Journal: Cell reports. Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Figure 2. Characterization of CD32-driven trogocytosis (A) 293T cells transiently expressing C-terminal GFP fusion proteins of FcgRs CD32A, CD32B, or CD32C or, as a control, the nucleocytoplasmic dNTPase SAMHD1 served as donors in co-cultures with CellTrace dye-stained SupT1 T target cells. All culture media contained IgG-depleted FCS. Shown are repre- sentative flow cytometry dot plots and the percentages of CD32+ and GFP+ target T cells. One experiment out of two is shown. (B) Schematic of topology determination of transferred CD32-GFP (top). Bottom: SupT1 T cells were co-cultured as described in (A) and stained with either an anti-GFP mAb or an isotype control antibody, both conjugated to Alexa 647, with or without prior cell permeabilization. One representative experiment is shown (n = 3). The illustration was created with BioRender.com. (C) 293T cells were co-transfected with plasmids encoding C-terminal GFP fusion proteins of CD32A, CD32B, or CD32C or, as a control, histone H2B-GFP, together with a plasmid encoding CCR5. After 2 days, cells were either left untreated or pre-treated with an anti-CD32 Ab or an isotype control Ab prior to co- cultivation with SupT1 T cells. One day later, the expression of GFP and CCR5 on the target T cells was determined by flow cytometry. Mean ± SEM are shown (n = 3). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). (D) Half-life of CD32 and CCR5 surface expression on SupT1 target cells following co-culture as in (A). Following 1 day of co-culture, SupT1 T cells positive for CD32-GFP were sorted by flow cytometry and kept in culture for an additional 9 days. The expression of CD32 (top) or CCR5 (bottom) on sorted cells was determined for up to 192 h of cultivation. One representative experiment is shown (n = 2). (E) Schematic of CD32B with important amino acids and motifs indicated. (F) Transfer of the indicated CD32B mutants, CD32A WT, CD32C WT, or H2B (GFP fusion proteins), assessed as in (A) (mean ± SEM; n = 4). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Dunnett). (G) Visualization of the material transfer from CD32B-GFP expressing 293T cells to LifeAct-mCherry-expressing SupT1 using live-cell imaging. 293T cells transiently expressing CD32B-GFP (green) were co-cultured with LifeAct-mCherry-expressing SupT1 cells (magenta), cultivated in IgG-depleted FCS and boosted with PGT151 antibody, and imaged using spinning disc microscopy for 4 h. The left panel shows the beginning of co-culture. (a) Labels the area with the first transfer event (middle panel). (b) Labels the area of the second transfer event (right panel). Dashed white box marks the area that is zoomed and depicted with individual time points before and after the transfer event (shown below). The time stamp (upper right corner, relative to the time frame which shows the transfer event (time 00:00) in zoom-ins). Scale bar, 10 mm. *p % 0.05; **p % 0.01; ***p % 0.001.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Expressing, Control, Staining, Cytometry, Cell Culture, Transfection, Plasmid Preparation, Comparison, Co-Culture Assay, Live Cell Imaging, Microscopy

Journal: Cell reports. Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Figure 3. CD32-driven trogocytosis is boosted by T cell-autoreactive antibodies associated with chronic HIV-1 infection (A) CD32 expression on CD4 T cells from peripheral blood of healthy donors (HD) (n = 23) and chronic HIV-1 infected patients (CHI) (n = 39). Median with 95% CI are shown. Asterisks indicate statistical significance by Mann-Whitney test. (B) 293T cells transiently co-expressing CD32B-GFP and CCR5 were pre-treated with the indicated patient sera before 1 day of co-culture with SupT1 T cells. Shown are the percentage of CD32B-GFP+ and CCR5+ target cells (median with 95% CI, each dot represents a different patient; see also Figure S7C). CHI, chronic HIV-1 infection; ART, anti-retroviral therapy; AHI, acute HIV-1 infection. Fiebig stages II-III of acute HIV-1 infection42; HIV-2, HIV type 2; HTLV-1, human T cell lymphotropic virus type 1; HCV, hepatitis C virus; DENV, dengue virus; YFV, yellow fever virus-vaccinated; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2; EC, Echinococcus multilocularis; SCH, Schistosoma spp.; TB, Mycobacterium tuberculosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; CG, cryoglobulinemia. Asterisks indicate statistical significance by Mann-Whitney test. (C) Percentage of GFP+ target cells after 1 day of co-culture with 293T cells as in (B). IgG was depleted from the sera of two healthy donor (HD) and two HIV-1 patient (CHI) samples from (B, pink and red) and input (original sera), flowthrough and eluate of the IgG depletion were used for pre-treatment of cells prior to co- culture. Mean of two donors from each category is shown. (D) Correlation of antibody binding to SupT1 T cells and CD32B-GFP trogocytosis as in (B), with sera from HIV-1 patients. P, Pearson correlation coefficient. (E) Binding of sera with high or low trogocytotic activity (pink and red dots in B) to primary CD4 T cells as detected with fluorochrome-coupled anti-human IgG Ab (median with 95% CI, CD4 T cells; n = 3). Kruskal-Wallis test with Dunn’s multiple-testing correction. (F) A panel of bNAbs was analyzed for binding to uninfected resting CD4 T cells (top) or activated CD4 T cells (bottom). Mean ± SEM; n = 3. Asterisks indicate statistical significance by one-way ANOVA (top) or three-way ANOVA (bottom). p values were corrected for multiple comparison (Dunnett). (G) Purified, CMV-encoded, soluble Fc-binding proteins gp34 and gp68, or control proteins gp34 non-binding mutant (mtrp; W65F) and soluble ICOSL (inducible T cell co-stimulator ligand) were added to 293T donor cells as in (A), in the presence of PGT151 Ab, and subsequently co-cultured with SupT1 T cells. CD32 transfer was evaluated as in (B). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). *p % 0.05, **p % 0.01, ***p % 0.001; n.s., not significant. (H) Schematic of the determinants of antibodies for trogocytosis enhancement.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Infection, Expressing, MANN-WHITNEY, Co-Culture Assay, Retroviral, Virus, Binding Assay, Activity Assay, Comparison, Control, Mutagenesis, Cell Culture

Journal: Cell

Article Title: Cone-shaped HIV-1 capsids are transported through intact nuclear pores

doi: 10.1016/j.cell.2021.01.025

Figure Lengend Snippet:

Article Snippet: Human T lymphoblast cells SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from Robert Doms, University of Pennsylvania, USA; certified by Eurofins according to DAkkS ISO 9001:2008) were cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher Scientific) and 0.3 μg/ml puromycin (Merck).

Techniques: Virus, Recombinant, Plasmid Preparation, shRNA, Control, Software

Journal: bioRxiv

Article Title: Experimental estimation of the effects of all amino-acid mutations to HIV Env

doi: 10.1101/067470

Figure Lengend Snippet: ( A ) We created libraries of HIV proviral plasmids with random codon mutations in env, and generated mutant viruses by transfecting these plasmid libraries into 293T cells. Since cells receive multiple plasmids, there may not be a link between viral genotype and phenotype at this stage. To establish this link and select for functional variants, we passaged the viruses twice at low multiplicity of infection (MOI) in SupT1 cells. We deep sequenced env before and after selection to quantify the enrichment or depletion of each mutation, and used these data to estimate the preference of each site for each amino acid. Each mutant library was paired with a control in which cells were transfected with a wildtype HIV proviral plasmid to generate initially wildtype viruses that were passaged in parallel with the mutant viruses. Deep sequencing of these wildtype controls enabled estimation of the rates of apparent mutations arising from deep sequencing and viral replication. ( B ) We performed the entire experiment in triplicate. Additionally, we passaged the replicate-3 transfection supernatant in duplicate (replicate 3b). We also performed the second passage of replicate 3b in duplicate (replicates 3b-1 and 3b-2).

Article Snippet: We then infected 10 8 SupT1 cells with 5 × 10 5 TZM-bl units of the mutant library transfection supernatant in a final volume of 100 mL SupT1 culture medium in a vented tissue-culture flask (Fisher Scientific, 14-826-80).

Techniques: Generated, Mutagenesis, Plasmid Preparation, Functional Assay, Infection, Selection, Transfection, Sequencing

Journal: bioRxiv

Article Title: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

doi: 10.1101/2021.09.14.460218

Figure Lengend Snippet: (a) Activated CD4 + T cells were infected with HIV-1*CA14 SiR (MOI∼0.8) for 24 h before DMSO/PF74 treatment for 1 h, fixation, and methanol extraction. Samples were immunostained against CA (green) and laminA (blue). Images show a single z slice through the cell. Enlargements show the particle marked by the arrowhead. Scale bars: 10 µm (overview) and 1 µm (enlargement). (b) Data analyzed from the experiment outlined in (a). The graph shows the number of CA positive foci per nucleus in cells infected with HIV-1* (n=35 cells, mean=0.85) or HIV-1*CA14 SiR (n= 73 cells, mean=0.51). Pooled data from 6 different blood donors are shown. Grey lines show median and interquartile lines. (c) CA(SiR) intensities of nuclear objects in infected and activated CD4 + T cells at an MOI∼0.8 (n=13; mean=12,485 ± 7,445 a.u.) and an MOI ∼8 (n=7; mean=39,502 ± 18,025 a.u.). MOI was determined in TZM-bl cells. Grey lines show median and interquartile lines. (d-f) Nuclear cone-shaped capsids detected by CLEM-ET. SupT1 cells were treated with 1 µM aphidicolin (APC) for 16 h to prevent cell division, before infection with HIV-1*CA14 SiR virions (2.3 µU RT/cell, corresponds to an MOI∼0.4 determined in TZM-bl cells). At 24 h p.i., cells were cryo-immobilized by high-pressure freezing, freeze substituted, and further processed for CLEM and ET as described in materials and methods. (d) SDCM image of a 250-nm thick resin section of the cell infected with HIV-1*CA14 SiR virions (magenta), post-stained with Hoechst (blue) and decorated with multi-fluorescent fiducials (Fd) for correlation. The arrowhead in the enlargement of the boxed region indicates a CA(SiR) signal within the Hoechst-stained nuclear region. Scale bars: 1 µm (overview) and 200 nm (enlargement). (e) Computational slices through tomographic reconstructions at the correlated region boxed in (d) with views highlighting the presence of clustered capsid-reminiscent structures (black arrowheads) in the nuclear region. Nu, nucleus; NPC, nuclear pore complex; NE, nuclear envelope. Scale bar: 100 nm. (f) Segmented and isosurface rendered structure of the cones detected in (e). Magenta: capsid, yellow: NE, cyan: NPC. See also supplementary movie 1.

Article Snippet: SupT1 cells were distributed in a 96-well plate (2×10 5 cells/well; U-bottom; Greiner Bio-one, 650180) and pre-incubated for 16 h with 1µm aphidicolin (APC; Merck).

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of SupT1 cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Binding Assay, Activation Assay, Construct, Transduction, Co-Culture Assay, Control

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Optimization of TetCAR surface expression and signaling. ( a ) Schematic overview of TetCAR constructs containing 41BB-ζ or CD28-ζ endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis. P values = FMC63-BBz versus Fab-Tet-BBz (***, 0.0003) or Fab-Tet-28z (***, 0.0002), FMC63-Tet-BBz versus Fab-Tet-BBz (ns, 0.089), FMC63-Tet-28z versus Fab-Tet-28z (*, 0.045). ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) control, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Šidák’s multiple comparisons within each group ± minocycline. P values for each construct + /- minocycline were: FMC63-Tet-BBz (**, 0.0023), FMC63-Tet-28z (***, 0.0003) and Fab-Tet-28z (*, 0.0279). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the TetCAR groups at 0 nM minocycline (with Tukey’s multiple comparisons) or within these groups ± minocycline (with Šidák’s multiple comparisons). P values were; between FMC63-Tet-BBz and Fab-Tet-BBz (**, 0.0097, IFN-γ) and between FMC63-Tet-28z and Fab-Tet-28z (****, < 0.0001, IFN-γ and **, 0.0017, IL-2). P values between the TetCAR constructs ± minocycline were: FMC63-Tet-BBz (***, 0.0007, IFN-γ), FMC63-Tet-28z (*, 0.0409, IFN-γ), Fab-Tet-BBz (****, < 0.0001, IFN-γ and *, 0.0152, IL-2) and Fab-Tet-28z (****, < 0.0001 both IFN-γ and IL-2).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Construct, Transduction, Staining, Marker, Co-Culture Assay, Control

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Reconfiguration of endodomain positions enhances TetCAR function. ( a ) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41BB or CD28 endodomains, with a TIP-CD3ζ or TIP-41BB-CD3ζ domains. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 5 donors from 2 experiments. Statistical analysis was through one-way ANOVA between the groups, p values were; between FMC63-BBz and each Fab-TetCAR (****, < 0.0001). Differences between Fab-TetCARs alone were analyzed by one-way ANOVA but were not significant. ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; FMC-Tet-BBz (*, 0.0119). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = 28-Tet-z (*, 0.0484), 28BB-Fab-Tet-z and 28BB-Fab-Tet-BBz (****, < 0.0001). P values for IL-2 = 28-Tet-z (*, 0.0150), 28BB-Fab-Tet-z (****, < 0.0001), 28-Fab-Tet-BBz (**, 0.0029) and 28BB-Fab-Tet-BBz (***, 0.0007). ( g ) Killing of NALM6 after 48 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; BB-Fab-Tet-z (**, 0.0083), BB-Fab-Tet-BBz (*, 0.0189) and 28-Fab-Tet-z, 28BB-Fab-Tet-z, 28-Fab-Tet-BBz and 28BB-Fab-Tet-BBz (****, < 0.0001). ( h ) IFN-γ and I) IL-2 release after 48 h of co-culture with NALM6 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = BB-Fab-Tet-z (*, 0.0433), 28-Fab-Tet-z (*, 0.0387), 28BB-Fab-Tet-z (****, < 0.0001) and 28BB-Fab-Tet-BBz (**, 0.0020). P values for IL-2 = 28BB-Fab-Tet-z (*, 0.0450).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Construct, Membrane, Transduction, Staining, Marker, Expressing, Co-Culture Assay

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: TetCAR activity can be fine-tuned in vitro with a dose-dependent response to minocycline. ( a ) Killing of SupT1-CD19-GFP after 24 h of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600 nM were added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, ± SD. n = 4 donors from 1 experiment. ( b ) IFN-γ and ( c ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at various minocycline doses. Data shows mean ± SD, n = 4 donors from 1 experiment. ( d ) IL-2 secretion from FMC63-BBz or 28BB-Fab-Tet-z CARs 1–5 h after co-culture with SupT1-CD19-GFP at a 2:1 E:T ratio. 100 nM minocycline was added to separate wells every hour. Data shows the mean (± SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. Color coded bars indicate the number of hours that the co-cultures were exposed to minocycline for. n = 4 donors from 2 independent experiments. ( e ) Cytotoxicity or ( f ) IL-2 secretion by 28BB-Fab-Tet-z CARs after coculture with SupT1-CD19 at a 1:1 E:T ratio. Inhibition by minocycline was removed by washing cells with complete media at 48, 24 and 2 h before addition of SupT1-CD19 targets. Wash steps are indicated by “[W]”. Data shows mean (± SD) % of live targets relative to NT T cells ( e ) or mean (± SD) IL-2 secretion ( f ) after 24 h. n = 3 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with multiple comparisons between the 28BB-Fab-Tet-z CAR under different conditions. P values for cytotoxicity ( e ) were: 48 h wash versus no wash (*, 0.0118), 24 h wash versus no wash (**, 0.0050) and no drug versus no wash (**, 0.0044). P values for IL-2 secretion ( f ) were: 48 h wash versus no wash (**, 0.0015), no drug versus no wash (**, 0.0044), no drug versus 2 h wash (*, 0.0103) and 48 h wash versus 2 h wash (**, 0.0033).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Activity Assay, In Vitro, Co-Culture Assay, Control, Inhibition

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Effector function of CD28-containing TetCAR matches 41BBζ control CAR. ( a ) Killing of SupT1-CD19-GFP after 24 h or NALM6 after 48 h of co-culture with CAR-T cells at 1:1–1:32 E:T ratio. Data shows mean ± SD, n = 4 donors from 2 independent experiments. ( b ) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1:2 E:T ratio for 7 days. To relevant wells, 400 nM of minocycline was added on day 0. Graphs show mean (± SD) number of RQR8 + T cells (filled bars) or total CD3 + T cells (white bars) for each target. n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n = 3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group ± minocycline (with Šidák’s multiple comparisons). P values for SupT1-CD19 were 28-Fab-Tet-z (**, 0.0028) and 28BB-Fab-Tet-z (**, 0.0050). P values for Raji were 28-Fab-Tet-z (*, 0.0320) and 28BB-Fab-Tet-z (*, 0.0304). ( c ) Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows geometric mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for Lag3 expression were 28-Fab-Tet-z (**, 0.0078) and 28BB-Fab-Tet-z (*, 0.0207). ( d ) Percentage of naïve (CD62L + , CD45RA + ), Tcm (central memory; CD62L + , CD45RA - ), Tem (effector memory; CD62L - , CD45RA - ) or Temra (terminally differentiated effector memory; CD62L - , CD45RA + ) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments.

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Control, Co-Culture Assay, Incubation, Cell Culture, Expressing

Journal: PLoS Biology

Article Title: The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

doi: 10.1371/journal.pbio.3001569

Figure Lengend Snippet: (A) Chemical genetic strategy to orthogonally regulate XBP1s and ATF6 in SupT1 DAX cells. (B–D) RNA sequencing (RNA-Seq) analysis of the transcriptomic consequences of (B) XBP1s, (C) ATF6, and (D) XBP1s/ATF6 induction. Transcripts that were differentially expressed under each condition based on a >1.5-fold change in expression level (for dox-, TMP-, or dox- and TMP-treated versus vehicle-treated cells) and a non-adjusted p -value < 10 −10 are separated by dashed lines and plotted in red, with select transcripts labeled. The lowest nonzero p -value recorded was 10 −291 ; therefore, p -values equal to 0 were replaced with p -value = 1.00 × 10 −300 for plotting purposes. Transcripts for which p -values could not be calculated owing to extremely low expression or noisy count distributions were excluded from plotting. (E–G) Comparison of transcript fold change upon (E) +XBP1s versus +ATF6, (F) +ATF6 versus +XBP1s/+ATF6, and (G) +XBP1s versus +XBP1s/+ATF6 remodeling of the endoplasmic reticulum proteostasis network. Only transcripts with false-discovery-rate-adjusted p -value < 0.05 and fold increase > 1 in both of the indicated conditions are plotted. Dashed lines indicate a 1.5-fold filter to assign genes as selectively induced by the proteostasis condition on the x -axis (red), y -axis (blue), or lacking selectivity (purple). Transcripts with fold increase < 1.2 in either proteostasis environment are colored in grey to indicate low differential expression. The complete RNA-Seq differential expression analysis is provided in . dox, doxycycline; TMP, trimethoprim.

Article Snippet: With stably engineered SupT1 DAX cells in hand, we anticipated that we could create 4 distinct ER proteostasis environments (basal, XBP1s-induced, ATF6-induced, and XBP1s/ATF6 co-induced) to assess potential consequences for Env mutational tolerance.

Techniques: RNA Sequencing, Expressing, Labeling, Comparison, Quantitative Proteomics

Journal: PLoS Biology

Article Title: The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

doi: 10.1371/journal.pbio.3001569

Figure Lengend Snippet: (A) Scheme for deep mutational scanning of Env in 4 distinct ER proteostasis environments (basal, +XBP1s, +ATF6, and +XBP1s/+ATF6). SupT1 DAX cells were pretreated with DMSO (basal), dox (+XBP1s), TMP (+ATF6), or both dox and TMP (+XBP1s/+ATF6) 18 h prior to infection with biological triplicate Env viral libraries. 4 d post-infection, cells were harvested, and non-integrated viral DNA was sequenced to quantify the diffsel of Env variants. (B) Diffsel for each amino acid variant can be visualized in a sequence logo plot. The black horizontal lines at the center represent the diffsel for the wild-type amino acid at that site, and the height of the amino acid letter abbreviations is proportional to the diffsel of that variant in the remodeled ER proteostasis environment relative to the basal environment. Variants that are relatively enriched in the indicated ER proteostasis environment (positive diffsel) are located above the black horizontal line. Variants that are relatively depleted in the indicated ER proteostasis environment (negative diffsel) are located below the black horizontal line. (C) Net site diffsel for all Env sites in 3 perturbed ER proteostasis environments, averaged over biological triplicates. The black horizontal lines on the violin plots indicate the median (solid line) and the first and the third quartiles (dashed lines) of the distribution. The significance of deviation from null (net site diffsel = 0, no selection) was tested using a 1-sample t test, with 2-tailed p -values shown. The mean of the distribution and the number of sites with net site diffsel >0 or <0 are listed below the distribution. (D and E) Correlation for net site diffsel values for (D) +XBP1s/+ATF6 versus +XBP1s and (E) +XBP1s/+ATF6 versus +ATF6, normalized to the basal proteostasis environment. Pearson correlation coefficients ( r ) and corresponding p -values are shown. Select sites with highly positive or highly negative net site diffsel values in both proteostasis environments are marked in red and labeled with site numbers. (F) Diffsel for individual Env variants in 3 perturbed ER proteostasis environments, averaged over biological triplicates. The black horizontal lines on the violin plots indicate the median (solid line) and the first and the third quartiles (dashed lines) of the distribution. The significance of deviation from null (diffsel = 0, no selection) was tested using a 1-sample t test, with 2-tailed p -values shown. The mean of the distribution and the number of sites with diffsel >0 and <0 are listed below the distribution. Diffsel values (C–F) are provided at https://github.com/yoon-jimin/2021_HIV_Env_DMS . diffsel, differential selection; dox, doxycycline; ER, endoplasmic reticulum; TMP, trimethoprim; WT, wild-type.

Article Snippet: With stably engineered SupT1 DAX cells in hand, we anticipated that we could create 4 distinct ER proteostasis environments (basal, XBP1s-induced, ATF6-induced, and XBP1s/ATF6 co-induced) to assess potential consequences for Env mutational tolerance.

Techniques: Infection, Variant Assay, Sequencing, Selection, Labeling