Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A) The first phase of generating a serum-free medium that can expand all human T cell subsets consisted of creating 10 prototype media by mixing different ratios of 3 base media that contain different concentrations of amino acids, vitamins, trace elements, antioxidants, metal ions, polyamines, and lipids. These prototype media were tested for their ability to expand activated primary human T cells using anti-CD3/CD28-coated beads and reiterated with a design of experiments (DOE) statistical quadratic model through Design-Expert 9.0.1 software with a desired response to maximally expand human T cells without serum supplementation. Phase 2 consisted of eliminating xenogeneic components, examining metabolites consumed in serum-supplemented X-VIVO-15 medium and prototype media from phase 1, and modifying media so that concentrations of metabolites are maintained upon feeding of activated T cells. The final phase focused on optimizing carbon sources, lipid concentrations, lentiviral transduction efficiency, and cytokine production post-activation on activated human T cells. (B) Total CD4 T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated by anti-CD3/CD28-coated beads in 1B2H medium containing optimal glucose, no glucose, or optimal glucose without glutamine. T cell proliferation was measured by CFSE dilution by flow cytometry. Data are representative of 3 independent experiments. (C–E) Primary human CD4 T cells were sort-purified into T N (CD25 − CD45RA + CCR7 + CD27 + ) (C), T CM (CD25 − CD45RO + CCR7 + CD27 + ) (D), and T EM cells (CD25 − CD45RO + CCR7 − CD27 − ) (E) and stimulated with anti-CD3/CD28-coated beads in 1B2H medium with or without 5% human serum (see for gating strategy). Cell expansion was monitored by Coulter counter on the indicated days. Data are representative of 2–3 donors and independent experiments. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test on day 9 population doublings; ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: Software, Transduction, Activation Assay, Labeling, Flow Cytometry, Purification, Two Tailed Test

Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A) The indicated subsets were stimulated with anti-CD3/CD28 coated beads in the presence of optimal (35 mM) or low (0.35 mM) glucose. Cell expansion was monitored by Coulter counter on the indicated days. Statistics were performed on day 9 population doublings. (B and C) T cell subsets that were expanded for 24 hr (B) with anti-CD3/CD28-coated beads or expanded for 9–11 days (C) with anti-CD3/CD28-coated beads before IFN-γ/IL-2 production was measured after PMA/ionomycin treatment. (D) CCR7 and CD27 expression measured on T cell subsets described in (A) 7 days after T cell expansion. (E and F) IFN-γ production by cells from the initial activation (E) and reactivation (F) are summarized from three independent experiments and donors (see for IL-2 and TNF-α quantification). (G and H) Quantification of CCR7 + CD27 + (G) and CCR7 – CD27 – (H) cells are summarized from three independent experiments. Error bars reflect SEM. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test. ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: Expressing, Activation Assay, Two Tailed Test

Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A and B) Total CD4 T cells were activated with anti-CD3/CD28 coated beads in optimal (35 mM), medium (3.5 mM), low (0.35 mM), or no glucose (0 mM) for 48 hr, and basal extracellular acidification rate (ECAR) (A) and basal oxygen consumption rate (OCR) (B) were measured by XF Seahorse Analyzer. (C and D) Total CD4 T cells were activated with anti-CD3/CD28-coated beads in optimal and low glucose and pre-treated in the presence of etomoxir (ET) or vehicle (DMSO) for 48 hr before basal OCR (D) and ECAR (C) was measured by XF Seahorse Analyzer. (E and F) Basal OCR (E) and basal ECAR (F) of indicated T cell subsets were quantified. (G) Lactate was measured in the media of indicated T cell subsets after 24, 48, or 72 hr of culture using high-performance liquid chromatography (HPLC). (H) Relative intracellular abundances of lactate from sorted activated T cell subsets at 48 hr by LC-MS, normalized by cell number and cell volume. (I–N) Basal OCR rates of T N (I), T CM (J), and T EM (K) and basal ECAR rates of T N (L), T CM (M), and T EM (N) were quantified. Error bars reflect SEM. All data are representative of 4 independent experiments and donors. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test or in case of multiple comparisons, one-way ANOVA followed by Tukey LSD; ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A) Total CD4 T cells were left unactivated or activated with anti-CD3/CD28-coated beads in optimal or low glucose. After 2 days of culture, T cells were examined using transmission electron microscopy. Arrow indicates presence of lipid droplets. Scale bars, 2 μm. (B) Total CD4 T cells were activated with anti-CD3/CD28-coated beads in optimal glucose for 48 hr and then transferred into medium with low glucose and cultured for up to an additional 48 hr. Bodipy 493/503 was used to stain the cells at the indicated time points after being transferred to low glucose for 0, 4, 24, or 48 hr. Fluorescence was visualized via confocal microscopy; 30–40 randomly selected cells per experiment were imaged (see for quantification). (C) Total CD4 T cells were activated with anti-CD3/CD28-coated beads for 3 or 6 days in optimal or low glucose. Cell lysates were probed for LC3B isoforms and β-actin (see for quantification). Data are representative of 3 independent experiments and donors. (D) T cells were stimulated with anti-CD3/CD28-coated beads and transduced with LC3B-mcherry. After 48 hr of activation, cells were stained with bodipy 493/503, and co-localization was visualized via confocal microscopy. 30–40 randomly selected cells per experiment were imaged. Data are representative of 3 independent experiments and donors. (E) Indicated subsets were stimulated with anti-CD3/CD28-coated beads in the presence of optimal or low glucose. After 2 days of culture, T cells were examined using transmission electron microscopy. Scale bar, 2 μm. (F and G) The number of lipid droplets per micrograph of total T cells (F) or indicated subsets (G) were quantified from 40 images per group per experiment in two independent experiments. Error bars reflect SEM. *p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey LSD; ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: Transmission Assay, Electron Microscopy, Cell Culture, Staining, Fluorescence, Confocal Microscopy, Transduction, Activation Assay

Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A) Model depicting how heavy glutamine is incorporated into citrate in an M+4, M+5, or M+6 manner and how each of those inform how glutamine is routed intracellularly. (B–D) Indicated T cell subsets were activated with anti-CD3/CD28-coated beads in optimal or low glucose supplemented with heavy glutamine for 48 hr. Percentage of M+4 (B), M+5 (C), or M+6 citrate (D) calculated from the total intracellular citrate pool of each subset by LC-MS is indicated. (E–G) Indicated T cell subsets were treated as in (B). Graphs show percentage of M+5 α-ketoglutarate (E), M+4 malate (F), and M+4 oxaloacetate (G) calculated from the total respective intracellular pools of each metabolite for each subset by LC-MS. (H–J) Indicated T cell subsets were treated as in (B). Percentages of M+0 (H), M+2 (I), and M+4 palmitate (J) were calculated from the total intracellular pool of palmitate for each subset by LC-MS. (K and L) Indicated T cell subsets were treated as in (B). Percentage of M+3 pyruvate (K) and M+3 lactate (L) from the total respective intracellular pool of each subset by LC-MS is indicated. (M) Ratios of lactate secreted and glucose consumed were calculated from moles of lactate secreted and moles of glucose consumed in supernatant of T cell subsets determined by HPLC 48 hr post-activation. Error bars reflect SEM. All data are representative of 3–4 independent experiments. See for overall relative metabolite abundances of each subset. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test; ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: Liquid Chromatography with Mass Spectroscopy, Activation Assay, Two Tailed Test

Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A and B) Total CD4 T cells were activated with anti-CD3/CD28-coated beads in optimal (A) or low glucose (B) in the presence of TOFA or with vehicle (DMSO) for 5 days. Total cell expansion is recorded on day 5 post-activation. (C) Indicated T cell subsets were activated with anti-CD3/CD28-coated beads in low glucose in the presence of TOFA or vehicle DMSO for 5 days. Cell expansion is recorded on day 5 post-activation. (D) Indicated T cell subsets were activated with anti-CD3/CD28 coated beads in optimal or low glucose and CD36 expression was measured at 48 hr post-activation. (E) Quantification of (D) from three independent experiments and donors. (F) Total CD4 T cells were activated with anti-CD3/CD28 beads in optimal or low glucose in media without any exogenous lipids (fat-free) or supplemented with exogenous lipids for 9 days. Cell expansion was monitored by Coulter counter on the days indicated. (G) Indicated T cell subsets were activated with anti-CD3/CD28-coated beads in low-glucose, fat-free medium with or without supplementation of exogenous lipids for 5 days. Cell expansion was recorded on day 5 post-activation. (H) Indicated subsets were treated with heavy palmitate for 24 hr. Percentages of M+2 acetyl CoA were calculated from the total acetyl-CoA pool by LC-MS. (I) Indicated T cell subsets were activated with anti-CD3/CD28-coated beads in fat-free medium in low glucose with or without exogenous lipids. Live cells were identified with Live/Dead Aqua by flow cytometry on day 5 post-activation. (J) Quantification of live cells from (H). Error bars reflect SEM. All data are representative of 3 independent experiments. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test or in case of multiple comparisons, one-way ANOVA followed by Tukey LSD; ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: Activation Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry, Two Tailed Test

Journal: Cell reports

Article Title: Differential Reliance on Lipid Metabolism as a Salvage Pathway Underlies Functional Differences of T Cell Subsets in Poor Nutrient Environments

doi: 10.1016/j.celrep.2018.03.084

Figure Lengend Snippet: (A) Indicated T cell subsets were activated with anti-CD3/CD28-coated beads in low glucose in the presence of vehicle (DMSO) or low-dose TOFA for 5 days before IFN-γ and IL-2 production was measured after PMA/ionomycin treatment. For optimal glucose data, see . (B and C) Quantification of IFN-γ (B) and IL-2 (C) production from 4 independent experiments. (D) Indicated T cell subsets were activated with anti-CD3/CD28-coated beads in low glucose in the presence of minimal, 1×, or 2×exogenous lipid concentrate in fat-free medium for 5 days post-activation before IFN-γ production was measured following PMA/ionomycin treatment. (E and F) Quantification of IFN-γ (E) and IL-2 (F) production from 3 independent experiments. Error bars reflect SEM. *p < 0.05, **p < 0.01, paired two-tailed Student’s t test; ns, not significant.

Article Snippet: Cells were activated with anti-CD3/CD28 beads in media supplemented with 8 mM [U- 13 C]-glutamine (Cambridge Isotope Laboratories, CNLM-1275-H-PK) for 48 hr when indicated.

Techniques: Activation Assay, Two Tailed Test

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

Journal: eLife

Article Title: Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation

doi: 10.7554/eLife.53725

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CD3 (armenian hamster, monoclonal) , Thermo Fisher Scientific , Cat # 14-0031-82, RRID: AB_467049 , T cell activation: 0.5 or 1 μg/ml as indicated in Materials and methods.

Techniques: Activation Assay, Staining, Blocking Assay, cDNA Synthesis, Protein Quantitation, Cell Isolation, Recombinant, Sequencing, Software