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: Journal of Virology

Article Title: Variation of HIV-1 Mutation Spectra among Cell Types

doi: 10.1128/JVI.03576-12

Figure Lengend Snippet: Mutant frequency of HIV-1 among selected cell lines a

Article Snippet: A3C mRNA expression levels suggested a possible correlation with the prevalence of proviral sequences harboring multiple G-to-A mutations from each cell line (i.e., CEM-GFP > U373-MAGI > 293T > SupT1).

Techniques: Mutagenesis

Journal: Journal of Virology

Article Title: Variation of HIV-1 Mutation Spectra among Cell Types

doi: 10.1128/JVI.03576-12

Figure Lengend Snippet: Proportion of HIV-1 proviral sequences recovered per infected cell line analyzed possessing multiple G-to-A mutations in the HSA mutation target gene a

Article Snippet: A3C mRNA expression levels suggested a possible correlation with the prevalence of proviral sequences harboring multiple G-to-A mutations from each cell line (i.e., CEM-GFP > U373-MAGI > 293T > SupT1).

Techniques: Infection, Mutagenesis

Journal: Journal of Virology

Article Title: Variation of HIV-1 Mutation Spectra among Cell Types

doi: 10.1128/JVI.03576-12

Figure Lengend Snippet: G-to-A mutational load and mutation location in HIV-1 proviruses with multiple G-to-A mutations. (A) G-to-A mutational load in HIV-1 proviruses from CEM-GFP, U373-MAGI, and 293T cells. Each provirus with a mutant HSA sequence containing with multiple G-to-A mutations is indicated by a black circle (CEM-GFP), black square (MAGI), or black triangle (293T). The average G-to-A mutational load and standard deviation are indicated. (B) Location of G-to-A mutations in recovered proviruses harboring multiple G-to-A mutations. The red, green, and blue circles above G residues indicate the locations of G-to-A mutations (one circle per mutation identified) in proviruses recovered from CEM-GFP, U373-MAGI, and 293T, respectively. The start and stop codons of the HSA gene are identified by black rectangular boxes. (C) Quantitative RT-PCR was performed to determine the relative levels of APOBEC3 mRNA expression among the cell lines under investigation (i.e., CEM-GFP, SupT1, U373-MAGI, and 293T). The asterisk indicates that the APOBEC3C level from 293T cells was set to 1, and all other values are relative to this measurement. The mRNA expression levels were normalized to TATA-binding protein (TBP) mRNA levels. For these analyses, data with a difference in efficiencies between the APOBEC3 standard and TBP standard were ≤10% and R2 ≥0.98. Threshold cycle (CT) values were determined using the regression method, and data were analyzed by E−ΔCT. Experiments were conducted in triplicate with the standard deviation indicated.

Article Snippet: A3C mRNA expression levels suggested a possible correlation with the prevalence of proviral sequences harboring multiple G-to-A mutations from each cell line (i.e., CEM-GFP > U373-MAGI > 293T > SupT1).

Techniques: Mutagenesis, Sequencing, Standard Deviation, Quantitative RT-PCR, Expressing, Binding Assay

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a Immunoblot showing expression of selected cellular factors in CEM-M7 and SupT1-Cas9 cells with or without the indicated IFNs (1000 U/ml). Whole-cell lysates were immunoblotted and stained with antibodies against the indicated proteins. Representative image of n = 2 (independent experiments). b Percentage of eGFP positive cells indicating infected CEM-M7-Cas9 cells at 4 days post infection electroporated with either the NT (gray) or GRN (red) sgRNA and infected with WT NL4-3. Bars represent the mean of infected cells at 2dpi relative to the control (100%) of n = 3 ± SEM (independent experiments). (lower panel) Representative WB showing GRN KO efficiency. c HEK293T cells were cotransfected with increasing amounts of GRN expression construct and proviral mutants of NL4-3 (black) or CH077 (red) lacking indicated accessory genes. Infectious virus yield was measured using the TZM-bl reporter cell infectivity. Each point represents the mean of n = 3 ± SEM (independent experiments). (lower panel) Representative WB indicating expression of Env, p55, p24 and GRN in virus supernatants or cell lysates. d HEK293T cells were transiently transfected with a luciferase reporter controlled by the HIV-1 LTR and expression constructs for GRN (red) in presence and absence of NL4-3 Tat or a vector control (black). Bars represent the mean of n = 3 ± SEM (independent experiments). e HEK293T cells were transiently transfected with different amount of GRN expression constructs with proviral constructs of NL4-3_eGFP (dark red), CH077_eGFP (red) or CH058_eGFP (light red) as indicated. At 48 h post transfection cells were analyzed by flow cytometry and mean fluorescence intensities (MFI) of eGFP in the eGFP+/GRN+ population relative to vector control (100%) quantified. Bars represent the mean of n = 3 ± SEM (independent experiments). f Representative WB and quantification of GRN KO (red) in primary CD4 + T cells. NT, gray. Bars represent the mean of n = 3 ± SD (independent donors). g CD4 + T cells were electroporated with sgRNA targeting GRN (red) or NT (gray), infected with the indicated WT HIV-1 strains and infectious virus yields determined by TZM-bl assays at 2–6 dpi. Dots represent the mean of n = 6 (NL4-3) or n = 3 (CH077) ± SEM (independent donors). b – f Student´s t test with Welch´s correction, two-sided. g Two-way Anova with Sidak´s multiple comparison. * p < 0.05, ** p < 0.001, *** p < 0.0001. Raw p values are provided in Supplementary Data .

Article Snippet: CD4 + T-cell lines (CEM-M7 and SupT1) were transduced with the lentiviruses using spinoculation.

Techniques: Western Blot, Expressing, Staining, Infection, Control, Construct, Virus, Transfection, Luciferase, Plasmid Preparation, Flow Cytometry, Fluorescence, Comparison

Journal: Nature Communications

Article Title: Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

doi: 10.1038/s41467-024-48228-x

Figure Lengend Snippet: a HEK293T cells transiently transfected with increasing amount of either CIITA, CC2D1B or CEACAM3 expression constructs and indicated proviral constructs (NL4-3, CH077, CH058, CH042 in different shades of gray). Infectious virus yield was measured using the TZM-bl reporter cell infectivity. Dots represent the mean of three independent experiments ±SEM. b – d CD4 + T cells were electroporated with either the sgRNA targeting CEACAM3 (brown), CIITA (purple), CC2D1B (petrol), or the NT control, infected with the indicated WT HIV-1 strains and infectious virus yields measured from 2 to 6 dpi by TZM-bl infection assays. Dots represent the mean of n = 6 ( b ) ±SEM, n = 3 ( c ) ±SEM or n = 2 ( d ) (independent donors). Examples from primary data in Supplementary Figs. and . e Percentage of p24 antigen in the supernatants of CD4 + T cells electroporated with either the sgRNA targeting CC2D1B (petrol) or NT (gray) control at 3 days post infection with VSV-G pseudo-typed Δ env NL4-3 or CH077. Bars represent the mean of n = 4 (NL4-3) or n = 3 (CH077) ± SEM (independent experiments). (lower panel) Representative WB showing CC2D1B KO efficiency. a , e Student´s t test with Welch´s correction, two-sided. b – d Two-way Anova with Sidak´s multiple comparison. * p < 0.05, ** p < 0.001, *** p < 0.0001. Raw p values are provided in Supplementary Data .

Article Snippet: CD4 + T-cell lines (CEM-M7 and SupT1) were transduced with the lentiviruses using spinoculation.

Techniques: Transfection, Expressing, Construct, Virus, Infection, Control, Comparison