activated T cell cultures. The median is shown as a blue line. *, P ≤ 0.05 (as determined by a Wilcoxon matched-pairs signed-rank test). APC cocultures during establishment of latency are shown by gray shading. (D) Numbers of induced EGFP-expressing cells following stimulation with monocytes, monocytes and anti-CD3 (20 μg/ml), and PMA (50 ng/ml)–ionomycin (500 ng/ml) compared to the level of EGFP expression following the same stimulation in cultures with CD4+ T cells alone or mitogen-treated T cells. Each point represents data for a single donor. The median is shown as a blue line. *, P ≤ 0.05 (as determined by a Wilcoxon matched-pairs signed-rank test). APC cocultures during the establishment of latency are shown by gray shading.

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: Infection, Expressing, Cell Culture, Activation Assay

Journal: Journal of Virology

Article Title: The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed

doi: 10.1128/JVI.02225-17

Figure Lengend Snippet: Summary of differences found in responses to reactivation between in vitro latency models

Article Snippet: The sorted non-EGFP-expressing cells were cocultured in medium supplemented with antivirals only or stimulated on plates coated with anti-CD3 (20 μg/ml of anti-human CD3ε, clone UCHT1; BD Biosciences), soluble anti-CD28 (3.6 μg/ml, clone L293; BD Biosciences), or anti-CD3/anti-CD28 beads (1:1 ratio; Dynal/Invitrogen) and mitogens, including PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) combined with ionomycin (500 ng/ml) or PHA (10 μg/ml; Thermo Fisher Scientific, Waltham, MA), Pan (30 nM; Selleck Chemicals, Houston, TX), Rom (40 nM; Selleck Chemicals), JQ1 (1 μM; Sigma-Aldrich), CTN (10 nM; Sigma-Aldrich), IL-7 (50 ng/ml; Sigma-Aldrich), or Bryo-1 (10 nM; Selleck).

Techniques: In Vitro, Expressing, Activation Assay